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Sample records for c-terminal alpha-thioesters based

  1. A Fmoc-compatible Method for the Solid-Phase Synthesis of Peptide C-Terminal (alpha)-Thioesters based on the Safety-Catch Hydrazine Linker

    Energy Technology Data Exchange (ETDEWEB)

    Camarero, J A; Hackel, B J; de Yoreo, J J; Mitchell, A R

    2003-11-22

    C-terminal peptide thioesters are key intermediates for the synthesis/semisynthesis of proteins and for the production of cyclic peptides by native chemical ligation. They can be synthetically prepared by solid-phase peptide synthesis (SPPS) methods or biosynthetically by protein splicing techniques. Until recently, the chemical synthesis of C-terminal a-thioester peptides by SPPS was largely restricted to the Boc/Benzyl methodology because of the poor stability of the thioester bond to the basic conditions employed for the deprotection of the N{sup {alpha}}-Fmoc group. In the present work, we describe a new method for the SPPS of C-terminal thioesters by Fmoc/t-Bu chemistry. This method is based on the use of an aryl hydrazide linker, which is totally stable to the Fmoc-SPPS conditions. Once the peptide synthesis has been completed, activation of the linker can be achieved by mild oxidation. This step transforms the hydrazide group into a highly reactive diazene intermediate which can react with different H-AA-SEt to yield the corresponding {alpha}-thioester peptide in good yields. This method has been successfully used for the generation of different thioester peptides, circular peptides and a fully functional SH3 protein domain.

  2. Synthesis of peptide .alpha.-thioesters

    Science.gov (United States)

    Camarero, Julio A [Livermore, CA; Mitchell, Alexander R [Livermore, CA; De Yoreo, James J [Clayton, CA

    2008-08-19

    Disclosed herein is a new method for the solid phase peptide synthesis (SPPS) of C-terminal peptide .alpha. thioesters using Fmoc/t-Bu chemistry. This method is based on the use of an aryl hydrazine linker, which is totally stable to conditions required for Fmoc-SPPS. When the peptide synthesis has been completed, activation of the linker is achieved by mild oxidation. The oxidation step converts the acyl-hydrazine group into a highly reactive acyl-diazene intermediate which reacts with an .alpha.-amino acid alkylthioester (H-AA-SR) to yield the corresponding peptide .alpha.-thioester in good yield. A variety of peptide thioesters, cyclic peptides and a fully functional Src homology 3 (SH3) protein domain have been successfully prepared.

  3. C-Terminal acetylene derivatized peptides via silyl-based alkyne immobilization.

    Science.gov (United States)

    Strack, Martin; Metzler-Nolte, Nils; Albada, H Bauke

    2013-06-21

    A new Silyl-based Alkyne Modifying (SAM)-linker for the synthesis of C-terminal acetylene-derivatized peptides is reported. The broad scope of this SAM2-linker is illustrated by manual synthesis of peptides that are side-chain protected, fully deprotected, and disulfide-bridged. Synthesis of a 14-meric (KLAKLAK)2 derivative by microwave-assisted automated SPPS and a one-pot cleavage click procedure yielding protected 1,2,3-triazole peptide conjugates are also described.

  4. Self-assemble nanoparticles based on polypeptides containing C-terminal luminescent Pt-cysteine complex

    Science.gov (United States)

    Vlakh, E. G.; Grachova, E. V.; Zhukovsky, D. D.; Hubina, A. V.; Mikhailova, A. S.; Shakirova, J. R.; Sharoyko, V. V.; Tunik, S. P.; Tennikova, T. B.

    2017-02-01

    The growing attention to the luminescent nanocarriers is strongly stimulated by their potential application as drug delivery systems and by the necessity to monitor their distribution in cells and tissues. In this communication we report on the synthesis of amphiphilic polypeptides bearing C-terminal phosphorescent label together with preparation of nanoparticles using the polypeptides obtained. The approach suggested is based on a unique and highly technological process where the new phosphorescent Pt-cysteine complex serves as initiator of the ring-opening polymerization of α-amino acid N-carboxyanhydrides to obtain the polypeptides bearing intact the platinum chromophore covalently bound to the polymer chain. It was established that the luminescent label retains unchanged its emission characteristics not only in the polypeptides but also in more complicated nanoaggregates such as the polymer derived amphiphilic block-copolymers and self-assembled nanoparticles. The phosphorescent nanoparticles display no cytotoxicity and hemolytic activity in the tested range of concentrations and easily internalize into living cells that makes possible in vivo cell visualization, including prospective application in time resolved imaging and drug delivery monitoring.

  5. MAS C-Terminal Tail Interacting Proteins Identified by Mass Spectrometry- Based Proteomic Approach.

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    Kalyan C Tirupula

    Full Text Available Propagation of signals from G protein-coupled receptors (GPCRs in cells is primarily mediated by protein-protein interactions. MAS is a GPCR that was initially discovered as an oncogene and is now known to play an important role in cardiovascular physiology. Current literature suggests that MAS interacts with common heterotrimeric G-proteins, but MAS interaction with proteins which might mediate G protein-independent or atypical signaling is unknown. In this study we hypothesized that MAS C-terminal tail (Ct is a major determinant of receptor-scaffold protein interactions mediating MAS signaling. Mass-spectrometry based proteomic analysis was used to comprehensively identify the proteins that interact with MAS Ct comprising the PDZ-binding motif (PDZ-BM. We identified both PDZ and non-PDZ proteins from human embryonic kidney cell line, mouse atrial cardiomyocyte cell line and human heart tissue to interact specifically with MAS Ct. For the first time our study provides a panel of PDZ and other proteins that potentially interact with MAS with high significance. A 'cardiac-specific finger print' of MAS interacting PDZ proteins was identified which includes DLG1, MAGI1 and SNTA. Cell based experiments with wild-type and mutant MAS lacking the PDZ-BM validated MAS interaction with PDZ proteins DLG1 and TJP2. Bioinformatics analysis suggested well-known multi-protein scaffold complexes involved in nitric oxide signaling (NOS, cell-cell signaling of neuromuscular junctions, synapses and epithelial cells. Majority of these protein hits were predicted to be part of disease categories comprising cancers and malignant tumors. We propose a 'MAS-signalosome' model to stimulate further research in understanding the molecular mechanism of MAS function. Identifying hierarchy of interactions of 'signalosome' components with MAS will be a necessary step in future to fully understand the physiological and pathological functions of this enigmatic receptor.

  6. MAS C-Terminal Tail Interacting Proteins Identified by Mass Spectrometry- Based Proteomic Approach.

    Science.gov (United States)

    Tirupula, Kalyan C; Zhang, Dongmei; Osbourne, Appledene; Chatterjee, Arunachal; Desnoyer, Russ; Willard, Belinda; Karnik, Sadashiva S

    2015-01-01

    Propagation of signals from G protein-coupled receptors (GPCRs) in cells is primarily mediated by protein-protein interactions. MAS is a GPCR that was initially discovered as an oncogene and is now known to play an important role in cardiovascular physiology. Current literature suggests that MAS interacts with common heterotrimeric G-proteins, but MAS interaction with proteins which might mediate G protein-independent or atypical signaling is unknown. In this study we hypothesized that MAS C-terminal tail (Ct) is a major determinant of receptor-scaffold protein interactions mediating MAS signaling. Mass-spectrometry based proteomic analysis was used to comprehensively identify the proteins that interact with MAS Ct comprising the PDZ-binding motif (PDZ-BM). We identified both PDZ and non-PDZ proteins from human embryonic kidney cell line, mouse atrial cardiomyocyte cell line and human heart tissue to interact specifically with MAS Ct. For the first time our study provides a panel of PDZ and other proteins that potentially interact with MAS with high significance. A 'cardiac-specific finger print' of MAS interacting PDZ proteins was identified which includes DLG1, MAGI1 and SNTA. Cell based experiments with wild-type and mutant MAS lacking the PDZ-BM validated MAS interaction with PDZ proteins DLG1 and TJP2. Bioinformatics analysis suggested well-known multi-protein scaffold complexes involved in nitric oxide signaling (NOS), cell-cell signaling of neuromuscular junctions, synapses and epithelial cells. Majority of these protein hits were predicted to be part of disease categories comprising cancers and malignant tumors. We propose a 'MAS-signalosome' model to stimulate further research in understanding the molecular mechanism of MAS function. Identifying hierarchy of interactions of 'signalosome' components with MAS will be a necessary step in future to fully understand the physiological and pathological functions of this enigmatic receptor.

  7. Small epitope-linker modules for PCR-based C-terminal tagging in Saccharomyces cerevisiae.

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    Funakoshi, Minoru; Hochstrasser, Mark

    2009-03-01

    PCR-mediated gene modification is a powerful approach to the functional analysis of genes in Saccharomyces cerevisiae. One application of this method is epitope-tagging of a gene to analyse the corresponding protein by immunological methods. However, the number of epitope tags available in a convenient format is still low, and interference with protein function by the epitope, particularly if it is large, is not uncommon. To address these limitations and broaden the utility of the method, we constructed a set of convenient template plasmids designed for PCR-based C-terminal tagging with 10 different, relatively short peptide sequences that are recognized by commercially available monoclonal antibodies. The encoded tags are FLAG, 3 x FLAG, T7, His-tag, Strep-tag II, S-tag, Myc, HSV, VSV-G and V5. The same pair of primers can be used to construct tagged alleles of a gene of interest with any of the 10 tags. In addition, a six-glycine linker sequence is inserted upstream of these tags to minimize the influence of the tag on the target protein and maximize its accessibility for antibody binding. Three marker genes, HIS3MX6, kanMX6 and hphMX4, are available for each epitope. We demonstrate the utility of the new tags for both immunoblotting and one-step affinity purification of the regulatory particle of the 26S proteasome. The set of plasmids has been deposited in the non-profit plasmid repository Addgene (http://www.addgene.org).

  8. Silyl-based alkyne-modifying linker for the preparation of C-terminal acetylene-derivatized protected peptides.

    Science.gov (United States)

    Strack, Martin; Langklotz, Sina; Bandow, Julia E; Metzler-Nolte, Nils; Albada, H Bauke

    2012-11-16

    A novel linker for the synthesis of C-terminal acetylene-functionalized protected peptides is described. This SAM1 linker is applied in the manual Fmoc-based solid-phase peptide synthesis of Leu-enkephalin and in microwave-assisted automated synthesis of Maculatin 2.1, an antibacterial peptide that contains 18 amino acid residues. For the cleavage, treatment with tetramethylammonium fluoride results in protected acetylene-derivatized peptides. Alternatively, a one-pot cleavage-click procedure affords the protected 1,2,3-triazole conjugate in high yields after purification.

  9. NMR-based homology model for the solution structure of the C-terminal globular domain of EMILIN1

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    Verdone, Giuliana [Istituto Biochimico Italiano ' G. Lorenzini' (Italy); Corazza, Alessandra [Universita di Udine, Dipartimento di Scienze e Tecnologie Biomediche - MATI Centre of Excellence (Italy); Colebrooke, Simon A. [University of Oxford, Department of Biochemistry (United Kingdom); Cicero, Daniel; Eliseo, Tommaso [Universita di Tor Vergata, Dipartimento di Chimica (Italy); Boyd, Jonathan [University of Oxford, Department of Biochemistry (United Kingdom); Doliana, Roberto [Centro di Riferimento Oncologico di Aviano, Divisione di Oncologia Sperimentale 2 (Italy); Fogolari, Federico; Viglino, Paolo; Colombatti, Alfonso [Universita di Udine, Dipartimento di Scienze e Tecnologie Biomediche - MATI Centre of Excellence (Italy); Campbell, Iain D. [University of Oxford, Department of Biochemistry (United Kingdom); Esposito, Gennaro [Universita di Udine, Dipartimento di Scienze e Tecnologie Biomediche - MATI Centre of Excellence (Italy)], E-mail: gesposito@mail.dstb.uniud.it

    2009-02-15

    EMILIN1 is a glycoprotein of elastic tissues that has been recently linked to the pathogenesis of hypertension. The protein is formed by different independently folded structural domains whose role has been partially elucidated. In this paper the solution structure, inferred from NMR-based homology modelling of the C-terminal trimeric globular C1q domain (gC1q) of EMILIN1, is reported. The high molecular weight and the homotrimeric structure of the protein required the combined use of highly deuterated {sup 15}N, {sup 13}C-labelled samples and TROSY experiments. Starting from a homology model, the protein structure was refined using heteronuclear residual dipolar couplings, chemical shift patterns, NOEs and H-exchange data. Analysis of the gC1q domain structure of EMILIN1 shows that each protomer of the trimer adopts a nine-stranded {beta} sandwich folding topology which is related to the conformation observed for other proteins of the family. Distinguishing features, however, include a missing edge-strand and an unstructured 19-residue loop. Although the current data do not allow this loop to be precisely defined, the available evidence is consistent with a flexible segment that protrudes from each subunit of the globular trimeric assembly and plays a key role in inter-molecular interactions between the EMILIN1 gC1q homotrimer and its integrin receptor {alpha}4{beta}1.

  10. Comparison of Peritoneal Low-Molecular-Weight-Protein-Removal in CCPD and CAPD Patients Based on C-Terminal Agrin Fragment Clearance

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    Dominik Steubl

    2016-03-01

    Full Text Available Background/Aims: This study compares the peritoneal elimination of the low-molecular-weight-protein (LMWP C-terminal agrin fragment (tCAF, size 22 kDa, a promising biomarker for kidney function, in continuous cycling peritoneal dialysis (CCPD and continuous ambulatory peritoneal dialysis (CAPD. Methods: 103 sets of serum, 24h-urine and dialysate samples were obtained in 15 CCPD (63 sets and 11 CAPD (40 sets patients. Total, renal and peritoneal substrate removals/clearances were measured/calculated for tCAF, creatinine, blood-urea-nitrogen (BUN, cystatin C and albumin and correlated with the peritoneal transport type. Results: Serum und urine concentrations of all biomarkers did not differ between both groups, urinary substrate removal was higher in CAPD patients for all biomarkers due to better residual renal function. Peritoneal substrate removal of tCAF and albumin were significantly higher in CAPD (tCAF: 35.3 vs. 19.3 µg/d, p2 body-surface-area, pConclusions: CAPD was superior to CCPD concerning peritoneal tCAF removal. This finding was pronounced in high-transporters and CAPD patients using icodextrin twice daily.

  11. Modules for C-terminal epitope tagging of Tetrahymena genes

    OpenAIRE

    Kataoka, Kensuke; Schoeberl, Ursula E.; Mochizuki, Kazufumi

    2010-01-01

    Although epitope tagging has been widely used for analyzing protein function in many organisms, there are few genetic tools for epitope tagging in Tetrahymena. In this study, we describe several C-terminal epitope tagging modules that can be used to express tagged proteins in Tetrahymena cells by both plasmid- and PCR-based strategies.

  12. Modules for C-terminal epitope tagging of Tetrahymena genes.

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    Kataoka, Kensuke; Schoeberl, Ursula E; Mochizuki, Kazufumi

    2010-09-01

    Although epitope tagging has been widely used for analyzing protein function in many organisms, there are few genetic tools for epitope tagging in Tetrahymena. In this study, we describe several C-terminal epitope tagging modules that can be used to express tagged proteins in Tetrahymena cells by both plasmid- and PCR-based strategies. (c) 2010 Elsevier B.V. All rights reserved.

  13. Plasmids with E2 epitope tags: tagging modules for N- and C-terminal PCR-based gene targeting in both budding and fission yeast, and inducible expression vectors for fission yeast.

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    Tamm, Tiina

    2009-01-01

    A single-step PCR-based epitope tagging enables fast and efficient gene targeting with various epitope tags. This report presents a series of plasmids for the E2 epitope tagging of proteins in Saccharomyces cerevisiae and Schizosaccharomyces pombe. E2Tags are 10-amino acids (epitope E2a: SSTSSDFRDR)- and 12 amino acids (epitope E2b: GVSSTSSDFRDR)-long peptides derived from the E2 protein of bovine papillomavirus type 1. The modules for C-terminal tagging with E2a and E2b epitopes were constructed by the modification of the pYM-series plasmid. The N-terminal E2a and E2b tagging modules were based on pOM-series plasmid. The pOM-series plasmids were selected for this study because of their use of the Cre-loxP recombination system. The latter enables a marker cassette to be removed after integration into the loci of interest and, thereafter, the tagged protein is expressed under its endogenous promoter. Specifically for fission yeast, high copy pREP plasmids containing the E2a epitope tag as an N-terminal or C-terminal tag were constructed. The properties of E2a and E2b epitopes and the sensitivity of two anti-E2 monoclonal antibodies (5E11 and 3F12) were tested using several S. cerevisiae and Sz. pombe E2-tagged strains.

  14. C-terminal functionalization of nylon-3 polymers: effects of C-terminal groups on antibacterial and hemolytic activities.

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    Zhang, Jihua; Markiewicz, Matthew J; Mowery, Brendan P; Weisblum, Bernard; Stahl, Shannon S; Gellman, Samuel H

    2012-02-13

    Nylon-3 polymers contain β-amino-acid-derived subunits and can be viewed as higher homologues of poly(α-amino acids). This structural relationship raises the possibility that nylon-3 polymers offer a platform for development of new materials with a variety of biological activities, a prospect that has recently begun to receive experimental support. Nylon-3 homo- and copolymers can be prepared via anionic ring-opening polymerization of β-lactams, and use of an N-acyl-β-lactam as coinitiator in the polymerization reaction allows placement of a specific functional group, borne by the N-acyl-β-lactam, at the N-terminus of each polymer chain. Controlling the unit at the C-termini of nylon-3 polymer chains, however, has been problematic. Here we describe a strategy for specifying C-terminal functionality that is based on the polymerization mechanism. After the anionic ring-opening polymerization is complete, we introduce a new β-lactam, approximately 1 equiv relative to the expected number of polymer chains. Because the polymer chains bear a reactive imide group at their C-termini, this new β-lactam should become attached at this position. If the terminating β-lactam bears a distinctive functional group, that functionality should be affixed to most or all C-termini in the reaction mixture. We use the new technique to compare the impact of N- and C-terminal placement of a critical hydrophobic fragment on the biological activity profile of nylon-3 copolymers. The synthetic advance described here should prove to be generally useful for tailoring the properties of nylon-3 materials.

  15. Updating the profile of C-terminal MECP2 deletions in Rett syndrome

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    Bebbington, A; Percy, A; Christodoulou, J; Ravine, D; Ho, G; Jacoby, P; Anderson, A; Pineda, M; Ben Zeev, B; Bahi-Buisson, N; Smeets, E; Leonard, H

    2014-01-01

    Objectives This study aimed to compare the phenotype of Rett syndrome cases with C-terminal deletions to that of cases with different MECP2 mutations and to examine the phenotypic variation within C-terminal deletions. Methods Cases were selected from InterRett, an international database and from the population-based Australian Rett Syndrome Database. Cases (n=832) were included if they had a pathogenic MECP2 mutation in which the nature of the amino acid change was known. Three severity scale systems were used, and individual aspects of the phenotype were also compared. Results Lower severity was associated with C-terminal deletions (n=79) compared to all other MECP2 mutations (e.g. Pineda scale C-terminals mean 15.0 (95% CI 14.0–16.0) vs 16.2 (15.9–16.5). Cases with C-terminal deletions were more likely to have a normal head circumference (odds ratio 3.22, 95% CI 1.53 – 6.79) and weight (odds ratio 2.97, 95% CI 1.25–5.76). Onset of stereotypies tended to be later (median age 2.5 years vs 2 years, pRett syndrome. PMID:19914908

  16. SPAN C - Terminal sterilization process analysis program

    Science.gov (United States)

    1969-01-01

    Computer program, SPAN-C, measures the dry heat thermal sterilization process applied to a planetary capsule and calculates the time required for heat application, steady state conditions, and cooling. The program is based on the logarithmic survival of micro-organisms. Temperature profiles must be input on cards.

  17. General inverse solid-phase synthesis method for C-terminally modified peptide mimetics.

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    Sasubilli, Ramakrishna; Gutheil, William G

    2004-01-01

    Peptide mimetics are of considerable interest as bioactive agents and drugs. C-terminally modified peptide mimetics are of particular interest given the synthetic versatility of the carboxyl group and its derivatives. A general approach to C-terminally modified peptide mimetics, based on a urethane attachment strategy and amino acid t-butyl ester-based N-to-C peptide synthesis, is described. This approach is compatible with the reaction conditions generally employed for solution-phase peptide mimetic synthesis. To develop and demonstrate this approach, it was employed for the solid-phase synthesis of peptide trifluoromethyl ketones, peptide boronic acids, and peptide hydroxamic acids. The development of a versatile general approach to C-terminally modified peptides using readily available starting materials provides a basis for the combinatorial and parallel solid-phase synthesis of these peptide mimetic classes for bioactive agent screening and also provides a basis for the further development of solid-phase C-terminal functional group elaboration strategies.

  18. Efficient, chemoselective synthesis of immunomicelles using single-domain antibodies with a C-terminal thioester

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    Raats Jos MH

    2009-07-01

    Full Text Available Abstract Background Classical bioconjugation strategies for generating antibody-functionalized nanoparticles are non-specific and typically result in heterogeneous compounds that can be compromised in activity. Expression systems based on self-cleavable intein domains allow the generation of recombinant proteins with a C-terminal thioester, providing a unique handle for site-specific conjugation using native chemical ligation (NCL. However, current methods to generate antibody fragments with C-terminal thioesters require cumbersome refolding procedures, effectively preventing application of NCL for antibody-mediated targeting and molecular imaging. Results Targeting to the periplasm of E. coli allowed efficient production of correctly-folded single-domain antibody (sdAb-intein fusions proteins. On column purification and 2-mercapthoethanesulfonic acid (MESNA-induced cleavage yielded single-domain antibodies with a reactive C-terminal MESNA thioester in good yields. These thioester-functionalized single-domain antibodies allowed synthesis of immunomicelles via native chemical ligation in a single step. Conclusion A novel procedure was developed to obtain soluble, well-folded single-domain antibodies with reactive C-terminal thioesters in good yields. These proteins are promising building blocks for the chemoselective functionalization via NCL of a broad range of nanoparticle scaffolds, including micelles, liposomes and dendrimers.

  19. Conserved C-terminal nascent peptide binding domain of HYPK ...

    Indian Academy of Sciences (India)

    Amino acid sequence analysis revealed 105 orthologs of human HYPK from plants, lower invertebrates to mammals. C-terminal part of HYPK was found to be particularly conserved and to contain nascent polypeptide-associated alpha subunit (NPAA) domain. This region experiences highest selection pressure, signifying ...

  20. Isolation of influenza virus A hemagglutinin C-terminal domain by hemagglutinin proteolysis in octylglucoside micelles.

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    Radyukhin, Victor A; Serebryakova, Marina V; Ksenofontov, Alexander L; Lukashina, Elena V; Baratova, Lyudmila A

    2006-01-01

    A method of isolation of hydrophobic membrane-bound C-terminal domain of influenza virus A hemagglutinin (HA) is suggested. The method is based on the insertion of HA into octylglucoside micelles followed by pepsin or thermolysin hydrolysis. Subsequent treatment of proteolytic digests with chloroform-hexafluoroisopropanol mixture resulted in the extraction of a few hydrophobic peptides into organic phase. Mass-spectrometry (MALDI-TOF) analysis revealed that the peptides with ion masses corresponding to the anchoring C-terminal domain with or without modifications predominated in the organic solution. The data obtained confirmed our speculation on the possibility of the suggested isolation scheme following from the strong interactions of anchoring domains in compact trimeric structure of HA spikes combined with micelle protection effect. Several appropriate peptides presence in the organic phase apparently arises from the presence of a few accessible proteolytic sites in HA transmembrane region.

  1. C-terminal region of MAP7 domain containing protein 3 (MAP7D3 promotes microtubule polymerization by binding at the C-terminal tail of tubulin.

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    Saroj Yadav

    Full Text Available MAP7 domain containing protein 3 (MAP7D3, a newly identified microtubule associated protein, has been shown to promote microtubule assembly and stability. Its microtubule binding region has been reported to consist of two coiled coil motifs located at the N-terminus. It possesses a MAP7 domain near the C-terminus and belongs to the microtubule associated protein 7 (MAP7 family. The MAP7 domain of MAP7 protein has been shown to bind to kinesin-1; however, the role of MAP7 domain in MAP7D3 remains unknown. Based on the bioinformatics analysis of MAP7D3, we hypothesized that the MAP7 domain of MAP7D3 may have microtubule binding activity. Indeed, we found that MAP7 domain of MAP7D3 bound to microtubules as well as enhanced the assembly of microtubules in vitro. Interestingly, a longer fragment MDCT that contained the MAP7 domain (MD with the C-terminal tail (CT of the protein promoted microtubule polymerization to a greater extent than MD and CT individually. MDCT stabilized microtubules against dilution induced disassembly. MDCT bound to reconstituted microtubules with an apparent dissociation constant of 3.0 ± 0.5 µM. An immunostaining experiment showed that MDCT localized along the length of the preassembled microtubules. Competition experiments with tau indicated that MDCT shares its binding site on microtubules with tau. Further, we present evidence indicating that MDCT binds to the C-terminal tail of tubulin. In addition, MDCT could bind to tubulin in HeLa cell extract. Here, we report a microtubule binding region in the C-terminal region of MAP7D3 that may have a role in regulating microtubule assembly dynamics.

  2. A vector system for efficient and economical switching of C-terminal epitope tags in Saccharomyces cerevisiae.

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    Sung, Min-Kyung; Ha, Cheol Woong; Huh, Won-Ki

    2008-04-01

    In Saccharomyces cerevisiae, one-step PCR-mediated modification of chromosomal genes allows fast and efficient tagging of yeast proteins with various epitopes at the C- or N-terminus. For many purposes, C-terminal tagging is advantageous in that the expression pattern of epitope tag is comparable to that of the authentic protein and the possibility for the tag to affect normal folding of polypeptide chain during translation is minimized. As experiments are getting complicated, it is often necessary to construct several fusion proteins tagged with various kinds of epitopes. Here, we describe development of a series of plasmids that allow efficient and economical switching of C-terminally tagged epitopes, using just one set of universal oligonucleotide primers. Containing a variety of epitopes (GFP, TAP, GST, Myc, HA and FLAG tag) and Kluyveromyces lactis URA3 gene as a selectable marker, the plasmids can be used to replace any MX6 module-based C-terminal epitope tag with one of the six epitopes. Furthermore, the plasmids also allow additional C-terminal epitope tagging of proteins in yeast cells that already carry MX6 module-based gene deletion or C-terminal epitope tag. (c) 2008 John Wiley & Sons, Ltd.

  3. A C-terminal Membrane Anchor Affects the Interactions of Prion Proteins with Lipid Membranes*

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    Chu, Nam K.; Shabbir, Waheed; Bove-Fenderson, Erin; Araman, Can; Lemmens-Gruber, Rosa; Harris, David A.; Becker, Christian F. W.

    2014-01-01

    Membrane attachment via a C-terminal glycosylphosphatidylinositol anchor is critical for conversion of PrPC into pathogenic PrPSc. Therefore the effects of the anchor on PrP structure and function need to be deciphered. Three PrP variants, including full-length PrP (residues 23–231, FL_PrP), N-terminally truncated PrP (residues 90–231, T_PrP), and PrP missing its central hydrophobic region (Δ105–125, ΔCR_PrP), were equipped with a C-terminal membrane anchor via a semisynthesis strategy. Analyses of the interactions of lipidated PrPs with phospholipid membranes demonstrated that C-terminal membrane attachment induces a different binding mode of PrP to membranes, distinct from that of non-lipidated PrPs, and influences the biochemical and conformational properties of PrPs. Additionally, fluorescence-based assays indicated pore formation by lipidated ΔCR_PrP, a variant that is known to be highly neurotoxic in transgenic mice. This finding was supported by using patch clamp electrophysiological measurements of cultured cells. These results provide new evidence for the role of the membrane anchor in PrP-lipid interactions, highlighting the importance of the N-terminal and the central hydrophobic domain in these interactions. PMID:25217642

  4. A C-terminal membrane anchor affects the interactions of prion proteins with lipid membranes.

    Science.gov (United States)

    Chu, Nam K; Shabbir, Waheed; Bove-Fenderson, Erin; Araman, Can; Lemmens-Gruber, Rosa; Harris, David A; Becker, Christian F W

    2014-10-24

    Membrane attachment via a C-terminal glycosylphosphatidylinositol anchor is critical for conversion of PrP(C) into pathogenic PrP(Sc). Therefore the effects of the anchor on PrP structure and function need to be deciphered. Three PrP variants, including full-length PrP (residues 23-231, FL_PrP), N-terminally truncated PrP (residues 90-231, T_PrP), and PrP missing its central hydrophobic region (Δ105-125, ΔCR_PrP), were equipped with a C-terminal membrane anchor via a semisynthesis strategy. Analyses of the interactions of lipidated PrPs with phospholipid membranes demonstrated that C-terminal membrane attachment induces a different binding mode of PrP to membranes, distinct from that of non-lipidated PrPs, and influences the biochemical and conformational properties of PrPs. Additionally, fluorescence-based assays indicated pore formation by lipidated ΔCR_PrP, a variant that is known to be highly neurotoxic in transgenic mice. This finding was supported by using patch clamp electrophysiological measurements of cultured cells. These results provide new evidence for the role of the membrane anchor in PrP-lipid interactions, highlighting the importance of the N-terminal and the central hydrophobic domain in these interactions. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. The effect of C-terminal amidation on the efficacy and selectivity of antimicrobial and anticancer peptides.

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    Dennison, Sarah Rachel; Harris, Frederick; Bhatt, Tailap; Singh, Jaipaul; Phoenix, David Andrew

    2009-12-01

    Cationic defence peptides show high therapeutic potential as antimicrobial and anticancer agents. Some of these peptides carry a C-terminal amide moiety which has been shown to be required for antimicrobial activity. However, whether this is a general requirement or whether C-terminal amidation is required for the anticancer activity of defence peptides is unclear. In response, this study analyses the toxicity of a series of C-terminally amidated defence peptides and their non-amidated isoforms to normal fibroblast cells, a variety of tumour cells and bacterial cells. The toxicities of these peptides to microbial and cancer cells were generally <200 microM. Peptides were either unaffected by C-terminal amidation or showed up to 10-fold decreases or increases in efficacy. However, these peptides all showed toxicity to normal fibroblast cells with levels (generally <150 microM) that were comparable to those of their antimicrobial and anticancer activities. In contrast to previous claims which have been based on analysis of single amidation events, the results of this study clearly show that the C-terminal amidation of defence peptides has a variable effect on their antimicrobial and anticancer efficacy and no clear effect on their selectivity for these cell types.

  6. Order of amino acids in C-terminal cysteine-containing peptide-based chelators influences cellular processing and biodistribution of 99mTc-labeled recombinant Affibody molecules.

    Science.gov (United States)

    Altai, Mohamed; Wållberg, Helena; Orlova, Anna; Rosestedt, Maria; Hosseinimehr, Seyed Jalal; Tolmachev, Vladimir; Ståhl, Stefan

    2012-05-01

    Affibody molecules constitute a novel class of molecular display selected affinity proteins based on non-immunoglobulin scaffold. Preclinical investigations and pilot clinical data have demonstrated that Affibody molecules provide high contrast imaging of tumor-associated molecular targets shortly after injection. The use of cysteine-containing peptide-based chelators at the C-terminus of recombinant Affibody molecules enabled site-specific labeling with the radionuclide 99mTc. Earlier studies have demonstrated that position, composition and the order of amino acids in peptide-based chelators influence labeling stability, cellular processing and biodistribution of Affibody molecules. To investigate the influence of the amino acid order, a series of anti-HER2 Affibody molecules, containing GSGC, GEGC and GKGC chelators have been prepared and characterized. The affinity to HER2, cellular processing of 99mTc-labeled Affibody molecules and their biodistribution were investigated. These properties were compared with that of the previously studied 99mTc-labeled Affibody molecules containing GGSC, GGEC and GGKC chelators. All variants displayed picomolar affinities to HER2. The substitution of a single amino acid in the chelator had an appreciable influence on the cellular processing of 99mTc. The biodistribution of all 99mTc-labeled Affibody molecules was in general comparable, with the main difference in uptake and retention of radioactivity in excretory organs. The hepatic accumulation of radioactivity was higher for the lysine-containing chelators and the renal retention of 99mTc was significantly affected by the amino acid composition of chelators. The order of amino acids influenced renal uptake of some conjugates at 1 h after injection, but the difference decreased at later time points. Such information can be helpful for the development of other scaffold protein-based imaging and therapeutic radiolabeled conjugates.

  7. Epimerization-free C-terminal peptide activation, elongation and cyclization

    NARCIS (Netherlands)

    Popović, S.

    2015-01-01

    C-terminal peptide activation and cyclization reactions are generally accompanied with epimerization (partial loss of C‐terminal stereointegrity). Therefore, the focus of this thesis was to develop epimerization-free methods for C-terminal peptide activation to enable C-terminal peptide elongation

  8. C-Terminal 23 kDa polypeptide of soybean Gly m Bd 28 K is a potential allergen.

    Science.gov (United States)

    Xiang, Ping; Haas, Eric J; Zeece, Michael G; Markwell, John; Sarath, Gautam

    2004-11-01

    Gly m Bd 28 K is a major soybean (Glycine max Merr.) glycoprotein allergen. It was originally identified as a 28 kDa polypeptide in soybean seed flour. However, the full-length protein is encoded by an open reading frame (ORF) of 473 amino acids, and contains a 23 kDa C-terminal polypeptide of as yet unknown allergenic and structural characteristics. IgE-binding (allergenic potential) of the Gly m Bd 28 K protein including the 23 kDa C-terminal portion as well as shorter fragments derived from the full-length ORF were evaluated using sera from soy-sensitive adults. All of these sera contained IgE that efficiently recognized the C-terminal region. Epitope mapping demonstrated that a dominant linear C-terminal IgE binding epitope resides between residues S256 and A270. Alanine scanning of this dominant epitope indicated that five amino acids, Y260, D261, D262, K264 and D266, contribute most towards IgE-binding. A model based on the structure of the beta subunit of soybean beta-conglycinin revealed that Gly m Bd 28 K contains two cupin domains. The dominant epitope is on the edge of the first beta-sheet of the C-terminal cupin domain and is present on a potentially solvent-accessible loop connecting the two cupin domains. Thus, the C-terminal 23 kDa polypeptide of Gly m Bd 28 K present in soy products is allergenic and apparently contains at least one immunodominant epitope near the edge of a cupin domain. This knowledge could be helpful in the future breeding of hypoallergenic soybeans.

  9. Paramembranous densities of 'C' terminal-motoneuron synapses in the spinal cord of the rat

    DEFF Research Database (Denmark)

    Schrøder, H D

    1979-01-01

    A category of large boutons forming synapses with the soma and proximal dendrites of spinal motoneurons was studied in glutaraldehyde-fixed, non-osmicated tissue stained with uranyl acetate and lead citrate. The identity of these boutons with 'C' boutons was indicated by their shape, frequency...... and distribution as well as by the ultrastructural characteristics of the boutons and the associated postsynaptic structures. In contrast to previous descriptions based on osmicated tissue, this study demonstrates that paramembranous densities are a feature of 'C' terminal-motoneuron synapses....

  10. One-step refolding and purification of disulfide-containing proteins with a C-terminal MESNA thioester

    Directory of Open Access Journals (Sweden)

    Merkx Maarten

    2008-10-01

    Full Text Available Abstract Background Expression systems based on self-cleavable intein domains allow the generation of recombinant proteins with a C-terminal thioester. This uniquely reactive C-terminus can be used in native chemical ligation reactions to introduce synthetic groups or to immobilize proteins on surfaces and nanoparticles. Unfortunately, common refolding procedures for recombinant proteins that contain disulfide bonds do not preserve the thioester functionality and therefore novel refolding procedures need to be developed. Results A novel redox buffer consisting of MESNA and diMESNA showed a refolding efficiency comparable to that of GSH/GSSG and prevented loss of the protein's thioester functionality. Moreover, introduction of the MESNA/diMESNA redox couple in the cleavage buffer allowed simultaneous on-column refolding of Ribonuclease A and intein-mediated cleavage to yield Ribonuclease A with a C-terminal MESNA-thioester. The C-terminal thioester was shown to be active in native chemical ligation. Conclusion An efficient method was developed for the production of disulfide bond containing proteins with C-terminal thioesters. Introduction of a MESNA/diMESNA redox couple resulted in simultaneous on-column refolding, purification and thioester generation of the model protein Ribonuclease A.

  11. C-terminal domains of bacterial proteases: structure, function and the biotechnological applications.

    Science.gov (United States)

    Huang, J; Wu, C; Liu, D; Yang, X; Wu, R; Zhang, J; Ma, C; He, H

    2017-01-01

    C-terminal domains widely exist in the C-terminal region of multidomain proteases. As a β-sandwich domain in multidomain protease, the C-terminal domain plays an important role in proteolysis including regulation of the secretory process, anchoring and swelling the substrate molecule, presenting as an inhibitor for the preprotease and adapting the protein structural flexibility and stability. In this review, the diversity, structural characteristics and biological function of C-terminal protease domains are described. Furthermore, the application prospects of C-terminal domains, including polycystic kidney disease, prepeptidase C-terminal and collagen-binding domain, in the area of medicine and biological artificial materials are also discussed. © 2016 The Society for Applied Microbiology.

  12. Structure of the C-terminal domain of nsp4 from feline coronavirus

    Energy Technology Data Exchange (ETDEWEB)

    Manolaridis, Ioannis; Wojdyla, Justyna A.; Panjikar, Santosh [EMBL Hamburg Outstation, c/o DESY, Notkestrasse 85, D-22603 Hamburg (Germany); Snijder, Eric J.; Gorbalenya, Alexander E. [Molecular Virology Laboratory, Department of Medical Microbiology, Center of Infectious Diseases, Leiden University Medical Center, PO Box 9600, 2300 RC Leiden (Netherlands); Berglind, Hanna; Nordlund, Pär [Division of Biophysics, Department of Medical Biochemistry and Biophysics, Scheeles väg 2, Karolinska Institute, SE-171 77 Stockholm (Sweden); Coutard, Bruno [Laboratoire Architecture et Fonction des Macromolécules Biologiques, UMR 6098, AFMB-CNRS-ESIL, Case 925, 163 Avenue de Luminy, 13288 Marseille (France); Tucker, Paul A., E-mail: tucker@embl-hamburg.de [EMBL Hamburg Outstation, c/o DESY, Notkestrasse 85, D-22603 Hamburg (Germany)

    2009-08-01

    The structure of the cytosolic C-terminal domain of nonstructural protein 4 from feline coronavirus has been determined and analyzed. Coronaviruses are a family of positive-stranded RNA viruses that includes important pathogens of humans and other animals. The large coronavirus genome (26–31 kb) encodes 15–16 nonstructural proteins (nsps) that are derived from two replicase polyproteins by autoproteolytic processing. The nsps assemble into the viral replication–transcription complex and nsp3, nsp4 and nsp6 are believed to anchor this enzyme complex to modified intracellular membranes. The largest part of the coronavirus nsp4 subunit is hydrophobic and is predicted to be embedded in the membranes. In this report, a conserved C-terminal domain (∼100 amino-acid residues) has been delineated that is predicted to face the cytoplasm and has been isolated as a soluble domain using library-based construct screening. A prototypical crystal structure at 2.8 Å resolution was obtained using nsp4 from feline coronavirus. Unmodified and SeMet-substituted proteins were crystallized under similar conditions, resulting in tetragonal crystals that belonged to space group P4{sub 3}. The phase problem was initially solved by single isomorphous replacement with anomalous scattering (SIRAS), followed by molecular replacement using a SIRAS-derived composite model. The structure consists of a single domain with a predominantly α-helical content displaying a unique fold that could be engaged in protein–protein interactions.

  13. Trypanosoma evansi: identification and characterization of a variant surface glycoprotein lacking cysteine residues in its C-terminal domain.

    Science.gov (United States)

    Jia, Yonggen; Zhao, Xinxin; Zou, Jingru; Suo, Xun

    2011-01-01

    African trypanosomes are flagellated unicellular parasites which proliferate extracellularly in the mammalian host blood-stream and tissue spaces. They evade the hosts' antibody-mediated lyses by sequentially changing their variant surface glycoprotein (VSG). VSG tightly coats the entire parasite body, serving as a physical barrier. In Trypanosoma brucei and the closely related species Trypanosoma evansi, Trypanosoma equiperdum, each VSG polypeptide can be divided into N- and C-terminal domains, based on cysteine distribution and sequence homology. N-terminal domain, the basis of antigenic variation, is hypervariable and contains all the exposed epitopes; C-terminal domain is relatively conserved and a full set of four or eight cysteines were generally observed. We cloned two genes from two distinct variants of T. evansi, utilizing RT-PCR with VSG-specific primers. One contained a VSG type A N-terminal domain followed a C-terminal domain lacking cysteine residues. To confirm that this gene is expressed as a functional VSG, the expression and localization of the corresponding gene product were characterized using Western blotting and immunofluorescent staining of living trypanosomes. Expression analysis showed that this protein was highly expressed, variant-specific, and had a ubiquitous cellular surface localization. All these results indicated that it was expressed as a functional VSG. Our finding showed that cysteine residues in VSG C-terminal domain were not essential; the conserved C-terminal domain generally in T. brucei like VSGs would possibly evolve for regulating the VSG expression. Copyright © 2010 Elsevier Inc. All rights reserved.

  14. Structural differences between C-terminal regions of tropomyosin isoforms

    Directory of Open Access Journals (Sweden)

    Małgorzata Śliwińska

    2013-10-01

    Full Text Available Tropomyosins are actin-binding regulatory proteins which overlap end-to-end along the filament. High resolution structures of the overlap regions were determined for muscle and non-muscle tropomyosins in the absence of actin. Conformations of the junction regions bound to actin are unknown. In this work, orientation of the overlap on actin alone and on actin–myosin complex was evaluated by measuring FRET distances between a donor (AEDANS attached to tropomyosin and an acceptor (DABMI bound to actin’s Cys374. Donor was attached to the Cys residue introduced by site-directed mutagenesis near the C-terminal half of the overlap. The recombinant alpha-tropomyosin isoforms used in this study – skeletal muscle skTM, non-muscle TM2 and TM5a, and chimeric TM1b9a had various amino acid sequences of the N- and C-termini involved in the end-to-end overlap. The donor-acceptor distances calculated for each isoform varied between 36.4 Å and 48.1 Å. Rigor binding of myosin S1 increased the apparent FRET distances of skTM and TM2, but decreased the distances separating TM5a and TM1b9a from actin. The results show that isoform-specific sequences of the end-to-end overlaps determine orientations and dynamics of tropomyosin isoforms on actin. This can be important for specificity of tropomyosin in the regulation of actin filament diverse functions.

  15. Selective enzymatic hydrolysis of C-terminal tert-butyl esters of peptides

    NARCIS (Netherlands)

    Eggen, I.F.; Boeriu, C.G.

    2007-01-01

    The present invention relates to a process for the selective enzymatic hydrolysis of C-terminal esters of peptide substrates in the synthesis of peptides, comprising hydrolysing C-terminal tert-butyl esters using the protease subtilisin. This process is useful in the production of protected or

  16. Selective enzymatic hydrolysis of C-terminal tert-butyl esters of peptides

    OpenAIRE

    Eggen, I.F.; Boeriu, C.G.

    2007-01-01

    The present invention relates to a process for the selective enzymatic hydrolysis of C-terminal esters of peptide substrates in the synthesis of peptides, comprising hydrolysing C-terminal tert-butyl esters using the protease subtilisin. This process is useful in the production of protected or unprotected peptides.

  17. C-terminals in the mouse branchiomotor nuclei originate from the magnocellular reticular formation.

    Science.gov (United States)

    Matsui, Toshiyasu; Hongo, Yu; Haizuka, Yoshinori; Kaida, Kenichi; Matsumura, George; Martin, Donna M; Kobayashi, Yasushi

    2013-08-26

    Large cholinergic synaptic boutons called "C-terminals" contact motoneurons and regulate their excitability. C-terminals in the spinal somatic motor nuclei originate from cholinergic interneurons in laminae VII and X that express a transcription factor Pitx2. Cranial motor nuclei contain another type of motoneuron: branchiomotor neurons. Although branchiomotor neurons receive abundant C-terminal projections, the neural source of these C-terminals remains unknown. In the present study, we first examined whether cholinergic neurons express Pitx2 in the reticular formation of the adult mouse brainstem, as in the spinal cord. Although Pitx2-positive cholinergic neurons were observed in the magnocellular reticular formation and region around the central canal in the caudal medulla, none was present more rostrally in the brainstem tegmentum. We next explored the origin of C-terminals in the branchiomotor nuclei by using biotinylated dextran amine (BDA). BDA injections into the magnocellular reticular formation of the medulla and pons resulted in the labeling of numerous C-terminals in the branchiomotor nuclei: the ambiguous, facial, and trigeminal motor nuclei. Our results revealed that the origins of C-terminals in the branchiomotor nuclei are cholinergic neurons in the magnocellular reticular formation not only in the caudal medulla, but also at more rostral levels of the brainstem, which lacks Pitx2-positive neurons. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  18. Requirement for the E1 Helicase C-Terminal Domain in Papillomavirus DNA Replication In Vivo.

    Science.gov (United States)

    Bergvall, Monika; Gagnon, David; Titolo, Steve; Lehoux, Michaël; D'Abramo, Claudia M; Melendy, Thomas; Archambault, Jacques

    2016-01-06

    The papillomavirus (PV) E1 helicase contains a conserved C-terminal domain (CTD), located next to its ATP-binding site, whose function in vivo is still poorly understood. The CTD is comprised of an alpha helix followed by an acidic region (AR) and a C-terminal extension termed the C-tail. Recent biochemical studies on bovine papillomavirus 1 (BPV1) E1 showed that the AR and C-tail regulate the oligomerization of the protein into a double hexamer at the origin. In this study, we assessed the importance of the CTD of human papillomavirus 11 (HPV11) E1 in vivo, using a cell-based DNA replication assay. Our results indicate that combined deletion of the AR and C-tail drastically reduces DNA replication, by 85%, and that further truncation into the alpha-helical region compromises the structural integrity of the E1 helicase domain and its interaction with E2. Surprisingly, removal of the C-tail alone or mutation of highly conserved residues within the domain still allows significant levels of DNA replication (55%). This is in contrast to the absolute requirement for the C-tail reported for BPV1 E1 in vitro and confirmed here in vivo. Characterization of chimeric proteins in which the AR and C-tail from HPV11 E1 were replaced by those of BPV1 indicated that while the function of the AR is transferable, that of the C-tail is not. Collectively, these findings define the contribution of the three CTD subdomains to the DNA replication activity of E1 in vivo and suggest that the function of the C-tail has evolved in a PV type-specific manner. While much is known about hexameric DNA helicases from superfamily 3, the papillomavirus E1 helicase contains a unique C-terminal domain (CTD) adjacent to its ATP-binding site. We show here that this CTD is important for the DNA replication activity of HPV11 E1 in vivo and that it can be divided into three functional subdomains that roughly correspond to the three conserved regions of the CTD: an alpha helix, needed for the structural

  19. Requirement for the E1 Helicase C-Terminal Domain in Papillomavirus DNA Replication In Vivo

    Science.gov (United States)

    Bergvall, Monika; Gagnon, David; Titolo, Steve; Lehoux, Michaël; D'Abramo, Claudia M.

    2016-01-01

    ABSTRACT The papillomavirus (PV) E1 helicase contains a conserved C-terminal domain (CTD), located next to its ATP-binding site, whose function in vivo is still poorly understood. The CTD is comprised of an alpha helix followed by an acidic region (AR) and a C-terminal extension termed the C-tail. Recent biochemical studies on bovine papillomavirus 1 (BPV1) E1 showed that the AR and C-tail regulate the oligomerization of the protein into a double hexamer at the origin. In this study, we assessed the importance of the CTD of human papillomavirus 11 (HPV11) E1 in vivo, using a cell-based DNA replication assay. Our results indicate that combined deletion of the AR and C-tail drastically reduces DNA replication, by 85%, and that further truncation into the alpha-helical region compromises the structural integrity of the E1 helicase domain and its interaction with E2. Surprisingly, removal of the C-tail alone or mutation of highly conserved residues within the domain still allows significant levels of DNA replication (55%). This is in contrast to the absolute requirement for the C-tail reported for BPV1 E1 in vitro and confirmed here in vivo. Characterization of chimeric proteins in which the AR and C-tail from HPV11 E1 were replaced by those of BPV1 indicated that while the function of the AR is transferable, that of the C-tail is not. Collectively, these findings define the contribution of the three CTD subdomains to the DNA replication activity of E1 in vivo and suggest that the function of the C-tail has evolved in a PV type-specific manner. IMPORTANCE While much is known about hexameric DNA helicases from superfamily 3, the papillomavirus E1 helicase contains a unique C-terminal domain (CTD) adjacent to its ATP-binding site. We show here that this CTD is important for the DNA replication activity of HPV11 E1 in vivo and that it can be divided into three functional subdomains that roughly correspond to the three conserved regions of the CTD: an alpha helix, needed

  20. Age-dependent loss of the C-terminal amino acid from alpha crystallin

    Science.gov (United States)

    Emmons, T.; Takemoto, L.; Spooner, B. S. (Principal Investigator)

    1992-01-01

    Antiserum made against the C-terminal region of alpha-A crystallin was used to monitor the purification of a tryptic peptide containing the C-terminus of the molecule from fetal versus adult bovine lenses. Mass spectral analysis of the peptide preparations obtained from these lenses demonstrated the presence of a peptide (T20) containing an intact C-terminus from fetal lenses and the presence of an additional peptide (T20') from older lenses that contained a cleaved C-terminal serine. These results demonstrate an age-dependent processing of alpha-A crystallin in the bovine lens, resulting in removal of the C-terminal amino acid residue.

  1. Mutations in the C-terminal region affect subcellular localization of crucian carp herpesvirus (CaHV) GPCR.

    Science.gov (United States)

    Wang, Jun; Gui, Lang; Chen, Zong-Yan; Zhang, Qi-Ya

    2016-08-01

    G protein-coupled receptors (GPCRs) are known as seven transmembrane domain receptors and consequently can mediate diverse biological functions via regulation of their subcellular localization. Crucian carp herpesvirus (CaHV) was recently isolated from infected fish with acute gill hemorrhage. CaHV GPCR of 349 amino acids (aa) was identified based on amino acid identity. A series of variants with truncation/deletion/substitution mutation in the C-terminal (aa 315-349) were constructed and expressed in fathead minnow (FHM) cells. The roles of three key C-terminal regions in subcellular localization of CaHV GPCR were determined. Lysine-315 (K-315) directed the aggregation of the protein preferentially at the nuclear side. Predicted N-myristoylation site (GGGWTR, aa 335-340) was responsible for punctate distribution in periplasm or throughout the cytoplasm. Predicted phosphorylation site (SSR, aa 327-329) and GGGWTR together determined the punctate distribution in cytoplasm. Detection of organelles localization by specific markers showed that the protein retaining K-315 colocalized with the Golgi apparatus. These experiments provided first evidence that different mutations of CaHV GPCR C-terminals have different affects on the subcellular localization of fish herpesvirus-encoded GPCRs. The study provided valuable information and new insights into the precise interactions between herpesvirus and fish cells, and could also provide useful targets for antiviral agents in aquaculture.

  2. Influence of C-terminal truncation of murine Serum amyloid A on fibril structure

    National Research Council Canada - National Science Library

    Matthies Rennegarbe; Inga Lenter; Angelika Schierhorn; Romy Sawilla; Christian Haupt

    2017-01-01

    .... While the protein precursor in humans and mice is the acute-phase reactant serum amyloid A (SAA) 1.1, the deposited fibrils consist mainly of C-terminally truncated SAA fragments, termed AA proteins...

  3. Versatile Peptide C-Terminal Functionalization via a Computationally Engineered Peptide Amidase

    NARCIS (Netherlands)

    Wu, Bian; Wijma, Hein J.; Song, Lu; Rozeboom, Henriette J.; Poloni, Claudia; Tian, Yue; Arif, Muhammad I.; Nuijens, Timo; Quaedflieg, Peter J. L. M.; Szymanski, Wiktor; Feringa, Ben L.; Janssen, Dick B.

    The properties of synthetic peptides, including potency, stability, and bioavailability, are strongly influenced by modification of the peptide chain termini. Unfortunately, generally applicable methods for selective and mild C-terminal peptide functionalization are lacking. In this work, we

  4. C-terminal Amidation of an Osteocalcin-derived Peptide Promotes Hydroxyapatite Crystallization*

    Science.gov (United States)

    Hosseini, Samaneh; Naderi-Manesh, Hossein; Mountassif, Driss; Cerruti, Marta; Vali, Hojatollah; Faghihi, Shahab

    2013-01-01

    Genesis of natural biocomposite-based materials, such as bone, cartilage, and teeth, involves interactions between organic and inorganic systems. Natural biopolymers, such as peptide motif sequences, can be used as a template to direct the nucleation and crystallization of hydroxyapatite (HA). In this study, a natural motif sequence consisting of 13 amino acids present in the first helix of osteocalcin was selected based on its calcium binding ability and used as substrate for nucleation of HA crystals. The acidic (acidic osteocalcin-derived peptide (OSC)) and amidic (amidic osteocalcin-derived peptide (OSN)) forms of this sequence were synthesized to investigate the effects of different C termini on the process of biomineralization. Electron microscopy analyses show the formation of plate-like HA crystals with random size and shape in the presence of OSN. In contrast, spherical amorphous calcium phosphate is formed in the presence of OSC. Circular dichroism experiments indicate conformational changes of amidic peptide to an open and regular structure as a consequence of interaction with calcium and phosphate. There is no conformational change detectable in OSC. It is concluded that HA crystal formation, which only occurred in OSN, is attributable to C-terminal amidation of a natural peptide derived from osteocalcin. It is also proposed that natural peptides with the ability to promote biomineralization have the potential to be utilized in hard tissue regeneration. PMID:23362258

  5. C-terminal amidation of an osteocalcin-derived peptide promotes hydroxyapatite crystallization.

    Science.gov (United States)

    Hosseini, Samaneh; Naderi-Manesh, Hossein; Mountassif, Driss; Cerruti, Marta; Vali, Hojatollah; Faghihi, Shahab

    2013-03-15

    Genesis of natural biocomposite-based materials, such as bone, cartilage, and teeth, involves interactions between organic and inorganic systems. Natural biopolymers, such as peptide motif sequences, can be used as a template to direct the nucleation and crystallization of hydroxyapatite (HA). In this study, a natural motif sequence consisting of 13 amino acids present in the first helix of osteocalcin was selected based on its calcium binding ability and used as substrate for nucleation of HA crystals. The acidic (acidic osteocalcin-derived peptide (OSC)) and amidic (amidic osteocalcin-derived peptide (OSN)) forms of this sequence were synthesized to investigate the effects of different C termini on the process of biomineralization. Electron microscopy analyses show the formation of plate-like HA crystals with random size and shape in the presence of OSN. In contrast, spherical amorphous calcium phosphate is formed in the presence of OSC. Circular dichroism experiments indicate conformational changes of amidic peptide to an open and regular structure as a consequence of interaction with calcium and phosphate. There is no conformational change detectable in OSC. It is concluded that HA crystal formation, which only occurred in OSN, is attributable to C-terminal amidation of a natural peptide derived from osteocalcin. It is also proposed that natural peptides with the ability to promote biomineralization have the potential to be utilized in hard tissue regeneration.

  6. C-terminal phosphorylation regulates the kinetics of a subset of melanopsin-mediated behaviors in mice.

    Science.gov (United States)

    Somasundaram, Preethi; Wyrick, Glenn R; Fernandez, Diego Carlos; Ghahari, Alireza; Pinhal, Cindy M; Simmonds Richardson, Melissa; Rupp, Alan C; Cui, Lihong; Wu, Zhijian; Brown, R Lane; Badea, Tudor Constantin; Hattar, Samer; Robinson, Phyllis R

    2017-03-07

    Intrinsically photosensitive retinal ganglion cells (ipRGCs) express the photopigment melanopsin and mediate several non-image-forming visual functions, including circadian photoentrainment and the pupillary light reflex (PLR). ipRGCs act as autonomous photoreceptors via the intrinsic melanopsin-based phototransduction pathway and as a relay for rod/cone input via synaptically driven responses. Under low light intensities, where only synaptically driven rod/cone input activates ipRGCs, the duration of the ipRGC response will be determined by the termination kinetics of the rod/cone circuits. Little is known, however, about the termination kinetics of the intrinsic melanopsin-based phototransduction pathway and its contribution to several melanopsin-mediated behaviors. Here, we show that C-terminal phosphorylation of melanopsin determines the recovery kinetics of the intrinsic melanopsin-based photoresponse in ipRGCs, the duration of the PLR, and the speed of reentrainment. In contrast, circadian phase alignment and direct effects of light on activity (masking) are not influenced by C-terminal phosphorylation of melanopsin. Electrophysiological measurements demonstrate that expression of a virally encoded melanopsin lacking all C-terminal phosphorylation sites (C terminus phosphonull) leads to a prolonged intrinsic light response. In addition, mice expressing the C terminus phosphonull in ipRGCs reentrain faster to a delayed light/dark cycle compared with mice expressing virally encoded WT melanopsin; however, the phase angle of entrainment and masking were indistinguishable. Importantly, a sustained PLR in the phosphonull animals is only observed at brighter light intensities that activate melanopsin phototransduction, but not at dimmer light intensities that activate only the rod/cone pathway. Taken together, our results highlight how the kinetics of the melanopsin photoresponse differentially regulate distinct light-mediated behaviors.

  7. The C-terminal domain of the Bloom syndrome DNA helicase is essential for genomic stability

    Directory of Open Access Journals (Sweden)

    Noonan James P

    2001-07-01

    Full Text Available Abstract Background Bloom syndrome is a rare cancer-prone disorder in which the cells of affected persons have a high frequency of somatic mutation and genomic instability. Bloom syndrome cells have a distinctive high frequency of sister chromatid exchange and quadriradial formation. BLM, the protein altered in BS, is a member of the RecQ DNA helicase family, whose members share an average of 40% identity in the helicase domain and have divergent N-terminal and C-terminal flanking regions of variable lengths. The BLM DNA helicase has been shown to localize to the ND10 (nuclear domain 10 or PML (promyelocytic leukemia nuclear bodies, where it associates with TOPIIIα, and to the nucleolus. Results This report demonstrates that the N-terminal domain of BLM is responsible for localization of the protein to the nuclear bodies, while the C-terminal domain directs the protein to the nucleolus. Deletions of the N-terminal domain of BLM have little effect on sister chromatid exchange frequency and chromosome stability as compared to helicase and C-terminal mutations which can increase SCE frequency and chromosome abnormalities. Conclusion The helicase activity and the C-terminal domain of BLM are critical for maintaining genomic stability as measured by the sister chromatid exchange assay. The localization of BLM into the nucleolus by the C-terminal domain appears to be more important to genomic stability than localization in the nuclear bodies.

  8. Development of a cysteine-deprived and C-terminally truncated GLP-1 receptor.

    Science.gov (United States)

    Underwood, Christina Rye; Knudsen, Lotte Bjerre; Garibay, Patrick W; Peters, Günther H; Reedtz-Runge, Steffen

    2013-11-01

    The glucagon-like peptide-1 receptor (GLP-1R) belongs to family B of the G-protein coupled receptors (GPCRs), and has become a promising target for the treatment of type 2 diabetes. Here we describe the development and characterization of a fully functional cysteine-deprived and C-terminally truncated GLP-1R. Single cysteines were initially substituted with alanine, and functionally redundant cysteines were subsequently changed simultaneously. Our results indicate that Cys(174), Cys(226), Cys(296) and Cys(403) are important for the GLP-1-mediated response, whereas Cys(236), Cys(329), Cys(341), Cys(347), Cys(438), Cys(458) and Cys(462) are not. Extensive deletions were made in the C-terminal tail of GLP-1R in order to determine the limit for truncation. As for other family B GPCRs, we observed a direct correlation between the length of the C-terminal tail and specific binding of (125)I-GLP-1, indicating that the membrane proximal part of the C-terminal is involved in receptor expression at the cell surface. The results show that seven cysteines and more than half of the C-terminal tail can be removed from GLP-1R without compromising GLP-1 binding or function. Copyright © 2013 Elsevier Inc. All rights reserved.

  9. Diuretic activity of C-terminal group analogues of the insect kinins in Acheta domesticus.

    Science.gov (United States)

    Nachman, R J; Coast, G M; Holman, G M; Beier, R C

    1995-01-01

    A series of insect kinin analogues, AFFPWG-X, modified at the C-terminal group, were evaluated in a cricket Malpighian tubule secretion bioassay. The results were compared with activity profiles observed in a cockroach hindgut myotropic bioassay for these analogues. Although the replacement of the C-terminal amide group with a negatively charged acid leads to a precipitious drop in diuretic activity, it can be partially restored with the introduction of ester groups such as methyl or benzyl. The presence of branched chain character in the C-terminal group or a C-terminal alpha-carbon-amide distance spanning five methylene group spacers is incompatible with the receptor interaction required for biological activity. Significant diuretic activity is retained with four or fewer methylene groups in this region. C-terminal group analogues containing -SCH3, -NHCH2CH2OCH3, or -OCH2(C6H5) offered the greatest retention of diuretic activity while providing increased hydrophobicity and/or steric bulk. The data are of potential value in the development of mimetic analogues of this insect neuropeptide family. Mimetic analogues are potentially valuable tools to insect neuroendocrinologists studying diuresis and/or engaged in the development of future pest management strategies.

  10. Automation of C-terminal sequence analysis of 2D-PAGE separated proteins

    Directory of Open Access Journals (Sweden)

    P.P. Moerman

    2014-06-01

    Full Text Available Experimental assignment of the protein termini remains essential to define the functional protein structure. Here, we report on the improvement of a proteomic C-terminal sequence analysis method. The approach aims to discriminate the C-terminal peptide in a CNBr-digest where Met-Xxx peptide bonds are cleaved in internal peptides ending at a homoserine lactone (hsl-derivative. pH-dependent partial opening of the lactone ring results in the formation of doublets for all internal peptides. C-terminal peptides are distinguished as singlet peaks by MALDI-TOF MS and MS/MS is then used for their identification. We present a fully automated protocol established on a robotic liquid-handling station.

  11. Protein and peptide alkoxyl radicals can give rise to C-terminal decarboxylation and backbone cleavage

    DEFF Research Database (Denmark)

    Davies, Michael Jonathan

    1996-01-01

    with k estimated as > or = 10(7) s(-1). With N-acetyl amino acids and dipeptides beta-scission of an alkoxyl radical at the C-terminal alpha-carbon results in C-terminal decarboxylation, with release of CO2.-; the corresponding amides undergo deamidation with release of .C(O)NH2. Cyclic dipeptides...... undergo analogous reactions with cleavage of the alpha-carbon to carbonyl-carbon bond and formation of .C(O)NHR radicals. With substrates with large aliphatic side chains, radicals from side-chain hydroperoxides are also observed. C-terminal decarboxylation and backbone fragmentation are also observed...... with larger peptides, amino acid homopolymers, and proteins. These observations suggest that alpha-carbon alkoxyl radicals may be key intermediates in the fragmentation of proteins in the presence of oxygen. The radicals released in these processes may react further to form O2.-, or redox cycle metal ions...

  12. Functional role of C-terminal domain of Thermus thermophilus leucyl-tRNA synthetase

    Directory of Open Access Journals (Sweden)

    Tukalo M. A.

    2010-11-01

    Full Text Available Aim. To study a role of C-terminal domain of T. thermophilus leucyl-tRNA synthetase (LeuRSTT in the reactions of aminoacylation and editing. Methods. A mutant of LeuRSTT without C- terminal domain (ΔС was obtained by the method of mutagenesis. The kinetic constants in aminoacylation reaction catalyzed by LeuRS and its mutant (ΔС were determined by the methods of equilibrium enzyme kinetics. To evaluate the contribution of C-terminal domain to interaction of the enzyme with tRNALeu, Kd of a complex between tRNA and LeuRSTT and its mutant ΔС was determined by fluorescence titration. Results. The C-terminal domain is shown to play a significant role in the aminoacylation and editing reactions of LeuRSTT and not essential for the activity in the reaction of amino acid activation. The kinetic parameters of aminoacylation of tRNALeu and tRNATyr by LeuRS and ΔС mutant were also determined, their analysis suggests that the C-domain is not critical for the manifestation of specificity of the enzyme in the recognition of homologous RNAs. At the same time a significant influence of the C-terminal domain on the value of catalytic constant was shown. At the domain deletion the kcat value is lower by 152-fold. Conclusion. The C-terminal domain of LeuRSTT is evolutionarily acquired to enhance the rate of catalysis in the aminoacylation and editing reactions, and makes no significant contribution to the specificity of the enzyme in the recognition of tRNA.

  13. Heparan sulfate regulates fibrillin-1 N- and C-terminal interactions

    DEFF Research Database (Denmark)

    Cain, Stuart A; Baldwin, Andrew K; Mahalingam, Yashithra

    2008-01-01

    in response to soluble PF1. Within domains encoded by exons 59-62 near the fibrillin-1 C terminus are novel conformation-dependent high affinity heparin and tropoelastin binding sites. Heparin disrupted tropoelastin binding but did not disrupt N- and C-terminal fibrillin-1 interactions. Thus, fibrillin-1 N......-terminal interactions with heparin/heparan sulfate directly influence cell behavior, whereas C-terminal interactions with heparin/heparan sulfate regulate elastin deposition. These data highlight how heparin/heparan sulfate controls fibrillin-1 interactions....

  14. Aggregation of thrombin-derived C-terminal fragments as a previously undisclosed host defense mechanism

    DEFF Research Database (Denmark)

    Petrlova, Jitka; Hansen, Finja C; van der Plas, Mariena J A

    2017-01-01

    Effective control of endotoxins and bacteria is crucial for normal wound healing. During injury, the key enzyme thrombin is formed, leading to generation of fibrin. Here, we show that human neutrophil elastase cleaves thrombin, generating 11-kDa thrombin-derived C-terminal peptides (TCPs), which ...

  15. Mutant Mice Lacking the p53 C-Terminal Domain Model Telomere Syndromes

    NARCIS (Netherlands)

    Simeonova, I.; Jaber, S.; Draskovic, I.; Bardot, B.; Fang, M.; Bouarich-Bourimi, R.; Lejour, V.; Charbonnier, L.; Soudais, C.; Bourdon, J.C.; Huerre, M.; Londono-Vallejo, A.; Toledo, F.

    2013-01-01

    Mutations in p53, although frequent in human cancers, have not been implicated in telomere-related syndromes. Here, we show that homozygous mutant mice expressing p53(Delta31), a p53 lacking the C-terminal domain, exhibit increased p53 activity and suffer from aplastic anemia and pulmonary fibrosis,

  16. Urinary uromodulin carries an intact ZP domain generated by a conserved C-terminal proteolytic cleavage.

    Science.gov (United States)

    Santambrogio, Sara; Cattaneo, Angela; Bernascone, Ilenia; Schwend, Thomas; Jovine, Luca; Bachi, Angela; Rampoldi, Luca

    2008-06-06

    Uromodulin (or Tamm-Horsfall protein) is the most abundant protein in human urine under physiological conditions. Little is known about the molecular mechanism of uromodulin secretion. By extensive Mass Spectrometry analyses we mapped the C-termini of human and murine urinary proteins demonstrating that urinary uromodulin is generated by a conserved C-terminal proteolytic cleavage and retains its entire ZP domain.

  17. C-terminal propeptide of the Caldariomyces fumago chloroperoxidase : an intramolecular chaperone?

    NARCIS (Netherlands)

    Conesa, A.; Weelink, G.; Hondel, C.A.M.J.J. van den; Punt, P.J.

    2001-01-01

    The Caldariomyces fumago chloroperoxidase (CPO) is synthesised as a 372-aa precursor which undergoes two proteolytic processing events: removal of a 21-aa N-terminal signal peptide and of a 52-aa C-terminal propeptide. The Aspergillus niger expression system developed for CPO was used to get insight

  18. C-terminal KDEL-modified cystatin C is retained in transfected CHO cells

    DEFF Research Database (Denmark)

    Johansen, Teit Eliot; Vogel, Charlotte Katrine; Schwartz, Thue W.

    1990-01-01

    The significance of a C-terminal tetrapeptide, Lys-Asp-Glu-Leu (KDEL), as a retention signal for the endoplasmatic reticulum was studied using cystatin C, a general thiol protease inhibitor, as the reporter protein. Clones of CHO cells were analyzed after stable transfection with eukaryotic...

  19. High-yield production of Streptavidin with native C-terminal in ...

    African Journals Online (AJOL)

    To increase the production yield of functional recombinant streptavidin in Escherichia coli, the effects of host strains and culture conditions on expression of streptavidin with native C terminal (CNSA, amino acid residues 13 to 159) were investigated. Results show that the CNSA, encoded by the CNSA gene, was produced ...

  20. A C-terminal Aldehyde Analog of the Insect Kinins Inhibits Diuresis in the Housefly

    Science.gov (United States)

    2006-09-21

    cricket Acheta domesticus Insect kinin analog Stimulation of Malpighian tubule fluid secretion—EC50 (10 9 M) (% maximal response) Arg-Phe-Phe-Pro-Trp...RJ, Coast GM, Holman GM, Beier RC. Diuretic activity of C-terminal group analogs of the insect kinins in Acheta domesticus . Peptides 1995;16:809–13

  1. CAPN3-mediated processing of C-terminal titin replaced by pathological cleavage in titinopathy.

    Science.gov (United States)

    Charton, Karine; Sarparanta, Jaakko; Vihola, Anna; Milic, Astrid; Jonson, Per Harald; Suel, Laurence; Luque, Helena; Boumela, Imène; Richard, Isabelle; Udd, Bjarne

    2015-07-01

    Mutations in the extreme C-terminus of titin (TTN), situated in the sarcomeric M-band, cause tibial muscular dystrophy (TMD) and limb-girdle muscular dystrophy 2J (LGMD2J). The mutations ultimately cause a loss of C-terminal titin, including a binding site for the protease calpain 3 (CAPN3), and lead to a secondary CAPN3 deficiency in LGMD2J muscle. CAPN3 has been previously shown to bind C-terminal titin and to use it as a substrate in vitro. Interestingly, mutations in CAPN3 underlie limb-girdle muscular dystrophy 2A (LGMD2A). Here, we aimed to clarify the relationship of CAPN3 and M-band titin in normal and pathological muscle. In vitro analyses identified several CAPN3 cleavage sites in C-terminal titin that were defined by protein sequencing. Furthermore, cleavage products were detected in normal muscle extracts by western blotting and in situ by immunofluorescence microscopy. The TMD/LGMD2J mutation FINmaj proved to alter this processing in vitro, while binding of CAPN3 to mutant titin was preserved. Unexpectedly, the pathological loss of M-band titin due to TMD/LGMD2J mutations was found to be independent of CAPN3, whereas the involvement of ubiquitous calpains is likely. We conclude that proteolytic processing of C-terminal titin by CAPN3 may have an important role in normal muscle, and that this process is disrupted in LGMD2A and in TMD/LGMD2J due to CAPN3 deficiency and to the loss of C-terminal titin, respectively. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  2. Contribution of chitinase A's C-terminal vacuolar sorting determinant to the study of soluble protein compartmentation.

    Science.gov (United States)

    Stigliano, Egidio; Di Sansebastiano, Gian-Pietro; Neuhaus, Jean-Marc

    2014-06-18

    Plant chitinases have been studied for their importance in the defense of crop plants from pathogen attacks and for their peculiar vacuolar sorting determinants. A peculiarity of the sequence of many family 19 chitinases is the presence of a C-terminal extension that seems to be important for their correct recognition by the vacuole sorting machinery. The 7 amino acids long C-terminal vacuolar sorting determinant (CtVSD) of tobacco chitinase A is necessary and sufficient for the transport to the vacuole. This VSD shares no homology with other CtVSDs such as the phaseolin's tetrapeptide AFVY (AlaPheValTyr) and it is also sorted by different mechanisms. While a receptor for this signal has not yet been convincingly identified, the research using the chitinase CtVSD has been very informative, leading to the observation of phenomena otherwise difficult to observe such as the presence of separate vacuoles in differentiating cells and the existence of a Golgi-independent route to the vacuole. Thanks to these new insights in the endoplasmic reticulum (ER)-to-vacuole transport, GFPChi (Green Fluorescent Protein carrying the chitinase A CtVSD) and other markers based on chitinase signals will continue to help the investigation of vacuolar biogenesis in plants.

  3. Contribution of Chitinase A’s C-Terminal Vacuolar Sorting Determinant to the Study of Soluble Protein Compartmentation

    Directory of Open Access Journals (Sweden)

    Egidio Stigliano

    2014-06-01

    Full Text Available Plant chitinases have been studied for their importance in the defense of crop plants from pathogen attacks and for their peculiar vacuolar sorting determinants. A peculiarity of the sequence of many family 19 chitinases is the presence of a C-terminal extension that seems to be important for their correct recognition by the vacuole sorting machinery. The 7 amino acids long C-terminal vacuolar sorting determinant (CtVSD of tobacco chitinase A is necessary and sufficient for the transport to the vacuole. This VSD shares no homology with other CtVSDs such as the phaseolin’s tetrapeptide AFVY (AlaPheValTyr and it is also sorted by different mechanisms. While a receptor for this signal has not yet been convincingly identified, the research using the chitinase CtVSD has been very informative, leading to the observation of phenomena otherwise difficult to observe such as the presence of separate vacuoles in differentiating cells and the existence of a Golgi-independent route to the vacuole. Thanks to these new insights in the endoplasmic reticulum (ER-to-vacuole transport, GFPChi (Green Fluorescent Protein carrying the chitinase A CtVSD and other markers based on chitinase signals will continue to help the investigation of vacuolar biogenesis in plants.

  4. The C-Terminal O-S Acyl Shift Pathway under Acidic Condition to Propose Peptide-Thioesters

    Directory of Open Access Journals (Sweden)

    Bo Mi Kim

    2016-11-01

    Full Text Available Peptide-thioester is a pivotal intermediate for peptide ligation and N-, C-terminal cyclization. In this study, desired pathway and the side products of two C-terminal handles, hydroxyethylthiol (HET and hydroxypropylthiol (HPT are described in different conditions as well as kinetic studies. In addition, a new mechanism of C-terminal residue racemization is proposed on the basis of differentiation of products derived from the two C-terminal handles in preparing peptide thioesters through an acid-catalyzed tandem thiol switch, first by an intramolecular O-S acyl shift, and then by an intermolecular S-S exchange.

  5. The C-terminal propeptide of a plant defensin confers cytoprotective and subcellular targeting functions.

    Science.gov (United States)

    Lay, Fung T; Poon, Simon; McKenna, James A; Connelly, Angela A; Barbeta, Barbara L; McGinness, Bruce S; Fox, Jennifer L; Daly, Norelle L; Craik, David J; Heath, Robyn L; Anderson, Marilyn A

    2014-02-05

    Plant defensins are small (45-54 amino acids), basic, cysteine-rich proteins that have a major role in innate immunity in plants. Many defensins are potent antifungal molecules and are being evaluated for their potential to create crop plants with sustainable disease resistance. Defensins are produced as precursor molecules which are directed into the secretory pathway and are divided into two classes based on the absence (class I) or presence (class II) of an acidic C-terminal propeptide (CTPP) of about 33 amino acids. The function of this CTPP had not been defined. By transgenically expressing the class II plant defensin NaD1 with and without its cognate CTPP we have demonstrated that NaD1 is phytotoxic to cotton plants when expressed without its CTPP. Transgenic cotton plants expressing constructs encoding the NaD1 precursor with the CTPP had the same morphology as non-transgenic plants but expression of NaD1 without the CTPP led to plants that were stunted, had crinkled leaves and were less viable. Immunofluorescence microscopy and transient expression of a green fluorescent protein (GFP)-CTPP chimera were used to confirm that the CTPP is sufficient for vacuolar targeting. Finally circular dichroism and NMR spectroscopy were used to show that the CTPP adopts a helical confirmation. In this report we have described the role of the CTPP on NaD1, a class II defensin from Nicotiana alata flowers. The CTPP of NaD1 is sufficient for vacuolar targeting and plays an important role in detoxification of the defensin as it moves through the plant secretory pathway. This work may have important implications for the use of defensins for disease protection in transgenic crops.

  6. Targeting the Hsp90 C-terminal domain to induce allosteric inhibition and selective client downregulation.

    Science.gov (United States)

    Goode, Kourtney M; Petrov, Dino P; Vickman, Renee E; Crist, Scott A; Pascuzzi, Pete E; Ratliff, Tim L; Davisson, V Jo; Hazbun, Tony R

    2017-08-01

    Inhibition of Hsp90 is desirable due to potential downregulation of oncogenic clients. Early generation inhibitors bind to the N-terminal domain (NTD) but C-terminal domain (CTD) inhibitors are a promising class because they do not induce a heat shock response. Here we present a new structural class of CTD binding molecules with a unique allosteric inhibition mechanism. A hit molecule, NSC145366, and structurally similar probes were assessed for inhibition of Hsp90 activities. A ligand-binding model was proposed indicating a novel Hsp90 CTD binding site. Client protein downregulation was also determined. NSC145366 interacts with the Hsp90 CTD and has anti-proliferative activity in tumor cell lines (GI50=0.2-1.9μM). NSC145366 increases Hsp90 oligomerization resulting in allosteric inhibition of NTD ATPase activity (IC50=119μM) but does not compete with NTD or CTD-ATP binding. Treatment of LNCaP prostate tumor cells resulted in selective client protein downregulation including AR and BRCA1 but without a heat shock response. Analogs had similar potencies in ATPase and chaperone activity assays and variable effects on oligomerization. In silico modeling predicted a binding site at the CTD dimer interface distinct from the nucleotide-binding site. A set of symmetrical scaffold molecules with bisphenol A cores induced allosteric inhibition of Hsp90. Experimental evidence and molecular modeling suggest that the binding site is independent of the CTD-ATP site and consistent with unique induction of allosteric effects. Allosteric inhibition of Hsp90 via a mechanism used by the NSC145366-based probes is a promising avenue for selective oncogenic client downregulation. Copyright © 2017. Published by Elsevier B.V.

  7. GBNV encoded movement protein (NSm) remodels ER network via C-terminal coiled coil domain

    Energy Technology Data Exchange (ETDEWEB)

    Singh, Pratibha; Savithri, H.S., E-mail: bchss@biochem.iisc.ernet.in

    2015-08-15

    Plant viruses exploit the host machinery for targeting the viral genome–movement protein complex to plasmodesmata (PD). The mechanism by which the non-structural protein m (NSm) of Groundnut bud necrosis virus (GBNV) is targeted to PD was investigated using Agrobacterium mediated transient expression of NSm and its fusion proteins in Nicotiana benthamiana. GFP:NSm formed punctuate structures that colocalized with mCherry:plasmodesmata localized protein 1a (PDLP 1a) confirming that GBNV NSm localizes to PD. Unlike in other movement proteins, the C-terminal coiled coil domain of GBNV NSm was shown to be involved in the localization of NSm to PD, as deletion of this domain resulted in the cytoplasmic localization of NSm. Treatment with Brefeldin A demonstrated the role of ER in targeting GFP NSm to PD. Furthermore, mCherry:NSm co-localized with ER–GFP (endoplasmic reticulum targeting peptide (HDEL peptide fused with GFP). Co-expression of NSm with ER–GFP showed that the ER-network was transformed into vesicles indicating that NSm interacts with ER and remodels it. Mutations in the conserved hydrophobic region of NSm (residues 130–138) did not abolish the formation of vesicles. Additionally, the conserved prolines at positions 140 and 142 were found to be essential for targeting the vesicles to the cell membrane. Further, systematic deletion of amino acid residues from N- and C-terminus demonstrated that N-terminal 203 amino acids are dispensable for the vesicle formation. On the other hand, the C-terminal coiled coil domain when expressed alone could also form vesicles. These results suggest that GBNV NSm remodels the ER network by forming vesicles via its interaction through the C-terminal coiled coil domain. Interestingly, NSm interacts with NP in vitro and coexpression of these two proteins in planta resulted in the relocalization of NP to PD and this relocalization was abolished when the N-terminal unfolded region of NSm was deleted. Thus, the NSm

  8. Presence and expression of hydrogenase specific C-terminal endopeptidases in cyanobacteria

    Directory of Open Access Journals (Sweden)

    Lindblad Peter

    2003-05-01

    Full Text Available Abstract Background Hydrogenases catalyze the simplest of all chemical reactions: the reduction of protons to molecular hydrogen or vice versa. Cyanobacteria can express an uptake, a bidirectional or both NiFe-hydrogenases. Maturation of those depends on accessory proteins encoded by hyp-genes. The last maturation step involves the cleavage of a ca. 30 amino acid long peptide from the large subunit by a C-terminal endopeptidase. Until know, nothing is known about the maturation of cyanobacterial NiFe-hydrogenases. The availability of three complete cyanobacterial genome sequences from strains with either only the uptake (Nostoc punctiforme ATCC 29133/PCC 73102, only the bidirectional (Synechocystis PCC 6803 or both NiFe-hydrogenases (Anabaena PCC 7120 prompted us to mine these genomes for hydrogenase maturation related genes. In this communication we focus on the presence and the expression of the NiFe-hydrogenases and the corresponding C-terminal endopeptidases, in the three strains mentioned above. Results We identified genes encoding putative cyanobacterial hydrogenase specific C-terminal endopeptidases in all analyzed cyanobacterial genomes. The genes are not part of any known hydrogenase related gene cluster. The derived amino acid sequences show only low similarity (28–41% to the well-analyzed hydrogenase specific C-terminal endopeptidase HybD from Escherichia coli, the crystal structure of which is known. However, computational secondary and tertiary structure modeling revealed the presence of conserved structural patterns around the highly conserved active site. Gene expression analysis shows that the endopeptidase encoding genes are expressed under both nitrogen-fixing and non-nitrogen-fixing conditions. Conclusion Anabaena PCC 7120 possesses two NiFe-hydrogenases and two hydrogenase specific C-terminal endopeptidases but only one set of hyp-genes. Thus, in contrast to the Hyp-proteins, the C-terminal endopeptidases are the only known

  9. Is the C-terminal insertional signal in Gram-negative bacterial outer membrane proteins species-specific or not?

    Directory of Open Access Journals (Sweden)

    Paramasivam Nagarajan

    2012-09-01

    Full Text Available Abstract Background In Gram-negative bacteria, the outer membrane is composed of an asymmetric lipid bilayer of phopspholipids and lipopolysaccharides, and the transmembrane proteins that reside in this membrane are almost exclusively β-barrel proteins. These proteins are inserted into the membrane by a highly conserved and essential machinery, the BAM complex. It recognizes its substrates, unfolded outer membrane proteins (OMPs, through a C-terminal motif that has been speculated to be species-specific, based on theoretical and experimental results from only two species, Escherichia coli and Neisseria meningitidis, where it was shown on the basis of individual sequences and motifs that OMPs from the one cannot easily be over expressed in the other, unless the C-terminal motif was adapted. In order to determine whether this species specificity is a general phenomenon, we undertook a large-scale bioinformatics study on all predicted OMPs from 437 fully sequenced proteobacterial strains. Results We were able to verify the incompatibility reported between Escherichia coli and Neisseria meningitidis, using clustering techniques based on the pairwise Hellinger distance between sequence spaces for the C-terminal motifs of individual organisms. We noticed that the amino acid position reported to be responsible for this incompatibility between Escherichia coli and Neisseria meningitidis does not play a major role for determining species specificity of OMP recognition by the BAM complex. Instead, we found that the signal is more diffuse, and that for most organism pairs, the difference between the signals is hard to detect. Notable exceptions are the Neisseriales, and Helicobacter spp. For both of these organism groups, we describe the specific sequence requirements that are at the basis of the observed difference. Conclusions Based on the finding that the differences between the recognition motifs of almost all organisms are small, we assume that

  10. C-Terminally modified peptides i>via cleavage of the HMBA linker by O-, N- or S-nucleophiles

    DEFF Research Database (Denmark)

    Hansen, Jonas; Diness, Frederik; Meldal, Morten Peter

    2016-01-01

    A large variety of C-terminally modified peptides was obtained by nucleophilic cleavage of the ester bond in solid phase linked peptide esters of 4-hydroxymethyl benzamide (HMBA). The developed methods provided peptides, C-terminally functionalized as esters, amides and thioesters, with high puri...

  11. Elevated fasting and postprandial C-terminal telopeptide after Roux-en-Y gastric bypass.

    Science.gov (United States)

    Maghsoodi, Negar; Alaghband-Zadeh, Jamshid; Cross, Gemma F; Werling, Malin; Fändriks, Lars; Docherty, Neil G; Olbers, Torsten; Dew, Tracy; Sherwood, Roy A; Vincent, Royce P; le Roux, Carel W

    2017-07-01

    Background Roux-en-Y gastric bypass increases circulating bile acid concentrations, known mediators of postprandial suppression of markers of bone resorption. Long-term data, however, indicate that Roux-en-Y gastric bypass confers an increased risk of bone loss on recipients. Methods Thirty-six obese individuals, median age 44 (26-64) with median body mass index at baseline of 42.5 (40.4-46) were studied before and 15 months after Roux-en-Y gastric bypass. After an overnight fast, patients received a 400 kcal mixed meal. Blood samples were collected premeal then at 30-min periods for 120 min. Pre and postmeal samples were analysed for total bile acids, parathyroid hormone and C-terminal telopeptide. Results Body weight loss post Roux-en-Y gastric bypass was associated with a median 4.9-fold increase in peak postprandial total bile acid concentration, and a median 2.4-fold increase in cumulative food evoked bile acid response. Median fasting parathyroid hormone, postprandial reduction in parathyroid hormone and total parathyroid hormone release over 120 min remained unchanged after surgery. After surgery, median fasting C-terminal telopeptide increased 2.3-fold, peak postprandial concentrations increased 3.8-fold and total release was increased 1.9-fold. Conclusions Fasting and postprandial total bile acids and C-terminal telopeptide are increased above reference range after Roux-en-Y gastric bypass. These changes occur in spite of improved vitamin D status with supplementation. These results suggest that post-Roux-en-Y gastric bypass increases in total bile acids do not effectively oppose an ongoing resorptive signal operative along the gut-bone axis. Serial measurement of C-terminal telopeptide may be of value as a risk marker for long-term skeletal pathology in patients post Roux-en-Y gastric bypass.

  12. Structural and Functional Characterization of the C-terminal Transmembrane Region of NBCe1-A*

    Science.gov (United States)

    Zhu, Quansheng; Kao, Liyo; Azimov, Rustam; Abuladze, Natalia; Newman, Debra; Pushkin, Alexander; Liu, Weixin; Chang, Connie; Kurtz, Ira

    2010-01-01

    NBCe1-A and AE1 both belong to the SLC4 HCO3− transporter family. The two transporters share 40% sequence homology in the C-terminal transmembrane region. In this study, we performed extensive substituted cysteine-scanning mutagenesis analysis of the C-terminal region of NBCe1-A covering amino acids Ala800–Lys967. Location of the introduced cysteines was determined by whole cell labeling with a membrane-permeant biotin maleimide and a membrane-impermeant 2-((5(6)-tetramethylrhodamine)carboxylamino) ethyl methanethiosulfonate (MTS-TAMRA) cysteine-reactive reagent. The results show that the extracellular surface of the NBCe1-A C-terminal transmembrane region is minimally exposed to aqueous media with Met858 accessible to both biotin maleimide and TAMRA and Thr926–Ala929 only to TAMRA labeling. The intracellular surface contains a highly exposed (Met813–Gly828) region and a cryptic (Met887–Arg904) connecting loop. The lipid/aqueous interface of the last transmembrane segment is at Asp960. Our data clearly determined that the C terminus of NBCe1-A contains 5 transmembrane segments with greater average size compared with AE1. Functional assays revealed only two residues in the region of Pro868–Leu967 (a functionally important region in AE1) that are highly sensitive to cysteine substitution. Our findings suggest that the C-terminal transmembrane region of NBCe1-A is tightly folded with unique structural and functional features that differ from AE1. PMID:20837482

  13. Structure of the Reston ebolavirus VP30 C-terminal domain

    OpenAIRE

    Clifton, Matthew C.; Kirchdoerfer, Robert N.; Atkins, Kateri; Abendroth, Jan; Raymond, Amy; Grice, Rena; Barnes, Steve; Moen, Spencer; Lorimer, Don; Edwards, Thomas E.; Peter J Myler; Saphire, Erica Ollmann

    2014-01-01

    The ebolaviruses can cause severe hemorrhagic fever. Essential to the ebolavirus life cycle is the protein VP30, which serves as a transcriptional cofactor. Here, the crystal structure of the C-terminal, NP-binding domain of VP30 from Reston ebolavirus is presented. Reston VP30 and Ebola VP30 both form homodimers, but the dimeric interfaces are rotated relative to each other, suggesting subtle inherent differences or flexibility in the dimeric interface.

  14. Structure of the Reston ebolavirus VP30 C-terminal domain.

    Science.gov (United States)

    Clifton, Matthew C; Kirchdoerfer, Robert N; Atkins, Kateri; Abendroth, Jan; Raymond, Amy; Grice, Rena; Barnes, Steve; Moen, Spencer; Lorimer, Don; Edwards, Thomas E; Myler, Peter J; Saphire, Erica Ollmann

    2014-04-01

    The ebolaviruses can cause severe hemorrhagic fever. Essential to the ebolavirus life cycle is the protein VP30, which serves as a transcriptional cofactor. Here, the crystal structure of the C-terminal, NP-binding domain of VP30 from Reston ebolavirus is presented. Reston VP30 and Ebola VP30 both form homodimers, but the dimeric interfaces are rotated relative to each other, suggesting subtle inherent differences or flexibility in the dimeric interface.

  15. Influenza A hemagglutinin C-terminal anchoring peptide: identification and mass spectrometric study.

    Science.gov (United States)

    Kordyukova, Larisa V; Ksenofontov, Aleksander L; Serebryakova, Marina V; Ovchinnikova, Tatyana V; Fedorova, Natalija V; Ivanova, Valeria T; Baratova, Ludmila A

    2004-08-01

    MALDI-TOF MS and N-terminal amino acid sequencing allowed us to identify several fragments of the C-terminal peptide of Influenza A hemagglutinin (HA) containing transmembrane domains (TMD). These fragments were detected in the organic phase of chloroform-methanol extracts from bromelain-treated virus particles. Heterogeneous fatty acylation of the C-terminus was revealed. Tritium bombardment technique might open an opportunity for 3D structural investigation of the HA TMD in situ.

  16. An intermediate region in C-terminal of phosphoprotein is required ...

    African Journals Online (AJOL)

    GREGORY

    2011-12-16

    Dec 16, 2011 ... replication or binds to assembled NP (NP-RNA or NPNC) to transcribe genome to produce the sub- genomic mRNAs. ... interactive region of P to NPNC was located within C-terminal half of P between amino acids 224 to 279. .... were grown at 37°C in LB broth until the culture reached A600 of about 0.6 to ...

  17. Structural and Functional Comparisons of Retroviral Envelope Protein C-Terminal Domains: Still Much to Learn

    Directory of Open Access Journals (Sweden)

    Jonathan D. Steckbeck

    2014-01-01

    Full Text Available Retroviruses are a family of viruses that cause a broad range of pathologies in animals and humans, from the apparently harmless, long-term genomic insertion of endogenous retroviruses, to tumors induced by the oncogenic retroviruses and acquired immunodeficiency syndrome (AIDS resulting from human immunodeficiency virus infection. Disease can be the result of diverse mechanisms, including tumorigenesis induced by viral oncogenes or immune destruction, leading to the gradual loss of CD4 T-cells. Of the virally encoded proteins common to all retroviruses, the envelope (Env displays perhaps the most diverse functionality. Env is primarily responsible for binding the cellular receptor and for effecting the fusion process, with these functions mediated by protein domains localized to the exterior of the virus. The remaining C-terminal domain may have the most variable functionality of all retroviral proteins. The C-terminal domains from three prototypical retroviruses are discussed, focusing on the different structures and functions, which include fusion activation, tumorigenesis and viral assembly and lifecycle influences. Despite these genetic and functional differences, however, the C-terminal domains of these viruses share a common feature in the modulation of Env ectodomain conformation. Despite their differences, perhaps each system still has information to share with the others.

  18. Development of a cysteine-deprived and C-terminally truncated GLP-1 receptor

    DEFF Research Database (Denmark)

    Underwood, Christina Rye; Knudsen, Lotte Bjerre; Garibay, Patrick W.

    2013-01-01

    The glucagon-like peptide-1 receptor (GLP-1R) belongs to family B of the G-protein coupled receptors (GPCRs), and has become a promising target for the treatment of type 2 diabetes. Here we describe the development and characterization of a fully functional cysteine-deprived and C-terminally trun......The glucagon-like peptide-1 receptor (GLP-1R) belongs to family B of the G-protein coupled receptors (GPCRs), and has become a promising target for the treatment of type 2 diabetes. Here we describe the development and characterization of a fully functional cysteine-deprived and C......, Cys458 and Cys462 are not. Extensive deletions were made in the C-terminal tail of GLP-1R in order to determine the limit for truncation. As for other family B GPCRs, we observed a direct correlation between the length of the C-terminal tail and specific binding of 125I-GLP-1, indicating...

  19. C-terminal fluorescent labeling impairs functionality of DNA mismatch repair proteins.

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    Angela Brieger

    Full Text Available The human DNA mismatch repair (MMR process is crucial to maintain the integrity of the genome and requires many different proteins which interact perfectly and coordinated. Germline mutations in MMR genes are responsible for the development of the hereditary form of colorectal cancer called Lynch syndrome. Various mutations mainly in two MMR proteins, MLH1 and MSH2, have been identified so far, whereas 55% are detected within MLH1, the essential component of the heterodimer MutLα (MLH1 and PMS2. Most of those MLH1 variants are pathogenic but the relevance of missense mutations often remains unclear. Many different recombinant systems are applied to filter out disease-associated proteins whereby fluorescent tagged proteins are frequently used. However, dye labeling might have deleterious effects on MutLα's functionality. Therefore, we analyzed the consequences of N- and C-terminal fluorescent labeling on expression level, cellular localization and MMR activity of MutLα. Besides significant influence of GFP- or Red-fusion on protein expression we detected incorrect shuttling of single expressed C-terminal GFP-tagged PMS2 into the nucleus and found that C-terminal dye labeling impaired MMR function of MutLα. In contrast, N-terminal tagged MutLαs retained correct functionality and can be recommended both for the analysis of cellular localization and MMR efficiency.

  20. Bacteriophage endolysin Lyt μ1/6: characterization of the C-terminal binding domain.

    Science.gov (United States)

    Tišáková, Lenka; Vidová, Barbora; Farkašovská, Jarmila; Godány, Andrej

    2014-01-01

    The gene product of orf50 from actinophage μ1/6 of Streptomyces aureofaciens is a putative endolysin, Lyt μ1/6. It has a two-domain modular structure, consisting of an N-terminal catalytic and a C-terminal cell wall binding domain (CBD). Comparative analysis of Streptomyces phage endolysins revealed that they all have a modular structure and contain functional C-terminal domains with conserved amino acids, probably associated with their binding function. A blast analysis of Lyt μ1/6 in conjunction with secondary and tertiary structure prediction disclosed the presence of a PG_binding_1 domain within the CBD. The sequence of the C-terminal domain of lyt μ1/6 and truncated forms of it were cloned and expressed in Escherichia coli. The ability of these CBD variants fused to GFP to bind to the surface of S. aureofaciens NMU was shown by specific binding assays. © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

  1. Resonance assignments and secondary structure of apolipoprotein E C-terminal domain in DHPC micelles.

    Science.gov (United States)

    Lo, Chi-Jen; Chyan, Chia-Lin; Chen, Yi-Chen; Chang, Chi-Fon; Huang, Hsien-Bin; Lin, Ta-Hsien

    2015-04-01

    Human apolipoprotein E (apoE) has been known to play a key role in the transport of plasma cholesterol and lipoprotein metabolism. It is an apolipoprotein of 299 amino acids with a molecular mass, ~34 kDa. ApoE has three major isoforms, apoE2, apoE3, and apoE4 which differ only at residue 112 or 158. ApoE consists of two independently folded domains (N-terminal and C-terminal domain) separated by a hinge region. The N-terminal domain and C-terminal domain of apoE are responsible for the binding to receptor and to lipid, respectively. Since the high resolution structures of apoE in lipids are still unavailable to date, we therefore aim to resolve the structures in lipids by NMR. Here, we reported the resonance assignments and secondary structure distribution of the C-terminal domain of wild-type human apoE (residue 195-299) in the micelles formed by dihexanoylphosphatidylcholine. Our results may provide a novel structural model of apoE in micelles and may shed new light on the molecular mechanisms underlying the apoE related biological processes.

  2. The flexible C-terminal arm of the Lassa arenavirus Z-protein mediates interactions with multiple binding partners.

    Science.gov (United States)

    May, Eric R; Armen, Roger S; Mannan, Aristotle M; Brooks, Charles L

    2010-08-01

    The arenavirus genome encodes for a Z-protein, which contains a RING domain that coordinates two zinc ions, and has been identified as having several functional roles at various stages of the virus life cycle. Z-protein binds to multiple host proteins and has been directly implicated in the promotion of viral budding, repression of mRNA translation, and apoptosis of infected cells. Using homology models of the Z-protein from Lassa strain arenavirus, replica exchange molecular dynamics (MD) was used to refine the structures, which were then subsequently clustered. Population-weighted ensembles of low-energy cluster representatives were predicted based upon optimal agreement of the chemical shifts computed with the SPARTA program with the experimental NMR chemical shifts. A member of the refined ensemble was identified to be a potential binder of budding factor Tsg101 based on its correspondence to the structure of the HIV-1 Gag late domain when bound to Tsg101. Members of these ensembles were docked against the crystal structure of human eIF4E translation initiation factor. Two plausible binding modes emerged based upon their agreement with experimental observation, favorable interaction energies and stability during MD trajectories. Mutations to Z are proposed that would either inhibit both binding mechanisms or selectively inhibit only one mode. The C-terminal domain conformation of the most populated member of the representative ensemble shielded protein-binding recognition motifs for Tsg101 and eIF4E and represents the most populated state free in solution. We propose that C-terminal flexibility is key for mediating the different functional states of the Z-protein. (c) 2010 Wiley-Liss, Inc.

  3. Synaptic Vesicle Tethering and the CaV2.2 Distal C-terminal

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    Fiona K Wong

    2014-03-01

    Full Text Available . Evidence that synaptic vesicles (SVs can be gated by a single voltage sensitive calcium channel (CaV2.2 predict a molecular linking mechanism or ‘tether’[Stanley 1993]. Recent studies have proposed that the SV binds to the distal C-terminal on the CaV2.2 calcium channel [Kaeser et al. 2011;Wong, Li, and Stanley 2013] while genetic analysis proposed a double tether mechanism via RIM: directly to the C terminus PDZ ligand domain or indirectly via a more proximal proline rich site [Kaeser et al. 2011]. Using a novel in vitro SV-PD binding assay, we reported that SVs bind to a fusion protein comprising the C-terminal distal third (C3, aa 2137-2357 [Wong, Li, and Stanley 2013]. Here we limit the binding site further to the last 58 aa, beyond the proline rich site, by the absence of SV capture by a truncated C3 fusion protein (aa 2137-2299. To test PDZ-dependent binding we generated two C terminus-mutant C3 fusion proteins and a mimetic blocking peptide (H-WC, aa 2349-2357 and validated these by elimination of MINT-1 or RIM binding. Persistence of SV capture with all three fusion proteins or with the full length C3 protein but in the presence of the blocking peptide, demonstrated that SVs can bind to the distal C-terminal via a PDZ-independent mechanism. These results were supported in situ by normal SV turnover in H-WC-loaded synaptosomes, as assayed by a novel peptide cryoloading method. Thus, SVs tether to the CaV2.2 C-terminal within a 49 aa region immediately prior to the terminus PDZ ligand domain. Long tethers that could reflect extended C termini were imaged by electron microscopy of synaptosome ghosts. To fully account for SV tethering we propose a model where SVs are initially captured, or ‘grabbed’, from the cytoplasm by a binding site on the distal region of the channel C-terminal and are then retracted to be ‘locked’ close to the channel by a second attachment mechanism in preparation for single channel domain gating.

  4. Probing the Impact of the EchinT C-Terminal Domain on Structure and Catalysis

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    S Bardaweel; J Pace; T Chou; V Cody; C Wagner

    2011-12-31

    Histidine triad nucleotide binding protein (Hint) is considered as the ancestor of the histidine triad protein superfamily and is highly conserved from bacteria to humans. Prokaryote genomes, including a wide array of both Gram-negative bacteria and Gram-positive bacteria, typically encode one Hint gene. The cellular function of Hint and the rationale for its evolutionary conservation in bacteria have remained a mystery. Despite its ubiquity and high sequence similarity to eukaryote Hint1 [Escherichia coli Hint (echinT) is 48% identical with human Hint1], prokaryote Hint has been reported in only a few studies. Here we report the first conformational information on the full-length N-terminal and C-terminal residues of Hint from the E. coli complex with GMP. Structural analysis of the echinT-GMP complex reveals that it crystallizes in the monoclinic space group P2{sub 1} with four homodimers in the asymmetric unit. Analysis of electron density for both the N-terminal residues and the C-terminal residues of the echinT-GMP complex indicates that the loops in some monomers can adopt more than one conformation. The observation of conformational flexibility in terminal loop regions could explain the presence of multiple homodimers in the asymmetric unit of this structure. To explore the impact of the echinT C-terminus on protein structure and catalysis, we conducted a series of catalytic radiolabeling and kinetic experiments on the C-terminal deletion mutants of echinT. In this study, we show that sequential deletion of the C-terminus likely has no effect on homodimerization and a modest effect on the secondary structure of echinT. However, we observed a significant impact on the folding structure, as reflected by a significant lowering of the T{sub m} value. Kinetic analysis reveals that the C-terminal deletion mutants are within an order of magnitude less efficient in catalysis compared to wild type, while the overall kinetic mechanism that proceeds through a fast step

  5. α-Helical to β-Helical Conformation Change in the C-Terminal of the Mammalian Prion Protein

    Science.gov (United States)

    Singh, Jesse; Whitford, Paul; Hayre, Natha; Cox, Daniel; Onuchic, José.

    2011-03-01

    We employ all-atom structure-based models with mixed basis contact maps to explore whether there are any significant geometric or energetic constraints limiting conjectured conformational transitions between the alpha-helical (α H) and the left handed beta helical (LHBH) conformations for the C-terminal (residues 166-226) of the mammalian prion protein. The LHBH structure has been proposed to describe infectious oligomers and one class of in vitro grown fibrils, as well as possibly self- templating the conversion of normal cellular prion protein to the infectious form. Our results confirm that the kinetics of the conformation change are not strongely limited by large scale geometry modification and there exists an overall preference for the LHBH conformation.

  6. Docking Studies of Binding of Ethambutol to the C-Terminal Domain of the Arabinosyltransferase from Mycobacterium tuberculosis

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    Guillermo Salgado-Moran

    2013-01-01

    Full Text Available The binding of ethambutol to the C-terminal domain of the arabinosyltransferase from Mycobacterium tuberculosis was studied. The analysis was performed using an in silico approach in order to find out, by docking calculations and energy descriptors, the conformer of Ethambutol that forms the most stable complex with the C-terminal domain of arabinosyltransferase. The complex shows that location of the Ethambutol coincides with the cocrystallization ligand position and that amino acid residues ASH1051, ASN740, ASP1052, and ARG1055 should be critical in the binding of Ethambutol to C-terminal domain EmbC.

  7. Apoptotic Activity of MeCP2 Is Enhanced by C-Terminal Truncating Mutations.

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    Alison A Williams

    Full Text Available Methyl-CpG binding protein 2 (MeCP2 is a widely abundant, multifunctional protein most highly expressed in post-mitotic neurons. Mutations causing Rett syndrome and related neurodevelopmental disorders have been identified along the entire MECP2 locus, but symptoms vary depending on mutation type and location. C-terminal mutations are prevalent, but little is known about the function of the MeCP2 C-terminus. We employ the genetic efficiency of Drosophila to provide evidence that expression of p.Arg294* (more commonly identified as R294X, a human MECP2 E2 mutant allele causing truncation of the C-terminal domains, promotes apoptosis of identified neurons in vivo. We confirm this novel finding in HEK293T cells and then use Drosophila to map the region critical for neuronal apoptosis to a small sequence at the end of the C-terminal domain. In vitro studies in mammalian systems previously indicated a role of the MeCP2 E2 isoform in apoptosis, which is facilitated by phosphorylation at serine 80 (S80 and decreased by interactions with the forkhead protein FoxG1. We confirm the roles of S80 phosphorylation and forkhead domain transcription factors in affecting MeCP2-induced apoptosis in Drosophila in vivo, thus indicating mechanistic conservation between flies and mammalian cells. Our findings are consistent with a model in which C- and N-terminal interactions are required for healthy function of MeCP2.

  8. C-Terminal Truncated α-Synuclein Fibrils Contain Strongly Twisted β-Sheets.

    Science.gov (United States)

    Iyer, Aditya; Roeters, Steven J; Kogan, Vladimir; Woutersen, Sander; Claessens, Mireille M A E; Subramaniam, Vinod

    2017-11-01

    C-terminal truncations of monomeric wild-type alpha-synuclein (henceforth WT-αS) have been shown to enhance the formation of amyloid aggregates both in vivo and in vitro and have been associated with accelerated progression of Parkinson's disease (PD). The correlation with PD may not solely be a result of faster aggregation, but also of which fibril polymorphs are preferentially formed when the C-terminal residues are deleted. Considering that different polymorphs are known to result in distinct pathologies, it is important to understand how these truncations affect the organization of αS into fibrils. Here we present high-resolution microscopy and advanced vibrational spectroscopy studies that indicate that the C-terminal truncation variant of αS, lacking residues 109-140 (henceforth referred to as 1-108-αS), forms amyloid fibrils with a distinct structure and morphology. The 1-108-αS fibrils have a unique negative circular dichroism band at ∼230 nm, a feature that differs from the canonical ∼218 nm band usually observed for amyloid fibrils. We show evidence that 1-108-αS fibrils consist of strongly twisted β-sheets with an increased inter-β-sheet distance and a higher solvent exposure than WT-αS fibrils, which is also indicated by the pronounced differences in the 1D-IR (FTIR), 2D-IR, and vibrational circular dichroism spectra. As a result of their distinct β-sheet structure, 1-108-αS fibrils resist incorporation of WT-αS monomers.

  9. Crystallization of the C-terminal globular domain of avian reovirus fibre

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    Raaij, Mark J. van, E-mail: vanraaij@usc.es [Departamento de Bioquímica y Biología Molecular, Facultad de Farmacia, Universidad de Santiago de Compostela, Campus Sur, E-15782 Santiago de Compostela (Spain); Unidad de Difracción de Rayos X, Laboratorio Integral de Dinámica y Estructura de Biomoléculas José R. Carracido, Edificio CACTUS, Universidad de Santiago de Compostela, Campus Sur, E-15782 Santiago de Compostela (Spain); Hermo Parrado, X. Lois; Guardado Calvo, Pablo [Departamento de Bioquímica y Biología Molecular, Facultad de Farmacia, Universidad de Santiago de Compostela, Campus Sur, E-15782 Santiago de Compostela (Spain); Fox, Gavin C. [Spanish CRG Beamline BM16, European Synchrotron Radiation Facility (ESRF), 6 Rue Jules Horowitz, BP 220, F-38043 Grenoble (France); Llamas-Saiz, Antonio L. [Unidad de Difracción de Rayos X, Laboratorio Integral de Dinámica y Estructura de Biomoléculas José R. Carracido, Edificio CACTUS, Universidad de Santiago de Compostela, Campus Sur, E-15782 Santiago de Compostela (Spain); Costas, Celina; Martínez-Costas, José; Benavente, Javier [Departamento de Bioquímica y Biología Molecular, Facultad de Farmacia, Universidad de Santiago de Compostela, Campus Sur, E-15782 Santiago de Compostela (Spain)

    2005-07-01

    Partial proteolysis of the avian reovirus cell-attachment protein σC yields a major homotrimeric C-terminal fragment that presumably contains the receptor-binding domain. This fragment has been crystallized in the presence and absence of zinc sulfate and cadmium sulfate. One of the crystal forms diffracts synchrotron X-rays to 2.2–2.3 Å. Avian reovirus fibre, a homotrimer of the σC protein, is responsible for primary host-cell attachment. Using the protease trypsin, a C-terminal σC fragment containing amino acids 156–326 has been generated which was subsequently purified and crystallized. Two different crystal forms were obtained, one grown in the absence of divalent cations and belonging to space group P6{sub 3}22 (unit-cell parameters a = 75.6, c = 243.1 Å) and one grown in the presence of either zinc or cadmium sulfate and belonging to space group P321 (unit-cell parameters a = 74.7, c = 74.5 Å and a = 73.1, c = 69.9 Å for the Zn{sup II}- and Cd{sup II}-grown crystals, respectively). The first crystal form diffracted synchrotron radiation to 3.0 Å resolution and the second form to 2.2–2.3 Å. Its closest related structure, the C-terminal fragment of mammalian reovirus fibre, has only 18% sequence identity and molecular-replacement attempts were unsuccessful. Therefore, a search is under way for suitable heavy-atom derivatives and attempts are being made to grow protein crystals containing selenomethionine instead of methionine.

  10. C-terminal tyrosine residues modulate the fusion activity of the Hendra virus fusion protein.

    Science.gov (United States)

    Popa, Andreea; Pager, Cara Teresia; Dutch, Rebecca Ellis

    2011-02-15

    The paramyxovirus family includes important human pathogens such as measles, mumps, respiratory syncytial virus, and the recently emerged, highly pathogenic Hendra and Nipah viruses. The viral fusion (F) protein plays critical roles in infection, promoting both the virus-cell membrane fusion events needed for viral entry as well as cell-cell fusion events leading to syncytia formation. We describe the surprising finding that addition of the short epitope HA tag to the cytoplasmic tail (CT) of the Hendra virus F protein leads to a significant increase in the extent of cell-cell membrane fusion. This increase was not due to alterations in surface expression, cleavage state, or association with lipid microdomains. Addition of a Myc tag of similar length did not alter Hendra F protein fusion activity, indicating that the observed stimulation was not solely a result of lengthening the CT. Three tyrosine residues within the HA tag were critical for the increase in the extent of fusion, suggesting C-terminal tyrosines may modulate Hendra fusion activity. The effects of addition of the HA tag varied with other fusion proteins, as parainfluenza virus 5 F-HA showed a decreased level of surface expression and no stimulation of fusion. These results indicate that additions to the C-terminal end of the F protein CT can modulate protein function in a sequence specific manner, reinforcing the need for careful analysis of epitope-tagged glycoproteins. In addition, our results implicate C-terminal tyrosine residues in the modulation of the membrane fusion reaction promoted by these viral glycoproteins.

  11. BS69/ZMYND11 C-Terminal Domains Bind and Inhibit EBNA2.

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    Matthew R Harter

    2016-02-01

    Full Text Available Epstein-Barr virus (EBV nuclear antigen 2 (EBNA2 plays an important role in driving immortalization of EBV-infected B cells through regulating the expression of many viral and cellular genes. We report a structural study of the tumor suppressor BS69/ZMYND11 C-terminal region, comprised of tandem coiled-coil-MYND domains (BS69CC-MYND, in complex with an EBNA2 peptide containing a PXLXP motif. The coiled-coil domain of BS69 self-associates to bring two separate MYND domains in close proximity, thereby enhancing the BS69 MYND-EBNA2 interaction. ITC analysis of BS69CC-MYND with a C-terminal fragment of EBNA2 further suggests that the BS69CC-MYND homodimer synergistically binds to the two EBNA2 PXLXP motifs that are respectively located in the conserved regions CR7 and CR8. Furthermore, we showed that EBNA2 interacts with BS69 and down-regulates its expression at both mRNA and protein levels in EBV-infected B cells. Ectopic BS69CC-MYND is recruited to viral target promoters through interactions with EBNA2, inhibits EBNA2-mediated transcription activation, and impairs proliferation of lymphoblastoid cell lines (LCLs. Substitution of critical residues in the MYND domain impairs the BS69-EBNA2 interaction and abolishes the BS69 inhibition of the EBNA2-mediated transactivation and LCL proliferation. This study identifies the BS69 C-terminal domains as an inhibitor of EBNA2, which may have important implications in development of novel therapeutic strategies against EBV infection.

  12. Two Disease-Causing SNAP-25B Mutations Selectively Impair SNARE C-terminal Assembly.

    Science.gov (United States)

    Rebane, Aleksander A; Wang, Bigeng; Ma, Lu; Qu, Hong; Coleman, Jeff; Krishnakumar, Shyam; Rothman, James E; Zhang, Yongli

    2018-02-16

    Synaptic exocytosis relies on assembly of three soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins into a parallel four-helix bundle to drive membrane fusion. SNARE assembly occurs by stepwise zippering of the vesicle-associated SNARE (v-SNARE) onto a binary SNARE complex on the target plasma membrane (t-SNARE). Zippering begins with slow N-terminal association followed by rapid C-terminal zippering, which serves as a power stroke to drive membrane fusion. SNARE mutations have been associated with numerous diseases, especially neurological disorders. It remains unclear how these mutations affect SNARE zippering, partly due to difficulties to quantify the energetics and kinetics of SNARE assembly. Here, we used single-molecule optical tweezers to measure the assembly energy and kinetics of SNARE complexes containing single mutations I67T/N in neuronal SNARE synaptosomal-associated protein of 25kDa (SNAP-25B), which disrupt neurotransmitter release and have been implicated in neurological disorders. We found that both mutations significantly reduced the energy of C-terminal zippering by ~10 kBT, but did not affect N-terminal assembly. In addition, we observed that both mutations lead to unfolding of the C-terminal region in the t-SNARE complex. Our findings suggest that both SNAP-25B mutations impair synaptic exocytosis by destabilizing SNARE assembly, rather than stabilizing SNARE assembly as previously proposed. Therefore, our measurements provide insights into the molecular mechanism of the disease caused by SNARE mutations. Copyright © 2017 Elsevier Ltd. All rights reserved.

  13. Groundnut bud necrosis virus encoded NSm associates with membranes via its C-terminal domain.

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    Pratibha Singh

    Full Text Available Groundnut Bud Necrosis Virus (GBNV is a tripartite ambisense RNA plant virus that belongs to serogroup IV of Tospovirus genus. Non-Structural protein-m (NSm, which functions as movement protein in tospoviruses, is encoded by the M RNA. In this communication, we demonstrate that despite the absence of any putative transmembrane domain, GBNV NSm associates with membranes when expressed in E. coli as well as in N. benthamiana. Incubation of refolded NSm with liposomes ranging in size from 200-250 nm resulted in changes in the secondary and tertiary structure of NSm. A similar behaviour was observed in the presence of anionic and zwitterionic detergents. Furthermore, the morphology of the liposomes was found to be modified in the presence of NSm. Deletion of coiled coil domain resulted in the inability of in planta expressed NSm to interact with membranes. Further, when the C-terminal coiled coil domain alone was expressed, it was found to be associated with membrane. These results demonstrate that NSm associates with membranes via the C-terminal coiled coil domain and such an association may be important for movement of viral RNA from cell to cell.

  14. Differentiation of odontoblasts is negatively regulated by MEPE via its C-terminal fragment.

    Science.gov (United States)

    Wang, Hanguo; Kawashima, Nobuyuki; Iwata, Takanori; Xu, Jing; Takahashi, Satomi; Sugiyama, Toshihiro; Suda, Hideaki

    2010-07-30

    Matrix extracellular phosphoglycoprotein (MEPE) is an extracellular matrix protein that is mainly expressed in mineralizing tissues, including the dental pulp. The purposes of this study were to clarify the localization of MEPE in the tooth germ and to investigate the roles of MEPE in the differentiation of odontoblasts. The immunohistochemical staining in the tooth germ of the upper first molars of male Wistar rats (postnatal day 3) revealed that MEPE was mainly localized in odontoblasts during dentinogenesis. Stable MEPE-overexpressing and MEPE-knockdown cell lines, which were established in odontoblast-lineage cells (OLCs), showed lower and higher differentiation capabilities, respectively. Eukaryotic proteins of the N-terminal fragment of MEPE produced in HEK cells had no effect on the differentiation of OLCs, whereas the C-terminal fragment containing an RGD sequence inhibited their differentiation. These results indicated that the C-terminal fragment of MEPE containing an RGD sequence, cleaved in odontoblasts, appeared to be the active form of MEPE, which may play important roles in dentinogenesis and pulpal homeostasis by keeping the odontoblasts in immature condition. Copyright 2010 Elsevier Inc. All rights reserved.

  15. Structure of the RecQ C-terminal domain of human Bloom syndrome protein.

    Science.gov (United States)

    Kim, Sun-Yong; Hakoshima, Toshio; Kitano, Ken

    2013-11-21

    Bloom syndrome is a rare genetic disorder characterized by genomic instability and cancer predisposition. The disease is caused by mutations of the Bloom syndrome protein (BLM). Here we report the crystal structure of a RecQ C-terminal (RQC) domain from human BLM. The structure reveals three novel features of BLM RQC which distinguish it from the previous structures of the Werner syndrome protein (WRN) and RECQ1. First, BLM RQC lacks an aromatic residue at the tip of the β-wing, a key element of the RecQ-family helicases used for DNA-strand separation. Second, a BLM-specific insertion between the N-terminal helices exhibits a looping-out structure that extends at right angles to the β-wing. Deletion mutagenesis of this insertion interfered with binding to Holliday junction. Third, the C-terminal region of BLM RQC adopts an extended structure running along the domain surface, which may facilitate the spatial positioning of an HRDC domain in the full-length protein.

  16. The spt5 C-terminal region recruits yeast 3' RNA cleavage factor I.

    Science.gov (United States)

    Mayer, Andreas; Schreieck, Amelie; Lidschreiber, Michael; Leike, Kristin; Martin, Dietmar E; Cramer, Patrick

    2012-04-01

    During transcription elongation, RNA polymerase II (Pol II) binds the general elongation factor Spt5. Spt5 contains a repetitive C-terminal region (CTR) that is required for cotranscriptional recruitment of the Paf1 complex (D. L. Lindstrom et al., Mol. Cell. Biol. 23:1368-1378, 2003; Z. Zhang, J. Fu, and D. S. Gilmour, Genes Dev. 19:1572-1580, 2005). Here we report a new role of the Spt5 CTR in the recruitment of 3' RNA-processing factors. Chromatin immunoprecipitation (ChIP) revealed that the Spt5 CTR is required for normal recruitment of pre-mRNA cleavage factor I (CFI) to the 3' ends of Saccharomyces cerevisiae genes. RNA contributes to CFI recruitment, as RNase treatment prior to ChIP further decreases CFI ChIP signals. Genome-wide ChIP profiling detected occupancy peaks of CFI subunits around 100 nucleotides downstream of the polyadenylation (pA) sites of genes. CFI recruitment to this defined region may result from simultaneous binding to the Spt5 CTR, to nascent RNA containing the pA sequence, and to the elongating Pol II isoform that is phosphorylated at serine 2 (S2) residues in its C-terminal domain (CTD). Consistent with this model, the CTR interacts with CFI in vitro but is not required for pA site recognition and transcription termination in vivo.

  17. Determination of C-Terminal δ-Catenin Responsible for Inducing Dendritic Morphogenesis.

    Science.gov (United States)

    Lee, Ho-Bin; He, Yongfeng; Yang, Gyeong-Su; Oh, Jin-A; Ha, Ji-Seon; Song, Oh-Hyuen; Lee, Do-Jin; Jung, Sang-Chul; Kim, Kyung Keun; Kim, Kwonseop; Kim, Hangun

    2015-08-01

    δ-Catenin induces dendritic morphogenesis in several cells and it was reported that deletion of C-terminal 207 amino acid of δ-catenin completely abolished the dendritic morphogenesis. However, exact domain responsible for inducing dendritic morphogenesis in C-terminus of δ-catenin was not mapped. Here, we report that expression of ΔC47 (lacking 47 amino acid of C-terminus: 1-1200), ΔC77 (lacking 77 amino acid of C-terminus: 1-1170) deletion mutants of δ-catenin induced the dendritic morphogenesis of HEK293T and NIH3T3 cells as full-length δ-catenin did. In agreement with previous report, ΔC207 deletion mutant did not show the dendritic morphogenesis of the cells. Interestingly, introducing 107 amino acid deletion of C-terminus (ΔC107 mutant: 1-1140) and 177 amino acid deletion of C-terminus (ΔC177 mutant: 1-1070) showed limited primary and secondary dendritic process and notable spine-like process formation. These results suggest that 1140-1170 amino acid of C-terminal δ-catenin is required for primary and secondary dendrite-like process formation.

  18. Effect of C-terminal domain truncation of Thermus thermophilus trehalose synthase on its substrate specificity.

    Science.gov (United States)

    Cho, Chang-Bae; Park, Da-Yeon; Lee, Soo-Bok

    2017-01-01

    The C-terminal domain of the three-domain-comprising trehalose synthase from Thermus thermophilus was truncated in order to study the effect on the enzyme's activity and substrate specificity. Compared with the wild-type (WT) enzyme, the two truncated enzymes (DM1 and DM2) showed lower maltose- and trehalose-converting activities and a different transglycosylation reaction mechanism. In the mutants, the glucose moiety cleaved from the maltose substrate was released from the enzyme and intercepted by external glucose oxidase, preventing the production of trehalose. The WT enzyme, however, retained the glucose in the active site to effectively produce trehalose. In addition, DM1 synthesized much higher amounts of mannose-containing disaccharide trehalose analog (Man-TA) than did the WT and DM2. The results suggest that the C-terminal domain in the WT enzyme is important for retaining the glucose moiety within the active site. The mutant enzymes could be used to produce Man-TA, a postulated inhibitor of gut disaccharidases. Copyright © 2016 Elsevier Inc. All rights reserved.

  19. C-terminal moiety of Tudor contains its in vivo activity in Drosophila.

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    Joël Anne

    Full Text Available BACKGROUND: In early Drosophila embryos, the germ plasm is localized to the posterior pole region and is partitioned into the germline progenitors, known as pole cells. Germ plasm, or pole plasm, contains the polar granules which form during oogenesis and are required for germline development. Components of these granules are also present in the perinuclear region of the nurse cells, the nuage. One such component is Tudor (Tud which is a large protein containing multiple Tudor domains. It was previously reported that specific Tudor domains are required for germ cell formation and Tud localization. METHODOLOGY/PRINCIPAL FINDINGS: In order to better understand the function of Tud the distribution and functional activity of fragments of Tud were analyzed. These fragments were fused to GFP and the fusion proteins were synthesized during oogenesis. Non-overlapping fragments of Tud were found to be able to localize to both the nuage and pole plasm. By introducing these fragments into a tud mutant background and testing their ability to rescue the tud phenotype, I determined that the C-terminal moiety contains the functional activity of Tud. Dividing this fragment into two parts reduces its localization in pole plasm and abolishes its activity. CONCLUSIONS/SIGNIFICANCE: I conclude that the C-terminal moiety of Tud contains all the information necessary for its localization in the nuage and pole plasm and its pole cell-forming activity. The present results challenge published data and may help refining the functional features of Tud.

  20. Identification of Novel Short C-Terminal Transcripts of Human SERPINA1 Gene.

    Science.gov (United States)

    Matamala, Nerea; Aggarwal, Nupur; Iadarola, Paolo; Fumagalli, Marco; Gomez-Mariano, Gema; Lara, Beatriz; Martinez, Maria Teresa; Cuesta, Isabel; Stolk, Jan; Janciauskiene, Sabina; Martinez-Delgado, Beatriz

    2017-01-01

    Human SERPINA1 gene is located on chromosome 14q31-32.3 and is organized into three (IA, IB, and IC) non-coding and four (II, III, IV, V) coding exons. This gene produces α1-antitrypsin (A1AT), a prototypical member of the serpin superfamily of proteins. We demonstrate that human peripheral blood leukocytes express not only a product corresponding to the transcript coding for the full-length A1AT protein but also two short transcripts (ST1C4 and ST1C5) of A1AT. In silico sequence analysis revealed that the last exon of the short transcripts contains an Open Reading Frame (ORF) and thus putatively can produce peptides. We found ST1C4 expression across different human tissues whereas ST1C5 was mainly restricted to leukocytes, specifically neutrophils. A high up-regulation (10-fold) of short transcripts was observed in isolated human blood neutrophils after activation with lipopolysaccharide. Parallel analyses by liquid chromatography-mass spectrometry identified peptides corresponding to C-terminal region of A1AT in supernatants of activated but not naïve neutrophils. Herein we report for the first time a tissue specific expression and regulation of short transcripts of SERPINA1 gene, and the presence of C-terminal peptides in supernatants from activated neutrophils, in vitro. This gives a novel insight into the studies on the transcription of SERPINA1 gene.

  1. Intrinsic Disorder of the C-Terminal Domain of Drosophila Methoprene-Tolerant Protein.

    Directory of Open Access Journals (Sweden)

    Marta Kolonko

    Full Text Available Methoprene tolerant protein (Met has recently been confirmed as the long-sought juvenile hormone (JH receptor. This protein plays a significant role in the cross-talk of the 20-hydroxyecdysone (20E and JH signalling pathways, which are important for control of insect development and maturation. Met belongs to the basic helix-loop-helix/Per-Arnt-Sim (bHLH-PAS family of transcription factors. In these proteins, bHLH domains are typically responsible for DNA binding and dimerization, whereas the PAS domains are crucial for the choice of dimerization partner and the specificity of target gene activation. The C-terminal region is usually responsible for the regulation of protein complex activity. The sequence of the Met C-terminal region (MetC is not homologous to any sequence deposited in the Protein Data Bank (PDB and has not been structurally characterized to date. In this study, we show that the MetC exhibits properties typical for an intrinsically disordered protein (IDP. The final averaged structure obtained with small angle X-ray scattering (SAXS experiments indicates that intrinsically disordered MetC exists in an extended conformation. This extended shape and the long unfolded regions characterise proteins with high flexibility and dynamics. Therefore, we suggest that the multiplicity of conformations adopted by the disordered MetC is crucial for its activity as a biological switch modulating the cross-talk of different signalling pathways in insects.

  2. Investigating the Roles of the C-Terminal Domain of Plasmodium falciparum GyrA.

    Directory of Open Access Journals (Sweden)

    Soshichiro Nagano

    Full Text Available Malaria remains as one of the most deadly diseases in developing countries. The Plasmodium causative agents of human malaria such as Plasmodium falciparum possess an organelle, the apicoplast, which is the result of secondary endosymbiosis and retains its own circular DNA. A type II topoisomerase, DNA gyrase, is present in the apicoplast. In prokaryotes this enzyme is a proven, effective target for antibacterial agents, and its discovery in P. falciparum opens up the prospect of exploiting it as a drug target. Basic characterisation of P. falciparum gyrase is important because there are significant sequence differences between it and the prokaryotic enzyme. However, it has proved difficult to obtain soluble protein. Here we have predicted a new domain boundary in P. falciparum GyrA that corresponds to the C-terminal domain of prokaryotic GyrA and successfully purified it in a soluble form. Biochemical analyses revealed many similarities between the C-terminal domains of GyrA from E. coli and P. falciparum, suggesting that despite its considerably larger size, the malarial protein carries out a similar DNA wrapping function. Removal of a unique Asn-rich region in the P. falciparum protein did not result in a significant change, suggesting it is dispensable for DNA wrapping.

  3. A short C-terminal sequence is necessary and sufficient for the targeting of chitinases to the plant vacuole.

    OpenAIRE

    Neuhaus, J.M. (John M.); Sticher, L.; Meins, F; Boller, T.

    1991-01-01

    Tobacco contains different isoforms of chitinase (EC 3.2.1.14), a hydrolase thought to be involved in the defense against pathogens. Deduced amino acid sequences for putatively vacuolar, basic chitinases differ from the homologous extracellular, acidic isoforms by the presence of a C-terminal extension. To examine the role of this C-terminal extension in protein sorting, Nicotiana silvestris plants were stably transformed with chimeric genes coding for tobacco basic chitinase A with and witho...

  4. C-terminal Src kinase-mediated EPIYA phosphorylation of Pragmin creates a feed-forward C-terminal Src kinase activation loop that promotes cell motility.

    Science.gov (United States)

    Senda, Yoshie; Murata-Kamiya, Naoko; Hatakeyama, Masanori

    2016-07-01

    Pragmin is one of the few mammalian proteins containing the Glu-Pro-Ile-Tyr-Ala (EPIYA) tyrosine-phosphorylation motif that was originally discovered in the Helicobacter pylori CagA oncoprotein. Following delivery into gastric epithelial cells by type IV secretion and subsequent tyrosine phosphorylation at the EPIYA motifs, CagA serves as an oncogenic scaffold/adaptor that promiscuously interacts with SH2 domain-containing mammalian proteins such as the Src homology 2 (SH2) domain-containing protein tyrosine phosphatase-2 (SHP2) and the C-terminal Src kinase (Csk), a negative regulator of Src family kinases. Like CagA, Pragmin also forms a physical complex with Csk. In the present study, we found that Pragmin directly binds to Csk by the tyrosine-phosphorylated EPIYA motif. The complex formation potentiates kinase activity of Csk, which in turn phosphorylates Pragmin on tyrosine-238 (Y238), Y343, and Y391. As Y391 of Pragmin comprises the EPIYA motif, Pragmin-Csk interaction creates a feed-forward regulatory loop of Csk activation. Together with the finding that Pragmin and Csk are colocalized to focal adhesions, these observations indicate that the Pragmin-Csk interaction, triggered by Pragmin EPIYA phosphorylation, robustly stimulates the kinase activity of Csk at focal adhesions, which direct cell-matrix adhesion that regulates cell morphology and cell motility. As a consequence, expression of Pragmin and/or Csk in epithelial cells induces an elongated cell shape with elevated cell scattering in a manner that is mutually dependent on Pragmin and Csk. Deregulation of the Pragmin-Csk axis may therefore induce aberrant cell migration that contributes to tumor invasion and metastasis. © 2016 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

  5. C-terminal hemocyanin from hemocytes of Penaeus vannamei interacts with ERK1/2 and undergoes serine phosphorylation.

    Science.gov (United States)

    Havanapan, Phattara-orn; Kanlaya, Rattiyaporn; Bourchookarn, Apichai; Krittanai, Chartchai; Thongboonkerd, Visith

    2009-05-01

    To understand molecular immune response of Penaeus vannamei during Taura syndrome virus (TSV) infection, expression and functional proteomics studies were performed on hemocyanin, which is a major abundant protein in shrimp hemocytes. Two-dimensional electrophoresis (2-DE) revealed up-regulation of several C-terminal fragments of hemocyanin, whereas the N-terminal fragments were down-regulated during TSV infection. 2-D Western blot analysis showed that the C-terminal hemocyanin fragments had more acidic isoelectric points (pI), whereas the N-terminal fragments had less acidic pI. Further analysis by NetPhos showed a greater number of serine phosphorylation sites in the C-terminal hemocyanin. Additionally, motif scan using Scansite revealed ERK D-domain, which is required for activation of ERK1/2 effector kinase, as a kinase-binding site at the 527th valine in the C-terminal hemocyanin, whereas neither motif nor functional domain was found in the N-terminus. Co-immunoprecipitation confirmed the interaction between the C-terminal hemocyanin and ERK1/2. 1-D Western blot analysis showed that ERK1/2 was also up-regulated during TSV infection. Our findings demonstrate for the first time that ERK1/2 signaling pathway may play an important role in molecular immune response of P. vannamei upon TSV infection through its interaction with the C-terminal hemocyanin.

  6. Nucleation process of a fibril precursor in the C-terminal segment of amyloid-β.

    Science.gov (United States)

    Baftizadeh, Fahimeh; Pietrucci, Fabio; Biarnés, Xevi; Laio, Alessandro

    2013-04-19

    By extended atomistic simulations in explicit solvent and bias-exchange metadynamics, we study the aggregation process of 18 chains of the C-terminal segment of amyloid-β, an intrinsically disordered protein involved in Alzheimer's disease and prone to form fibrils. Starting from a disordered aggregate, we are able to observe the formation of an ordered nucleus rich in beta sheets. The rate limiting step in the nucleation pathway involves crossing a barrier of approximately 40 kcal/mol and is associated with the formation of a very specific interdigitation of the side chains belonging to different sheets. This structural pattern is different from the one observed experimentally in a microcrystal of the same system, indicating that the structure of a "nascent" fibril may differ from the one of an "extended" fibril.

  7. The C-terminal region of E1A: a molecular tool for cellular cartography.

    Science.gov (United States)

    Yousef, Ahmed F; Fonseca, Gregory J; Cohen, Michael J; Mymryk, Joe S

    2012-04-01

    The adenovirus E1A proteins function via protein-protein interactions. By making many connections with the cellular protein network, individual modules of this virally encoded hub reprogram numerous aspects of cell function and behavior. Although many of these interactions have been thoroughly studied, those mediated by the C-terminal region of E1A are less well understood. This review focuses on how this region of E1A affects cell cycle progression, apoptosis, senescence, transformation, and conversion of cells to an epithelial state through interactions with CTBP1/2, DYRK1A/B, FOXK1/2, and importin-α. Furthermore, novel potential pathways that the C-terminus of E1A influences through these connections with the cellular interaction network are discussed.

  8. C-terminal region of Mad2 plays an important role during mitotic spindle checkpoint in fission yeast Schizosaccharomyces pombe.

    Science.gov (United States)

    Singh, Gaurav Kumar; Karade, Sharanbasappa Shrimant; Ranjan, Rajeev; Ahamad, Nafees; Ahmed, Shakil

    2017-02-01

    The mitotic arrest deficiency 2 (Mad2) protein is an essential component of the spindle assembly checkpoint that interacts with Cdc20/Slp1 and inhibit its ability to activate anaphase promoting complex/cyclosome (APC/C). In bladder cancer cell line the C-terminal residue of the mad2 gene has been found to be deleted. In this study we tried to understand the role of the C-terminal region of mad2 on the spindle checkpoint function. To envisage the role of C-terminal region of Mad2, we truncated 25 residues of Mad2 C-terminal region in fission yeast S.pombe and characterized its effect on spindle assembly checkpoint function. The cells containing C-terminal truncation of Mad2 exhibit sensitivity towards microtubule destabilizing agent suggesting perturbation of spindle assembly checkpoint. Further, the C-terminal truncation of Mad2 exhibit reduced viability in the nda3-KM311 mutant background at non-permissive temperature. Truncation in mad2 gene also affects its foci forming ability at unattached kinetochore suggesting that the mad2-∆CT mutant is unable to maintain spindle checkpoint activation. However, in response to the defective microtubule, only brief delay of mitotic progression was observed in Mad2 C-terminal truncation mutant. In addition we have shown that the deletion of two β strands of Mad2 protein abolishes its ability to interact with APC activator protein Slp1/Cdc20. We purpose that the truncation of two β strands (β7 and β8) of Mad2 destabilize the safety belt and affect the Cdc20-Mad2 interaction leading to defects in the spindle checkpoint activation.

  9. Loss of Smad4 function in pancreatic tumors: C-terminal truncation leads to decreased stability.

    Science.gov (United States)

    Maurice, D; Pierreux, C E; Howell, M; Wilentz, R E; Owen, M J; Hill, C S

    2001-11-16

    At early stages of tumorigenesis, the transforming growth factor-beta (TGF-beta) signaling pathway is thought to have tumor suppressor activity as a result of its ability to arrest the growth of epithelial cells. Smad4 plays a pivotal role in the TGF-beta signaling pathway and has been identified as a tumor suppressor, being mutated or deleted in approximately 50% of pancreatic carcinomas and 15% of colorectal cancers. A nonsense mutation generating a C-terminal truncation of 38 amino acids in the Smad4 protein has been identified in a pancreatic adenocarcinoma (Hahn, S. A., Schutte, M., Hoque, A. T., Moskaluk, C. A., da Costa, L. T., Rozenblum, E., Weinstein, C. L., Fischer, A., Yeo, C. J., Hruban, R. H., and Kern, S. E. (1996) Science 271, 350-353), and here we investigate the functional consequences of this mutation. We demonstrate that the C-terminal truncation prevents Smad4 homomeric complex formation and heteromeric complex formation with activated Smad2. Furthermore, the mutant protein is unable to be recruited to DNA by transcription factors and hence cannot form transcriptionally active DNA-binding complexes. These observations are supported by molecular modeling, which indicates that the truncation removes residues critical for homomeric and heteromeric Smad complex formation. We go on to show that the mutant Smad4 is highly unstable compared with wild type Smad4 and is rapidly degraded through the ubiquitin-proteasome pathway. Consistent with this, we demonstrate that the pancreatic adenocarcinoma harboring this mutated allele, in conjunction with loss of the other allele, expresses no Smad4 protein. Thus we conclude that these tumors completely lack Smad4 activity.

  10. PrPSc-Specific Antibody Reveals C-Terminal Conformational Differences between Prion Strains.

    Science.gov (United States)

    Saijo, Eri; Hughson, Andrew G; Raymond, Gregory J; Suzuki, Akio; Horiuchi, Motohiro; Caughey, Byron

    2016-05-15

    Understanding the structure of PrP(Sc) and its strain variation has been one of the major challenges in prion disease biology. To study the strain-dependent conformations of PrP(Sc), we purified proteinase-resistant PrP(Sc) (PrP(RES)) from mouse brains with three different murine-adapted scrapie strains (Chandler, 22L, and Me7) and systematically tested the accessibility of epitopes of a wide range of anti-PrP and anti-PrP(Sc) specific antibodies by indirect enzyme-linked immunosorbent assay (ELISA). We found that epitopes of most anti-PrP antibodies were hidden in the folded structure of PrP(RES), even though these epitopes are revealed with guanidine denaturation. However, reactivities to a PrP(Sc)-specific conformational C-terminal antibody showed significant differences among the three different prion strains. Our results provide evidence for strain-dependent conformational variation near the C termini of molecules within PrP(Sc) multimers. It has long been apparent that prion strains can have different conformations near the N terminus of the PrP(Sc) protease-resistant core. Here, we show that a C-terminal conformational PrP(Sc)-specific antibody reacts differently to three murine-adapted scrapie strains. These results suggest, in turn, that conformational differences in the C terminus of PrP(Sc) also contribute to the phenotypic distinction between prion strains. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  11. Screening for Small Molecule Inhibitors of Statin-Induced APP C-terminal Toxic Fragment Production.

    Science.gov (United States)

    Poksay, Karen S; Sheffler, Douglas J; Spilman, Patricia; Campagna, Jesus; Jagodzinska, Barbara; Descamps, Olivier; Gorostiza, Olivia; Matalis, Alex; Mullenix, Michael; Bredesen, Dale E; Cosford, Nicholas D P; John, Varghese

    2017-01-01

    Alzheimer's disease (AD) is characterized by neuronal and synaptic loss. One process that could contribute to this loss is the intracellular caspase cleavage of the amyloid precursor protein (APP) resulting in release of the toxic C-terminal 31-amino acid peptide APP-C31 along with the production of APPΔC31, full-length APP minus the C-terminal 31 amino acids. We previously found that a mutation in APP that prevents this caspase cleavage ameliorated synaptic loss and cognitive impairment in a murine AD model. Thus, inhibition of this cleavage is a reasonable target for new therapeutic development. In order to identify small molecules that inhibit the generation of APP-C31, we first used an APPΔC31 cleavage site-specific antibody to develop an AlphaLISA to screen several chemical compound libraries for the level of N-terminal fragment production. This antibody was also used to develop an ELISA for validation studies. In both high throughput screening (HTS) and validation testing, the ability of compounds to inhibit simvastatin- (HTS) or cerivastatin- (validation studies) induced caspase cleavage at the APP-D720 cleavage site was determined in Chinese hamster ovary (CHO) cells stably transfected with wildtype (wt) human APP (CHO-7W). Several compounds, as well as control pan-caspase inhibitor Q-VD-OPh, inhibited APPΔC31 production (measured fragment) and rescued cell death in a dose-dependent manner. The effective compounds fell into several classes including SERCA inhibitors, inhibitors of Wnt signaling, and calcium channel antagonists. Further studies are underway to evaluate the efficacy of lead compounds - identified here using cells and tissues expressing wt human APP - in mouse models of AD expressing mutated human APP, as well as to identify additional compounds and determine the mechanisms by which they exert their effects.

  12. Characteristic NH3 and CO losses from sodiated peptides C-terminated by glutamine residues.

    Science.gov (United States)

    Guan, Xinshu; Wang, Bing; Wang, Huixin; Liu, Jinrong; Li, Ying; Guo, Xinhua

    2017-04-15

    Under certain conditions some amino acid (AA) residues undergo special reactions in the gas phase, generating characteristic neutral losses and product ions. Taking these special fragments into account and understanding the effect of AA residues on peptide cleavages will consummate database search algorithms and manual data interpretation in peptide sequencing by mass spectrometry (MS). In this study, the details of the characteristic NH3 and CO losses of glutamine (Gln) residues located at the C-terminus of peptides are presented. A number of selected peptides were fragmented under collision-induced dissociation (CID) in electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-QTOF-MS). Density functional theory (DFT) quantum mechanical calculations at the B3LYP/6-31+G(d,p) level were carried out to optimize the geometry of peptide ions and provide energy barriers of ions in each step during fragmentations. Two characteristic peaks appear near the precursor ions of sodiated Gln C-terminated peptides, suggesting the loss of neutral NH3 and CO via a two-step process. The proposed mechanism of their formation is as follows: after losing NH3 , a non-classical bn   * ion is formed with a glutaric anhydride structure that further dissociates to lose CO. The sodiated peptides show more intensive peaks corresponding to the loss of neutral molecules than the protonated ones. This type of neutral loss can also occur at the Gln residue that is rearranged to the C-terminus of sodiated peptides. The experiments and calculations suggest that the two-step characteristic NH3 and CO loss of sodiated peptides is energetically favored, and can be applied to identify C-terminated Gln residues. This study provides a mechanistic insight into the role of sodium ion during peptide fragmentation. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

  13. The C-terminal region of alpha-crystallin: involvement in protection against heat-induced denaturation

    Science.gov (United States)

    Takemoto, L.; Emmons, T.; Horwitz, J.; Spooner, B. S. (Principal Investigator)

    1993-01-01

    Recent studies have demonstrated that the alpha-crystallins can protect other proteins against heat-induced denaturation and aggregation. To determine the possible involvement of the C-terminal region in this activity, the alpha-crystallins were subjected to limited tryptic digestion, and the amount of cleavage from the N-terminal and C-terminal regions of the alpha-A and alpha-B crystallin chains was assessed using antisera specific for these regions. Limited tryptic digestion resulted in cleavage only from the C-terminal region of alpha-A crystallin. This trypsin-treated alpha-A crystallin preparation showed a decreased ability to protect proteins from heat-induced aggregation using an in vitro assay. Together, these results demonstrate that the C-terminal region of alpha-A crystallin is important for its ability to protect against heat-induced aggregation, which is consistent with the hypothesis that post-translational changes that are known to occur at the C-terminal region may have significant effects on the ability of alpha-A crystallin to protect against protein denaturation in vivo.

  14. Specific recognition of the C-terminal end of A beta 42 by a high affinity monoclonal antibody

    DEFF Research Database (Denmark)

    Axelsen, Trine Veje; Holm, Arne; Birkelund, Svend

    2009-01-01

    The neurotoxic peptide A beta(42) is derived from the amyloid precursor protein by proteolytic cleavage and is deposited in the brain of patients suffering from Alzheimer's disease (AD). In this study we generate a high affinity monoclonal antibody that targets the C-terminal end of A beta(42......) with high specificity. By this is meant that the paratope of the antibody must enclose the C-terminal end of A beta(42) including the carboxy-group of amino acid 42, and not just recognize a linear epitope in the C-terminal part of A beta. This has been accomplished by using a unique antigen construct made...... by the Ligand Presenting Assembly technology (LPA technology). This strategy results in dimeric presentation of the free C-terminal end of A beta(42). The generated Mab A beta1.1 is indeed specific for the C-terminal end of A beta(42) to which it binds with high affinity. Mab A beta1.1 recognizes the epitope...

  15. C-Terminal Binding Protein: A Molecular Link between Metabolic Imbalance and Epigenetic Regulation in Breast Cancer

    Directory of Open Access Journals (Sweden)

    Jung S. Byun

    2013-01-01

    Full Text Available The prevalence of obesity has given rise to significant global concerns as numerous population-based studies demonstrate an incontrovertible association between obesity and breast cancer. Mechanisms proposed to account for this linkage include exaggerated levels of carbohydrate substrates, elevated levels of circulating mitogenic hormones, and inflammatory cytokines that impinge on epithelial programming in many tissues. Moreover, recently many scientists have rediscovered the observation, first described by Otto Warburg nearly a century ago, that most cancer cells undergo a dramatic metabolic shift in energy utilization and expenditure that fuels and supports the cellular expansion associated with malignant proliferation. This shift in substrate oxidation comes at the cost of sharp changes in the levels of the high energy intermediate, nicotinamide adenine dinucleotide (NADH. In this review, we discuss a novel example of how shifts in the concentration and flux of substrates metabolized and generated during carbohydrate metabolism represent components of a signaling network that can influence epigenetic regulatory events in the nucleus. We refer to this regulatory process as “metabolic transduction” and describe how the C-terminal binding protein (CtBP family of NADH-dependent nuclear regulators represents a primary example of how cellular metabolic status can influence epigenetic control of cellular function and fate.

  16. The Truncated C-terminal Fragment of Mutant ATXN3 Disrupts Mitochondria Dynamics in Spinocerebellar Ataxia Type 3 Models

    Directory of Open Access Journals (Sweden)

    Jung-Yu Hsu

    2017-06-01

    Full Text Available Spinocerebellar ataxia type 3 (SCA3, known as Machado-Joseph disease, is an autosomal dominant disease caused by an abnormal expansion of polyglutamine in ATXN3 gene, leading to neurodegeneration in SCA3 patients. Similar to other neurodegenerative diseases, the dysfunction of mitochondria is observed to cause neuronal death in SCA3 patients. Based on previous studies, proteolytic cleavage of mutant ATXN3 is found to produce truncated C-terminal fragments in SCA3 models. However, whether these truncated mutant fragments disturb mitochondrial functions and result in pathological death is still unclear. Here, we used neuroblastoma cell and transgenic mouse models to examine the effects of truncated mutant ATXN3 on mitochondria functions. In different models, we observed truncated mutant ATXN3 accelerated the formation of aggregates, which translocated into the nucleus to form intranuclear aggregates. In addition, truncated mutant ATXN3 caused more mitochondrial fission, and decreased the expression of mitochondrial fusion markers, including Mfn-1 and Mfn-2. Furthermore, truncated mutant ATXN3 decreased the mitochondrial membrane potential, increased reactive oxygen species and finally increased cell death rate. In transgenic mouse models, truncated mutant ATXN3 also led to more mitochondrial dysfunction, neurodegeneration and cell death in the cerebellums. This study supports the toxic fragment hypothesis in SCA3, and also provides evidence that truncated mutant ATXN3 is severer than full-length mutant one in vitro and in vivo.

  17. Solution structure and dynamics of C-terminal regulatory domain of Vibrio vulnificus extracellular metalloprotease

    Energy Technology Data Exchange (ETDEWEB)

    Yun, Ji-Hye; Kim, Heeyoun [Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749 (Korea, Republic of); Park, Jung Eun [Department of Biotechnology, College of Natural Sciences, Chosun University, Gwangju 501-759 (Korea, Republic of); Lee, Jung Sup, E-mail: jsplee@mail.chosun.ac.kr [Department of Biotechnology, College of Natural Sciences, Chosun University, Gwangju 501-759 (Korea, Republic of); Lee, Weontae, E-mail: wlee@spin.yonsei.ac.kr [Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749 (Korea, Republic of)

    2013-01-11

    Highlights: Black-Right-Pointing-Pointer We have determined solution structures of vEP C-terminal regulatory domain. Black-Right-Pointing-Pointer vEP C-ter100 has a compact {beta}-barrel structure with eight anti-parallel {beta}-strands. Black-Right-Pointing-Pointer Solution structure of vEP C-ter100 shares its molecular topology with that of the collagen-binding domain of collagenase. Black-Right-Pointing-Pointer Residues in the {beta}3 region of vEP C-ter100 might be important in putative ligand/receptor binding. Black-Right-Pointing-Pointer vEP C-ter100 interacts strongly with iron ion. -- Abstract: An extracellular metalloprotease (vEP) secreted by Vibrio vulnificus ATCC29307 is a 45-kDa proteolytic enzyme that has prothrombin activation and fibrinolytic activities during bacterial infection. The action of vEP could result in clotting that could serve to protect the bacteria from the host defense machinery. Very recently, we showed that the C-terminal propeptide (C-ter100), which is unique to vEP, is involved in regulation of vEP activity. To understand the structural basis of this function of vEP C-ter100, we have determined the solution structure and backbone dynamics using multidimensional nuclear magnetic resonance spectroscopy. The solution structure shows that vEP C-ter100 is composed of eight anti-parallel {beta}-strands with a unique fold that has a compact {beta}-barrel formation which stabilized by hydrophobic and hydrogen bonding networks. Protein dynamics shows that the overall structure, including loops, is very rigid and stabilized. By structural database analysis, we found that vEP C-ter100 shares its topology with that of the collagen-binding domain of collagenase, despite low sequence homology between the two domains. Fluorescence assay reveals that vEP C-ter100 interacts strongly with iron (Fe{sup 3+}). These findings suggest that vEP protease might recruit substrate molecules, such as collagen, by binding at C-ter100 and that vEP participates

  18. Specific recognition of the C-terminal end of A beta 42 by a high affinity monoclonal antibody

    DEFF Research Database (Denmark)

    Axelsen, T.V.; Holm, A.; Birkelund, S.

    2009-01-01

    ) with high specificity. By this is meant that the paratope of the antibody must enclose the C-terminal end of A beta(42) including the carboxy-group of amino acid 42, and not just recognize a linear epitope in the C-terminal part of A beta. This has been accomplished by using a unique antigen construct made...... in human AD tissue and stains plaques with high specificity. Therefore the monoclonal antibody can thus be useful in the histological investigations of the AD pathology Udgivelsesdato: 2009/7...

  19. Pigs produce only a single form of CGRP, part of which is processed to N- and C-terminal fragments

    DEFF Research Database (Denmark)

    Rasmussen, T N; Bersani, M; Johnsen, A H

    1994-01-01

    Using radioimmunoassays with two different antisera, one directed towards the C-terminal and one towards the mid part of porcine and human alpha-CGRP, respectively, we isolated three immunoreactive peptides from acid/ethanol extracts of porcine spinal cord by means of HPLC. By amino acid sequence...... to detect any second full-length form of CGRP. Thus, we conclude that only a single form of full-length CGRP is found in pigs and that this peptide may be cleaved to produce potentially bioactive N- and C-terminal fragments....

  20. Physical association of GPR54 C-terminal with protein phosphatase 2A.

    Science.gov (United States)

    Evans, Barry J; Wang, Zixuan; Mobley, La'Tonya; Khosravi, Davood; Fujii, Nobutaka; Navenot, Jean-Marc; Peiper, Stephen C

    2008-12-26

    KiSS1 was discovered as a metastasis suppressor gene and subsequently found to encode kisspeptins (KP), ligands for a G protein coupled receptor (GPCR), GPR54. This ligand-receptor pair was later shown to play a critical role in the neuro-endocrine regulation of puberty. The C-terminal cytoplasmic (C-ter) domain of GPR54 contains a segment rich in proline and arginine residues that corresponds to the primary structure of four overlapping SH3 binding motifs. Yeast two hybrid experiments identified the catalytic subunit of protein phosphatase 2A (PP2A-C) as an interacting protein. Pull-down experiments with GST fusion proteins containing the GPR54 C-ter confirmed binding to PP2A-C in cell lysates and these complexes contained phosphatase activity. The proline arginine rich segment is necessary for these interactions. The GPR54 C-ter bound directly to purified recombinant PP2A-C, indicating the GPR54 C-ter may form complexes involving the catalytic subunit of PP2A that regulate phosphorylation of critical signaling intermediates.

  1. Structural Aspects of N-Glycosylations and the C-terminal Region in Human Glypican-1.

    Science.gov (United States)

    Awad, Wael; Adamczyk, Barbara; Örnros, Jessica; Karlsson, Niclas G; Mani, Katrin; Logan, Derek T

    2015-09-18

    Glypicans are multifunctional cell surface proteoglycans involved in several important cellular signaling pathways. Glypican-1 (Gpc1) is the predominant heparan sulfate proteoglycan in the developing and adult human brain. The two N-linked glycans and the C-terminal domain that attach the core protein to the cell membrane are not resolved in the Gpc1 crystal structure. Therefore, we have studied Gpc1 using crystallography, small angle x-ray scattering, and chromatographic approaches to elucidate the composition, structure, and function of the N-glycans and the C terminus and also the topology of Gpc1 with respect to the membrane. The C terminus is shown to be highly flexible in solution, but it orients the core protein transverse to the membrane, directing a surface evolutionarily conserved in Gpc1 orthologs toward the membrane, where it may interact with signaling molecules and/or membrane receptors on the cell surface, or even the enzymes involved in heparan sulfate substitution in the Golgi apparatus. Furthermore, the N-glycans are shown to extend the protein stability and lifetime by protection against proteolysis and aggregation. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  2. Conservation and divergence of C-terminal domain structure in the retinoblastoma protein family

    Energy Technology Data Exchange (ETDEWEB)

    Liban, Tyler J.; Medina, Edgar M.; Tripathi, Sarvind; Sengupta, Satyaki; Henry, R. William; Buchler, Nicolas E.; Rubin, Seth M. (UCSC); (Duke); (MSU)

    2017-04-24

    The retinoblastoma protein (Rb) and the homologous pocket proteins p107 and p130 negatively regulate cell proliferation by binding and inhibiting members of the E2F transcription factor family. The structural features that distinguish Rb from other pocket proteins have been unclear but are critical for understanding their functional diversity and determining why Rb has unique tumor suppressor activities. We describe here important differences in how the Rb and p107 C-terminal domains (CTDs) associate with the coiled-coil and marked-box domains (CMs) of E2Fs. We find that although CTD–CM binding is conserved across protein families, Rb and p107 CTDs show clear preferences for different E2Fs. A crystal structure of the p107 CTD bound to E2F5 and its dimer partner DP1 reveals the molecular basis for pocket protein–E2F binding specificity and how cyclin-dependent kinases differentially regulate pocket proteins through CTD phosphorylation. Our structural and biochemical data together with phylogenetic analyses of Rb and E2F proteins support the conclusion that Rb evolved specific structural motifs that confer its unique capacity to bind with high affinity those E2Fs that are the most potent activators of the cell cycle.

  3. Characterization of AWAP IV, the C-terminal domain of the avian protein AWAK.

    Science.gov (United States)

    Townes, C L; Milona, P; Hall, J

    2006-04-01

    AWAP IV constitutes the C-terminal domain of the larger 81 kDa protein AWAK [Avian WAP (whey acidic protein) domain- and Kunitz domain-containing], which is predicted, through conserved domain database searching, to contain at least four WAP domains and one Kunitz domain. RT (reverse transcription)-PCR analyses revealed mRNA transcripts encoding AWAP IV in the small intestinal and kidney tissues of 5-day-old Salmonella-infected chicks. Time-kill antimicrobial assays using rAWAP IV (recombinant AWAP IV) cell lysate indicated antimicrobial activity against gram-positive and gram-negative bacteria including Salmonella, Streptococcus and Staphylococcus spp. In addition, permeabilization of the outer membrane of Salmonella, as shown by the NPN (N-phenyl-1-naphthylamine) fluorescent probe assay, supported the ability of rAWAP IV to disrupt prokaryotic membranes. WAP domains can function as inhibitors of serine protease activity, and the microbial serine proteases subtilisin and proteinase K were inhibited by rAWAP IV cell lysate. However, at comparable concentrations, no significant inhibition of the mammalian serine protease elastase was observed. The combined broad-spectrum antibacterial and anti-protease activities of AWAP IV suggest a novel role in the avian innate defence mechanisms operating against microbial infection.

  4. C-terminal domains implicated in the functional surface expression of potassium channels

    Science.gov (United States)

    Jenke, Marc; Sánchez, Araceli; Monje, Francisco; Stühmer, Walter; Weseloh, Rüdiger M.; Pardo, Luis A.

    2003-01-01

    A short C-terminal domain is required for correct tetrameric assembly in some potassium channels. Here, we show that this domain forms a coiled coil that determines not only the stability but also the selectivity of the multimerization. Synthetic peptides comprising the sequence of this domain in Eag1 and other channels are able to form highly stable tetrameric coiled coils and display selective heteromultimeric interactions. We show that loss of function caused by disruption of this domain in Herg1 can be rescued by introducing the equivalent domain from Eag1, and that this chimeric protein can form heteromultimers with Eag1 while wild-type Erg1 cannot. Additionally, a short endoplasmic reticulum retention sequence closely preceding the coiled coil plays a crucial role for surface expression. Both domains appear to co-operate to form fully functional channels on the cell surface and are a frequent finding in ion channels. Many pathological phenotypes may be attributed to mutations affecting one or both domains. PMID:12554641

  5. Solution structure of the RecQ C-terminal domain of human Bloom syndrome protein.

    Science.gov (United States)

    Park, Chin-Ju; Ko, Junsang; Ryu, Kyoung-Seok; Choi, Byong-Seok

    2014-02-01

    RecQ C-terminal (RQC) domain is known as the main DNA binding module of RecQ helicases such as Bloom syndrome protein (BLM) and Werner syndrome protein (WRN) that recognizes various DNA structures. Even though BLM is able to resolve various DNA structures similarly to WRN, BLM has different binding preferences for DNA substrates from WRN. In this study, we determined the solution structure of the RQC domain of human BLM. The structure shares the common winged-helix motif with other RQC domains. However, half of the N-terminal has unstructured regions (α1-α2 loop and α3 region), and the aromatic side chain on the top of the β-hairpin, which is important for DNA duplex strand separation in other RQC domains, is substituted with a negatively charged residue (D1165) followed by the polar residue (Q1166). The structurally distinctive features of the RQC domain of human BLM suggest that the DNA binding modes of the BLM RQC domain may be different from those of other RQC domains.

  6. The C-Terminal Region of G72 Increases D-Amino Acid Oxidase Activity

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    Sunny Li-Yun Chang

    2013-12-01

    Full Text Available The schizophrenia-related protein G72 plays a unique role in the regulation of D-amino acid oxidase (DAO in great apes. Several psychiatric diseases, including schizophrenia and bipolar disorder, are linked to overexpression of DAO and G72. Whether G72 plays a positive or negative regulatory role in DAO activity, however, has been controversial. Exploring the molecular basis of the relationship between G72 and DAO is thus important to understand how G72 regulates DAO activity. We performed yeast two-hybrid experiments and determined enzymatic activity to identify potential sites in G72 involved in binding DAO. Our results demonstrate that residues 123–153 and 138–153 in the long isoform of G72 bind to DAO and enhance its activity by 22% and 32%, respectively. A docking exercise indicated that these G72 peptides can interact with loops in DAO that abut the entrance of the tunnel that substrate and cofactor must traverse to reach the active site. We propose that a unique gating mechanism underlies the ability of G72 to increase the activity of DAO. Because upregulation of DAO activity decreases d-serine levels, which may lead to psychiatric abnormalities, our results suggest a molecular mechanism involving interaction between DAO and the C-terminal region of G72 that can regulate N-methyl-d-aspartate receptor-mediated neurotransmission.

  7. C-terminal engineering of CXCL12 and CCL5 chemokines: functional characterization by electrophysiological recordings.

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    Antoine Picciocchi

    Full Text Available Chemokines are chemotactic cytokines comprised of 70-100 amino acids. The chemokines CXCL12 and CCL5 are the endogenous ligands of the CXCR4 and CCR5 G protein-coupled receptors that are also HIV co-receptors. Biochemical, structural and functional studies of receptors are ligand-consuming and the cost of commercial chemokines hinders their use in such studies. Here, we describe methods for the expression, refolding, purification, and functional characterization of CXCL12 and CCL5 constructs incorporating C-terminal epitope tags. The model tags used were hexahistidines and Strep-Tag for affinity purification, and the double lanthanoid binding tag for fluorescence imaging and crystal structure resolution. The ability of modified and purified chemokines to bind and activate CXCR4 and CCR5 receptors was tested in Xenopus oocytes expressing the receptors, together with a Kir3 G-protein activated K(+ channel that served as a reporter of receptor activation. Results demonstrate that tags greatly influence the biochemical properties of the recombinant chemokines. Besides, despite the absence of any evidence for CXCL12 or CCL5 C-terminus involvement in receptor binding and activation, we demonstrated unpredictable effects of tag insertion on the ligand apparent affinity and efficacy or on the ligand dissociation. These tagged chemokines should constitute useful tools for the selective purification of properly-folded chemokines receptors and the study of their native quaternary structures.

  8. Impedance Analysis of Ovarian Cancer Cells upon Challenge with C-terminal Clostridium Perfringens Enterotoxin

    Science.gov (United States)

    Gordon, Geoffrey; Lo, Chun-Min

    2007-03-01

    Both in vitro and animal studies in breast, prostate, and ovarian cancers have shown that clostridium perfringens enterotoxin (CPE), which binds to CLDN4, may have an important therapeutic benefit, as it is rapidly cytotoxic in tissues overexpressing CLDN4. This study sought to evaluate the ability of C-terminal clostridium perfringens enterotoxin (C-CPE), a CLDN4-targetting molecule, to disrupt tight junction barrier function. Electric cell-substrate impedance sensing (ECIS) was used to measure both junctional resistance and average cell-substrate separation of ovarian cancer cell lines after exposure to C-CPE. A total of 14 ovarian cancer cell lines were used, and included cell lines derived from serous, mucinous, and clear cells. Our results showed that junctional resistance increases as CLDN4 expression increases. In addition, C-CPE is non-cytotoxic in ovarian cancer cells expressing CLDN4. However, exposure to C-CPE results in a significant (p<0.05) dose- and CLDN4-dependent decrease in junctional resistance and an increase in cell-substrate separation. Treatment of ovarian cancer cell lines with C-CPE disrupts tight junction barrier function.

  9. Effect of C-Terminal S-Palmitoylation on D2 Dopamine Receptor Trafficking and Stability.

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    Brittany Ebersole

    Full Text Available We have used bioorthogonal click chemistry (BCC, a sensitive non-isotopic labeling method, to analyze the palmitoylation status of the D2 dopamine receptor (D2R, a G protein-coupled receptor (GPCR crucial for regulation of processes such as mood, reward, and motor control. By analyzing a series of D2R constructs containing mutations in cysteine residues, we found that palmitoylation of the D2R most likely occurs on the C-terminal cysteine residue (C443 of the polypeptide. D2Rs in which C443 was deleted showed significantly reduced palmitoylation levels, plasma membrane expression, and protein stability compared to wild-type D2Rs. Rather, the C443 deletion mutant appeared to accumulate in the Golgi, indicating that palmitoylation of the D2R is important for cell surface expression of the receptor. Using the full-length D2R as bait in a membrane yeast two-hybrid (MYTH screen, we identified the palmitoyl acyltransferase (PAT zDHHC4 as a D2R interacting protein. Co-immunoprecipitation analysis revealed that several other PATs, including zDHHC3 and zDHHC8, also interacted with the D2R and that each of the three PATs was capable of affecting the palmitoylation status of the D2R. Finally, biochemical analyses using D2R mutants and the palmitoylation blocker, 2-bromopalmitate indicate that palmitoylation of the receptor plays a role in stability of the D2R.

  10. A C-terminal PDZ domain-binding sequence is required for striatal distribution of the dopamine transporter

    DEFF Research Database (Denmark)

    Rickhag, Karl Mattias; Hansen, Freja Herborg; Sørensen, Gunnar

    2013-01-01

    The dopamine transporter mediates reuptake of dopamine from the synaptic cleft. The cellular mechanisms controlling dopamine transporter levels in striatal nerve terminals remain poorly understood. The dopamine transporters contain a C-terminal PDZ (PSD-95/Discs-large/ZO-1) domain-binding sequence...

  11. Pseudomonas aeruginosa elastase cleaves a C-terminal peptide from human thrombin that inhibits host inflammatory responses

    DEFF Research Database (Denmark)

    van der Plas, Mariena J A; Bhongir, Ravi K V; Kjellström, Sven

    2016-01-01

    Pseudomonas aeruginosa is an opportunistic pathogen known for its immune evasive abilities amongst others by degradation of a large variety of host proteins. Here we show that digestion of thrombin by P. aeruginosa elastase leads to the release of the C-terminal thrombin-derived peptide FYT21...

  12. NMR assignments of SPOC domain of the human transcriptional corepressor SHARP in complex with a C-terminal SMRT peptide.

    Science.gov (United States)

    Mikami, Suzuka; Kanaba, Teppei; Ito, Yutaka; Mishima, Masaki

    2013-10-01

    The transcriptional corepressor SMRT/HDAC1-associated repressor protein (SHARP) recruits histone deacetylases. Human SHARP protein is thought to function in processes involving steroid hormone responses and the Notch signaling pathway. SHARP consists of RNA recognition motifs (RRMs) in the N-terminal region and the spen paralog and ortholog C-terminal (SPOC) domain in the C-terminal region. It is known that the SPOC domain binds the LSD motif in the C-terminal tail of corepressors silencing mediator for retinoid and thyroid receptor (SMRT)/nuclear receptor corepressor (NcoR). We are interested in delineating the mechanism by which the SPOC domain recognizes the LSD motif of the C-terminal tail of SMRT/NcoR. To this end, we are investigating the tertiary structure of the SPOC/SMRT peptide using NMR. Herein, we report on the (1)H, (13)C and (15)N resonance assignments of the SPOC domain in complex with a SMRT peptide, which contributes towards a structural understanding of the SPOC/SMRT peptide and its molecular recognition.

  13. Sol–gel immobilization of Alcalase from Bacillus licheniformis for application in the synthesis of C-terminal peptide amides

    NARCIS (Netherlands)

    Corici, L.N.; Frissen, A.E.; Zoelen, van D.J.; Eggen, I.F.; Peter, F.; Davidescu, C.M.; Boeriu, C.G.

    2011-01-01

    Alcalase 2.4L FG, a commercial preparation of Subtilisin A, was physically entrapped in glass sol–gel matrices using alkoxysilanes of different types mixed with tetramethoxysilane (TMOS). The materials were used for catalyzing C-terminal amidation of Z-Ala-Phe-OMe in a mixture of tert-butanol/DMF.

  14. Design and synthesis of peptide YY analogues with c-terminal backbone amide-to-ester modifications

    DEFF Research Database (Denmark)

    Albertsen, Louise; Andersen, J.J.; Paulsson, J.F.

    2013-01-01

    Peptide YY (PYY) is a gut hormone that activates the G protein-coupled neuropeptide Y (NPY) receptors, and because of its appetite reducing actions, it is evaluated as an antiobesity drug candidate. The C-terminal tail of PYY is crucial for activation of the NPY receptors. Here, we describe...

  15. C-terminal Src Kinase Gates Homeostatic Synaptic Plasticity and Regulates Fasciclin II Expression at the Drosophila Neuromuscular Junction.

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    Ashlyn M Spring

    2016-02-01

    Full Text Available Forms of homeostatic plasticity stabilize neuronal outputs and promote physiologically favorable synapse function. A well-studied homeostatic system operates at the Drosophila melanogaster larval neuromuscular junction (NMJ. At the NMJ, impairment of postsynaptic glutamate receptor activity is offset by a compensatory increase in presynaptic neurotransmitter release. We aim to elucidate how this process operates on a molecular level and is preserved throughout development. In this study, we identified a tyrosine kinase-driven signaling system that sustains homeostatic control of NMJ function. We identified C-terminal Src Kinase (Csk as a potential regulator of synaptic homeostasis through an RNAi- and electrophysiology-based genetic screen. We found that Csk loss-of-function mutations impaired the sustained expression of homeostatic plasticity at the NMJ, without drastically altering synapse growth or baseline neurotransmission. Muscle-specific overexpression of Src Family Kinase (SFK substrates that are negatively regulated by Csk also impaired NMJ homeostasis. Surprisingly, we found that transgenic Csk-YFP can support homeostatic plasticity at the NMJ when expressed either in the muscle or in the nerve. However, only muscle-expressed Csk-YFP was able to localize to NMJ structures. By immunostaining, we found that Csk mutant NMJs had dysregulated expression of the Neural Cell Adhesion Molecule homolog Fasciclin II (FasII. By immunoblotting, we found that levels of a specific isoform of FasII were decreased in homeostatically challenged GluRIIA mutant animals-but markedly increased in Csk mutant animals. Additionally, we found that postsynaptic overexpression of FasII from its endogenous locus was sufficient to impair synaptic homeostasis, and genetically reducing FasII levels in Csk mutants fully restored synaptic homeostasis. Based on these data, we propose that Csk and its SFK substrates impinge upon homeostatic control of NMJ function by

  16. Effect of copper variation in yeast hydrolysate on C-terminal lysine levels of a monoclonal antibody.

    Science.gov (United States)

    Mitchelson, Fernie G; Mondia, Jessica P; Hughes, Erik H

    2017-03-01

    The ability to control charge heterogeneity in monoclonal antibodies is important to demonstrate product quality comparability and consistency. This article addresses the control of C-terminal lysine processing through copper supplementation to yeast hydrolysate powder, a raw material used in the cell culture process. Large-scale production of a murine cell line exhibited variation in the C-terminal lysine levels of the monoclonal antibody. Analysis of process data showed that this variation correlated well with shifts in cell lactate metabolism and pH levels of the production culture. Small-scale studies demonstrated sensitivity of the cells to copper, where a single low dose of copper to the culture impacted cell lactate metabolism and C-terminal lysine processing. Subsequent analytical tests indicated that the yeast hydrolysate powder, added to the basal media and nutrient feed in the process, contained varying levels of trace copper across lots. The measured copper concentrations in yeast hydrolysate lots correlated well with the variation in lactate and pH trends and C-terminal lysine levels of the batches in manufacturing. Small-scale studies further demonstrated that copper supplementation to yeast hydrolysate lots with low concentrations of copper can shift the metabolic performance and C-terminal lysine levels of these cultures to match the control, high copper cultures. Hence, a strategy of monitoring, and if necessary supplementing, copper in yeast-hydrolysate powders resulted in the ability to control and ensure product quality consistency. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:463-468, 2017. © 2017 American Institute of Chemical Engineers.

  17. Structural model of the TRPP2/PKD1 C-terminal coiled-coil complex produced by a combined computational and experimental approach.

    Science.gov (United States)

    Zhu, Jiang; Yu, Yong; Ulbrich, Maximilian H; Li, Ming-hui; Isacoff, Ehud Y; Honig, Barry; Yang, Jian

    2011-06-21

    Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in TRPP2 and PKD1, which form an ion channel/receptor complex containing three TRPP2 and one PKD1. A TRPP2 C-terminal coiled-coil trimer, critical for the assembly of this complex, associates with a single PKD1 C-terminal coiled-coil. Many ADPKD pathogenic mutations result in the abolishment of the TRPP2/PKD1 coiled-coil complex. To gain molecular and functional insights into this heterotetrameric complex, we computationally constructed a structural model by using a two-step docking strategy, based on a known crystal structure of the TRPP2 coiled-coil trimer. The model shows that this tetrameric complex has a novel di-trimer configuration: An upstream trimer made of three TRPP2 helices and a downstream trimer made of two TRPP2 helices and one PKD1 helix. Mutagenesis and biochemical analysis identified critical TRPP2/PKD1 interface contacts essential for the heteromeric coiled-coil complex. Mutation of these interface positions in the full-length proteins showed that these interactions were critical for the assembly of the full-length complex in cells. Our results provide a means to specifically weaken the TRPP2 and PKD1 association, thus facilitating future in vitro and in vivo studies on the functional importance of this association.

  18. A C-terminal Myb extension domain defines a novel family of double-strand telomeric DNA-binding proteins in Arabidopsis.

    Science.gov (United States)

    Karamysheva, Zemfira N; Surovtseva, Yulia V; Vespa, Laurent; Shakirov, Eugene V; Shippen, Dorothy E

    2004-11-12

    Little is known about the protein composition of plant telomeres. We queried the Arabidopsis thaliana genome data base in search of genes with similarity to the human telomere proteins hTRF1 and hTRF2. hTRF1/hTRF2 are distinguished by the presence of a single Myb-like domain in their C terminus that is required for telomeric DNA binding in vitro. Twelve Arabidopsis genes fitting this criterion, dubbed TRF-like (TRFL), fell into two distinct gene families. Notably, TRFL family 1 possessed a highly conserved region C-terminal to the Myb domain called Myb-extension (Myb-ext) that is absent in TRFL family 2 and hTRF1/hTRF2. Immunoprecipitation experiments revealed that recombinant proteins from TRFL family 1, but not those from family 2, formed homodimers and heterodimers in vitro. DNA binding studies with isolated C-terminal fragments from TRFL family 1 proteins, but not family 2, showed specific binding to double-stranded plant telomeric DNA in vitro. Removal of the Myb-ext domain from TRFL1, a family 1 member, abolished DNA binding. However, when the Myb-ext domain was introduced into the corresponding region in TRFL3, a family 2 member, telomeric DNA binding was observed. Thus, Myb-ext is required for binding plant telomeric DNA and defines a novel class of proteins in Arabidopsis.

  19. Cdc15 Phosphorylates the C-terminal Domain of RNA Polymerase II for Transcription during Mitosis.

    Science.gov (United States)

    Singh, Amit Kumar; Rastogi, Shivangi; Shukla, Harish; Asalam, Mohd; Rath, Srikanta Kumar; Akhtar, Md Sohail

    2017-03-31

    In eukaryotes, the basal transcription in interphase is orchestrated through the regulation by kinases (Kin28, Bur1, and Ctk1) and phosphatases (Ssu72, Rtr1, and Fcp1), which act through the post-translational modification of the C-terminal domain (CTD) of the largest subunit of RNA polymerase II. The CTD comprises the repeated Tyr-Ser-Pro-Thr-Ser-Pro-Ser motif with potential epigenetic modification sites. Despite the observation of transcription and periodic expression of genes during mitosis with entailing CTD phosphorylation and dephosphorylation, the associated CTD specific kinase(s) and its role in transcription remains unknown. Here we have identified Cdc15 as a potential kinase phosphorylating Ser-2 and Ser-5 of CTD for transcription during mitosis in the budding yeast. The phosphorylation of CTD by Cdc15 is independent of any prior Ser phosphorylation(s). The inactivation of Cdc15 causes reduction of global CTD phosphorylation during mitosis and affects the expression of genes whose transcript levels peak during mitosis. Cdc15 also influences the complete transcription of clb2 gene and phosphorylates Ser-5 at the promoter and Ser-2 toward the 3' end of the gene. The observation that Cdc15 could phosphorylate Ser-5, as well as Ser-2, during transcription in mitosis is in contrast to the phosphorylation marks put by the kinases in interphase (G1, S, and G2), where Cdck7/Kin28 phosphorylates Ser-5 at promoter and Bur1/Ctk1 phosphorylates Ser-2 at the 3' end of the genes. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. C-terminal tensin-like protein is a novel prognostic marker for primary melanoma patients.

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    Cecilia Sjoestroem

    Full Text Available C-terminal tensin-like protein (Cten is a focal adhesion protein originally identified as a tumor suppressor in prostate cancer. It has since been found to be overexpressed and function as an oncogene in numerous other cancers, but the expression status of Cten in melanoma is still unknown.Using tissue microarrays containing 562 melanocytic lesions, we evaluated Cten protein expression by immunohistochemistry. The association between Cten expression and patient survival was examined using Kaplan-Meier survival analysis, and univariate and multivariate Cox regression analyses were used to estimate the crude and adjusted hazard ratios.Strong Cten expression was detected in 7%, 24%, 41%, and 46% of normal nevi, dysplastic nevi, primary melanoma, and metastatic melanoma samples, respectively, and Cten expression was found to be significantly higher in dysplastic nevi compared to normal nevi (P = 0.046, and in primary melanoma compared to dysplastic nevi (P = 0.003, but no difference was observed between metastatic and primary melanoma. Cten staining also correlated with AJCC stages (P = 0.015 and primary tumor thickness (P = 0.002, with Cten expression being induced in the transition from thin (<1 mm to thick (≥1 mm melanomas. Strong Cten expression was significantly associated with a worse 5-year overall (P = 0.008 and disease-specific survival (P = 0.004 for primary melanoma patients, and multivariate Cox regression analysis revealed that Cten expression was an independent prognostic marker for these patients (P = 0.038 for overall survival; P = 0.021 for disease-specific survival.Our findings indicate that induction of Cten protein expression is a relatively early event in melanoma progression, and that Cten has the potential to serve as a prognostic marker for primary melanoma patients.

  1. Cooperative cold denaturation: the case of the C-terminal domain of ribosomal protein L9.

    Science.gov (United States)

    Luan, Bowu; Shan, Bing; Baiz, Carlos; Tokmakoff, Andrei; Raleigh, Daniel P

    2013-04-09

    Cold denaturation is a general property of globular proteins, but it is difficult to directly characterize because the transition temperature of protein cold denaturation, T(c), is often below the freezing point of water. As a result, studies of protein cold denaturation are often facilitated by addition of denaturants, using destabilizing pHs or extremes of pressure, or reverse micelle encapsulation, and there are few studies of cold-induced unfolding under near native conditions. The thermal and denaturant-induced unfolding of single-domain proteins is usually cooperative, but the cooperativity of cold denaturation is controversial. The issue is of both fundamental and practical importance because cold unfolding may reveal information about otherwise inaccessible partially unfolded states and because many therapeutic proteins need to be stabilized against cold unfolding. It is thus desirable to obtain more information about the process under nonperturbing conditions. The ability to access cold denaturation in native buffer is also very useful for characterizing protein thermodynamics, especially when other methods are not applicable. In this work, we study a point mutant of the C-terminal domain of ribosomal protein L9 (CTL9), which has a T(c) above 0 °C. The mutant was designed to allow the study of cold denaturation under near native conditions. The cold denaturation process of I98A CTL9 was characterized by nuclear magnetic resonance, circular dichroism, and Fourier transform infrared spectroscopy. The results are consistent with apparently cooperative, two-state cold unfolding. Small-angle X-ray scattering studies show that the unfolded state expands as the temperature is lowered.

  2. The C-terminal binding protein (CTBP-1) regulates dorsal SMD axonal morphology in Caenorhabditis elegans.

    Science.gov (United States)

    Reid, A; Sherry, T J; Yücel, D; Llamosas, E; Nicholas, H R

    2015-12-17

    C-terminal binding proteins (CtBPs) are transcriptional co-repressors which cooperate with a variety of transcription factors to repress gene expression. Caenorhabditis elegans CTBP-1 expression has been observed in the nervous system and hypodermis. In C. elegans, CTBP-1 regulates several processes including Acute Functional Tolerance to ethanol and functions in the nervous system to modulate both lifespan and expression of a lipase gene called lips-7. Incorrect structure and/or function of the nervous system can lead to behavioral changes. Here, we demonstrate reduced exploration behavior in ctbp-1 mutants. Our examination of a subset of neurons involved in regulating locomotion revealed that the axonal morphology of dorsal SMD (SMDD) neurons is altered in ctbp-1 mutants at the fourth larval (L4) stage. Expressing CTBP-1 under the control of the endogenous ctbp-1 promoter rescued both the exploration behavior phenotype and defective SMDD axon structure in ctbp-1 mutants at the L4 stage. Interestingly, the pre-synaptic marker RAB-3 was found to localize to the mispositioned portion of SMDD axons in a ctbp-1 mutant. Further analysis of SMDD axonal morphology at days 1, 3 and 5 of adulthood revealed that the number of ctbp-1 mutants showing an SMDD axonal morphology defect increases in early adulthood and the observed defect appears to be qualitatively more severe. CTBP-1 is prominently expressed in the nervous system with weak expression detected in the hypodermis. Surprisingly, solely expressing CTBP-1a in the nervous system or hypodermis did not restore correct SMDD axonal structure in a ctbp-1 mutant. Our results demonstrate a role for CTBP-1 in exploration behavior and the regulation of SMDD axonal morphology in C. elegans. Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.

  3. Factor H C-Terminal Domains Are Critical for Regulation of Platelet/Granulocyte Aggregate Formation

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    Adam Z. Blatt

    2017-11-01

    Full Text Available Platelet/granulocyte aggregates (PGAs increase thromboinflammation in the vasculature, and PGA formation is tightly controlled by the complement alternative pathway (AP negative regulator, Factor H (FH. Mutations in FH are associated with the prothrombotic disease atypical hemolytic uremic syndrome (aHUS, yet it is unknown whether increased PGA formation contributes to the thrombosis seen in patients with aHUS. Here, flow cytometry assays were used to evaluate the effects of aHUS-related mutations on FH regulation of PGA formation and characterize the mechanism. Utilizing recombinant fragments of FH spanning the entire length of the protein, we mapped the regions of FH most critical for limiting AP activity on the surface of isolated human platelets and neutrophils, as well as the regions most critical for regulating PGA formation in human whole blood stimulated with thrombin receptor-activating peptide (TRAP. FH domains 19–20 were the most critical for limiting AP activity on platelets, neutrophils, and at the platelet/granulocyte interface. The role of FH in PGA formation was attributed to its ability to regulate AP-mediated C5a generation. AHUS-related mutations in domains 19–20 caused differential effects on control of PGA formation and AP activity on platelets and neutrophils. Our data indicate FH C-terminal domains are key for regulating PGA formation, thus increased FH protection may have a beneficial impact on diseases characterized by increased PGA formation, such as cardiovascular disease. Additionally, aHUS-related mutations in domains 19–20 have varying effects on control of TRAP-mediated PGA formation, suggesting that some, but not all, aHUS-related mutations may cause increased PGA formation that contributes to excessive thrombosis in patients with aHUS.

  4. The C-terminal sequence of IFITM1 regulates its anti-HIV-1 activity.

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    Rui Jia

    Full Text Available The interferon-inducible transmembrane (IFITM proteins inhibit a wide range of viruses. We previously reported the inhibition of human immunodeficiency virus type 1 (HIV-1 strain BH10 by human IFITM1, 2 and 3. It is unknown whether other HIV-1 strains are similarly inhibited by IFITMs and whether there exists viral countermeasure to overcome IFITM inhibition. We report here that the HIV-1 NL4-3 strain (HIV-1NL4-3 is not restricted by IFITM1 and its viral envelope glycoprotein is partly responsible for this insensitivity. However, HIV-1NL4-3 is profoundly inhibited by an IFITM1 mutant, known as Δ(117-125, which is deleted of 9 amino acids at the C-terminus. In contrast to the wild type IFITM1, which does not affect HIV-1 entry, the Δ(117-125 mutant diminishes HIV-1NL4-3 entry by 3-fold. This inhibition correlates with the predominant localization of Δ(117-125 to the plasma membrane where HIV-1 entry occurs. In spite of strong conservation of IFITM1 among most species, mouse IFITM1 is 19 amino acids shorter at its C-terminus as compared to human IFITM1 and, like the human IFITM1 mutant Δ(117-125, mouse IFITM1 also inhibits HIV-1 entry. This is the first report illustrating the role of viral envelope protein in overcoming IFITM1 restriction. The results also demonstrate the importance of the C-terminal region of IFITM1 in modulating the antiviral function through controlling protein subcellular localization.

  5. Evolutionary origins of C-terminal (GPPn 3-hydroxyproline formation in vertebrate tendon collagen.

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    David M Hudson

    Full Text Available Approximately half the proline residues in fibrillar collagen are hydroxylated. The predominant form is 4-hydroxyproline, which helps fold and stabilize the triple helix. A minor form, 3-hydroxyproline, still has no clear function. Using peptide mass spectrometry, we recently revealed several previously unknown molecular sites of 3-hydroxyproline in fibrillar collagen chains. In fibril-forming A-clade collagen chains, four new partially occupied 3-hydroxyproline sites were found (A2, A3, A4 and (GPPn in addition to the fully occupied A1 site at Pro986. The C-terminal (GPPn motif has five consecutive GPP triplets in α1(I, four in α2(I and three in α1(II, all subject to 3-hydroxylation. The evolutionary origins of this substrate sequence were investigated by surveying the pattern of its 3-hydroxyproline occupancy from early chordates through amphibians, birds and mammals. Different tissue sources of type I collagen (tendon, bone and skin and type II collagen (cartilage and notochord were examined by mass spectrometry. The (GPPn domain was found to be a major substrate for 3-hydroxylation only in vertebrate fibrillar collagens. In higher vertebrates (mouse, bovine and human, up to five 3-hydroxyproline residues per (GPPn motif were found in α1(I and four in α2(I, with an average of two residues per chain. In vertebrate type I collagen the modification exhibited clear tissue specificity, with 3-hydroxyproline prominent only in tendon. The occupancy also showed developmental changes in Achilles tendon, with increasing 3-hydroxyproline levels with age. The biological significance is unclear but the level of 3-hydroxylation at the (GPPn site appears to have increased as tendons evolved and shows both tendon type and developmental variations within a species.

  6. Sequence conservation in the C-terminal region of spider silk proteins (Spidroin) from Nephila clavipes (Tetragnathidae) and Araneus bicentenarius (Araneidae).

    Science.gov (United States)

    Beckwitt, R; Arcidiacono, S

    1994-03-04

    The polymerase chain reaction (PCR) has been used to amplify the portion of the Spidroin 1 gene that codes for the C-terminal part of the silk protein of the spider Nephila clavipes. Along with some substitution mutations of minor consequence, the PCR-derived sequence reveals an additional base missing from the previously published Nephila Spidroin 1 sequence. Comparison of the PCR-derived sequence with the equivalent region of Spidroin 2 indicates that the insertion of this single base results in greatly increased similarity in the resulting amino acid sequences of Spidroin 1 and Spidroin 2 (75% over 97 amino acids). The same PCR primers also amplified a fragment of the same length from Araneus bicentenarius. This sequence is also very similar to Spidroin 1 of Nephila (71% over 238 bases excluding the PCR primers, which translates into 76% over 79 amino acids).

  7. The presence and in vivo biosynthesis of fragments of CPP (the C-terminal glycopeptide of the rat vasopressin precursor) in the hypothalamo-neurohypophyseal system

    NARCIS (Netherlands)

    Seger, M.A.; Burbach, J.P.H.

    1987-01-01

    The existence and rate of formation of fragments of the 39-residue C-terminal glycopeptide of propressophysin (CPP1–39) was investigated in the hypothalamo-neurohypophyseal system. Newly-prepared antisera to CPP were used to confirm the existence of smaller C-terminal fragments derived from CPP1–39.

  8. Process for the conversion of C-terminal peptide esters or acids to amides employing subtilisin in the presence of ammonium salts

    NARCIS (Netherlands)

    Eggen, I.F.; Boeriu, C.G.

    2008-01-01

    The present invention relates to a process for the amidation of C-terminal esters or acids of peptide substrates in solution-phase synthesis of peptides, comprising amidating one or more peptide substrates comprising C-terminal esters or acids using the protease subtilisin in any suitable form in

  9. C-Terminal Domain Swapping of SSB Changes the Size of the ssDNA Binding Site

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    Yen-Hua Huang

    2014-01-01

    Full Text Available Single-stranded DNA-binding protein (SSB plays an important role in DNA metabolism, including DNA replication, repair, and recombination, and is therefore essential for cell survival. Bacterial SSB consists of an N-terminal ssDNA-binding/oligomerization domain and a flexible C-terminal protein-protein interaction domain. We characterized the ssDNA-binding properties of Klebsiella pneumoniae SSB (KpSSB, Salmonella enterica Serovar Typhimurium LT2 SSB (StSSB, Pseudomonas aeruginosa PAO1 SSB (PaSSB, and two chimeric KpSSB proteins, namely, KpSSBnStSSBc and KpSSBnPaSSBc. The C-terminal domain of StSSB or PaSSB was exchanged with that of KpSSB through protein chimeragenesis. By using the electrophoretic mobility shift assay, we characterized the stoichiometry of KpSSB, StSSB, PaSSB, KpSSBnStSSBc, and KpSSBnPaSSBc, complexed with a series of ssDNA homopolymers. The binding site sizes were determined to be 26±2, 21±2, 29±2, 21±2, and 29±2 nucleotides (nt, respectively. Comparison of the binding site sizes of KpSSB, KpSSBnStSSBc, and KpSSBnPaSSBc showed that the C-terminal domain swapping of SSB changes the size of the binding site. Our observations suggest that not only the conserved N-terminal domain but also the C-terminal domain of SSB is an important determinant for ssDNA binding.

  10. The linkage between binding of the C-terminal domain of hirudin and amidase activity in human alpha-thrombin.

    OpenAIRE

    de Cristofaro, R; Rocca, B; Bizzi, B; Landolfi, R

    1993-01-01

    A method derived from the analysis of viscosity effects on the hydrolysis of the amide substrates D-phenylalanylpipecolyl-arginine-p-nitroaniline, tosylglycylprolylarginine-p-nitroanaline and cyclohexylglycylalanylarginine-p-nitroalanine by human alpha-thrombin was developed to dissect the Michaelis-Menten parameters Km and kcat into the individual rate constants of the binding, acylation and deacylation reactions. This method was used to analyse the effect of the C-terminal hirudin (residues...

  11. Hemoglobin Cochin-Port-Royal: consequences of the replacement of the beta chain C-terminal by an arginine.

    Science.gov (United States)

    Wajcman, H; Kilmartin, J V; Najman, A; Labie, D

    1975-08-19

    Hemoglobin Cochin Port-Royal beta 146 (HC3) His yields Arg is the second example in which the beta C-terminal residue is replaced. Owing to the known importance of His beta 146 in the co-operative effects of hemoglobin, the functional properties of this variant were carefully studied. It had a normal Hill coefficient but a reduced alkaline Bohr effect. However, the reduction in Bohr effect is less than the halving predicted from previous mutants and modified hemoglobins.

  12. TREX1 C-terminal frameshift mutations in the systemic variant of retinal vasculopathy with cerebral leukodystrophy.

    Science.gov (United States)

    DiFrancesco, Jacopo C; Novara, Francesca; Zuffardi, Orsetta; Forlino, Antonella; Gioia, Roberta; Cossu, Federica; Bolognesi, Martino; Andreoni, Simona; Saracchi, Enrico; Frigeni, Barbara; Stellato, Tiziana; Tolnay, Markus; Winkler, David T; Remida, Paolo; Isimbaldi, Giuseppe; Ferrarese, Carlo

    2015-02-01

    Retinal vasculopathy with cerebral leukodystrophy (RVCL) is an adult-onset disorder caused by C-terminal heterozygous frameshift (fs) mutations in the human 3'-5' DNA exonuclease TREX1. Hereditary systemic angiopathy (HSA) is considered a variant of RVCL with systemic involvement of unknown genetic cause, described in a unique family so far. Here we describe the second case of RVCL with systemic involvement, characterized by cerebral calcifications and pseudotumoral lesions, retinopathy, osteonecrosis, renal and hepatic failure. The genetic screening of TREX1 in this patient revealed the novel heterozygous T270fs mutation on the C-terminal region. On the same gene, we found the V235fs mutation, formerly shown in RVCL, in one patient previously reported with HSA. These mutations lead to important alterations of the C-terminal of the protein, with the loss of the transmembrane helix (T270fs) and the insertion of a premature stop codon, resulting in a truncated protein (V235fs). Functional analysis of T270fs-mutated fibroblasts showed a prevalent localization of the protein in the cytosol, rather than in the perinuclear region. RVCL with systemic involvement is an extremely rare condition, whose diagnosis is complex due to multiorgan manifestations, unusual radiological and histopathological findings, not easily attributable to a single disease. It should be suspected in young adults with systemic microangiopathy involving retina, liver, kidney, bones and brain. Here we confirm the causative role played by TREX1 autosomal dominant fs mutations disrupting the C-terminal of the protein, providing a model for the study of stroke in young adults.

  13. The C-terminal domain of CENP-C displays multiple and critical functions for mammalian centromere formation.

    Directory of Open Access Journals (Sweden)

    Stefania Trazzi

    2009-06-01

    Full Text Available CENP-C is a fundamental component of functional centromeres. The elucidation of its structure-function relationship with centromeric DNA and other kinetochore proteins is critical to the understanding of centromere assembly. CENP-C carries two regions, the central and the C-terminal domains, both of which are important for the ability of CENP-C to associate with the centromeric DNA. However, while the central region is largely divergent in CENP-C homologues, the C-terminal moiety contains two regions that are highly conserved from yeast to humans, named Mif2p homology domains (blocks II and III. The activity of these two domains in human CENP-C is not well defined. In this study we performed a functional dissection of C-terminal CENP-C region analyzing the role of single Mif2p homology domains through in vivo and in vitro assays. By immunofluorescence and Chromatin immunoprecipitation assay (ChIP we were able to elucidate the ability of the Mif2p homology domain II to target centromere and contact alpha satellite DNA. We also investigate the interactions with other conserved inner kinetochore proteins by means of coimmunoprecipitation and bimolecular fluorescence complementation on cell nuclei. We found that the C-terminal region of CENP-C (Mif2p homology domain III displays multiple activities ranging from the ability to form higher order structures like homo-dimers and homo-oligomers, to mediate interaction with CENP-A and histone H3. Overall, our findings support a model in which the Mif2p homology domains of CENP-C, by virtue of their ability to establish multiple contacts with DNA and centromere proteins, play a critical role in the structuring of kinethocore chromatin.

  14. The C-terminal domain of Clostridium perfringens alpha toxin as a vaccine candidate against bovine necrohemorrhagic enteritis.

    Science.gov (United States)

    Goossens, Evy; Verherstraeten, Stefanie; Valgaeren, Bonnie R; Pardon, Bart; Timbermont, Leen; Schauvliege, Stijn; Rodrigo-Mocholí, Diego; Haesebrouck, Freddy; Ducatelle, Richard; Deprez, Piet R; Van Immerseel, Filip

    2016-04-27

    Bovine necrohemorrhagic enteritis is caused by Clostridium perfringens and leads to sudden death. Alpha toxin, together with perfringolysin O, has been identified as the principal toxin involved in the pathogenesis. We assessed the potential of alpha toxin as a vaccine antigen. Using an intestinal loop model in calves, we investigated the protection afforded by antisera raised against native alpha toxin or its non-toxic C-terminal fragment against C. perfringens-induced intestinal necrosis. Immunization of calves with either of the vaccine preparations induced a strong antibody response. The resulting antisera were able to neutralize the alpha toxin activity and the C. perfringens-induced endothelial cytotoxicity in vitro. The antisera raised against the native toxin had a stronger neutralizing activity than those against the C-terminal fragment. However, antibodies against alpha toxin alone were not sufficient to completely neutralize the C. perfringens-induced necrosis in the intestinal loop model. The development of a multivalent vaccine combining the C-terminal fragment of alpha toxin with other C. perfringens virulence factors might be necessary for complete protection against bovine necrohemorrhagic enteritis.

  15. Crystal Structure of the C-terminal Domain of Splicing Factor Prp8 Carrying Retinitis Pigmentosa Mutants

    Energy Technology Data Exchange (ETDEWEB)

    Zhang,L.; Shen, J.; Guarnieri, M.; Heroux, A.; Yang, K.; Zhao, R.

    2007-01-01

    Prp8 is a critical pre-mRNA splicing factor. Prp8 is proposed to help form and stabilize the spliceosome catalytic core and to be an important regulator of spliceosome activation. Mutations in human Prp8 (hPrp8) cause a severe form of the genetic disorder retinitis pigmentosa, RP13. Understanding the molecular mechanism of Prp8's function in pre-mRNA splicing and RP13 has been hindered by its large size (over 2000 amino acids) and remarkably low-sequence similarity with other proteins. Here we present the crystal structure of the C-terminal domain (the last 273 residues) of Caenorhabditis elegans Prp8 (cPrp8). The core of the C-terminal domain is an / structure that forms the MPN (Mpr1, Pad1 N-terminal) fold but without Zn{sup 2+} coordination. We propose that the C-terminal domain is a protein interaction domain instead of a Zn{sup 2+}-dependent metalloenzyme as proposed for some MPN proteins. Mapping of RP13 mutants on the Prp8 structure suggests that these residues constitute a binding surface between Prp8 and other partner(s), and the disruption of this interaction provides a plausible molecular mechanism for RP13.

  16. Insights into the oligomerization process of the C-terminal domain of human plasma membrane Ca²+-ATPase.

    Science.gov (United States)

    Benetti, Federico; Mičetić, Ivan; Carsughi, Flavio; Spinozzi, Francesco; Bubacco, Luigi; Beltramini, Mariano

    2011-02-15

    Plasma membrane calcium pumps (PMCAs) sustain a primary transport system for the specific removal of cytosolic calcium ions from eukaryotic cells. PMCAs are characterized by the presence of a C-terminal domain referred to as a regulatory domain. This domain is target of several regulatory mechanisms: activation by Ca²+-calmodulin complex and acidic phospholipids, phosphorylation by kinase A and C, proteolysis by calpain and oligomerization. As far as oligomerization is concerned, the C-terminal domain seems to be crucial for this process. We have cloned the C-terminal domain of the human PMCA isoform 1b, and characterized its properties in solution. The expressed protein maintains its tendency to oligomerize in aqueous solutions, but it is dissociated by amphipathic molecules such as diacylglycerol and sodium dodecyl sulphate. The presence of sodium dodecyl sulphate stabilizes the domain as a compact structure in monomeric form retaining the secondary structure elements, as shown by small angle neutron scattering and circular dichroism measurements. The importance of oligomerization for the regulation of PMCA activity and intracellular calcium concentration is discussed. Copyright © 2010 Elsevier Inc. All rights reserved.

  17. The C-terminal part of microcin B is crucial for DNA gyrase inhibition and antibiotic uptake by sensitive cells.

    Science.gov (United States)

    Shkundina, Irina; Serebryakova, Marina; Severinov, Konstantin

    2014-05-01

    Microcin B (McB) is a ribosomally synthesized antibacterial peptide. It contains up to nine oxazole and thiazole heterocycles that are introduced posttranslationally and are required for activity. McB inhibits the DNA gyrase, a validated drug target. Previous structure-activity analyses indicated that two fused heterocycles located in the central part of McB are important for antibacterial action and gyrase inhibition. Here, we used site-specific mutagenesis of the McB precursor gene to assess the functional significance of the C-terminal part of McB that is located past the second fused heterocycle and contains two single heterocycles as well as an unmodified four-amino-acid C-terminal tail. We found that removal of unmodified C-terminal amino acids of McB, while having no effect on fused heterocycles, has a very strong negative effect on activity in vivo and in vitro. In fact, even nonconservative point substitutions in the last McB amino acid have a very strong effect by simultaneously decreasing uptake and ability to inhibit the gyrase. The results highlight the importance of unmodified McB amino acids for function and open the way for creation of recombinant McB derivatives with an altered or expanded spectrum of antibacterial action.

  18. Insights into the Structure of Dimeric RNA Helicase CsdA and Indispensable Role of Its C-Terminal Regions.

    Science.gov (United States)

    Xu, Ling; Wang, Lijun; Peng, Junhui; Li, Fudong; Wu, Lijie; Zhang, Beibei; Lv, Mengqi; Zhang, Jiahai; Gong, Qingguo; Zhang, Rongguang; Zuo, Xiaobing; Zhang, Zhiyong; Wu, Jihui; Tang, Yajun; Shi, Yunyu

    2017-12-05

    CsdA has been proposed to be essential for the biogenesis of ribosome and gene regulation after cold shock. However, the structure of CsdA and the function of its long C-terminal regions are still unclear. Here, we solved all of the domain structures of CsdA and found two previously uncharacterized auxiliary domains: a dimerization domain (DD) and an RNA-binding domain (RBD). Small-angle X-ray scattering experiments helped to track the conformational flexibilities of the helicase core domains and C-terminal regions. Biochemical assays revealed that DD is indispensable for stabilizing the CsdA dimeric structure. We also demonstrate for the first time that CsdA functions as a stable dimer at low temperature. The C-terminal regions are critical for RNA binding and efficient enzymatic activities. CsdA_RBD could specifically bind to the regions with a preference for single-stranded G-rich RNA, which may help to bring the helicase core to unwind the adjacent duplex. Copyright © 2017 Elsevier Ltd. All rights reserved.

  19. Influence of C-terminal tail deletion on structure and stability of hyperthermophile Sulfolobus tokodaii RNase HI.

    Science.gov (United States)

    Chen, Lin; Zhang, Ji-Long; Zheng, Qing-Chuan; Chu, Wen-Ting; Xue, Qiao; Zhang, Hong-Xing; Sun, Chia-Chung

    2013-06-01

    The C-terminus tail (G144-T149) of the hyperthermophile Sulfolobus tokodaii (Sto-RNase HI) plays an important role in this protein's hyperstabilization and may therefore be a good protein stability tag. Detailed understanding of the structural and dynamic effects of C-terminus tail deletion is required for gaining insights into the thermal stability mechanism of Sto-RNase HI. Focused on Sulfolobus tokodaii RNase HI (Sto-RNase HI) and its derivative lacking the C-terminal tail (ΔC6 Sto-RNase HI) (PDB codes: 2EHG and 3ALY), we applied molecular dynamics (MD) simulations at four different temperatures (300, 375, 475, and 500 K) to examine the effect of the C-terminal tail on the hyperstabilization of Sto-RNase HI and to investigate the unfolding process of Sto-RNase HI and ΔC6 Sto-RNase HI. The simulations suggest that the C-terminal tail has significant impact in hyperstabilization of Sto-RNase HI and the unfolding of these two proteins evolves along dissimilar pathways. Essential dynamics analysis indicates that the essential subspaces of the two proteins at different temperatures are non-overlapping within the trajectories and they exhibit different directions of motion. Our work can give important information to understand the three-state folding mechanism of Sto-RNase HI and to offer alternative strategies to improve the protein stability.

  20. Tandem duplications in the C-terminal domain of the mesotocin receptor exclusively identified among East Eurasian thrushes.

    Science.gov (United States)

    Abe, Hideaki; Nishiumi, Isao; Inoue-Murayama, Miho

    2013-12-01

    Mesotocin is a neurohypophyseal hormone found in some non-mammalian vertebrates, including birds, reptiles, and amphibians. In this study, we identified and characterized 18-amino acid duplications in the C-terminal domain of the mesotocin receptor (MTR), specifically found in Turdus thrushes (Aves: Passeriforms: Turdidae). These duplicated elements are located in the distal part of the C-terminal tails of MTR and consist of amino acids that are highly conserved among major vertebrates. Intraspecific polymorphisms in a variable number of tandem duplications are commonly found in East Eurasian Turdus, but not in any other genus of Turdidae. Moreover, the genus Turdus can be further classified into 2 groups according to the presence or absence of a 3-amino acid deletion just adjacent to the putative palmitoylation site in the cytoplasmic C-terminal tail. The phylogeny presented here strongly supports the conspecific group of 4 East Eurasian thrushes (Turdus pallidus, T. chrysolaus, T. obscurus, and T. celaenops). Our findings, therefore, provide a new synapomorphy that can be used for phylogenetic assumptions and shed a light on the history of diversification within Eurasian Turdus clades.

  1. Conformational effects of a common codon 751 polymorphism on the C-terminal domain of the xeroderma pigmentosum D protein

    Directory of Open Access Journals (Sweden)

    Monaco Regina

    2009-01-01

    Full Text Available Aim: The xeroderma pigmentosum D (XPD protein is a DNA helicase involved in the repair of DNA damage, including nucleotide excision repair (NER and transcription-coupled repair (TCR. The C-terminal domain of XPD has been implicated in interactions with other components of the TFIIH complex, and it is also the site of a common genetic polymorphism in XPD at amino acid residue 751 (Lys->Gln. Some evidence suggests that this polymorphism may alter DNA repair capacity and increase cancer risk. The aim of this study was to investigate whether these effects could be attributable to conformational changes in XPD induced by the polymorphism. Materials and Methods: Molecular dynamics techniques were used to predict the structure of the wild-type and polymorphic forms of the C-terminal domain of XPD and differences in structure produced by the polymorphic substitution were determined. Results: The results indicate that, although the general configuration of both proteins is similar, the substitution produces a significant conformational change immediately N-terminal to the site of the polymorphism. Conclusion: These results provide support for the hypothesis that this polymorphism in XPD could affect DNA repair capability, and hence cancer risk, by altering the structure of the C-terminal domain.

  2. Mode of inhibition of HIV-1 Integrase by a C-terminal domain-specific monoclonal antibody*

    Directory of Open Access Journals (Sweden)

    Merkel George

    2006-06-01

    Full Text Available Abstract Background To further our understanding of the structure and function of HIV-1 integrase (IN we developed and characterized a library of monoclonal antibodies (mAbs directed against this protein. One of these antibodies, mAb33, which is specific for the C-terminal domain, was found to inhibit HIV-1 IN processing activity in vitro; a corresponding Fv fragment was able to inhibit HIV-1 integration in vivo. Our subsequent studies, using heteronuclear nuclear magnetic resonance spectroscopy, identified six solvent accessible residues on the surface of the C-terminal domain that were immobilized upon binding of the antibody, which were proposed to comprise the epitope. Here we test this hypothesis by measuring the affinity of mAb33 to HIV-1 proteins that contain Ala substitutions in each of these positions. To gain additional insight into the mode of inhibition we also measured the DNA binding capacity and enzymatic activities of the Ala substituted proteins. Results We found that Ala substitution of any one of five of the putative epitope residues, F223, R224, Y226, I267, and I268, caused a decrease in the affinity of the mAb33 for HIV-1 IN, confirming the prediction from NMR data. Although IN derivatives with Ala substitutions in or near the mAb33 epitope exhibited decreased enzymatic activity, none of the epitope substitutions compromised DNA binding to full length HIV-1 IN, as measured by surface plasmon resonance spectroscopy. Two of these derivatives, IN (I276A and IN (I267A/I268A, exhibited both increased DNA binding affinity and uncharacteristic dissociation kinetics; these proteins also exhibited non-specific nuclease activity. Results from these investigations are discussed in the context of current models for how the C-terminal domain interacts with substrate DNA. Conclusion It is unlikely that inhibition of HIV-1 IN activity by mAb33 is caused by direct interaction with residues that are essential for substrate binding. Rather

  3. Structure of the C-Terminal Half of UvrC Reveals an RNase H Endonuclease Domain with an Argonaute-like Catalytic Triad

    Energy Technology Data Exchange (ETDEWEB)

    Karakas,E.; Truglio, J.; Croteau, D.; Rhau, B.; Wang, L.; Van Houten, B.; Kisker, C.

    2007-01-01

    Removal and repair of DNA damage by the nucleotide excision repair pathway requires two sequential incision reactions, which are achieved by the endonuclease UvrC in eubacteria. Here, we describe the crystal structure of the C-terminal half of UvrC, which contains the catalytic domain responsible for 5' incision and a helix-hairpin-helix-domain that is implicated in DNA binding. Surprisingly, the 5' catalytic domain shares structural homology with RNase H despite the lack of sequence homology and contains an uncommon DDH triad. The structure also reveals two highly conserved patches on the surface of the protein, which are not related to the active site. Mutations of residues in one of these patches led to the inability of the enzyme to bind DNA and severely compromised both incision reactions. Based on our results, we suggest a model of how UvrC forms a productive protein-DNA complex to excise the damage from DNA.

  4. C-terminal motif prediction in eukaryotic proteomes using comparative genomics and statistical over-representation across protein families

    Directory of Open Access Journals (Sweden)

    Cutler Sean R

    2007-06-01

    Full Text Available Abstract Background The carboxy termini of proteins are a frequent site of activity for a variety of biologically important functions, ranging from post-translational modification to protein targeting. Several short peptide motifs involved in protein sorting roles and dependent upon their proximity to the C-terminus for proper function have already been characterized. As a limited number of such motifs have been identified, the potential exists for genome-wide statistical analysis and comparative genomics to reveal novel peptide signatures functioning in a C-terminal dependent manner. We have applied a novel methodology to the prediction of C-terminal-anchored peptide motifs involving a simple z-statistic and several techniques for improving the signal-to-noise ratio. Results We examined the statistical over-representation of position-specific C-terminal tripeptides in 7 eukaryotic proteomes. Sequence randomization models and simple-sequence masking were applied to the successful reduction of background noise. Similarly, as C-terminal homology among members of large protein families may artificially inflate tripeptide counts in an irrelevant and obfuscating manner, gene-family clustering was performed prior to the analysis in order to assess tripeptide over-representation across protein families as opposed to across all proteins. Finally, comparative genomics was used to identify tripeptides significantly occurring in multiple species. This approach has been able to predict, to our knowledge, all C-terminally anchored targeting motifs present in the literature. These include the PTS1 peroxisomal targeting signal (SKL*, the ER-retention signal (K/HDEL*, the ER-retrieval signal for membrane bound proteins (KKxx*, the prenylation signal (CC* and the CaaX box prenylation motif. In addition to a high statistical over-representation of these known motifs, a collection of significant tripeptides with a high propensity for biological function exists

  5. Skin-Derived C-Terminal Filaggrin-2 Fragments Are Pseudomonas aeruginosa-Directed Antimicrobials Targeting Bacterial Replication.

    Directory of Open Access Journals (Sweden)

    Britta Hansmann

    2015-09-01

    Full Text Available Soil- and waterborne bacteria such as Pseudomonas aeruginosa are constantly challenging body surfaces. Since infections of healthy skin are unexpectedly rare, we hypothesized that the outermost epidermis, the stratum corneum, and sweat glands directly control the growth of P. aeruginosa by surface-provided antimicrobials. Due to its high abundance in the upper epidermis and eccrine sweat glands, filaggrin-2 (FLG2, a water-insoluble 248 kDa S100 fused-type protein, might possess these innate effector functions. Indeed, recombinant FLG2 C-terminal protein fragments display potent antimicrobial activity against P. aeruginosa and other Pseudomonads. Moreover, upon cultivation on stratum corneum, P. aeruginosa release FLG2 C-terminus-containing FLG2 fragments from insoluble material, indicating liberation of antimicrobially active FLG2 fragments by the bacteria themselves. Analyses of the underlying antimicrobial mechanism reveal that FLG2 C-terminal fragments do not induce pore formation, as known for many other antimicrobial peptides, but membrane blebbing, suggesting an alternative mode of action. The association of the FLG2 fragment with the inner membrane of treated bacteria and its DNA-binding implicated an interference with the bacterial replication that was confirmed by in vitro and in vivo replication assays. Probably through in situ-activation by soil- and waterborne bacteria such as Pseudomonads, FLG2 interferes with the bacterial replication, terminates their growth on skin surface and thus may contributes to the skin's antimicrobial defense shield. The apparent absence of FLG2 at certain body surfaces, as in the lung or of burned skin, would explain their higher susceptibility towards Pseudomonas infections and make FLG2 C-terminal fragments and their derivatives candidates for new Pseudomonas-targeting antimicrobials.

  6. C-terminal diversity within the p53 family accounts for differences in DNA binding and transcriptional activity

    OpenAIRE

    Sauer, Markus; Bretz, Anne Catherine; Beinoraviciute-Kellner, Rasa; Beitzinger, Michaela; Burek, Christof; Rosenwald, Andreas; Harms, Gregory S.; Stiewe, Thorsten

    2008-01-01

    The p53 family is known as a family of transcription factors with functions in tumor suppression and development. Whereas the central DNA-binding domain is highly conserved among the three family members p53, p63 and p73, the C-terminal domains (CTDs) are diverse and subject to alternative splicing and post-translational modification. Here we demonstrate that the CTDs strongly influence DNA binding and transcriptional activity: while p53 and the p73 isoform p73γ have basic CTDs and form weak ...

  7. Downstream signaling mechanism of the C-terminal activation domain of transcriptional coactivator CoCoA

    OpenAIRE

    Kim, Jeong Hoon; Yang, Catherine K.; Stallcup, Michael R.

    2006-01-01

    The coiled-coil coactivator (CoCoA) is a transcriptional coactivator for nuclear receptors and enhances nuclear receptor function by the interaction with the bHLH-PAS domain (AD3) of p160 coactivators. The C-terminal activation domain (AD) of CoCoA possesses strong transactivation activity and is required for the coactivator function of CoCoA with nuclear receptors. To understand how CoCoA AD transmits its activating signal to the transcription machinery, we defined specific subregions, amino...

  8. Asparagine 326 in the extremely C-terminal region of XRCC4 is essential for the cell survival after irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Wanotayan, Rujira; Fukuchi, Mikoto; Imamichi, Shoji; Sharma, Mukesh Kumar; Matsumoto, Yoshihisa, E-mail: yoshim@nr.titech.ac.jp

    2015-02-20

    XRCC4 is one of the crucial proteins in the repair of DNA double-strand break (DSB) through non-homologous end-joining (NHEJ). As XRCC4 consists of 336 amino acids, N-terminal 200 amino acids include domains for dimerization and for association with DNA ligase IV and XLF and shown to be essential for XRCC4 function in DSB repair and V(D)J recombination. On the other hand, the role of the remaining C-terminal region of XRCC4 is not well understood. In the present study, we noticed that a stretch of ∼20 amino acids located at the extreme C-terminus of XRCC4 is highly conserved among vertebrate species. To explore its possible importance, series of mutants in this region were constructed and assessed for the functionality in terms of ability to rescue radiosensitivity of M10 cells lacking XRCC4. Among 13 mutants, M10 transfectant with N326L mutant (M10-XRCC4{sup N326L}) showed elevated radiosensitivity. N326L protein showed defective nuclear localization. N326L sequence matched the consensus sequence of nuclear export signal. Leptomycin B treatment accumulated XRCC4{sup N326L} in the nucleus but only partially rescued radiosensitivity of M10-XRCC4{sup N326L}. These results collectively indicated that the functional defects of XRCC4{sup N326L} might be partially, but not solely, due to its exclusion from nucleus by synthetic nuclear export signal. Further mutation of XRCC4 Asn326 to other amino acids, i.e., alanine, aspartic acid or glutamine did not affect the nuclear localization but still exhibited radiosensitivity. The present results indicated the importance of the extremely C-terminal region of XRCC4 and, especially, Asn326 therein. - Highlights: • Extremely C-terminal region of XRCC4 is highly conserved among vertebrate species. • XRCC4 C-terminal point mutants, R325F and N326L, are functionally deficient in terms of survival after irradiation. • N326L localizes to the cytoplasm because of synthetic nuclear export signal. • Leptomycin B restores the

  9. Side-chain anchoring strategy for solid-phase synthesis of peptide acids with C-terminal cysteine.

    Science.gov (United States)

    Barany, George; Han, Yongxin; Hargittai, Balazs; Liu, Rong-Qiang; Varkey, Jaya T

    2003-01-01

    Many naturally occurring peptide acids, e.g., somatostatins, conotoxins, and defensins, contain a cysteine residue at the C-terminus. Furthermore, installation of C-terminal cysteine onto epitopic peptide sequences as a preliminary to conjugating such structures to carrier proteins is a valuable tactic for antibody preparation. Anchoring of N(alpha)-Fmoc, S-protected C-terminal cysteine as an ester onto the support for solid-phase peptide synthesis is known to sometimes occur in low yields, has attendant risks of racemization, and may also result in conversion to a C-terminal 3-(1-piperidinyl)alanine residue as the peptide chain grows by Fmoc chemistry. These problems are documented for several current strategies, but can be circumvented by the title anchoring strategy, which features the following: (a). conversion of the eventual C-terminal cysteine residue, with Fmoc for N(alpha)-amino protection and tert-butyl for C(alpha)-carboxyl protection, to a corresponding S-xanthenyl ((2)XAL(4)) preformed handle derivative; and (b). attachment of the resultant preformed handle to amino-containing supports. This approach uses key intermediates that are similar to previously reported Fmoc-XAL handles, and builds on earlier experience with Xan and related protection for cysteine. Implementation of this strategy is documented here with syntheses of three small model peptides, as well as the tetradecapeptide somatostatin. Anchoring occurs without racemization, and the absence of 3-(1-piperidinyl)alanine formation is inferred by retention of chains on the support throughout the cycles of Fmoc chemistry. Fully deprotected peptides, including free sulfhydryl peptides, are released from the support in excellent yield by using cocktails containing a high concentration (i.e., 80-90%) of TFA plus appropriate thiols or silanes as scavengers. High-yield release of partially protected peptides is achieved by treatment with cocktails containing a low concentration (i.e., 1-5%) of TFA. In

  10. Human IgG is produced in a pro-form that requires clipping of C-terminal lysines for maximal complement activation

    DEFF Research Database (Denmark)

    van den Bremer, E. T. J.; Beurskens, F. J.; Voorhorst, M.

    2015-01-01

    G hexamerization at the cell surface. Here we demonstrate that C-terminal lysines may interfere with this process, leading to suboptimal C1q binding and CDC of cells opsonized with C-terminal lysine-containing IgG. After we removed these lysines with a carboxypeptidase, maximal complement activation was observed...... are produced in a pro-form in which charged C-termini interfere with IgG hexamer formation, C1q binding and CDC. To allow maximal complement activation, C-terminal lysine processing is required to release the antibody's full cytotoxic potential.......Human IgG is produced with C-terminal lysines that are cleaved off in circulation. The function of this modification was unknown and generally thought not to affect antibody function. We recently reported that efficient C1q binding and complement-dependent cytotoxicity (CDC) requires Ig...

  11. C-Terminal to Intact Fibroblast Growth Factor 23 Ratio in Relation to Estimated Glomerular Filtration Rate in Elderly Population

    Directory of Open Access Journals (Sweden)

    Maria Bożentowicz-Wikarek

    2016-08-01

    Full Text Available Background/Aims: An analytical equivalence between intact fibroblasts growth factor(iFGF23 and C-terminal(cFGF23 assays is logically expected, however, numerous studies demonstrate lack of a strong association between them. Previously, we have demonstrated the increase in cFGF23 slightly precedes the increase of iFGF23 with the impairment of kidney excretory function; without actually analyzing the ratio between both assays, which are postulated to be affected by declining kidney function. Therefore, the aim of this study was to analyze the ratio between C and iFGF23 in relation to the estimated glomerular filtration rate (eGFR in an elderly population. Methods: We analysed the variability of c/iFGF23 ratio in the population of 3264 elderly PolSenior study participants (≥ 65years in the relation to eGFR calculated according full Modification of Diet in Renal Disease, serum levels of C-reactive protein (hs-CRP, and iron. Results: The log10(c/i FGF23 ratio increased in the subsequent CKD stages. Serum iron and CRP levels reduced the log10 and increased it with age in multivariate regression analysis. Conclusions: Our results suggest impairment in the cleavage of the C-terminal FGF23 fragments with the deterioration of kidney excretory function and age in the elderly population. Inflammation and low serum iron level seems to diminish degradation capacity of FGF23 fragments.

  12. The C-terminal extension of human telomerase reverse transcriptase is necessary for high affinity binding to telomeric DNA.

    Science.gov (United States)

    Tomlinson, Christopher G; Holien, Jessica K; Mathias, Jordan A T; Parker, Michael W; Bryan, Tracy M

    2016-01-01

    The ribonucleoprotein enzyme telomerase maintains telomeres and is essential for cellular immortality in most cancers. Insight into the telomerase mechanism can be gained from short telomere syndromes, in which mutation of telomerase components manifests in telomere dysfunction. We carried out detailed kinetic analyses and molecular modelling of a disease-associated mutant in the C-terminal extension of the reverse transcriptase subunit of human telomerase. The kinetic analyses revealed that the mutation substantially impacts the affinity of telomerase for telomeric DNA, but the magnitude of this impact varies for primers with different 3' ends. Molecular dynamics simulations corroborate this finding, revealing that the mutation results in greater movement of a nearby loop, impacting the DNA-RNA helix differentially with different DNA primers. Thus, the data indicate that this region is the location of one of the enzyme conformational changes responsible for the long-standing observation that off-rates of telomerase vary with telomeric 3' end sequence. Our data provide a molecular basis for a disease-associated telomerase mutation, and the first direct evidence for a role of the C-terminal extension in DNA binding affinity, a function analogous to the "thumb" domain of retroviral reverse transcriptases. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  13. Function and control of RNA polymerase II C-terminal domain phosphorylation in vertebrate transcription and RNA processing.

    Science.gov (United States)

    Hsin, Jing-Ping; Xiang, Kehui; Manley, James L

    2014-07-01

    The C-terminal domain of the RNA polymerase II largest subunit (the Rpb1 CTD) is composed of tandem heptad repeats of the consensus sequence Y(1)S(2)P(3)T(4)S(5)P(6)S(7). We reported previously that Thr 4 is phosphorylated and functions in histone mRNA 3'-end formation in chicken DT40 cells. Here, we have extended our studies on Thr 4 and to other CTD mutations by using these cells. We found that an Rpb1 derivative containing only the N-terminal half of the CTD, as well as a similar derivative containing all-consensus repeats (26r), conferred full viability, while the C-terminal half, with more-divergent repeats, did not, reflecting a strong and specific defect in snRNA 3'-end formation. Mutation in 26r of all Ser 2 (S2A) or Ser 5 (S5A) residues resulted in lethality, while Ser 7 (S7A) mutants were fully viable. While S2A and S5A cells displayed defects in transcription and RNA processing, S7A cells behaved identically to 26r cells in all respects. Finally, we found that Thr 4 was phosphorylated by cyclin-dependent kinase 9 in cells and dephosphorylated both in vitro and in vivo by the phosphatase Fcp1. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  14. Insights into the Functional Roles of N-Terminal and C-Terminal Domains of Helicobacter pylori DprA.

    Directory of Open Access Journals (Sweden)

    Gajendradhar R Dwivedi

    Full Text Available DNA processing protein A (DprA plays a crucial role in the process of natural transformation. This is accomplished through binding and subsequent protection of incoming foreign DNA during the process of internalization. DprA along with Single stranded DNA binding protein A (SsbA acts as an accessory factor for RecA mediated DNA strand exchange. H. pylori DprA (HpDprA is divided into an N-terminal domain and a C- terminal domain. In the present study, individual domains of HpDprA have been characterized for their ability to bind single stranded (ssDNA and double stranded DNA (dsDNA. Oligomeric studies revealed that HpDprA possesses two sites for dimerization which enables HpDprA to form large and tightly packed complexes with ss and dsDNA. While the N-terminal domain was found to be sufficient for binding with ss or ds DNA, C-terminal domain has an important role in the assembly of poly-nucleoprotein complex. Using site directed mutagenesis approach, we show that a pocket comprising positively charged amino acids in the N-terminal domain has an important role in the binding of ss and dsDNA. Together, a functional cross talk between the two domains of HpDprA facilitating the binding and formation of higher order complex with DNA is discussed.

  15. Crystal structures reveal a thiol protease-like catalytic triad in the C-terminal region of Pasteurella multocida toxin.

    Science.gov (United States)

    Kitadokoro, Kengo; Kamitani, Shigeki; Miyazawa, Masayuki; Hanajima-Ozawa, Miyuki; Fukui, Aya; Miyake, Masami; Horiguchi, Yasuhiko

    2007-03-20

    Pasteurella multocida toxin (PMT), one of the virulence factors produced by the bacteria, exerts its toxicity by up-regulating various signaling cascades downstream of the heterotrimeric GTPases Gq and G12/13 in an unknown fashion. Here, we present the crystal structure of the C-terminal region (residues 575-1,285) of PMT, which carries an intracellularly active moiety. The overall structure of C-terminal region of PMT displays a Trojan horse-like shape, composed of three domains with a "feet"-,"body"-, and "head"-type arrangement, which were designated C1, C2, and C3 from the N to the C terminus, respectively. The C1 domain, showing marked similarity in steric structure to the N-terminal domain of Clostridium difficile toxin B, was found to lead the toxin molecule to the plasma membrane. The C3 domain possesses the Cys-His-Asp catalytic triad that is organized only when the Cys is released from a disulfide bond. The steric alignment of the triad corresponded well to that of papain or other enzymes carrying Cys-His-Asp. PMT toxicities on target cells were completely abrogated when one of the amino acids constituting the triad was mutated. Our results indicate that PMT is an enzyme toxin carrying the cysteine protease-like catalytic triad dependent on the redox state and functions on the cytoplasmic face of the plasma membrane of target cells.

  16. Hevea brasiliensis prohevein possesses a conserved C-terminal domain with amyloid-like properties in vitro.

    Science.gov (United States)

    Berthelot, Karine; Lecomte, Sophie; Coulary-Salin, Bénédicte; Bentaleb, Ahmed; Peruch, Frédéric

    2016-04-01

    Prohevein is a wound-induced protein and a main allergen from latex of Hevea brasiliensis (rubber tree). This 187 amino-acid protein is cleaved in two fragments: a N-terminal 43 amino-acids called hevein, a lectin bearing a chitin-binding motif with antifungal properties and a C-terminal domain (C-ter) far less characterized. We provide here new insights on the characteristics of prohevein, hevein and C-terminal domain. Using complementary biochemical (ThT/CR/chitin binding, agglutination) and structural (modeling, ATR-FTIR, TEM, WAXS) approaches, we show that this domain clearly displays all the characteristics of an amyloid-like proteins in vitro, that could confer agglutination activity in synergy with its chitin-binding activity. Additionally, this C-ter domain is highly conserved and present in numerous plant prohevein-like proteins or pathogenesis-related (PR and WIN) proteins. This could be the hallmark of the eventual presence of proteins with amyloid properties in plants, that could potentially play a role in defense through aggregation properties. Copyright © 2016 Elsevier B.V. All rights reserved.

  17. Production and characterization of N- and C-terminally truncated Mtx2: a mosquitocidal toxin from Bacillus sphaericus.

    Science.gov (United States)

    Phannachet, Kulwadee; Raksat, Ponlawoot; Limvuttegrijeerat, Thidarat; Promdonkoy, Boonhiang

    2010-12-01

    Mosquitocidal toxin 2 (Mtx2) is a mosquito-larvicidal protein produced during vegetative stage of Bacillus sphaericus. The toxin consists of 292 amino acids and has a molecular weight of 31.8 kDa. To determine the active core region of the toxin, amino acids at N- and C-termini were sequentially removed. Deletion up to 23 amino acids from the N-terminus (Met1-Tyr23) did not significantly affect protein production and the toxin activity, whereas removal of 26 amino acids from the N-terminus (Met1-Lys26) completely abolished toxicity even though the protein production remained unchanged. Deletion of only 5 amino acids from the C-terminal end yielded the protein that could not be solubilized and rendered the toxin inactive. The results demonstrated that the C-terminal end of Mtx2 is required for proper folding and toxicity. Amino acids at the N-terminus up to Tyr23 did not play a significant role in protein production and toxicity whereas amino acids between Thr24 and Lys26 are required for full toxicity.

  18. PCSK9-mediated degradation of the LDL receptor generates a 17 kDa C-terminal LDL receptor fragment.

    Science.gov (United States)

    Tveten, Kristian; Strøm, Thea Bismo; Berge, Knut Erik; Leren, Trond P

    2013-06-01

    Proprotein convertase subtilisin/kexin type 9 (PCSK9) binds to the LDL receptor (LDLR) at the cell surface and reroutes the internalized LDLR to intracellular degradation. In this study, we have shown that PCSK9-mediated degradation of the full-length 160 kDa LDLR generates a 17 kDa C-terminal LDLR fragment. This fragment was not generated from mutant LDLRs resistant to PCSK9-mediated degradation or when degradation was prevented by chemicals such as ammonium chloride or the cysteine cathepsin inhibitor E64d. The observation that the 17 kDa fragment was only detected when the cells were cultured in the presence of the γ-secretase inhibitor DAPT indicates that this 17 kDa fragment undergoes γ-secretase cleavage within the transmembrane domain. The failure to detect the complementary 143 kDa ectodomain fragment is likely to be due to its rapid degradation in the endosomal lumen. The 17 kDa C-terminal LDLR fragment was also generated from a Class 5 mutant LDLR undergoing intracellular degradation. Thus, one may speculate that an LDLR with bound PCSK9 and a Class 5 LDLR with bound LDL are degraded by a similar mechanism that could involve ectodomain cleavage in the endosome.

  19. An alternative outer membrane secretion mechanism for an autotransporter protein lacking a C-terminal stable core.

    Science.gov (United States)

    Besingi, Richard N; Chaney, Julie L; Clark, Patricia L

    2013-12-01

    Autotransporter (AT) proteins are a broad class of virulence factors from Gram-negative pathogens. AT outer membrane (OM) secretion appears simple in many regards, yet the mechanism that enables transport of the central AT 'passenger' across the OM remains unclear. OM secretion efficiency for two AT passengers is enhanced by approximately 20 kDa stable core at the C-terminus of the passenger, but studies on a broader range of AT proteins are needed in order to determine whether a stability difference between the passenger N- and C-terminus represents a truly common mechanistic feature. Yersinia pestis YapV is homologous to Shigella flexneri IcsA, and like IcsA, YapV recruits mammalian neural Wiskott-Aldrich syndrome protein (N-WASP). In vitro, the purified YapV passenger is functional and rich in β-sheet structure, but lacks a approximately 20 kDa C-terminal stable core. However, the N-terminal 49 residues of the YapV passenger globally destabilize the entire YapV passenger, enhancing its OM secretion efficiency. These results indicate that the contributions of AT passenger sequences to OM secretion efficiency extend beyond a C-terminal stable core, and highlight a role of the passenger N-terminus in reducing passenger stability in order to facilitate OM secretion of some AT proteins. © 2013 John Wiley & Sons Ltd.

  20. An isoform of Arabidopsis myosin XI interacts with small GTPases in its C-terminal tail region

    Science.gov (United States)

    Hashimoto, Kohsuke; Igarashi, Hisako; Mano, Shoji; Takenaka, Chikako; Shiina, Takashi; Yamaguchi, Masatoshi; Demura, Taku; Nishimura, Mikio; Shimmen, Teruo; Yokota, Etsuo

    2008-01-01

    Myosin XI, a class of myosins expressed in plants is believed to be responsible for cytoplasmic streaming and the translocation of organelles and vesicles. To gain further insight into the translocation of organelles and vesicles by myosin XI, an isoform of Arabidopsis myosin XI, MYA2, was chosen and its role in peroxisome targeting was examined. Using the yeast two-hybrid screening method, two small GTPases, AtRabD1 and AtRabC2a, were identified as factors that interact with the C-terminal tail region of MYA2. Both recombinant AtRabs tagged with His bound to the recombinant C-terminal tail region of MYA2 tagged with GST in a GTP-dependent manner. Furthermore, AtRabC2a was localized on peroxisomes, when its CFP-tagged form was expressed transiently in protoplasts prepared from Arabidopsis leaf tissue. It is suggested that MYA2 targets the peroxisome through an interaction with AtRabC2a. PMID:18703495

  1. Solution structure and Rpn1 interaction of the UBL domain of human RNA polymerase II C-terminal domain phosphatase.

    Directory of Open Access Journals (Sweden)

    Ji-Hye Yun

    Full Text Available The ubiquitin-like modifier (UBL domain of ubiquitin-like domain proteins (UDPs interacts specifically with subunits of the 26 S proteasome. A novel UDP, ubiquitin-like domain-containing C-terminal domain phosphatase (UBLCP1, has been identified as an interacting partner of the 26 S proteasome. We determined the high-resolution solution structure of the UBL domain of human UBLCP1 by nuclear magnetic resonance spectroscopy. The UBL domain of hUBLCP1 has a unique β-strand (β3 and β3-α2 loop, instead of the canonical β4 observed in other UBL domains. The molecular topology and secondary structures are different from those of known UBL domains including that of fly UBLCP1. Data from backbone dynamics shows that the β3-α2 loop is relatively rigid although it might have intrinsic dynamic profile. The positively charged residues of the β3-α2 loop are involved in interacting with the C-terminal leucine-rich repeat-like domain of Rpn1.

  2. The C-terminal region Mesd peptide mimics full-length Mesd and acts as an inhibitor of Wnt/β-catenin signaling in cancer cells.

    Directory of Open Access Journals (Sweden)

    Cuihong Lin

    Full Text Available While Mesd was discovered as a specialized molecular endoplasmic reticulum chaperone for the Wnt co-receptors LRP5 and LRP6, recombinant Mesd protein is able to bind to mature LRP5 and LRP6 on the cell surface and acts as a universal antagonist of LRP5/6 modulators. In our previous study, we found that the C-terminal region of Mesd, which is absent in sequences from invertebrates, is necessary and sufficient for binding to mature LRP6 on the cell surface. In the present studies, we further characterized the interaction between the C-terminal region Mesd peptide and LRP5/6. We found that Mesd C-terminal region-derived peptides block Mesd binding to LRP5 at the cell surface too. We also showed that there are two LRP5/6 binding sites within Mesd C-terminal region which contain several positively charged residues. Moreover, we demonstrated that the Mesd C-terminal region peptide, like the full-length Mesd protein, blocked Wnt 3A- and Rspodin1-induced Wnt/β-catenin signaling in LRP5- and LRP6- expressing cells, suppressed Wnt/β-catenin signaling in human breast HS578T cells and prostate cancer PC-3 cells, and inhibited cancer cell proliferation, although the full-length Mesd protein is more potent than its peptide. Finally, we found that treatment of the full-length Mesd protein and its C-terminal region peptide significantly increased chemotherapy agent Adriamycin-induced cytotoxicity in HS578T and PC-3 cells. Together, our results suggest that Mesd C-terminal region constitutes the major LRP5/6-binding domain, and that Mesd protein and its C-terminal region peptide have a potential therapeutic value in cancer.

  3. Structural and dynamic properties of the C-terminal region of the Escherichia coli RNA chaperone Hfq: integrative experimental and computational studies.

    Science.gov (United States)

    Wen, Bin; Wang, Weiwei; Zhang, Jiahai; Gong, Qingguo; Shi, Yunyu; Wu, Jihui; Zhang, Zhiyong

    2017-08-09

    In Escherichia coli, hexameric Hfq is an important RNA chaperone that facilitates small RNA-mediated post-transcriptional regulation. The Hfq monomer consists of an evolutionarily conserved Sm domain (residues 1-65) and a flexible C-terminal region (residues 66-102). It has been recognized that the existence of the C-terminal region is important for the function of Hfq, but its detailed structural and dynamic properties remain elusive due to its disordered nature. In this work, using integrative experimental techniques, such as nuclear magnetic resonance spectroscopy and small-angle X-ray scattering, as well as multi-scale computational simulations, new insights into the structure and dynamics of the C-terminal region in the context of the Hfq hexamer are provided. Although the C-terminal region is intrinsically disordered, some residues (83-86) are motionally restricted. The hexameric core may affect the secondary structure propensity of the C-terminal region, due to transient interactions between them. The residues at the rim and the proximal side of the core have significantly more transient contacts with the C-terminal region than those residues at the distal side, which may facilitate the function of the C-terminal region in the release of double-stranded RNAs and the cycling of small non-coding RNAs. Structure ensembles constructed by fitting the experimental data also support that the C-terminal region prefers to locate at the proximal side. From multi-scale simulations, we propose that the C-terminal region may play a dual role of steric effect (especially at the proximal side) and recruitment (at the both sides) in the binding process of RNA substrates. Interestingly, we have found that these motionally restricted residues may serve as important binding sites for the incoming RNAs that is probably driven by favorable electrostatic interactions. These integrative studies may aid in our understanding of the functional role of the C-terminal region of Hfq.

  4. Crystal structure of the C-terminal globular domain of the third paralog of the Archaeoglobus fulgidus oligosaccharyltransferases.

    Science.gov (United States)

    Matsumoto, Shunsuke; Shimada, Atsushi; Kohda, Daisuke

    2013-07-01

    Protein N-glycosylation occurs in the three domains of life. Oligosaccharyltransferase (OST) transfers an oligosaccharide chain to the asparagine residue in the N-glycosylation sequons. The catalytic subunits of the OST enzyme are STT3 in eukaryotes, AglB in archaea and PglB in eubacteria. The genome of a hyperthermophilic archaeon, Archaeoglobus fulgidus, encodes three paralogous AglB proteins. We previously solved the crystal structures of the C-terminal globular domains of two paralogs, AglB-Short 1 and AglB-Short 2. We determined the crystal structure of the C-terminal globular domain of the third AglB paralog, AglB-Long, at 1.9 Å resolutions. The crystallization of the fusion protein with maltose binding protein (MBP) afforded high quality protein crystals. Two MBP-AglB-L molecules formed a swapped dimer in the crystal. Since the fusion protein behaved as a monomer upon gel filtration, we reconstituted the monomer structure from the swapped dimer by exchanging the swapped segments. The C-terminal domain of A. fulgidus AglB-L includes a structural unit common to AglB-S1 and AglB-S2. This structural unit contains the evolutionally conserved WWDYG and DK motifs. The present structure revealed that A. fulgidus AglB-L contained a variant type of the DK motif with a short insertion, and confirmed that the second signature residue, Lys, of the DK motif participates in the formation of a pocket that binds to the serine and threonine residues at the +2 position of the N-glycosylation sequon. The structure of A. fulgidus AglB-L, together with the two previously solved structures of AglB-S1 and AglB-S2, provides a complete overview of the three AglB paralogs encoded in the A. fulgidus genome. All three AglBs contain a variant type of the DK motif. This finding supports a previously proposed rule: The STT3/AglB/PglB paralogs in one organism always contain the same type of Ser/Thr-binding pocket. The present structure will be useful as a search model for molecular

  5. C-Terminally Truncated Derivatives of Escherichia coli Hfq Are Proficient in Riboregulation

    DEFF Research Database (Denmark)

    Olsen, Anders Steno; Møller-Jensen, Jakob; Brennan, Richard G

    2010-01-01

    The prokaryotic Sm-like protein Hfq plays an essential role in the stability and function of trans-encoded small regulatory RNAs in enterobacteria that function in posttranscriptional control by base-pairing with cognate target mRNAs. Hfq associates with both regulatory RNA and target RNA, and its...

  6. Highly efficient synthetic method onpyroacm resin using the boc SPPS protocol for C-terminal cysteine peptide synthesis

    Energy Technology Data Exchange (ETDEWEB)

    Juvekar, Vinayak; Kim, Kang Tae; Gong, Young Dae [Innovative Drug Library Research Center, Dept. of Chemistry, College of Science, Dongguk University, Seoul (Korea, Republic of)

    2017-01-15

    A very effective process on Pyroacm resin was developed for solid-phase peptide synthesis (SPPS) of C-terminal cysteine and cysteine ester peptides. The process uses cysteine side chain anchoring to the Pyroacm resin and the Boc protocol for SPPS. The Pyroacm resin showed remarkable stability under standard trifluoromethanesulfonic acid (TFMSA) cleavage condition. TFMSA cleavage of protecting groups generates a peptide-linked resin, which can be subjected to peptide modification reactions. Finally, the peptide can be cleaved from the resin using methoxycarbonylsulfenyl chloride. The utility of this protocol was demonstrated by its applications to the synthesis of model peptides, key intermediates in the preparation of natural products riparin 1.2 and a-factor.

  7. Phage Endolysin: A Way To Understand A Binding Function Of C-Terminal Domains A Mini Review

    Directory of Open Access Journals (Sweden)

    Jarábková Veronika

    2015-12-01

    Full Text Available Endolysins are bacteriophage-encoded peptidoglycan hydrolases, which are synthesized in the end of phage reproduction cycle, in an infected host cell. Usually, for endolysins from phages that infect Gram-positive bacteria, a modular structure is typical. Therefore, these are composed of at least two separate functional domains: an N-terminal catalytic domain (EAD and a C-terminal cell wall binding domain (CBD. Specific ligand recognition of CBDs and following peptidoglycan (PG binding mostly allows a rapid lytic activity of an EAD. Here we briefly characterize phage endolysin CBDs in conjuction with their domain architecture, (nonnecessity for the following lytic activity and a high/low specificity of their ligands as well. Such an overall assessment of CBDs may help to find new ways to widen opportunities in their protein design to create ‛designer recombinant endolysins’ with diverse applications.

  8. Biological Activity and Antidiabetic Potential of C-Terminal Octapeptide Fragments of the Gut-Derived Hormone Xenin.

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    Christine M Martin

    Full Text Available Xenin is a peptide that is co-secreted with the incretin hormone, glucose-dependent insulinotropic polypeptide (GIP, from intestinal K-cells in response to feeding. Studies demonstrate that xenin has appetite suppressive effects and modulates glucose-induced insulin secretion. The present study was undertaken to determine the bioactivity and antidiabetic properties of two C-terminal fragment xenin peptides, namely xenin 18-25 and xenin 18-25 Gln. In BRIN-BD11 cells, both xenin fragment peptides concentration-dependently stimulated insulin secretion, with similar efficacy as the parent peptide. Neither fragment peptide had any effect on acute feeding behaviour at elevated doses of 500 nmol/kg bw. When administered together with glucose to normal mice at 25 nmol/kg bw, the overall insulin secretory effect was significantly enhanced in both xenin 18-25 and xenin 18-25 Gln treated mice, with better moderation of blood glucose levels. Twice daily administration of xenin 18-25 or xenin 18-25 Gln for 21 days in high fat fed mice did not affect energy intake, body weight, circulating blood glucose or body fat stores. However, circulating plasma insulin concentrations had a tendency to be elevated, particularly in xenin 18-25 Gln mice. Both treatment regimens significantly improved insulin sensitivity by the end of the treatment period. In addition, sustained treatment with xenin 18-25 Gln significantly reduced the overall glycaemic excursion and augmented the insulinotropic response to an exogenous glucose challenge on day 21. In harmony with this, GIP-mediated glucose-lowering and insulin-releasing effects were substantially improved by twice daily xenin 18-25 Gln treatment. Overall, these data provide evidence that C-terminal octapeptide fragments of xenin, such as xenin 18-25 Gln, have potential therapeutic utility for type 2 diabetes.

  9. Transactivation activity and nucleocytoplasmic transport of β-catenin are independently regulated by its C-terminal end.

    Science.gov (United States)

    Maturana, J L; Niechi, I; Silva, E; Huerta, H; Cataldo, R; Härtel, S; Barros, L F; Galindo, M; Tapia, J C

    2015-11-15

    The key protein in the canonical Wnt pathway is β-catenin, which is phosphorylated both in absence and presence of Wnt signals by different kinases. Upon activation in the cytoplasm, β-catenin can enter into the nucleus to transactivate target gene expression, many of which are cancer-related genes. The mechanism governing β-catenin's nucleocytoplasmic transport has been recently unvealed, although phosphorylation at its C-terminal end and its functional consequences are not completely understood. Serine 646 of β-catenin is a putative CK2 phosphorylation site and lies in a region which has been proposed to be important for its nucleocytoplasmic transport and transactivation activity. This residue was mutated to aspartic acid mimicking CK2-phosphorylation and its effects on β-catenin activity as well as localization were explored. β-Catenin S6464D did not show significant differences in both transcriptional activity and nuclear localization compared to the wild-type form, but displayed a characteristic granular nuclear pattern. Three-dimensional models of nuclei were constructed which showed differences in number and volume of granules, being those from β-catenin S646D more and smaller than the wild-type form. FRAP microscopy was used to compare nuclear export of both proteins which showed a slightly higher but not significant retention of β-catenin S646D. Altogether, these results show that C-terminal phosphorylation of β-catenin seems to be related with its nucleocytoplasmic transport but not transactivation activity. Copyright © 2015 Elsevier B.V. All rights reserved.

  10. NMR determines transient structure and dynamics in the disordered C-terminal domain of WASp interacting protein.

    Science.gov (United States)

    Haba, Noam Y; Gross, Renana; Novacek, Jiri; Shaked, Hadassa; Zidek, Lukas; Barda-Saad, Mira; Chill, Jordan H

    2013-07-16

    WASp-interacting protein (WIP) is a 503-residue proline-rich polypeptide expressed in human T cells. The WIP C-terminal domain binds to Wiskott-Aldrich syndrome protein (WASp) and regulates its activation and degradation, and the WIP-WASp interaction has been shown to be critical for actin polymerization and implicated in the onset of WAS and X-linked thrombocytopenia. WIP is predicted to be an intrinsically disordered protein, a class of polypeptides that are of great interest because they violate the traditional structure-function paradigm. In this first (to our knowledge) study of WIP in its unbound state, we used NMR to investigate the biophysical behavior of WIP(C), a C-terminal domain fragment of WIP that includes residues 407-503 and contains the WASp-binding site. In light of the poor spectral dispersion exhibited by WIP(C) and the high occurrence (25%) of proline residues, we employed 5D-NMR(13)C-detected NMR experiments with nonuniform sampling to accomplish full resonance assignment. Secondary chemical-shift analysis, (15)N relaxation rates, and protection from solvent exchange all concurred in detecting transient structure located in motifs that span the WASp-binding site. Residues 446-456 exhibited a propensity for helical conformation, and an extended conformation followed by a short, capped helix was observed for residues 468-478. The (13)C-detected approach allows chemical-shift assignment in the WIP(C) polyproline stretches and thus sheds light on their conformation and dynamics. The effects of temperature on chemical shifts referenced to a denatured sample of the polypeptide demonstrate that heating reduces the structural character of WIP(C). Thus, we conclude that the disordered WIP(C) fragment is comprised of regions with latent structure connected by flexible loops, an architecture with implications for binding affinity and function. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  11. Regulation of fibrinolysis by C-terminal lysines operates through plasminogen and plasmin but not tissue-type plasminogen activator.

    Science.gov (United States)

    Silva, M M C G; Thelwell, C; Williams, S C; Longstaff, C

    2012-11-01

    Binding of tissue-type plasminogen (Pgn) activator (t-PA) and Pgn to fibrin regulates plasmin generation, but there is no consistent, quantitative understanding of the individual contribution of t-PA finger and kringle 2 domains to the regulation of fibrinolysis. Kringle domains bind to lysines in fibrin, and this interaction can be studied by competition with lysine analogs and removal of C-terminal lysines by carboxypeptidase B (CPB). High-throughput, precise clot lysis assays incorporating the lysine analog tranexamic acid (TA) or CPB and genetically engineered variants of t-PA were performed. In particular, wild-type (WT) t-PA (F-G-K1-K2-P) and a domain-switched variant K1K1t-PA (F-G-K1-K1-P) that lacks kringle 2 but retains normal t-PA structure were compared to probe the importance of fibrin lysine binding by t-PA kringle 2. WT t-PA showed higher rates of fibrinolysis than K1K1t-PA, but the inhibitory effects of TA or CPB were very similar for WT t-PA and the variant t-PA (fibrinolysis was also inhibited by TA, even though Pgn activation could be stimulated. Fibrin treated with factor XIIIa (FXIIIa) generates crosslinked degradation products, but these did not affect the results obtained with WT t-PA and K1K1t-PA. t-PA kringle 2 has a minor role in the initial interaction of t-PA and fibrin, but stimulation of fibrinolysis by C-terminal lysines (or inhibition by carboxypeptidases or TA) operates through Pgn and plasmin binding, not through t-PA. This is also true when fibrin is crosslinked by treatment with FXIIIa. © 2012 International Society on Thrombosis and Haemostasis.

  12. Evolutionary Divergence of the C-terminal Domain of Complexin Accounts for Functional Disparities between Vertebrate and Invertebrate Complexins

    Directory of Open Access Journals (Sweden)

    Rachel T. Wragg

    2017-05-01

    Full Text Available Complexin is a critical presynaptic protein that regulates both spontaneous and calcium-triggered neurotransmitter release in all synapses. Although the SNARE-binding central helix of complexin is highly conserved and required for all known complexin functions, the remainder of the protein has profoundly diverged across the animal kingdom. Striking disparities in complexin inhibitory activity are observed between vertebrate and invertebrate complexins but little is known about the source of these differences or their relevance to the underlying mechanism of complexin regulation. We found that mouse complexin 1 (mCpx1 failed to inhibit neurotransmitter secretion in Caenorhabditis elegans neuromuscular junctions lacking the worm complexin 1 (CPX-1. This lack of inhibition stemmed from differences in the C-terminal domain (CTD of mCpx1. Previous studies revealed that the CTD selectively binds to highly curved membranes and directs complexin to synaptic vesicles. Although mouse and worm complexin have similar lipid binding affinity, their last few amino acids differ in both hydrophobicity and in lipid binding conformation, and these differences strongly impacted CPX-1 inhibitory function. Moreover, function was not maintained if a critical amphipathic helix in the worm CPX-1 CTD was replaced with the corresponding mCpx1 amphipathic helix. Invertebrate complexins generally shared more C-terminal similarity with vertebrate complexin 3 and 4 isoforms, and the amphipathic region of mouse complexin 3 significantly restored inhibitory function to worm CPX-1. We hypothesize that the CTD of complexin is essential in conferring an inhibitory function to complexin, and that this inhibitory activity has been attenuated in the vertebrate complexin 1 and 2 isoforms. Thus, evolutionary changes in the complexin CTD differentially shape its synaptic role across phylogeny.

  13. Characterization of a novel mutant KCNQ1 channel subunit lacking a large part of the C-terminal domain.

    Science.gov (United States)

    Kimoto, Katsuya; Kinoshita, Koshi; Yokoyama, Tomoki; Hata, Yukiko; Komatsu, Takuto; Tsushima, Eikichi; Nishide, Kohki; Yamaguchi, Yoshiaki; Mizumaki, Koichi; Tabata, Toshihide; Inoue, Hiroshi; Nishida, Naoki; Fukurotani, Kenkichi

    2013-10-18

    A mutation of KCNQ1 gene encoding the alpha subunit of the channel mediating the slow delayed rectifier K(+) current in cardiomyocytes may cause severe arrhythmic disorders. We identified KCNQ1(Y461X), a novel mutant gene encoding KCNQ1 subunit whose C-terminal domain is truncated at tyrosine 461 from a man with a mild QT interval prolongation. We made whole-cell voltage-clamp recordings from HEK-293T cells transfected with either of wild-type KCNQ1 [KCNQ1(WT)], KCNQ1(Y461X), or their mixture plus KCNE1 auxiliary subunit gene. The KCNQ1(Y461X)-transfected cells showed no delayed rectifying current. The cells transfected with both KCNQ1(WT) and KCNQ1(Y461X) showed the delayed rectifying current that is thought to be mediated largely by homomeric channel consisting of KCNQ1(WT) subunit because its voltage-dependence of activation, activation rate, and deactivation rate were similar to the current in the KCNQ1(WT)-transfected cells. The immunoblots of HEK-293T cell-derived lysates showed that KCNQ1(Y461X) subunit cannot form channel tetramers by itself or with KCNQ1(WT) subunit. Moreover, immunocytochemical analysis in HEK-293T cells showed that the surface expression level of KCNQ1(Y461X) subunit was very low with or without KCNQ1(WT) subunit. These findings suggest that the massive loss of the C-terminal domain of KCNQ1 subunit impairs the assembly, trafficking, and function of the mutant subunit-containing channels, whereas the mutant subunit does not interfere with the functional expression of the homomeric wild-type channel. Therefore, the homozygous but not heterozygous inheritance of KCNQ1(Y461X) might cause major arrhythmic disorders. This study provides a new insight into the structure-function relation of KCNQ1 channel and treatments of cardiac channelopathies. Copyright © 2013 Elsevier Inc. All rights reserved.

  14. Role of the C-terminal extension peptide of plastid located glutamine synthetase from Medicago truncatula: Crucial for enzyme activity and needless for protein import into the plastids.

    Science.gov (United States)

    Ferreira, Maria João; Vale, Diogo; Cunha, Luis; Melo, Paula

    2017-02-01

    Glutamine synthetase (GS), a key enzyme in plant nitrogen metabolism, is encoded by a small family of highly homologous nuclear genes that produce cytosolic (GS1) and plastidic (GS2) isoforms. Compared to GS1, GS2 proteins have two extension peptides, one at the N- and the other at the C-terminus, which show a high degree of conservation among plant species. It has long been known that the N-terminal peptide acts as a transit peptide, targeting the protein to the plastids however, the function of the C-terminal extension is still unknown. To investigate whether the C-terminal extension influences the activity of the enzyme, we produced a C-terminal truncated version of Medicago truncatula GS2a in Escherechia coli and studied its catalytic properties. The activity of the truncated protein was found to be lower than that of MtGS2a and with less affinity for glutamate. The importance of the C-terminal extension for the protein import into the chloroplast was also assessed by transient expression of fluorescently-tagged MtGS2a truncated at the C-terminus, which was correctly detected in the chloroplast. The results obtained in this work demonstrate that the C-terminal extension of M. truncatula GS2a is important for the activity of the enzyme and does not contain crucial information for the import process. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  15. The C-terminal region controls correct folding of genus Trametes pyranose 2-oxidases.

    Science.gov (United States)

    Maresová, Helena; Palyzová, Andrea; Kyslík, Pavel

    2007-06-30

    The pyranose 2-oxidases from Trametes ochracea and Trametes pubescens share markedly similar amino acid sequences with identity of 93.4%. When expressed from the recombinant plasmids based on the same vector in the Escherichia coli host strain BL21(DE3) at higher growth temperatures, they differ strikingly in the formation of the inclusion bodies. Upon overexpression in the cultures performed at 28 degrees C, the specific activity of pyranose 2-oxidase from T. pubescens was eight times higher than that from T. ochracea: 93% of pyranose 2-oxidase from T. ochracea and only 15% of that from T. pubescens was present in the form of inclusion bodies. To ascertain the cause of this difference, both cloned genes were shuffled. Site-directed recombination of p2o cDNAs revealed that DNA constructs ending with 3' end of p2o cDNA from T. pubescens code for proteins that are folded into an active form to the greater extent, regardless of the gene expression level. "In silicio" analysis of physico-chemical properties of the protein sequences of pyranose 2-oxidases revealed that the sequence of amino acid residues 368-430, constituting the small, head domain of pyranose 2-oxidase from T. pubescens, affects positively the enzyme folding at higher cultivation temperatures. The domain differs in six amino acid residues from that of T. ochracea.

  16. [C-terminal lysosome targeting domain of CD63 modifies cellular localization of rabies virus glycoprotein].

    Science.gov (United States)

    Starodubova, E S; Kuzmenko, Y V; Latanova, A A; Preobrazhenskaya, O V; Karpov, V L

    2017-01-01

    The glycoprotein of rabies virus is the central antigen elicited the immune response to infection; therefore, the majority of developing anti-rabies vaccines are based on this protein. In order to increase the efficacy of DNA immunogen encoding rabies virus glycoprotein, the construction of chimeric protein with the CD63 domain has been proposed. The CD63 is a transmembrane protein localized on the cell surface and in lysosomes. The lysosome targeting motif GYEVM is located at its C-terminus. We used the domain that bears this motif (c-CD63) to generate chimeric glycoprotein in order to relocalize it into lysosomes. Here, it was shown that, in cells transfected with plasmid that encodes glycoprotein with c-CD63 motif at the C-terminus, the chimeric protein was predominantly observed in lysosomes and at the cell membrane where the unmodified glycoprotein is localized in the endoplasmic reticulum and at the cell surface. We suppose that current modification of the glycoprotein may improve the immunogenicity of anti-rabies DNA vaccines due to more efficient antibody production.

  17. Structural features discriminate androgen receptor N/C terminal and coactivator interactions.

    Science.gov (United States)

    Askew, Emily B; Minges, John T; Hnat, Andrew T; Wilson, Elizabeth M

    2012-01-30

    Human androgen receptor (AR) transcriptional activity involves interdomain and coactivator interactions with the agonist-bound AR ligand binding domain (LBD). Structural determinants of the AR NH(2)- and carboxyl-terminal interaction between the AR NH(2)-terminal FXXLF motif and activation function 2 (AF2) in the LBD were shown previously by crystallography. In this report, we provide evidence for a region in AR LBD helix 12 outside the AF2 binding cleft that facilitates interactions with the FXXLF and LXXLL motifs. Mutagenesis of glutamine 902 to alanine in AR LBD helix 12 (Q902A) disrupted AR FXXLF motif binding to AF2, but enhanced coactivator LXXLL motif binding. Functional compensation for defective FXXLF motif binding by AR-Q902A was suggested by the slower dissociation rate of bound androgen. Functional importance of glutamine 902 was indicated by the charged residue germline mutation Q902R that caused partial androgen insensitivity, and a similar somatic mutation Q902K reported in prostate cancer, both of which increased the androgen dissociation rate and decreased AR transcriptional activity. High affinity equilibrium androgen binding was retained by alanine substitution mutations at Tyr-739 in AR LBD helix 5 or Lys-905 in helix 12 structurally adjacent to AF2, whereas transcriptional activity decreased and the androgen dissociation increased. Deleterious effects of these loss of function mutations were rescued by the helix stabilizing AR prostate cancer somatic mutation H874Y. Sequence NH(2)-terminal to the AR FXXLF motif contributed to the AR NH(2)- and carboxyl-terminal interaction based on greater AR-2-30 FXXLF motif peptide binding to the agonist-bound AR LBD than a shorter AR-20-30 FXXLF motif peptide. We conclude that helix 12 residues outside the AF2 binding cleft modulate AR transcriptional activity by providing flexibility to accommodate FXXLF or LXXLL motif binding. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  18. Crystallization of the C-terminal head domain of the avian adenovirus CELO long fibre

    Energy Technology Data Exchange (ETDEWEB)

    Guardado Calvo, Pablo [Departamento de Bioquímica y Biología Molecular, Facultad de Farmacia, Universidad de Santiago de Compostela, Campus Sur, E-15782 Santiago de Compostela (Spain); Llamas-Saiz, Antonio L. [Unidad de Difracción de Rayos X, Laboratorio Integral de Dinámica y Estructura de Biomoléculas José R. Carracido, Edificio CACTUS, Universidad de Santiago de Compostela, Campus Sur, E-15782 Santiago de Compostela (Spain); Langlois, Patrick [Agence Francaise de Securité Sanitaire des Aliments, Unité Génétique Virale et Biosecurité, Site Les Croix, BP 53, F-22440 Ploufragan (France); Raaij, Mark J. van, E-mail: vanraaij@usc.es [Departamento de Bioquímica y Biología Molecular, Facultad de Farmacia, Universidad de Santiago de Compostela, Campus Sur, E-15782 Santiago de Compostela (Spain); Unidad de Difracción de Rayos X, Laboratorio Integral de Dinámica y Estructura de Biomoléculas José R. Carracido, Edificio CACTUS, Universidad de Santiago de Compostela, Campus Sur, E-15782 Santiago de Compostela (Spain)

    2006-05-01

    Avian adenovirus long-fibre head trimers were expressed, purified and crystallized. The crystals belong to space group C2 (unit-cell parameters a = 216.5, b = 59.2, c = 57.5 Å, β = 101.3°). A complete highly redundant data set was collected to 2.2 Å resolution at 100 K using a rotating-anode X-ray source. Avian adenovirus CELO contains two different fibres: fibre 1, the long fibre, and fibre 2, the short fibre. The short fibre is responsible for binding to an unknown avian receptor and is essential for infection of birds. The long fibre is not essential, but is known to bind the coxsackievirus and adenovirus receptor protein. Both trimeric fibres are attached to the same penton base, of which each icosahedral virus contains 12 copies. The short fibre extends straight outwards, while the long fibre emerges at an angle. The carboxy-terminal amino acids 579–793 of the avian adenovirus long fibre have been expressed with an amino-terminal hexahistidine tag and the expressed trimeric protein has been purified by nickel-affinity chromatography and crystallized. Crystals were grown at low pH using PEG 10 000 as precipitant and belonged to space group C2. The crystals diffracted rotating-anode Cu Kα radiation to at least 1.9 Å resolution and a complete data set was collected from a single crystal to 2.2 Å resolution. Unit-cell parameters were a = 216.5, b = 59.2, c = 57.5 Å, β = 101.3°, suggesting one trimer per asymmetric unit and a solvent content of 46%. The long fibre head does not have significant sequence homology to any other protein of known structure and molecular-replacement attempts with known fibre-head structures were unsuccessful. However, a map calculated using SIRAS phasing shows a clear trimer with a shape similar to known adenovirus fibre-head structures. Structure solution is in progress.

  19. Epoxysuccinyl peptide-derived cathepsin B inhibitors: modulating membrane permeability by conjugation with the C-terminal heptapeptide segment of penetratin.

    Science.gov (United States)

    Schaschke, Norbert; Deluca, Dominga; Assfalg-Machleidt, Irmgard; Höhneke, Clara; Sommerhoff, Christian P; Machleidt, Werner

    2002-05-01

    Besides its physiological role in lysosomal protein breakdown, extralysosomal cathepsin B has recently been implicated in apoptotic cell death. Highly specific irreversible cathepsin B inhibitors that are readily cell-permeant should be useful tools to elucidate the effects of cathepsin B in the cytosol. We have covalently functionalised the poorly cell-permeant epoxysuccinyl-based cathepsin B inhibitor [R-Gly-Gly-Leu-(2S,3S)-tEps-Leu-Pro-OH; R=OMe] with the C-terminal heptapeptide segment of penetratin (R=epsilonAhx-Arg-Arg-Nle-Lys-Trp-Lys-Lys-NH2). The high inhibitory potency and selectivity for cathepsin B versus cathepsin L of the parent compound was not affected by the conjugation with the penetratin heptapeptide. The conjugate was shown to efficiently penetrate into MCF-7 cells as an active inhibitor, thereby circumventing an intracellular activation step that is required by other inhibitors, such as the prodrug-like epoxysuccinyl peptides E64d and CA074Me.

  20. The C-terminal tails of heterotrimeric kinesin-2 motor subunits directly bind to α-tubulin1: Possible implications for cilia-specific tubulin entry.

    Science.gov (United States)

    Girotra, Mukul; Srivastava, Shalini; Kulkarni, Anuttama; Barbora, Ayan; Bobra, Kratika; Ghosal, Debnath; Devan, Pavithra; Aher, Amol; Jain, Akanksha; Panda, Dulal; Ray, Krishanu

    2017-02-01

    The assembly of microtubule-based cytoskeleton propels the cilia and flagella growth. Previous studies have indicated that the kinesin-2 family motors transport tubulin into the cilia through intraflagellar transport. Here, we report a direct interaction between the C-terminal tail fragments of heterotrimeric kinesin-2 and α-tubulin1 isoforms in vitro. Blot overlay screen, affinity purification from tissue extracts, cosedimentation with subtilisin-treated microtubule and LC-ESI-MS/MS characterization of the tail-fragment-associated tubulin identified an association between the tail domains and α-tubulin1A/D isotype. The interaction was confirmed by Forster's resonance energy transfer assay in tissue-cultured cells. The overexpression of the recombinant tails in NIH3T3 cells affected the primary cilia growth, which was rescued by coexpression of a α-tubulin1 transgene. Furthermore, fluorescent recovery after photobleach analysis in the olfactory cilia of Drosophila indicated that tubulin is transported in a non-particulate form requiring kinesin-2. These results provide additional new insight into the mechanisms underlying selective tubulin isoform enrichment in the cilia. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  1. Mucosal C-terminal maltase-glucoamylase hydrolyzes large size starch digestion products that may contribute to rapid postprandial glucose generation.

    Science.gov (United States)

    Lee, Byung-Hoo; Lin, Amy Hui-Mei; Nichols, Buford L; Jones, Kyra; Rose, David R; Quezada-Calvillo, Roberto; Hamaker, Bruce R

    2014-05-01

    The four mucosal α-glucosidases, which differ in their digestive roles, generate glucose from glycemic carbohydrates and accordingly can be viewed as a control point for rate of glucose delivery to the body. In this study, individual recombinant enzymes were used to understand how α-glucan oligomers are digested by each enzyme, and how intermediate α-amylolyzed starches are hydrolyzed, to elucidate a strategy for moderating the glycemic spike of rapidly digestible starchy foods. The C-terminal maltase-glucoamylase (ctMGAM, commonly termed "glucoamylase") was able to rapidly hydrolyze longer maltooligosaccharides, such as maltotetraose and maltopentaose, to glucose. Moreover, it was found to convert larger size maltodextrins, as would be produced early in α-amylase digestion of starch, efficiently to glucose. It is postulated that ctMGAM has the additional capacity to hydrolyze large α-amylase products that are produced immediately on starch digestion in the duodenum and contribute to the rapid generation of glucose from starch-based meals. The findings suggest that partial inhibition of ctMGAM, such as by natural inhibitors found in foods, might be used to moderate the early stage of high glycemic response, as well as to extend digestion distally; thereby having relevance in regulating glucose delivery to the body. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. Rare RNF213 variants in the C-terminal region encompassing the RING-finger domain are associated with moyamoya angiopathy in Caucasians.

    Science.gov (United States)

    Guey, Stéphanie; Kraemer, Markus; Hervé, Dominique; Ludwig, Thomas; Kossorotoff, Manoëlle; Bergametti, Françoise; Schwitalla, Jan Claudius; Choi, Simone; Broseus, Lucile; Callebaut, Isabelle; Genin, Emmanuelle; Tournier-Lasserve, Elisabeth

    2017-08-01

    Moyamoya angiopathy (MMA) is a cerebral angiopathy affecting the terminal part of internal carotid arteries. Its prevalence is 10 times higher in Japan and Korea than in Europe. In East Asian countries, moyamoya is strongly associated to the R4810K variant in the RNF213 gene that encodes for a protein containing a RING-finger and two AAA+ domains. This variant has never been detected in Caucasian MMA patients, but several rare RNF213 variants have been reported in Caucasian cases. Using a collapsing test based on exome data from 68 European MMA probands and 573 ethnically matched controls, we showed a significant association between rare missense RNF213 variants and MMA in European patients (odds ratio (OR)=2.24, 95% confidence interval (CI)=(1.19-4.11), P=0.01). Variants specific to cases had higher pathogenicity predictive scores (median of 24.2 in cases versus 9.4 in controls, P=0.029) and preferentially clustered in a C-terminal hotspot encompassing the RING-finger domain of RNF213 (P<10(-3)). This association was even stronger when restricting the analysis to childhood-onset and familial cases (OR=4.54, 95% CI=(1.80-11.34), P=1.1 × 10(-3)). All clinically affected relatives who were genotyped were carriers. However, the need for additional factors to develop MMA is strongly suggested by the fact that only 25% of mutation carrier relatives were clinically affected.

  3. Contribution of the C-terminal regions of promyelocytic leukemia protein (PML) isoforms II and V to PML nuclear body formation.

    Science.gov (United States)

    Geng, Yunyun; Monajembashi, Shamci; Shao, Anwen; Cui, Di; He, Weiyong; Chen, Zhongzhou; Hemmerich, Peter; Tang, Jun

    2012-08-31

    Promyelocytic leukemia protein (PML) nuclear bodies are dynamic and heterogeneous nuclear protein complexes implicated in various important functions, most notably tumor suppression. PML is the structural component of PML nuclear bodies and has several nuclear splice isoforms that share a common N-terminal region but differ in their C termini. Previous studies have suggested that the coiled-coil motif within the N-terminal region is sufficient for PML nuclear body formation by mediating homo/multi-dimerization of PML molecules. However, it has not been investigated whether any of the C-terminal variants of PML may contribute to PML body assembly. Here we report that the unique C-terminal domains of PML-II and PML-V can target to PML-NBs independent of their N-terminal region. Strikingly, both domains can form nuclear bodies in the absence of endogenous PML. The C-terminal domain of PML-II interacts transiently with unknown binding sites at PML nuclear bodies, whereas the C-terminal domain of PML-V exhibits hyperstable binding to PML bodies via homo-dimerization. This strong interaction is mediated by a putative α-helix in the C terminus of PML-V. Moreover, nuclear bodies assembled from the C-terminal domain of PML-V also recruit additional PML body components, including Daxx and Sp100. These observations establish the C-terminal domain of PML-V as an additional important contributor to the assembly mechanism(s) of PML bodies.

  4. The C-terminal Region and SUMOylation of Cockayne Syndrome Group B Protein Play Critical Roles in Transcription-coupled Nucleotide Excision Repair.

    Science.gov (United States)

    Sin, Yooksil; Tanaka, Kiyoji; Saijo, Masafumi

    2016-01-15

    Cockayne syndrome (CS) is a recessive disorder that results in deficiencies in transcription-coupled nucleotide excision repair (TC-NER), a subpathway of nucleotide excision repair, and cells from CS patients exhibit hypersensitivity to UV light. CS group B protein (CSB), which is the gene product of one of the genes responsible for CS, belongs to the SWI2/SNF2 DNA-dependent ATPase family and has an ATPase domain and an ubiquitin-binding domain (UBD) in the central region and the C-terminal region, respectively. The C-terminal region containing the UBD is essential for the functions of CSB. In this study, we generated several CSB deletion mutants and analyzed the functions of the C-terminal region of CSB in TC-NER. Not only the UBD but also the C-terminal 30-amino acid residues were required for UV light resistance and TC-NER. This region was needed for the interaction of CSB with RNA polymerase II, the translocation of CS group A protein to the nuclear matrix, and the association of CSB with chromatin after UV irradiation. CSB was modified by small ubiquitin-like modifier 2/3 in a UV light-dependent manner. This modification was abolished in a CSB mutant lacking the C-terminal 30 amino acid residues. However, the substitution of lysine residues in this region with arginine did not affect SUMOylation or TC-NER. By contrast, substitution of a lysine residue in the N-terminal region with arginine decreased SUMOylation and resulted in cells with defects in TC-NER. These results indicate that both the most C-terminal region and SUMOylation are important for the functions of CSB in TC-NER. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. The C-terminal Region and SUMOylation of Cockayne Syndrome Group B Protein Play Critical Roles in Transcription-coupled Nucleotide Excision Repair*

    Science.gov (United States)

    Sin, Yooksil; Tanaka, Kiyoji; Saijo, Masafumi

    2016-01-01

    Cockayne syndrome (CS) is a recessive disorder that results in deficiencies in transcription-coupled nucleotide excision repair (TC-NER), a subpathway of nucleotide excision repair, and cells from CS patients exhibit hypersensitivity to UV light. CS group B protein (CSB), which is the gene product of one of the genes responsible for CS, belongs to the SWI2/SNF2 DNA-dependent ATPase family and has an ATPase domain and an ubiquitin-binding domain (UBD) in the central region and the C-terminal region, respectively. The C-terminal region containing the UBD is essential for the functions of CSB. In this study, we generated several CSB deletion mutants and analyzed the functions of the C-terminal region of CSB in TC-NER. Not only the UBD but also the C-terminal 30-amino acid residues were required for UV light resistance and TC-NER. This region was needed for the interaction of CSB with RNA polymerase II, the translocation of CS group A protein to the nuclear matrix, and the association of CSB with chromatin after UV irradiation. CSB was modified by small ubiquitin-like modifier 2/3 in a UV light-dependent manner. This modification was abolished in a CSB mutant lacking the C-terminal 30 amino acid residues. However, the substitution of lysine residues in this region with arginine did not affect SUMOylation or TC-NER. By contrast, substitution of a lysine residue in the N-terminal region with arginine decreased SUMOylation and resulted in cells with defects in TC-NER. These results indicate that both the most C-terminal region and SUMOylation are important for the functions of CSB in TC-NER. PMID:26620705

  6. Structure determination of Murine Norovirus NS6 proteases with C-terminal extensions designed to probe protease–substrate interactions

    Directory of Open Access Journals (Sweden)

    Humberto Fernandes

    2015-02-01

    Full Text Available Noroviruses are positive-sense single-stranded RNA viruses. They encode an NS6 protease that cleaves a viral polyprotein at specific sites to produce mature viral proteins. In an earlier study we obtained crystals of murine norovirus (MNV NS6 protease in which crystal contacts were mediated by specific insertion of the C-terminus of one protein (which contains residues P5-P1 of the NS6-7 cleavage junction into the peptide binding site of an adjacent molecule, forming an adventitious protease-product complex. We sought to reproduce this crystal form to investigate protease–substrate complexes by extending the C-terminus of NS6 construct to include residues on the C-terminal (P′ side of the cleavage junction. We report the crystallization and crystal structure determination of inactive mutants of murine norovirus NS6 protease with C-terminal extensions of one, two and four residues from the N-terminus of the adjacent NS7 protein (NS6 1′, NS6 2′, NS6 4′. We also determined the structure of a chimeric extended NS6 protease in which the P4-P4′ sequence of the NS6-7 cleavage site was replaced with the corresponding sequence from the NS2-3 cleavage junction (NS6 4′ 2|3.The constructs NS6 1′ and NS6 2′ yielded crystals that diffracted anisotropically. We found that, although the uncorrected data could be phased by molecular replacement, refinement of the structures stalled unless the data were ellipsoidally truncated and corrected with anisotropic B-factors. These corrections significantly improved phasing by molecular replacement and subsequent refinement.The refined structures of all four extended NS6 proteases are very similar in structure to the mature MNV NS6—and in one case reveal additional details of a surface loop. Although the packing arrangement observed showed some similarities to those observed in the adventitious protease-product crystals reported previously, in no case were specific protease–substrate interactions

  7. WXG100 protein superfamily consists of three subfamilies and exhibits an α-helical C-terminal conserved residue pattern.

    Directory of Open Access Journals (Sweden)

    Christian Poulsen

    Full Text Available Members of the WXG100 protein superfamily form homo- or heterodimeric complexes. The most studied proteins among them are the secreted T-cell antigens CFP-10 (10 kDa culture filtrate protein, EsxB and ESAT-6 (6 kDa early secreted antigen target, EsxA from Mycobacterium tuberculosis. They are encoded on an operon within a gene cluster, named as ESX-1, that encodes for the Type VII secretion system (T7SS. WXG100 proteins are secreted in a full-length form and it is known that they adopt a four-helix bundle structure. In the current work we discuss the evolutionary relationship between the homo- and heterodimeric WXG100 proteins, the basis of the oligomeric state and the key structural features of the conserved sequence pattern of WXG100 proteins. We performed an iterative bioinformatics analysis of the WXG100 protein superfamily and correlated this with the atomic structures of the representative WXG100 proteins. We find, firstly, that the WXG100 protein superfamily consists of three subfamilies: CFP-10-, ESAT-6- and sagEsxA-like proteins (EsxA proteins similar to that of Streptococcus agalactiae. Secondly, that the heterodimeric complexes probably evolved from a homodimeric precursor. Thirdly, that the genes of hetero-dimeric WXG100 proteins are always encoded in bi-cistronic operons and finally, by combining the sequence alignments with the X-ray data we identify a conserved C-terminal sequence pattern. The side chains of these conserved residues decorate the same side of the C-terminal α-helix and therefore form a distinct surface. Our results lead to a putatively extended T7SS secretion signal which combines two reported T7SS recognition characteristics: Firstly that the T7SS secretion signal is localized at the C-terminus of T7SS substrates and secondly that the conserved residues YxxxD/E are essential for T7SS activity. Furthermore, we propose that the specific α-helical surface formed by the conserved sequence pattern including Yxxx

  8. WXG100 Protein Superfamily Consists of Three Subfamilies and Exhibits an α-Helical C-Terminal Conserved Residue Pattern

    Science.gov (United States)

    Poulsen, Christian; Panjikar, Santosh; Holton, Simon J.; Wilmanns, Matthias; Song, Young-Hwa

    2014-01-01

    Members of the WXG100 protein superfamily form homo- or heterodimeric complexes. The most studied proteins among them are the secreted T-cell antigens CFP-10 (10 kDa culture filtrate protein, EsxB) and ESAT-6 (6 kDa early secreted antigen target, EsxA) from Mycobacterium tuberculosis. They are encoded on an operon within a gene cluster, named as ESX-1, that encodes for the Type VII secretion system (T7SS). WXG100 proteins are secreted in a full-length form and it is known that they adopt a four-helix bundle structure. In the current work we discuss the evolutionary relationship between the homo- and heterodimeric WXG100 proteins, the basis of the oligomeric state and the key structural features of the conserved sequence pattern of WXG100 proteins. We performed an iterative bioinformatics analysis of the WXG100 protein superfamily and correlated this with the atomic structures of the representative WXG100 proteins. We find, firstly, that the WXG100 protein superfamily consists of three subfamilies: CFP-10-, ESAT-6- and sagEsxA-like proteins (EsxA proteins similar to that of Streptococcus agalactiae). Secondly, that the heterodimeric complexes probably evolved from a homodimeric precursor. Thirdly, that the genes of hetero-dimeric WXG100 proteins are always encoded in bi-cistronic operons and finally, by combining the sequence alignments with the X-ray data we identify a conserved C-terminal sequence pattern. The side chains of these conserved residues decorate the same side of the C-terminal α-helix and therefore form a distinct surface. Our results lead to a putatively extended T7SS secretion signal which combines two reported T7SS recognition characteristics: Firstly that the T7SS secretion signal is localized at the C-terminus of T7SS substrates and secondly that the conserved residues YxxxD/E are essential for T7SS activity. Furthermore, we propose that the specific α-helical surface formed by the conserved sequence pattern including YxxxD/E motif is a key

  9. The C-terminal extension peptide of non-photoconvertible water-soluble chlorophyll-binding proteins (Class II WSCPs) affects their solubility and stability: comparative analyses of the biochemical and chlorophyll-binding properties of recombinant Brassica, Raphanus and Lepidium WSCPs with or without their C-terminal extension peptides.

    Science.gov (United States)

    Takahashi, Shigekazu; Uchida, Akira; Nakayama, Katsumi; Satoh, Hiroyuki

    2014-02-01

    Numerous members of the Brassicaceae possess non-photoconvertible water-soluble chlorophyll (Chl)-binding proteins (Class II WSCPs), which function as Chl scavengers during cell disruption caused by wounding, pest/pathogen attacks, and/or environmental stress. Class II WSCPs have two extension peptides, one at the N-terminus and one at the C-terminus. The N-terminal peptide acts as a signal peptide, targeting the protein to the endoplasmic reticulum body, a unique defensive organelle found only in the Brassicaceae. However, the physiological and biochemical functions of the C-terminal extension peptide had not been characterized previously. To investigate the function of the C-terminal extension peptide, we produced expression constructs of recombinant WSCPs with or without the C-terminal extension peptide. The WSCPs used were of Brussels sprouts (Brassica oleracea), Japanese wild radish (Raphanus sativus) and Virginia pepperweed (Lepidium virginicum). The solubility of all of the WSCPs with the C-terminal extension peptide was drastically lower than that of the recombinant WSCPs without the C-terminal extension peptide. In addition, the stability of the reconstituted WSCPs complexes with the C-terminal extension peptide was altered compared with that of the proteins without the C-terminal extension peptide. These finding indicate that the C-terminal extension peptide affects not only the solubility, but also the stability of Class II WSCP. Furthermore, we characterized the Chl-binding properties of the recombinant WSCP from Japanese wild radish (RshWSCP-His) in a 40 % methanol solution. An electrophoretic mobility shift assay revealed that RshWSCP-His required a half-molar ratio of Chls to form a tetramer.

  10. The C-terminal aqueous-exposed domain of the 45 kDa subunit of the particulate methane monooxygenase in Methylococcus capsulatus (Bath) is a Cu(I) sponge.

    Science.gov (United States)

    Yu, Steve S-F; Ji, Cheng-Zhi; Wu, Ya Ping; Lee, Tsu-Lin; Lai, Chien-Hung; Lin, Su-Ching; Yang, Zong-Lin; Wang, Vincent C-C; Chen, Kelvin H-C; Chan, Sunney I

    2007-12-04

    The crystal structure of the particulate methane monooxygenase (pMMO) from Methylococcus capsulatus (Bath) has been reported recently [Lieberman, R. L., and Rosenzweig, A. C. (2005) Crystal structure of a membrane-bound metalloenzyme that catalyses the biological oxidation of methane, Nature 434, 177-182]. Subsequent work has shown that the preparation on which the X-ray analysis is based might be missing many of the important metal cofactors, including the putative trinuclear copper cluster at the active site as well as ca. 10 copper ions (E-clusters) that have been proposed to serve as a buffer of reducing equivalents to re-reduce the copper atoms at the active site following the catalytic chemistry [Chan, S. I., Wang, V. C.-C., Lai, J. C.-H., Yu, S. S.-F., Chen, P. P.-Y., Chen, K. H.-C., Chen, C.-L., and Chan, M. K. (2007) Redox potentiometry studies of particulate methane monooxygenase: Support for a trinuclear copper cluster active site, Angew. Chem., Int. Ed. 46, 1992-1994]. Since the aqueous-exposed domains of the 45 kDa subunit (PmoB) have been suggested to be the putative binding domains for the E-cluster copper ions, we have cloned and overexpressed in Escherichia coli the two aqueous-exposed subdomains toward the N- and C-termini of the subunit: the N-terminal subdomain (residues 54-178) and the C-terminal subdomain (residues 257-394 and 282-414). The recombinant C-terminal water-exposed subdomain is shown to behave like a Cu(I) sponge, taking up to ca. 10 Cu(I) ions cooperatively when cupric ions are added to the protein fragment in the presence of dithiothreitol or ascorbate. In addition, circular dichroism measurements reveal that the C-terminal subdomain folds into a beta-sheet structure in the presence of Cu(I). The propensity for the C-terminal subdomain to bind Cu(I) is consistent with the high redox potential(s) determined for the E-cluster copper ions in the pMMO. These properties of the E-clusters are in accordance with the function proposed

  11. Bio-molecular architects: a scaffold provided by the C-terminal domain of eukaryotic RNA polymerase II.

    Science.gov (United States)

    Zhang, Mengmeng; Gill, Gordon N; Zhang, Yan

    2010-01-01

    In eukaryotic cells, the transcription of genes is accurately orchestrated both spatially and temporally by the C-terminal domain of RNA polymerase II (CTD). The CTD provides a dynamic platform to recruit different regulators of the transcription apparatus. Different posttranslational modifications are precisely applied to specific sites of the CTD to coordinate transcription process. Regulators of the RNA polymerase II must identify specific sites in the CTD for cellular survival, metabolism, and development. Even though the CTD is disordered in the eukaryotic RNA polymerase II crystal structures due to its intrinsic flexibility, recent advances in the complex structural analysis of the CTD with its binding partners provide essential clues for understanding how selectivity is achieved for individual site recognition. The recent discoveries of the interactions between the CTD and histone modification enzymes disclose an important role of the CTD in epigenetic control of the eukaryotic gene expression. The intersection of the CTD code with the histone code discloses an intriguing yet complicated network for eukaryotic transcriptional regulation.

  12. C-Terminal Domain of Hemocyanin, a Major Antimicrobial Protein from Litopenaeus vannamei: Structural Homology with Immunoglobulins and Molecular Diversity

    Directory of Open Access Journals (Sweden)

    Yue-Ling Zhang

    2017-06-01

    Full Text Available Invertebrates rely heavily on immune-like molecules with highly diversified variability so as to counteract infections. However, the mechanisms and the relationship between this variability and functionalities are not well understood. Here, we showed that the C-terminal domain of hemocyanin (HMC from shrimp Litopenaeus vannamei contained an evolutionary conserved domain with highly variable genetic sequence, which is structurally homologous to immunoglobulin (Ig. This domain is responsible for recognizing and binding to bacteria or red blood cells, initiating agglutination and hemolysis. Furthermore, when HMC is separated into three fractions using anti-human IgM, IgG, or IgA, the subpopulation, which reacted with anti-human IgM (HMC-M, showed the most significant antimicrobial activity. The high potency of HMC-M is a consequence of glycosylation, as it contains high abundance of α-d-mannose relative to α-d-glucose and N-acetyl-d-galactosamine. Thus, the removal of these glycans abolished the antimicrobial activity of HMC-M. Our results present a comprehensive investigation of the role of HMC in fighting against infections through genetic variability and epigenetic modification.

  13. Synapse associated protein 102 (SAP102 binds the C-terminal part of the scaffolding protein neurobeachin.

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    Juliane Lauks

    Full Text Available Neurobeachin (Nbea is a multidomain scaffold protein abundant in the brain, where it is highly expressed during development. Nbea-null mice have severe defects in neuromuscular synaptic transmission resulting in lethal paralysis of the newborns. Recently, it became clear that Nbea is important also for the functioning of central synapses, where it is suggested to play a role in trafficking membrane proteins to both, the pre- and post-synaptic sites. So far, only few binding partners of Nbea have been found and the precise mechanism of their trafficking remains unclear. Here, we used mass spectrometry to identify SAP102, a MAGUK protein implicated in trafficking of the ionotropic glutamate AMPA- and NMDA-type receptors during synaptogenesis, as a novel Nbea interacting protein in mouse brain. Experiments in heterologous cells confirmed this interaction and revealed that SAP102 binds to the C-terminal part of Nbea that contains the DUF, PH, BEACH and WD40 domains. Furthermore, we discovered that introducing a mutation in Nbea's PH domain, which disrupts its interaction with the BEACH domain, abolishes this binding, thereby creating an excellent starting point to further investigate Nbea-SAP102 function in the central nervous system.

  14. The Stability Enhancement of Nitrile Hydratase from Bordetella petrii by Swapping the C-terminal Domain of β subunit.

    Science.gov (United States)

    Sun, Weifeng; Zhu, Longbao; Chen, Xianggui; Wu, Lunjie; Zhou, Zhemin; Liu, Yi

    2016-04-01

    The thermal stability of most nitrile hydratases (NHase) is poor, which has been enhanced to some extent by molecular modifications in several specific regions of the C-terminal domain (C-domain) of β subunit of NHase. Since the C-domain could be present as a naturally separate domain in a few NHases, the whole C-domain is proposed to be related to the NHase stability. The chimeric NHase (SBpNHase) from the thermal-sensitive BpNHase (NHase from Bordetella petrii) and the relatively thermal-stable PtNHase (NHase from Pseudonocardia thermophila) was constructed by swapping the corresponding C-domains. After 30 min incubation at 50 °C, the original BpNHase nearly lost its activity, while the SBpNHase retained 50 % residual activity, compared with the melting temperature (Tm) (50 °C) of the original BpNHase, that of the SBpNHase was 55 °C. The SBpNHase with higher thermal stability would be useful for the thermal stability enhancement of NHase and for the understanding of the relationship between the stability of NHase and its structure.

  15. Functional analysis of the C-terminal region of human adenovirus E1A reveals a misidentified nuclear localization signal

    Energy Technology Data Exchange (ETDEWEB)

    Cohen, Michael J.; King, Cason R.; Dikeakos, Jimmy D. [Department of Microbiology and Immunology, The University of Western Ontario, A4-833 London Regional Cancer Centre, 800 Commissioners Road E., London, Ontario, N6A 4L6 Canada (Canada); Mymryk, Joe S., E-mail: jmymryk@uwo.ca [Department of Microbiology and Immunology, The University of Western Ontario, A4-833 London Regional Cancer Centre, 800 Commissioners Road E., London, Ontario, N6A 4L6 Canada (Canada); Department of Oncology, The University of Western Ontario, London Regional Cancer Centre, Ontario (Canada)

    2014-11-15

    The immortalizing function of the human adenovirus 5 E1A oncoprotein requires efficient localization to the nucleus. In 1987, a consensus monopartite nuclear localization sequence (NLS) was identified at the C-terminus of E1A. Since that time, various experiments have suggested that other regions of E1A influence nuclear import. In addition, a novel bipartite NLS was recently predicted at the C-terminal region of E1A in silico. In this study, we used immunofluorescence microscopy and co-immunoprecipitation analysis with importin-α to verify that full nuclear localization of E1A requires the well characterized NLS spanning residues 285–289, as well as a second basic patch situated between residues 258 and 263 ({sup 258}RVGGRRQAVECIEDLLNEPGQPLDLSCKRPRP{sup 289}). Thus, the originally described NLS located at the C-terminus of E1A is actually a bipartite signal, which had been misidentified in the existing literature as a monopartite signal, altering our understanding of one of the oldest documented NLSs. - Highlights: • Human adenovirus E1A is localized to the nucleus. • The C-terminus of E1A contains a bipartite nuclear localization signal (NLS). • This signal was previously misidentified to be a monopartite NLS. • Key basic amino acid residues within this sequence are highly conserved.

  16. Plasmids for C-terminal tagging in Saccharomyces cerevisiae that contain improved GFP proteins, Envy and Ivy.

    Science.gov (United States)

    Slubowski, Christian J; Funk, Alyssa D; Roesner, Joseph M; Paulissen, Scott M; Huang, Linda S

    2015-04-01

    Green fluorescent protein (GFP) has become an invaluable tool in biological research. Many GFP variants have been created that differ in brightness, photostability, and folding robustness. We have created two hybrid GFP variants, Envy and Ivy, which we placed in a vector for the C-terminal tagging of yeast proteins by PCR-mediated recombination. The Envy GFP variant combines mutations found in the robustly folding SuperfolderGFP and GFPγ, while the Ivy GFP variant is a hybrid of GFPγ and the yellow-green GFP variant, Clover. We compared Envy and Ivy to EGFP, SuperfolderGFP and GFPγ and found that Envy is brighter than the other GFP variants at both 30°C and 37°C, while Ivy is the most photostable. Envy and Ivy are recognized by a commonly used anti-GFP antibody, and both variants can be immunoprecipitated using the GFP TRAP Camelidae antibody nanotrap technology. Because Envy is brighter than the other GFP variants and is as photostable as GFPγ, we suggest that Envy should be the preferred GFP variant, while Ivy may be used in cases where photostability is of the utmost importance. Copyright © 2015 John Wiley & Sons, Ltd.

  17. Aurora A mediates cross-talk between N- and C-terminal post-translational modifications of p53

    Science.gov (United States)

    Raines, Sally Anne; Milner, Jo

    2011-01-01

    The serine/threonine protein kinase Aurora A is known to interact with and phosphorylate tumor suppressor p53 at Serine 215 (S215), inhibiting the transcriptional activity of p53. We show that Aurora A positively regulates human p53 protein levels and, using isogenic p53 wild-type and p53-null colorectal carcinoma cells, further show that p53 regulates human Aurora A protein expression. S215 is located in the DNA-binding core of p53 and at the center of the cryptic epitope for PAb240 antibody, which is used to detect mutant and denatured p53. Following denaturing SDS PAGE, the PAb240 epitope was detectable by immunoblotting in only two out of eight cell lines. The efficacy of novel p53-targeted anticancer therapies may be influenced by the conformational state of p53, therefore, the initial determination of p53 status may be relevant. We found no correlation between phosphorylation of p53 at S215 and PAb240 antibody recognition. However, phosphorylation at S37 was positively associated with PAb240 reactivity. More importantly, we provide the first evidence of Aurora A-mediated cross-talk between N- and C-terminal p53 post-translational modifications. As p53 and Aurora A are targets for anticancer therapy the impact of their reciprocal relationship and Aurora A-induced post-translational modification of p53 should be considered. PMID:22157150

  18. Probing the interaction of the p53 C-terminal domain to the histone demethylase LSD1.

    Science.gov (United States)

    Speranzini, Valentina; Ciossani, Giuseppe; Marabelli, Chiara; Mattevi, Andrea

    2017-10-15

    The p53 transcription factor plays a central role in the regulation of the expression of several genes, and itself is post-translationally regulated through its different domains. Of particular relevance for p53 function is its intrinsically disordered C-terminal domain (CTD), representing a hotspot for post-translational modifications and a docking site for transcriptional regulators. For example, the histone H3 lysine demethylase 1 (LSD1) interacts with p53 via the p53-CTD for mutual regulation. To biochemically and functionally characterize this complex, we evaluated the in vitro interactions of LSD1 with several p53-CTD peptides differing in length and modifications. Binding was demonstrated through thermal shift, enzymatic and fluorescence polarization assays, but no enzymatic activity could be detected on methylated p53-CTD peptides in vitro. These experiments were performed using the wild-type enzyme and LSD1 variants that are mutated on three active-site residues. We found that LSD1 demethylase activity is inhibited by p53-CTD. We also noted that the association between the two proteins is mediated by mostly non-specific electrostatic interactions involving conserved active-site residues of LSD1 and a highly charged segment of the p53-CTD. We conclude that p53-CTD inhibits LSD1 activity and that the direct association between the two proteins can contribute to their functional cross-talk. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. The identification of putative RNA polymerase II C-terminal domain associated proteins in red and green algae.

    Science.gov (United States)

    Yang, Chunlin; Hager, Paul W; Stiller, John W

    2014-01-01

    A tandemly repeated C-terminal domain (CTD) of the largest subunit of RNA polymerase II is functionally essential and strongly conserved in many organisms, including animal, yeast and plant models. Although present in simple, ancestral red algae, CTD tandem repeats have undergone extensive modifications and degeneration during the evolutionary transition to developmentally complex rhodophytes. In contrast, CTD repeats are conserved in both green algae and their more complex land plant relatives. Understanding the mechanistic differences that underlie these variant patterns of CTD evolution requires knowledge of CTD-associated proteins in these 2 lineages. To provide an initial baseline comparison, we bound potential phospho-CTD associated proteins (PCAPs) to artificially synthesized and phosphorylated CTD repeats from the unicellular red alga Cyanidioschyzon merolae and green alga Chlamydomonas reinhardtii. Our results indicate that red and green algae share a number of PCAPs, including kinases and proteins involved in mRNA export. There also are important taxon-specific differences, including mRNA splicing-related PCAPs recovered from Chlamydomonas but not Cyanidioschyzon, consistent with the relative intron densities in green and red algae. Our results also offer the first experimental indication that different proteins bind 2 distinct types of repeats in Cyanidioschyzon, suggesting a division of function between the proximal and distal CTD, similar to patterns identified in more developmentally complex model organisms.

  20. Ubiquitin C-terminal hydrolase-L3 regulates EMT process and cancer metastasis in prostate cell lines.

    Science.gov (United States)

    Song, Hyun Min; Lee, Jae Eun; Kim, Jung Hwa

    2014-09-26

    Ubiquitin C-terminal hydrolase-L3 (UCH-L3) is among the deubiquitinating enzymes (DUBs) that cleave ubiquitin (Ub) from Ub precursors or protein substrates. Many DUBs have been shown to participate in cancer progression in various tissues. However, the mechanism and role of UCH-L3 in carcinogenesis has largely been unknown until recently. Here we investigated the implication of UCH-L3 in prostate cancer progression. Interestingly, UCH-L3 is upregulated in normal or non-metastatic prostate cancer cells and is downregulated in metastatic prostate cancer cell lines. Notably, knockdown of UCH-L3 in normal prostate cell line RWPE1 promotes epithelial-to-mesenchymal transition (EMT), an important process for cancer cell invasion and metastasis. The induction of EMT by UCH-L3 knockdown results in an increase of cell migration and invasion. Yet, to the contrary, overexpression of UCH-L3 in highly metastatic prostate cancer cell line PC3 reverses EMT but the active site mutant UCH-L3 did not. Collectively, our findings identify UCH-L3 as a novel EMT regulator in prostate cells and highlight UCH-L3 as a potential therapeutic target for preventing metastatic prostate cancer. Copyright © 2014 Elsevier Inc. All rights reserved.

  1. Bio-molecular architects: a scaffold provided by the C-terminal domain of eukaryotic RNA polymerase II

    Directory of Open Access Journals (Sweden)

    Yan Zhang

    2010-08-01

    Full Text Available In eukaryotic cells, the transcription of genes is accurately orchestrated both spatially and temporally by the C-terminal domain of RNA polymerase II (CTD. The CTD provides a dynamic platform to recruit different regulators of the transcription apparatus. Different posttranslational modifications are precisely applied to specific sites of the CTD to coordinate transcription process. Regulators of the RNA polymerase II must identify specific sites in the CTD for cellular survival, metabolism, and development. Even though the CTD is disordered in the eukaryotic RNA polymerase II crystal structures due to its intrinsic flexibility, recent advances in the complex structural analysis of the CTD with its binding partners provide essential clues for understanding how selectivity is achieved for individual site recognition. The recent discoveries of the interactions between the CTD and histone modification enzymes disclose an important role of the CTD in epigenetic control of the eukaryotic gene expression. The intersection of the CTD code with the histone code discloses an intriguing yet complicated network for eukaryotic transcriptional regulation.

  2. Cloning, purification and preliminary X-ray analysis of the C-terminal domain of Helicobacter pylori MotB

    Energy Technology Data Exchange (ETDEWEB)

    Roujeinikova, Anna, E-mail: anna.roujeinikova@manchester.ac.uk [Manchester Interdisciplinary Biocentre, Faculty of Life Sciences, University of Manchester, 131 Princess Street, Manchester M1 7DN (United Kingdom)

    2008-04-01

    The cloning, overexpression, purification, crystallization and preliminary X-ray diffraction analysis of a putative peptidoglycan-binding domain of H. pylori MotB, a stator component of the bacterial flagellar motor, are reported. The C-terminal domain of MotB (MotB-C) contains a putative peptidoglycan-binding motif and is believed to anchor the MotA/MotB stator unit of the bacterial flagellar motor to the cell wall. Crystals of Helicobacter pylori MotB-C (138 amino-acid residues) were obtained by the hanging-drop vapour-diffusion method using polyethylene glycol as a precipitant. These crystals belong to space group P2{sub 1}, with unit-cell parameters a = 50.8, b = 89.5, c = 66.3 Å, β = 112.5°. The crystals diffract X-rays to at least 1.6 Å resolution using a synchrotron-radiation source. Self-rotation function and Matthews coefficient calculations suggest that the asymmetric unit contains one tetramer with 222 point-group symmetry. The anomalous difference Patterson maps calculated for an ytterbium-derivative crystal using diffraction data at a wavelength of 1.38 Å showed significant peaks on the v = 1/2 Harker section, suggesting that ab initio phase information could be derived from the MAD data.

  3. C-terminal diversity within the p53 family accounts for differences in DNA binding and transcriptional activity

    Science.gov (United States)

    Sauer, Markus; Bretz, Anne Catherine; Beinoraviciute-Kellner, Rasa; Beitzinger, Michaela; Burek, Christof; Rosenwald, Andreas; Harms, Gregory S.; Stiewe, Thorsten

    2008-01-01

    The p53 family is known as a family of transcription factors with functions in tumor suppression and development. Whereas the central DNA-binding domain is highly conserved among the three family members p53, p63 and p73, the C-terminal domains (CTDs) are diverse and subject to alternative splicing and post-translational modification. Here we demonstrate that the CTDs strongly influence DNA binding and transcriptional activity: while p53 and the p73 isoform p73γ have basic CTDs and form weak sequence-specific protein–DNA complexes, the major p73 isoforms have neutral CTDs and bind DNA strongly. A basic CTD has been previously shown to enable sliding along the DNA backbone and to facilitate the search for binding sites in the complex genome. Our experiments, however, reveal that a basic CTD also reduces protein–DNA complex stability, intranuclear mobility, promoter occupancy in vivo, target gene activation and induction of cell cycle arrest or apoptosis. A basic CTD therefore provides both positive and negative regulatory functions presumably to enable rapid switching of protein activity in response to stress. The different DNA-binding characteristics of the p53 family members could therefore reflect their predominant role in the cellular stress response (p53) or developmental processes (p73). PMID:18267967

  4. Hepatitis B Virus Core Protein Phosphorylation Sites Affect Capsid Stability and Transient Exposure of the C-terminal Domain.

    Science.gov (United States)

    Selzer, Lisa; Kant, Ravi; Wang, Joseph C-Y; Bothner, Brian; Zlotnick, Adam

    2015-11-20

    Hepatitis B virus core protein has 183 amino acids divided into an assembly domain and an arginine-rich C-terminal domain (CTD) that regulates essential functions including genome packaging, reverse transcription, and intracellular trafficking. Here, we investigated the CTD in empty hepatitis B virus (HBV) T=4 capsids. We examined wild-type core protein (Cp183-WT) and a mutant core protein (Cp183-EEE), in which three CTD serines are replaced with glutamate to mimic phosphorylated protein. We found that Cp183-WT capsids were less stable than Cp183-EEE capsids. When we tested CTD sensitivity to trypsin, we detected two different populations of CTDs differentiated by their rate of trypsin cleavage. Interestingly, CTDs from Cp183-EEE capsids exhibited a much slower rate of proteolytic cleavage when compared with CTDs of Cp183-WT capsids. Cryo-electron microscopy studies of trypsin-digested capsids show that CTDs at five-fold symmetry vertices are most protected. We hypothesize that electrostatic interactions between glutamates and arginines in Cp183-EEE, particularly at five-fold, increase capsid stability and reduce CTD exposure. Our studies show that quasi-equivalent CTDs exhibit different rates of exposure and thus might perform distinct functions during the hepatitis B virus lifecycle. Our results demonstrate a structural role for CTD phosphorylation and indicate crosstalk between CTDs within a capsid particle. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. Fructose-1,6-bisphosphate aldolase of Neisseria meningitidis binds human plasminogen via its C-terminal lysine residue.

    Science.gov (United States)

    Shams, Fariza; Oldfield, Neil J; Lai, Si Kei; Tunio, Sarfraz A; Wooldridge, Karl G; Turner, David P J

    2016-04-01

    Neisseria meningitidis is a leading cause of fatal sepsis and meningitis worldwide. As for commensal species of human neisseriae, N. meningitidis inhabits the human nasopharynx and asymptomatic colonization is ubiquitous. Only rarely does the organism invade and survive in the bloodstream leading to disease. Moonlighting proteins perform two or more autonomous, often dissimilar, functions using a single polypeptide chain. They have been increasingly reported on the surface of both prokaryotic and eukaryotic organisms and shown to interact with a variety of host ligands. In some organisms moonlighting proteins perform virulence-related functions, and they may play a role in the pathogenesis of N. meningitidis. Fructose-1,6-bisphosphate aldolase (FBA) was previously shown to be surface-exposed in meningococci and involved in adhesion to host cells. In this study, FBA was shown to be present on the surface of both pathogenic and commensal neisseriae, and surface localization and anchoring was demonstrated to be independent of aldolase activity. Importantly, meningococcal FBA was found to bind to human glu-plasminogen in a dose-dependent manner. Site-directed mutagenesis demonstrated that the C-terminal lysine residue of FBA was required for this interaction, whereas subterminal lysine residues were not involved. © 2016 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

  6. A Superhelical Spiral in the Escherichia coli DNA Gyrase A C-terminal Domain Imparts Unidirectional Supercoiling Bias

    Energy Technology Data Exchange (ETDEWEB)

    Ruthenburg,A.; Graybosch, D.; Huetsch, J.; Verdine, G.

    2005-01-01

    DNA gyrase is unique among type II topoisomerases in that its DNA supercoiling activity is unidirectional. The C-terminal domain of the gyrase A subunit (GyrA-CTD) is required for this supercoiling bias. We report here the x-ray structure of the Escherichia coli GyrA-CTD (Protein Data Bank code 1ZI0). The E. coli GyrA-CTD adopts a circular-shaped {beta}-pinwheel fold first seen in the Borrelia burgdorferi GyrA-CTD. However, whereas the B. burgdorferi GyrA-CTD is flat, the E. coli GyrA-CTD is spiral. DNA relaxation assays reveal that the E. coli GyrA-CTD wraps DNA inducing substantial (+) superhelicity, while the B. burgdorferi GyrA-CTD introduces a more modest (+) superhelicity. The observation of a superhelical spiral in the present structure and that of the Bacillus stearothermophilus ParC-CTD structure suggests unexpected similarities in substrate selectivity between gyrase and Topo IV enzymes. We propose a model wherein the right-handed ((+) solenoidal) wrapping of DNA around the E. coli GyrA-CTD enforces unidirectional (-) DNA supercoiling.

  7. Functional insight into the C-terminal extension of halolysin SptA from haloarchaeon Natrinema sp. J7.

    Directory of Open Access Journals (Sweden)

    Zhisheng Xu

    Full Text Available Halolysin SptA from haloarchaeon Natrinema sp. J7 consists of a subtilisin-like catalytic domain and a C-terminal extension (CTE containing two cysteine residues. In this report, we have investigated the function of the CTE using recombinant enzymes expressed in Haloferax volcanii WFD11. Deletion of the CTE greatly reduced but did not abolish protease activity, which suggests that the CTE is not essential for enzyme folding. Mutational analysis suggests that residues Cys303 and Cys338 within the CTE form a disulfide bond that make this domain resistant to autocleavage and proteolysis under hypotonic conditions. Characterization of full-length and CTE-truncation enzymes indicates the CTE not only confers extra stability to the enzyme but also assists enzyme activity on protein substrates by facilitating binding at high salinities. Interestingly, homology modeling of the CTE yields a β-jelly roll-like structure similar to those seen in Claudin-binding domain of Clostridium perfringens enterotoxin (clostridial C-CPE and collagen binding domain (CBD, and the CTE also possesses collagen-binding activity, making it a potential candidate as an anchoring unit in drug delivery systems.

  8. Tight intramolecular regulation of the human Upf1 helicase by its N- and C-terminal domains.

    Science.gov (United States)

    Fiorini, Francesca; Boudvillain, Marc; Le Hir, Hervé

    2013-02-01

    The RNA helicase Upf1 is a multifaceted eukaryotic enzyme involved in DNA replication, telomere metabolism and several mRNA degradation pathways. Upf1 plays a central role in nonsense-mediated mRNA decay (NMD), a surveillance process in which it links premature translation termination to mRNA degradation with its conserved partners Upf2 and Upf3. In human, both the ATP-dependent RNA helicase activity and the phosphorylation of Upf1 are essential for NMD. Upf1 activation occurs when Upf2 binds its N-terminal domain, switching the enzyme to the active form. Here, we uncovered that the C-terminal domain of Upf1, conserved in higher eukaryotes and containing several essential phosphorylation sites, also inhibits the flanking helicase domain. With different biochemical approaches we show that this domain, named SQ, directly interacts with the helicase domain to impede ATP hydrolysis and RNA unwinding. The phosphorylation sites in the distal half of the SQ domain are not directly involved in this inhibition. Therefore, in the absence of multiple binding partners, Upf1 is securely maintained in an inactive state by two intramolecular inhibition mechanisms. This study underlines the tight and intricate regulation pathways required to activate multifunctional RNA helicases like Upf1.

  9. Structure of the S1 subunit C-terminal domain from bat-derived coronavirus HKU5 spike protein.

    Science.gov (United States)

    Han, Xue; Qi, Jianxun; Song, Hao; Wang, Qihui; Zhang, Yanfang; Wu, Ying; Lu, Guangwen; Yuen, Kwok-Yung; Shi, Yi; Gao, George F

    2017-07-01

    Accumulating evidence indicates that MERS-CoV originated from bat coronaviruses (BatCoVs). Previously, we demonstrated that both MERS-CoV and BatCoV HKU4 use CD26 as a receptor, but how the BatCoVs evolved to bind CD26 is an intriguing question. Here, we solved the crystal structure of the S1 subunit C-terminal domain of HKU5 (HKU5-CTD), another BatCoV that is phylogenetically related to MERS-CoV but cannot bind to CD26. We observed that the conserved core subdomain and those of other betacoronaviruses (betaCoVs) have a similar topology of the external subdomain, indicating the same ancestor of lineage C betaCoVs. However, two deletions in two respective loops located in HKU5-CTD result in conformational variations in CD26-binding interface and are responsible for the non-binding of HKU5-CTD to CD26. Combined with sequence variation in the HKU5-CTD receptor binding interface, we propose the necessity for surveilling the mutation in BatCoV HKU5 spike protein in case of bat-to-human interspecies transmission. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. The BARD1 C-Terminal Domain Structure and Interactions with Polyadenylation Factor CstF-50

    Energy Technology Data Exchange (ETDEWEB)

    Edwards, Ross A.; Lee, Megan S.; Tsutakawa, Susan E.; Williams, R. Scott; Tainer, John A.; Glover, J. N. Mark

    2009-07-13

    The BARD1 N-terminal RING domain binds BRCA1 while the BARD1 C-terminal ankyrin and tandem BRCT repeat domains bind CstF-50 to modulate mRNA processing and RNAP II stability in response to DNA damage. Here we characterize the BARD1 structural biochemistry responsible for CstF- 50 binding. The crystal structure of the BARD1 BRCT domain uncovers a degenerate phosphopeptide binding pocket lacking the key arginine required for phosphopeptide interactions in other BRCT proteins.Small angle X-ray scattering together with limited proteolysis results indicates that ankyrin and BRCT domains are linked by a flexible tether and do not adopt a fixed orientation relative to one another. Protein pull-down experiments utilizing a series of purified BARD1 deletion mutants indicate that interactions between the CstF-50 WD-40 domain and BARD1 involve the ankyrin-BRCT linker but do not require ankyrin or BRCT domains. The structural plasticity imparted by the ANK-BRCT linker helps to explain the regulated assembly of different protein BARD1 complexes with distinct functions in DNA damage signaling including BARD1-dependent induction of apoptosis plus p53 stabilization and interactions. BARD1 architecture and plasticity imparted by the ANK-BRCT linker are suitable to allow the BARD1 C-terminus to act as a hub with multiple binding sites to integrate diverse DNA damage signals directly to RNA polymerase.

  11. Directed Evolution of Recombinant C-Terminal Truncated Staphylococcus epidermidis Lipase AT2 for the Enhancement of Thermostability

    Directory of Open Access Journals (Sweden)

    Jiivittha Veno

    2017-11-01

    Full Text Available In the industrial processes, lipases are expected to operate at temperatures above 45 °C and could retain activity in organic solvents. Hence, a C-terminal truncated lipase from Staphylococcus epidermis AT2 (rT-M386 was engineered by directed evolution. A mutant with glycine-to-cysteine substitution (G210C demonstrated a remarkable improvement of thermostability, whereby the mutation enhanced the activity five-fold when compared to the rT-M386 at 50 °C. The rT-M386 and G210C lipases were purified concurrently using GST-affinity chromatography. The biochemical and biophysical properties of both enzymes were investigated. The G210C lipase showed a higher optimum temperature (45 °C and displayed a more prolonged half-life in the range of 40–60 °C as compared to rT-M386. Both lipases exhibited optimal activity and stability at pH 8. The G210C showed the highest stability in the presence of polar organic solvents at 50 °C compared to the rT-M386. Denatured protein analysis presented a significant change in the molecular ellipticity value above 60 °C, which verified the experimental result on the temperature and thermostability profile of G210C.

  12. Directed Evolution of Recombinant C-Terminal Truncated Staphylococcus epidermidis Lipase AT2 for the Enhancement of Thermostability

    Science.gov (United States)

    Veno, Jiivittha; Ahmad Kamarudin, Nor Hafizah; Mohamad Ali, Mohd Shukuri; Masomian, Malihe

    2017-01-01

    In the industrial processes, lipases are expected to operate at temperatures above 45 °C and could retain activity in organic solvents. Hence, a C-terminal truncated lipase from Staphylococcus epidermis AT2 (rT-M386) was engineered by directed evolution. A mutant with glycine-to-cysteine substitution (G210C) demonstrated a remarkable improvement of thermostability, whereby the mutation enhanced the activity five-fold when compared to the rT-M386 at 50 °C. The rT-M386 and G210C lipases were purified concurrently using GST-affinity chromatography. The biochemical and biophysical properties of both enzymes were investigated. The G210C lipase showed a higher optimum temperature (45 °C) and displayed a more prolonged half-life in the range of 40–60 °C as compared to rT-M386. Both lipases exhibited optimal activity and stability at pH 8. The G210C showed the highest stability in the presence of polar organic solvents at 50 °C compared to the rT-M386. Denatured protein analysis presented a significant change in the molecular ellipticity value above 60 °C, which verified the experimental result on the temperature and thermostability profile of G210C. PMID:29113034

  13. The C-terminal domain of Tetrahymena thermophila telomerase holoenzyme protein p65 induces multiple structural changes in telomerase RNA

    Science.gov (United States)

    Akiyama, Benjamin M.; Loper, John; Najarro, Kevin; Stone, Michael D.

    2012-01-01

    The unique cellular activity of the telomerase reverse transcriptase ribonucleoprotein (RNP) requires proper assembly of protein and RNA components into a functional complex. In the ciliate model organism Tetrahymena thermophila, the La-domain protein p65 is required for in vivo assembly of telomerase. Single-molecule and biochemical studies have shown that p65 promotes efficient RNA assembly with the telomerase reverse transcriptase (TERT) protein, in part by inducing a bend in the conserved stem IV region of telomerase RNA (TER). The domain architecture of p65 consists of an N-terminal domain, a La-RRM motif, and a C-terminal domain (CTD). Using single-molecule Förster resonance energy transfer (smFRET), we demonstrate the p65CTD is necessary for the RNA remodeling activity of the protein and is sufficient to induce a substantial conformational change in stem IV of TER. Moreover, nuclease protection assays directly map the site of p65CTD interaction to stem IV and reveal that, in addition to bending stem IV, p65 binding reorganizes nucleotides that comprise the low-affinity TERT binding site within stem–loop IV. PMID:22315458

  14. Neuroprotective effects of inositol 1,4,5-trisphosphate receptor C-terminal fragment in a Huntington's disease mouse model.

    Science.gov (United States)

    Tang, Tie-Shan; Guo, Caixia; Wang, Hongyu; Chen, Xi; Bezprozvanny, Ilya

    2009-02-04

    Huntington's disease (HD) is a dominantly inherited, progressive neurodegenerative disease caused by an expanded polyglutamine tract in huntingtin protein (Htt). Medium spiny striatal neurons (MSNs) are primarily affected in HD. Mutant huntingtin protein (Htt(exp)) specifically binds to and activates type 1 inositol 1,4,5-trisphosphate receptor (InsP(3)R1), an intracellular Ca(2+) release channel. Htt(exp)-InsP(3)R1 association is mediated by a cytosolic C-terminal tail of InsP(3)R1 (a 122-aa-long IC10 fragment). To evaluate an importance of Htt(exp) association with InsP(3)R1 for HD pathology, we generated lentiviral and adeno-associated viruses expressing GFP-IC10 fusion protein and performed a series of experiments with YAC128 HD transgenic mouse. Infection with Lenti-GFP-IC10 virus stabilized Ca(2+) signaling in cultured YAC128 MSNs and protected YAC128 MSNs from glutamate-induced apoptosis. Intrastriatal injections of AAV1-GFP-IC10 significantly alleviated motor deficits and reduced MSN loss and shrinkage in YAC128 mice. Our results demonstrate an importance of InsP(3)R1-Htt(exp) association for HD pathogenesis and suggested that InsP(3)R1 is a potential therapeutic target for HD. Our data also support potential use of IC10 peptide as a novel HD therapeutic agent.

  15. Resolving hot spots in the C-terminal dimerization domain that determine the stability of the molecular chaperone Hsp90.

    Directory of Open Access Journals (Sweden)

    Emanuele Ciglia

    Full Text Available Human heat shock protein of 90 kDa (hHsp90 is a homodimer that has an essential role in facilitating malignant transformation at the molecular level. Inhibiting hHsp90 function is a validated approach for treating different types of tumors. Inhibiting the dimerization of hHsp90 via its C-terminal domain (CTD should provide a novel way to therapeutically interfere with hHsp90 function. Here, we predicted hot spot residues that cluster in the CTD dimerization interface by a structural decomposition of the effective energy of binding computed by the MM-GBSA approach and confirmed these predictions using in silico alanine scanning with DrugScore(PPI. Mutation of these residues to alanine caused a significant decrease in the melting temperature according to differential scanning fluorimetry experiments, indicating a reduced stability of the mutant hHsp90 complexes. Size exclusion chromatography and multi-angle light scattering studies demonstrate that the reduced stability of the mutant hHsp90 correlates with a lower complex stoichiometry due to the disruption of the dimerization interface. These results suggest that the identified hot spot residues can be used as a pharmacophoric template for identifying and designing small-molecule inhibitors of hHsp90 dimerization.

  16. A Novel Fold in the Tral Relaxase-Helicase C-Terminal Domain Is Essential for Conjugative DNA Transfer

    Energy Technology Data Exchange (ETDEWEB)

    Guogas, Laura M.; Kennedy, Sarah A.; Lee, Jin-Hyup; Redinbo, Matthew R.; (UNC)

    2009-06-04

    TraI relaxase-helicase is the central catalytic component of the multiprotein relaxosome complex responsible for conjugative DNA transfer (CDT) between bacterial cells. CDT is a primary mechanism for the lateral propagation of microbial genetic material, including the spread of antibiotic resistance genes. The 2.4-{angstrom} resolution crystal structure of the C-terminal domain of the multifunctional Escherichia coli F (fertility) plasmid TraI protein is presented, and specific structural regions essential for CDT are identified. The crystal structure reveals a novel fold composed of a 28-residue N-terminal {alpha}-domain connected by a proline-rich loop to a compact {alpha}/{beta}-domain. Both the globular nature of the {alpha}/{beta}-domain and the presence as well as rigidity of the proline-rich loop are required for DNA transfer and single-stranded DNA binding. Taken together, these data establish the specific structural features of this noncatalytic domain that are essential to DNA conjugation.

  17. Differential cellulolytic activity of native-form and C-terminal tagged-form cellulase derived from coptotermes formosanus and expressed in E. coli

    Science.gov (United States)

    The endogenous cellulase gene (CfEG3a) of Coptotermes formosanus, an economically important pest termite, was cloned and overexpressed in both native form (nCfEG) and C-terminal His-tagged form (tCfEG) in E.coli. Both forms of recombinant cellulases showed hydrolytic activity on cellulosic substrate...

  18. The ubiquitin C-terminal hydrolase UCH-L1 promotes bacterial invasion by altering the dynamics of the actin cytoskeleton

    DEFF Research Database (Denmark)

    Basseres, Eugene; Coppotelli, Giuseppe; Pfirrmann, Thorsten

    2010-01-01

    Invasion of eukaryotic target cells by pathogenic bacteria requires extensive remodelling of the membrane and actin cytoskeleton. Here we show that the remodelling process is regulated by the ubiquitin C-terminal hydrolase UCH-L1 that promotes the invasion of epithelial cells by Listeria...

  19. Evaluation of heavy chain C-terminal deletions on productivity and product quality of monoclonal antibodies in Chinese hamster ovary (CHO) cells.

    Science.gov (United States)

    Hu, Zhilan; Tang, Danming; Misaghi, Shahram; Jiang, Guoying; Yu, Christopher; Yim, Mandy; Shaw, David; Snedecor, Brad; Laird, Michael W; Shen, Amy

    2017-05-01

    Monoclonal antibodies (mAbs) have been well established as potent therapeutic agents and are used to treat many different diseases. During cell culture production, antibody charge variants can be generated by cleavage of heavy chain (HC) C-terminal lysine and proline amidation. Differences in levels of charge variants during manufacturing process changes make it challenging to demonstrate process comparability. In order to reduce heterogeneity and achieve consistent product quality, we generated and expressed antibodies with deletion of either HC C-terminal lysine (-K) or lysine and glycine (-GK). Interestingly, clones that express antibodies lacking HC C-terminal lysine (-K) had considerably lower specific productivities compared to clones that expressed either wild type antibodies (WT) or antibodies lacking HC glycine and lysine (-GK). While no measurable differences in antibody HC and LC mRNA levels, glycosylation and secretion were observed, our analysis suggests that the lower specific productivity of clones expressing antibody lacking HC C-terminal lysine was due to slower antibody HC synthesis and faster antibody degradation. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:786-794, 2017. © 2017 American Institute of Chemical Engineers.

  20. Solution structure and DNA-binding properties of the C-terminal domain of UvrC from E.coli

    NARCIS (Netherlands)

    Singh, S.; Folkers, G.E.|info:eu-repo/dai/nl/162277202; Bonvin, A.M.J.J.|info:eu-repo/dai/nl/113691238; Boelens, R.|info:eu-repo/dai/nl/070151407; Wechselberger, R.W.|info:eu-repo/dai/nl/304829005; Niztayev, A.; Kaptein, R.|info:eu-repo/dai/nl/074334603

    2002-01-01

    The C-terminal domain of the UvrC protein (UvrC CTD) is essential for 5' incision in the prokaryotic nucleotide excision repair process. We have determined the three-dimensional structure of the UvrC CTD using heteronuclear NMR techniques. The structure shows two helix±hairpin±helix (HhH) motifs

  1. Structure of the DNA-bound BRCA1 C-terminal region from human replication factor C p140 and model of the protein-DNA complex

    NARCIS (Netherlands)

    Kobayashi, M.; AB, E.; Bonvin, A.M.J.J.|info:eu-repo/dai/nl/113691238; Siegal, G.

    2010-01-01

    BRCA1 C-terminal domain (BRCT)-containing proteins are found widely throughout the animal and bacteria kingdoms where they are exclusively involved in cell cycle regulation and DNA metabolism. Whereas most BRCT domains are involved in protein-protein interactions, a small subset has bona fide DNA

  2. Biophysical Evidence for Intrinsic Disorder in the C-terminal Tails of the Epidermal Growth Factor Receptor (EGFR) and HER3 Receptor Tyrosine Kinases.

    Science.gov (United States)

    Keppel, Theodore R; Sarpong, Kwabena; Murray, Elisa M; Monsey, John; Zhu, Jian; Bose, Ron

    2017-01-13

    The epidermal growth factor receptor (EGFR)/ErbB family of receptor tyrosine kinases includes oncogenes important in the progression of breast and other cancers, and they are targets for many drug development strategies. Each member of the ErbB family possesses a unique, structurally uncharacterized C-terminal tail that plays an important role in autophosphorylation and signal propagation. To determine whether these C-terminal tails are intrinsically disordered regions, we conducted a battery of biophysical experiments on the EGFR and HER3 tails. Using hydrogen/deuterium exchange mass spectrometry, we measured the conformational dynamics of intracellular half constructs and compared the tails with the ordered kinase domains. The C-terminal tails demonstrate more rapid deuterium exchange behavior when compared with the kinase domains. Next, we expressed and purified EGFR and HER3 tail-only constructs. Results from circular dichroism spectroscopy, size exclusion chromatography with multiangle light scattering, dynamic light scattering, analytical ultracentrifugation, and small angle X-ray scattering each provide evidence that the EGFR and HER3 C-terminal tails are intrinsically disordered with extended, non-globular structure in solution. The intrinsic disorder and extended conformation of these tails may be important for their function by increasing the capture radius and reducing the thermodynamic barriers for binding of downstream signaling proteins. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  3. Two Distinct Binding Modes Define the Interaction of Brox with the C-Terminal Tails of CHMP5 and CHMP4B

    Energy Technology Data Exchange (ETDEWEB)

    Mu, Ruiling; Dussupt, Vincent; Jiang, Jiansheng; Sette, Paola; Rudd, Victoria; Chuenchor, Watchalee; Bello, Nana F.; Bouamr, Fadila; Xiao, Tsan Sam (NIH)

    2012-05-21

    Interactions of the CHMP protein carboxyl terminal tails with effector proteins play important roles in retroviral budding, cytokinesis, and multivesicular body biogenesis. Here we demonstrate that hydrophobic residues at the CHMP4B C-terminal amphipathic {alpha} helix bind a concave surface of Brox, a mammalian paralog of Alix. Unexpectedly, CHMP5 was also found to bind Brox and specifically recruit endogenous Brox to detergent-resistant membrane fractions through its C-terminal 20 residues. Instead of an {alpha} helix, the CHMP5 C-terminal tail adopts a tandem {beta}-hairpin structure that binds Brox at the same site as CHMP4B. Additional Brox:CHMP5 interface is furnished by a unique CHMP5 hydrophobic pocket engaging the Brox residue Y348 that is not conserved among the Bro1 domains. Our studies thus unveil a {beta}-hairpin conformation of the CHMP5 protein C-terminal tail, and provide insights into the overlapping but distinct binding profiles of ESCRT-III and the Bro1 domain proteins.

  4. Truncation of the C-terminal region of Toscana Virus NSs protein is critical for interferon-β antagonism and protein stability

    National Research Council Canada - National Science Library

    Gori Savellini, Gianni; Gandolfo, Claudia; Cusi, Maria Grazia

    2015-01-01

    .... To this aim, two C-terminal truncated NSs proteins, Δ1C-NSs (aa 1-284) and Δ2C-NSs (aa 1-287) were tested. Only Δ1C-NSs did not present any inhibitory effect on RIG-I and it showed a greater stability than the whole NSs protein...

  5. Mutations in the C-terminal domain of ALSV (Avian Leukemia and Sarcoma Viruses) integrase alter the concerted DNA integration process in vitro.

    Science.gov (United States)

    Moreau, Karen; Faure, Claudine; Violot, Sébastien; Verdier, Gérard; Ronfort, Corinne

    2003-11-01

    Integrase (IN) is the retroviral enzyme responsible for the integration of the DNA copy of the retroviral genome into the host cell DNA. The C-terminal domain of IN is involved in DNA binding and enzyme multimerization. We previously performed single amino acid substitutions in the C-terminal domain of the avian leukemia and sarcoma viruses (ALSV) IN. Here, we modelled these IN mutants and analysed their ability to mediate concerted DNA integration (in an in vitro assay) as well as to form dimers (by size exclusion chromatography and protein-protein cross-linking). Mutations of residues located at the dimer interface (V239, L240, Y246, V257 and K266) have the greatest effects on the activity of the IN. Among them: (a) the L240A mutation resulted in a decrease of integration efficiency that was concomitant with a decrease of IN dimerization; (b) the V239A, V249A and K266A mutants preferentially mediated non-concerted DNA integration rather than concerted DNA integration although they were found as dimers. Other mutations (V260E and Y246W/DeltaC25) highlight the role of the C-terminal domain in the general folding of the enzyme and, hence, on its activity. This study points to the important role of residues at the IN C-terminal domain in the folding and dimerization of the enzyme as well as in the concerted DNA integration of viral DNA ends.

  6. N-Terminal Domains in Two-Domain Proteins Are Biased to Be Shorter and Predicted to Fold Faster Than Their C-Terminal Counterparts

    Directory of Open Access Journals (Sweden)

    Etai Jacob

    2013-04-01

    Full Text Available Computational analysis of proteomes in all kingdoms of life reveals a strong tendency for N-terminal domains in two-domain proteins to have shorter sequences than their neighboring C-terminal domains. Given that folding rates are affected by chain length, we asked whether the tendency for N-terminal domains to be shorter than their neighboring C-terminal domains reflects selection for faster-folding N-terminal domains. Calculations of absolute contact order, another predictor of folding rate, provide additional evidence that N-terminal domains tend to fold faster than their neighboring C-terminal domains. A possible explanation for this bias, which is more pronounced in prokaryotes than in eukaryotes, is that faster folding of N-terminal domains reduces the risk for protein aggregation during folding by preventing formation of nonnative interdomain interactions. This explanation is supported by our finding that two-domain proteins with a shorter N-terminal domain are much more abundant than those with a shorter C-terminal domain.

  7. Dissection of influenza A virus M1 protein: pH-dependent oligomerization of N-terminal domain and dimerization of C-terminal domain.

    Science.gov (United States)

    Zhang, Ke; Wang, Zhao; Liu, Xiaoling; Yin, Changcheng; Basit, Zeshan; Xia, Bin; Liu, Wenjun

    2012-01-01

    The matrix 1 (M1) protein of Influenza A virus plays many critical roles throughout the virus life cycle. The oligomerization of M1 is essential for the formation of the viral matrix layer during the assembly and budding process. In the present study, we report that M1 can oligomerize in vitro, and that the oligomerization is pH-dependent. The N-terminal domain of M1 alone exists as multiple-order oligomers at pH 7.4, and the C-terminal domain alone forms an exclusively stable dimer. As a result, intact M1 can display different forms of oligomers and dimer is the smallest oligomerization state, at neutral pH. At pH 5.0, oligomers of the N-terminal domain completely dissociate into monomers, while the C-terminal domain remains in dimeric form. As a result, oligomers of intact M1 dissociate into a stable dimer at acidic pH. Oligomerization of M1 involves both the N- and C-terminal domains. The N-terminal domain determines the pH-dependent oligomerization characteristic, and C-terminal domain forms a stable dimer, which contributes to the dimerization of M1. The present study will help to unveil the mechanisms of influenza A virus assembly and uncoating process.

  8. Hydrophobic benzyl amines as supports for liquid-phase C-terminal amidated peptide synthesis: application to the preparation of ABT-510.

    Science.gov (United States)

    Matsumoto, Emiko; Fujita, Yuko; Okada, Yohei; Kauppinen, Esko I; Kamiya, Hidehiro; Chiba, Kazuhiro

    2015-09-01

    C-terminal amidation is one of the most common modification of peptides and frequently found in bioactive peptides. However, the C-terminal modification must be creative, because current chemical synthetic techniques of peptides are dominated by the use of C-terminal protecting supports. Therefore, it must be carried out after the removal of such supports, complicating reaction work-up and product isolation. In this context, hydrophobic benzyl amines were successfully added to the growing toolbox of soluble tag-assisted liquid-phase peptide synthesis as supports, leading to the total synthesis of ABT-510 (2). Although an ethyl amide-forming type was used in the present work, different types of hydrophobic benzyl amines could also be simply designed and prepared through versatile reductive aminations in one step. The standard acidic treatment used in the final deprotection step for peptide synthesis gave the desired C-terminal secondary amidated peptide with no epimerization. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.

  9. Identification of chronic heart failure patients with a high 12-month mortality risk using biomarkers including plasma C-terminal pro-endothelin-1.

    Directory of Open Access Journals (Sweden)

    Ewa A Jankowska

    Full Text Available OBJECTIVES: We hypothesised that assessment of plasma C-terminal pro-endothelin-1 (CT-proET-1, a stable endothelin-1 precursor fragment, is of prognostic value in patients with chronic heart failure (CHF, beyond other prognosticators, including N-terminal pro-B-type natriuretic peptide (NT-proBNP. METHODS: We examined 491 patients with systolic CHF (age: 63±11 years, 91% men, New York Heart Association [NYHA] class [I/II/III/IV]: 9%/45%/38%/8%, 69% ischemic etiology. Plasma CT-proET-1 was detected using a chemiluminescence immunoassay. RESULTS: Increasing CT-proET-1 was a predictor of increased cardiovascular mortality at 12-months of follow-up (standardized hazard ratio 1.42, 95% confidence interval [CI] 1.04-1.95, p = 0.03 after adjusting for NT-proBNP, left ventricular ejection fraction (LVEF, age, creatinine, NYHA class. In receiver operating characteristic curve analysis, areas under curve for 12-month follow-up were similar for CT-proET-1 and NT-proBNP (p = 0.40. Both NT-proBNP and CT-proET-1 added prognostic value to a base model that included LVEF, age, creatinine, and NYHA class. Adding CT-proET-1 to the base model had stronger prognostic power (p<0.01 than adding NT-proBNP (p<0.01. Adding CT-proET-1 to NT-proBNP in this model yielded further prognostic information (p = 0.02. CONCLUSIONS: Plasma CT-proET-1 constitutes a novel predictor of increased 12-month cardiovascular mortality in patients with CHF. High CT-proET-1 together with high NT-proBNP enable to identify patients with CHF and particularly unfavourable outcomes.

  10. DNA requirements for interaction of the C-terminal region of Ku80 with the DNA-dependent protein kinase catalytic subunit (DNA-PKcs).

    Science.gov (United States)

    Radhakrishnan, Sarvan Kumar; Lees-Miller, Susan P

    2017-09-01

    Non-homologous end joining (NHEJ) is the major pathway for the repair of ionizing radiation induced DNA double strand breaks (DSBs) in human cells. Critical to NHEJ is the DNA-dependent interaction of the Ku70/80 heterodimer with the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) to form the DNA-PK holoenzyme. However, precisely how Ku recruits DNA-PKcs to DSBs ends to enhance its kinase activity has remained enigmatic, with contradictory findings reported in the literature. Here we address the role of the Ku80 C-terminal region (CTR) in the DNA-dependent interaction of Ku70/80 with DNA-PKcs using purified components and defined DNA structures. Our results show that the Ku80 CTR is required for interaction with DNA-PKcs on short segments of blunt ended 25bp dsDNA or 25bp dsDNA with a 15-base poly dA single stranded (ss) DNA extension, but this requirement is less stringent on longer dsDNA molecules (35bp blunt ended dsDNA) or 25bp duplex DNA with either a 15-base poly dT or poly dC ssDNA extension. Moreover, the DNA-PKcs-Ku complex preferentially forms on 25 bp DNA with a poly-pyrimidine ssDNA extension.Our work clarifies the role of the Ku80 CTR and dsDNA ends on the interaction of DNA-PKcs with Ku and provides key information to guide assembly and biology of NHEJ complexes. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. C-terminal in Sp1-like artificial zinc-finger proteins plays crucial roles in determining their DNA binding affinity.

    Science.gov (United States)

    Zhang, Baozhen; Xiang, Shengyan; Yin, Yanru; Gu, Liankun; Deng, Dajun

    2013-12-01

    It is well known that the C-terminal zinc-finger-3 in transcription factor Sp1 contributes more than the N-terminal zinc-finger-1 in determining Sp1's DNA binding capacity. Sp1-like artificial poly-zinc-finger proteins (ZFPs) are powerful biotechnological tools for gene-specific recognization and manipulation. It is important to understand whether the C-terminal fingers in the Sp1-like artificial ZFPs remain crucial for their DNA binding ability. Recently, a set of p16 promoter-specific seven-ZFPs (7ZFPs) has been constructed to reactivate the expression of methylation-silenced p16. These 7ZFPs contain one N-terminal three-zinc-finger domain of Sp1 (3ZF), two Sp1-like two-zinc-finger domains derived from the Sp1 finger-2 and finger-3 (2ZF) in the middle and C-terminal regions. In the present study, sets of variants for several representative 7ZFPs with the p16-binding affinity were further constructed. This was accomplished through finger replacements and key amino acid mutations in the N-terminal fingers, C-terminal fingers, and linker peptide, respectively. Their p16-binding activity was analysed using gel mobility shift assays. Results showed that the motif replacement or a key amino acid mutation (S > R) at position +2 of the α-helix in the C-terminal 2ZF domain completely abolished their p16-binding affinity. Deletion of three amino acids in a consensus linker (TGEKP > TG) between finger-7 and the 6 × Histidine-tag in the C-terminal also dramatically abolished their binding affinity. In contrast, the replacement of the finger-3 in the N-terminal 3ZF domain did not affect their binding affinity, but decreased their binding stability. Altogether, the present study show that the C-terminal region may play crucial roles in determining the DNA binding affinity of Sp1-like artificial ZFPs.

  12. Contribution of the C-terminal tri-lysine regions of human immunodeficiency virus type 1 integrase for efficient reverse transcription and viral DNA nuclear import

    Directory of Open Access Journals (Sweden)

    Fowke Keith R

    2005-10-01

    Full Text Available Abstract Background In addition to mediating the integration process, HIV-1 integrase (IN has also been implicated in different steps during viral life cycle including reverse transcription and viral DNA nuclear import. Although the karyophilic property of HIV-1 IN has been well demonstrated using a variety of experimental approaches, the definition of domain(s and/or motif(s within the protein that mediate viral DNA nuclear import and its mechanism are still disputed and controversial. In this study, we performed mutagenic analyses to investigate the contribution of different regions in the C-terminal domain of HIV-1 IN to protein nuclear localization as well as their effects on virus infection. Results Our analysis showed that replacing lysine residues in two highly conserved tri-lysine regions, which are located within previously described Region C (235WKGPAKLLWKGEGAVV and sequence Q (211KELQKQITK in the C-terminal domain of HIV-1 IN, impaired protein nuclear accumulation, while mutations for RK263,4 had no significant effect. Analysis of their effects on viral infection in a VSV-G pseudotyped RT/IN trans-complemented HIV-1 single cycle replication system revealed that all three C-terminal mutant viruses (KK215,9AA, KK240,4AE and RK263,4AA exhibited more severe defect of induction of β-Gal positive cells and luciferase activity than an IN class 1 mutant D64E in HeLa-CD4-CCR5-β-Gal cells, and in dividing as well as non-dividing C8166 T cells, suggesting that some viral defects are occurring prior to viral integration. Furthermore, by analyzing viral DNA synthesis and the nucleus-associated viral DNA level, the results clearly showed that, although all three C-terminal mutants inhibited viral reverse transcription to different extents, the KK240,4AE mutant exhibited most profound effect on this step, whereas KK215,9AA significantly impaired viral DNA nuclear import. In addition, our analysis could not detect viral DNA integration in each C-terminal

  13. Three-dimensional structure of a Streptomyces sviceus GNAT acetyltransferase with similarity to the C-terminal domain of the human GH84 O-GlcNAcase

    Energy Technology Data Exchange (ETDEWEB)

    He, Yuan [Northwest University, Xi’an 710069 (China); The University of York, York YO10 5DD (United Kingdom); Roth, Christian; Turkenburg, Johan P.; Davies, Gideon J., E-mail: gideon.davies@york.ac.uk [The University of York, York YO10 5DD (United Kingdom); Northwest University, Xi’an 710069 (China)

    2014-01-01

    The crystal structure of a bacterial acetyltransferase with 27% sequence identity to the C-terminal domain of human O-GlcNAcase has been solved at 1.5 Å resolution. This S. sviceus protein is compared with known GCN5-related acetyltransferases, adding to the diversity observed in this superfamily. The mammalian O-GlcNAc hydrolysing enzyme O-GlcNAcase (OGA) is a multi-domain protein with glycoside hydrolase activity in the N-terminus and with a C-terminal domain that has low sequence similarity to known acetyltransferases, prompting speculation, albeit controversial, that the C-terminal domain may function as a histone acetyltransferase (HAT). There are currently scarce data available regarding the structure and function of this C-terminal region. Here, a bacterial homologue of the human OGA C-terminal domain, an acetyltransferase protein (accession No. ZP-05014886) from Streptomyces sviceus (SsAT), was cloned and its crystal structure was solved to high resolution. The structure reveals a conserved protein core that has considerable structural homology to the acetyl-CoA (AcCoA) binding site of GCN5-related acetyltransferases (GNATs). Calorimetric data further confirm that SsAT is indeed able to bind AcCoA in solution with micromolar affinity. Detailed structural analysis provided insight into the binding of AcCoA. An acceptor-binding cavity was identified, indicating that the physiological substrate of SsAT may be a small molecule. Consistent with recently published work, the SsAT structure further questions a HAT function for the human OGA domain.

  14. An overview of the sequence features of N- and C-terminal segments of the human chemokine receptors.

    Science.gov (United States)

    Raucci, Raffaele; Costantini, Susan; Castello, Giuseppe; Colonna, Giovanni

    2014-12-01

    Chemokine receptors play a crucial role in the cellular signaling enrolling extracellular ligands chemotactic proteins which recruit immune cells. They possess seven trans-membrane helices, an extracellular N-terminal region with three extracellular hydrophilic loops being important for search and recognition of specific ligand(s), and an intracellular C-terminal region with three intracellular loops that couple G-proteins. Although the functional aspects of the terminal segments of the extra-and intra-cellular G proteins are universally identified, the molecular basis on which they rest are still unclear because they are not definable by means of X-rays due to their high mobility and are not easy to study in the membrane. The purpose of this work is to define which physical-chemical properties of the terminal segments of the human chemokine receptors are at the basis of their functional mechanisms. Therefore, we have evaluated their physical-chemical properties in terms of amino acid composition, local flexibility, disorder propensity, net charge distribution and putative sites of post-translational modifications. Our results support the conclusion that all 19 C-terminal and N-terminal segments of human chemokine receptors are very flexible due to the systematic presence of intrinsic disorder. Although, the purpose of this plasticity clearly appears that of controlling and modulating the binding of ligands, we provide evidence that the overlap of linearly charged stretches, intrinsic disorder and post-translational modification sites, consistently found in these motives, is a necessary feature to exert the function. The role of the intrinsic disorder has been discussed considering the structural information coming from intrinsically disordered model compounds which support the view that the chemokine terminals have to be considered as strong polyampholytes or polyelectrolytes where conformational ensembles and structural transitions between them are modulated by

  15. Chemical and thermal unfolding of a global staphylococcal virulence regulator with a flexible C-terminal end.

    Directory of Open Access Journals (Sweden)

    Avisek Mahapa

    Full Text Available SarA, a Staphylococcus aureus-specific dimeric protein, modulates the expression of numerous proteins including various virulence factors. Interestingly, S. aureus synthesizes multiple SarA paralogs seemingly for optimizing the expression of its virulence factors. To understand the domain structure/flexibility and the folding/unfolding mechanism of the SarA protein family, we have studied a recombinant SarA (designated rSarA using various in vitro probes. Limited proteolysis of rSarA and the subsequent analysis of the resulting protein fragments suggested it to be a single-domain protein with a long, flexible C-terminal end. rSarA was unfolded by different mechanisms in the presence of different chemical and physical denaturants. While urea-induced unfolding of rSarA occurred successively via the formation of a dimeric and a monomeric intermediate, GdnCl-induced unfolding of this protein proceeded through the production of two dimeric intermediates. The surface hydrophobicity and the structures of the intermediates were not identical and also differed significantly from those of native rSarA. Of the intermediates, the GdnCl-generated intermediates not only possessed a molten globule-like structure but also exhibited resistance to dissociation during their unfolding. Compared to the native rSarA, the intermediate that was originated at lower GdnCl concentration carried a compact shape, whereas, other intermediates owned a swelled shape. The chemical-induced unfolding, unlike thermal unfolding of rSarA, was completely reversible in nature.

  16. Functional C-TERMINALLY ENCODED PEPTIDE (CEP) plant hormone domains evolved de novo in the plant parasite Rotylenchulus reniformis.

    Science.gov (United States)

    Eves-Van Den Akker, Sebastian; Lilley, Catherine J; Yusup, Hazijah B; Jones, John T; Urwin, Peter E

    2016-10-01

    Sedentary plant-parasitic nematodes (PPNs) induce and maintain an intimate relationship with their host, stimulating cells adjacent to root vascular tissue to re-differentiate into unique and metabolically active 'feeding sites'. The interaction between PPNs and their host is mediated by nematode effectors. We describe the discovery of a large and diverse family of effector genes, encoding C-TERMINALLY ENCODED PEPTIDE (CEP) plant hormone mimics (RrCEPs), in the syncytia-forming plant parasite Rotylenchulus reniformis. The particular attributes of RrCEPs distinguish them from all other CEPs, regardless of origin. Together with the distant phylogenetic relationship of R. reniformis to the only other CEP-encoding nematode genus identified to date (Meloidogyne), this suggests that CEPs probably evolved de novo in R. reniformis. We have characterized the first member of this large gene family (RrCEP1), demonstrating its significant up-regulation during the plant-nematode interaction and expression in the effector-producing pharyngeal gland cell. All internal CEP domains of multi-domain RrCEPs are followed by di-basic residues, suggesting a mechanism for cleavage. A synthetic peptide corresponding to RrCEP1 domain 1 is biologically active and capable of up-regulating plant nitrate transporter (AtNRT2.1) expression, whilst simultaneously reducing primary root elongation. When a non-CEP-containing, syncytia-forming PPN species (Heterodera schachtii) infects Arabidopsis in a CEP-rich environment, a smaller feeding site is produced. We hypothesize that CEPs of R. reniformis represent a two-fold adaptation to sustained biotrophy in this species: (i) increasing host nitrate uptake, whilst (ii) limiting the size of the syncytial feeding site produced. © 2016 The Authors. Molecular Plant Pathology Published by British Society for Plant Pathology and John Wiley & Sons Ltd.

  17. BRCA1 Expression Is Epigenetically Repressed in Sporadic Ovarian Cancer Cells by Overexpression of C-Terminal Binding Protein 2

    Directory of Open Access Journals (Sweden)

    Taymaa May

    2013-06-01

    Full Text Available INTRODUCTION: Ovarian cancer is the leading cause of mortality from gynecological malignancy despite advancements in novel therapeutics. We have recently demonstrated that the transcriptional co-repressor C-terminal binding protein 2 (CtBP2 is overexpressed in epithelial ovarian carcinoma. MATERIALS AND METHODS: Reverse-transcribed cDNA from CtBP2 wild-type and knockdown ovarian cancer cell lines was hybridized to Affymetrix Gene 1.0 ST microarrays, and differentially expressed genes were studied. Immunohistochemical analysis of CtBP2 and BRCA1 staining of ovarian tissues was performed. Chromatin immunoprecipitation (ChIP and luciferase assays were carried out. The effect of the drugs 4-methylthio-2-oxobutyric acid (MTOB and poly(ADP-ribose polymerase (PARP inhibitor Olaparib on CtBP2 wild-type and knockdown cell lines was examined using methylthiazol tetrazolium assays and an xCELLigence System. RESULTS: Eighty-five genes involved in DNA repair, mitotic checkpoint, nucleosome assembly, and the BRCA1 network were differentially regulated by CtBP2 expression. ChIP and luciferase reporter assays using a BRCA1 promoter-regulated luciferase construct indicated that the CtBP2 complex binds the BRCA1 promoter and represses BRCA1 transcription. Immunohistochemistry illustrated a significant inverse CtBP2 and BRCA1 expression in a panel of malignant ovarian tumor tissues. The CtBP2 inhibitor MTOB suppressed ovarian cancer cell survival in a CtBP2-dependent manner. Ovarian cancer cells with CtBP2 knockdown did not display increased sensitivity to the PARP inhibitor Olaparib. CONCLUSION: CtBP2 is an ovarian cancer oncogene that may play a significant role in epigenetically silencing BRCA1 function in sporadic epithelial ovarian cancer. CtBP2-specific inhibitors, such as MTOB, may be effective adjunct therapies in the management of patients with CtBP2-positive ovarian carcinoma.

  18. Functional Characterization of C-terminal Ryanodine Receptor 1 Variants Associated with Central Core Disease or Malignant Hyperthermia.

    Science.gov (United States)

    Parker, Remai; Schiemann, Anja H; Langton, Elaine; Bulger, Terasa; Pollock, Neil; Bjorksten, Andrew; Gillies, Robyn; Hutchinson, David; Roxburgh, Richard; Stowell, Kathryn M

    2017-01-01

    Central core disease and malignant hyperthermia are human disorders of skeletal muscle resulting from aberrant Ca2+ handling. Most malignant hyperthermia and central core disease cases are associated with amino acid changes in the type 1 ryanodine receptor (RyR1), the skeletal muscle Ca2+-release channel. Malignant hyperthermia exhibits a gain-of-function phenotype, and central core disease results from loss of channel function. For a variant to be classified as pathogenic, functional studies must demonstrate a correlation with the pathophysiology of malignant hyperthermia or central core disease. We assessed the pathogenicity of four C-terminal variants of the ryanodine receptor using functional analysis. The variants were identified in families affected by either malignant hyperthermia or central core disease. Four variants were introduced separately into human cDNA encoding the skeletal muscle ryanodine receptor. Following transient expression in HEK-293T cells, functional studies were carried out using calcium release assays in response to an agonist. Two previously characterized variants and wild-type skeletal muscle ryanodine receptor were used as controls. The p.Met4640Ile variant associated with central core disease showed no difference in calcium release compared to wild-type. The p.Val4849Ile variant associated with malignant hyperthermia was more sensitive to agonist than wild-type but did not reach statistical significance and two variants (p.Phe4857Ser and p.Asp4918Asn) associated with central core disease were completely inactive. The p.Val4849Ile variant should be considered a risk factor for malignant hyperthermia, while the p.Phe4857Ser and p.Asp4918Asn variants should be classified as pathogenic for central core disease.

  19. Computational and experimental studies of the interaction between phospho-peptides and the C-terminal domain of BRCA1

    Science.gov (United States)

    Anisimov, Victor M.; Ziemys, Arturas; Kizhake, Smitha; Yuan, Ziyan; Natarajan, Amarnath; Cavasotto, Claudio N.

    2011-11-01

    The C-terminal domain of BRCA1(BRCT) is involved in the DNA repair pathway by recognizing the pSXXF motif in interacting proteins. It has been reported that short peptides containing this motif bind to BRCA1(BRCT) in the micromolar range with high specificity. In this work, the binding of pSXXF peptides has been studied computationally and experimentally in order to characterize their interaction with BRCA1(BRCT). Elucidation of the contacts that drive the protein-ligand interaction is critical for the development of high affinity small-molecule BRCA1 inhibitors. Molecular dynamics (MD) simulations revealed the key role of threonine at the peptide P+2 position in providing structural rigidity to the ligand in the bound state. The mutation at P+1 had minor effects. Peptide extension at the N-terminal position with the naphthyl amino acid exhibited a modest increase in binding affinity, what could be explained by the dispersion interaction of the naphthyl side-chain with a hydrophobic patch. Three in silico end-point methods were considered for the calculation of binding free energy. The Molecular Mechanics Poisson-Boltzmann Surface Area and the Solvated Interaction Energy methods gave reasonable agreement with experimental data, exhibiting a Pearlman predictive index of 0.71 and 0.78, respectively. The MM-quantum mechanics-surface area method yielded improved results, which was characterized by a Pearlman index of 0.78. The correlation coefficients were 0.59, 0.61 and 0.69, respectively. The ability to apply a QM level of theory within an end-point binding free energy protocol may provide a way for a consistent improvement of accuracy in computer-aided drug design.

  20. Topology of the C-terminal tail of HIV-1 gp41: differential exposure of the Kennedy epitope on cell and viral membranes.

    Directory of Open Access Journals (Sweden)

    Jonathan D Steckbeck

    2010-12-01

    Full Text Available The C-terminal tail (CTT of the HIV-1 gp41 envelope (Env protein is increasingly recognized as an important determinant of Env structure and functional properties, including fusogenicity and antigenicity. While the CTT has been commonly referred to as the "intracytoplasmic domain" based on the assumption of an exclusive localization inside the membrane lipid bilayer, early antigenicity studies and recent biochemical analyses have produced a credible case for surface exposure of specific CTT sequences, including the classical "Kennedy epitope" (KE of gp41, leading to an alternative model of gp41 topology with multiple membrane-spanning domains. The current study was designed to test these conflicting models of CTT topology by characterizing the exposure of native CTT sequences and substituted VSV-G epitope tags in cell- and virion-associated Env to reference monoclonal antibodies (MAbs. Surface staining and FACS analysis of intact, Env-expressing cells demonstrated that the KE is accessible to binding by MAbs directed to both an inserted VSV-G epitope tag and the native KE sequence. Importantly, the VSV-G tag was only reactive when inserted into the KE; no reactivity was observed in cells expressing Env with the VSV-G tag inserted into the LLP2 domain. In contrast to cell-surface expressed Env, no binding of KE-directed MAbs was observed to Env on the surface of intact virions using either immune precipitation or surface plasmon resonance spectroscopy. These data indicate apparently distinct CTT topologies for virion- and cell-associated Env species and add to the case for a reconsideration of CTT topology that is more complex than currently envisioned.

  1. Osteopontin and the C-terminal peptide of thrombospondin-4 compete for CD44 binding and have opposite effects on CD133+ cell colony formation

    Directory of Open Access Journals (Sweden)

    Dobocan Monica C

    2009-10-01

    Full Text Available Abstract Background C21, the C-terminal peptide of thrombospondin-4, has growth promoting activity and was discovered as one of several erythropoietin-dependent endothelial proteins. C21 stimulates red cell formation in anemic mice and is a growth factor for CD34+ and CD36+ hematopoietic cells, skin fibroblasts and kidney epithelial cells. ROD1 has been identified as an intracellular mediator. Nothing is known about the existence of putative C21 receptors on plasma membranes of target cells. Findings We analyzed the nature of C21-binding proteins in cell lysates of skin fibroblasts using C21 affinity columns. The membrane receptor CD44 was identified as C21-binding protein by mass spectrometry. We were unable to demonstrate any direct involvement of CD44 on cell growth or the effect of C21 on cell proliferation. A soluble form of CD44 was synthesized in insect cells and purified from culture supernatants with a combination of PVDF filtration in the presence of ammonium sulphate and HPLC. Both osteopontin and hyaluronic acid competitively displaced Biotin-C21 binding to CD44. In a colony-forming assay using primitive CD133+ hematopoietic stem cells from cord blood, osteopontin and C21 had opposite effects and C21 reduced the inhibitory action of osteopontin. Conclusion CD44 is a C21-binding membrane protein. We could not demonstrate an involvement of CD44 in the proliferative action of C21. Nevertheless, based on the antagonism of C21 and osteopontin in hematopoietic precursors, we speculate that C21 could indirectly have a major impact on hematopoietic stem cell proliferation, by hindering osteopontin membrane binding at the level of the bone marrow niche.

  2. Heteronuclear multidimensional NMR and homology modelling studies of the C-terminal nucleotide-binding domain of the human mitochondrial ABC transporter ABCB6

    Energy Technology Data Exchange (ETDEWEB)

    Kurashima-Ito, Kaori [RIKEN, Cellular and Molecular Biology Laboratory (Japan); Ikeya, Teppei [National Institute of Advanced Industrial Science and Technology (AIST), (Japan); Senbongi, Hiroshi [Mitochondrial Diseases Group, MRC Dunn Human NutritionUnit (United Kingdom); Tochio, Hidehito [International Graduate School of Arts and Sciences, Supramolecular Biology, Yokohama City University, Molecular Biophysics Laboratory (Japan); Mikawa, Tsutomu [RIKEN, Cellular and Molecular Biology Laboratory (Japan); Shibata, Takehiko [RIKEN, Shibata Distinguished Senior Scientist Laboratory (Japan); Ito, Yutaka [RIKEN, Cellular and Molecular Biology Laboratory (Japan)], E-mail: ito-yutaka@center.tmu.ac.jp

    2006-05-15

    Human ATP-binding cassette, sub-family B, member 6 (ABCB6) is a mitochondrial ABC transporter, and presumably contributes to iron homeostasis. Aimed at understanding the structural basis for the conformational changes accompanying the substrate-transportation cycle, we have studied the C-terminal nucleotide-binding domain of ABCB6 (ABCB6-C) in both the nucleotide-free and ADP-bound states by heteronuclear multidimensional NMR and homology modelling. A non-linear sampling scheme was utilised for indirectly acquired {sup 13}C and {sup 15}N dimensions of all 3D triple-resonance NMR experiments, in order to overcome the instability and the low solubility of ABCB6-C. The backbone resonances for approximately 25% of non-proline residues, which are mostly distributed around the functionally important loops and in the Helical domain, were not observed for nucleotide-free form of ABCB6-C. From the pH, temperature and magnetic field strength dependencies of the resonance intensities, we concluded that this incompleteness in the assignments is mainly due to the exchange between multiple conformations at an intermediate rate on the NMR timescale. These localised conformational dynamics remained in ADP-bound ABCB6-C except for the loops responsible for adenine base and {alpha}/{beta}-phosphate binding. These results revealed that the localised dynamic cooperativity, which was recently proposed for a prokaryotic ABC MJ1267, also exists in a higher eukaryotic ABC, and is presumably shared by all members of the ABC family. Since the Helical domain is the putative interface to the transmembrane domain, this cooperativity may explain the coupled functions between domains in the substrate-transportation cycle.

  3. The C-terminal residue of phage Vp16 PDF, the smallest peptide deformylase, acts as an offset element locking the active conformation.

    Science.gov (United States)

    Grzela, Renata; Nusbaum, Julien; Fieulaine, Sonia; Lavecchia, Francesco; Bienvenut, Willy V; Dian, Cyril; Meinnel, Thierry; Giglione, Carmela

    2017-09-08

    Prokaryotic proteins must be deformylated before the removal of their first methionine. Peptide deformylase (PDF) is indispensable and guarantees this mechanism. Recent metagenomics studies revealed new idiosyncratic PDF forms as the most abundant family of viral sequences. Little is known regarding these viral PDFs, including the capacity of the corresponding encoded proteins to ensure deformylase activity. We provide here the first evidence that viral PDFs, including the shortest PDF identified to date, Vp16 PDF, display deformylase activity in vivo, despite the absence of the key ribosome-interacting C-terminal region. Moreover, characterization of phage Vp16 PDF underscores unexpected structural and molecular features with the C-terminal Isoleucine residue significantly contributing to deformylase activity both in vitro and in vivo. This residue fully compensates for the absence of the usual long C-domain. Taken together, these data elucidate an unexpected mechanism of enzyme natural evolution and adaptation within viral sequences.

  4. Inactivation of Mechanically Activated Piezo1 Ion Channels Is Determined by the C-Terminal Extracellular Domain and the Inner Pore Helix

    Directory of Open Access Journals (Sweden)

    Jason Wu

    2017-11-01

    Full Text Available Piezo proteins form mechanically activated ion channels that are responsible for our sense of light touch, proprioception, and vascular blood flow. Upon activation by mechanical stimuli, Piezo channels rapidly inactivate in a voltage-dependent manner through an unknown mechanism. Inactivation of Piezo channels is physiologically important, as it modulates overall mechanical sensitivity, gives rise to frequency filtering of repetitive mechanical stimuli, and is itself the target of numerous human disease-related channelopathies that are not well understood mechanistically. Here, we identify the globular C-terminal extracellular domain as a structure that is sufficient to confer the time course of inactivation and a single positively charged lysine residue at the adjacent inner pore helix as being required for its voltage dependence. Our results are consistent with a mechanism for inactivation that is mediated through voltage-dependent conformations of the inner pore helix and allosteric coupling with the C-terminal extracellular domain.

  5. Expression, purification, crystallization and preliminary X-ray analysis of a C-terminal fragment of the Epstein–Barr virus ZEBRA protein

    Energy Technology Data Exchange (ETDEWEB)

    Morand, Patrice [European Molecular Biology Laboratory, Grenoble Outstation, BP 181, 38042 Grenoble CEDEX 9 (France); Laboratoire de Virologie Moléculaire et Structurale, EA 2939, Université Joseph Fourier, Grenoble (France); Budayova-Spano, Monika [European Molecular Biology Laboratory, Grenoble Outstation, BP 181, 38042 Grenoble CEDEX 9 (France); Perrissin, Monique [Laboratoire de Virologie Moléculaire et Structurale, EA 2939, Université Joseph Fourier, Grenoble (France); Müller, Christoph W., E-mail: mueller@embl-grenoble.fr; Petosa, Carlo [European Molecular Biology Laboratory, Grenoble Outstation, BP 181, 38042 Grenoble CEDEX 9 (France)

    2006-03-01

    A C-terminal fragment of the Epstein–Barr virus lytic switch protein ZEBRA has been crystallized in complex with DNA. A C-terminal fragment of the Epstein–Barr virus immediate-early transcription factor ZEBRA has been expressed as a recombinant protein in Escherichia coli and purified to homogeneity. The fragment behaves as a dimer in solution, consistent with the presence of a basic region leucine-zipper (bZIP) domain. Crystals of the fragment in complex with a DNA duplex were grown by the hanging-drop vapour-diffusion technique using polyethylene glycol 4000 and magnesium acetate as crystallization agents. Crystals diffract to better than 2.5 Å resolution using synchrotron radiation (λ = 0.976 Å). Crystals belong to space group C2, with unit-cell parameters a = 94.2, b = 26.5, c = 98.1 Å, β = 103.9°.

  6. The C-terminal domain of human grp94 protects the catalytic subunit of protein kinase CK2 (CK2alpha) against thermal aggregation. Role of disulfide bonds

    DEFF Research Database (Denmark)

    Roher, N; Miró, F; Boldyreff, B

    2001-01-01

    The C-terminal domain (residues 518-803) of the 94 kDa glucose regulated protein (grp94) was expressed in Escherichia coli as a fusion protein with a His6-N-terminal tag (grp94-CT). This truncated form of grp94 formed dimers and oligomers that could be dissociated into monomers by treatment...... ratios of 4 : 1. The presence of dithiothreitol markedly reduced the anti-aggregation effects of grp94-CT on CK2alpha without altering the solubility of the chaperone. It is concluded that the chaperone activity of the C-terminal domain of human grp94 requires the maintenance of its quaternary structure...... (dimers and oligomers), which seems to be stabilised by disulphide bonds....

  7. C-terminally truncated constitutively active androgen receptor variants and their biologic and clinical significance in castration-resistant prostate cancer.

    Science.gov (United States)

    Azoitei, Anca; Merseburger, Axel S; Godau, Beate; Hoda, M Raschid; Schmid, Evi; Cronauer, Marcus V

    2017-02-01

    A mechanism allowing castration resistant prostate cancer cells to escape the effects of conventional anti-hormonal treatments is the synthesis of constitutively active, C-terminally truncated androgen receptor (AR)-variants. Lacking the entire or vast parts of the ligand binding domain, the intended target of traditional endocrine therapies, these AR-variants (termed ARΔLBD) are insensitive to all traditional treatments including second generation compounds like abiraterone, enzalutamide or ARN-509. Although ARΔLBD are predominantly products of alternative splicing, they can also be products of nonsense mutations or proteolytic cleavage. In this review, we will discuss the etiology and function of c-terminally truncated AR-variants and their clinical significance as markers/targets for the treatment of castration resistant prostate cancer. Copyright © 2016 Elsevier Ltd. All rights reserved.

  8. The C-terminal domain of BinA is responsible for Bacillus sphaericus binary toxin BinA-BinB interaction.

    Science.gov (United States)

    Limpanawat, Suweeraya; Promdonkoy, Boonhiang; Boonserm, Panadda

    2009-11-01

    The binary toxin (Bin) from Bacillus sphaericus consists of two polypeptides, BinA (42 kDa) and BinB (51 kDa) that work together to kill susceptible mosquito larvae. To investigate the functional regions of BinA involved in the interaction with BinB, four BinA truncated fragments, from both N- and C- termini, were constructed and expressed in Escherichia coli. Neither individual nor a mixture of fragments of BinA showed larvicidal activity against Culex quinquefasciatus larvae even using a high dose of toxins. Far-Western dot blot analysis showed strong binding of both C-terminal fragments (17 and 28 kDa) to BinB protein. This is the first report to demonstrate that the C-terminal part of BinA plays an important role for the interaction with BinB.

  9. The binding site for regulatory 14-3-3 protein in plant plasma membrane H+-ATPase: Involvement of a region promoting phosphorylation-independent interaction in addition to the phosphorylation-dependent C-terminal end

    DEFF Research Database (Denmark)

    Fuglsang, Anja T; Borch, Jonas; Bych, Katrine

    2003-01-01

    ) in the extreme C-terminal end of the H+-ATPase interacts with the binding cleft of 14-3-3 protein (Wurtele, M., Jelich-Ottmann, C., Wittinghofer, A., and Oecking, C. (2003) EMBO J. 22, 987-994). We report binding of 14-3-3 protein to a nonphosphorylated peptide representing the 34 C-terminal residues...

  10. C-terminal Domain Modulates the Nucleic Acid Chaperone Activity of Human T-cell Leukemia Virus Type 1 Nucleocapsid Protein via an Electrostatic Mechanism*

    OpenAIRE

    Qualley, Dominic F.; Stewart-Maynard, Kristen M.; Wang, Fei; Mitra, Mithun; Gorelick, Robert J.; Rouzina, Ioulia; Williams, Mark C.; Musier-Forsyth, Karin

    2009-01-01

    Retroviral nucleocapsid (NC) proteins are molecular chaperones that facilitate nucleic acid (NA) remodeling events critical in viral replication processes such as reverse transcription. Surprisingly, the NC protein from human T-cell leukemia virus type 1 (HTLV-1) is an extremely poor NA chaperone. Using bulk and single molecule methods, we find that removal of the anionic C-terminal domain (CTD) of HTLV-1 NC results in a protein with chaperone properties comparable with that of other retrovir...

  11. Generation of the beta-amyloid peptide and the amyloid precursor protein C-terminal fragment gamma are potentiated by FE65L1.

    Science.gov (United States)

    Chang, Yang; Tesco, Giuseppina; Jeong, William J; Lindsley, Loren; Eckman, Elizabeth A; Eckman, Christopher B; Tanzi, Rudolph E; Guénette, Suzanne Y

    2003-12-19

    Members of the FE65 family of adaptor proteins, FE65, FE65L1, and FE65L2, bind the C-terminal region of the amyloid precursor protein (APP). Overexpression of FE65 and FE65L1 was previously reported to increase the levels of alpha-secretase-derived APP (APPs alpha). Increased beta-amyloid (A beta) generation was also observed in cells showing the FE65-dependent increase in APPs alpha. To understand the mechanism for the observed increase in both A beta and APPs alpha given that alpha-secretase cleavage of a single APP molecule precludes A beta generation, we examined the effects of FE65L1 overexpression on APP C-terminal fragments (APP CTFs). Our data show that FE65L1 potentiates gamma-secretase processing of APP CTFs, including the amyloidogenic CTF C99, accounting for the ability of FE65L1 to increase generation of APP C-terminal domain and A beta 40. The FE65L1 modulation of these processing events requires binding of FE65L1 to APP and APP CTFs and is not because of a direct effect on gamma-secretase activity, because Notch intracellular domain generation is not altered by FE65L1. Furthermore, enhanced APP CTF processing can be detected in early endosome vesicles but not in endoplasmic reticulum or Golgi membranes, suggesting that the effects of FE65L1 occur at or near the plasma membrane. Finally, although FE65L1 increases APP C-terminal domain production, it does not mediate the APP-dependent transcriptional activation observed with FE65.

  12. The C-terminal random coil region tunes the Ca²⁺-binding affinity of S100A4 through conformational activation.

    Directory of Open Access Journals (Sweden)

    Annette Duelli

    Full Text Available S100A4 interacts with many binding partners upon Ca2+ activation and is strongly associated with increased metastasis formation. In order to understand the role of the C-terminal random coil for the protein function we examined how small angle X-ray scattering of the wild-type S100A4 and its C-terminal deletion mutant (residues 1-88, Δ13 changes upon Ca2+ binding. We found that the scattering intensity of wild-type S100A4 changes substantially in the 0.15-0.25 Å-1 q-range whereas a similar change is not visible in the C-terminus deleted mutant. Ensemble optimization SAXS modeling indicates that the entire C-terminus is extended when Ca2+ is bound. Pulsed field gradient NMR measurements provide further support as the hydrodynamic radius in the wild-type protein increases upon Ca2+ binding while the radius of Δ13 mutant does not change. Molecular dynamics simulations provide a rational explanation of the structural transition: the positively charged C-terminal residues associate with the negatively charged residues of the Ca2+-free EF-hands and these interactions loosen up considerably upon Ca2+-binding. As a consequence the Δ13 mutant has increased Ca2+ affinity and is constantly loaded at Ca2+ concentration ranges typically present in cells. The activation of the entire C-terminal random coil may play a role in mediating interaction with selected partner proteins of S100A4.

  13. N- and C-terminal truncations of a GH10 xylanase significantly increase its activity and thermostability but decrease its SDS resistance.

    Science.gov (United States)

    Zheng, Fei; Huang, Jingxuan; Liu, Xingchen; Hu, Hang; Long, Liangkun; Chen, Kaixiang; Ding, Shaojun

    2016-04-01

    XynII from Volvariella volvacea has high sodium dodecyl sulfate (SDS) resistance, with the potential for industrial applications under harsh conditions. It consists of a single glycoside hydrolase family 10 (GH10) catalytic domain but contains an additional unique 10 and 4 amino acid residues at the N- and C-terminus, respectively. In this study, five XynII derivatives with N- and/or C-terminus deletions were constructed to determine the effects of these regions on enzyme activity, substrate specificity, thermostability, and SDS resistance. Our results revealed that N- and/or C-terminal truncations significantly increased enzyme activity and thermostability, but reduced SDS resistance. Specifically, the XynIIΔNC4 mutant had 2.53-fold more catalytic efficiency (k cat/K m) towards beechwood xylan than wild-type and 3.0-fold more thermostability (t 1/2 [55°C]). XynIIΔNC4 displayed 3.33-, 4.38-, 1.37-, and 1.98-fold more activity against xylotriose, xylotetraose, xylopentaose, and xylohexaose, respectively, than XynII did. However, its half-life (t 1/2) in 4 % SDS was only 1.72 h, while that of XynII was 4.65 h. Circular dichroism analysis revealed that deletion of N- and C-terminal segments caused minor changes in secondary structure. Our observations suggest that the extra N- and C-terminal segments in wild-type XynII evolved to strengthen the interaction between these regions of the protein, making the local structure more rigid and reducing structural flexibility. In this way, N- and C-terminal truncations increased the thermostability and activity of XynII on different xylans and linear xylooligosaccharides, but reduced its resistance to SDS.

  14. Structural investigation of a C-terminal EphA2 receptor mutant: Does mutation affect the structure and interaction properties of the Sam domain?

    Science.gov (United States)

    Mercurio, Flavia A; Costantini, Susan; Di Natale, Concetta; Pirone, Luciano; Guariniello, Stefano; Scognamiglio, Pasqualina L; Marasco, Daniela; Pedone, Emilia M; Leone, Marilisa

    2017-09-01

    Ephrin A2 receptor (EphA2) plays a key role in cancer, it is up-regulated in several types of tumors and the process of ligand-induced receptor endocytosis, followed by degradation, is considered as a potential path to diminish tumor malignancy. Protein modulators of this mechanism are recruited at the cytosolic Sterile alpha motif (Sam) domain of EphA2 (EphA2-Sam) through heterotypic Sam-Sam associations. These interactions engage the C-terminal helix of EphA2 and close loop regions (the so called End Helix side). In addition, several studies report on destabilizing mutations in EphA2 related to cataract formation and located in/or close to the Sam domain. Herein, we analyzed from a structural point of view, one of these mutants characterized by the insertion of a novel 39 residue long polypeptide at the C-terminus of EphA2-Sam. A 3D structural model was built by computational methods and revealed partial disorder in the acquired C-terminal tail and a few residues participating in an α-helix and two short β-strands. We investigated by CD and NMR studies the conformational properties in solution of two peptides encompassing the whole C-terminal tail and its predicted helical region, respectively. NMR binding experiments demonstrated that these peptides do not interact relevantly with either EphA2-Sam or its interactor Ship2-Sam. Molecular dynamics (MD) simulations further indicated that the EphA2 mutant could be represented only through a conformational ensemble and that the C-terminal tail should not largely wrap the EphA2-Sam End-Helix interface and affect binding to other Sam domains. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. Crystal Structure of the C-terminal Region of Streptococcus mutans Antigen I/II and Characterization of Salivary Agglutinin Adherence Domains

    Energy Technology Data Exchange (ETDEWEB)

    Larson, Matthew R.; Rajashankar, Kanagalaghatta R.; Crowley, Paula J.; Kelly, Charles; Mitchell, Tim J.; Brady, L. Jeannine; Deivanayagam, Champion (King); (Cornell); (UAB); (Glasgow); (Florida)

    2012-05-29

    The Streptococcus mutans antigen I/II (AgI/II) is a cell surface-localized protein that adheres to salivary components and extracellular matrix molecules. Here we report the 2.5 {angstrom} resolution crystal structure of the complete C-terminal region of AgI/II. The C-terminal region is comprised of three major domains: C{sub 1}, C{sub 2}, and C{sub 3}. Each domain adopts a DE-variant IgG fold, with two {beta}-sheets whose A and F strands are linked through an intramolecular isopeptide bond. The adherence of the C-terminal AgI/II fragments to the putative tooth surface receptor salivary agglutinin (SAG), as monitored by surface plasmon resonance, indicated that the minimal region of binding was contained within the first and second DE-variant-IgG domains (C{sub 1} and C{sub 2}) of the C terminus. The minimal C-terminal region that could inhibit S. mutans adherence to SAG was also confirmed to be within the C{sub 1} and C{sub 2} domains. Competition experiments demonstrated that the C- and N-terminal regions of AgI/II adhere to distinct sites on SAG. A cleft formed at the intersection between these C{sub 1} and C{sub 2} domains bound glucose molecules from the cryo-protectant solution, revealing a putative binding site for its highly glycosylated receptor SAG. Finally, electron microscopy images confirmed the elongated structure of AgI/II and enabled building a composite tertiary model that encompasses its two distinct binding regions.

  16. The C-terminal polyproline-containing region of ELMO contributes to an increase in the life-time of the ELMO-DOCK complex.

    Science.gov (United States)

    Sévajol, Marion; Reiser, Jean-Baptiste; Chouquet, Anne; Pérard, Julien; Ayala, Isabel; Gans, Pierre; Kleman, Jean-Philippe; Housset, Dominique

    2012-03-01

    The eukaryotic Engulfment and CellMotility (ELMO) proteins form an evolutionary conserved family of key regulators which play a central role in Rho-dependent biological processes such as engulfment and cell motility/migration. ELMO proteins interact with a subset of Downstream of Crk (DOCK) family members, a new type of guanine exchange factors (GEF) for Rac and cdc42 GTPases. The physiological function of DOCK is to facilitate actin remodeling, a process which occurs only in presence of ELMO. Several studies have determined that the last 200 C-terminal residues of ELMO1 and the first 180 N-terminal residues of DOCK180 are responsible for the ELMO-DOCK interaction. However, the precise role of the different domains and motifs identified in these regions has remained elusive. Divergent functional, biochemical and structural data have been reported regarding the contribution of the C-terminal end of ELMO, comprising its polyproline motif, and of the DOCK SH3 domain. In the present study, we have investigated the contribution of the C-terminal end of ELMO1 to the interaction between ELMO1 and the SH3 domain of DOCK180 using nuclear magnetic resonance spectroscopy and surface plasmon resonance. Our data presented here demonstrate the ability of the SH3 domain of DOCK180 to interact with ELMO1, regardless of the presence of the polyproline-containing C-terminal end. However, the presence of the polyproline region leads to a significant increase in the half-life of the ELMO1-DOCK180 complex, along with a moderate increase on the affinity. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

  17. Functional analysis reveals the possible role of the C-terminal sequences and PI motif in the function of lily (Lilium longiflorum) PISTILLATA (PI) orthologues

    Science.gov (United States)

    Chen, Ming-Kun; Hsieh, Wen-Ping; Yang, Chang-Hsien

    2012-01-01

    Two lily (Lilium longiflorum) PISTILLATA (PI) genes, Lily MADS Box Gene 8 and 9 (LMADS8/9), were characterized. LMADS9 lacked 29 C-terminal amino acids including the PI motif that was present in LMADS8. Both LMADS8/9 mRNAs were prevalent in the first and second whorl tepals during all stages of development and were expressed in the stamen only in young flower buds. LMADS8/9 could both form homodimers, but the ability of LMADS8 homodimers to bind to CArG1 was relatively stronger than that of LMADS9 homodimers. 35S:LMADS8 completely, and 35S:LMADS9 only partially, rescued the second whorl petal formation and partially converted the first whorl sepal into a petal-like structure in Arabidopsis pi-1 mutants. Ectopic expression of LMADS8-C (with deletion of the 29 amino acids of the C-terminal sequence) or LMADS8-PI (with only the PI motif deleted) only partially rescued petal formation in pi mutants, which was similar to what was observed in 35S:LMADS9/pi plants. In contrast, 35:LMADS9+L8C (with the addition of the 29 amino acids of the LMADS8 C-terminal sequence) or 35S:LMADS9+L8PI (with the addition of the LMADS8 PI motif) demonstrated an increased ability to rescue petal formation in pi mutants, which was similar to what was observed in 35S:LMADS8/pi plants. Furthermore, ectopic expression of LMADS8-M (with the MADS domain truncated) generated more severe dominant negative phenotypes than those seen in 35S:LMADS9-M flowers. These results revealed that the 29 amino acids including the PI motif in the C-terminal region of the lily PI orthologue are valuable for its function in regulating perianth organ formation. PMID:22068145

  18. Binding of a truncated form of lecithin:retinol acyltransferase and its N- and C-terminal peptides to lipid monolayers.

    Science.gov (United States)

    Bussières, Sylvain; Cantin, Line; Desbat, Bernard; Salesse, Christian

    2012-02-21

    Lecithin:retinol acyltransferase (LRAT) is a 230 amino acid membrane-associated protein which catalyzes the esterification of all-trans-retinol into all-trans-retinyl ester. A truncated form of LRAT (tLRAT), which contains the residues required for catalysis but which is lacking the N- and C-terminal hydrophobic segments, was produced to study its membrane binding properties. Measurements of the maximum insertion pressure of tLRAT, which is higher than the estimated lateral pressure of membranes, and the positive synergy factor a argue in favor of a strong binding of tLRAT to phospholipid monolayers. Moreover, the binding, secondary structure and orientation of the peptides corresponding to its N- and C-terminal hydrophobic segments of LRAT have been studied by circular dichroism and polarization-modulation infrared reflection absorption spectroscopy in monolayers. The results show that these peptides spontaneously bind to lipid monolayers and adopt an α-helical secondary structure. On the basis of these data, a new membrane topology model of LRAT is proposed where its N- and C-terminal segments allow to anchor this protein to the lipid bilayer.

  19. Roles of C-Terminal Region of Yeast and Human Rad52 in Rad51-Nucleoprotein Filament Formation and ssDNA Annealing.

    Directory of Open Access Journals (Sweden)

    Nilesh V Khade

    Full Text Available Yeast Rad52 (yRad52 has two important functions at homologous DNA recombination (HR; annealing complementary single-strand DNA (ssDNA molecules and recruiting Rad51 recombinase onto ssDNA (recombination mediator activity. Its human homolog (hRAD52 has a lesser role in HR, and apparently lacks mediator activity. Here we show that yRad52 can load human Rad51 (hRAD51 onto ssDNA complexed with yeast RPA in vitro. This is biochemically equivalent to mediator activity because it depends on the C-terminal Rad51-binding region of yRad52 and on functional Rad52-RPA interaction. It has been reported that the N-terminal two thirds of both yRad52 and hRAD52 is essential for binding to and annealing ssDNA. Although a second DNA binding region has been found in the C-terminal region of yRad52, its role in ssDNA annealing is not clear. In this paper, we also show that the C-terminal region of yRad52, but not of hRAD52, is involved in ssDNA annealing. This suggests that the second DNA binding site is required for the efficient ssDNA annealing by yRad52. We propose an updated model of Rad52-mediated ssDNA annealing.

  20. iPreny-PseAAC: Identify C-terminal Cysteine Prenylation Sites in Proteins by Incorporating Two Tiers of Sequence Couplings into PseAAC.

    Science.gov (United States)

    Xu, Yan; Wang, Zu; Li, Chunhui; Chou, Kuo-Chen

    2017-01-01

    Occurring at the cysteine residue in the C-terminal of a protein, prenylation is a special kind of post-translational modification (PTM), which may play a key role for statin in altering immune function. Therefore, knowledge of the prenylation sites in proteins is important for drug development as well as for in-depth understanding the biological process concerned. Given a query protein whose C-terminal contains some cysteine residues, which one can be of prenylation or none of them can be prenylated? To address this problem, we have developed a new predictor, called "iPreny-PseAAC", by incorporating two tiers of sequence pair coupling effects into the general form of PseAAC (pseudo amino acid composition). It has been observed by four different cross-validation approaches that all the important indexes in reflecting its prediction quality are quite high and fully consistent to each other. It is anticipated that the iPreny-PseAAC predictor holds very high potential to become a useful high throughput tool in identifying protein C-terminal cysteine prenylation sites and the other relevant areas. To maximize the convenience for most experimental biologists, the webserver for the new predictor has been established at http://app.aporc.org/iPreny-PseAAC/, by which users can easily get their desired results without needing to go through the mathematical details involved in this paper. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  1. The AvrM effector from flax rust has a structured C-terminal domain and interacts directly with the M resistance protein.

    Science.gov (United States)

    Catanzariti, Ann-Maree; Dodds, Peter N; Ve, Thomas; Kobe, Bostjan; Ellis, Jeffrey G; Staskawicz, Brian J

    2010-01-01

    In plant immunity, recognition of pathogen effectors by plant resistance proteins leads to the activation of plant defenses and a localized cell death response. The AvrM effector from flax rust is a small secreted protein that is recognized by the M resistance protein in flax. Here, we investigate the mechanism of M-AvrM recognition and show that these two proteins directly interact in a yeast two-hybrid assay, and that this interaction correlates with the recognition specificity observed for each of the different AvrM variants. We further characterize this interaction by demonstrating that the C-terminal domain of AvrM is required for M-dependent cell death, and show that this domain also interacts with the M protein in yeast. We investigate the role of C-terminal differences among the different AvrM proteins for their involvement in this interaction and establish that M recognition is hindered by an additional 34 amino acids present at the C terminus of several AvrM variants. Structural characterization of recombinant AvrM-A protein revealed a globular C-terminal domain that dimerizes.

  2. Amyloidogenic Properties of a D/N Mutated 12 Amino Acid Fragment of the C-Terminal Domain of the Cholesteryl-Ester Transfer Protein (CETP

    Directory of Open Access Journals (Sweden)

    Victor García-González

    2011-03-01

    Full Text Available The cholesteryl-ester transfer protein (CETP facilitates the transfer of cholesterol esters and triglycerides between lipoproteins in plasma where the critical site for its function is situated in the C-terminal domain. Our group has previously shown that this domain presents conformational changes in a non-lipid environment when the mutation D470N is introduced. Using a series of peptides derived from this C-terminal domain, the present study shows that these changes favor the induction of a secondary β-structure as characterized by spectroscopic analysis and fluorescence techniques. From this type of secondary structure, the formation of peptide aggregates and fibrillar structures with amyloid characteristics induced cytotoxicity in microglial cells in culture. These supramolecular structures promote cell cytotoxicity through the formation of reactive oxygen species (ROS and change the balance of a series of proteins that control the process of endocytosis, similar to that observed when β-amyloid fibrils are employed. Therefore, a fine balance between the highly dynamic secondary structure of the C-terminal domain of CETP, the net charge, and the physicochemical characteristics of the surrounding microenvironment define the type of secondary structure acquired. Changes in this balance might favor misfolding in this region, which would alter the lipid transfer capacity conducted by CETP, favoring its propensity to substitute its physiological function.

  3. Inappropriate Expression of an NLP Effector in Colletotrichum orbiculare Impairs Infection on Cucurbitaceae Cultivars via Plant Recognition of the C-Terminal Region.

    Science.gov (United States)

    Azmi, Nur Sabrina Ahmad; Singkaravanit-Ogawa, Suthitar; Ikeda, Kyoko; Kitakura, Saeko; Inoue, Yoshihiro; Narusaka, Yoshihiro; Shirasu, Ken; Kaido, Masanori; Mise, Kazuyuki; Takano, Yoshitaka

    2018-01-01

    The hemibiotrophic pathogen Colletotrichum orbiculare preferentially expresses a necrosis and ethylene-inducing peptide 1 (Nep1)-like protein named NLP1 during the switch to necrotrophy. Here, we report that the constitutive expression of NLP1 in C. orbiculare blocks pathogen infection in multiple Cucurbitaceae cultivars via their enhanced defense responses. NLP1 has a cytotoxic activity that induces cell death in Nicotiana benthamiana. However, C. orbiculare transgenic lines constitutively expressing a mutant NLP1 lacking the cytotoxic activity still failed to infect cucumber, indicating no clear relationship between cytotoxic activity and the NLP1-dependent enhanced defense. NLP1 also possesses the microbe-associated molecular pattern (MAMP) sequence called nlp24, recognized by Arabidopsis thaliana at its central region, similar to NLPs of other pathogens. Surprisingly, inappropriate expression of a mutant NLP1 lacking the MAMP signature is also effective for blocking pathogen infection, uncoupling the infection block from the corresponding MAMP. Notably, the deletion analyses of NLP1 suggested that the C-terminal region of NLP1 is critical to enhance defense in cucumber. The expression of mCherry fused with the C-terminal 32 amino acids of NLP1 was enough to trigger the defense of cucurbits, revealing that the C-terminal region of the NLP1 protein is recognized by cucurbits and, then, terminates C. orbiculare infection.

  4. Structure of the C-terminal heme-binding domain of THAP domain containing protein 4 from Homo sapiens

    Energy Technology Data Exchange (ETDEWEB)

    Bianchetti, Christopher M.; Bingman, Craig A.; Phillips, Jr., George N. (UW)

    2012-03-15

    The thanatos (the Greek god of death)-associated protein (THAP) domain is a sequence-specific DNA-binding domain that contains a C2-CH (Cys-Xaa{sub 2-4}-Cys-Xaa{sub 35-50}-Cys-Xaa{sub 2}-His) zinc finger that is similar to the DNA domain of the P element transposase from Drosophila. THAP-containing proteins have been observed in the proteome of humans, pigs, cows, chickens, zebrafish, Drosophila, C. elegans, and Xenopus. To date, there are no known THAP domain proteins in plants, yeast, or bacteria. There are 12 identified human THAP domain-containing proteins (THAP0-11). In all human THAP protein, the THAP domain is located at the N-terminus and is {approx}90 residues in length. Although all of the human THAP-containing proteins have a homologous N-terminus, there is extensive variation in both the predicted structure and length of the remaining protein. Even though the exact function of these THAP proteins is not well defined, there is evidence that they play a role in cell proliferation, apoptosis, cell cycle modulation, chromatin modification, and transcriptional regulation. THAP-containing proteins have also been implicated in a number of human disease states including heart disease, neurological defects, and several types of cancers. Human THAP4 is a 577-residue protein of unknown function that is proposed to bind DNA in a sequence-specific manner similar to THAP1 and has been found to be upregulated in response to heat shock. THAP4 is expressed in a relatively uniform manner in a broad range of tissues and appears to be upregulated in lymphoma cells and highly expressed in heart cells. The C-terminal domain of THAP4 (residues 415-577), designated here as cTHAP4, is evolutionarily conserved and is observed in all known THAP4 orthologs. Several single-domain proteins lacking a THAP domain are found in plants and bacteria and show significant levels of homology to cTHAP4. It appears that cTHAP4 belongs to a large class of proteins that have yet to be fully

  5. Analysis of Tb{sup 3+}- and melittin-binding with the C-terminal domain of centrin in Euplotes octocarinatus

    Energy Technology Data Exchange (ETDEWEB)

    Zhao Yaqin; Diao Xiuling; Yan Jun; Feng Yanan [Key Laboratory of Chemical Biology and Microcular Engineering of Ministry of Education, Shanxi University, Taiyuan 030006 (China); Wang Zhijun [Chemical Department, Changzhi University, Changzhi 046011 (China); Liang Aihua, E-mail: aliang@sxu.edu.cn [Key Laboratory of Chemical Biology and Microcular Engineering of Ministry of Education, Shanxi University, Taiyuan 030006 (China); Yang Binsheng, E-mail: yangbs@sxu.edu.cn [Key Laboratory of Chemical Biology and Microcular Engineering of Ministry of Education, Shanxi University, Taiyuan 030006 (China)

    2012-04-15

    Centrin is a low molecular mass (20 KDa) protein that belongs to the EF-hand superfamily. In this work, the interaction between the Tb{sup 3+}-saturated C-terminal domain of Euplotes octocarinatus centrin (Tb{sub 2}-C-EoCen) and 2-p-toluidinylnaphthalene-6-sulfonate (TNS) was investigated using difference UV-vis spectra and the fluorescence spectra methods. In 100 mM N-2-hydroxy-ethylpiperazine-N-2-ethanesulfonic acid (Hepes) at pH 7.4, with the addition of Tb{sub 2}-C-EoCen, four new peaks were observed at 265 nm, 278 nm, 317 nm and 360 nm by absorptivity compared with blank solution of TNS. At the same time, the reaction could be measured by fluorescence spectra. The fluorescence emission of TNS was shifted from 480 nm to 445 nm in the presence of Tb{sub 2}-C-EoCen. Meanwhile, its fluorescence intensity was increased markedly. The 1:1 stoichiometric ratio of C-EoCen to TNS was confirmed by fluorescence titration curves. The conditional binding constants of TNS with C-EoCen and Tb{sub 2}-C-EoCen were calculated to be log K{sub (C-EoCen-TNS)}=5.32{+-}0.04 M{sup -1} and log K{sub (Tb2-C-EoCen-TNS)}=5.58{+-}0.12 M{sup -1}, respectively. In addition, the protein of Tb{sub 2}-C-EoCen binding with melittin was also studied. Based on the fluorescence titration curves, the 1:1 stoichiometric ratio of Tb{sub 2}-C-EoCen to melittin was confirmed. And the conditional binding constant of C-EoCen with melittin was calculated to be log Ka Prime =6.79{+-}0.17 M{sup -1}. - Highlights: Black-Right-Pointing-Pointer Tb{sup 3+} induced conformational changes of protein C-EoCen from closed state to open state. Black-Right-Pointing-Pointer Conformational changes resulted in the exposure of hydrophobic surfaces on C-EoCen. Black-Right-Pointing-Pointer Tb{sub 2}-C-EoCen may bind with target peptide melittin.

  6. Anti-migratory effect of vinflunine in endothelial and glioblastoma cells is associated with changes in EB1 C-terminal detyrosinated/tyrosinated status.

    Directory of Open Access Journals (Sweden)

    Amandine Rovini

    Full Text Available We previously showed that vinflunine, a microtubule-targeting drug of the Vinca-alkaloid family exerted its anti-angiogenic/anti-migratory activities through an increase in microtubule dynamics and an inhibition of microtubule targeting to adhesion sites. Such effect was associated with a reduction of EB1 comet length at microtubule (+ ends. In this work we first showed that the pro-angiogenic vascular endothelial growth factor VEGF suppressed microtubule dynamics in living Human Umbilical Vein Endothelial Cells (HUVECs, increased EB1 comet length by 40%, and induced EB1 to bind all along the microtubules, without modifying its expression level. Such microtubule (+ end stabilization occurred close to the plasma membrane in the vicinity of focal adhesion as shown by TIRF microscopy experiments. Vinflunine completely abolished the effect of VEGF on EB1 comets. Interestingly, we found a correlation between the reduction of EB1 comet length by vinflunine and the inhibition of cell migration. By using 2D gel electrophoresis we demonstrated for the first time that EB1 underwent several post-translational modifications in endothelial and tumor cells. Particularly, the C-terminal EEY sequence was poorly detectable in control and VEGF-treated HUVECs suggesting the existence of a non-tyrosinated form of EB1. By using specific antibodies that specifically recognized and discriminated the native tyrosinated form of EB1 and a putative C-terminal detyrosinated form, we showed that a detyrosinated form of EB1 exists in HUVECs and tumor cells. Interestingly, vinflunine decreased the level of the detyrosinated form and increased the native tyrosinated form of EB1. Using 3-L-Nitrotyrosine incorporation experiments, we concluded that the EB1 C-terminal modifications result from a detyrosination/retyrosination cycle as described for tubulin. Altogether, our results show that vinflunine inhibits endothelial cell migration through an alteration of EB1 comet length

  7. Interaction between the C-terminal region of human myelin basic protein and calmodulin: analysis of complex formation and solution structure

    Directory of Open Access Journals (Sweden)

    Hayashi Nobuhiro

    2008-02-01

    Full Text Available Abstract Background The myelin sheath is a multilamellar membrane structure wrapped around the axon, enabling the saltatory conduction of nerve impulses in vertebrates. Myelin basic protein, one of the most abundant myelin-specific proteins, is an intrinsically disordered protein that has been shown to bind calmodulin. In this study, we focus on a 19-mer synthetic peptide from the predicted calmodulin-binding segment near the C-terminus of human myelin basic protein. Results The interaction of native human myelin basic protein with calmodulin was confirmed by affinity chromatography. The binding of the myelin basic protein peptide to calmodulin was tested with isothermal titration calorimetry (ITC in different temperatures, and Kd was observed to be in the low μM range, as previously observed for full-length myelin basic protein. Surface plasmon resonance showed that the peptide bound to calmodulin, and binding was accompanied by a conformational change; furthermore, gel filtration chromatography indicated a decrease in the hydrodynamic radius of calmodulin in the presence of the peptide. NMR spectroscopy was used to map the binding area to reside mainly within the hydrophobic pocket of the C-terminal lobe of calmodulin. The solution structure obtained by small-angle X-ray scattering indicates binding of the myelin basic protein peptide into the interlobal groove of calmodulin, while calmodulin remains in an extended conformation. Conclusion Taken together, our results give a detailed structural insight into the interaction of calmodulin with a C-terminal segment of a major myelin protein, the myelin basic protein. The used 19-mer peptide interacts mainly with the C-terminal lobe of calmodulin, and a conformational change accompanies binding, suggesting a novel mode of calmodulin-target protein interaction. Calmodulin does not collapse and wrap around the peptide tightly; instead, it remains in an extended conformation in the solution structure

  8. Display of cell surface sites for fibronectin assembly is modulated by cell adherence to (1)F3 and C-terminal modules of fibronectin.

    Science.gov (United States)

    Xu, Jielin; Bae, Eunnyung; Zhang, Qinghong; Annis, Douglas S; Erickson, Harold P; Mosher, Deane F

    2009-01-01

    Fibronectin-null cells assemble soluble fibronectin shortly after adherence to a substrate coated with intact fibronectin but not when adherent to the cell-binding domain of fibronectin (modules (7)F3-(10)F3). Interactions of adherent cells with regions of adsorbed fibronectin other than modules (7)F3-(10)F3, therefore, are required for early display of the cell surface sites that initiate and direct fibronectin assembly. To identify these regions, coatings of proteolytically derived or recombinant pieces of fibronectin containing modules in addition to (7)F3-(10)F3 were tested for effects on fibronectin assembly by adherent fibronectin-null fibroblasts. Pieces as large as one comprising modules (2)F3-(14)F3, which include the heparin-binding and cell adhesion domains, were not effective in supporting fibronectin assembly. Addition of module (1)F3 or the C-terminal modules to modules (2)F3-(14)F3 resulted in some activity, and addition of both (1)F3 and the C-terminal modules resulted in a construct, (1)F3-C, that best mimicked the activity of a coating of intact fibronectin. Constructs (1)F3-C V0, (1)F3-C V64, and (1)F3-C Delta(V(15)F3(10)F1) were all able to support fibronectin assembly, suggesting that (1)F3 through (11)F1 and/or (12)F1 were important for activity. Coatings in which the active parts of (1)F3-C were present in different proteins were much less active than intact (1)F3-C. These results suggest that (1)F3 acts together with C-terminal modules to induce display of fibronectin assembly sites on adherent cells.

  9. A functional C-terminal TRAF3-binding site in MAVS participates in positive and negative regulation of the IFN antiviral response.

    Science.gov (United States)

    Paz, Suzanne; Vilasco, Myriam; Werden, Steven J; Arguello, Meztli; Joseph-Pillai, Deshanthe; Zhao, Tiejun; Nguyen, Thi Lien-Anh; Sun, Qiang; Meurs, Eliane F; Lin, Rongtuan; Hiscott, John

    2011-06-01

    Recognition of viral RNA structures by the cytosolic sensor retinoic acid-inducible gene-I (RIG-I) results in the activation of signaling cascades that culminate with the generation of the type I interferon (IFN) antiviral response. Onset of antiviral and inflammatory responses to viral pathogens necessitates the regulated spatiotemporal recruitment of signaling adapters, kinases and transcriptional proteins to the mitochondrial antiviral signaling protein (MAVS). We previously demonstrated that the serine/threonine kinase IKKε is recruited to the C-terminal region of MAVS following Sendai or vesicular stomatitis virus (VSV) infection, mediated by Lys63-linked polyubiquitination of MAVS at Lys500, resulting in inhibition of downstream IFN signaling (Paz et al, Mol Cell Biol, 2009). In this study, we demonstrate that C-terminus of MAVS harbors a novel TRAF3-binding site in the aa450-468 region of MAVS. A consensus TRAF-interacting motif (TIM), 455-PEENEY-460, within this site is required for TRAF3 binding and activation of IFN antiviral response genes, whereas mutation of the TIM eliminates TRAF3 binding and the downstream IFN response. Reconstitution of MAVS(-/-) mouse embryo fibroblasts with a construct expressing a TIM-mutated version of MAVS failed to restore the antiviral response or block VSV replication, whereas wild-type MAVS reconstituted antiviral inhibition of VSV replication. Furthermore, recruitment of IKKε to an adjacent C-terminal site (aa 468-540) in MAVS via Lys500 ubiquitination decreased TRAF3 binding and protein stability, thus contributing to IKKε-mediated shutdown of the IFN response. This study demonstrates that MAVS harbors a functional C-terminal TRAF3-binding site that participates in positive and negative regulation of the IFN antiviral response.

  10. A protein kinase binds the C-terminal domain of the readthrough protein of Turnip yellows virus and regulates virus accumulation

    Energy Technology Data Exchange (ETDEWEB)

    Rodriguez-Medina, Caren; Boissinot, Sylvaine [UMR 1131 SVQV INRA-UDS, 28 rue de Herrlisheim, 68021 Colmar (France); Chapuis, Sophie [Institut de Biologie Moléculaire des Plantes, Laboratoire propre du CNRS conventionné avec l’Université de Strasbourg, 12 rue du Général Zimmer, 67084 Strasbourg (France); Gereige, Dalya; Rastegar, Maryam; Erdinger, Monique [UMR 1131 SVQV INRA-UDS, 28 rue de Herrlisheim, 68021 Colmar (France); Revers, Frédéric [INRA, Université de Bordeaux, UMR 1332 de Biologie du Fruit et Pathologie, 33882 Villenave d’Ornon (France); Ziegler-Graff, Véronique [Institut de Biologie Moléculaire des Plantes, Laboratoire propre du CNRS conventionné avec l’Université de Strasbourg, 12 rue du Général Zimmer, 67084 Strasbourg (France); Brault, Véronique, E-mail: veronique.brault@colmar.inra.fr [UMR 1131 SVQV INRA-UDS, 28 rue de Herrlisheim, 68021 Colmar (France)

    2015-12-15

    Turnip yellows virus (TuYV), a phloem-limited virus, encodes a 74 kDa protein known as the readthrough protein (RT) involved in virus movement. We show here that a TuYV mutant deleted of the C-terminal part of the RT protein (TuYV-∆RT{sub Cter}) was affected in long-distance trafficking in a host-specific manner. By using the C-terminal domain of the RT protein as a bait in a yeast two-hybrid screen of a phloem cDNA library from Arabidopsis thaliana we identified the calcineurin B-like protein-interacting protein kinase-7 (AtCIPK7). Transient expression of a GFP:CIPK7 fusion protein in virus-inoculated Nicotiana benthamiana leaves led to local increase of wild-type TuYV accumulation, but not that of TuYV-∆RT{sub Cter}. Surprisingly, elevated virus titer in inoculated leaves did not result in higher TuYV accumulation in systemic leaves, which indicates that virus long-distance movement was not affected. Since GFP:CIPK7 was localized in or near plasmodesmata, CIPK7 could negatively regulate TuYV export from infected cells. - Highlights: • The C-terminal domain of TuYV-RT is required for long-distance movement. • CIPK7 from Arabidopsis interacts with RT{sub Cter} in yeast and in plants. • CIPK7 overexpression increases virus titer locally but not virus systemic movement. • CIPK7 localizes to plasmodesmata. • CIPK7 could be a defense protein regulating virus export.

  11. A variant of the bovine noradrenaline transporter reveals the importance of the C-terminal region for correct targeting to the membrane and functional expression.

    OpenAIRE

    Burton, L D; Kippenberger, A G; Van Lingen, B.; Brüss, M; Bönisch, H.; Christie, D L

    1998-01-01

    We have characterized a cDNA clone which encodes a variant (bNAT2) of the bovine noradrenaline transporter. This cDNA differs from the previously identified bovine noradrenaline transporter (bNAT1) in the sequence encoding part of the cytoplasmic-facing C-terminus and the 3'-untranslated region. The bNAT1 and bNAT2 cDNA clones are encoded by a 5.8 and 3.6 kb mRNA species respectively. The bNAT1 and bNAT2 proteins, which are identical apart from their C-terminal 31 and 18 residues, were stably...

  12. γ-Tubulin and the C-Terminal Motor Domain Kinesin-like Protein, KLPA, Function in the Establishment of Spindle Bipolarity in Aspergillus nidulans

    OpenAIRE

    Prigozhina, Natalie L.; Walker, Richard A.; Oakley, C. Elizabeth; Oakley, Berl R.

    2001-01-01

    Previous research has found that a γ-tubulin mutation in Schizosaccharomyces pombe is synthetically lethal with a deletion of the C-terminal motor domain kinesin-like protein gene pkl1, but the lethality of the double mutant prevents a phenotypic analysis of the synthetic interaction. We have investigated interactions between klpA1, a deletion of an Aspergillus nidulans homolog of pkl1, and mutations in the mipA, γ-tubulin gene. We find that klpA1 dramatically increases the cold sensitivity a...

  13. The 14-3-3 protein interacts directly with the C-terminal region of the plant plasma membrane H(+)-ATPase

    DEFF Research Database (Denmark)

    Jahn, T.; Fuglsang, A.T.; Olsson, A.

    1997-01-01

    Accumulating evidence suggests that 14-3-3 proteins are involved in the regulation of plant plasma membrane H(+)-ATPase activity. However, it is not known whether the 14-3-3 protein interacts directly or indirectly with the H(+)-ATPase. In this study, detergent-solubilized plasma membrane H(+)-AT...... plasma membrane H(+)-ATPase. We propose that the 14-3-3 protein is a natural ligand of the plasma membrane H(+)-ATPase, regulating proton pumping by displacing the C-terminal autoinhibitory domain of the H(+)-ATPase....

  14. The functional importance of the extreme C-terminal tail in the gene 2 organellar Ca(2+)-transport ATPase (SERCA2a/b).

    OpenAIRE

    Verboomen, H; Wuytack, F; van den Bosch, L; Mertens, L.; Casteels, R

    1994-01-01

    Ca(2+)-uptake experiments in microsomal fractions from transfected COS-1 cells have revealed a functional difference between the non-muscle SERCA2b Ca2+ pump and its muscle-specific SERCA2a splice variant. Structurally, the two pumps differ only in their C-terminal tail. The last four amino acids of SERCA2a are replaced in SERCA2b by a 49-residue-long peptide chain containing a very hydrophobic stretch which could be an additional transmembrane segment. The functionally important subdomains i...

  15. Identification of potent 11mer glucagon-like peptide-1 receptor agonist peptides with novel C-terminal amino acids: Homohomophenylalanine analogs.

    Science.gov (United States)

    Haque, Tasir S; Lee, Ving G; Riexinger, Douglas; Lei, Ming; Malmstrom, Sarah; Xin, Li; Han, Songping; Mapelli, Claudio; Cooper, Christopher B; Zhang, Ge; Ewing, William R; Krupinski, John

    2010-05-01

    We report the identification of potent agonists of the Glucagon-Like Peptide-1 Receptor (GLP-1R). These compounds are short, 11 amino acid peptides containing several unnatural amino acids, including (in particular) analogs of homohomophenylalanine (hhPhe) at the C-terminal position. Typically the functional activity of the more potent peptides in this class is in the low picomolar range in an in vitro cAMP assay, with one example demonstrating excellent in vivo activity in an ob/ob mouse model of diabetes. Copyright (c) 2010 Elsevier Inc. All rights reserved.

  16. Contribution of the C-terminal region within the catalytic core domain of HIV-1 integrase to yeast lethality, chromatin binding and viral replication

    Directory of Open Access Journals (Sweden)

    Belhumeur Pierre

    2008-11-01

    Full Text Available Abstract Background HIV-1 integrase (IN is a key viral enzymatic molecule required for the integration of the viral cDNA into the genome. Additionally, HIV-1 IN has been shown to play important roles in several other steps during the viral life cycle, including reverse transcription, nuclear import and chromatin targeting. Interestingly, previous studies have demonstrated that the expression of HIV-1 IN induces the lethal phenotype in some strains of Saccharomyces cerevisiae. In this study, we performed mutagenic analyses of the C-terminal region of the catalytic core domain of HIV-1 IN in order to delineate the critical amino acid(s and/or motif(s required for the induction of the lethal phenotype in the yeast strain HP16, and to further elucidate the molecular mechanism which causes this phenotype. Results Our study identified three HIV-1 IN mutants, V165A, A179P and KR186,7AA, located in the C-terminal region of the catalytic core domain of IN that do not induce the lethal phenotype in yeast. Chromatin binding assays in yeast and mammalian cells demonstrated that these IN mutants were impaired for the ability to bind chromatin. Additionally, we determined that while these IN mutants failed to interact with LEDGF/p75, they retained the ability to bind Integrase interactor 1. Furthermore, we observed that VSV-G-pseudotyped HIV-1 containing these IN mutants was unable to replicate in the C8166 T cell line and this defect was partially rescued by complementation with the catalytically inactive D64E IN mutant. Conclusion Overall, this study demonstrates that three mutations located in the C-terminal region of the catalytic core domain of HIV-1 IN inhibit the IN-induced lethal phenotype in yeast by inhibiting the binding of IN to the host chromatin. These results demonstrate that the C-terminal region of the catalytic core domain of HIV-1 IN is important for binding to host chromatin and is crucial for both viral replication and the promotion of

  17. The lone S41 family C-terminal processing protease in Staphylococcus aureus is localized to the cell wall and contributes to virulence

    Science.gov (United States)

    Carroll, Ronan K.; Rivera, Frances E.; Cavaco, Courtney K.; Johnson, Grant M.; Martin, David

    2014-01-01

    Staphylococcus aureus is a versatile pathogen of humans and a continued public health concern due to the rise and spread of multidrug-resistant strains. As part of an ongoing investigation into the pathogenic mechanisms of this organism we previously demonstrated that an intracellular N-terminal processing protease is required for S. aureus virulence. Following on from this, here we examine the role of CtpA, the lone C-terminal processing protease of S. aureus. CtpA, a member of the S41 family, is a serine protease whose homologues in Gram-negative bacteria have been implicated in a range of biological functions, including pathogenesis. We demonstrate that S. aureus CtpA is localized to the bacterial cell wall and expression of the ctpA gene is maximal upon exposure to conditions encountered during infection. Disruption of the ctpA gene leads to decreased heat tolerance and increased sensitivity when exposed to components of the host immune system. Finally we demonstrate that the ctpA− mutant strain is attenuated for virulence in a murine model of infection. Our results represent the first characterization of a C-terminal processing protease in a pathogenic Gram-positive bacterium and show that it plays a critical role during infection. PMID:24928312

  18. Effect of pH on the structure of the recombinant C-terminal domain of Nephila clavipes dragline silk protein.

    Science.gov (United States)

    Gauthier, Martin; Leclerc, Jérémie; Lefèvre, Thierry; Gagné, Stéphane M; Auger, Michèle

    2014-12-08

    Spider silk proteins undergo a complex series of molecular events before being converted into an outstanding hierarchically organized fiber. Recent literature has underlined the crucial role of the C-terminal domain in silk protein stability and fiber formation. However, the effect of pH remains to be clarified. We have thus developed an efficient purification protocol to obtain stable native-like recombinant MaSp1 C-terminal domain of Nephila clavipes (NCCTD). Its structure was investigated as a function of pH using circular dichroism, fluorescence and solution NMR spectroscopy. The results show that the NCCTD structure is very sensitive to pH and suggest that a molten globule state occurs at pH 5.0 and below. Electronic microscopy images also indicate fiber formation at low pH and coarser globular particles at more basic pH. The results are consistent with a spinning process model where the NCCTD acts as an aggregation nucleus favoring the β-aggregation of the hydrophobic polyalanine repeats upon spinning.

  19. Influence of N- and/or C-terminal regions on activity, expression, characteristics and structure of lipase from Geobacillus sp. 95.

    Science.gov (United States)

    Gudiukaitė, Renata; Gegeckas, Audrius; Kazlauskas, Darius; Citavicius, Donaldas

    2014-01-01

    GD-95 lipase from Geobacillus sp. strain 95 and its modified variants lacking N-terminal signal peptide and/or 10 or 20 C-terminal amino acids were successfully cloned, expressed and purified. To our knowledge, GD-95 lipase precursor (Pre-GD-95) is the first Geobacillus lipase possessing more than 80% lipolytic activity at 5 °C. It has maximum activity at 55 °C and displays a broad pH activity range. GD-95 lipase was shown to hydrolyze p-NP dodecanoate, tricaprylin and canola oil better than other analyzed substrates. Structural and sequence alignments of bacterial lipases and GD-95 lipase revealed that the C-terminus forms an α helix, which is a conserved structure in lipases from Pseudomonas, Clostridium or Staphylococcus bacteria. This work demonstrates that 10 and 20 C-terminal amino acids of GD-95 lipase significantly affect stability and other physicochemical properties of this enzyme, which has never been reported before and can help create lipases with more specific properties for industrial application. GD-95 lipase and its modified variants GD-95-10 can be successfully applied to biofuel production, in leather and pulp industries, for the production of cosmetics or perfumes. These lipases are potential biocatalysts in processes, which require extreme conditions: low or high temperature, strongly acidic or alkaline environment and various organic solvents.

  20. Extensive de novo solid-state NMR assignments of the 33 kDa C-terminal domain of the Ure2 prion

    Energy Technology Data Exchange (ETDEWEB)

    Habenstein, Birgit [UMR 5086 CNRS/Universite de Lyon 1, Institut de Biologie et Chimie des Proteines (France); Wasmer, Christian [Harvard Medical School (United States); Bousset, Luc; Sourigues, Yannick [UPR 3082 CNRS, Laboratoire d' Enzymologie et Biochimie Structurales (France); Schuetz, Anne [ETH Zurich, Physical Chemistry (Switzerland); Loquet, Antoine [Max Planck Institute for Biophysical Chemistry (Germany); Meier, Beat H., E-mail: beme@ethz.ch [ETH Zurich, Physical Chemistry (Switzerland); Melki, Ronald, E-mail: melki@lebs.cnrs-gif.fr [UPR 3082 CNRS, Laboratoire d' Enzymologie et Biochimie Structurales (France); Boeckmann, Anja, E-mail: a.bockmann@ibcp.fr [UMR 5086 CNRS/Universite de Lyon 1, Institut de Biologie et Chimie des Proteines (France)

    2011-11-15

    We present the de novo resonance assignments for the crystalline 33 kDa C-terminal domain of the Ure2 prion using an optimized set of five 3D solid-state NMR spectra. We obtained, using a single uniformly {sup 13}C, {sup 15}N labeled protein sample, sequential chemical-shift information for 74% of the N, C{alpha}, C{beta} triples, and for 80% of further side-chain resonances for these spin systems. We describe the procedures and protocols devised, and discuss possibilities and limitations of the assignment of this largest protein assigned today by solid-state NMR, and for which no solution-state NMR shifts were available. A comparison of the NMR chemical shifts with crystallographic data reveals that regions with high crystallographic B-factors are particularly difficult to assign. While the secondary structure elements derived from the chemical shift data correspond mainly to those present in the X-ray crystal structure, we detect an additional helical element and structural variability in the protein crystal, most probably originating from the different molecules in the asymmetric unit, with the observation of doubled resonances in several parts, including entire stretches, of the protein. Our results provide the point of departure towards an atomic-resolution structural analysis of the C-terminal Ure2p domain in the context of the full-length prion fibrils.

  1. Addition of thiols to the double bond of dipeptide C-terminal dehydroalanine as a source of new inhibitors of cathepsin C.

    Science.gov (United States)

    Lenartowicz, Paweł; Makowski, Maciej; Oszywa, Bartosz; Haremza, Kinga; Latajka, Rafał; Pawełczak, Małgorzata; Kafarski, Paweł

    2017-08-01

    Addition of thiols to double bond of glycyl-dehydroalanine and phenyl-dehydroalanine esters provided micromolar inhibitors of cathepsin C. The structure-activity studies indicated that dipeptides containing N-terminal phenylalanine exhibit higher affinity towards the enzyme. A series of C-terminal S-substituted cysteines are responsible for varying interaction with S1 binding pocket of cathepsin C. Depending on diastereomer these compounds most likely act as slowly reacting substrates or competitive inhibitors. This was proved by TLC analysis of the medium in which interaction of methyl (S)-phenylalanyl-(R,S)-(S-adamantyl)cysteinate (7i) with the enzyme was studied. Molecular modeling enabled to establish their mode of binding showed that S2 pocket is long and narrow and accommodates phenyl group of phenylalanine while significantly spacious sites located at the surface of the enzyme (one of them being S1 pocket) bind the adamantyl moiety oriented in different direction for each stereoisomer. Finally replacement of carboxymethyl moiety of methyl (S)-phenylalanyl-(R,S)-(S-phenyl)cysteinate (7c) with nitrile group provided about 650-times more potent inhibitor of cathepsin C indicating that the studied C-terminal S-substituted cysteines are good activity probes for S1 binding pocket of this enzyme. Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  2. Order through disorder: hyper-mobile C-terminal residues stabilize the folded state of a helical peptide. a molecular dynamics study.

    Directory of Open Access Journals (Sweden)

    Kalliopi K Patapati

    Full Text Available Conventional wisdom has it that the presence of disordered regions in the three-dimensional structures of polypeptides not only does not contribute significantly to the thermodynamic stability of their folded state, but, on the contrary, that the presence of disorder leads to a decrease of the corresponding proteins' stability. We have performed extensive 3.4 µs long folding simulations (in explicit solvent and with full electrostatics of an undecamer peptide of experimentally known helical structure, both with and without its disordered (four residue long C-terminal tail. Our simulations clearly indicate that the presence of the apparently disordered (in structural terms C-terminal tail, increases the thermodynamic stability of the peptide's folded (helical state. These results show that at least for the case of relatively short peptides, the interplay between thermodynamic stability and the apparent structural stability can be rather subtle, with even disordered regions contributing significantly to the stability of the folded state. Our results have clear implications for the understanding of peptide energetics and the design of foldable peptides.

  3. Characterization of two novel bacterial type A exo-chitobiose hydrolases having C-terminal 5/12-type carbohydrate-binding modules

    DEFF Research Database (Denmark)

    Binti Jamek, Shariza; Nyffenegger, Christian; Muschiol, Jan

    2017-01-01

    "exo-chitobiose hydrolases." In this study, the chitinase type A from Serratia marcescens (SmaChiA) was used as a template for identifying two novel exo-chitobiose hydrolase type A enzymes, FbalChi18A and MvarChi18A, originating from the marine organisms Ferrimonas balearica and Microbulbifer....../α barrel domain of each of the new enzymes showed individual differences, but ~69% identity of each to that of SmaChiA and highly conserved active site residues. Superposition of a model substrate on 3D structural models of the catalytic domain of the enzymes corroborated exo-chitobiose hydrolase type...... A activity for FbalChi18A and MvarChi18A, i.e., substrate attack from the reducing end. A main feature of both of the new enzymes was the presence of C-terminal 5/12 type carbohydrate-binding modules (SmaChiA has no C-terminal carbohydrate binding module). These new enzymes may be useful tools...

  4. Cognitive Decline Typical of Frontotemporal Lobar Degeneration in Transgenic Mice Expressing the 25-kDa C-Terminal Fragment of TDP-43

    Science.gov (United States)

    Caccamo, Antonella; Majumder, Smita; Oddo, Salvatore

    2012-01-01

    Transactive response DNA-binding protein 43 (TDP-43) is the pathological signature protein in several neurodegenerative disorders, including the majority of frontotemporal lobar degeneration cases (FTLD-TDP), motor neuron disease, and amyotrophic lateral sclerosis. Pathological TDP-43 is mislocalized from its nuclear location to the cytoplasm, where it accumulates and is proteolytically cleaved to form C-terminal fragments. Although the 25-kDa C-terminal fragment of TDP-43 (TDP-25) accumulates in affected brain regions, its role in the disease pathogenesis remains elusive. To address this problem, we have generated a novel transgenic mouse that selectively expresses TDP-25 in neurons. We show that transgenic mice expressing TDP-25 develop cognitive deficits associated with the build-up of soluble TDP-25. These cognitive deficits are independent of TDP-43–positive inclusions and occur without overt neurodegeneration. Additionally, we show that the expression of TDP-25 is sufficient to alter the processing of endogenous full-length TDP-43. These studies represent the first in vivo demonstration of a pathological role for TDP-25 and strongly suggest that the onset of cognitive deficits in TDP-43 proteinopathies is independent of TDP-43 inclusions. These data provide a framework for understanding the molecular mechanisms underlying the onset of cognitive deficits in FTLD-TDP and other TDP-43 proteinopathies; thus, the TDP-25 transgenic mice represent a unique tool to reach this goal. PMID:22067910

  5. The C-Terminal RpoN Domain of sigma54 Forms an unpredictedHelix-Turn-Helix Motif Similar to domains of sigma70

    Energy Technology Data Exchange (ETDEWEB)

    Doucleff, Michaeleen; Malak, Lawrence T.; Pelton, Jeffrey G.; Wemmer, David E.

    2005-11-01

    The ''{delta}'' subunit of prokaryotic RNA-polymerase allows gene-specific transcription initiation. Two {sigma} families have been identified, {sigma}{sup 70} and {sigma}{sup 54}, which use distinct mechanisms to initiate transcription and share no detectable sequence homology. Although the {sigma}{sup 70}-type factors have been well characterized structurally by x-ray crystallography, no high-resolution structural information is available for the {sigma}{sup 54}-type factors. Here we present the NMR derived structure of the C-terminal domain of {sigma}{sup 54} from Aquifex aeolicus. This domain (Thr323 to Gly389), which contains the highly conserved RpoN box sequence, consists of a poorly structured N-terminal tail followed by a three-helix bundle, which is surprisingly similar to domains of the {sigma}{sup 70}-type proteins. Residues of the RpoN box, which have previously been shown to be critical for DNA binding, form the second helix of an unpredicted helix-turn-helix motif. This structure's homology with other DNA binding proteins, combined with previous biochemical data, suggest how the C-terminal domain of {sigma}{sup 54} binds to DNA.

  6. Design and verification of halogen-bonding system at the complex interface of human fertilization-related MUP PDZ5 domain with CAMK's C-terminal peptide.

    Science.gov (United States)

    Wang, Juan; Guo, Yunjie; Zhang, Xue

    2017-11-21

    Calmodulin-dependent protein kinase (CAMK) is physiologically activated in fertilized human oocytes and is involved in the Ca2+ response pathways that link the fertilization calmodulin signal to meiosis resumption and cortical granule exocytosis. The kinase has an unstructured C-terminal tail that can be recognized and bound by the PDZ5 domain of its cognate partner, the multi-PDZ domain protein (MUP). In the current study, we reported a rational biomolecular design of halogen-bonding system at the complex interface of CAMK's C-terminal peptide with MUP PDZ5 domain by using high-level computational approaches. Four organic halogens were employed as atom probes to explore the structural geometry and energetic property of designed halogen bonds in the PDZ5-peptide complex. It was found that the heavier halogen elements such as bromine Br and iodine I can confer stronger halogen bond but would cause bad atomic contacts and overlaps at the complex interface, while fluorine F cannot form effective halogen bond in the complex. In addition, the halogen substitution at different positions of peptide's aromatic ring would result in distinct effects on the halogen-bonding system. The computational findings were then verified by using fluorescence analysis; it is indicated that the halogen type and substitution position play critical role in the interaction strength of halogen bonds, and thus the PDZ5-peptide binding affinity can be improved considerably by optimizing their combination. Copyright © 2017 Elsevier Ltd. All rights reserved.

  7. Purification and Characterization of Peptides Inhibiting MMP-1 Activity with C Terminate of Gly-Leu from Simulated Gastrointestinal Digestion Hydrolysates of Tilapia (Oreochromis niloticus) Skin Gelatin.

    Science.gov (United States)

    Liping, Sun; Qiuming, Liu; Jian, Fan; Xiao, Li; Yongliang, Zhuang

    2018-01-24

    Tilapia skin gelatin hydrolysates (TSGHs) were prepared by simulated gastrointestinal digestion and separated by gel filtration and semi-preparative reversed-phase high-performance liquid chromatography. The anti-photoaging effects were evaluated using an ultraviolet radiation B (UVB)-induced mouse embryonic fibroblast (MEF) photoaging model in vitro. Three fractions from TSGHs with high inhibitory intercellular matrix metalloproteinase-1 (MMP-1) activities and reactive oxygen species (ROS) production were obtained. Three key peptides, GYTGL, LGATGL, and VLGL, were identified, and their C terminate was Gly-Leu. Three peptides were synthesized and exhibited a significant inhibition of intercellular MMP-1 activity and ROS production. Furthermore, three peptides inhibiting MMP-1 activities were evaluated through their docking of S 1 ' and S 3 ' active pockets of MMP-1. Hydrogen bonds and C terminate Gly-Leu played important roles. Finally, the protective effects of three peptides on intercellular collagen in UVB-induced MEFs were compared. Our results indicated that tilapia gelatin peptides exhibited potential activities to prevent and regulate photoaging.

  8. A C-terminally truncated form of β-catenin acts as a novel regulator of Wnt/β-catenin signaling in planarians.

    Directory of Open Access Journals (Sweden)

    Hanxia Su

    2017-10-01

    Full Text Available β-Catenin, the core element of the Wnt/β-catenin pathway, is a multifunctional and evolutionarily conserved protein which performs essential roles in a variety of developmental and homeostatic processes. Despite its crucial roles, the mechanisms that control its context-specific functions in time and space remain largely unknown. The Wnt/β-catenin pathway has been extensively studied in planarians, flatworms with the ability to regenerate and remodel the whole body, providing a 'whole animal' developmental framework to approach this question. Here we identify a C-terminally truncated β-catenin (β-catenin4, generated by gene duplication, that is required for planarian photoreceptor cell specification. Our results indicate that the role of β-catenin4 is to modulate the activity of β-catenin1, the planarian β-catenin involved in Wnt signal transduction in the nucleus, mediated by the transcription factor TCF-2. This inhibitory form of β-catenin, expressed in specific cell types, would provide a novel mechanism to modulate nuclear β-catenin signaling levels. Genomic searches and in vitro analysis suggest that the existence of a C-terminally truncated form of β-catenin could be an evolutionarily conserved mechanism to achieve a fine-tuned regulation of Wnt/β-catenin signaling in specific cellular contexts.

  9. A C-terminally truncated form of β-catenin acts as a novel regulator of Wnt/β-catenin signaling in planarians.

    Science.gov (United States)

    Su, Hanxia; Sureda-Gomez, Miquel; Rabaneda-Lombarte, Neus; Gelabert, Maria; Xie, Jianlei; Wu, Wei; Adell, Teresa

    2017-10-01

    β-Catenin, the core element of the Wnt/β-catenin pathway, is a multifunctional and evolutionarily conserved protein which performs essential roles in a variety of developmental and homeostatic processes. Despite its crucial roles, the mechanisms that control its context-specific functions in time and space remain largely unknown. The Wnt/β-catenin pathway has been extensively studied in planarians, flatworms with the ability to regenerate and remodel the whole body, providing a 'whole animal' developmental framework to approach this question. Here we identify a C-terminally truncated β-catenin (β-catenin4), generated by gene duplication, that is required for planarian photoreceptor cell specification. Our results indicate that the role of β-catenin4 is to modulate the activity of β-catenin1, the planarian β-catenin involved in Wnt signal transduction in the nucleus, mediated by the transcription factor TCF-2. This inhibitory form of β-catenin, expressed in specific cell types, would provide a novel mechanism to modulate nuclear β-catenin signaling levels. Genomic searches and in vitro analysis suggest that the existence of a C-terminally truncated form of β-catenin could be an evolutionarily conserved mechanism to achieve a fine-tuned regulation of Wnt/β-catenin signaling in specific cellular contexts.

  10. High-resolution crystal structure reveals a HEPN domain at the C-terminal region of S. cerevisiae RNA endonuclease Swt1

    Energy Technology Data Exchange (ETDEWEB)

    Peng, Shuxia, E-mail: pengsx@ihep.ac.cn; Zhou, Ke; Wang, Wenjia; Gao, Zengqiang; Dong, Yuhui; Liu, Quansheng

    2014-10-31

    Highlights: • Crystal structure of the C-terminal (CT) domain of Swt1 was determined at 2.3 Å. • Structure of the CT domain was identified as HEPN domain superfamily member. • Low-resolution envelope of Swt1 full-length in solution was analyzed by SAXS. • The middle and CT domains gave good fit to SAXS structural model. - Abstract: Swt1 is an RNA endonuclease that plays an important role in quality control of nuclear messenger ribonucleoprotein particles (mRNPs) in eukaryotes; however, its structural details remain to be elucidated. Here, we report the crystal structure of the C-terminal (CT) domain of Swt1 from Saccharomyces cerevisiae, which shares common characteristics of higher eukaryotes and prokaryotes nucleotide binding (HEPN) domain superfamily. To study in detail the full-length protein structure, we analyzed the low-resolution architecture of Swt1 in solution using small angle X-ray scattering (SAXS) method. Both the CT domain and middle domain exhibited a good fit upon superimposing onto the molecular envelope of Swt1. Our study provides the necessary structural information for detailed analysis of the functional role of Swt1, and its importance in the process of nuclear mRNP surveillance.

  11. Ultrahigh and High Resolution Structures and Mutational Analysis of Monomeric Streptococcus pyogenes SpeB Reveal a Functional Role for the Glycine-rich C-terminal Loop

    Energy Technology Data Exchange (ETDEWEB)

    González-Páez, Gonzalo E.; Wolan, Dennis W. (Scripps)

    2012-09-05

    Cysteine protease SpeB is secreted from Streptococcus pyogenes and has been studied as a potential virulence factor since its identification almost 70 years ago. Here, we report the crystal structures of apo mature SpeB to 1.06 {angstrom} resolution as well as complexes with the general cysteine protease inhibitor trans-epoxysuccinyl-L-leucylamido(4-guanidino)butane and a novel substrate mimetic peptide inhibitor. These structures uncover conformational changes associated with maturation of SpeB from the inactive zymogen to its active form and identify the residues required for substrate binding. With the use of a newly developed fluorogenic tripeptide substrate to measure SpeB activity, we determined IC{sub 50} values for trans-epoxysuccinyl-L-leucylamido(4-guanidino)butane and our new peptide inhibitor and the effects of mutations within the C-terminal active site loop. The structures and mutational analysis suggest that the conformational movements of the glycine-rich C-terminal loop are important for the recognition and recruitment of biological substrates and release of hydrolyzed products.

  12. Design, synthesis, and evaluation of Trolox-conjugated amyloid-β C-terminal peptides for therapeutic intervention in an in vitro model of Alzheimer's disease.

    Science.gov (United States)

    Arai, Takuya; Ohno, Akiko; Kazunori, Mori; Kakizawa, Taeko; Kuwata, Hiroshi; Ozawa, Toshihiko; Shibanuma, Motoko; Hara, Shuntaro; Ishida, Seiichi; Kurihara, Masaaki; Miyata, Naoki; Nakagawa, Hidehiko; Fukuhara, Kiyoshi

    2016-09-15

    Two hallmarks of Alzheimer's disease (AD) observed in the brains of patients with the disease include oxidative injury and deposition of protein aggregates comprised of amyloid-β (Aβ) variants. To inhibit these toxic processes, we synthesized antioxidant-conjugated peptides comprised of Trolox and various C-terminal motifs of Aβ variants, TxAβx-n (x=34, 36, 38, 40; n=40, 42, 43). Most of these compounds were found to exhibit anti-aggregation activities. Among them, TxAβ36-42 significantly inhibited Aβ1-42 aggregation, showed potent antioxidant activity, and protected SH-SY5Y cells from Aβ1-42-induced cytotoxicity. Thus, this method represents a promising strategy for developing multifunctional AD therapeutic agents. Copyright © 2016 Elsevier Ltd. All rights reserved.

  13. Activation of the plasma membrane Na/H antiporter salt-overly-sensitive 1 (SOS1) by phosphorylation of an auto-inhibitory C-terminal domain

    KAUST Repository

    Quintero, Francisco J.

    2011-01-24

    The plasma membrane sodium/proton exchanger Salt-Overly-Sensitive 1 (SOS1) is a critical salt tolerance determinant in plants. The SOS2-SOS3 calcium-dependent protein kinase complex upregulates SOS1 activity, but the mechanistic details of this crucial event remain unresolved. Here we show that SOS1 is maintained in a resting state by a C-terminal auto-inhibitory domain that is the target of SOS2-SOS3. The auto-inhibitory domain interacts intramolecularly with an adjacent domain of SOS1 that is essential for activity. SOS1 is relieved from auto-inhibition upon phosphorylation of the auto-inhibitory domain by SOS2-SOS3. Mutation of the SOS2 phosphorylation and recognition site impeded the activation of SOS1 in vivo and in vitro. Additional amino acid residues critically important for SOS1 activity and regulation were identified in a genetic screen for hypermorphic alleles.

  14. Homologies between the non-collagenous C-terminal (NC1) globular domains of the alpha 1 and alpha 2 subunits of type-IV collagen.

    Science.gov (United States)

    Kaytes, P S; Theriault, N Y; Vogeli, G

    1987-01-01

    The non-collagenous C-terminal globular domain (NC1) of type-IV collagen has the dual role of initiating triple-helix formation among the subunits and of crosslinking two collagen molecules during basement-membrane meshwork formation. By cloning a cDNA for the NC1 domain of the alpha 2(IV) collagen chain, we have found a high degree of homology (63% for nucleotides, 66% for amino acids) between the NC1 of the alpha 2 and alpha 1 chains of type-IV collagen. All cysteine residues are conserved. This high degree of homology is not found within the helical portion where the homology is 41% for amino acids (only 14% if the obligatory glycine is not used for this analysis). We propose that this high degree of homology within the non-collagenous domain indicates a close evolutionary relationship maintained by functional restraints between the two chains of type IV collagen.

  15. NMR Structure of the C-Terminal Transmembrane Domain of the HDL Receptor, SR-BI, and a Functionally Relevant Leucine Zipper Motif.

    Science.gov (United States)

    Chadwick, Alexandra C; Jensen, Davin R; Hanson, Paul J; Lange, Philip T; Proudfoot, Sarah C; Peterson, Francis C; Volkman, Brian F; Sahoo, Daisy

    2017-03-07

    The interaction of high-density lipoprotein (HDL) with its receptor, scavenger receptor BI (SR-BI), is critical for lowering plasma cholesterol levels and reducing the risk for cardiovascular disease. The HDL/SR-BI complex facilitates delivery of cholesterol into cells and is likely mediated by receptor dimerization. This work describes the use of nuclear magnetic resonance (NMR) spectroscopy to generate the first high-resolution structure of the C-terminal transmembrane domain of SR-BI. This region of SR-BI harbors a leucine zipper dimerization motif, which when mutated impairs the ability of the receptor to bind HDL and mediate cholesterol delivery. These losses in function correlate with the inability of SR-BI to form dimers. We also identify juxtamembrane regions of the extracellular domain of SR-BI that may interact with the lipid surface to facilitate cholesterol transport functions of the receptor. Copyright © 2017 Elsevier Ltd. All rights reserved.

  16. Full-length Gαq-phospholipase C-β3 structure reveals interfaces of the C-terminal coiled-coil domain

    Energy Technology Data Exchange (ETDEWEB)

    Lyon, Angeline M.; Dutta, Somnath; Boguth, Cassandra A.; Skiniotis, Georgios; Tesmer, John J.G. [Michigan

    2014-08-21

    Phospholipase C-β (PLCβ) is directly activated by Gαq, but the molecular basis for how its distal C-terminal domain (CTD) contributes to maximal activity is poorly understood. Herein we present both the crystal structure and cryo-EM three-dimensional reconstructions of human full-length PLCβ3 in complex with mouse Gαq. The distal CTD forms an extended monomeric helical bundle consisting of three antiparallel segments with structural similarity to membrane-binding bin-amphiphysin-Rvs (BAR) domains. Sequence conservation of the distal CTD suggests putative membrane and protein interaction sites, the latter of which bind the N-terminal helix of Gαq in both the crystal structure and cryo-EM reconstructions. Functional analysis suggests that the distal CTD has roles in membrane targeting and in optimizing the orientation of the catalytic core at the membrane for maximal rates of lipid hydrolysis.

  17. An autoinhibitory helix in the C-terminal region of phospholipase C-[beta] mediates G[alpaha subscript q] activation

    Energy Technology Data Exchange (ETDEWEB)

    Lyon, Angeline M.; Tesmer, Valerie M.; Dhamsania, Vishan D.; Thal, David M.; Gutierrez, Joanne; Chowdhury, Shoaib; Suddala, Krishna C.; Northup, John K.; Tesmer, John J.G. (Michigan); (NIH)

    2012-03-16

    The enzyme phospholipase C-{beta} (PLC{beta}) is a crucial regulator of intracellular calcium levels whose activity is controlled by heptahelical receptors that couple to members of the G{sub q} family of heterotrimeric G proteins. We have determined atomic structures of two invertebrate homologs of PLC{beta} (PLC21) from cephalopod retina and identified a helix from the C-terminal regulatory region that interacts with a conserved surface of the catalytic core of the enzyme. Mutations designed to disrupt the analogous interaction in human PLC{beta}3 considerably increase basal activity and diminish stimulation by G{alpha}{sub q}. G{alpha}{sub q} binding requires displacement of the autoinhibitory helix from the catalytic core, thus providing an allosteric mechanism for activation of PLC{beta}.

  18. Solution structure of the Arabidopsis thaliana telomeric repeat-binding protein DNA binding domain: a new fold with an additional C-terminal helix.

    Science.gov (United States)

    Sue, Shih-Che; Hsiao, Hsin-Hao; Chung, Ben C-P; Cheng, Ying-Hsien; Hsueh, Kuang-Lung; Chen, Chung Mong; Ho, Chia Hsing; Huang, Tai-Huang

    2006-02-10

    The double-stranded telomeric repeat-binding protein (TRP) AtTRP1 is isolated from Arabidopsis thaliana. Using gel retardation assays, we defined the C-terminal 97 amino acid residues, Gln464 to Val560 (AtTRP1(464-560)), as the minimal structured telomeric repeat-binding domain. This region contains a typical Myb DNA-binding motif and a C-terminal extension of 40 amino acid residues. The monomeric AtTRP1(464-560) binds to a 13-mer DNA duplex containing a single repeat of an A.thaliana telomeric DNA sequence (GGTTTAG) in a 1:1 complex, with a K(D) approximately 10(-6)-10(-7) M. Nuclear magnetic resonance (NMR) examination revealed that the solution structure of AtTRP1(464-560) is a novel four-helix tetrahedron rather than the three-helix bundle structure found in typical Myb motifs and other TRPs. Binding of the 13-mer DNA duplex to AtTRP1(464-560) induced significant chemical shift perturbations of protein amide resonances, which suggests that helix 3 (H3) and the flexible loop connecting H3 and H4 are essential for telomeric DNA sequence recognition. Furthermore, similar to that in hTRF1, the N-terminal arm likely contributes to or stabilizes DNA binding. Sequence comparisons suggested that the four-helix structure and the involvement of the loop residues in DNA binding may be features unique to plant TRPs.

  19. Viperin is induced following dengue virus type-2 (DENV-2 infection and has anti-viral actions requiring the C-terminal end of viperin.

    Directory of Open Access Journals (Sweden)

    Karla J Helbig

    Full Text Available The host protein viperin is an interferon stimulated gene (ISG that is up-regulated during a number of viral infections. In this study we have shown that dengue virus type-2 (DENV-2 infection significantly induced viperin, co-incident with production of viral RNA and via a mechanism requiring retinoic acid-inducible gene I (RIG-I. Viperin did not inhibit DENV-2 entry but DENV-2 RNA and infectious virus release was inhibited in viperin expressing cells. Conversely, DENV-2 replicated to higher tires earlier in viperin shRNA expressing cells. The anti-DENV effect of viperin was mediated by residues within the C-terminal 17 amino acids of viperin and did not require the N-terminal residues, including the helix domain, leucine zipper and S-adenosylmethionine (SAM motifs known to be involved in viperin intracellular membrane association. Viperin showed co-localisation with lipid droplet markers, and was co-localised and interacted with DENV-2 capsid (CA, NS3 and viral RNA. The ability of viperin to interact with DENV-2 NS3 was associated with its anti-viral activity, while co-localisation of viperin with lipid droplets was not. Thus, DENV-2 infection induces viperin which has anti-viral properties residing in the C-terminal region of the protein that act to restrict early DENV-2 RNA production/accumulation, potentially via interaction of viperin with DENV-2 NS3 and replication complexes. These anti-DENV-2 actions of viperin show both contrasts and similarities with other described anti-viral mechanisms of viperin action and highlight the diverse nature of this unique anti-viral host protein.

  20. Distinct repeat motifs at the C-terminal region of CagA of Helicobacter pylori strains isolated from diseased patients and asymptomatic individuals in West Bengal, India

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    Chattopadhyay Santanu

    2012-05-01

    Full Text Available Abstract Background Infection with Helicobacter pylori strains that express CagA is associated with gastritis, peptic ulcer disease, and gastric adenocarcinoma. The biological function of CagA depends on tyrosine phosphorylation by a cellular kinase. The phosphate acceptor tyrosine moiety is present within the EPIYA motif at the C-terminal region of the protein. This region is highly polymorphic due to variations in the number of EPIYA motifs and the polymorphism found in spacer regions among EPIYA motifs. The aim of this study was to analyze the polymorphism at the C-terminal end of CagA and to evaluate its association with the clinical status of the host in West Bengal, India. Results Seventy-seven H. pylori strains isolated from patients with various clinical statuses were used to characterize the C-ternimal polymorphic region of CagA. Our analysis showed that there is no correlation between the previously described CagA types and various disease outcomes in Indian context. Further analyses of different CagA structures revealed that the repeat units in the spacer sequences within the EPIYA motifs are actually more discrete than the previously proposed models of CagA variants. Conclusion Our analyses suggest that EPIYA motifs as well as the spacer sequence units are present as distinct insertions and deletions, which possibly have arisen from extensive recombination events. Moreover, we have identified several new CagA types, which could not be typed by the existing systems and therefore, we have proposed a new typing system. We hypothesize that a cagA gene encoding higher number EPIYA motifs may perhaps have arisen from cagA genes that encode lesser EPIYA motifs by acquisition of DNA segments through recombination events.

  1. A novel COL4A1 frameshift mutation in familial kidney disease: the importance of the C-terminal NC1 domain of type IV collagen

    Science.gov (United States)

    Gale, Daniel P.; Oygar, D. Deren; Lin, Fujun; Oygar, P. Derin; Khan, Nadia; Connor, Thomas M.F.; Lapsley, Marta; Maxwell, Patrick H.; Neild, Guy H.

    2016-01-01

    Background Hereditary microscopic haematuria often segregates with mutations of COL4A3, COL4A4 or COL4A5 but in half of families a gene is not identified. We investigated a Cypriot family with autosomal dominant microscopic haematuria with renal failure and kidney cysts. Methods We used genome-wide linkage analysis, whole exome sequencing and cosegregation analyses. Results We identified a novel frameshift mutation, c.4611_4612insG:p.T1537fs, in exon 49 of COL4A1. This mutation predicts truncation of the protein with disruption of the C-terminal part of the NC1 domain. We confirmed its presence in 20 family members, 17 with confirmed haematuria, 5 of whom also had stage 4 or 5 chronic kidney disease. Eleven family members exhibited kidney cysts (55% of those with the mutation), but muscle cramps or cerebral aneurysms were not observed and serum creatine kinase was normal in all individuals tested. Conclusions Missense mutations of COL4A1 that encode the CB3 [IV] segment of the triple helical domain (exons 24 and 25) are associated with HANAC syndrome (hereditary angiopathy, nephropathy, aneurysms and cramps). Missense mutations of COL4A1 that disrupt the NC1 domain are associated with antenatal cerebral haemorrhage and porencephaly, but not kidney disease. Our findings extend the spectrum of COL4A1 mutations linked with renal disease and demonstrate that the highly conserved C-terminal part of the NC1 domain of the α1 chain of type IV collagen is important in the integrity of glomerular basement membrane in humans. PMID:27190376

  2. Localization and trafficking of an isoform of the AtPRA1 family to the Golgi apparatus depend on both N- and C-terminal sequence motifs.

    Science.gov (United States)

    Jung, Chan Jin; Lee, Myoung Hui; Min, Myung Ki; Hwang, Inhwan

    2011-02-01

    Prenylated Rab acceptors (PRAs) bind to prenylated Rab proteins and possibly aid in targeting Rabs to their respective compartments. In Arabidopsis, 19 isoforms of PRA1 have been identified and, depending upon the isoforms, they localize to the endoplasmic reticulum (ER), Golgi apparatus and endosomes. Here, we investigated the localization and trafficking of AtPRA1.B6, an isoform of the Arabidopsis PRA1 family. In colocalization experiments with various organellar markers, AtPRA1.B6 tagged with hemagglutinin (HA) at the N-terminus localized to the Golgi apparatus in protoplasts and transgenic plants. The valine residue at the C-terminal end and an EEE motif in the C-terminal cytoplasmic domain were critical for anterograde trafficking from the ER to the Golgi apparatus. The N-terminal region contained a sequence motif for retention of AtPRA1.B6 at the Golgi apparatus. In addition, anterograde trafficking of AtPRA1.B6 from the ER to the Golgi apparatus was highly sensitive to the HA:AtPRA1.B6 level. The region that contains the sequence motif for Golgi retention also conferred the abundance-dependent trafficking inhibition. On the basis of these results, we propose that AtPRA1.B6 localizes to the Golgi apparatus and its ER-to-Golgi trafficking and localization to the Golgi apparatus are regulated by multiple sequence motifs in both the C- and N-terminal cytoplasmic domains. © 2010 John Wiley & Sons A/S.

  3. The C-terminal domain of the Arabinosyltransferase Mycobacterium tuberculosis EmbC is a lectin-like carbohydrate binding module.

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    Luke J Alderwick

    2011-02-01

    Full Text Available The D-arabinan-containing polymers arabinogalactan (AG and lipoarabinomannan (LAM are essential components of the unique cell envelope of the pathogen Mycobacterium tuberculosis. Biosynthesis of AG and LAM involves a series of membrane-embedded arabinofuranosyl (Araf transferases whose structures are largely uncharacterised, despite the fact that several of them are pharmacological targets of ethambutol, a frontline drug in tuberculosis therapy. Herein, we present the crystal structure of the C-terminal hydrophilic domain of the ethambutol-sensitive Araf transferase M. tuberculosis EmbC, which is essential for LAM synthesis. The structure of the C-terminal domain of EmbC (EmbC(CT encompasses two sub-domains of different folds, of which subdomain II shows distinct similarity to lectin-like carbohydrate-binding modules (CBM. Co-crystallisation with a cell wall-derived di-arabinoside acceptor analogue and structural comparison with ligand-bound CBMs suggest that EmbC(CT contains two separate carbohydrate binding sites, associated with subdomains I and II, respectively. Single-residue substitution of conserved tryptophan residues (Trp868, Trp985 at these respective sites inhibited EmbC-catalysed extension of LAM. The same substitutions differentially abrogated binding of di- and penta-arabinofuranoside acceptor analogues to EmbC(CT, linking the loss of activity to compromised acceptor substrate binding, indicating the presence of two separate carbohydrate binding sites, and demonstrating that subdomain II indeed functions as a carbohydrate-binding module. This work provides the first step towards unravelling the structure and function of a GT-C-type glycosyltransferase that is essential in M. tuberculosis.

  4. Evaluation of the C-Terminal Fragment of Entamoeba histolytica Gal/GalNAc Lectin Intermediate Subunit as a Vaccine Candidate against Amebic Liver Abscess.

    Science.gov (United States)

    Min, Xiangyang; Feng, Meng; Guan, Yue; Man, Suqin; Fu, Yongfeng; Cheng, Xunjia; Tachibana, Hiroshi

    2016-01-01

    Entamoeba histolytica is an intestinal protozoan parasite that causes amoebiasis, including amebic dysentery and liver abscesses. E. histolytica invades host tissues by adhering onto cells and phagocytosing them depending on the adaptation and expression of pathogenic factors, including Gal/GalNAc lectin. We have previously reported that E. histolytica possesses multiple CXXC sequence motifs, with the intermediate subunit of Gal/GalNAc lectin (i.e., Igl) as a key factor affecting the amoeba's pathogenicity. The present work showed the effect of immunization with recombinant Igl on amebic liver abscess formation and the corresponding immunological properties. A prokaryotic expression system was used to prepare the full-length Igl and the N-terminal, middle, and C-terminal fragments (C-Igl) of Igl. Vaccine efficacy was assessed by challenging hamsters with an intrahepatic injection of E. histolytica trophozoites. Hamsters intramuscularly immunized with full-length Igl and C-Igl were found to be 92% and 96% immune to liver abscess formation, respectively. Immune-response evaluation revealed that C-Igl can generate significant humoral immune responses, with high levels of antibodies in sera from immunized hamsters inhibiting 80% of trophozoites adherence to mammalian cells and inducing 80% more complement-mediated lysis of trophozoites compared with the control. C-Igl was further assessed for its cellular response by cytokine-gene qPCR analysis. The productions of IL-4 (8.4-fold) and IL-10 (2-fold) in the spleen cells of immunized hamsters were enhanced after in vitro stimulation. IL-4 expression was also supported by increased programmed cell death 1 ligand 1 gene. Immunobiochemical characterization strongly suggests the potential of recombinant Igl, especially the C-terminal fragment, as a vaccine candidate against amoebiasis. Moreover, protection through Th2-cell participation enabled effective humoral immunity against amebic liver abscesses.

  5. RAD51AP2, a novel vertebrate- and meiotic-specific protein, sharesa conserved RAD51-interacting C-terminal domain with RAD51AP1/PIR51

    Energy Technology Data Exchange (ETDEWEB)

    Kovalenko, Oleg V.; Wiese, Claudia; Schild, David

    2006-07-25

    Many interacting proteins regulate and/or assist the activities of RAD51, a recombinase which plays a critical role in both DNA repair and meiotic recombination. Yeast two-hybrid screening of a human testis cDNA library revealed a new protein, RAD51AP2 (RAD51 Associated Protein 2), that interacts strongly with RAD51. A full-length cDNA clone predicts a novel vertebrate specific protein of 1159 residues, and the RAD51AP2 transcript was observed only in meiotic tissue (i.e. adult testis and fetal ovary), suggesting a meiotic-specific function for RAD51AP2. In HEK293 cells the interaction of RAD51 with an ectopically-expressed recombinant large fragment of RAD51AP2 requires the C-terminal 57 residues of RAD51AP2. This RAD51-binding region shows 81% homology to the C-terminus of RAD51AP1/PIR51, an otherwise totally unrelated RAD51-binding partner that is ubiquitously expressed. Analyses using truncations and point mutations in both RAD51AP1 and RAD51AP2 demonstrate that these proteins use the same structural motif for RAD51 binding. RAD54 shares some homology with this RAD51-binding motif, but this homologous region plays only an accessory role to the adjacent main RAD51-interacting region, which has been narrowed here to 40 amino acids. A novel protein, RAD51AP2, has been discovered that interacts with RAD51 through a C-terminal motif also present in RAD51AP1.

  6. Identification of a novel binding site between HIV type 1 Nef C-terminal flexible loop and AP2 required for Nef-mediated CD4 downregulation.

    Science.gov (United States)

    Jin, Yong-Jiu; Cai, Catherine Yi; Mezei, Mihaly; Ohlmeyer, Michael; Sanchez, Roberto; Burakoff, Steven J

    2013-04-01

    HIV-1 Nef is an accessory protein necessary for HIV-1 virulence and rapid AIDS development. Nef promotes viral replication and infection by connecting CD4 and several other cell surface receptors to the clathrin adaptor protein AP2, resulting in the internalization and degradation of the receptors interacting with Nef. We investigated how Nef can mediate constitutive receptor endocytosis through the interaction of the dileucine motif in its C-terminal flexible loop (C-loop) with AP2, whereas AP2 binding of the transmembrane receptors usually results in an equilibrated (recycled) endocytosis. Our results indicated that in addition to the dileucine motif, there is a second motif in the Nef C-loop involved in the Nef-AP2 interaction. Nef-mediated CD4 downregulation was impaired when the residue in the hydrophobic region in the Nef C-loop (LL165HPMSLHGM173) was mutated to a basic residue K/R or an acidic residue E/D or to the rigid residue P, or when M168L170, L170H171, or G172M173 was mutated to AA. A pull-down assay indicated that AP2 was not coprecipitated with Nef mutants that did not downregulate CD4. Molecular modeling of the Nef C-terminal flexible loop in complex with AP2 suggests that M168L170 occupies a pocket in the AP2 σ2 subunit. Our data suggest a new model in the Nef-AP2 interaction in which the hydrophobic region in the Nef C-loop with the dileucine (L164L165) motif and M168L170 motif binds to AP2(σ2), while the acidic motif E174 and D175 binds to AP2(α), which explains how Nef through the flexible loop connects CD4 to AP2 for constitutive CD4 downregulation.

  7. Identification of a Novel TGF-β-Binding Site in the Zona Pellucida C-terminal (ZP-C) Domain of TGF-β-Receptor-3 (TGFR-3).

    Science.gov (United States)

    Diestel, Uschi; Resch, Marcus; Meinhardt, Kathrin; Weiler, Sigrid; Hellmann, Tina V; Mueller, Thomas D; Nickel, Joachim; Eichler, Jutta; Muller, Yves A

    2013-01-01

    The zona pellucida (ZP) domain is present in extracellular proteins such as the zona pellucida proteins and tectorins and participates in the formation of polymeric protein networks. However, the ZP domain also occurs in the cytokine signaling co-receptor transforming growth factor β (TGF-β) receptor type 3 (TGFR-3, also known as betaglycan) where it contributes to cytokine ligand recognition. Currently it is unclear how the ZP domain architecture enables this dual functionality. Here, we identify a novel major TGF-β-binding site in the FG loop of the C-terminal subdomain of the murine TGFR-3 ZP domain (ZP-C) using protein crystallography, limited proteolysis experiments, surface plasmon resonance measurements and synthetic peptides. In the murine 2.7 Å crystal structure that we are presenting here, the FG-loop is disordered, however, well-ordered in a recently reported homologous rat ZP-C structure. Surprisingly, the adjacent external hydrophobic patch (EHP) segment is registered differently in the rat and murine structures suggesting that this segment only loosely associates with the remaining ZP-C fold. Such a flexible and temporarily-modulated association of the EHP segment with the ZP domain has been proposed to control the polymerization of ZP domain-containing proteins. Our findings suggest that this flexibility also extends to the ZP domain of TGFR-3 and might facilitate co-receptor ligand interaction and presentation via the adjacent FG-loop. This hints that a similar C-terminal region of the ZP domain architecture possibly regulates both the polymerization of extracellular matrix proteins and cytokine ligand recognition of TGFR-3.

  8. A 21-kDa C-terminal fragment of protein-disulfide isomerase has isomerase, chaperone, and anti-chaperone activities.

    Science.gov (United States)

    Puig, A; Primm, T P; Surendran, R; Lee, J C; Ballard, K D; Orkiszewski, R S; Makarov, V; Gilbert, H F

    1997-12-26

    A catalyst of disulfide formation and isomerization during protein folding, protein-disulfide isomerase (PDI) has two catalytic sites housed in two domains homologous to thioredoxin, one near the N terminus and the other near the C terminus. The thioredoxin domains, by themselves, can catalyze disulfide formation, but they are unable to catalyze disulfide isomerizations (Darby, N. J. and Creighton, T. E. (1995) Biochemistry 34, 11725-11735). A 21-kDa, C-terminal fragment of PDI (amino acids 308-491), termed weePDI, comprises the C-terminal third of the molecule. The kcat for ribonuclease oxidative folding by weePDI is 0.26 +/- 0.02 min-1, 3-fold lower than the wild-type enzyme but indistinguishable from the activity of a full-length mutant of PDI in which both active site cysteines of the N-terminal thioredoxin domain have been mutated to serine. Eliminating the ability of weePDI to escape easily from covalent complexes with substrate by mutating the active site cysteine nearer the C terminus to serine has a large effect on the isomerase activity of weePDI compared with its effect on the full-length enzyme. weePDI also displays chaperone and anti-chaperone activity characteristic of the full-length molecule. As isolated, weePDI is a disulfide-linked dimer in which the single cysteine (Cys-326) outside active site cross-links two weePDI monomers. The presence of the intermolecular disulfide decreases the activity by more than 2-fold. The results imply that the functions of the core thioredoxin domains of PDI and other members of the thioredoxin superfamily might be modified quite easily by the addition of relatively small accessory domains.

  9. Charge neutralization as the major factor for the assembly of nucleocapsid-like particles from C-terminal truncated hepatitis C virus core protein

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    Theo Luiz Ferraz de Souza

    2016-11-01

    Full Text Available Background Hepatitis C virus (HCV core protein, in addition to its structural role to form the nucleocapsid assembly, plays a critical role in HCV pathogenesis by interfering in several cellular processes, including microRNA and mRNA homeostasis. The C-terminal truncated HCV core protein (C124 is intrinsically unstructured in solution and is able to interact with unspecific nucleic acids, in the micromolar range, and to assemble into nucleocapsid-like particles (NLPs in vitro. The specificity and propensity of C124 to the assembly and its implications on HCV pathogenesis are not well understood. Methods Spectroscopic techniques, transmission electron microscopy and calorimetry were used to better understand the propensity of C124 to fold or to multimerize into NLPs when subjected to different conditions or in the presence of unspecific nucleic acids of equivalent size to cellular microRNAs. Results The structural analysis indicated that C124 has low propensity to self-folding. On the other hand, for the first time, we show that C124, in the absence of nucleic acids, multimerizes into empty NLPs when subjected to a pH close to its isoelectric point (pH ≈ 12, indicating that assembly is mainly driven by charge neutralization. Isothermal calorimetry data showed that the assembly of NLPs promoted by nucleic acids is enthalpy driven. Additionally, data obtained from fluorescence correlation spectroscopy show that C124, in nanomolar range, was able to interact and to sequester a large number of short unspecific nucleic acids into NLPs. Discussion Together, our data showed that the charge neutralization is the major factor for the nucleocapsid-like particles assembly from C-terminal truncated HCV core protein. This finding suggests that HCV core protein may physically interact with unspecific cellular polyanions, which may correspond to microRNAs and mRNAs in a host cell infected by HCV, triggering their confinement into infectious particles.

  10. Lack of a 5.9 kDa peptide C-terminal fragment of fibrinogen α chain precedes fibrosis progression in patients with liver disease.

    Science.gov (United States)

    Marfà, Santiago; Crespo, Gonzalo; Reichenbach, Vedrana; Forns, Xavier; Casals, Gregori; Morales-Ruiz, Manuel; Navasa, Miquel; Jiménez, Wladimiro

    2014-01-01

    Early detection of fibrosis progression is of major relevance for the diagnosis and management of patients with liver disease. This study was designed to find non-invasive biomarkers for fibrosis in a clinical context where this process occurs rapidly, HCV-positive patients who underwent liver transplantation (LT). We analyzed 93 LT patients with HCV recurrence, 41 non-LT patients with liver disease showing a fibrosis stage F≥1 and 9 patients without HCV recurrence who received antiviral treatment before LT, as control group. Blood obtained from 16 healthy subjects was also analyzed. Serum samples were fractionated by ion exchange chromatography and their proteomic profile was analyzed by SELDI-TOF-MS. Characterization of the peptide of interest was performed by ion chromatography and electrophoresis, followed by tandem mass spectrometry identification. Marked differences were observed between the serum proteome profile of LT patients with early fibrosis recurrence and non-recurrent LT patients. A robust peak intensity located at 5905 m/z was the distinguishing feature of non-recurrent LT patients. However, the same peak was barely detected in recurrent LT patients. Similar results were found when comparing samples of healthy subjects with those of non-LT fibrotic patients, indicating that our findings were not related to either LT or HCV infection. Using tandem mass-spectrometry, we identified the protein peak as a C-terminal fragment of the fibrinogen α chain. Cell culture experiments demonstrated that TGF-β reduces α-fibrinogen mRNA expression and 5905 m/z peak intensity in HepG2 cells, suggesting that TGF-β activity regulates the circulating levels of this protein fragment. In conclusion, we identified a 5.9 kDa C-terminal fragment of the fibrinogen α chain as an early serum biomarker of fibrogenic processes in patients with liver disease.

  11. The C-terminal region of A-kinase anchor protein 350 (AKAP350A) enables formation of microtubule-nucleation centers and interacts with pericentriolar proteins.

    Science.gov (United States)

    Kolobova, Elena; Roland, Joseph T; Lapierre, Lynne A; Williams, Janice A; Mason, Twila A; Goldenring, James R

    2017-12-15

    Microtubules in animal cells assemble (nucleate) from both the centrosome and the cis-Golgi cisternae. A-kinase anchor protein 350 kDa (AKAP350A, also called AKAP450/CG-NAP/AKAP9) is a large scaffolding protein located at both the centrosome and Golgi apparatus. Previous findings have suggested that AKAP350 is important for microtubule dynamics at both locations, but how this scaffolding protein assembles microtubule nucleation machinery is unclear. Here, we found that overexpression of the C-terminal third of AKAP350A, enhanced GFP-AKAP350A(2691-3907), induces the formation of multiple microtubule-nucleation centers (MTNCs). Nevertheless, these induced MTNCs lacked "true" centriole proteins, such as Cep135. Mapping analysis with AKAP350A truncations demonstrated that AKAP350A contains discrete regions responsible for promoting or inhibiting the formation of multiple MTNCs. Moreover, GFP-AKAP350A(2691-3907) recruited several pericentriolar proteins to MTNCs, including γ-tubulin, pericentrin, Cep68, Cep170, and Cdk5RAP2. Proteomic analysis indicated that Cdk5RAP2 and Cep170 both interact with the microtubule nucleation-promoting region of AKAP350A, whereas Cep68 interacts with the distal C-terminal AKAP350A region. Yeast two-hybrid assays established a direct interaction of Cep170 with AKAP350A. Super-resolution and deconvolution microscopy analyses were performed to define the association of AKAP350A with centrosomes, and these studies disclosed that AKAP350A spans the bridge between centrioles, co-localizing with rootletin and Cep68 in the linker region. siRNA-mediated depletion of AKAP350A caused displacement of both Cep68 and Cep170 from the centrosome. These results suggest that AKAP350A acts as a scaffold for factors involved in microtubule nucleation at the centrosome and coordinates the assembly of protein complexes associating with the intercentriolar bridge.

  12. Development and characterization of a novel C-terminal inhibitor of Hsp90 in androgen dependent and independent prostate cancer cells

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    Eskew Jeffery D

    2011-10-01

    Full Text Available Abstract Background The molecular chaperone, heat shock protein 90 (Hsp90 has been shown to be overexpressed in a number of cancers, including prostate cancer, making it an important target for drug discovery. Unfortunately, results with N-terminal inhibitors from initial clinical trials have been disappointing, as toxicity and resistance resulting from induction of the heat shock response (HSR has led to both scheduling and administration concerns. Therefore, Hsp90 inhibitors that do not induce the heat shock response represent a promising new direction for the treatment of prostate cancer. Herein, the development of a C-terminal Hsp90 inhibitor, KU174, is described, which demonstrates anti-cancer activity in prostate cancer cells in the absence of a HSR and describe a novel approach to characterize Hsp90 inhibition in cancer cells. Methods PC3-MM2 and LNCaP-LN3 cells were used in both direct and indirect in vitro Hsp90 inhibition assays (DARTS, Surface Plasmon Resonance, co-immunoprecipitation, luciferase, Western blot, anti-proliferative, cytotoxicity and size exclusion chromatography to characterize the effects of KU174 in prostate cancer cells. Pilot in vivo efficacy studies were also conducted with KU174 in PC3-MM2 xenograft studies. Results KU174 exhibits robust anti-proliferative and cytotoxic activity along with client protein degradation and disruption of Hsp90 native complexes without induction of a HSR. Furthermore, KU174 demonstrates direct binding to the Hsp90 protein and Hsp90 complexes in cancer cells. In addition, in pilot in-vivo proof-of-concept studies KU174 demonstrates efficacy at 75 mg/kg in a PC3-MM2 rat tumor model. Conclusions Overall, these findings suggest C-terminal Hsp90 inhibitors have potential as therapeutic agents for the treatment of prostate cancer.

  13. C-terminal β9-strand of the cyclic nucleotide-binding homology domain stabilizes activated states of Kv11.1 channels.

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    Chai Ann Ng

    Full Text Available Kv11.1 potassium channels are important for regulation of the normal rhythm of the heartbeat. Reduced activity of Kv11.1 channels causes long QT syndrome type 2, a disorder that increases the risk of cardiac arrhythmias and sudden cardiac arrest. Kv11.1 channels are members of the KCNH subfamily of voltage-gated K(+ channels. However, they also share many similarities with the cyclic nucleotide gated ion channel family, including having a cyclic nucleotide-binding homology (cNBH domain. Kv11.1 channels, however, are not directly regulated by cyclic nucleotides. Recently, crystal structures of the cNBH domain from mEAG and zELK channels, both members of the KCNH family of voltage-gated potassium channels, revealed that a C-terminal β9-strand in the cNBH domain occupied the putative cyclic nucleotide-binding site thereby precluding binding of cyclic nucleotides. Here we show that mutations to residues in the β9-strand affect the stability of the open state relative to the closed state of Kv11.1 channels. We also show that disrupting the structure of the β9-strand reduces the stability of the inactivated state relative to the open state. Clinical mutations located in this β9-strand result in reduced trafficking efficiency, which suggests that binding of the C-terminal β9-strand to the putative cyclic nucleotide-binding pocket is also important for assembly and trafficking of Kv11.1 channels.

  14. Lack of a 5.9 kDa peptide C-terminal fragment of fibrinogen α chain precedes fibrosis progression in patients with liver disease.

    Directory of Open Access Journals (Sweden)

    Santiago Marfà

    Full Text Available Early detection of fibrosis progression is of major relevance for the diagnosis and management of patients with liver disease. This study was designed to find non-invasive biomarkers for fibrosis in a clinical context where this process occurs rapidly, HCV-positive patients who underwent liver transplantation (LT. We analyzed 93 LT patients with HCV recurrence, 41 non-LT patients with liver disease showing a fibrosis stage F≥1 and 9 patients without HCV recurrence who received antiviral treatment before LT, as control group. Blood obtained from 16 healthy subjects was also analyzed. Serum samples were fractionated by ion exchange chromatography and their proteomic profile was analyzed by SELDI-TOF-MS. Characterization of the peptide of interest was performed by ion chromatography and electrophoresis, followed by tandem mass spectrometry identification. Marked differences were observed between the serum proteome profile of LT patients with early fibrosis recurrence and non-recurrent LT patients. A robust peak intensity located at 5905 m/z was the distinguishing feature of non-recurrent LT patients. However, the same peak was barely detected in recurrent LT patients. Similar results were found when comparing samples of healthy subjects with those of non-LT fibrotic patients, indicating that our findings were not related to either LT or HCV infection. Using tandem mass-spectrometry, we identified the protein peak as a C-terminal fragment of the fibrinogen α chain. Cell culture experiments demonstrated that TGF-β reduces α-fibrinogen mRNA expression and 5905 m/z peak intensity in HepG2 cells, suggesting that TGF-β activity regulates the circulating levels of this protein fragment. In conclusion, we identified a 5.9 kDa C-terminal fragment of the fibrinogen α chain as an early serum biomarker of fibrogenic processes in patients with liver disease.

  15. N- and C-terminal flanking regions modulate light-induced signal transduction in the LOV2 domain of the blue light sensor phototropin 1 from Avena sativa.

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    Halavaty, Andrei S; Moffat, Keith

    2007-12-11

    Light sensing by photoreceptors controls phototropism, chloroplast movement, stomatal opening, and leaf expansion in plants. Understanding the molecular mechanism by which these processes are regulated requires a quantitative description of photoreceptor dynamics. We focus on a light-driven signal transduction mechanism in the LOV2 domain (LOV, light, oxygen, voltage) of the blue light photoreceptor phototropin 1 from Avena sativa (oat). High-resolution crystal structures of the dark and light states of an oat LOV2 construct including residues Leu404 through Leu546 (LOV2 (404-546)) have been determined at 105 and 293 K. In all four structures, LOV2 (404-546) exhibits the typical Per-ARNT-Sim (PAS) fold, flanked by an additional conserved N-terminal turn-helix-turn motif and a C-terminal flanking region containing an amphipathic Jalpha helix. These regions dock on the LOV2 core domain and bury several hydrophobic residues of the central beta-sheet of the core domain that would otherwise be exposed to solvent. Light structures of LOV2 (404-546) reveal that formation of the covalent bond between Cys450 and the C4a atom of the flavin mononucleotide (FMN) results in local rearrangement of the hydrogen-bonding network in the FMN binding pocket. These rearrangements are associated with disruption of the Asn414-Asp515 hydrogen bond on the surface of the protein and displacement of the N- and C-terminal flanking regions of LOV2 (404-546), both of which constitute a structural signal.

  16. Tubulin C-terminal post-translational modifications do not occur in wood forming tissue of Populus

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    Hao Hu

    2016-10-01

    Full Text Available Cortical microtubules (MTs are evolutionarily conserved cytoskeletal components with specialized roles in plants, including regulation of cell wall biogenesis. MT functions and dynamics are dictated by the composition of their monomeric subunits, α- (TUA and β-tubulins (TUB, which in animals and protists are subject to both transcriptional regulation and post-translational modifications (PTM. While spatiotemporal regulation of tubulin gene expression has been reported in plants, whether and to what extent tubulin PTMs occur in these species remain poorly understood. We chose the woody perennial Populus for investigation of tubulin PTMs in this study, with a particular focus on developing xylem where high tubulin transcript levels support MT-dependent secondary cell wall deposition. Mass spectrometry and immunodetection concurred that detyrosination, nontyrosination and glutamylation were essentially absent in tubulins isolated from wood-forming tissues of Populus deltoides and P. tremula  alba. Label-free quantification of tubulin isotypes and RNA-Seq estimation of tubulin transcript abundance were largely consistent with transcriptional regulation. However, two TUB isoypes were detected at noticeably lower levels than expected based on RNA-Seq transcript abundance in both Populus species. These findings led us to conclude that MT composition during wood formation depends exclusively on transcriptional and, to a lesser extent, translational regulation of tubulin isotypes.

  17. The C-terminal Helix of Pseudomonas aeruginosa Elongation Factor Ts Tunes EF-Tu Dynamics to Modulate Nucleotide Exchange*

    Science.gov (United States)

    De Laurentiis, Evelina Ines; Mercier, Evan; Wieden, Hans-Joachim

    2016-01-01

    Little is known about the conservation of critical kinetic parameters and the mechanistic strategies of elongation factor (EF) Ts-catalyzed nucleotide exchange in EF-Tu in bacteria and particularly in clinically relevant pathogens. EF-Tu from the clinically relevant pathogen Pseudomonas aeruginosa shares over 84% sequence identity with the corresponding elongation factor from Escherichia coli. Interestingly, the functionally closely linked EF-Ts only shares 55% sequence identity. To identify any differences in the nucleotide binding properties, as well as in the EF-Ts-mediated nucleotide exchange reaction, we performed a comparative rapid kinetics and mutagenesis analysis of the nucleotide exchange mechanism for both the E. coli and P. aeruginosa systems, identifying helix 13 of EF-Ts as a previously unnoticed regulatory element in the nucleotide exchange mechanism with species-specific elements. Our findings support the base side-first entry of the nucleotide into the binding pocket of the EF-Tu·EF-Ts binary complex, followed by displacement of helix 13 and rapid binding of the phosphate side of the nucleotide, ultimately leading to the release of EF-Ts. PMID:27624934

  18. Reducing C-terminal-truncated alpha-synuclein by immunotherapy attenuates neurodegeneration and propagation in Parkinson's disease-like models.

    Science.gov (United States)

    Games, Dora; Valera, Elvira; Spencer, Brian; Rockenstein, Edward; Mante, Michael; Adame, Anthony; Patrick, Christina; Ubhi, Kiren; Nuber, Silke; Sacayon, Patricia; Zago, Wagner; Seubert, Peter; Barbour, Robin; Schenk, Dale; Masliah, Eliezer

    2014-07-09

    Parkinson's disease (PD) and dementia with Lewy bodies (DLB) are common neurodegenerative disorders of the aging population, characterized by progressive and abnormal accumulation of α-synuclein (α-syn). Recent studies have shown that C-terminus (CT) truncation and propagation of α-syn play a role in the pathogenesis of PD/DLB. Therefore, we explored the effect of passive immunization against the CT of α-syn in the mThy1-α-syn transgenic (tg) mouse model, which resembles the striato-nigral and motor deficits of PD. Mice were immunized with the new monoclonal antibodies 1H7, 5C1, or 5D12, all directed against the CT of α-syn. CT α-syn antibodies attenuated synaptic and axonal pathology, reduced the accumulation of CT-truncated α-syn (CT-α-syn) in axons, rescued the loss of tyrosine hydroxylase fibers in striatum, and improved motor and memory deficits. Among them, 1H7 and 5C1 were most effective at decreasing levels of CT-α-syn and higher-molecular-weight aggregates. Furthermore, in vitro studies showed that preincubation of recombinant α-syn with 1H7 and 5C1 prevented CT cleavage of α-syn. In a cell-based system, CT antibodies reduced cell-to-cell propagation of full-length α-syn, but not of the CT-α-syn that lacked the 118-126 aa recognition site needed for antibody binding. Furthermore, the results obtained after lentiviral expression of α-syn suggest that antibodies might be blocking the extracellular truncation of α-syn by calpain-1. Together, these results demonstrate that antibodies against the CT of α-syn reduce levels of CT-truncated fragments of the protein and its propagation, thus ameliorating PD-like pathology and improving behavioral and motor functions in a mouse model of this disease. Copyright © 2014 the authors 0270-6474/14/349441-14$15.00/0.

  19. Ubiquitin C-Terminal Hydrolase L1 (UCH-L1) Promotes Hippocampus-Dependent Memory via Its Deubiquitinating Effect on TrkB.

    Science.gov (United States)

    Guo, Yun-Yun; Lu, Yi; Zheng, Yuan; Chen, Xiao-Rong; Dong, Jun-Lu; Yuan, Rong-Rong; Huang, Shu-Hong; Yu, Hui; Wang, Yue; Chen, Zhe-Yu; Su, Bo

    2017-06-21

    Multiple studies have established that brain-derived neurotrophic factor (BDNF) plays a critical role in the regulation of synaptic plasticity via its receptor, TrkB. In addition to being phosphorylated, TrkB has also been demonstrated to be ubiquitinated. However, the mechanisms of TrkB ubiquitination and its biological functions remain poorly understood. In this study, we demonstrate that ubiquitin C-terminal hydrolase L1 (UCH-L1) promotes contextual fear conditioning learning and memory via the regulation of ubiquitination of TrkB. We provide evidence that UCH-L1 can deubiquitinate TrkB directly. K460 in the juxtamembane domain of TrkB is the primary ubiquitination site and is regulated by UCH-L1. By using a peptide that competitively inhibits the association between UCH-L1 and TrkB, we show that the blockade of UCH-L1-regulated TrkB deubiquitination leads to increased BDNF-induced TrkB internalization and consequently directs the internalized TrkB to the degradation pathway, resulting in increased degradation of surface TrkB and attenuation of TrkB activation and its downstream signaling pathways. Moreover, injection of the peptide into the DG region of mice impairs hippocampus-dependent memory. Together, our results suggest that the ubiquitination of TrkB is a mechanism that controls its downstream signaling pathways via the regulation of its endocytosis and postendocytic trafficking and that UCH-L1 mediates the deubiquitination of TrkB and could be a potential target for the modulation of hippocampus-dependent memory.SIGNIFICANCE STATEMENT Ubiquitin C-terminal hydrolase L1 (UCH-L1) has been demonstrated to play important roles in the regulation of synaptic plasticity and learning and memory. TrkB, the receptor for brain-derived neurotrophic factor, has also been shown to be a potent regulator of synaptic plasticity. In this study, we demonstrate that UCH-L1 functions as a deubiquitinase for TrkB. The blockage of UCH-L1-regulated deubiquitination of Trk

  20. Porphyromonas gingivalis C-terminal signal peptidase PG0026 and HagA interact with outer membrane protein PG27/LptO.

    Science.gov (United States)

    Saiki, K; Konishi, K

    2014-02-01

    Outer membrane protein PG27 is essential for secretion/maturation of conserved C-terminal domain (CTD) proteins such as gingipains, HagA, and PG0026. To determine the binding partner(s) of PG27, we used a Porphyromonas gingivalis mutant strain, 83K48, which expressed functional histidine-tagged PG27. Purification of histidine-tagged PG27 from 83K48 found that 136-kDa and 264-kDa proteins accompanied histidine-tagged PG27. Mass spectrometry revealed the 136-kDa protein and 264-kDa protein to be PG0026 and PG1837 (HagA), respectively. PG0026 is a C-terminal signal peptidase which cleaves the CTDs of other CTD proteins. A cross-linking and a native electrophoresis studies suggested the interaction between histidine-tagged PG27 and HagA and the interaction between histidine-tagged PG27 and PG0026. We constructed Porphyromonas gingivalis gene disrupting mutants, and characterized them. PG0026 was required for the full activities of gingipains, whereas HagA was not. A mutation disrupting PG0026 affected localization of PG27, but a mutation disrupting PG1837 showed little effect on the expression and localizations of PG27 and PG0026. To determine the functional role of HagA, we used PG1837-disruptant 83K54 which expressed functional histidine-tagged PG27. Histidine-tagged PG27 in 83K54 was co-purified with not only PG0026 but also several contaminated proteins. These results suggest that PG0026 interacts with PG27 in the absence of HagA, and that the binding state of a PG27-PG0026 complex was affected and stabilized by HagA. Taken together, these data suggest that secreted PG0026 anchors to the cell by interacting with PG27, is stabilized by HagA, and functions in processing of other CTD proteins such as gingipains. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  1. Caspase-3-derived C-terminal product of synphilin-1 displays antiapoptotic function via modulation of the p53-dependent cell death pathway.

    Science.gov (United States)

    Giaime, Emilie; Sunyach, Claire; Herrant, Magali; Grosso, Sébastien; Auberger, Patrick; McLean, Pamela J; Checler, Frédéric; da Costa, Cristine Alves

    2006-04-28

    Parkinson disease is the second most frequent neurodegenerative disorder after Alzheimer disease. A subset of genetic forms of Parkinson disease has been attributed to alpha-synuclein, a synaptic protein with remarkable chaperone properties. Synphilin-1 is a cytoplasmic protein that has been identified as a partner of alpha-synuclein (Engelender, S., Kaminsky, Z., Guo, X., Sharp, A. H., Amaravi, R. K., Kleiderlein, J. J., Margolis, R. L., Troncoso, J. C., Lanahan, A. A., Worley, P. F., Dawson, V. L., Dawson, T. M., and Ross, C. A. (1999) Nat. Gen. 22, 110-114), but its function remains totally unknown. We show here for the first time that synphilin-1 displays an antiapoptotic function in the control of cell death. We have established transient and stable transfectants overexpressing wild-type synphilin-1 in human embryonic kidney 293 cells, telecephalon-specific murine 1 neurons, and SH-SY5Y neuroblastoma cells, and we show that both cell systems display lower responsiveness to staurosporine and 6-hydroxydopamine. Thus, synphilin-1 reduces procaspase-3 hydrolysis and thereby caspase-3 activity and decreases poly(ADP-ribose) polymerase cleavage, two main indicators of apoptotic cell death. Furthermore, we establish that synphilin-1 drastically reduces p53 transcriptional activity and expression and lowers p53 promoter transactivation and mRNA levels. Interestingly, we demonstrate that synphilin-1 catabolism is enhanced by staurosporine and blocked by caspase-3 inhibitors. Accordingly, we show by transcription/translation assay that recombinant caspase-3 and, to a lesser extent, caspase-6 but not caspase-7 hydrolyze synphilin-1. Furthermore, we demonstrate that mutated synphilin-1, in which a consensus caspase-3 target sequence has been disrupted, resists proteolysis by cellular and recombinant caspases and displays drastically reduced antiapoptotic phenotype. We further show that the caspase-3-derived C-terminal fragment of synphilin-1 was probably responsible for

  2. Casein is an essential cofactor in autoantibody reactivity directed against the C-terminal SmD1 peptide AA 83-119 in systemic lupus erythematosus.

    Science.gov (United States)

    Riemekasten, Gabriela; Marell, Jeannette; Hentschel, Christian; Klein, Rolf; Burmester, Gerd-R; Schoessler, Werner; Hiepe, Falk

    2002-12-01

    The C-terminal peptide SmD1(83-119) has been identified as an important autoantigen in systemic lupus erythematosus (SLE). ELISA studies have shown that roughly 70% of all sera from patients with SLE react with this peptide. Previous findings revealed that the addition of blocking agents and sample dilution buffers influences the discrimination between positive and negative anti-SmD1(83-119) sera in SLE. The aim of the present study was to identify possible cofactors in the anti-SmD1(83-119) reactivity. We therefore tested SLE sera (n=6) for anti-SmD1(83-119) reactivity by ELISA and analysed the effects of different blocking agents (1% skim milk, 1% gelatin, and 1% BSA). In our investigation, lipids were extracted from skim milk using dichlomethane, and the putative fraction was tested to assess the assay's ability to discriminate between positive and negative sera. The effects of enzymatic digestion by casein were analyzed, and different concentrations of casein were used to determine the role of this protein in the detection of anti-SmD1(83-119) antibodies by ELISA. Furthermore, rabbits were immunized with SmD1(83-119) adsorbed to casein and control proteins. One percent skim milk was the most effective blocking agent and sample dilution buffer for the discrimination between positive and negative sera. As demonstrated by SDS electrophoresis, the discriminative capacity was influenced by enzymatic digestion of skim milk proteins, but not by lipid extraction. Differences in anti-SmD1(83-119) reactivity upon variation of the casein concentration suggest that the protein plays a significant role in the detection of anti-SmD1(83-119) antibodies. However, our immunisation studies did not show any effect of casein on anti-SmD1(83-119) reactivity, suggesting that it has no immunogenic effect on the anti-SmD1(83-119) response. In conclusion, casein seems to be an important cofactor in autoantibody reactivity directed against the C-terminal SmD1(83-119) peptide and

  3. Structure-function analysis of mouse Sry reveals dual essential roles of the C-terminal polyglutamine tract in sex determination.

    Science.gov (United States)

    Zhao, Liang; Ng, Ee Ting; Davidson, Tara-Lynne; Longmuss, Enya; Urschitz, Johann; Elston, Marlee; Moisyadi, Stefan; Bowles, Josephine; Koopman, Peter

    2014-08-12

    The mammalian sex-determining factor SRY comprises a conserved high-mobility group (HMG) box DNA-binding domain and poorly conserved regions outside the HMG box. Mouse Sry is unusual in that it includes a C-terminal polyglutamine (polyQ) tract that is absent in nonrodent SRY proteins, and yet, paradoxically, is essential for male sex determination. To dissect the molecular functions of this domain, we generated a series of Sry mutants, and studied their biochemical properties in cell lines and transgenic mouse embryos. Sry protein lacking the polyQ domain was unstable, due to proteasomal degradation. Replacing this domain with irrelevant sequences stabilized the protein but failed to restore Sry's ability to up-regulate its key target gene SRY-box 9 (Sox9) and its sex-determining function in vivo. These functions were restored only when a VP16 transactivation domain was substituted. We conclude that the polyQ domain has important roles in protein stabilization and transcriptional activation, both of which are essential for male sex determination in mice. Our data disprove the hypothesis that the conserved HMG box domain is the only functional domain of Sry, and highlight an evolutionary paradox whereby mouse Sry has evolved a novel bifunctional module to activate Sox9 directly, whereas SRY proteins in other taxa, including humans, seem to lack this ability, presumably making them dependent on partner proteins(s) to provide this function.

  4. Role of p54 RNA helicase activity and its C-terminal domain in translational repression, P-body localization and assembly.

    Science.gov (United States)

    Minshall, Nicola; Kress, Michel; Weil, Dominique; Standart, Nancy

    2009-05-01

    The RNA helicase p54 (DDX6, Dhh1, Me31B, Cgh-1, RCK) is a prototypic component of P-(rocessing) bodies in cells ranging from yeast to human. Previously, we have shown that it is also a component of the large cytoplasmic polyadenylation element-binding protein translation repressor complex in Xenopus oocytes and that when tethered to the 3' untranslated region, Xp54 represses reporter mRNA translation. Here, we examine the role of the p54 helicase activity in translational repression and in P-body formation. Mutagenesis of conserved p54 helicase motifs activates translation in the tethered function assay, reduces accumulation of p54 in P-bodies in HeLa cells, and inhibits its capacity to assemble P-bodies in p54-depleted cells. Similar results were obtained in four helicase motifs implicated in ATP binding and in coupling ATPase and RNA binding activities. This is accompanied by changes in the interaction of the mutant p54 with the oocyte repressor complex components. Surprisingly, the C-terminal D2 domain alone is sufficient for translational repression and complete accumulation in P-bodies, although it is deficient for P-body assembly. We propose a novel RNA helicase model, in which the D2 domain acts as a protein binding platform and the ATPase/helicase activity allows protein complex remodeling that dictates the balance between repressors and an activator of translation.

  5. The C-terminal domains of NF-H and NF-M subunits maintain axonal neurofilament content by blocking turnover of the stationary neurofilament network.

    Directory of Open Access Journals (Sweden)

    Mala V Rao

    Full Text Available Newly synthesized neurofilaments or protofilaments are incorporated into a highly stable stationary cytoskeleton network as they are transported along axons. Although the heavily phosphorylated carboxyl-terminal tail domains of the heavy and medium neurofilament (NF subunits have been proposed to contribute to this process and particularly to stability of this structure, their function is still obscure. Here we show in NF-H/M tail deletion [NF-(H/M(tailΔ] mice that the deletion of both of these domains selectively lowers NF levels 3-6 fold along optic axons without altering either rates of subunit synthesis or the rate of slow axonal transport of NF. Pulse labeling studies carried out over 90 days revealed a significantly faster rate of disappearance of NF from the stationary NF network of optic axons in NF-(H/M(tailΔ mice. Faster NF disappearance was accompanied by elevated levels of NF-L proteolytic fragments in NF-(H/M(tailΔ axons. We conclude that NF-H and NF-M C-terminal domains do not normally regulate NF transport rates as previously proposed, but instead increase the proteolytic resistance of NF, thereby stabilizing the stationary neurofilament cytoskeleton along axons.

  6. The C-terminal part of the surface-associated protein MopE of the methanotroph Methylococcus capsulatus (Bath) is secreted into the growth medium.

    Science.gov (United States)

    Fjellbirkeland, A; Kruger, P G; Bemanian, V; Høgh, B T; Murrell, J C; Jensen, H B

    2001-09-01

    A protein with an apparent molecular mass of 46 kDa was detected as the major polypeptide in the culture medium of the biotechnologically important methanotrophic bacterium Methylococcus capsulatus (Bath). The protein cross-reacted with polyclonal antibodies raised against the outer-membrane-associated protein MopE. The antiserum was used to identify a positive clone from a lambda gt11 library. The nucleotide sequence determined for the clone demonstrated that MopE and the secreted protein are encoded by the same gene, and that the secreted protein represents an N-terminally truncated form of MopE. By using monospecific antibodies against MopE in immunogold electron microscopy, the protein was localized at the cell surface and cell periphery. The mopE gene was expressed in Escherichia coli. The MopE protein synthesized was found in the periplasmic space of E. coli. No protein with sequence similarity over the entire length of MopE was detected in the databases, but some sequence similarity to the copper-repressible CorA protein of the methanotroph Methylomicrobium albus (Berson and Lidstrom 1997) was observed for the C-terminal region of MopE.

  7. C-Terminal carbohydrate-binding module 9_2 fused to the N-terminus of GH11 xylanase from Aspergillus niger.

    Science.gov (United States)

    Xu, Wenxuan; Liu, Yajuan; Ye, Yanxin; Liu, Meng; Han, Laichuang; Song, Andong; Liu, Liangwei

    2016-10-01

    The 9_2 carbohydrate-binding module (C2) locates natively at the C-terminus of the GH10 thermophilic xylanase from Thermotoga marimita. When fused to the C-terminus, C2 improved thermostability of a GH11 xylanase (Xyn) from Aspergillus niger. However, a question is whether the C-terminal C2 would have a thermostabilizing effect when fused to the N-terminus of a catalytic module. A chimeric enzyme, C2-Xyn, was created by step-extension PCR, cloned in pET21a(+), and expressed in E. coli BL21(DE3). The C2-Xyn exhibited a 2 °C higher optimal temperature, a 2.8-fold longer thermostability, and a 4.5-fold higher catalytic efficiency on beechwood xylan than the Xyn. The C2-Xyn exhibited a similar affinity for binding to beechwood xylan and a higher affinity for oat-spelt xylan than Xyn. C2 is a thermostabilizing carbohydrate-binding module and provides a model of fusion at an enzymatic terminus inconsistent with the modular natural terminal location.

  8. Emerging role of N- and C-terminal interactions in stabilizing (β/α8 fold with special emphasis on Family 10 xylanases

    Directory of Open Access Journals (Sweden)

    Amit Bhardwaj

    2012-09-01

    Full Text Available Xylanases belong to an important class of industrial enzymes. Various xylanases have been purified and characterized from a plethora of organisms including bacteria, marine algae, plants, protozoans, insects, snails and crustaceans. Depending on the source, the enzymatic activity of xylanases varies considerably under various physico-chemical conditions such as temperature, pH, high salt and in the presence of proteases. Family 10 or glycosyl hydrolase 10 (GH10 xylanases are one of the well characterized and thoroughly studied classes of industrial enzymes. The TIM-barrel fold structure which is ubiquitous in nature is one of the characteristics of family 10 xylanases. Family 10 xylanases have been used as a “model system” due to their TIM-barrel fold to dissect and understand protein stability under various conditions. A better understanding of structure-stability-function relationships of family 10 xylanases allows one to apply these governing molecular rules to engineer other TIM-barrel fold proteins to improve their stability and retain function(s under adverse conditions. In this review, we discuss the implications of N-and C-terminal interactions, observed in family 10 xylanases on protein stability under extreme conditions. The role of metal binding and aromatic clusters in protein stability is also discussed. Studying and understanding family 10 xylanase structure and function, can contribute to our protein engineering knowledge.

  9. Synchrotron radiation circular dichroism spectroscopy-defined structure of the C-terminal domain of NaChBac and its role in channel assembly

    Science.gov (United States)

    Powl, Andrew M.; O’Reilly, Andrias O.; Miles, Andrew J.; Wallace, B. A.

    2010-01-01

    Extramembranous domains play important roles in the structure and function of membrane proteins, contributing to protein stability, forming association domains, and binding ancillary subunits and ligands. However, these domains are generally flexible, making them difficult or unsuitable targets for obtaining high-resolution X-ray and NMR structural information. In this study we show that the highly sensitive method of synchrotron radiation circular dichroism (SRCD) spectroscopy can be used as a powerful tool to investigate the structure of the extramembranous C-terminal domain (CTD) of the prokaryotic voltage-gated sodium channel (NaV) from Bacillus halodurans, NaChBac. Sequence analyses predict its CTD will consist of an unordered region followed by an α-helix, which has a propensity to form a multimeric coiled-coil motif, and which could form an association domain in the homotetrameric NaChBac channel. By creating a number of shortened constructs we have shown experimentally that the CTD does indeed contain a stretch of ∼20 α-helical residues preceded by a nonhelical region adjacent to the final transmembrane segment and that the efficiency of assembly of channels in the membrane progressively decreases as the CTD residues are removed. Analyses of the CTDs of 32 putative prokaryotic NaV sequences suggest that a CTD helical bundle is a structural feature conserved throughout the bacterial sodium channel family. PMID:20663949

  10. Structure–function analysis of mouse Sry reveals dual essential roles of the C-terminal polyglutamine tract in sex determination

    Science.gov (United States)

    Zhao, Liang; Ng, Ee Ting; Davidson, Tara-Lynne; Longmuss, Enya; Urschitz, Johann; Elston, Marlee; Moisyadi, Stefan; Bowles, Josephine; Koopman, Peter

    2014-01-01

    The mammalian sex-determining factor SRY comprises a conserved high-mobility group (HMG) box DNA-binding domain and poorly conserved regions outside the HMG box. Mouse Sry is unusual in that it includes a C-terminal polyglutamine (polyQ) tract that is absent in nonrodent SRY proteins, and yet, paradoxically, is essential for male sex determination. To dissect the molecular functions of this domain, we generated a series of Sry mutants, and studied their biochemical properties in cell lines and transgenic mouse embryos. Sry protein lacking the polyQ domain was unstable, due to proteasomal degradation. Replacing this domain with irrelevant sequences stabilized the protein but failed to restore Sry’s ability to up-regulate its key target gene SRY-box 9 (Sox9) and its sex-determining function in vivo. These functions were restored only when a VP16 transactivation domain was substituted. We conclude that the polyQ domain has important roles in protein stabilization and transcriptional activation, both of which are essential for male sex determination in mice. Our data disprove the hypothesis that the conserved HMG box domain is the only functional domain of Sry, and highlight an evolutionary paradox whereby mouse Sry has evolved a novel bifunctional module to activate Sox9 directly, whereas SRY proteins in other taxa, including humans, seem to lack this ability, presumably making them dependent on partner proteins(s) to provide this function. PMID:25074915

  11. Hydrogen-induced nanotunnel structure on the C-terminated β-SiC(0 0 1)-c(2 × 2) surface investigated by ab-initio calculations

    Energy Technology Data Exchange (ETDEWEB)

    Rosso, E.F. [Departamento de Química, Universidade Federal de Santa Maria, 97105-900, Santa Maria, RS (Brazil); Baierle, R.J., E-mail: rbaierle@smail.ufsm.br [Departamento de Física, Universidade Federal de Santa Maria, 97105-900, Santa Maria, RS (Brazil); Orellana, W. [Departamento de Ciencias Físicas, Universidad Andres Bello, República 220, 837-0134 Santiago (Chile); Miwa, R.H. [Instituto de Física, Universidade Federal de Uberlândia, 38408-100, Uberlândia, MG (Brazil)

    2015-12-01

    Graphical abstract: - Highlights: • β-SiC(0 0 1) surface. • Hydrogenation. • DFT calculations – PBE and HSE06 functional for the exchange correlation term. • Nanotunnel formation. • Electronic confinement. - Abstract: The structural and electronic properties of pristine and H-passivated C-terminated β-SiC(0 0 1)-c(2 × 2) surface are addressed by ab initio calculations. Here, we verify the formation of C chains composed by double-bonded dimers rows (C=C), separated by triple-bonded bridged dimers (C≡C). The surface states near the bandgap are confined along the C=C dimer rows, with no electronic contribution from the C≡C bridged dimers. After hydrogenation, the C-chains are strongly modified, forming subsurface voids or nanotunnel (NT) structures. By considering a plausible set of energy release steps for increasing hydrogenation, we obtain a C-rich NT ruled by the C=C dimer rows. Somewhat similar to that recently reported on the Si-rich termination, but 0.8 eV lower in energy. The electronic band structures of both Si-rich and C-rich NTs have been examined within the hybrid HSE06 functional, which are compared with those previously reported using a semilocal functional.

  12. The application of an alanine-substituted mutant of the C-terminal fragment of Clostridium perfringens enterotoxin as a mucosal vaccine in mice.

    Science.gov (United States)

    Suzuki, Hidehiko; Kondoh, Masuo; Kakutani, Hideki; Yamane, Seiji; Uchida, Hiroshi; Hamakubo, Takao; Yagi, Kiyohito

    2012-01-01

    Efficient delivery of antigen to mucosal immune tissues is an essential part of mucosal vaccination. Claudin-4 is expressed on the epithelial cells that cover the mucosal immune tissues. We previously found that claudin-4-targeting is a promising strategy for mucosal vaccination by using a claudin-4 binder, the C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE). Substitution of Asn and Ser at positions 309 and 313, respectively, with alanine increased the affinity of C-CPE for claudin-4. However, application of the C-CPE mutant as a mucosal vaccine has never been tried. Here, we investigated whether the C-CPE mutant could serve as a mucosal vaccine. We used ovalbumin (OVA) as a model antigen and fused the C-CPE mutant to it. The resultant fusion protein was bound to claudin-4. When mice were immunized with the C-CPE mutant-fused OVA, OVA-specific serum IgG and nasal IgA increased relative to levels in mice immunized with a C-CPE-fused antigen. Immunization with the C-CPE mutant-fused OVA activated Th1- and Th2-type responses and led to increased anti-tumor activity against OVA-expressing thymoma cells relative to that of mice immunized with the C-CPE-fused antigen. These findings suggest that the alanine-substituted C-CPE mutant shows promise as a claudin-targeted mucosal vaccine. Copyright © 2011 Elsevier Ltd. All rights reserved.

  13. Identification of two pentatricopeptide repeat genes required for RNA editing and zinc binding by C-terminal cytidine deaminase-like domains.

    Science.gov (United States)

    Hayes, Michael L; Giang, Karolyn; Berhane, Beniam; Mulligan, R Michael

    2013-12-20

    Many transcripts expressed from plant organelle genomes are modified by C-to-U RNA editing. Nuclear encoded pentatricopeptide repeat (PPR) proteins are required as RNA binding specificity determinants in the RNA editing mechanism. Bioinformatic analysis has shown that most of the Arabidopsis PPR proteins necessary for RNA editing events include a C-terminal portion that shares structural characteristics with a superfamily of deaminases. The DYW deaminase domain includes a highly conserved zinc binding motif that shares characteristics with cytidine deaminases. The Arabidopsis PPR genes, ELI1 and DOT4, both have DYW deaminase domains and are required for single RNA editing events in chloroplasts. The ELI1 DYW deaminase domain was expressed as a recombinant protein in Escherichia coli and was shown to bind two zinc atoms per polypeptide. Thus, the DYW deaminase domain binds a zinc metal ion, as expected for a cytidine deaminase, and is potentially the catalytic component of an editing complex. Genetic complementation experiments demonstrate that large portions of the DYW deaminase domain of ELI1 may be eliminated, but the truncated genes retain the ability to restore editing site conversion in a mutant plant. These results suggest that the catalytic activity can be supplied in trans by uncharacterized protein(s) of the editosome.

  14. Characterization of human cyclin-dependent kinase 12 (CDK12) and CDK13 complexes in C-terminal domain phosphorylation, gene transcription, and RNA processing.

    Science.gov (United States)

    Liang, Kaiwei; Gao, Xin; Gilmore, Joshua M; Florens, Laurence; Washburn, Michael P; Smith, Edwin; Shilatifard, Ali

    2015-03-01

    Cyclin-dependent kinase 9 (CDK9) and CDK12 have each been demonstrated to phosphorylate the RNA polymerase II C-terminal domain (CTD) at serine 2 of the heptad repeat, both in vitro and in vivo. CDK9, as part of P-TEFb and the super elongation complex (SEC), is by far the best characterized of CDK9, CDK12, and CDK13. We employed both in vitro and in vivo assays to further investigate the molecular properties of CDK12 and its paralog CDK13. We isolated Flag-tagged CDK12 and CDK13 and found that they associate with numerous RNA processing factors. Although knockdown of CDK12, CDK13, or their cyclin partner CCNK did not affect the bulk CTD phosphorylation levels in HCT116 cells, transcriptome sequencing (RNA-seq) analysis revealed that CDK12 and CDK13 losses in HCT116 cells preferentially affect expression of DNA damage response and snoRNA genes, respectively. CDK12 and CDK13 depletion also leads to a loss of expression of RNA processing factors and to defects in RNA processing. These findings suggest that in addition to implementing CTD phosphorylation, CDK12 and CDK13 may affect RNA processing through direct physical interactions with RNA processing factors and by regulating their expression. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  15. A novel fold in the TraI relaxase-helicase c-terminal domain is essential for conjugative DNA transfer.

    Science.gov (United States)

    Guogas, Laura M; Kennedy, Sarah A; Lee, Jin-Hyup; Redinbo, Matthew R

    2009-02-20

    TraI relaxase-helicase is the central catalytic component of the multiprotein relaxosome complex responsible for conjugative DNA transfer (CDT) between bacterial cells. CDT is a primary mechanism for the lateral propagation of microbial genetic material, including the spread of antibiotic resistance genes. The 2.4-A resolution crystal structure of the C-terminal domain of the multifunctional Escherichia coli F (fertility) plasmid TraI protein is presented, and specific structural regions essential for CDT are identified. The crystal structure reveals a novel fold composed of a 28-residue N-terminal alpha-domain connected by a proline-rich loop to a compact alpha/beta-domain. Both the globular nature of the alpha/beta-domain and the presence as well as rigidity of the proline-rich loop are required for DNA transfer and single-stranded DNA binding. Taken together, these data establish the specific structural features of this noncatalytic domain that are essential to DNA conjugation.

  16. A C-terminal fragment of Agouti-related protein increases feeding and antagonizes the effect of alpha-melanocyte stimulating hormone in vivo.

    Science.gov (United States)

    Rossi, M; Kim, M S; Morgan, D G; Small, C J; Edwards, C M; Sunter, D; Abusnana, S; Goldstone, A P; Russell, S H; Stanley, S A; Smith, D M; Yagaloff, K; Ghatei, M A; Bloom, S R

    1998-10-01

    Agouti-related protein (Agrp) is present in rat and human hypothalamus and is structurally related to agouti protein. Overexpression of either of these proteins results in obesity. However the effect of exogenous Agrp and its in vivo interaction with alpha-melanocyte stimulating hormone (alphaMSH), the likely endogenous melanocortin 3 and 4 receptor (MC3-R and MC4-R) agonist, have not been demonstrated. We report that 1 nmol of Agrp(83-132), a C-terminal fragment of Agrp, when administered intracerebroventricularly (ICV) into rats, increased food intake over a 24-h period (23.0+/-1.4 g saline vs 32.9+/-2.3 g Agrp, pinhibition of cAMP activation to that previously reported for the full length peptide. In conclusion, Agrp(83-132) administered i.c.v. increases feeding with long lasting effects and is able to inhibit the action of alphaMSH. This interaction may be mediated by the MC3-R and/or MC4-R.

  17. Identification and solution structure of a highly conserved C-terminal domain within ORF1p required for retrotransposition of long interspersed nuclear element-1.

    Science.gov (United States)

    Januszyk, Kurt; Li, Patrick Wai-Lun; Villareal, Valerie; Branciforte, Dan; Wu, Haihong; Xie, Yongming; Feigon, Juli; Loo, Joseph A; Martin, Sandra L; Clubb, Robert T

    2007-08-24

    Long interspersed nuclear element-1 (LINE-1 or L1) retrotransposons comprise a large fraction of the human and mouse genomes. The mobility of these successful elements requires the protein encoded by open reading frame-1 (ORF1p), which binds single-stranded RNA with high affinity and functions as a nucleic acid chaperone. In this report, we have used limited proteolysis, filter binding, and NMR spectroscopy to characterize the global structure of ORF1p and the three-dimensional structure of a highly conserved RNA binding domain. ORF1p contains three structured regions, a coiled-coil domain, a middle domain of unknown function, and a C-terminal domain (CTD). We show that high affinity RNA binding by ORF1p requires the CTD and residues within an amino acid protease-sensitive segment that joins the CTD to the middle domain. Insights in the mechanism of RNA binding were obtained by determining the solution structure of the CTD, which is shown to adopt a novel fold consisting of a three-stranded beta sheet that is packed against three alpha-helices. An RNA binding surface on the CTD has been localized using chemical shift perturbation experiments and is proximal to residues previously shown to be essential for retrotransposition, RNA binding, and chaperone activity. A similar structure and mechanism of RNA binding is expected for all vertebrate long interspersed nuclear element-1 elements, since residues encoding the middle, protease-sensitive segment, and CTD are highly conserved.

  18. In Sup35p filaments (the [PSI+] prion), the globular C-terminal domains are widely offset from the amyloid fibril backbone

    Energy Technology Data Exchange (ETDEWEB)

    Baxa, U.; Wall, J.; Keller, P. W.; Cheng, N.; Steven, A. C.

    2011-01-01

    In yeast cells infected with the [PSI+] prion, Sup35p forms aggregates and its activity in translation termination is downregulated. Transfection experiments have shown that Sup35p filaments assembled in vitro are infectious, suggesting that they reproduce or closely resemble the prion. We have used several EM techniques to study the molecular architecture of filaments, seeking clues as to the mechanism of downregulation. Sup35p has an N-terminal 'prion' domain; a highly charged middle (M-)domain; and a C-terminal domain with the translation termination activity. By negative staining, cryo-EM and scanning transmission EM (STEM), filaments of full-length Sup35p show a thin backbone fibril surrounded by a diffuse 65-nm-wide cloud of globular C-domains. In diameter ({approx}8 nm) and appearance, the backbones resemble amyloid fibrils of N-domains alone. STEM mass-per-unit-length data yield -1 subunit per 0.47 nm for N-fibrils, NM-filaments and Sup35p filaments, further supporting the fibril backbone model. The 30 nm radial span of decorating C-domains indicates that the M-domains assume highly extended conformations, offering an explanation for the residual Sup35p activity in infected cells, whereby the C-domains remain free enough to interact with ribosomes.

  19. Overexpression, purification and crystallization of the two C-terminal domains of the bifunctional cellulase ctCel9D-Cel44A from Clostridium thermocellum

    Energy Technology Data Exchange (ETDEWEB)

    Najmudin, Shabir [REQUIMTE, Departamento de Química, FCT-UNL, 2829-516 Caparica (Portugal); Guerreiro, Catarina I. P. D.; Ferreira, Luís M. A. [CIISA - Faculdade de Medicina Veterinária, Universidade Técnica de Lisboa, Avenida da Universidade Técnica, 1300-477 Lisboa (Portugal); Romão, Maria J. C. [REQUIMTE, Departamento de Química, FCT-UNL, 2829-516 Caparica (Portugal); Fontes, Carlos M. G. A.; Prates, José A. M., E-mail: japrates@fmv.utl.pt [CIISA - Faculdade de Medicina Veterinária, Universidade Técnica de Lisboa, Avenida da Universidade Técnica, 1300-477 Lisboa (Portugal); REQUIMTE, Departamento de Química, FCT-UNL, 2829-516 Caparica (Portugal)

    2005-12-01

    The two C-terminal domains of the cellulase ctCel9D-Cel44A from C. thermocellum cellulosome have been crystallized in tetragonal space group P4{sub 3}2{sub 1}2 and X-ray diffraction data have been collected to 2.1 and 2.8 Å from native and seleno-l-methionine-derivative crystals, respectively. Clostridium thermocellum produces a highly organized multi-enzyme complex of cellulases and hemicellulases for the hydrolysis of plant cell-wall polysaccharides, which is termed the cellulosome. The bifunctional multi-modular cellulase ctCel9D-Cel44A is one of the largest components of the C. thermocellum cellulosome. The enzyme contains two internal catalytic domains belonging to glycoside hydrolase families 9 and 44. The C-terminus of this cellulase, comprising a polycystic kidney-disease module (PKD) and a carbohydrate-binding module (CBM44), has been crystallized. The crystals belong to the tetragonal space group P4{sub 3}2{sub 1}2, containing a single molecule in the asymmetric unit. Native and seleno-l-methionine-derivative crystals diffracted to 2.1 and 2.8 Å, respectively.

  20. Confirmation of the immunoreactivity of monoclonal anti-human C-terminal EGFR antibodies in bronze Corydoras Corydoras aeneus (Callichthyidae Teleostei) by Western Blot method.

    Science.gov (United States)

    Mytych, Jennifer; Satora, Leszek; Kozioł, Katarzyna

    2017-12-12

    Bronze corydoras (Corydoras aeneus) uses the distal part of the intestine as accessory respiratory organ. Our previous study showed the presence of epidermal growth factor receptor (EGFR) cytoplasmic domain in the digestive tract of the bronze corydoras. In this study, using Western Blot method, we validated the results presented in the previous research. In detail, results of Western Blot analysis on digestive and respiratory part of bronze corydoras intestine homogenates confirmed the immunoreactivity of anti-cytoplasmic domain (C-terminal) human EGFR antibodies with protein band of approximately 180kDa (EGFR molecular weight). This indicates a high homology of EGFR domain between these species and the possibility of such antibody use in bronze corydoras. Statistically significantly higher EGFR expression was observed in the respiratory part of intestine when compared to the digestive part. This implies higher proliferation activity and angiogenesis of epithelium in this part of intestine, creating conditions for air respiration. Therefore, Corydoras aeneus may be considered as a model organism for the molecular studies of the mechanisms of epithelial proliferation initiation and inhibition depending on hypoxia and normoxia. Copyright © 2017. Published by Elsevier GmbH.

  1. A C-terminal Hydrophobic, Solvent-protected Core and a Flexible N-terminus are Potentially Required for Human Papillomavirus 18 E7 Protein Functionality

    Energy Technology Data Exchange (ETDEWEB)

    Liu, S.; Tian, Y; Greenaway, F; Sun, M

    2010-01-01

    The oncogenic potential of the high-risk human papillomavirus (HPV) relies on the expression of genes specifying the E7 and E6 proteins. To investigate further the variation in oligomeric structure that has been reported for different E7 proteins, an HPV-18 E7 cloned from a Hispanic woman with cervical intraepithelial neoplasia was purified to homogeneity most probably as a stable monomeric protein in aqueous solution. We determined that one zinc ion is present per HPV-18 E7 monomer by amino acid and inductively coupled plasma-atomic emission spectroscopy analysis. Intrinsic fluorescence and circular dichroism spectroscopic results indicate that the zinc ion is important for the correct folding and thermal stability of HPV-18 E7. Hydroxyl radical mediated protein footprinting coupled to mass spectrometry and other biochemical and biophysical data indicate that near the C-terminus, the four cysteines of the two Cys-X{sub 2}-Cys motifs that are coordinated to the zinc ion form a solvent inaccessible core. The N-terminal LXCXE pRb binding motif region is hydroxyl radical accessible and conformationally flexible. Both factors, the relative flexibility of the pRb binding motif at the N-terminus and the C-terminal metal-binding hydrophobic solvent-protected core, combine together and facilitate the biological functions of HPV-18 E7.

  2. Ubiquitin C-terminal hydrolase-activity is involved in sperm acrosomal function and anti-polyspermy defense during porcine fertilization.

    Science.gov (United States)

    Yi, Young-Joo; Manandhar, Gaurishankar; Sutovsky, Miriam; Li, Rongfeng; Jonáková, Vera; Oko, Richard; Park, Chang-Sik; Prather, Randall S; Sutovsky, Peter

    2007-11-01

    The 26S proteasome, which is a multi-subunit protease with specificity for substrate proteins that are postranslationally modified by ubiquitination, has been implicated in acrosomal function and sperm-zona pellucida (ZP) penetration during mammalian fertilization. Ubiquitin C-terminal hydrolases (UCHs) are responsible for the removal of polyubiquitin chains during substrate priming for proteasomal proteolysis. The inhibition of deubiquitination increases the rate of proteasomal proteolysis. Consequently, we have hypothesized that inhibition of sperm acrosome-borne UCHs increases the rate of sperm-ZP penetration and polyspermy during porcine in vitro fertilization (IVF). Ubiquitin aldehyde (UA), which is a specific nonpermeating UCH inhibitor, significantly (P polyspermy during porcine IVF and reduced (P polyspermy during IVF, consistent with the UA-induced polyspermy surge. In the oocyte, UCHL3 was primarily associated with the meiotic spindle. Sperm-borne UCHL3 was localized to the acrosomal surface and coimmunoprecipitated with a peripheral acrosomal membrane protein, spermadhesin AQN1. Recombinant UCHs, UCHL3, and isopeptidase T reduced polyspermy when added to the fertilization medium. UCHL1 was detected in the oocyte cortex but not on the sperm surface, and was partially degraded 6-8 h after fertilization. Enucleated oocyte-somatic cell electrofusion caused polarized redistribution of cortical UCHL1. We conclude that sperm-acrosomal UCHs are involved in sperm-ZP interactions and antipolyspermy defense. Modulation of UCH activity could facilitate the management of polyspermy during IVF and provide insights into male infertility.

  3. Accumulation of C-terminal fragments of transactive response DNA-binding protein 43 leads to synaptic loss and cognitive deficits in human TDP-43 transgenic mice.

    Science.gov (United States)

    Medina, David X; Orr, Miranda E; Oddo, Salvatore

    2014-01-01

    Accumulation of the transactive response DNA-binding protein 43 (TDP-43) is a major hallmark of several neurodegenerative disorders, collectively known as TDP-43 proteinopathies. The most common TDP-43 proteinopathies, frontotemporal lobar degeneration with TDP-43-positive inclusions, and amyotrophic lateral sclerosis, share overlapping neuropathological and clinical phenotypes. The development and detailed analysis of animal models of TDP-43 proteinopathies are critical for understanding the pathogenesis of these disorders. Transgenic mice overexpressing mutant human TDP-43 (herein referred to as hTDP-43) are characterized by neurodegeneration and reduced life span. However, little is known about the behavioral phenotype of these mice. Here we report the novel finding that hTDP-43 mice develop deficits in cognition, motor performance, and coordination. We show that these behavioral deficits are associated with the accumulation of nuclear and cytosolic TDP-43 C-terminal fragments, a decrease in endogenous TDP-43 levels, and synaptic loss. Our findings provide critical insights into disease pathology, and will help guide future preclinical studies aimed at testing the effects of potential therapeutic agents on the onset and progression of TDP-43 proteinopathies. Copyright © 2014 Elsevier Inc. All rights reserved.

  4. Novel phenotypic variant in the MYH7 spectrum due to a stop-loss mutation in the C-terminal region: a case report.

    Science.gov (United States)

    Bánfai, Zsolt; Hadzsiev, Kinga; Pál, Endre; Komlósi, Katalin; Melegh, Márton; Balikó, László; Melegh, Béla

    2017-09-19

    Defects of the slow myosin heavy chain isoform coding MYH7 gene primarily cause skeletal myopathies including Laing Distal Myopathy, Myosin Storage Myopathy and are also responsible for cardiomyopathies. Scapuloperoneal and limb-girdle muscle weakness, congenital fiber type disproportion, multi-minicore disease were also reported in connection of MYH7. Pathogeneses of the defects in the head and proximal rod region of the protein are well described. However, the C-terminal mutations of the MYH7 gene are less known. Moreover, only two articles describe the phenotypic impact of the elongated mature protein product caused by termination signal loss. Here we present a male patient with an unusual phenotypic variant of early-onset and predominant involvement of neck muscles with muscle biopsy indicating myopathy and sarcoplasmic storage material. Cardiomyopathic involvements could not be observed. Sequencing of MYH7 gene revealed a stop-loss mutation on the 3-prime end of the rod region, which causes the elongation of the mature protein. The elongated protein likely disrupts the functions of the sarcomere by multiple functional abnormalities. This elongation could also affect the thick filament degradation leading to protein deposition and accumulation in the sarcomere, resulting in the severe myopathy of certain axial muscles. The phenotypic expression of the detected novel MYH7 genotype could strengthen and further expand our knowledge about mutations affecting the structure of MyHCI by termination signal loss in the MYH7 gene.

  5. The C-terminal region of the non-structural protein 2B from Hepatitis A Virus demonstrates lipid-specific viroporin-like activity

    Science.gov (United States)

    Shukla, Ashutosh; Dey, Debajit; Banerjee, Kamalika; Nain, Anshu; Banerjee, Manidipa

    2015-10-01

    Viroporins are virally encoded, membrane-active proteins, which enhance viral replication and assist in egress of viruses from host cells. The 2B proteins in the picornaviridae family are known to have viroporin-like properties, and play critical roles during virus replication. The 2B protein of Hepatitis A Virus (2B), an unusual picornavirus, is somewhat dissimilar from its analogues in several respects. HAV 2B is approximately 2.5 times the length of other 2B proteins, and does not disrupt calcium homeostasis or glycoprotein trafficking. Additionally, its membrane penetrating properties are not yet clearly established. Here we show that the membrane interacting activity of HAV 2B is localized in its C-terminal region, which contains an alpha-helical hairpin motif. We show that this region is capable of forming small pores in membranes and demonstrates lipid specific activity, which partially rationalizes the intracellular localization of full-length 2B. Using a combination of biochemical assays and molecular dynamics simulation studies, we also show that HAV 2B demonstrates a marked propensity to dimerize in a crowded environment, and probably interacts with membranes in a multimeric form, a hallmark of other picornavirus viroporins. In sum, our study clearly establishes HAV 2B as a bona fide viroporin in the picornaviridae family.

  6. 3.3 Å structure of Niemann–Pick C1 protein reveals insights into the function of the C-terminal luminal domain in cholesterol transport

    Energy Technology Data Exchange (ETDEWEB)

    Li, Xiaochun; Lu, Feiran; Trinh, Michael N.; Schmiege, Philip; Seemann, Joachim; Wang, Jiawei; Blobel, Günter

    2017-08-07

    Niemann–Pick C1 (NPC1) and NPC2 proteins are indispensable for the export of LDL-derived cholesterol from late endosomes. Mutations in these proteins result in Niemann–Pick type C disease, a lysosomal storage disease. Despite recent reports of the NPC1 structure depicting its overall architecture, the function of its C-terminal luminal domain (CTD) remains poorly understood even though 45% of NPC disease-causing mutations are in this domain. Here, we report a crystal structure at 3.3 Å resolution of NPC1* (residues 314–1,278), which—in contrast to previous lower resolution structures—features the entire CTD well resolved. Notably, all eight cysteines of the CTD form four disulfide bonds, one of which (C909–C914) enforces a specific loop that in turn mediates an interaction with a loop of the N-terminal domain (NTD). Importantly, this loop and its interaction with the NTD were not observed in any previous structures due to the lower resolution. Our mutagenesis experiments highlight the physiological relevance of the CTD–NTD interaction, which might function to keep the NTD in the proper orientation for receiving cholesterol from NPC2. Additionally, this structure allows us to more precisely map all of the disease-causing mutations, allowing future molecular insights into the pathogenesis of NPC disease.

  7. C-Terminal Repeats of Clostridium difficile Toxin A Induce Production of Chemokine and Adhesion Molecules in Endothelial Cells and Promote Migration of Leukocytes▿

    Science.gov (United States)

    Yeh, Chiou-Yueh; Lin, Chun-Nan; Chang, Chuan-Fa; Lin, Chun-Hung; Lien, Huei-Ting; Chen, Jen-Yang; Chia, Jean-San

    2008-01-01

    The C-terminal repeating sequences of Clostridium difficile toxin A (designated ARU) are homologous to the carbohydrate-binding domain of streptococcal glucosyltransferases (GTFs) that were recently identified as potent modulins. To test the hypothesis that ARU might exert a similar biological activity on endothelial cells, recombinant ARU (rARU), which was noncytotoxic to cell cultures, was analyzed using human umbilical vein endothelial cells. The rARU could bind directly to endothelial cells in a serum- and calcium-dependent manner and induce the production of interleukin-6 (IL-6), IL-8, and monocyte chemoattractant protein 1 in a dose-dependent manner. An oligosaccharide binding assay indicated that rARU, but not GTFC, binds preferentially to Lewis antigens and 3′HSO3-containing oligosaccharides. Binding of rARU to human endothelial or intestinal cells correlated directly with the expression of Lewis Y antigen. Bound rARU directly activated mitogen-activated protein kinases and the NF-κB signaling pathway in endothelial cells to release biologically active chemokines and adhesion molecules that promoted migration in a transwell assay and the adherence of polymorphonuclear and mononuclear cells to the endothelial cells. These results suggest that ARU may bind to multiple carbohydrate motifs to exert its biological activity on human endothelial cells. PMID:18160482

  8. p53 searches on DNA by rotation-uncoupled sliding at C-terminal tails and restricted hopping of core domains.

    Science.gov (United States)

    Terakawa, Tsuyoshi; Kenzaki, Hiroo; Takada, Shoji

    2012-09-05

    The tumor suppressor p53 is a transcription factor that searches its cognate sites on DNA. During the search, the roles and interplay of its two DNA binding domains, the folded core domain and the disordered C-terminal domain (CTD), have been controversial. Here, we performed molecular simulations of p53 at various salt concentrations finding that, at physiological salt concentration, p53 diffuses along nonspecific DNA via rotation-uncoupled sliding with its CTD, whereas the core domain repeats dissociation and association. This is in perfect agreement with a recent single molecule experiment. In the simulation of tetrameric full-length p53, two DNA binding domains both bound to nonspecific DNA in a characteristic form at low salt concentration, whereas at physiological salt concentration, only CTD kept bound to DNA and the core domain frequently hopped on DNA. Simulations of a construct that lacks the core domain (TetCD) clarified rotation-uncoupled diffusion on nonspecific DNA. At low salt concentration, the diffusion constant due to sliding was dependent on the salt concentration, which differs from the prediction of a classic theory of transcription factors. At physiological salt concentration, it was independent of the salt concentration, in harmony with experiments. Moreover, we found that the sliding via the CTD follows the helical pitch of DNA (i.e., rotation-coupled sliding) at low salt concentration while it is virtually uncoupled to the helical pitch, a hallmark of rotation-uncoupled sliding at physiological salt concentration.

  9. EMERGING ROLE OF N- AND C-TERMINAL INTERACTIONS IN STABILIZING (β;/α8 FOLD WITH SPECIAL EMPHASIS ON FAMILY 10 XYLANASES

    Directory of Open Access Journals (Sweden)

    Amit Bhardwaj

    2012-09-01

    Full Text Available Xylanases belong to an important class of industrial enzymes. Various xylanases have been purified and characterized from a plethora of organisms including bacteria, marine algae, plants, protozoans, insects, snails and crustaceans. Depending on the source, the enzymatic activity of xylanases varies considerably under various physico-chemical conditions such as temperature, pH, high salt and in the presence of proteases. Family 10 or glycosyl hydrolase 10 (GH10 xylanases are one of the well characterized and thoroughly studied classes of industrial enzymes. The TIM-barrel fold structure which is ubiquitous in nature is one of the characteristics of family 10 xylanases. Family 10 xylanases have been used as a “model system” due to their TIM-barrel fold to dissect and understand protein stability under various conditions. A better understanding of structure-stability-function relationships of family 10 xylanases allows one to apply these governing molecular rules to engineer other TIM-barrel fold proteins to improve their stability and retain function(s under adverse conditions. In this review, we discuss the implications of N-and C-terminal interactions, observed in family 10 xylanases on protein stability under extreme conditions. The role of metal binding and aromatic clusters in protein stability is also discussed. Studying and understanding family 10 xylanase structure and function, can contribute to our protein engineering knowledge.

  10. Synthetic peptides derived from the C-terminal region of Lys49 phospholipase A2 homologues from viperidae snake venoms: biomimetic activities and potential applications.

    Science.gov (United States)

    Lomonte, Bruno; Angulo, Yamileth; Moreno, Edgardo

    2010-01-01

    Lys49-phospholipase A(2) homologues constitute a large family of toxins present in the venoms of viperid snake species, which despite lacking catalytic activity, cause significant skeletal muscle necrosis. The main structural determinants of this toxic effect have been experimentally mapped to a region near their C-terminus (115-129), which combines cationic and hydrophobic/aromatic amino acid residues. Short (13-mer) synthetic peptides representing this C-terminal region can mimick several of the effects of Lys49 PLA(2) homologues. In addition to their ability to damage muscle cells, these peptides display antibacterial, antiendotoxic, antifungal, antiparasite, and antitumor activities, as well as VEGF-receptor 2 (KDR)-binding and heparin-binding properties. Modifications of their sequences have shown possibilities to enhance their effects upon prokaryotic cells, while decreasing toxicity for eukaryotic cells. This review presents an updated summary on the biomimetic actions exerted by such peptides, and highlights their potential value as molecular tools or as drug leads in diverse biomedical areas.

  11. Ral GTPase interacts with the N-terminal in addition to the C-terminal region of PLC-delta1.

    Science.gov (United States)

    Grujic, Ognjen; Bhullar, Rajinder P

    2009-06-12

    Previously, we have shown that RalA, a calmodulin (CaM)-binding protein, binds to the C2 region in the C-terminal of PLC-delta1, and increases its enzymatic activity. Since PLC-delta1 contains a CaM-like region in its N-terminus, we have investigated if RalA can also bind to the N-terminus of PLC-delta1. Therefore, we created a GST-PLC-delta1 construct consisting of the first 294 amino acids of PLC-delta1 (GST-PLC-delta1(1-294)). In vitro binding experiments confirmed that PLC-delta1(1-294) was capable of binding directly to RalA. W-7 coupled to polyacrylamide beads bound pure PLC-delta1, demonstrating that PLC-delta1 contains a CaM-like region. Competition assays with W-7, peptides representing RalA and the newly identified RalB CaM-binding regions, or the IQ peptide from PLC-delta1 were able to inhibit RalA binding to PLC-delta1(1-294). This study demonstrates that there are two binding sites for RalA in PLC-delta1 and provides further insight into the role of Ral GTPase in the regulation of PLC-delta1 function.

  12. γ-Tubulin and the C-Terminal Motor Domain Kinesin-like Protein, KLPA, Function in the Establishment of Spindle Bipolarity in Aspergillus nidulans

    Science.gov (United States)

    Prigozhina, Natalie L.; Walker, Richard A.; Oakley, C. Elizabeth; Oakley, Berl R.

    2001-01-01

    Previous research has found that a γ-tubulin mutation in Schizosaccharomyces pombe is synthetically lethal with a deletion of the C-terminal motor domain kinesin-like protein gene pkl1, but the lethality of the double mutant prevents a phenotypic analysis of the synthetic interaction. We have investigated interactions between klpA1, a deletion of an Aspergillus nidulans homolog of pkl1, and mutations in the mipA, γ-tubulin gene. We find that klpA1 dramatically increases the cold sensitivity and slightly reduces the growth rate at all temperatures, of three mipA alleles. In synchronized cells we find that klpA1 causes a substantial but transient inhibition of the establishment of spindle bipolarity. At a restrictive temperature, mipAD123 causes a slight, transient inhibition of spindle bipolarity and a more significant inhibition of anaphase A. In the mipAD123/klpA1 strain, formation of bipolar spindles is more strongly inhibited than in the klpA1 single mutant and many spindles apparently never become bipolar. These results indicate, surprisingly, that γ-tubulin and the klpA kinesin have overlapping roles in the establishment of spindle bipolarity. We propose a model to account for these data. PMID:11598200

  13. Effect of the C-terminal domain of Vibrio proteolyticus chitinase A on the chitinolytic activity in association with pH changes.

    Science.gov (United States)

    Itoi, S; Kanomata, Y; Uchida, S; Kadokura, K; Nishio, T; Oku, T; Sugita, H

    2012-05-01

    To reveal the cause of the difference in activity of chitinase A from Vibrio proteolyticus and chitinase A from a strain of Vibrio carchariae (a junior synonym of Vibrio harveyi), we investigated the pH-dependent activity of full-length V. proteolyticus chitinase A and a truncated recombinant corresponding to the V. harveyi form of chitinase A. After overexpression in Escherichia coli strain DH5α, the full-length and truncated recombinant chitinases were purified by ammonium sulphate precipitation and anion exchange column chromatography. Chitinase activity was measured at various pH values using α-crystal and colloidal chitins as the substrate. The pH-dependent patterns of the relative specific activities for α-crystal chitin differed between the full-length and truncated recombinant chitinases, whereas those for colloidal chitin were similar to each other. The difference in the activity of V. proteolyticus chitinase A and V. harveyi chitinase A might be partly due to a change in the pH dependence of the chitinase activities against α-crystal chitin, resulting from C-terminal processing. The present results are important findings for not only ecological studies on the genus Vibrio in association with survival strategies, but also phylogenetic studies. © 2012 The Authors. Letters in Applied Microbiology © 2012 The Society for Applied Microbiology.

  14. Repressor of temperate mycobacteriophage L1 harbors a stable C-terminal domain and binds to different asymmetric operator DNAs with variable affinity

    Directory of Open Access Journals (Sweden)

    Mandal Nitai C

    2007-06-01

    Full Text Available Abstract Background Lysogenic mode of life cycle of a temperate bacteriophage is generally maintained by a protein called 'repressor'. Repressor proteins of temperate lambdoid phages bind to a few symmetric operator DNAs in order to regulate their gene expression. In contrast, repressor molecules of temperate mycobacteriophages and some other phages bind to multiple asymmetric operator DNAs. Very little is known at present about the structure-function relationship of any mycobacteriophage repressor. Results Using highly purified repressor (CI of temperate mycobacteriophage L1, we have demonstrated here that L1 CI harbors an N-terminal domain (NTD and a C-terminal domain (CTD which are separated by a small hinge region. Interestingly, CTD is more compact than NTD at 25°C. Both CTD and CI contain significant amount of α-helix at 30°C but unfold partly at 42°C. At nearly 200 nM concentration, both proteins form appreciable amount of dimers in solution. Additional studies reveal that CI binds to O64 and OL types of asymmetric operators of L1 with variable affinity at 25°C. Interestingly, repressor – operator interaction is affected drastically at 42°C. The conformational change of CI is most possibly responsible for its reduced operator binding affinity at 42°C. Conclusion Repressors encoded by mycobacteriophages differ significantly from the repressor proteins of λ and related phages at functional level but at structural level they are nearly similar.

  15. Inherited germline TP53 mutation encodes a protein with an aberrant C-terminal motif in a case of pediatric adrenocortical tumor.

    Science.gov (United States)

    Pinto, Emilia M; Ribeiro, Raul C; Kletter, Gad B; Lawrence, John P; Jenkins, Jesse J; Wang, Jinling; Shurtleff, Sheila; McGregor, Lisa; Kriwacki, Richard W; Zambetti, Gerard P

    2011-03-01

    Childhood adrenocortical tumor (ACT), a very rare malignancy, has an annual worldwide incidence of about 0.3 per million children younger than 15 years. The association between inherited germline mutations of the TP53 gene and an increased predisposition to ACT was described in the context of the Li-Fraumeni syndrome. In fact, about two-thirds of children with ACT have a TP53 mutation. However, less than 10% of pediatric ACT cases occur in Li-Fraumeni syndrome, suggesting that inherited low-penetrance TP53 mutations play an important role in pediatric adrenal cortex tumorigenesis. We identified a novel inherited germline TP53 mutation affecting the acceptor splice site at intron 10 in a child with an ACT and no family history of cancer. The lack of family history of cancer and previous information about the carcinogenic potential of the mutation led us to further characterize it. Bioinformatics analysis showed that the non-natural and highly hydrophobic C-terminal segment of the frame-shifted mutant p53 protein may disrupt its tumor suppressor function by causing misfolding and aggregation. Our findings highlight the clinical and genetic counseling dilemmas that arise when an inherited TP53 mutation is found in a child with ACT without relatives with Li-Fraumeni-component tumors.

  16. The binding of lupus-derived autoantibodies to the C-terminal peptide (83-119) of the major SmD1 autoantigen can be mediated by double-stranded DNA and nucleosomes.

    NARCIS (Netherlands)

    Dieker, J.W.C.; Bavel, C.C.A.W. van; Riemekasten, G.; Berden, J.H.M.; Vlag, J. van der

    2006-01-01

    OBJECTIVES: To evaluate the binding of lupus-derived autoantibodies, double-stranded DNA and nucleosomes to the positively charged C-terminal SmD1(residues 83-119) peptide and the full-length SmD protein. METHODS: The binding of lupus-derived monoclonal antibodies, sera from patients with systemic

  17. CD97 inhibits cell migration in human fibrosarcoma cells by modulating TIMP-2/MT1- MMP/MMP-2 activity--role of GPS autoproteolysis and functional cooperation between the N- and C-terminal fragments

    NARCIS (Netherlands)

    Hsiao, Cheng-Chih; Wang, Wen-Chih; Kuo, Wan-Lin; Chen, Hsin-Yi; Chen, Tse-Ching; Hamann, Jörg; Lin, Hsi-Hsien

    2014-01-01

    CD97 is a tumor-associated adhesion-class G-protein-coupled receptor involved in modulating cell migration. Adhesion-class G-protein-coupled receptors are characterized by proteolytic cleavage at a G-protein-coupled receptor proteolysis site (GPS) into an N-terminal fragment (NTF) and a C-terminal

  18. Fusion of the C-terminal triskaidecapeptide of hirudin variant 3 to alpha1-proteinase inhibitor M358R increases the serpin-mediated rate of thrombin inhibition

    Science.gov (United States)

    2013-01-01

    Background Alpha-1 proteinase inhibitor (API) is a plasma serpin superfamily member that inhibits neutrophil elastase; variant API M358R inhibits thrombin and activated protein C (APC). Fusing residues 1-75 of another serpin, heparin cofactor II (HCII), to API M358R (in HAPI M358R) was previously shown to accelerate thrombin inhibition over API M358R by conferring thrombin exosite 1 binding properties. We hypothesized that replacing HCII 1-75 region with the 13 C-terminal residues (triskaidecapeptide) of hirudin variant 3 (HV354-66) would further enhance the inhibitory potency of API M358R fusion proteins. We therefore expressed HV3API M358R (HV354-66 fused to API M358R) and HV3API RCL5 (HV354-66 fused to API F352A/L353V/E354V/A355I/I356A/I460L/M358R) API M358R) as N-terminally hexahistidine-tagged polypeptides in E. coli. Results HV3API M358R inhibited thrombin 3.3-fold more rapidly than API M358R; for HV3API RCL5 the rate enhancement was 1.9-fold versus API RCL5; neither protein inhibited thrombin as rapidly as HAPI M358R. While the thrombin/Activated Protein C rate constant ratio was 77-fold higher for HV3API RCL5 than for HV3API M358R, most of the increased specificity derived from the API F352A/L353V/E354V/A355I/I356A/I460L API RCL 5 mutations, since API RCL5 remained 3-fold more specific than HV3API RCL5. An HV3 54-66 peptide doubled the Thrombin Clotting Time (TCT) and halved the binding of thrombin to immobilized HCII 1-75 at lower concentrations than free HCII 1-75. HV3API RCL5 bound active site-inhibited FPR-chloromethyl ketone-thrombin more effectively than HAPI RCL5. Transferring the position of the fused HV3 triskaidecapeptide to the C-terminus of API M358R decreased the rate of thrombin inhibition relative to that mediated by HV3API M358R by 11-to 14-fold. Conclusions Fusing the C-terminal triskaidecapeptide of HV3 to API M358R-containing serpins significantly increased their effectiveness as thrombin inhibitors, but the enhancement was less than that

  19. C-terminal domain modulates the nucleic acid chaperone activity of human T-cell leukemia virus type 1 nucleocapsid protein via an electrostatic mechanism.

    Science.gov (United States)

    Qualley, Dominic F; Stewart-Maynard, Kristen M; Wang, Fei; Mitra, Mithun; Gorelick, Robert J; Rouzina, Ioulia; Williams, Mark C; Musier-Forsyth, Karin

    2010-01-01

    Retroviral nucleocapsid (NC) proteins are molecular chaperones that facilitate nucleic acid (NA) remodeling events critical in viral replication processes such as reverse transcription. Surprisingly, the NC protein from human T-cell leukemia virus type 1 (HTLV-1) is an extremely poor NA chaperone. Using bulk and single molecule methods, we find that removal of the anionic C-terminal domain (CTD) of HTLV-1 NC results in a protein with chaperone properties comparable with that of other retroviral NCs. Increasing the ionic strength of the solution also improves the chaperone activity of full-length HTLV-1 NC. To determine how the CTD negatively modulates the chaperone activity of HTLV-1 NC, we quantified the thermodynamics and kinetics of wild-type and mutant HTLV-1 NC/NA interactions. The wild-type protein exhibits very slow dissociation kinetics, and removal of the CTD or mutations that eliminate acidic residues dramatically increase the protein/DNA interaction kinetics. Taken together, these results suggest that the anionic CTD interacts with the cationic N-terminal domain intramolecularly when HTLV-1 NC is not bound to nucleic acids, and similar interactions occur between neighboring molecules when NC is NA-bound. The intramolecular N-terminal domain-CTD attraction slows down the association of the HTLV-1 NC with NA, whereas the intermolecular interaction leads to multimerization of HTLV-1 NC on the NA. The latter inhibits both NA/NC aggregation and rapid protein dissociation from single-stranded DNA. These features make HTLV-1 NC a poor NA chaperone, despite its robust duplex destabilizing capability.

  20. A variant of the bovine noradrenaline transporter reveals the importance of the C-terminal region for correct targeting to the membrane and functional expression.

    Science.gov (United States)

    Burton, L D; Kippenberger, A G; Lingen, B; Brüss, M; Bönisch, H; Christie, D L

    1998-03-01

    We have characterized a cDNA clone which encodes a variant (bNAT2) of the bovine noradrenaline transporter. This cDNA differs from the previously identified bovine noradrenaline transporter (bNAT1) in the sequence encoding part of the cytoplasmic-facing C-terminus and the 3'-untranslated region. The bNAT1 and bNAT2 cDNA clones are encoded by a 5.8 and 3.6 kb mRNA species respectively. The bNAT1 and bNAT2 proteins, which are identical apart from their C-terminal 31 and 18 residues, were stably expressed in HEK293 cells. Cells expressing bNAT1 showed a high level of desipramine-sensitive [3H]noradrenaline uptake activity, whereas no activity was present in bNAT2 cells. The bNAT1 and bNAT2 proteins were present as major 80 and 50 kDa species respectively. Cells expressing bNAT1 showed strong immunostaining of the plasma membrane, whereas bNAT2 was present in the endoplasmic reticulum/Golgi region. Treatment of membrane samples from bNAT1 cells with peptide N-glycosidase F resulted in the formation of a predominantly 50 kDa species, but little effect was observed after similar treatment of bNAT2 cell membranes. These results indicate that bNAT2 is retained in the endoplasmic reticulum and that the glycosylation of this variant differs from that of bNAT1. The characterization of bNAT2 and its comparison with bNAT1 highlight the importance of the cytoplasmic-facing C-terminus for the intracellular trafficking of neurotransmitter transporters.

  1. Detailed topology mapping reveals substantial exposure of the "cytoplasmic" C-terminal tail (CTT sequences in HIV-1 Env proteins at the cell surface.

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    Jonathan D Steckbeck

    Full Text Available Substantial controversy surrounds the membrane topology of the HIV-1 gp41 C-terminal tail (CTT. While few studies have been designed to directly address the topology of the CTT, results from envelope (Env protein trafficking studies suggest that the CTT sequence is cytoplasmically localized, as interactions with intracellular binding partners are required for proper Env targeting. However, previous studies from our lab demonstrate the exposure of a short CTT sequence, the Kennedy epitope, at the plasma membrane of intact Env-expressing cells, the exposure of which is not observed on viral particles. To address the topology of the entire CTT sequence, we serially replaced CTT sequences with a VSV-G epitope tag sequence and examined reactivity of cell- and virion-surface Env to an anti-VSV-G monoclonal antibody. Our results demonstrate that the majority of the CTT sequence is accessible to antibody binding on the surface of Env expressing cells, and that the CTT-exposed Env constitutes 20-50% of the cell-surface Env. Cell surface CTT exposure was also apparent in virus-infected cells. Passive transfer of Env through cell culture media to Env negative (non-transfected cells was not responsible for the apparent cell surface CTT exposure. In contrast to the cell surface results, CTT-exposed Env was not detected on infectious pseudoviral particles containing VSV-G-substituted Env. Finally, a monoclonal antibody directed to the Kennedy epitope neutralized virus in a temperature-dependent manner in a post-attachment neutralization assay. Collectively, these results suggest that the membrane topology of the HIV gp41 CTT is more complex than the widely accepted intracytoplasmic model.

  2. Ras GAP-related and C-terminal domain-dependent localization and tumorigenic activities of IQGAP1 in melanoma cells.

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    Michael Reimer

    Full Text Available IQGAP1 interacts with a number of binding partners through a calponin homology domain (CHD, a WW motif, IQ repeats, a Ras GAP-related domain (GRD, and a conserved C-terminal (CT domain. Among various biological and cellular functions, IQGAP1 is known to play a role in actin cytoskeleton dynamics during membrane ruffling and lamellipodium protrusion. In addition, phosphorylation near the CT domain is thought to control IQGAP1 activity through regulation of intramolecular interaction. In a previous study, we discovered that IQGAP1 preferentially localizes to retracting areas in B16F10 mouse melanoma cells, not areas of membrane ruffling and lamellipodium protrusion. Nothing is known of the domains needed for retraction localization and very little is known of IQGAP1 function in the actin cytoskeleton of melanoma cells. Thus, we examined localization of IQGAP1 mutants to retracting areas, and characterized knock down phenotypes on tissue culture plastic and physiologic-stiffness hydrogels. Localization of IQGAP1 mutants (S1441E/S1443D, S1441A/S1443A, ΔCHD, ΔGRD or ΔCT to retracting and protruding cell edges were measured. In retracting areas there was a decrease in S1441A/S1443A, ΔGRD and ΔCT localization, a minor decrease in ΔCHD localization, and normal localization of the S1441E/S1443D mutant. In areas of cell protrusion just behind the lamellipodium leading edge, we surprisingly observed both ΔGRD and ΔCT localization, and increased number of microtubules. IQGAP1 knock down caused loss of cell polarity on laminin-coated glass, decreased proliferation on tissue culture polystyrene, and abnormal spheroid growth on laminin-coated hydrogels. We propose that the GRD and CT domains regulate IQGAP1 localization to retracting actin networks to promote a tumorigenic role in melanoma cells.

  3. The Small C-terminal Domain Phosphatase 1 Inhibits Cancer Cell Migration and Invasion by Dephosphorylating Ser(P)68-Twist1 to Accelerate Twist1 Protein Degradation.

    Science.gov (United States)

    Sun, Tong; Fu, Junjiang; Shen, Tao; Lin, Xia; Liao, Lan; Feng, Xin-Hua; Xu, Jianming

    2016-05-27

    Twist1 is a basic helix-loop-helix transcription factor that strongly promotes epithelial-to-mesenchymal transition, migration, invasion, and metastasis of cancer cells. The MAPK-phosphorylated Twist1 on its serine 68 (Ser(P)(68)-Twist1) has a significantly enhanced stability and function to drive cancer cell invasion and metastasis. However, the phosphatase that dephosphorylates Ser(P)(68)-Twist1 and destabilizes Twist1 has not been identified and characterized. In this study, we screened a serine/threonine phosphatase cDNA expression library in HEK293T cells with ectopically coexpressed Twist1. We found that the small C-terminal domain phosphatase 1 (SCP1) specifically dephosphorylates Ser(P)(68)-Twist1 in both cell-free reactions and living cells. SCP1 uses its amino acid residues 43-63 to interact with the N terminus of Twist1. Increased SCP1 expression in cells decreased Ser(P)(68)-Twist1 and total Twist1 proteins, whereas knockdown of SCP1 increased Ser(P)(68)-Twist1 and total Twist1 proteins. Furthermore, the levels of SCP1 are negatively correlated with Twist1 protein levels in several cancer cell lines. SCP1-dephosphorylated Twist1 undergoes fast degradation via the ubiquitin-proteasome pathway. Importantly, an increase in SCP1 expression in breast cancer cells with either endogenous or ectopically expressed Twist1 largely inhibits the Twist1-induced epithelial-to-mesenchymal transition phenotype and the migration and invasion capabilities of these cells. These results indicate that SCP1 is the phosphatase that counterregulates the MAPK-mediated phosphorylation of Ser(68)-Twist1. Thus, an increase in SCP1 expression and activity may be a useful strategy for eliminating the detrimental roles of Twist1 in cancer cells. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  4. Bipartite Topology of Treponema pallidum Repeat Proteins C/D and I: OUTER MEMBRANE INSERTION, TRIMERIZATION, AND PORIN FUNCTION REQUIRE A C-TERMINAL β-BARREL DOMAIN.

    Science.gov (United States)

    Anand, Arvind; LeDoyt, Morgan; Karanian, Carson; Luthra, Amit; Koszelak-Rosenblum, Mary; Malkowski, Michael G; Puthenveetil, Robbins; Vinogradova, Olga; Radolf, Justin D

    2015-05-08

    We previously identified Treponema pallidum repeat proteins TprC/D, TprF, and TprI as candidate outer membrane proteins (OMPs) and subsequently demonstrated that TprC is not only a rare OMP but also forms trimers and has porin activity. We also reported that TprC contains N- and C-terminal domains (TprC(N) and TprC(C)) orthologous to regions in the major outer sheath protein (MOSP(N) and MOSP(C)) of Treponema denticola and that TprC(C) is solely responsible for β-barrel formation, trimerization, and porin function by the full-length protein. Herein, we show that TprI also possesses bipartite architecture, trimeric structure, and porin function and that the MOSP(C)-like domains of native TprC and TprI are surface-exposed in T. pallidum, whereas their MOSP(N)-like domains are tethered within the periplasm. TprF, which does not contain a MOSP(C)-like domain, lacks amphiphilicity and porin activity, adopts an extended inflexible structure, and, in T. pallidum, is tightly bound to the protoplasmic cylinder. By thermal denaturation, the MOSP(N) and MOSP(C)-like domains of TprC and TprI are highly thermostable, endowing the full-length proteins with impressive conformational stability. When expressed in Escherichia coli with PelB signal sequences, TprC and TprI localize to the outer membrane, adopting bipartite topologies, whereas TprF is periplasmic. We propose that the MOSP(N)-like domains enhance the structural integrity of the cell envelope by anchoring the β-barrels within the periplasm. In addition to being bona fide T. pallidum rare outer membrane proteins, TprC/D and TprI represent a new class of dual function, bipartite bacterial OMP. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. A conserved glycine residue in the C-terminal region of human ATG9A is required for its transport from the endoplasmic reticulum to the Golgi apparatus.

    Science.gov (United States)

    Staudt, Catherine; Gilis, Florentine; Tevel, Virginie; Jadot, Michel; Boonen, Marielle

    2016-10-14

    ATG9A is the only polytopic protein of the mammalian autophagy-related protein family whose members regulate autophagosome formation during macroautophagy. At steady state, ATG9A localizes to several intracellular sites, including the Golgi apparatus, endosomes and the plasma membrane, and it redistributes towards autophagosomes upon autophagy induction. Interestingly, the transport of yeast Atg9 to the pre-autophagosomal structure depends on its self-association, which is mediated by a short amino acid motif located in the C-terminal region of the protein. Here, we investigated whether the residues that align with this motif in human ATG9A (V(515)-C(519)) are also required for its trafficking in mammalian cells. Interestingly, our findings support that human ATG9A self-interacts as well, and that this process promotes transport of ATG9A molecules through the Golgi apparatus. Furthermore, our data reveal that the transport of ATG9A out of the ER is severely impacted after mutation of the conserved V(515)-C(519) motif. Nevertheless, the mutated ATG9A molecules could still interact with each other, indicating that the molecular mechanism of self-interaction differs in mammalian cells compared to yeast. Using sequential amino acid substitutions of glycine 516 and cysteine 519, we found that the stability of ATG9A relies on both of these residues, but that only the former is required for efficient transport of human ATG9A from the endoplasmic reticulum to the Golgi apparatus. Copyright © 2016 Elsevier Inc. All rights reserved.

  6. A new role for the neuronal ubiquitin C-terminal hydrolase-L1 (UCH-L1) in podocyte process formation and podocyte injury in human glomerulopathies.

    Science.gov (United States)

    Meyer-Schwesinger, C; Meyer, T N; Münster, S; Klug, P; Saleem, M; Helmchen, U; Stahl, R A K

    2009-02-01

    Glomerular epithelial cell (podocyte) injury is characterized by foot process retraction, slit diaphragm reorganization, and degradation of podocyte-specific proteins. However, the mechanisms underlying podocyte injury are largely unknown. The ubiquitin C-terminal hydrolase-L1 (UCH-L1) is a key modulator of ubiquitin modification in neurons. Like neurons, UCH-L1 expression was associated with an undifferentiated status in cultured human podocytes, whereas differentiation and arborization decreased UCH-L1 and monoUb expression. Inhibition of UCH-L1 induced time and concentration-dependent process formation with alpha-actinin-4 distribution to the cell membrane and processes. An immunohistochemical approach was used to evaluate whether UCH-L1 expression was associated with podocyte injury in 15 different human glomerular diseases. Whereas normal kidneys expressed no UCH-L1 and little ubiquitin, a subset of human glomerulopathies associated with podocyte foot process effacement (membranous nephropathy, SLE class V, FSGS) de novo expressed UCH-L1 in podocyte cell bodies, nuclei, and processes. Interestingly, UCH-L1 expression correlated with podocyte ubiquitin content and internalization of the podocyte-specific proteins nephrin and alpha-actinin-4. In contrast, minimal change glomerulonephritis, a reversible disease, demonstrated minimal UCH-L1 and ubiquitin expression with intact alpha-actinin-4 but internalized nephrin. Glomerular kidney diseases typically not associated with foot process effacement (SLE class IV, ANCA+ necrotizing GN, amyloidosis, IgA nephritis) expressed intermediate to no UCH-L1 and ubiquitin. These studies show a role for UCH-L1 and ubiquitin modification in podocyte differentiation and injury.

  7. The arginine residue within the C-terminal active core of Bombyx mori pheromone biosynthesis-activating neuropeptide (PBAN is essential for receptor binding and activation

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    Takeshi eKawai

    2012-03-01

    Full Text Available In most lepidopteran insects, the biosynthesis of sex pheromones is regulated by pheromone biosynthesis activating neuropeptide (PBAN. Bombyx mori PBAN (BomPBAN consists of 33 amino acid residues and contains a C-terminus FSPRLamide motif as the active core. Among neuropeptides containing the FXPRLamide motif, the arginine (Arg, R residue two positions from the C-terminus is highly conserved across several neuropeptides, which can be designated as RXamide peptides. The purpose of this study was to reveal the role of the Arg residue in the BomPBAN active core. We synthesized a ten-residue peptide corresponding to the C-terminal part of BomPBAN with a series of point mutants at the 2nd position (ie, Arg from the C-terminus, termed the C2 position, and measured their efficacy in stimulating Ca2+ influx in insect cells concomitantly expressing a fluorescent PBAN receptor chimera (PBANR-EGFP and loaded with the fluorescent Ca2+ indicator, Fura Red-AM. PBAN analogs with the C2 position replaced with alanine (Ala, A, aspartic acid (Asp, D, serine (Ser, S or L-2-aminooctanoic acid (Aoc decreased PBAN-like activity. RC2A (SKTRYFSPALamide and RC2D (SKTRYFSPDLamide had the lowest activity and could not inhibit the activity of PBAN C10 (SKTRYFSPRLamide. We also prepared Rhodamine Red-labeled PBAN analogs of the mutants and examined their ability to bind PBANR. In contrast to 100 nM Rhodamine Red-PBAN C10, none of the mutants at the same concentration exhibited PBANR binding. Taken together, our results demonstrate that the C2 Arg residue in BomPBAN is essential for PBANR binding and activation.

  8. Cytoplasmic location of α1A voltage-gated calcium channel C-terminal fragment (Cav2.1-CTF aggregate is sufficient to cause cell death.

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    Makoto Takahashi

    Full Text Available The human α1A voltage-dependent calcium channel (Cav2.1 is a pore-forming essential subunit embedded in the plasma membrane. Its cytoplasmic carboxyl(C-tail contains a small poly-glutamine (Q tract, whose length is normally 4∼19 Q, but when expanded up to 20∼33Q, the tract causes an autosomal-dominant neurodegenerative disorder, spinocerebellar ataxia type 6 (SCA6. A recent study has shown that a 75-kDa C-terminal fragment (CTF containing the polyQ tract remains soluble in normal brains, but becomes insoluble mainly in the cytoplasm with additional localization to the nuclei of human SCA6 Purkinje cells. However, the mechanism by which the CTF aggregation leads to neurodegeneration is completely elusive, particularly whether the CTF exerts more toxicity in the nucleus or in the cytoplasm. We tagged recombinant (rCTF with either nuclear-localization or nuclear-export signal, created doxycyclin-inducible rat pheochromocytoma (PC12 cell lines, and found that the CTF is more toxic in the cytoplasm than in the nucleus, the observations being more obvious with Q28 (disease range than with Q13 (normal-length. Surprisingly, the CTF aggregates co-localized both with cAMP response element-binding protein (CREB and phosphorylated-CREB (p-CREB in the cytoplasm, and Western blot analysis showed that the quantity of CREB and p-CREB were both decreased in the nucleus when the rCTF formed aggregates in the cytoplasm. In human brains, polyQ aggregates also co-localized with CREB in the cytoplasm of SCA6 Purkinje cells, but not in other conditions. Collectively, the cytoplasmic Cav2.1-CTF aggregates are sufficient to cause cell death, and one of the pathogenic mechanisms may be abnormal CREB trafficking in the cytoplasm and reduced CREB and p-CREB levels in the nuclei.

  9. Downregulation of 5-HT7 Serotonin Receptors by the Atypical Antipsychotics Clozapine and Olanzapine. Role of Motifs in the C-Terminal Domain and Interaction with GASP-1.

    Science.gov (United States)

    Manfra, Ornella; Van Craenenbroeck, Kathleen; Skieterska, Kamila; Frimurer, Thomas; Schwartz, Thue W; Levy, Finn Olav; Andressen, Kjetil Wessel

    2015-07-15

    The human 5-HT7 serotonin receptor, a G-protein-coupled receptor (GPCR), activates adenylyl cyclase constitutively and upon agonist activation. Biased ligands differentially activate 5-HT7 serotonin receptor desensitization, internalization and degradation in addition to G protein activation. We have previously found that the atypical antipsychotics clozapine and olanzapine inhibited G protein activation and, surprisingly, induced both internalization and lysosomal degradation of 5-HT7 receptors. Here, we aimed to determine the mechanism of clozapine- and olanzapine-mediated degradation of 5-HT7 receptors. In the C-terminus of the 5-HT7 receptor, we identified two YXXΦ motifs, LR residues, and a palmitoylated cysteine anchor as potential sites involved in receptor trafficking to lysosomes followed by receptor degradation. Mutating either of these sites inhibited clozapine- and olanzapine-mediated degradation of 5-HT7 receptors and also interfered with G protein activation. In addition, we tested whether receptor degradation was mediated by the GPCR-associated sorting protein-1 (GASP-1). We show that GASP-1 binds the 5-HT7 receptor and regulates the clozapine-mediated degradation. Mutations of the identified motifs and residues, located in or close to Helix-VIII of the 5-HT7 receptor, modified antipsychotic-stimulated binding of proteins (such as GASP-1), possibly by altering the flexibility of Helix-VIII, and also interfered with G protein activation. Taken together, our data demonstrate that binding of clozapine or olanzapine to the 5-HT7 receptor leads to antagonist-mediated lysosomal degradation by exposing key residues in the C-terminal tail that interact with GASP-1.

  10. Reduced protein turnover mediates functional deficits in transgenic mice expressing the 25 kDa C-terminal fragment of TDP-43.

    Science.gov (United States)

    Caccamo, Antonella; Shaw, Darren M; Guarino, Francesca; Messina, Angela; Walker, Aaron W; Oddo, Salvatore

    2015-08-15

    Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD-TDP) are two neurodegenerative disorders characterized by the accumulation of TDP-43. TDP-43 is proteolitically cleaved to generate two major C-terminal fragments of 35 and 25 kDa. The latter, known as TDP-25, is a consistent feature of FTLD-TDP and ALS; however, little is known about its role in disease pathogenesis. We have previously developed transgenic mice overexpressing low levels of TDP-25 (TgTDP-25(+/0)), which at 6 months of age show mild cognitive impairments and no motor deficits. To better understand the role of TDP-25 in the pathogenesis of ALS and FTLD-TDP, we generated TDP-25 homozygous mice (TgTDP-25(+/+)), thereby further increasing TDP-25 expression. We found a gene-dosage effect on cognitive and motor function at 15 months of age, as the TgTDP-25(+/+) showed more severe spatial and working memory deficits as well as worse motor performance than TgTDP-25(+/0) mice. These behavioral deficits were associated with increased soluble levels of TDP-25 in the nucleus and cytosol. Notably, high TDP-25 levels were also linked to reduced autophagy induction and proteasome function, two events that have been associated with both ALS and FTLD-TDP. In summary, we present strong in vivo evidence that high levels of TDP-25 are sufficient to cause behavioral deficits and reduce function of two of the major protein turnover systems: autophagy and proteasome. These mice represent a new tool to study the role of TDP-25 in the pathogenesis of ALS and FTLD-TDP. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  11. {sup 1}H and {sup 15}N resonance assignment, secondary structure and dynamic behaviour of the C-terminal domain of human papillomavirus oncoprotein E6

    Energy Technology Data Exchange (ETDEWEB)

    Nomine, Yves; Charbonnier, Sebastian [Laboratoire d' Immunotechnologie, CNRS UMR 7100, Ecole Superieure de Biotechnologie de Strasbourg (France); Miguet, Laurent; Potier, Noelle; Dorsselaer, Alain Van [Laboratoire de Spectrometrie de Masse Bio-Organique, CNRS UMR 7509, Faculte de Chimie (France); Atkinson, R. Andrew [Laboratoire de RMN, CNRS UMR 7104, Ecole Superieure de Biotechnologie de Strasbourg (France); Trave, Gilles [Laboratoire d' Immunotechnologie, CNRS UMR 7100, Ecole Superieure de Biotechnologie de Strasbourg (France)], E-mail: trave@esbs.u-strasbg.fr; Kieffer, Bruno [Laboratoire de RMN, CNRS UMR 7104, Ecole Superieure de Biotechnologie de Strasbourg (France)], E-mail: kieffer@esbs.u-strasbg.fr

    2005-02-15

    E6 is a viral oncoprotein implicated in cervical cancers, produced by human papillomaviruses (HPVs). E6 contains two putative zinc-binding domains of about 75 residues each. The difficulty in producing recombinant E6 has long hindered the obtention of structural data. Recently, we described the expression and purification of E6-C 4C/4S, a stable, folded mutant of the C-terminal domain of HPV16 E6. Here, we have produced {sup 15}N-labelled samples of E6-C 4C/4S for structural studies by NMR. We have assigned most {sup 1}H and {sup 15}N resonances and identified the elements of secondary structure of the domain. The domain displays an original {alpha}/{beta} topology with roughly equal proportions of {alpha}-helix and {beta}-sheet. The PDZ-binding region of E6, located at the extreme C-terminus of the domain, is in a random conformation. Mass spectrometry demonstrated the presence of one zinc ion per protein molecule. Kinetics of replacement of zinc by cadmium followed by {sup 1}H,{sup 15}N-HSQC experiments revealed specific frequency changes for the zinc-binding cysteines and their immediate neighbours. NMR spectra were affected by severe line-broadening effects which seriously hindered the assignment work. Investigation of these effects by {sup 15}N relaxation experiments showed that they are due to heterogeneous dynamic behaviour with {mu}s-ms time scale motions occurring in localised regions of the monomeric domain.

  12. Evidence against Extracellular Exposure of a Highly Immunogenic Region in the C-Terminal Domain of the Simian Immunodeficiency Virus gp41 Transmembrane Protein

    Science.gov (United States)

    Postler, Thomas S.; Martinez-Navio, José M.; Yuste, Eloísa

    2012-01-01

    The generally accepted model for human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein topology includes a single membrane-spanning domain. An alternate model has been proposed which features multiple membrane-spanning domains. Consistent with the alternate model, a high percentage of HIV-1-infected individuals produce unusually robust antibody responses to a region of envelope, the so-called “Kennedy epitope,” that in the conventional model should be in the cytoplasm. Here we show analogous, robust antibody responses in simian immunodeficiency virus SIVmac239-infected rhesus macaques to a region of SIVmac239 envelope located in the C-terminal domain, which in the conventional model should be inside the cell. Sera from SIV-infected rhesus macaques consistently reacted with overlapping oligopeptides corresponding to a region located within the cytoplasmic domain of gp41 by the generally accepted model, at intensities comparable to those observed for immunodominant areas of the surface component gp120. Rabbit serum raised against this highly immunogenic region (HIR) reacted with SIV envelope in cell surface-staining experiments, as did monoclonal anti-HIR antibodies isolated from an SIVmac239-infected rhesus macaque. However, control experiments demonstrated that this surface staining could be explained in whole or in part by the release of envelope protein from expressing cells into the supernatant and the subsequent attachment to the surfaces of cells in the culture. Serum and monoclonal antibodies directed against the HIR failed to neutralize even the highly neutralization-sensitive strain SIVmac316. Furthermore, a potential N-linked glycosylation site located close to the HIR and postulated to be outside the cell in the alternate model was not glycosylated. An artificially introduced glycosylation site within the HIR was also not utilized for glycosylation. Together, these data support the conventional model of SIV envelope as a type Ia

  13. Interaction of the C-terminal peptide of pulmonary surfactant protein B (SP-B with a bicellar lipid mixture containing anionic lipid.

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    Alexander Sylvester

    Full Text Available The hydrophobic lung surfactant SP-B is essential for respiration. SP-B promotes spreading and adsorption of surfactant at the alveolar air-water interface and may facilitate connections between the surface layer and underlying lamellar reservoirs of surfactant material. SP-B63-78 is a cationic and amphipathic helical peptide containing the C-terminal helix of SP-B. (2H NMR has been used to examine the effect of SP-B63-78 on the phase behavior and dynamics of bicellar lipid dispersions containing the longer chain phospholipids DMPC-d 54 and DMPG and the shorter chain lipid DHPC mixed with a 3∶1∶1 molar ratio. Below the gel-to-liquid crystal phase transition temperature of the longer chain components, bicellar mixtures form small, rapidly reorienting disk-like particles with shorter chain lipid components predominantly found around the highly curved particle edges. With increasing temperature, the particles coalesce into larger magnetically-oriented structures and then into more extended lamellar phases. The susceptibility of bicellar particles to coalescence and large scale reorganization makes them an interesting platform in which to study peptide-induced interactions between lipid assemblies. SP-B63-78 is found to lower the temperature at which the orientable phase transforms to the more extended lamellar phase. The peptide also changes the spectrum of motions contributing to quadrupole echo decay in the lamellar phase. The way in which the peptide alters interactions between bilayered micelle structures may provide some insight into some aspects of the role of full-length SP-B in maintaining a functional surfactant layer in lungs.

  14. The C-terminal end of parainfluenza virus 5 NP protein is important for virus-like particle production and M-NP protein interaction.

    Science.gov (United States)

    Schmitt, Phuong Tieu; Ray, Greeshma; Schmitt, Anthony P

    2010-12-01

    Enveloped virus particles are formed by budding from infected-cell membranes. For paramyxoviruses, viral matrix (M) proteins are key drivers of virus assembly and budding. However, other paramyxovirus proteins, including glycoproteins, nucleocapsid (NP or N) proteins, and C proteins, are also important for particle formation in some cases. To investigate the role of NP protein in parainfluenza virus 5 (PIV5) particle formation, NP protein truncation and substitution mutants were analyzed. Alterations near the C-terminal end of NP protein completely disrupted its virus-like particle (VLP) production function and significantly impaired M-NP protein interaction. Recombinant viruses with altered NP proteins were generated, and these viruses acquired second-site mutations. Recombinant viruses propagated in Vero cells acquired mutations that mainly affected components of the viral polymerase, while recombinant viruses propagated in MDBK cells acquired mutations that mainly affected the viral M protein. Two of the Vero-propagated viruses acquired the same mutation, V/P(S157F), found previously to be responsible for elevated viral gene expression induced by a well-characterized variant of PIV5, P/V-CPI(-). Vero-propagated viruses caused elevated viral protein synthesis and spread rapidly through infected monolayers by direct cell-cell fusion, bypassing the need to bud infectious virions. Both Vero- and MDBK-propagated viruses exhibited infectivity defects and altered polypeptide composition, consistent with poor incorporation of viral ribonucleoprotein complexes (RNPs) into budding virions. Second-site mutations affecting M protein restored interaction with altered NP proteins in some cases and improved VLP production. These results suggest that multiple avenues are available to paramyxoviruses for overcoming defects in M-NP protein interaction.

  15. Novel C-Terminal Heat Shock Protein 90 Inhibitors (KU711 and Ku757 Are Effective in Targeting Head and Neck Squamous Cell Carcinoma Cancer Stem cells

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    C. Subramanian

    2017-12-01

    Full Text Available Advanced head and neck squamous cell carcinoma (HNSCC remains a therapeutic challenge due to the development of therapy resistance. Several studies have implicated the development of cancer stem cells as a possible mechanism for therapy resistance in HNSCC. Heat shock protein 90’s (Hsp90’s molecular chaperone function is implicated in pathways of resistance in HNSCC. Therefore, in the present study, we investigated the efficacy of novel C-terminal Hsp90 inhibitors (KU711 and KU757 in targeting HNSCC cancer stem cells (CSCs. Treatment of HNSCC human cell lines MDA1986, UMSCC 22B, and UMSCC 22B cisplatin-resistant cells with the KU compounds indicated complete blockage of self-renewal for the resistant and parent cell lines starting from 20 μM KU711 and 1 μM KU757. Dose-dependent decrease in the cancer stem cell markers CD44, ALDH, and CD44/ALDH double-positive cells was observed for all cell lines after treatment with KU711 and KU757. When cells were treated with either drug, migration and invasion were downregulated greater than 90% even at the lowest concentrations of 20 μM KU711 and 1 μM KU757. Western blot showed >90% reduction in client protein “stemness” marker BMI-1 and mesenchymal marker vimentin, as well as increase in epithelial marker E-cadherin for both cell lines, indicating epithelial to mesenchymal transition quiescence. Several CSC-mediated miRNAs that play a critical role in HNSCC therapy resistance were also downregulated with KU treatment. In vivo, KU compounds were effective in decreasing tumor growth with no observed toxicity. Taken together, these results indicate that KU compounds are effective therapeutics for targeting HNSCC CSCs.

  16. Unmasking the roles of N- and C-terminal flanking sequences from exon 1 of huntingtin as modulators of polyglutamine aggregation.

    Science.gov (United States)

    Crick, Scott L; Ruff, Kiersten M; Garai, Kanchan; Frieden, Carl; Pappu, Rohit V

    2013-12-10

    Huntington disease is caused by mutational expansion of the CAG trinucleotide within exon 1 of the huntingtin (Htt) gene. Exon 1 spanning N-terminal fragments (NTFs) of the Htt protein result from aberrant splicing of transcripts of mutant Htt. NTFs typically encompass a polyglutamine tract flanked by an N-terminal 17-residue amphipathic stretch (N17) and a C-terminal 38-residue proline-rich stretch (C38). We present results from in vitro biophysical studies that quantify the driving forces for and mechanisms of polyglutamine aggregation as modulated by N17 and C38. Although N17 is highly soluble by itself, it lowers the saturation concentration of soluble NTFs and increases the driving force, vis-à-vis homopolymeric polyglutamine, for forming insoluble aggregates. Kinetically, N17 accelerates fibril formation and destabilizes nonfibrillar intermediates. C38 is also highly soluble by itself, and it lends its high intrinsic solubility to lower the driving force for forming insoluble aggregates by increasing the saturation concentration of soluble NTFs. In NTFs with both modules, N17 and C38 act synergistically to destabilize nonfibrillar intermediates (N17 effect) and lower the driving force for forming insoluble aggregates (C38 effect). Morphological studies show that N17 and C38 promote the formation of ordered fibrils by NTFs. Homopolymeric polyglutamine forms a mixture of amorphous aggregates and fibrils, and its aggregation mechanisms involve early formation of heterogeneous distributions of nonfibrillar species. We propose that N17 and C38 act as gatekeepers that control the intrinsic heterogeneities of polyglutamine aggregation. This provides a biophysical explanation for the modulation of in vivo NTF toxicities by N17 and C38.

  17. Mitrocomin from the jellyfish Mitrocoma cellularia with deleted C-terminal tyrosine reveals a higher bioluminescence activity compared to wild type photoprotein

    Energy Technology Data Exchange (ETDEWEB)

    Burakova, Ludmila P.; Natashin, Pavel V.; Markova, Svetlana V.; Eremeeva, Elena V.; Malikova, Natalia P.; Cheng, Chongyun; Liu, Zhi-Jie; Vysotski, Eugene S.

    2016-09-01

    The full-length cDNA genes encoding five new isoforms of Ca2 +-regulated photoprotein mitrocomin from a small tissue sample of the outer bell margin containing photocytes of only one specimen of the luminous jellyfish Mitrocoma cellularia were cloned, sequenced, and characterized after their expression in Escherichia coli and subsequent purification. The analysis of cDNA nucleotide sequences encoding mitrocomin isoforms allowed suggestion that two isoforms might be the products of two allelic genes differing in one amino acid residue (64R/Q) whereas other isotypes appear as a result of transcriptional mutations. In addition, the crystal structure of mitrocomin was determined at 1.30 Å resolution which expectedly revealed a high similarity with the structures of other hydromedusan photoproteins. Although mitrocomin isoforms reveal a high degree of identity of amino acid sequences, they vary in specific bioluminescence activities. At that, all isotypes displayed the identical bioluminescence spectra (473–474 nm with no shoulder at 400 nm). Fluorescence spectra of Ca2 +-discharged mitrocomins were almost identical to their light emission spectra similar to the case of Ca2 +-discharged aequorin, but different from Ca2 +-discharged obelins and clytin which fluorescence is red-shifted by 25–30 nm from bioluminescence spectra. The main distinction of mitrocomin from other hydromedusan photoproteins is an additional Tyr at the C-terminus. Using site-directed mutagenesis, we showed that this Tyr is not important for bioluminescence because its deletion even increases specific activity and efficiency of apo-mitrocomin conversion into active photoprotein, in contrast to C-terminal Pro of other photoproteins. Since genes in a population generally exist as different isoforms, it makes us anticipate the cloning of even more isoforms of mitrocomin and other hydromedusan photoproteins with different bioluminescence properties.

  18. Transglutaminase 2 strongly binds to an extracellular matrix component other than fibronectin via its second C-terminal beta-barrel domain.

    Science.gov (United States)

    Stamnaes, Jorunn; Cardoso, Inês; Iversen, Rasmus; Sollid, Ludvig M

    2016-11-01

    Transglutaminase 2 (TG2) is a ubiquitous crosslinking enzyme present in both intra- and extracellular in many cell types and tissues. TG2 is upregulated upon cellular stress or injury, and extracellular TG2 is implicated in several human diseases, including celiac disease. However, incomplete knowledge about extracellular TG2 biology limits our understanding of how TG2 is involved in disease. Here, we demonstrate that binding of TG2 to the ECM of small intestinal tissue sections is the sum of binding to fibronectin (FN) via its N-terminal domain and binding to an abundant, novel extracellular matrix (ECM) interaction partner via its second C-terminal beta-barrel domain. The latter interaction dominates and gives rise to the characteristic reticular staining pattern of extracellular TG2. Of relevance for celiac disease, we show that self-multimerized TG2 does not efficiently deposit in the intestinal ECM, and TG2 complexes may thus become free-floating antigens in tissues in contrast to monomeric TG2 that would readily become sequestered by the ECM. Upon injection of monoclonal antibody targeting the FN-binding site, we observe antibody deposition on extracellular TG2 in cryosections, suggesting that the FN-binding site of TG2 is exposed in vivo. This would explain how and why celiac autoantibodies recognizing the FN-binding site of TG2 can bind TG2 in vitro, in situ as well as in vivo. © 2016 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.

  19. TAL effectors target the C-terminal domain of RNA polymerase II (CTD by inhibiting the prolyl-isomerase activity of a CTD-associated cyclophilin.

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    Mariane Noronha Domingues

    Full Text Available Transcriptional activator-like (TAL effectors of plant pathogenic bacteria function as transcription factors in plant cells. However, how TAL effectors control transcription in the host is presently unknown. Previously, we showed that TAL effectors of the citrus canker pathogen Xanthomonas citri, named PthAs, targeted the citrus protein complex comprising the thioredoxin CsTdx, ubiquitin-conjugating enzymes CsUev/Ubc13 and cyclophilin CsCyp. Here we show that CsCyp complements the function of Cpr1 and Ess1, two yeast cyclophilins that regulate transcription by the isomerization of proline residues of the regulatory C-terminal domain (CTD of RNA polymerase II. We also demonstrate that CsCyp, CsTdx, CsUev and four PthA variants interact with the citrus CTD and that CsCyp co-immunoprecipitate with the CTD in citrus cell extracts and with PthA2 transiently expressed in sweet orange epicotyls. The interactions of CsCyp with the CTD and PthA2 were inhibited by cyclosporin A (CsA, a cyclophilin inhibitor. Moreover, we present evidence that PthA2 inhibits the peptidyl-prolyl cis-trans isomerase (PPIase activity of CsCyp in a similar fashion as CsA, and that silencing of CsCyp, as well as treatments with CsA, enhance canker lesions in X. citri-infected leaves. Given that CsCyp appears to function as a negative regulator of cell growth and that Ess1 negatively regulates transcription elongation in yeast, we propose that PthAs activate host transcription by inhibiting the PPIase activity of CsCyp on the CTD.

  20. A590T mutation in KCNQ1 C-terminal helix D decreases IKs channel trafficking and function but not Yotiao interaction.

    Science.gov (United States)

    Kinoshita, Koshi; Komatsu, Takuto; Nishide, Kohki; Hata, Yukiko; Hisajima, Nozomi; Takahashi, Hiroyuki; Kimoto, Katsuya; Aonuma, Kei; Tsushima, Eikichi; Tabata, Toshihide; Yoshida, Tomoyuki; Mori, Hisashi; Nishida, Kunihiro; Yamaguchi, Yoshiaki; Ichida, Fukiko; Fukurotani, Kenkichi; Inoue, Hiroshi; Nishida, Naoki

    2014-07-01

    KCNQ1 encodes the α subunit of the voltage-gated channel that mediates the cardiac slow delayed rectifier K(+) current (IKs). Here, we report a KCNQ1 allele encoding an A590T mutation [KCNQ1(A590T)] found in a 39-year-old female with a mild QT prolongation. A590 is located in the C-terminal α helical region of KCNQ1 that mediates subunit tetramerization, membrane trafficking, and interaction with Yotiao. This interaction is known to be required for the proper modulation of IKs by cAMP. Since previous studies reported that mutations in the vicinity of A590 impair IKs channel surface expression and function, we examined whether and how the A590T mutation affects the IKs channel. Electrophysiological measurements in HEK-293T cells showed that the A590T mutation caused a reduction in IKs density and a right-shift of the current-voltage relation of channel activation. Immunocytochemical and immunoblot analyses showed the reduced cell surface expression of KCNQ1(A590T) subunit and its rescue by coexpression of the wild-type KCNQ1 [KCNQ1(WT)] subunit. Moreover, KCNQ1(A590T) subunit interacted with Yotiao and had a cAMP-responsiveness comparable to that of KCNQ1(WT) subunit. These findings indicate that the A590 of KCNQ1 subunit plays important roles in the maintenance of channel surface expression and function via a novel mechanism independent of interaction with Yotiao. Copyright © 2014 Elsevier Ltd. All rights reserved.

  1. A novel C-terminal domain of RecJ is critical for interaction with HerA in Deinococcus radiodurans

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    Kaiying eCheng

    2015-11-01

    Full Text Available Homologous recombination (HR generates error-free repair products, which plays an important role in double strand break repair and replication fork rescue processes. DNA end resection, the critical step in HR, is usually performed by a series of nuclease/helicase. RecJ was identified as a 5’-3’ exonuclease involved in bacterial DNA end resection. Typical RecJ possesses a conserved DHH domain, a DHHA1 domain, and an oligonucleotide/oligosaccharide-binding (OB fold. However, RecJs from Deinococcus-Thermus phylum, such as Deinococcus radiodurans RecJ (DrRecJ, possess an extra C-terminal domain (CTD, of which the function has not been characterized. Here, we showed that a CTD-deletion of DrRecJ (DrRecJΔC could not restore drrecJ mutant growth and mitomycin C (MMC-sensitive phenotypes, indicating that this domain is essential for DrRecJ in vivo. DrRecJΔC displayed reduced DNA nuclease activity and DNA binding ability. Direct interaction was identified between DrRecJ-CTD and DrHerA, which stimulates DrRecJ nuclease activity by enhancing its DNA binding affinity. Moreover, DrNurA nuclease, another partner of DrHerA, inhibited the stimulation of DrHerA on DrRecJ nuclease activity by interaction with DrHerA. Opposing growth and MMC-resistance phenotypes between the recJ and nurA mutants were observed. A novel modulation mechanism among DrRecJ, DrHerA, and DrNurA was also suggested.

  2. Constitutive endocytosis and turnover of the neuronal glycine transporter GlyT2 is dependent on ubiquitination of a C-terminal lysine cluster.

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    Jaime de Juan-Sanz

    Full Text Available Inhibitory glycinergic neurotransmission is terminated by sodium and chloride-dependent plasma membrane glycine transporters (GlyTs. The mainly glial glycine transporter GlyT1 is primarily responsible for the completion of inhibitory neurotransmission and the neuronal glycine transporter GlyT2 mediates the reuptake of the neurotransmitter that is used to refill synaptic vesicles in the terminal, a fundamental role in the physiology and pathology of glycinergic neurotransmission. Indeed, inhibitory glycinergic neurotransmission is modulated by the exocytosis and endocytosis of GlyT2. We previously reported that constitutive and Protein Kinase C (PKC-regulated endocytosis of GlyT2 is mediated by clathrin and that PKC accelerates GlyT2 endocytosis by increasing its ubiquitination. However, the role of ubiquitination in the constitutive endocytosis and turnover of this protein remains unexplored. Here, we show that ubiquitination of a C-terminus four lysine cluster of GlyT2 is required for constitutive endocytosis, sorting into the slow recycling pathway and turnover of the transporter. Ubiquitination negatively modulates the turnover of GlyT2, such that increased ubiquitination driven by PKC activation accelerates transporter degradation rate shortening its half-life while decreased ubiquitination increases transporter stability. Finally, ubiquitination of GlyT2 in neurons is highly responsive to the free pool of ubiquitin, suggesting that the deubiquitinating enzyme (DUB ubiquitin C-terminal hydrolase-L1 (UCHL1, as the major regulator of neuronal ubiquitin homeostasis, indirectly modulates the turnover of GlyT2. Our results contribute to the elucidation of the mechanisms underlying the dynamic trafficking of this important neuronal protein which has pathological relevance since mutations in the GlyT2 gene (SLC6A5 are the second most common cause of human hyperekplexia.

  3. Insights into PG-binding, conformational change, and dimerization of the OmpA C-terminal domains from Salmonella enterica serovar Typhimurium and Borrelia burgdorferi: Characterization of OmpA C-Terminal Domain

    Energy Technology Data Exchange (ETDEWEB)

    Tan, Kemin [Center for Structural Genomics of Infectious Diseases, University of Chicago, 5735 South Ellis Avenue Chicago Illinois 60637; Midwest Center for Structural Genomics, Argonne National Laboratory, Argonne Illinois 60439; Structural Biology Center, Biosciences, Argonne National Laboratory, Argonne Illinois 60439; Deatherage Kaiser, Brooke L. [National Security Directorate, Pacific Northwest National Laboratory, Richland Washington 99352; Wu, Ruiying [Midwest Center for Structural Genomics, Argonne National Laboratory, Argonne Illinois 60439; Cuff, Marianne [Midwest Center for Structural Genomics, Argonne National Laboratory, Argonne Illinois 60439; Fan, Yao [Midwest Center for Structural Genomics, Argonne National Laboratory, Argonne Illinois 60439; Bigelow, Lance [Midwest Center for Structural Genomics, Argonne National Laboratory, Argonne Illinois 60439; Jedrzejczak, Robert P. [Center for Structural Genomics of Infectious Diseases, University of Chicago, 5735 South Ellis Avenue Chicago Illinois 60637; Adkins, Joshua N. [Earth and Biological Sciences Directorate, Pacific Northwest National Laboratory, Richland Washington 99352; Cort, John R. [Earth and Biological Sciences Directorate, Pacific Northwest National Laboratory, Richland Washington 99352; Babnigg, Gyorgy [Center for Structural Genomics of Infectious Diseases, University of Chicago, 5735 South Ellis Avenue Chicago Illinois 60637; Midwest Center for Structural Genomics, Argonne National Laboratory, Argonne Illinois 60439; Joachimiak, Andrzej [Center for Structural Genomics of Infectious Diseases, University of Chicago, 5735 South Ellis Avenue Chicago Illinois 60637; Midwest Center for Structural Genomics, Argonne National Laboratory, Argonne Illinois 60439; Structural Biology Center, Biosciences, Argonne National Laboratory, Argonne Illinois 60439

    2017-06-19

    S. Typhimurium can induce both humoral and cell-mediated responses when establishing itself in the host. These responses are primarily stimulated against the lipopolysaccharide and major outer membrane (OM) proteins of the bacterium. OmpA is one of these major OM proteins. It comprises a N-terminal eight-stranded -barrel membrane domain and a C-terminal so-called OmpA C-terminal domain (OmpACTD). The OmpACTD and its homologs are believed to bind to peptidoglycan (PG) within the periplasm, maintaining bacterial osmotic homeostasis and modulating the permeability and integrity of the outer membrane. Here we present the structures of two forms of the OmpACTD of S. Typhimurium (STOmpACTD) and one structure of the less-studied OmpACTD of Borrelia burgdorferi (BbOmpACTD). In the open form of STOmpACTD, an aspartic acid residue from a long 2-3 loop points into the binding pocket, suggesting that an anion group such as a carboxylate group from PG is favored at the binding site. In the closed form of STOmpACTD and in the structure of BbOmpACTD, a sulfate group from the crystallization buffer is tightly bound at the equivalent site. The differences between the closed and open forms of STOmpACTD, suggest a large conformational change that includes an extension of 3 helix by ordering a part of 2-3 loop. We suggest that the sulfate anion observed in these structures mimics the carboxylate group of PG when bound to STOmpACTD. In addition, the binding of PG or a ligand mimic may enhance dimerization of STOmpACTD, or possibly that of full length STOmpA.

  4. Expression and actions of corticotropin-releasing factor/diuretic hormone-like peptide (CDLP) and teneurin C-terminal associated peptide (TCAP) in the vase tunicate, Ciona intestinalis: Antagonism of the feeding response.

    Science.gov (United States)

    D'Aquila, Andrea L; Hsieh, Alan Hwa-Ruey; Hsieh, Adam Hwa-Ming; De Almeida, Reuben; Lovejoy, Sabine R; Lovejoy, David A

    2017-05-15

    Teneurin C-terminal associated peptide (TCAP) is a neuropeptide that bears some structural similarity to the corticotropin-releasing factor (CRF) family of peptides. TCAP and CRF are both implicated in the regulation of stress-related behaviors, as established in rodent models. However, in vertebrates, both TCAP and CRF possess three additional paralogous forms making vertebrate models difficult to assess with respect to TCAP-CRF interaction. As a urochordate, this species possesses single homologs of TCAP and of a CRF/Diuretic-like peptide (CDLP) in the genome, thereby establishing Ciona intestinalis as an excellent model organism to examine the interaction of these peptide systems. However, the lack of C. intestinalis synthetic peptides and specific antisera has complicated experimentation. We, therefore, prepared synthetic versions of CDLP and TCAP to prepare specific antisera and to investigate their bioactivity in this species. To analyze stress-related behaviors, a novel behavioral assay was used to characterize different types of contraction-based behaviors, using buccal opening contractions, cloacal opening contractions, lateral contractions, longitudinal contractions and expulsions. Protein and mRNA expression data indicate that the mature versions of both peptides are present in a number of tissues. With respect to behavioral activity, both TCAP- and CDLP-treated animals had distinct contraction profiles under ambient conditions. Moreover, food stimulation tests revealed that whereas CDLP-treated animals displayed a strong expulsion behavior in response to feeding, TCAP-treated animals did not show this effect. These actions are consistent with previous studies done in vertebrates. Copyright © 2016. Published by Elsevier Inc.

  5. Cellulose microfibril crystallinity is reduced by mutating C-terminal transmembrane region residues CESA1{sup A903V} and CESA3{sup T942I} of cellulose synthase

    Energy Technology Data Exchange (ETDEWEB)

    Harris, Darby; Corbin, Kendall; Wang, Tuo; Gutierrez, Ryan; Bertolo, Ana; Petti, Caroalberto; Smilgies, Detlef-M; Estevez, Jose Manuel; Bonetta, Dario; Urbanowicz, Breeanna; Ehrhardt, David; Somerville, Chris; Rose, Jocelyn; Hong, Mei; DeBolt, Seth

    2012-01-08

    The mechanisms underlying the biosynthesis of cellulose in plants are complex and still poorly understood. A central question concerns the mechanism of microfibril structure and how this is linked to the catalytic polymerization action of cellulose synthase (CESA). Furthermore, it remains unclear whether modification of cellulose microfibril structure can be achieved genetically, which could be transformative in a bio-based economy. To explore these processes in planta, we developed a chemical genetic toolbox of pharmacological inhibitors and corresponding resistance-conferring point mutations in the C-terminal transmembrane domain region of CESA1{sup A903V} and CESA3{sup T942I} in Arabidopsis thaliana. Using {sup 13}C solid-state nuclear magnetic resonance spectroscopy and X-ray diffraction, we show that the cellulose microfibrils displayed reduced width and an additional cellulose C4 peak indicative of a degree of crystallinity that is intermediate between the surface and interior glucans of wild type, suggesting a difference in glucan chain association during microfibril formation. Consistent with measurements of lower microfibril crystallinity, cellulose extracts from mutated CESA1{sup A903V} and CESA3{sup T942I} displayed greater saccharification efficiency than wild type. Using live-cell imaging to track fluorescently labeled CESA, we found that these mutants show increased CESA velocities in the plasma membrane, an indication of increased polymerization rate. Collectively, these data suggest that CESA1{sup A903V} and CESA3{sup T942I} have modified microfibril structure in terms of crystallinity and suggest that in plants, as in bacteria, crystallization biophysically limits polymerization.

  6. Elevated Plasma C-Terminal Endothelin-1 Precursor Fragment Concentrations Are Associated with Less Anxiety in Patients with Cardiovascular Risk Factors. Results from the Observational DIAST-CHF Study.

    Science.gov (United States)

    Meyer, Thomas; Chavanon, Mira-Lynn; Herrrmann-Lingen, Christoph; Roggenthien, Maren; Nolte, Kathleen; Pieske, Burkert; Wachter, Rolf; Edelmann, Frank

    2015-01-01

    The role of endothelin-1 (ET-1) in the neurobiology of anxiety is unknown, therefore, we assessed in the observational multicenter DIAST-CHF study whether the C-terminal ET-1 precursor fragment (CT-proET-1) is linked to anxiety. Plasma concentrations of CT-proET-1 were measured in a total of 1,410 patients presenting with cardiovascular risk factors (mean age 66.91±8.2 years, 49.3% males, mean left ventricular ejection fraction 60.0±8.2%) who had completed the Hospital Anxiety and Depression Scale (HADS) questionnaire. Among the total study cohort (n = 1,410), there were 118 subjects (8.4%) with an HADS anxiety score above the cut-off level of 11 suggestive of clinically relevant anxiety. Plasma CT-proET-1 levels were significantly lower in the group of anxious patients as compared to non-anxious patients (p = 0.013). In regression models adjusted for sex, age, systolic blood pressure, and diameters of left atrium and ventricle, plasma CT-proET-1 was again linked to anxiety (Exp(β) = 0.247, 95%-confidence interval [95%-CI] = 0.067-0.914, p = 0.036). Given the high prevalence of depressive disorders in anxious patients, we additionally included the HADS depression score as an independent variable in the models and found that CT-proET-1 remained a significant predictor of anxiety, independent of comorbid depression (Exp(β) = 0.114, 95%-CI = 0.023-0.566, p = 0.008). Our data from a population-based study in outpatients with cardiovascular risk factors revealed that circulating CT-proET-1 levels are negatively associated with anxiety. Further investigations are required to clarify the putative anxiolytic effect of ET-1 or its precursor molecules in humans and to decipher its mechanistic pathways.

  7. Structural model of dodecameric heat-shock protein Hsp21: Flexible N-terminal arms interact with client proteins while C-terminal tails maintain the dodecamer and chaperone activity.

    Science.gov (United States)

    Rutsdottir, Gudrun; Härmark, Johan; Weide, Yoran; Hebert, Hans; Rasmussen, Morten I; Wernersson, Sven; Respondek, Michal; Akke, Mikael; Højrup, Peter; Koeck, Philip J B; Söderberg, Christopher A G; Emanuelsson, Cecilia

    2017-05-12

    Small heat-shock proteins (sHsps) prevent aggregation of thermosensitive client proteins in a first line of defense against cellular stress. The mechanisms by which they perform this function have been hard to define due to limited structural information; currently, there is only one high-resolution structure of a plant sHsp published, that of the cytosolic Hsp16.9. We took interest in Hsp21, a chloroplast-localized sHsp crucial for plant stress resistance, which has even longer N-terminal arms than Hsp16.9, with a functionally important and conserved methionine-rich motif. To provide a framework for investigating structure-function relationships of Hsp21 and understanding these sequence variations, we developed a structural model of Hsp21 based on homology modeling, cryo-EM, cross-linking mass spectrometry, NMR, and small-angle X-ray scattering. Our data suggest a dodecameric arrangement of two trimer-of-dimer discs stabilized by the C-terminal tails, possibly through tail-to-tail interactions between the discs, mediated through extended IXVXI motifs. Our model further suggests that six N-terminal arms are located on the outside of the dodecamer, accessible for interaction with client proteins, and distinct from previous undefined or inwardly facing arms. To test the importance of the IXVXI motif, we created the point mutant V181A, which, as expected, disrupts the Hsp21 dodecamer and decreases chaperone activity. Finally, our data emphasize that sHsp chaperone efficiency depends on oligomerization and that client interactions can occur both with and without oligomer dissociation. These results provide a generalizable workflow to explore sHsps, expand our understanding of sHsp structural motifs, and provide a testable Hsp21 structure model to inform future investigations. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. The C-terminal Six Amino Acids of the FNT Channel FocA Are Required for Formate Translocation But Not Homopentamer Integrity.

    Science.gov (United States)

    Hunger, Doreen; Röcker, Marie; Falke, Dörte; Lilie, Hauke; Sawers, R Gary

    2017-01-01

    FocA is the archetype of the pentameric formate-nitrite transporter (FNT) superfamily of channels, members of which translocate small organic and inorganic anions across the cytoplasmic membrane of microorganisms. The N- and C-termini of each protomer are cytoplasmically oriented. A Y-L-R motif is found immediately after transmembrane helix 6 at the C-terminus of FNT proteins related to FocA, or those with a role in formate translocation. Previous in vivo studies had revealed that formate translocation through FocA was controlled by interaction with the formate-producing glycyl-radical enzyme pyruvate formate-lyase (PflB) or its structural and functional homolog, TdcE. In this study we analyzed the effect on in vivo formate export and import, as well as on the stability of the homopentamer in the membrane, of successively removing amino acid residues from the C-terminus of FocA. Removal of up to five amino acids was without consequence for either formate translocation or oligomer stability. Removal of a sixth residue (R280) prevented formate uptake by FocA in a strain lacking PflB and significantly reduced, but did not prevent, formate export. Sensitivity to the toxic formate analog hypophosphite, which is also transported into the cell by FocA, was also relieved. Circular dichroism spectroscopy and blue-native PAGE analysis revealed, however, that this variant had near identical secondary and quaternary structural properties to those of native FocA. Interaction with the glycyl radical enzyme, TdcE, was also unaffected by removal of the C-terminal 6 amino acid residues, indicating that impaired interaction with TdcE was not the reason for impaired formate translocation. Removal of a further residue (L279) severely restricted formate export, the stability of the protein and its ability to form homopentamers. Together, these studies revealed that the Y278-L279-R280 motif at the C-terminus is essential for bidirectional formate translocation by FocA, but that L279 is

  9. Ca2+-dependent photocrosslinking of tropomyosin residue 146 to residues 157-163 in the C-terminal domain of troponin I in reconstituted skeletal muscle thin filaments

    Science.gov (United States)

    Mudalige, Wasana A.K.A.; Tao, Terence C.; Lehrer, Sherwin S.

    2009-01-01

    Summary The Ca2+-dependent interaction of troponin I (TnI) with actin•tropomyosin (Actin•Tm) in the muscle thin filament is a critical step in the regulation of muscle contraction. Previous studies have suggested that, in the absence of Ca2+, TnI interacts with Tm as well as actin in the reconstituted muscle thin filament, maintaining Tm at the outer domain of actin and blocking myosin-actin interaction. To obtain direct evidence for this Tm-TnI interaction we performed photochemical crosslinking studies using Tm labeled with 4-maleimidobenzophenone (BPmal) at position 146 or 174 (Tm146* or Tm174*, respectively), reconstituted with actin and troponin (composed of TnI; troponin T, TnT; and troponin C, TnC) or with actin and TnI. After near uv-irradiation, SDS gels of the Tm*146-containing thin filament showed 3 new high molecular weight bands determined to be crosslinked products Tm*146-TnI, Tm*146-TnC and Tm*146-TnT using fluorescence-labeled TnI, mass spectrometry and Western blots. While Tm*146-TnI was produced only in the absence of Ca2+, the production of the other crosslinked species did not show a Ca2+ dependence. Tm*174 mainly crosslinked to TnT. In the absence of actin a similar crosslinking pattern was obtained with a much lower yield. A tryptic peptide from Tm*146-TnI of MW 2601.2 Da that was not present in the tryptic peptides of Tm*146 or TnI was identified using HPLC and MALDI-TOF. This was shown, using absorption and fluorescence spectroscopy, to be the BPmal-labeled peptide from Tm crosslinked to TnI peptide 157-163. These data showing that a region in the C-terminal domain of TnI interacts with Tm in the absence of Ca2+ support the hypothesis that a TnI-Tm interaction maintains Tm at the outer domain of actin, and will help efforts to localize Tn in the actin•Tm muscle thin filament. PMID:19379756

  10. Septal membrane localization by C-terminal amphipathic α-helices of MinD in Bacillus subtilis mutant cells lacking MinJ or DivIVA.

    Science.gov (United States)

    Ishikawa, Kazuki; Matsuoka, Satoshi; Hara, Hiroshi; Matsumoto, Kouji

    2017-10-18

    The Min system, which inhibits assembly of the cytokinetic protein FtsZ, is largely responsible for positioning the division site in rod-shaped bacteria. It has been reported that MinJ, which bridges DivIVA and MinD, is targeted to the cell poles by an interaction with DivIVA, and that MinJ in turn recruits MinCD to the cell poles. MinC, however, is located primarily at active division sites at mid-cell when expressed from its native promoter. Surprisingly, we found that Bacillus subtilis MinD is located at nascent septal membranes and at an asymmetric site on lateral membranes between nascent septal membranes in filamentous cells lacking MinJ or DivIVA. Bacillus subtilis MinD has two amphipathic α-helices rich in basic amino acid residues at its C-terminus; one of these, named MTS1 here, is the counterpart of the membrane targeting sequence (MTS) in Escherichia coli MinD while the other, named MTS-like sequence (MTSL), is the nearest helix to MTS1. These amphipathic helices were located independently at nascent septal membranes in cells lacking MinJ or DivIVA, whereas elimination of the helices from the wild type protein reduced its localization considerably. MinD variants with altered MTS1 and MTSL, in which basic amino acid residues were replaced with proline or acidic residues, were not located at nascent septal membranes, indicating that the binding to the nascent septal membranes requires basic residues and a helical structure. The septal localization of MTSL, but not of MTS1, was dependent on host cell MinD. These results suggest that MinD is targeted to nascent septal membranes via its C-terminal amphipathic α-helices in B. subtilis cells lacking MinJ or DivIVA. Moreover, the diffuse distribution of MinD lacking both MTSs suggests that only a small fraction of MinD depends on MinJ for its localization to nascent septal membranes.

  11. MicroRNA-212 Regulates the Expression of Olfactomedin 1 and C-Terminal Binding Protein 1 in Human Endometrial Epithelial Cells to Enhance Spheroid Attachment In Vitro.

    Science.gov (United States)

    Kottawatta, Kottawattage S A; So, Kam-Hei; Kodithuwakku, Suranga P; Ng, Ernest H Y; Yeung, William S B; Lee, Kai-Fai

    2015-11-01

    Successful embryo implantation requires a synchronized dialogue between a competent blastocyst and the receptive endometrium, which occurs in a limited time period known as the "window of implantation." Recent studies suggested that down-regulation of olfactomedin 1 (OLFM1) in the endometrium and fallopian tube is associated with receptive endometrium and tubal ectopic pregnancy in humans. Interestingly, the human chorionic gonadotropin (hCG) induces miR-212 expression, which modulates OLFM1 and C-terminal binding protein 1 (CTBP1) expressions in mouse granulosa cells. Therefore, we hypothesized that embryo-derived hCG would increase miR-212 expression and down-regulate OLFM1 and CTBP1 expressions to favor embryo attachment onto the female reproductive tract. We found that hCG stimulated the expression of miR-212 and down-regulated OLFM1 but not CTBP1 mRNA in both human endometrial (Ishikawa) and fallopian (OE-E6/E7) epithelial cells. However, hCG suppressed the expression of OLFM1 and CTBP1 proteins in both cell lines. The 3'UTR of both OLFM1 and CTBP1 contained binding sites for miR-212. The miR-212 precursor suppressed luciferase expression, whereas the miR-212 inhibitor stimulated luciferase expression of the wild-type (WT)-OLFM1 and WT-CTBP1 reporter constructs. Furthermore, hCG (25 IU/ml) treatments stimulated trophoblastic (Jeg-3) spheroid (blastocyst surrogate) attachment onto Ishikawa and OE-E6/E7 cells. Transfection of miR-212 precursor increased Jeg-3 spheroid attachment onto Ishikawa cells and decreased OLFM1 and CTBP1 protein expressions, whereas the opposite occurred with miR-212 inhibitor. Taken together, hCG stimulated miR-212, which in turn down-regulated OLFM1 and CTBP1 expression in fallopian and endometrial epithelial cells to favor spheroid attachment. © 2015 by the Society for the Study of Reproduction, Inc.

  12. The C-terminal 18 Amino Acid Region of Dengue Virus NS5 Regulates its Subcellular Localization and Contains a Conserved Arginine Residue Essential for Infectious Virus Production.

    Directory of Open Access Journals (Sweden)

    Moon Y F Tay

    2016-09-01

    Full Text Available Dengue virus NS5 is the most highly conserved amongst the viral non-structural proteins and is responsible for capping, methylation and replication of the flavivirus RNA genome. Interactions of NS5 with host proteins also modulate host immune responses. Although replication occurs in the cytoplasm, an unusual characteristic of DENV2 NS5 is that it localizes to the nucleus during infection with no clear role in replication or pathogenesis. We examined NS5 of DENV1 and 2, which exhibit the most prominent difference in nuclear localization, employing a combination of functional and structural analyses. Extensive gene swapping between DENV1 and 2 NS5 identified that the C-terminal 18 residues (Cter18 alone was sufficient to direct the protein to the cytoplasm or nucleus, respectively. The low micromolar binding affinity between NS5 Cter18 and the nuclear import receptor importin-alpha (Impα, allowed their molecular complex to be purified, crystallised and visualized at 2.2 Å resolution using x-ray crystallography. Structure-guided mutational analysis of this region in GFP-NS5 clones of DENV1 or 2 and in a DENV2 infectious clone reveal residues important for NS5 subcellular localization. Notably, the trans conformation adopted by Pro-884 allows proper presentation for binding Impα and mutating this proline to Thr, as present in DENV1 NS5, results in mislocalizaion of NS5 to the cytoplasm without compromising virus fitness. In contrast, a single mutation to alanine at NS5 position R888, a residue conserved in all flaviviruses, resulted in a completely non-viable virus, and the R888K mutation led to a severely attenuated phentoype, even though NS5 was located in the nucleus. R888 forms a hydrogen bond with Y838 that is also conserved in all flaviviruses. Our data suggests an evolutionarily conserved function for NS5 Cter18, possibly in RNA interactions that are critical for replication, that is independent of its role in subcellular localization.

  13. Adaptive immunity against Leishmania nucleoside hydrolase maps its c-terminal domain as the target of the CD4+ T cell-driven protective response.

    Science.gov (United States)

    Nico, Dirlei; Claser, Carla; Borja-Cabrera, Gulnara P; Travassos, Luiz R; Palatnik, Marcos; Soares, Irene da Silva; Rodrigues, Mauricio Martins; Palatnik-de-Sousa, Clarisa B

    2010-11-09

    Nucleoside hydrolases (NHs) show homology among parasite protozoa, fungi and bacteria. They are vital protagonists in the establishment of early infection and, therefore, are excellent candidates for the pathogen recognition by adaptive immune responses. Immune protection against NHs would prevent disease at the early infection of several pathogens. We have identified the domain of the NH of L. donovani (NH36) responsible for its immunogenicity and protective efficacy against murine visceral leishmaniasis (VL). Using recombinant generated peptides covering the whole NH36 sequence and saponin we demonstrate that protection against L. chagasi is related to its C-terminal domain (amino-acids 199-314) and is mediated mainly by a CD4+ T cell driven response with a lower contribution of CD8+ T cells. Immunization with this peptide exceeds in 36.73±12.33% the protective response induced by the cognate NH36 protein. Increases in IgM, IgG2a, IgG1 and IgG2b antibodies, CD4+ T cell proportions, IFN-γ secretion, ratios of IFN-γ/IL-10 producing CD4+ and CD8+ T cells and percents of antibody binding inhibition by synthetic predicted epitopes were detected in F3 vaccinated mice. The increases in DTH and in ratios of TNFα/IL-10 CD4+ producing cells were however the strong correlates of protection which was confirmed by in vivo depletion with monoclonal antibodies, algorithm predicted CD4 and CD8 epitopes and a pronounced decrease in parasite load (90.5-88.23%; p = 0.011) that was long-lasting. No decrease in parasite load was detected after vaccination with the N-domain of NH36, in spite of the induction of IFN-γ/IL-10 expression by CD4+ T cells after challenge. Both peptides reduced the size of footpad lesions, but only the C-domain reduced the parasite load of mice challenged with L. amazonensis. The identification of the target of the immune response to NH36 represents a basis for the rationale development of a bivalent vaccine against leishmaniasis and for multivalent

  14. Adaptive immunity against Leishmania nucleoside hydrolase maps its c-terminal domain as the target of the CD4+ T cell-driven protective response.

    Directory of Open Access Journals (Sweden)

    Dirlei Nico

    Full Text Available Nucleoside hydrolases (NHs show homology among parasite protozoa, fungi and bacteria. They are vital protagonists in the establishment of early infection and, therefore, are excellent candidates for the pathogen recognition by adaptive immune responses. Immune protection against NHs would prevent disease at the early infection of several pathogens. We have identified the domain of the NH of L. donovani (NH36 responsible for its immunogenicity and protective efficacy against murine visceral leishmaniasis (VL. Using recombinant generated peptides covering the whole NH36 sequence and saponin we demonstrate that protection against L. chagasi is related to its C-terminal domain (amino-acids 199-314 and is mediated mainly by a CD4+ T cell driven response with a lower contribution of CD8+ T cells. Immunization with this peptide exceeds in 36.73±12.33% the protective response induced by the cognate NH36 protein. Increases in IgM, IgG2a, IgG1 and IgG2b antibodies, CD4+ T cell proportions, IFN-γ secretion, ratios of IFN-γ/IL-10 producing CD4+ and CD8+ T cells and percents of antibody binding inhibition by synthetic predicted epitopes were detected in F3 vaccinated mice. The increases in DTH and in ratios of TNFα/IL-10 CD4+ producing cells were however the strong correlates of protection which was confirmed by in vivo depletion with monoclonal antibodies, algorithm predicted CD4 and CD8 epitopes and a pronounced decrease in parasite load (90.5-88.23%; p = 0.011 that was long-lasting. No decrease in parasite load was detected after vaccination with the N-domain of NH36, in spite of the induction of IFN-γ/IL-10 expression by CD4+ T cells after challenge. Both peptides reduced the size of footpad lesions, but only the C-domain reduced the parasite load of mice challenged with L. amazonensis. The identification of the target of the immune response to NH36 represents a basis for the rationale development of a bivalent vaccine against leishmaniasis and

  15. Identification of critical amino acids in the proximal C-terminal of TREK-2 K+ channel for activation by acidic pHi and ATP-dependent inhibition.

    Science.gov (United States)

    Woo, Joohan; Jun, Young Keul; Zhang, Yin-Hua; Nam, Joo Hyun; Shin, Dong Hoon; Kim, Sung Joon

    2018-02-01

    TWIK-related two-pore domain K+ channels (TREKs) are regulated by intracellular pH (pHi) and Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). Previously, Glu306 in proximal C-terminal (pCt) of mouse TREK-1 was identified as the pHi-sensing residue. The direction of PI(4,5)P2 sensitivity is controversial, and we have recently shown that TREKs are inhibited by intracellular ATP via endogenous PI(4,5)P2 formation. Here we investigate the anionic and cationic residues of pCt for the pHi and ATP-sensitivity in human TREK-2 (hTREK-2). In inside-out patch clamp recordings (ITREK-2,i-o), acidic pHi-induced activation was absent in E332A and was partly attenuated in E335A. Neutralization of cationic Lys (K330A) also eliminated the acidic pHi sensitivity of ITREK-2,i-o. Unlike the inhibition of wild-type (WT) ITREK-2,i-o by intracellular ATP, neither E332A nor K330A was sensitive to ATP. Nevertheless, exogenous PI(4,5)P2 (10 μM) abolished ITREK-2 i-o in all the above mutants as well as in WT, indicating unspecific inhibition by exogenous PI(4,5)P2. In whole-cell recordings of TREK-2 (ITREK-2,w-c), K330A and E332A showed higher or fully active basal activity, showing attenuated or insignificant activation by 2-APB, arachidonic acid, or acidic pHe 6.9. ITREK-1,w-c of WT is largely suppressed by pHe 6.9, and the inhibition is slightly attenuated in K312A and E315A. The results show concerted roles of the oppositely charged Lys and Glu in pCt for the ATP-dependent low basal activity and pHi sensitivity.

  16. Swapping the N- and C-terminal domains of human apolipoprotein E3 and AI reveals insights into their structure/activity relationship.

    Directory of Open Access Journals (Sweden)

    Mark T Lek

    Full Text Available Apolipoprotein (apo E3 and apoAI are exchangeable apolipoproteins that play a dominant role in regulating plasma lipoprotein metabolism. ApoE3 (299 residues is composed of an N-terminal (NT domain bearing a 4-helix bundle and a C-terminal (CT domain bearing a series of amphipathic α-helices. ApoAI (243 residues also comprises a highly helical NT domain and a less structured CT tail. The objective of this study was to understand their structural and functional role by generating domain swapped chimeras: apoE3-NT/apoAI-CT and apoAI-NT/apoE-CT. The bacterially overexpressed chimeras were purified by affinity chromatography and their identity confirmed by immunoblotting and mass spectrometry. Their α-helical content was comparable to that of the parent proteins. ApoE3-NT/apoAI-CT retained the denaturation profile of apoE3 NT domain, with apoAI CT tail eliciting a relatively unstructured state; its lipid binding ability improved dramatically compared to apoE3 indicative of a significant role of apoAI CT tail in lipid binding interaction. The LDL receptor interaction and ability to promote ABCA1-mediated cholesterol efflux of apoE3-NT/apoAI-CT was comparable to that of apoE3. In contrast, apoAI-NT/apoE-CT elicited an unfolding pattern and lipid binding ability that were similar to that of apoAI. As expected, DMPC/apoAI-NT/apoE-CT discoidal particles did not elicit LDLr binding ability, and promoted SR-B1 mediated cellular uptake of lipids to a limited extent. However, apoAI-NT/apoE-CT displayed an enhanced ability to promote cholesterol efflux compared to apoAI, indicative of a significant role for apoE CT domain in mediating this function. Together, these results indicate that the functional attributes of apoAI and apoE3 can be conferred on each other and that NT-CT domain interactions significantly modulate their structure and function.

  17. VGF and Its C-Terminal Peptide TLQP-62 Regulate Memory Formation in Hippocampus via a BDNF-TrkB-Dependent Mechanism

    Science.gov (United States)

    Lin, Wei-Jye; Jiang, Cheng; Sadahiro, Masato; Bozdagi, Ozlem; Vulchanova, Lucy; Alberini, Cristina M.

    2015-01-01

    Regulated expression and secretion of BDNF, which activates TrkB receptor signaling, is known to play a critical role in cognition. Identification of additional modulators of cognitive behavior that regulate activity-dependent BDNF secretion and/or potentiate TrkB receptor signaling would therefore be of considerable interest. In this study, we show in the adult mouse hippocampus that expression of the granin family gene Vgf and secretion of its C-terminal VGF-derived peptide TLQP-62 are required for fear memory formation. We found that hippocampal VGF expression and TLQP-62 levels were transiently induced after fear memory training and that sequestering secreted TLQP-62 peptide in the hippocampus immediately after training impaired memory formation. Reduced VGF expression was found to impair learning-evoked Rac1 induction and phosphorylation of the synaptic plasticity markers cofilin and synapsin in the adult mouse hippocampus. Moreover, TLQP-62 induced acute, transient activation of the TrkB receptor and subsequent CREB phosphorylation in hippocampal slice preparations and its administration immediately after training enhanced long-term memory formation. A critical role of BDNF-TrkB signaling as a downstream effector in VGF/TLQP-62-mediated memory consolidation was further revealed by posttraining activation of BDNF-TrkB signaling, which rescued impaired fear memory resulting from hippocampal administration of anti-VGF antibodies or germline VGF ablation in mice. We propose that VGF is a critical component of a positive BDNF-TrkB regulatory loop and, upon its induced expression by memory training, the TLQP-62 peptide rapidly reinforces BDNF-TrkB signaling, regulating hippocampal memory consolidation. SIGNIFICANCE STATEMENT Identification of the cellular and molecular mechanisms that regulate long-term memory formation and storage may provide alternative treatment modalities for degenerative and neuropsychiatric memory disorders. The neurotrophin BDNF plays a

  18. The mitogenic function of hepatitis B virus X protein resides within amino acids 51 to 140 and is modulated by N- and C-terminal regulatory regions.

    Science.gov (United States)

    Li, Huajie; Chi, Chia-Yi; Lee, Sook; Andrisani, Ourania M

    2006-11-01

    The hepatitis B virus (HBV) X protein (pX) is implicated in hepatocarcinogenesis by an unknown mechanism. pX variants encoded by HBV genomes found integrated in genomic DNA from liver tumors of patients with hepatocellular carcinoma (HCC) generally lack amino acids 134 to 154. Since deregulation of mitogenic pathways is linked to oncogenic transformation, herein we define the pX region required for mitogenic pathway activation. A series of pX deletions was used to construct tetracycline-regulated pX-expressing cell lines. The activation of the mitogenic pathways by these pX deletions expressed in the constructed cell lines was measured by transient transreporter assays, effects on endogenous cyclin A expression, and apoptosis. Conditional expression of pX51-140 in AML12 clone 4 cell line activates the mitogenic pathways, induces endogenous cyclin A expression, and sensitizes cells to apoptosis, similar to wild-type (WT) pX. By contrast, pX1-115 is inactive, supporting the idea that amino acids 116 to 140 are required for mitogenic pathway activation. Moreover, this pX deletion analysis demonstrates that WT pX function is modulated by two regions spanning amino acids 1 to 78 and 141 to 154. The N-terminal X1-78, expressed via a retroviral vector in WT pX-expressing 4pX-1 cells, coimmunoprecipitates with WT pX, indicating this pX region participates in protein-protein interactions leading to pX oligomerization. Interestingly, pX1-78 interferes with WT pX in mediating mitogenic pathway activation, endogenous gene expression, and apoptosis. The C-terminal pX region spanning amino acids 141 to 154 decreases pX stability, determined by pulse-chase studies of WT pX and pX1-140, suggesting that increased stability of naturally occurring pX variants lacking amino acids 134 to 154 may play a role in HCC development.

  19. Neuronal Biomarker Ubiquitin C-Terminal Hydrolase Detects Traumatic Intracranial Lesions on Computed Tomography in Children and Youth with Mild Traumatic Brain Injury.

    Science.gov (United States)

    Papa, Linda; Mittal, Manoj K; Ramirez, Jose; Silvestri, Salvatore; Giordano, Philip; Braga, Carolina F; Tan, Ciara N; Ameli, Neema J; Lopez, Marco A; Haeussler, Crystal A; Mendez Giordano, Diego; Zonfrillo, Mark R

    2017-07-01

    This study examined the performance of serum ubiquitin C-terminal hydrolase (UCH-L1) in detecting traumatic intracranial lesions on computed tomography (CT) scan (+CT) in children and youth with mild and moderate TBI (mmTBI) and assessed its performance in trauma control patients without head trauma. This prospective cohort study enrolled children and youth presenting to three level 1 trauma centers after blunt head trauma and a Glasgow Coma Scale (GCS) score of 9-15 as well as trauma control patients with GCS 15 that did not have blunt head trauma. The primary outcome measure was the presence of intracranial lesions on initial CT scan. Blood samples were obtained in all patients within 6 h of injury and measured by enzyme-linked immunosorbent assay ELISA for UCH-L1 (ng/mL). A total of 256 children and youth were enrolled in the study and had serum samples drawn within 6 h of injury for analysis; 196 had blunt head trauma and 60 were trauma controls. CT scan of the head was performed in 151 patients and traumatic intracranial lesions on CT scan were evident in 17 (11%), all of whom had a GCS of 13-15. The area under the receiver operating characteristic curve (AUC) for UCH-L1 in detecting children and youth with traumatic intracranial lesions on CT was 0.83 (95% confidence interval [CI], 0.73-0.93). In those presenting with a GCS of 15, the AUC for detecting lesions was 0.83 (95% CI, 0.72-0.94). Similarly, in children under 5 years of age, the AUC was 0.79 (95% CI, 0.59-1.00). Performance for detecting intracranial lesions at a UCH-L1 cut-off level of 0.18 ng/mL yielded a sensitivity of 100%, a specificity of 47%, and a negative predictive value of 100%. UCH-L1 showed good performance in infants and toddlers younger than 5 years and performed well in children and youth with a GCS score of 15. Before clinical application, further study in larger cohort of children and youth with mild TBI is warranted.

  20. The C-terminal region of Rad52 is essential for Rad52 nuclear and nucleolar localization, and accumulation at DNA damage sites immediately after irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Koike, Manabu, E-mail: m_koike@nirs.go.jp [DNA Repair Gene Res., National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555 (Japan); Yutoku, Yasutomo [DNA Repair Gene Res., National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555 (Japan); Graduate School of Science, Chiba University, Yayoicho, Inage-ku, Chiba 263-8522 (Japan); Koike, Aki [DNA Repair Gene Res., National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555 (Japan)

    2013-05-31

    Highlights: •Rad52 might play a key role in the repair of DSB immediately after irradiation. •EYFP-Rad52 accumulates rapidly at DSB sites and colocalizes with Ku80. •Accumulation of Rad52 at DSB sites is independent of the core NHEJ factors. •Localization and recruitment of Rad52 to DSB sites are dependent on the Rad52 CTR. •Basic amino acids in Rad52 CTR are highly conserved among vertebrate species. -- Abstract: Rad52 plays essential roles in homologous recombination (HR) and repair of DNA double-strand breaks (DSBs) in Saccharomyces cerevisiae. However, in vertebrates, knockouts of the Rad52 gene show no hypersensitivity to agents that induce DSBs. Rad52 localizes in the nucleus and forms foci at a late stage following irradiation. Ku70 and Ku80, which play an essential role in nonhomologous DNA-end-joining (NHEJ), are essential for the accumulation of other core NHEJ factors, e.g., XRCC4, and a HR-related factor, e.g., BRCA1. Here, we show that the subcellular localization of EYFP-Rad52(1–418) changes dynamically during the cell cycle. In addition, EYFP-Rad52(1–418) accumulates rapidly at microirradiated sites and colocalizes with the DSB sensor protein Ku80. Moreover, the accumulation of EYFP-Rad52(1–418) at DSB sites is independent of the core NHEJ factors, i.e., Ku80 and XRCC4. Furthermore, we observed that EYFP-Rad52(1–418) localizes in nucleoli in CHO-K1 cells and XRCC4-deficient cells, but not in Ku80-deficient cells. We also found that Rad52 nuclear localization, nucleolar localization, and accumulation at DSB sites are dependent on eight amino acids (411–418) at the end of the C-terminal region of Rad52 (Rad52 CTR). Furthermore, basic amino acids on Rad52 CTR are highly conserved among mammalian, avian, and fish homologues, suggesting that Rad52 CTR is important for the regulation and function of Rad52 in vertebrates. These findings also suggest that the mechanism underlying the regulation of subcellular localization of Rad52 is

  1. Structure and function of the C-terminal domain of MrpA in the Bacillus subtilis Mrp-antiporter complex--the evolutionary progenitor of the long horizontal helix in complex I.

    Science.gov (United States)

    Virzintiene, Egle; Moparthi, Vamsi K; Al-Eryani, Yusra; Shumbe, Leonard; Górecki, Kamil; Hägerhäll, Cecilia

    2013-10-11

    MrpA and MrpD are homologous to NuoL, NuoM and NuoN in complex I over the first 14 transmembrane helices. In this work, the C-terminal domain of MrpA, outside this conserved area, was investigated. The transmembrane orientation was found to correspond to that of NuoJ in complex I. We have previously demonstrated that the subunit NuoK is homologous to MrpC. The function of the MrpA C-terminus was tested by expression in a previously used Bacillus subtilis model system. At neutral pH, the truncated MrpA still worked, but at pH 8.4, where Mrp-complex formation is needed for function, the C-terminal domain of MrpA was absolutely required. Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  2. Dandelion PPO-1/PPO-2 domain-swaps: the C-terminal domain modulates the pH optimum and the linker affects SDS-mediated activation and stability.

    Science.gov (United States)

    Leufken, Christine M; Moerschbacher, Bruno M; Dirks-Hofmeister, Mareike E

    2015-02-01

    Plant polyphenol oxidases (PPOs) have a conserved three-domain structure: (i) the N-terminal domain (containing the active site) is connected via (ii) a linker to (iii) the C-terminal domain. The latter covers the active site, thereby maintaining the enzyme in a latent state. Activation can be achieved with SDS but little is known about the mechanism. We prepared domain-swap variants of dandelion PPO-1 and PPO-2 to test the specific functions of individual domains and their impact on enzyme characteristics. Our experiments revealed that the C-terminal domain modulates the pH optimum curve and has a strong influence on the optimal pH value. The linker determines the SDS concentration required for full activation. It also influences the SDS concentration required for half maximal activation (kSDS) and the stability of the enzyme during prolonged incubation in buffers containing SDS, but the N-terminal domain has the strongest effect on these parameters. The N-terminal domain also determines the IC50 of SDS and the stability in buffers containing or lacking SDS. We propose that the linker and C-terminal domain fine-tune the activation of plant PPOs. The C-terminal domain adjusts the pH optimum and the linker probably contains an SDS-binding/interaction site that influences inactivation and determines the SDS concentration required for activation. For the first time, we have determined the influence of the three PPO domains on enzyme activation and stability providing insight into the regulation and activation mechanisms of type-3 copper proteins in general. Copyright © 2014 Elsevier B.V. All rights reserved.

  3. Structural and Regulatory Elements of HCV NS5B Polymerase – β-Loop and C-Terminal Tail – Are Required for Activity of Allosteric Thumb Site II Inhibitors

    Science.gov (United States)

    Boyce, Sarah E.; Tirunagari, Neeraj; Niedziela-Majka, Anita; Perry, Jason; Wong, Melanie; Kan, Elaine; Lagpacan, Leanna; Barauskas, Ona; Hung, Magdeleine; Fenaux, Martijn; Appleby, Todd; Watkins, William J.; Schmitz, Uli; Sakowicz, Roman

    2014-01-01

    Elucidation of the mechanism of action of the HCV NS5B polymerase thumb site II inhibitors has presented a challenge. Current opinion holds that these allosteric inhibitors stabilize the closed, inactive enzyme conformation, but how this inhibition is accomplished mechanistically is not well understood. Here, using a panel of NS5B proteins with mutations in key regulatory motifs of NS5B – the C-terminal tail and β-loop – in conjunction with a diverse set of NS5B allosteric inhibitors, we show that thumb site II inhibitors possess a distinct mechanism of action. A combination of enzyme activity studies and direct binding assays reveals that these inhibitors require both regulatory elements to maintain the polymerase inhibitory activity. Removal of either element has little impact on the binding affinity of thumb site II inhibitors, but significantly reduces their potency. NS5B in complex with a thumb site II inhibitor displays a characteristic melting profile that suggests stabilization not only of the thumb domain but also the whole polymerase. Successive truncations of the C-terminal tail and/or removal of the β-loop lead to progressive destabilization of the protein. Furthermore, the thermal unfolding transitions characteristic for thumb site II inhibitor – NS5B complex are absent in the inhibitor – bound constructs in which interactions between C-terminal tail and β-loop are abolished, pointing to the pivotal role of both regulatory elements in communication between domains. Taken together, a comprehensive picture of inhibition by compounds binding to thumb site II emerges: inhibitor binding provides stabilization of the entire polymerase in an inactive, closed conformation, propagated via coupled interactions between the C-terminal tail and β-loop. PMID:24416288

  4. Identification and characterization of the first mutation (Arg776Cys) in the C-terminal domain of the Human Molybdenum Cofactor Sulfurase (HMCS) associated with type II classical xanthinuria.

    Science.gov (United States)

    Peretz, Hava; Naamati, Meirav Shtauber; Levartovsky, David; Lagziel, Ayala; Shani, Esther; Horn, Ivona; Shalev, Hanna; Landau, Daniel

    2007-05-01

    Classical xanthinuria type II is an autosomal recessive disorder characterized by deficiency of xanthine dehydrogenase and aldehyde oxidase activities due to lack of a common sulfido-olybdenum cofactor (MoCo). Two mutations, both in the N-terminal domain of the Human Molybdenum Cofactor Sulfurase (HMCS), were reported in patients with type II xanthinuria. Whereas the N-terminal domain of HMCS was demonstrated to have cysteine desulfurase activity, the C-terminal domain hypothetically transfers the sulfur to the MoCo. We describe the first mutation in the C-terminal domain of HMCS identified in a Bedouin-Arab child presenting with urolithiasis and in an asymptomatic Jewish female. Patients were diagnosed with type II xanthinuria by homozygosity mapping and/or allopurinol loading test. The Bedouin-Arab child was homozygous for a c.2326C>T (p.Arg776Cys) mutation, while the female patient was compound heterozygous for this and a novel c.1034insA (p.Gln347fsStop379) mutation in the N-terminal domain of HMCS. Cosegregation of the homozygous mutant genotype with hypouricemia and hypouricosuria was demonstrated in the Bedouin family. Haplotype analysis indicated that p.Arg776Cys is a recurrent mutation. Arg776 together with six surrounding amino acid residues were found fully conserved and predicted to be buried in homologous eukaryotic MoCo sulfurases. Moreover, Arg776 is conserved in a diversity of eukaryotic and prokaryotic proteins that posses a domain homologous to the C-terminal domain of HMCS. Our findings suggest that Arg776 is essential for a core structure of the C-terminal domain of the HMCS and identification of a mutation at this site may contribute clarifying the mechanism of MoCo sulfuration.

  5. Oligomerization-induced conformational change in the C-terminal region of Nel-like molecule 1 (NELL1) protein is necessary for the efficient mediation of murine MC3T3-E1 cell adhesion and spreading.

    Science.gov (United States)

    Nakamura, Yoko; Hasebe, Ai; Takahashi, Kaneyoshi; Iijima, Masumi; Yoshimoto, Nobuo; Maturana, Andrés D; Ting, Kang; Kuroda, Shun'ichi; Niimi, Tomoaki

    2014-04-04

    NELL1 is a large oligomeric secretory glycoprotein that functions as an osteoinductive factor. NELL1 contains several conserved domains, has structural similarities to thrombospondin 1, and supports osteoblastic cell adhesion through integrins. To define the structural requirements for NELL1-mediated cell adhesion, we prepared a series of recombinant NELL1 proteins (intact, deleted, and cysteine-mutant) from a mammalian expression system and tested their activities. A deletion analysis demonstrated that the C-terminal cysteine-rich region of NELL1 is critical for the cell adhesion activity of NELL1. Reducing agent treatment decreased the cell adhesion activity of full-length NELL1 but not of its C-terminal fragments, suggesting that the intramolecular disulfide bonds within this region are not functionally necessary but that other disulfide linkages in the N-terminal region of NELL1 may be involved in cell adhesion activity. By replacing cysteine residues with serines around the coiled-coil domain of NELL1, which is responsible for oligomerization, we created a mutant NELL1 protein that was unable to form homo-oligomers, and this monomeric mutant showed substantially lower cell adhesion activity than intact NELL1. These results suggest that an oligomerization-induced conformational change in the C-terminal region of NELL1 is important for the efficient mediation of cell adhesion and spreading by NELL1.

  6. Oligomerization-induced Conformational Change in the C-terminal Region of Nel-like Molecule 1 (NELL1) Protein Is Necessary for the Efficient Mediation of Murine MC3T3-E1 Cell Adhesion and Spreading*

    Science.gov (United States)

    Nakamura, Yoko; Hasebe, Ai; Takahashi, Kaneyoshi; Iijima, Masumi; Yoshimoto, Nobuo; Maturana, Andrés D.; Ting, Kang; Kuroda, Shun'ichi; Niimi, Tomoaki

    2014-01-01

    NELL1 is a large oligomeric secretory glycoprotein that functions as an osteoinductive factor. NELL1 contains several conserved domains, has structural similarities to thrombospondin 1, and supports osteoblastic cell adhesion through integrins. To define the structural requirements for NELL1-mediated cell adhesion, we prepared a series of recombinant NELL1 proteins (intact, deleted, and cysteine-mutant) from a mammalian expression system and tested their activities. A deletion analysis demonstrated that the C-terminal cysteine-rich region of NELL1 is critical for the cell adhesion activity of NELL1. Reducing agent treatment decreased the cell adhesion activity of full-length NELL1 but not of its C-terminal fragments, suggesting that the intramolecular disulfide bonds within this region are not functionally necessary but that other disulfide linkages in the N-terminal region of NELL1 may be involved in cell adhesion activity. By replacing cysteine residues with serines around the coiled-coil domain of NELL1, which is responsible for oligomerization, we created a mutant NELL1 protein that was unable to form homo-oligomers, and this monomeric mutant showed substantially lower cell adhesion activity than intact NELL1. These results suggest that an oligomerization-induced conformational change in the C-terminal region of NELL1 is important for the efficient mediation of cell adhesion and spreading by NELL1. PMID:24563467

  7. AKT and GSK-3 are necessary for direct ezrin binding to NHE3 as part of a C-terminal stimulatory complex: role of a novel Ser-rich NHE3 C-terminal motif in NHE3 activity and trafficking.

    Science.gov (United States)

    Singh, Varsha; Lin, Rong; Yang, Jianbo; Cha, Boyoung; Sarker, Rafiquel; Tse, Chung Ming; Donowitz, Mark

    2014-02-28

    Basal activity of the BB Na(+)/H(+) exchanger NHE3 requires multiprotein complexes that form on its C terminus. One complex stimulates basal NHE3 activity and contains ezrin and phosphoinositides as major components; how it stimulates NHE3 activity is not known. This study tested the hypothesis that ezrin dynamically associates with this complex, which sets ezrin binding. NHE3 activity was reduced by an Akti. This effect was eliminated if ezrin binding to NHE3 was inhibited by a point mutant. Recombinant AKT phosphorylated NHE3 C terminus in the domain ezrin directly binds. This domain (amino acids 475-589) is predicted to be α-helical and contains a conserved cluster of three serines (Ser(515), Ser(522), and Ser(526)). Point mutations of two of these (S515A, S515D, or S526A) reduced basal NHE3 activity and surface expression and had no Akti inhibition. S526D had NHE3 activity equal to wild type with normal Akti inhibition. Ezrin binding to NHE3 was regulated by Akt, being eliminated by Akti. NHE3-S515A and -S526D did not bind ezrin; NHE3-S515D had reduced ezrin binding; NHE3-S526D bound ezrin normally. NHE3-Ser(526) is predicted to be a GSK-3 kinase phosphorylation site. A GSK-3 inhibitor reduced basal NHE3 activity as well as ezrin-NHE3 binding, and this effect was eliminated in NHE3-S526A and -S526D mutants. The conclusions were: 1) NHE3 basal activity is regulated by a signaling complex that is controlled by sequential effects of two kinases, Akt and GSK-3, which act on a Ser cluster in the same NHE3 C-terminal domain that binds ezrin; and 2) these kinases regulate the dynamic association of ezrin with NHE3 to affect basal NHE3 activity.

  8. Insights into PG-binding, conformational change, and dimerization of the OmpA C-terminal domains from Salmonella enterica serovar Typhimurium and Borrelia burgdorferi: Characterization of OmpA C-Terminal Domain

    Energy Technology Data Exchange (ETDEWEB)

    Tan, Kemin [Center for Structural Genomics of Infectious Diseases, University of Chicago, 5735 South Ellis Avenue Chicago Illinois 60637; Midwest Center for Structural Genomics, Argonne National Laboratory, Argonne Illinois 60439; Structural Biology Center, Biosciences, Argonne National Laboratory, Argonne Illinois 60439; Deatherage Kaiser, Brooke L. [National Security Directorate, Pacific Northwest National Laboratory, Richland Washington 99352; Wu, Ruiying [Midwest Center for Structural Genomics, Argonne National Laboratory, Argonne Illinois 60439; Cuff, Marianne [Midwest Center for Structural Genomics, Argonne National Laboratory, Argonne Illinois 60439; Fan, Yao [Midwest Center for Structural Genomics, Argonne National Laboratory, Argonne Illinois 60439; Bigelow, Lance [Midwest Center for Structural Genomics, Argonne National Laboratory, Argonne Illinois 60439; Jedrzejczak, Robert P. [Center for Structural Genomics of Infectious Diseases, University of Chicago, 5735 South Ellis Avenue Chicago Illinois 60637; Adkins, Joshua N. [Earth and Biological Sciences Directorate, Pacific Northwest National Laboratory, Richland Washington 99352; Cort, John R. [Earth and Biological Sciences Directorate, Pacific Northwest National Laboratory, Richland Washington 99352; Babnigg, Gyorgy [Center for Structural Genomics of Infectious Diseases, University of Chicago, 5735 South Ellis Avenue Chicago Illinois 60637; Midwest Center for Structural Genomics, Argonne National Laboratory, Argonne Illinois 60439; Joachimiak, Andrzej [Center for Structural Genomics of Infectious Diseases, University of Chicago, 5735 South Ellis Avenue Chicago Illinois 60637; Midwest Center for Structural Genomics, Argonne National Laboratory, Argonne Illinois 60439; Structural Biology Center, Biosciences, Argonne National Laboratory, Argonne Illinois 60439

    2017-06-19

    S. Typhimurium can induce both humoral and cell-mediated responses when establishing itself in the host. These responses are primarily stimulated against the lipopolysaccharide and major outer membrane (OM) proteins. OmpA is one of these major OM proteins. It comprises a N-terminal eight-stranded b-barrel trans membrane domain and a C-terminal domain (OmpACTD). The OmpACTD and its homologs are believed to bind to peptidoglycan (PG) within the periplasm, maintaining bacterial osmotic homeostasis and modulating the permeability and integrity of the OM. Here we present the first crystal structures of the OmpACTD from two pathogens: S. Typhimurium (STOmpACTD) in open and closed forms and causative agent of Lyme Disease Borrelia burgdorferi (BbOmpACTD), in closed form. In the open form of STOmpACTD, an aspartic acid residue from a long b2-a3 loop points into the binding pocket, suggesting that an anion group such as a carboxylate group from PG is favored at the binding site. In the closed form of STOmpACTD and in the structure of BbOmpACTD, a sulfate group from the crystallization buffer is tightly bound at the binding site. The differences between the closed and open forms of STOmpACTD, suggest a large conformational change that includes an extension of a3 helix by ordering a part of b2-a3 loop. We propose that the sulfate anion observed in these structures mimics the carboxylate group of PG when bound to STOmpACTD suggesting PG-anchoring mechanism. In addition, the binding of PG or a ligand mimic may enhance dimerization of STOmpACTD, or possibly that of full length STOmpA.

  9. The C-terminal N-glycosylation sites of the human alpha1,3/4-fucosyltransferase III, -V, and -VI (hFucTIII, -V, adn -VI) are necessary for the expression of full enzyme activity.

    Science.gov (United States)

    Christensen, L L; Jensen, U B; Bross, P; Orntoft, T F

    2000-09-01

    The alpha1,3/4-fucosyltransferases are involved in the synthesis of fucosylated cell surface glycoconjugates. Human alpha1,3/4-fucosyltransferase III, -V, and -VI (hFucTIII, -V, and -VI) contain two conserved C-terminal N-glycosylation sites (hFucTIII: Asn154 and Asn185; hFucTV: Asn167 and Asn198; and hFucTVI: Asn153 and Asn184). In the present study, we have analyzed the functional role of these potential N-glycosylation sites, laying the main emphasis on the sites in hFucTIII. Tunicamycin treatment completely abolished hFucTIII enzyme activity while castanospermine treatment diminished hFucTIII enzyme activity to approximately 40% of the activity of the native enzyme. To further analyze the role of the conserved N-glycosylation sites in hFucTIII, -V, and -VI, we made a series of mutant genomic DNAs in which the asparagine residues in the potential C-terminal N-glycosylation sites were replaced by glutamine. Subsequently, the hFucTIII, -V, and -VI wild type and the mutants were expressed in COS-7 cells. All the mutants exhibited lower enzyme activity than the wild type and elimination of individual sites had different effects on the activity. The mutations did not affect the protein level of the mutants in the cells, but reduced the molecular mass as predicted. Kinetic analysis of hFucTIII revealed that lack of glycosylation at Asn185 did not change the Km values for the oligosaccharide acceptor and the nucleotide sugar donor. The present study demonstrates that hFucTIII, -V, and -VI require N-glycosylation at the two conserved C-terminal N-glycosylation sites for expression of full enzyme activity.

  10. Molecular basis of cannabinoid CB1 receptor coupling to the G protein heterotrimer Gαiβγ: identification of key CB1 contacts with the C-terminal helix α5 of Gαi.

    Science.gov (United States)

    Shim, Joong-Youn; Ahn, Kwang H; Kendall, Debra A

    2013-11-08

    The cannabinoid (CB1) receptor is a member of the rhodopsin-like G protein-coupled receptor superfamily. The human CB1 receptor, which is among the most expressed receptors in the brain, has been implicated in several disease states, including drug addiction, anxiety, depression, obesity, and chronic pain. Different classes of CB1 agonists evoke signaling pathways through the activation of specific subtypes of G proteins. The molecular basis of CB1 receptor coupling to its cognate G protein is unknown. As a first step toward understanding CB1 receptor-mediated G protein signaling, we have constructed a ternary complex structural model of the CB1 receptor and Gi heterotrimer (CB1-Gi), guided by the x-ray structure of β2-adrenergic receptor (β2AR) in complex with Gs (β2AR-Gs), through 824-ns duration molecular dynamics simulations in a fully hydrated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine bilayer environment. We identified a group of residues at the juxtamembrane regions of the intracellular loops 2 and 3 (IC2 and IC3) of the CB1 receptor, including Ile-218(3.54), Tyr-224(IC2), Asp-338(6.30), Arg-340(6.32), Leu-341(6.33), and Thr-344(6.36), as potential key contacts with the extreme C-terminal helix α5 of Gαi. Ala mutations of these residues at the receptor-Gi interface resulted in little G protein coupling activity, consistent with the present model of the CB1-Gi complex, which suggests tight interactions between CB1 and the extreme C-terminal helix α5 of Gαi. The model also suggests that unique conformational changes in the extreme C-terminal helix α5 of Gα play a crucial role in the receptor-mediated G protein activation.

  11. Free-fatty acid receptor-4 (FFA4) modulates ROS generation and COX-2 expression via the C-terminal β-arrestin phosphosensor in Raw 264.7 macrophages.

    Science.gov (United States)

    Cheshmehkani, Ameneh; Senatorov, Ilya S; Dhuguru, Jyothi; Ghoneim, Ola; Moniri, Nader H

    2017-12-15

    Agonism of the G protein-coupled free-fatty acid receptor-4 (FFA4) has been shown to promote numerous anti-inflammatory effects in macrophages that arise due to interaction with β-arrestin partner proteins. Humans express functionally distinct short and long FFA4 splice variants, such that FFA4-S signals through Gαq/11 and β-arrestin, while FFA4-L is intrinsically biased solely towards β-arrestin signaling. Recently, we and others have shown that phosphorylation of the FFA4 C-terminal tail is responsible for β-arrestin interactability and signaling. Given the significance of β-arrestin in the anti-inflammatory function of FFA4, the goal of this study was to examine the role of the C-terminal β-arrestin phosphosensor in FFA4 signaling induced by PMA and LPS in murine Raw 264.7 macrophages. Our data reveal for the first time that both FFA4 isoforms modulate PMA-induced ROS generation, and that abolishment of the FFA4-S, but not FFA4-L C-terminal phosphosensor, is detrimental to this effect. Furthermore, we show that while both isoforms reduce PMA-induced expression of COX-2, removal of the FFA4-S phosphosensor significantly decreases this response, suggesting that these effects of FFA4-S are β-arrestin mediated. On the contrary, FFA4-S, as well as the truncated C-terminal congener lacking the β-arrestin phosphosensor were both able to reduce LPS-induced NF-κB activity and ERK1/2 phosphorylation. However, FFA4-L and its corresponding mutant were incapable of modulating either, suggesting that these responses are mediated by G protein coupling. Taken together, our data reveal important structure-function and signaling differences between the two FFA4 isoforms, and for the first time link FFA4 to modulation of ROS in macrophages. Copyright © 2017 Elsevier Inc. All rights reserved.

  12. The role of the Brr5/Ysh1 C-terminal domain and its homolog Syc1 in mRNA 3′-end processing in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Nasser, Tommy; Tacahashi, Zhelkovsky A.; He, X.

    2006-01-01

    subunit Pap1. Because of similarity to other nucleases in the metallo-beta-lactamase family, the Brr5/Ysh1 subunit has been proposed to be the endonuclease. The C-terminal domain of Brr5 lies outside of beta-lactamase homology, and its function has not been elucidated. We show here that this region of Brr......5 is necessary for cell viability and mRNA 3'-end processing. It is highly homologous to another CPF subunit, Syc1. Syc1 is not essential, but its removal improves the growth of other processing mutants at restrictive temperatures and restores in vitro processing activity to cleavage...

  13. The RNA Polymerase II C-Terminal Domain Phosphatase-Like Protein FIERY2/CPL1 Interacts with eIF4AIII and Is Essential for Nonsense-Mediated mRNA Decay in Arabidopsis

    KAUST Repository

    Cui, Peng

    2016-02-18

    © 2016 American Society of Plant Biologists. All rights reserved. Nonsense-mediated decay (NMD) is a posttranscriptional surveillance mechanism in eukaryotes that recognizes and degrades transcripts with premature translation-termination codons. The RNA polymerase II C-terminal domain phosphatase-like protein FIERY2 (FRY2; also known as C-TERMINAL DOMAIN PHOSPHATASE-LIKE1 [CPL1]) plays multiple roles in RNA processing in Arabidopsis thaliana. Here, we found that FRY2/CPL1 interacts with two NMD factors, eIF4AIII and UPF3, and is involved in the dephosphorylation of eIF4AIII. This dephosphorylation retains eIF4AIII in the nucleus and limits its accumulation in the cytoplasm. By analyzing RNA-seq data combined with quantitative RT-PCR validation, we found that a subset of alternatively spliced transcripts and 59-extended mRNAs with NMD-eliciting features accumulated in the fry2-1 mutant, cycloheximidetreated wild type, and upf3 mutant plants, indicating that FRY2 is essential for the degradation of these NMD transcripts.

  14. Structure of the TPR domain of AIP: lack of client protein interaction with the C-terminal α-7 helix of the TPR domain of AIP is sufficient for pituitary adenoma predisposition.

    Directory of Open Access Journals (Sweden)

    Rhodri M L Morgan

    Full Text Available Mutations of the aryl hydrocarbon receptor interacting protein (AIP have been associated with familial isolated pituitary adenomas predisposing to young-onset acromegaly and gigantism. The precise tumorigenic mechanism is not well understood as AIP interacts with a large number of independent proteins as well as three chaperone systems, HSP90, HSP70 and TOMM20. We have determined the structure of the TPR domain of AIP at high resolution, which has allowed a detailed analysis of how disease-associated mutations impact on the structural integrity of the TPR domain. A subset of C-terminal α-7 helix (Cα-7h mutations, R304* (nonsense mutation, R304Q, Q307* and R325Q, a known site for AhR and PDE4A5 client-protein interaction, occur beyond those that interact with the conserved MEEVD and EDDVE sequences of HSP90 and TOMM20. These C-terminal AIP mutations appear to only disrupt client-protein binding to the Cα-7h, while chaperone binding remains unaffected, suggesting that failure of client-protein interaction with the Cα-7h is sufficient to predispose to pituitary adenoma. We have also identified a molecular switch in the AIP TPR-domain that allows recognition of both the conserved HSP90 motif, MEEVD, and the equivalent sequence (EDDVE of TOMM20.

  15. Generation of H9 T-cells stably expressing a membrane-bound form of the cytoplasmic tail of the Env-glycoprotein: lack of transcomplementation of defective HIV-1 virions encoding C-terminally truncated Env

    Directory of Open Access Journals (Sweden)

    Bosch Valerie

    2006-05-01

    Full Text Available Abstract H9-T-cells do not support the replication of mutant HIV-1 encoding Env protein lacking its long cytoplasmic C-terminal domain (Env-CT. Here we describe the generation of a H9-T-cell population constitutively expressing the HIV-1 Env-CT protein domain anchored in the cellular membrane by it homologous membrane-spanning domain (TMD. We confirmed that the Env-TMD-CT protein was associated with cellular membranes, that its expression did not have any obvious cytotoxic effects on the cells and that it did not affect wild-type HIV-1 replication. However, as measured in both a single-round assay as well as in spreading infections, replication competence of mutant pNL-Tr712, lacking the Env-CT, was not restored in this H9 T-cell population. This means that the Env-CT per se cannot transcomplement the replication block of HIV-1 virions encoding C-terminally truncated Env proteins and suggests that the Env-CT likely exerts its function only in the context of the complete Env protein.

  16. C-terminal splice variants of P/Q-type Ca(2+) channel CaV2.1 α1 subunits are differentially regulated by Rab3-interacting molecule proteins.

    Science.gov (United States)

    Hirano, Mitsuru; Takada, Yoshinori; Wong, Chee Fah; Yamaguchi, Kazuma; Kotani, Hiroshi; Kurokawa, Tatsuki; Mori, Masayuki X; Snutch, Terrance P; Ronjat, Michel; De Waard, Michel; Mori, Yasuo

    2017-06-02

    Voltage-dependent Ca(2+) channels (VDCCs) mediate neurotransmitter release controlled by presynaptic proteins such as the scaffolding proteins Rab3-interacting molecules (RIMs). RIMs confer sustained activity and anchoring of synaptic vesicles to the VDCCs. Multiple sites on the VDCC α1 and β subunits have been reported to mediate the RIMs-VDCC interaction, but their significance is unclear. Because alternative splicing of exons 44 and 47 in the P/Q-type VDCC α1 subunit CaV2.1 gene generates major variants of the CaV2.1 C-terminal region, known for associating with presynaptic proteins, we focused here on the protein regions encoded by these two exons. Co-immunoprecipitation experiments indicated that the C-terminal domain (CTD) encoded by CaV2.1 exons 40-47 interacts with the α-RIMs, RIM1α and RIM2α, and this interaction was abolished by alternative splicing that deletes the protein regions encoded by exons 44 and 47. Electrophysiological characterization of VDCC currents revealed that the suppressive effect of RIM2α on voltage-dependent inactivation (VDI) was stronger than that of RIM1α for the CaV2.1 variant containing the region encoded by exons 44 and 47. Importantly, in the CaV2.1 variant in which exons 44 and 47 were deleted, strong RIM2α-mediated VDI suppression was attenuated to a level comparable with that of RIM1α-mediated VDI suppression, which was unaffected by the exclusion of exons 44 and 47. Studies of deletion mutants of the exon 47 region identified 17 amino acid residues on the C-terminal side of a polyglutamine stretch as being essential for the potentiated VDI suppression characteristic of RIM2α. These results suggest that the interactions of the CaV2.1 CTD with RIMs enable CaV2.1 proteins to distinguish α-RIM isoforms in VDI suppression of P/Q-type VDCC currents. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  17. Two enzymes which catalyze the amidation of peptide C-terminals are synthesized by a single mRNA. Peptide C mattan amid ka hanno wo shokubaisuru futatsu no koso wa ippon no mRNA yori goseisareru

    Energy Technology Data Exchange (ETDEWEB)

    Kato, I.; Yonekura, H.; Okamoto, H. (Tohoku Univ., Sendai (Japan))

    1991-10-25

    Recent findings by the authors are reviewed on the amidation that forms amid structure essential to physiological activities in C-terminals of peptide hormones such as oxytocin,VIP,PP. It is noted that the amidation had been considered to be catalyzed by peptidylglycine{alpha} -amidating monooxyganase ( PAM ) and that the authors investigated the PAM function by expression of PAM cDNA isolated from rat pituitary and its deletion mutant into COS-7 cells, reaching to the important findings of a singl PMA mRNA encoding two enzymes, namely one at 5 {prime} side, peptidylglicine {alpha} hydroxylase which catalyses the conversion of C-termianl glycine on peptide to the hydroxylated form ( the first step of amidation ),and another at 3{prime} side, {alpha}- hydroxylglycine amidating dealkylase which catalyzes the conversion of hydroxylated glycine to the amidated form ( the second step of amidation). 19 refs., 4 figs.

  18. Variable protection against experimental broiler necrotic enteritis after immunization with the C-terminal fragment of Clostridium perfringens alpha-toxin and a non-toxic NetB variant.

    Science.gov (United States)

    Fernandes da Costa, Sérgio P; Mot, Dorien; Geeraerts, Sofie; Bokori-Brown, Monika; Van Immerseel, Filip; Titball, Richard W

    2016-06-01

    Necrotic enteritis toxin B (NetB) is a pore-forming toxin produced by Clostridium perfringens and has been shown to play a key role in avian necrotic enteritis, a disease causing significant costs to the poultry production industry worldwide. The aim of this work was to determine whether immunization with a non-toxic variant of NetB (NetB W262A) and the C-terminal fragment of C. perfringens alpha-toxin (CPA247-370) would provide protection against experimental necrotic enteritis. Immunized birds with either antigen or a combination of antigens developed serum antibody levels against NetB and CPA. When CPA247-370 and NetB W262A were used in combination as immunogens, an increased protection was observed after oral challenge by individual dosing, but not after in-feed-challenge.

  19. The 18-kilodalton Chlamydia trachomatis histone H1-like protein (Hc1) contains a potential N-terminal dimerization site and a C-terminal nucleic acid-binding domain

    DEFF Research Database (Denmark)

    Pedersen, LB; Birkelund, Svend; Holm, A

    1996-01-01

    , in part, be due to Hc1-mediated alterations of DNA topology. To locate putative functional domains within Hc1, polypeptides Hc1(2-57) and Hc1(53-125), corresponding to the N- and C-terminal parts of Hc1, respectively, were generated. By chemical cross-linking with ethylene glycol-bis (succinic acid N...... retardation assays, Hc1(53-125) was shown to contain a domain capable of binding both DNA and RNA. Under the same conditions, Hc1(2-57) had no nucleic acid-binding activity. Electron microscopy of Hc1-DNA and Hc1(53-125)-DNA complexes revealed differences suggesting that the N-terminal part of Hc1 may affect...

  20. Direct interaction of Gβγ with a C-terminal Gβγ-binding domain of the Ca2+ channel α1 subunit is responsible for channel inhibition by G protein-coupled receptors

    Science.gov (United States)

    Qin, Ning; Platano, Daniela; Olcese, Riccardo; Stefani, Enrico; Birnbaumer, Lutz

    1997-01-01

    Several classes of voltage-gated Ca2+ channels (VGCCs) are inhibited by G proteins activated by receptors for neurotransmitters and neuromodulatory peptides. Evidence has accumulated to indicate that for non-L-type Ca2+ channels the executing arm of the activated G protein is its βγ dimer (Gβγ). We report below the existence of two Gβγ-binding sites on the A-, B-, and E-type α1 subunits that form non-L-type Ca2+ channels. One, reported previously, is in loop 1 connecting transmembrane domains I and II. The second is located approximately in the middle of the ca. 600-aa-long C-terminal tails. Both Gβγ-binding regions also bind the Ca2+ channel β subunit (CCβ), which, when overexpressed, interferes with inhibition by activated G proteins. Replacement in α1E of loop 1 with that of the G protein-insensitive and Gβγ-binding-negative loop 1 of α1C did not abolish inhibition by G proteins, but the exchange of the α1E C terminus with that of α1C did. This and properties of α1E C-terminal truncations indicated that the Gβγ-binding site mediating the inhibition of Ca2+ channel activity is the one in the C terminus. Binding of Gβγ to this site was inhibited by an α1-binding domain of CCβ, thus providing an explanation for the functional antagonism existing between CCβ and G protein inhibition. The data do not support proposals that Gβγ inhibits α1 function by interacting with the site located in the loop I–II linker. These results define the molecular mechanism by which presynaptic G protein-coupled receptors inhibit neurotransmission. PMID:9238069

  1. Direct interaction of gbetagamma with a C-terminal gbetagamma-binding domain of the Ca2+ channel alpha1 subunit is responsible for channel inhibition by G protein-coupled receptors.

    Science.gov (United States)

    Qin, N; Platano, D; Olcese, R; Stefani, E; Birnbaumer, L

    1997-08-05

    Several classes of voltage-gated Ca2+ channels (VGCCs) are inhibited by G proteins activated by receptors for neurotransmitters and neuromodulatory peptides. Evidence has accumulated to indicate that for non-L-type Ca2+ channels the executing arm of the activated G protein is its betagamma dimer (Gbetagamma). We report below the existence of two Gbetagamma-binding sites on the A-, B-, and E-type alpha1 subunits that form non-L-type Ca2+ channels. One, reported previously, is in loop 1 connecting transmembrane domains I and II. The second is located approximately in the middle of the ca. 600-aa-long C-terminal tails. Both Gbetagamma-binding regions also bind the Ca2+ channel beta subunit (CCbeta), which, when overexpressed, interferes with inhibition by activated G proteins. Replacement in alpha1E of loop 1 with that of the G protein-insensitive and Gbetagamma-binding-negative loop 1 of alpha1C did not abolish inhibition by G proteins, but the exchange of the alpha1E C terminus with that of alpha1C did. This and properties of alpha1E C-terminal truncations indicated that the Gbetagamma-binding site mediating the inhibition of Ca2+ channel activity is the one in the C terminus. Binding of Gbetagamma to this site was inhibited by an alpha1-binding domain of CCbeta, thus providing an explanation for the functional antagonism existing between CCbeta and G protein inhibition. The data do not support proposals that Gbetagamma inhibits alpha1 function by interacting with the site located in the loop I-II linker. These results define the molecular mechanism by which presynaptic G protein-coupled receptors inhibit neurotransmission.

  2. A naturally occurring C-terminal fragment of the prion protein (PrP) delays disease and acts as a dominant-negative inhibitor of PrPSc formation.

    Science.gov (United States)

    Westergard, Laura; Turnbaugh, Jessie A; Harris, David A

    2011-12-23

    The cellular prion protein (PrPC) undergoes constitutive proteolytic cleavage between residues 111/112 to yield a soluble N-terminal fragment (N1) and a membrane-anchored C-terminal fragment (C1). The C1 fragment represents the major proteolytic fragment of PrPC in brain and several cell types. To explore the role of C1 in prion disease, we generated Tg(C1) transgenic mice expressing this fragment (PrP(Δ23-111)) in the presence and absence of endogenous PrP. In contrast to several other N-terminally deleted forms of PrP, the C1 fragment does not cause a spontaneous neurological disease in the absence of endogenous PrP. Tg(C1) mice inoculated with scrapie prions remain healthy and do not accumulate protease-resistant PrP, demonstrating that C1 is not a substrate for conversion to PrPSc (the disease-associated isoform). Interestingly, Tg(C1) mice co-expressing C1 along with wild-type PrP (either endogenous or encoded by a second transgene) become ill after scrapie inoculation, but with a dramatically delayed time course compared with mice lacking C1. In addition, accumulation of PrPSc was markedly slowed in these animals. Similar effects were produced by a shorter C-terminal fragment of PrP(Δ23-134). These results demonstrate that C1 acts as dominant-negative inhibitor of PrPSc formation and accumulation of neurotoxic forms of PrP. Thus, C1, a naturally occurring fragment of PrPC, might play a modulatory role during the course of prion diseases. In addition, enhancing production of C1, or exogenously administering this fragment, represents a potential therapeutic strategy for the treatment of prion diseases.

  3. Interaction of LDL receptor-related protein 4 (LRP4) with postsynaptic scaffold proteins via its C-terminal PDZ domain-binding motif, and its regulation by Ca/calmodulin-dependent protein kinase II.

    Science.gov (United States)

    Tian, Qing-Bao; Suzuki, Tatsuo; Yamauchi, Takashi; Sakagami, Hiroyuki; Yoshimura, Yoshiyuki; Miyazawa, Shoko; Nakayama, Kohzo; Saitoh, Fuminori; Zhang, Jing-Ping; Lu, Yonghao; Kondo, Hisatake; Endo, Shogo

    2006-06-01

    We cloned here a full-length cDNA of Dem26[Tian et al. (1999)Mol. Brain Res., 72, 147-157], a member of the low-density lipoprotein (LDL) receptor gene family from the rat brain. We originally named the corresponding protein synaptic LDL receptor-related protein (synLRP) [Tian et al. (2002) Soc. Neurosci. Abstr., 28, 405] and have renamed it LRP4 to accord it systematic nomenclature (GenBank(TM) accession no. AB073317). LRP4 protein interacted with postsynaptic scaffold proteins such as postsynaptic density (PSD)-95 via its C-terminal tail sequence, and associated with N-methyl-D-aspartate (NMDA)-type glutamate receptor subunit. The mRNA of LRP4 was localized to dendrites, as well as somas, of neuronal cells, and the full-length protein of 250 kDa was highly concentrated in the brain and localized to various subcellular compartments in the brain, including synaptic fractions. Immunocytochemical study using cultured cortical neurons suggested surface localization in the neuronal cells both in somas and dendrites. Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) phosphorylated the C-terminal cytoplasmic region of LRP4 at Ser1887 and Ser1900, and the phosphorylation at the latter site suppressed the interaction of the protein with PSD-95 and synapse-associated protein 97 (SAP97). These findings suggest a postsynaptic role for LRP4, a putative endocytic multiligand receptor, and a mechanism in which CaMKII regulates PDZ-dependent protein-protein interactions and receptor dynamics.

  4. The stretch of C-terminal acidic amino acids of translational release factor eRF1 is a primary binding site for eRF3 of fission yeast.

    Science.gov (United States)

    Ito, K; Ebihara, K; Nakamura, Y

    1998-08-01

    Translation termination in eukaryotes requires a codon-specific (class-I) release factor, eRF1, and a GTP/GDP-dependent (class-II) release factor, eRF3. The model of "molecular mimicry between release factors and tRNA" predicts that eRF1 mimics tRNA to read the stop codon and that eRF3 mimics elongation factor EF-Tu to bring eRF1 to the A site of the ribosome for termination of protein synthesis. In this study, we set up three systems, in vitro affinity binding, a yeast two-hybrid system, and in vitro competition assay, to determine the eRF3-binding site of eRF1 using the fission yeast Schizosaccharomyces pombe proteins and creating systematic deletions in eRF1. The in vitro affinity binding experiments demonstrated that the predicted tRNA-mimicry truncation of eRF1 (Sup45) forms a stable complex with eRF3 (Sup35). All three test systems revealed that the most critical binding site is located at the C-terminal region of eRF1, which is conserved among eukaryotic eRF1s and rich in acidic amino acids. To our surprise, however, the C-terminal deletion eRF1 seems to be sufficient for cell viability in spite of the severe defect in eRF3 binding when expressed in a temperature-sensitive sup45 mutant of the budding yeast, Saccharomyces cerevisiae. These results cannot be accounted for by the simple "eRF3-EF-Tu mimicry" model, but may provide new insight into the eRF3 function for translation termination in eukaryotes.

  5. Organization of the multifunctional enzyme type 1: interaction between N- and C-terminal domains is required for the hydratase-1/isomerase activity.

    OpenAIRE

    Kiema, Tiila-Riikka; Taskinen, Jukka P; Päivi L Pirilä; Koivuranta, Kari T; Wierenga, Rik K; Hiltunen, J. Kalervo

    2002-01-01

    Rat peroxisomal multifunctional enzyme type 1 (perMFE-1) is a monomeric protein of beta-oxidation. We have defined five functional domains (A, B, C, D and E) in the perMFE-1 based on comparison of the amino acid sequence with homologous proteins from databases and structural data of the hydratase-1/isomerases (H1/I) and (3 S )-hydroxyacyl-CoA dehydrogenases (HAD). Domain A (residues 1-190) comprises the H1/I fold and catalyses both 2-enoyl-CoA hydratase-1 and Delta(3)-Delta(2)-enoyl-CoA isome...

  6. The Structure of the RNA m5C Methyltransferase YebU from Escherichia coli Reveals a C-terminal RNA-recruiting PUA Domain

    DEFF Research Database (Denmark)

    Hallberg, B. Martin; Ericsson, Ulrika B.; Johnson, Kenneth A

    2006-01-01

    Nucleotide methylations are the most common type of rRNA modification in bacteria, and are introduced post-transcriptionally by a wide variety of site-specific enzymes. Three 5-methylcytidine (m(5)C) bases are found in the rRNAs of Escherichia coli and one of these, at nucleotide 1407 in 16 S r...... by X-ray crystallography, and we present a molecular model for how YebU specifically recognizes, binds and methylates its ribosomal substrate. The YebU protein has an N-terminal SAM-binding catalytic domain with structural similarity to the equivalent domains in several other m(5)C RNA MTases including...

  7. Formation of C-terminally truncated version of the Taz1 protein employs cleavage-box structure in mRNA

    Energy Technology Data Exchange (ETDEWEB)

    Gunisova, Stanislava; Bartosova, Zdenka [Department of Genetics, Comenius University, Faculty of Natural Sciences, 842 15 Bratislava (Slovakia); Kramara, Juraj; Nosek, Jozef [Department of Biochemistry, Comenius University, Faculty of Natural Sciences, 842 15 Bratislava (Slovakia); Tomaska, Lubomir, E-mail: tomaska@fns.uniba.sk [Department of Genetics, Comenius University, Faculty of Natural Sciences, 842 15 Bratislava (Slovakia)

    2010-02-12

    When expressed in various hosts the taz1{sup +} gene encoding the fission yeast telomere-binding protein produces two forms of polypeptides: full-length (Taz1p) and truncated (Taz1p{Delta}C) version lacking almost entire Myb-domain. Whereas Taz1p binds telomeric DNA in vitro, Taz1p{Delta}C forms long filaments unable of DNA binding. The formation of Taz1p{Delta}C is a result of neither site-specific proteolysis, nor premature termination of transcription. In silico analysis of the taz1{sup +} RNA transcript revealed a stem-loop structure at the site of cleavage (cleavage box; CB). In order to explore whether it possesses inherent destabilizing effects, we cloned CB sequence into the open reading frame (ORF) of glutathione-S-transferase (GST) and observed that when expressed in Escherichia coli the engineered gene produced two forms of the reporter protein. The formation of the truncated version of GST was abolished, when CB was replaced with recoded sequence containing synonymous codons thus indicating that the truncation is based on structural properties of taz1{sup +} mRNA.

  8. Both the Hydrophobicity and a Positively Charged Region Flanking the C-Terminal Region of the Transmembrane Domain of Signal-Anchored Proteins Play Critical Roles in Determining Their Targeting Specificity to the Endoplasmic Reticulum or Endosymbiotic Organelles in Arabidopsis Cells[W

    Science.gov (United States)

    Lee, Junho; Lee, Hyunkyung; Kim, Jinho; Lee, Sumin; Kim, Dae Heon; Kim, Sanguk; Hwang, Inhwan

    2011-01-01

    Proteins localized to various cellular and subcellular membranes play pivotal roles in numerous cellular activities. Accordingly, in eukaryotic cells, the biogenesis of organellar proteins is an essential process requiring their correct localization among various cellular and subcellular membranes. Localization of these proteins is determined by either cotranslational or posttranslational mechanisms, depending on the final destination. However, it is not fully understood how the targeting specificity of membrane proteins is determined in plant cells. Here, we investigate the mechanism by which signal-anchored (SA) proteins are differentially targeted to the endoplasmic reticulum (ER) or endosymbiotic organelles using in vivo targeting, subcellular fractionation, and bioinformatics approaches. For targeting SA proteins to endosymbiotic organelles, the C-terminal positively charged region (CPR) flanking the transmembrane domain (TMD) is necessary but not sufficient. The hydrophobicity of the TMD in CPR-containing proteins also plays a critical role in determining targeting specificity; TMDs with a hydrophobicity value >0.4 on the Wimley and White scale are targeted primarily to the ER, whereas TMDs with lower values are targeted to endosymbiotic organelles. Based on these data, we propose that the CPR and the hydrophobicity of the TMD play a critical role in determining the targeting specificity between the ER and endosymbiotic organelles. PMID:21515817

  9. A substrate specificity-determining unit of three Lin12-Notch repeat modules is formed in trans within the pappalysin-1 dimer and requires a sequence stretch C-terminal to the third module

    DEFF Research Database (Denmark)

    Weyer, Kathrin; Boldt, Henning Bünsow; Poulsen, Christine Bruun

    2007-01-01

    Members of the pappalysin family of metzincin metalloproteinases, pregnancy-associated plasma protein-A (PAPP-A, pappalysin-1) and PAPP-A2 (pappalysin-2), regulate the bioavailability of insulin-like growth factors (IGFs) by specific proteolytic inactivation of IGF-binding proteins (IGFBPs). PAPP-A...... cleaves IGFBP-4 and IGFBP-5, whereas PAPP-A2 cleaves only IGFBP-5. The pappalysins contain three Lin12-Notch repeat (LNR1-3) modules, previously considered unique to the Notch receptor family in which they function to regulate receptor cleavage. In contrast to the Notch receptor where three LNR modules...... are tandemly arranged, LNR3 is separated by more than 1000 residues from LNR1-2 in the pappalysin sequence. Each of the three LNR modules of PAPP-A is required for proteolysis of IGFBP-4, but not IGFBP-5. However, we here find that a C-terminal truncated variant of PAPP-A, which lacks LNR3 and therefore...

  10. Two functions of the C-terminal domain of Escherichia coli Rob: mediating "sequestration-dispersal" as a novel off-on switch for regulating Rob's activity as a transcription activator and preventing degradation of Rob by Lon protease.

    Science.gov (United States)

    Griffith, Kevin L; Fitzpatrick, M Megan; Keen, Edward F; Wolf, Richard E

    2009-05-08

    In Escherichia coli, Rob activates transcription of the SoxRS/MarA/Rob regulon. Previous work revealed that Rob resides in three to four immunostainable foci, that dipyridyl and bile salts are inducers of its activity, and that inducers bind to Rob's C-terminal domain (CTD). We propose that sequestration inactivates Rob by blocking its access to the transcriptional machinery and that inducers activate Rob by mediating its dispersal, allowing interaction with RNA polymerase. To test "sequestration-dispersal" as a new mechanism for regulating the activity of transcriptional activators, we fused Rob's CTD to SoxS and used indirect immunofluorescence microscopy to determine the effect of inducers on SoxS-Rob's cellular localization. Unlike native SoxS, which is uniformly distributed throughout the cell, SoxS-Rob is sequestered without an inducer, but is rapidly dispersed when cells are treated with an inducer. In this manner, Rob's CTD serves as an anti-sigma factor in regulating the co-sigma-factor-like activity of SoxS when fused to it. Rob's CTD also protects its N-terminus from Lon protease, since Lon's normally rapid degradation of SoxS is blocked in the chimera. Accordingly, Rob's CTD has novel regulatory properties that can be bestowed on another E. coli protein.

  11. Small C-terminal Domain Phosphatase 3 Dephosphorylates the Linker Sites of Receptor-regulated Smads (R-Smads) to Ensure Transforming Growth Factor β (TGFβ)-mediated Germ Layer Induction in Xenopus Embryos.

    Science.gov (United States)

    Sun, Guanni; Hu, Zhirui; Min, Zheying; Yan, Xiaohua; Guan, Zhenpo; Su, Hanxia; Fu, Yu; Ma, Xiaopeng; Chen, Ye-Guang; Zhang, Michael Q; Tao, Qinghua; Wu, Wei

    2015-07-10

    Germ layer induction is one of the earliest events shortly after fertilization that initiates body formation of vertebrate embryos. In Xenopus, the maternally deposited transcriptional factor VegT promotes the expression of zygotic Nodal/Activin ligands that further form a morphogen gradient along the vegetal-animal axis and trigger the induction of the three germ layers. Here we found that SCP3 (small C-terminal domain phosphatase 3) is maternally expressed and vegetally enriched in Xenopus embryos and is essential for the timely induction of germ layers. SCP3 is required for the full activation of Nodal/Activin and bone morphogenetic protein signals and functions via dephosphorylation in the linker regions of receptor-regulated Smads. Consistently, the linker regions of receptor-regulated Smads are heavily phosphorylated in fertilized eggs, and this phosphorylation is gradually removed when embryos approach the midblastula transition. Knockdown of maternal SCP3 attenuates these dephosphorylation events and the activation of Nodal/Activin and bone morphogenetic protein signals after midblastula transition. This study thus suggested that the maternal SCP3 serves as a vegetally enriched, intrinsic factor to ensure a prepared status of Smads for their activation by the upcoming ligands during germ layer induction of Xenopus embryos. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  12. Proteolysis-induced N-terminal ectodomain shedding of the integral membrane glycoprotein CUB domain-containing protein 1 (CDCP1) is accompanied by tyrosine phosphorylation of its C-terminal domain and recruitment of Src and PKCdelta.

    Science.gov (United States)

    He, Yaowu; Wortmann, Andreas; Burke, Les J; Reid, Janet C; Adams, Mark N; Abdul-Jabbar, Ibtissam; Quigley, James P; Leduc, Richard; Kirchhofer, Daniel; Hooper, John D

    2010-08-20

    CUB-domain-containing protein 1 (CDCP1) is an integral membrane glycoprotein with potential as a marker and therapeutic target for a number of cancers. Here we examine mechanisms regulating cellular processing of CDCP1. By analyzing cell lines exclusively passaged non-enzymatically and through use of a panel of protease inhibitors, we demonstrate that full-length 135 kDa CDCP1 is post-translationally processed in a range of cell lines by a mechanism involving serine protease activity, generating a C-terminal 70-kDa fragment. Immunopurification and N-terminal sequencing of this cell-retained fragment and detailed mutagenesis, show that proteolytic processing of CDCP1 occurs at two sites, Arg-368 and Lys-369. We show that the serine protease matriptase is an efficient, but not essential, cellular processor of CDCP1 at Arg-368. Importantly, we also demonstrate that proteolysis induces tyrosine phosphorylation of 70-kDa CDCP1 and recruitment of Src and PKCdelta to this fragment. In addition, Western blot and mass spectroscopy analyses show that an N-terminal 65-kDa CDCP1 ectodomain is shed intact from the cell surface. These data provide new insights into mechanisms regulating CDCP1 and suggest that the biological role of this protein and, potentially, its function in cancer, may be mediated by both 70-kDa cell retained and 65-kDa shed fragments, as well as the full-length 135-kDa protein.

  13. The Chikungunya Virus Capsid Protein Contains Linear B Cell Epitopes in the N- and C-Terminal Regions that are Dependent on an Intact C-Terminus for Antibody Recognition

    Directory of Open Access Journals (Sweden)

    Lucas Y. H. Goh

    2015-06-01

    Full Text Available Chikungunya virus (CHIKV is an arthropod-borne agent that causes severe arthritic disease in humans and is considered a serious health threat in areas where competent mosquito vectors are prevalent. CHIKV has recently been responsible for several millions of cases of disease, involving over 40 countries. The recent re-emergence of CHIKV and its potential threat to human health has stimulated interest in better understanding of the biology and pathogenesis of the virus, and requirement for improved treatment, prevention and control measures. In this study, we mapped the binding sites of a panel of eleven monoclonal antibodies (mAbs previously generated towards the capsid protein (CP of CHIKV. Using N- and C-terminally truncated recombinant forms of the CHIKV CP, two putative binding regions, between residues 1–35 and 140–210, were identified. Competitive binding also revealed that five of the CP-specific mAbs recognized a series of overlapping epitopes in the latter domain. We also identified a smaller, N-terminally truncated product of native CP that may represent an alternative translation product of the CHIKV 26S RNA and have potential functional significance during CHIKV replication. Our data also provides evidence that the C-terminus of CP is required for authentic antigenic structure of CP. This study shows that these anti-CP mAbs will be valuable research tools for further investigating the structure and function of the CHIKV CP.

  14. The Chikungunya Virus Capsid Protein Contains Linear B Cell Epitopes in the N- and C-Terminal Regions that are Dependent on an Intact C-Terminus for Antibody Recognition

    Science.gov (United States)

    Goh, Lucas Y. H.; Hobson-Peters, Jody; Prow, Natalie A.; Baker, Kelly; Piyasena, Thisun B. H.; Taylor, Carmel T.; Rana, Ashok; Hastie, Marcus L.; Gorman, Jeff J.; Hall, Roy A.

    2015-01-01

    Chikungunya virus (CHIKV) is an arthropod-borne agent that causes severe arthritic disease in humans and is considered a serious health threat in areas where competent mosquito vectors are prevalent. CHIKV has recently been responsible for several millions of cases of disease, involving over 40 countries. The recent re-emergence of CHIKV and its potential threat to human health has stimulated interest in better understanding of the biology and pathogenesis of the virus, and requirement for improved treatment, prevention and control measures. In this study, we mapped the binding sites of a panel of eleven monoclonal antibodies (mAbs) previously generated towards the capsid protein (CP) of CHIKV. Using N- and C-terminally truncated recombinant forms of the CHIKV CP, two putative binding regions, between residues 1–35 and 140–210, were identified. Competitive binding also revealed that five of the CP-specific mAbs recognized a series of overlapping epitopes in the latter domain. We also identified a smaller, N-terminally truncated product of native CP that may represent an alternative translation product of the CHIKV 26S RNA and have potential functional significance during CHIKV replication. Our data also provides evidence that the C-terminus of CP is required for authentic antigenic structure of CP. This study shows that these anti-CP mAbs will be valuable research tools for further investigating the structure and function of the CHIKV CP. PMID:26061335

  15. Interactions between the Leucine-zipper Motif of cGMP-Dependent Protein Kinase and the C-terminal Region of the Targeting Subunit of Myosin Light Chain Phosphatase*

    Science.gov (United States)

    Lee, Eunhee; Hayes, David B.; Langsetmo, Knut; Sundberg, Eric J.; Tao, Terence C.

    2007-01-01

    Summary Nitric oxide induces vasodilation by elevating the production of cGMP, an activator of cGMP-dependent protein kinase (PKG). PKG subsequently causes smooth muscle relaxation in part via activation of myosin light chain phosphatase (MLCP). To date, the interaction between PKG and the targeting subunit of MLCP (MYPT1) is not fully understood. Earlier studies by one group of workers showed that the binding of PKG to MYPT1 is mediated by the leucine-zipper motifs at the N- and C-termini, respectively, of the two proteins. Another group, however, reported that binding of PKG to MYPT1 did not require the leucine-zipper motif of MYPT1. In this work we fully characterized the interaction between PKG and MYPT1 using biophysical techniques. For this purpose we constructed a recombinant PKG peptide corresponding to a predicted coiled coil region that contains the leucine-zipper motif. We further constructed various C-terminal MYPT1 peptides bearing various combinations of a predicted coiled coil region, extensions preceding this coiled coil region, and the leucine-zipper motif. Our results show, firstly, that while the leucine-zipper motif at the N-terminus of PKG forms a homodimeric coiled coil, the one at the C-terminus of MYPT1 is monomeric and non-helical. Secondly, the leucine-zipper motif of PKG binds to that of MYPT1 to form a heterodimer. Thirdly, when the leucine-zipper motif of MYPT1 is absent, the PKG leucine-zipper motif binds to the coiled coil region and upstream segments of MYPT1 via formation of a heterotetramer. These results provide rationalization of some of the findings by others using alternative binding analyses. PMID:17904578

  16. Plasma concentrations of the vasoactive peptide fragments mid-regional pro-adrenomedullin, C-terminal pro-endothelin 1 and copeptin in hemodialysis patients: associated factors and prediction of mortality.

    Directory of Open Access Journals (Sweden)

    Ferruh Artunc

    Full Text Available Vasopressin, endothelin and adrenomedullin are vasoactive peptides that regulate vascular tone and might play a role in hypertensive diseases. Recently, laboratory assays have been developed to measure stable fragments of vasopressin, endothelin and adrenomedullin. Little is known about their diagnostic and prognostic value in hemodialysis patients. In this study, we measured the plasma concentration of copeptin, mid-regional-pro-adrenomedullin (MR-pro-ADM and C-terminal pro-endothelin 1 (CT-pro-ET1 in stable ambulatory hemodialysis patients (n = 239 and investigated their associations with clinical factors and mortality. In all patients enrolled, the plasma concentrations of copeptin, MR-pro-ADM and CT-pro-ET1 were largely elevated with a median concentration of 132 pmol/L (interquartile range [IQR] 78-192 for copeptin, 1.26 nmol/L (IQR 1.02-1.80 for MR-pro-ADM and 149 pmol/L (IQR 121-181 for CT-pro-ET1. The plasma concentrations of all vasoactive peptide fragments correlated with time on dialysis and plasma β2-microglobulin concentration and were negatively correlated to residual diuresis. The plasma concentration of MR-pro-ADM was a strong predictor of all-cause (univariate hazard ratio for a 10-fold increase 9.94 [3.14;32], p<0.0001 and cardiovascular mortality (hazard ratio 34.87 [5.58;217], p = 0.0001 within a 3.8-year follow-up. The associations remained stable in models adjusted for dialysis specific factors and were attenuated in a full model adjusted for all prognostic factors. Plasma copeptin concentration was weakly associated with cardiovascular mortality (only in univariate analysis and CT-pro-ET1 was not associated with mortality at all. In conclusion, vasoactive peptide fragments are elevated in hemodialysis patients because of accumulation and, most likely, increased release. Increased concentrations of MR-pro-ADM are predictive of mortality.

  17. Novel aggregate formation of a frame-shift mutant protein of tissue-nonspecific alkaline phosphatase is ascribed to three cysteine residues in the C-terminal extension. Retarded secretion and proteasomal degradation.

    Science.gov (United States)

    Komaru, Keiichi; Ishida, Yoko; Amaya, Yoshihiro; Goseki-Sone, Masae; Orimo, Hideo; Oda, Kimimitsu

    2005-04-01

    In the majority of hypophosphatasia patients, reductions in the serum levels of alkaline phosphatase activity are caused by various missense mutations in the tissue-nonspecific alkaline phosphatase (TNSALP) gene. A unique frame-shift mutation due to a deletion of T at cDNA number 1559 [TNSALP (1559delT)] has been reported only in Japanese patients with high allele frequency. In this study, we examined the molecular phenotype of TNSALP (1559delT) using in vitro translation/translocation system and COS-1 cells transiently expressing this mutant protein. We showed that the mutant protein not only has a larger molecular size than the wild type enzyme by approximately 12 kDa, reflecting an 80 amino acid-long extension at its C-terminus, but that it also lacks a glycosylphosphatidylinositol anchor. In support of this, alkaline phosphatase activity of the cells expressing TNSALP (1559delT) was localized at the juxtanucleus position, but not on the cell surface. However, only a limited amount of the newly synthesized protein was released into the medium and the rest was polyubiquitinated, followed by degradation in the proteasome. SDS/PAGE and analysis by sucrose-density-gradient analysis indicated that TNSALP (1559delT) forms a disulfide-bonded high-molecular-mass aggregate. Interestingly, the aggregate form of TNSALP (1559delT) exhibited a significant enzyme activity. When all three cysteines at positions of 506, 521 and 577 of TNSALP (1559delT) were replaced with serines, the aggregation disappeared and instead this modified mutant protein formed a noncovalently associated dimer, strongly indicating that these cysteine residues in the C-terminal region are solely responsible for aggregate formation by cross-linking the catalytically active dimers. Thus, complete absence of TNSALP on cell surfaces provides a plausible explanation for a severe lethal phenotype of a homozygote hypophosphatasia patient carrying TNSALP (1559delT).

  18. Epstein-Barr Virus Latent Membrane Protein 1 (LMP1) C-Terminal-Activating Region 3 Contributes to LMP1-Mediated Cellular Migration via Its Interaction with Ubc9 ▿

    Science.gov (United States)

    Bentz, Gretchen L.; Whitehurst, Christopher B.; Pagano, Joseph S.

    2011-01-01

    Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1), the principal viral oncoprotein and a member of the tumor necrosis factor receptor superfamily, is a constitutively active membrane signaling protein that regulates multiple signal transduction pathways via its C-terminal-activating region 1 (CTAR1) and CTAR2, and also the less-studied CTAR3. Because protein sumoylation among other posttranslational modifications may regulate many signaling pathways induced by LMP1, we investigated whether during EBV latency LMP1 regulates sumoylation processes that control cellular activation and cellular responses. By immunoprecipitation experiments, we show that LMP1 interacts with Ubc9, the single reported SUMO-conjugating enzyme. Requirements for LMP1-Ubc9 interactions include enzymatically active Ubc9: expression of inactive Ubc9 (Ubc9 C93S) inhibited the LMP1-Ubc9 interaction. LMP1 CTAR3, but not CTAR1 and CTAR2, participated in the LMP1-Ubc9 interaction, and amino acid sequences found in CTAR3, including the JAK-interacting motif, contributed to this interaction. Furthermore, LMP1 expression coincided with increased sumoylation of cellular proteins, and disruption of the Ubc9-LMP1 CTAR3 interaction almost completely abrogated LMP1-induced protein sumoylation, suggesting that this interaction promotes the sumoylation of downstream targets. Additional consequences of the disruption of the LMP1 CTAR3-Ubc9 interaction revealed effects on cellular migration, a hallmark of oncogenesis. Together, these data demonstrate that LMP1 CTAR3 does in fact function in intracellular signaling and leads to biological effects. We propose that LMP1, by interaction with Ubc9, modulates sumoylation processes, which regulate signal transduction pathways that affect phenotypic changes associated with oncogenesis. PMID:21795333

  19. Epstein-Barr virus latent membrane protein 1 (LMP1) C-terminal-activating region 3 contributes to LMP1-mediated cellular migration via its interaction with Ubc9.

    Science.gov (United States)

    Bentz, Gretchen L; Whitehurst, Christopher B; Pagano, Joseph S

    2011-10-01

    Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1), the principal viral oncoprotein and a member of the tumor necrosis factor receptor superfamily, is a constitutively active membrane signaling protein that regulates multiple signal transduction pathways via its C-terminal-activating region 1 (CTAR1) and CTAR2, and also the less-studied CTAR3. Because protein sumoylation among other posttranslational modifications may regulate many signaling pathways induced by LMP1, we investigated whether during EBV latency LMP1 regulates sumoylation processes that control cellular activation and cellular responses. By immunoprecipitation experiments, we show that LMP1 interacts with Ubc9, the single reported SUMO-conjugating enzyme. Requirements for LMP1-Ubc9 interactions include enzymatically active Ubc9: expression of inactive Ubc9 (Ubc9 C93S) inhibited the LMP1-Ubc9 interaction. LMP1 CTAR3, but not CTAR1 and CTAR2, participated in the LMP1-Ubc9 interaction, and amino acid sequences found in CTAR3, including the JAK-interacting motif, contributed to this interaction. Furthermore, LMP1 expression coincided with increased sumoylation of cellular proteins, and disruption of the Ubc9-LMP1 CTAR3 interaction almost completely abrogated LMP1-induced protein sumoylation, suggesting that this interaction promotes the sumoylation of downstream targets. Additional consequences of the disruption of the LMP1 CTAR3-Ubc9 interaction revealed effects on cellular migration, a hallmark of oncogenesis. Together, these data demonstrate that LMP1 CTAR3 does in fact function in intracellular signaling and leads to biological effects. We propose that LMP1, by interaction with Ubc9, modulates sumoylation processes, which regulate signal transduction pathways that affect phenotypic changes associated with oncogenesis.

  20. Characterization of the Wild-Type and Truncated Forms of a Neutral GH10 Xylanase from Coprinus cinereus: Roles of C-Terminal Basic Amino Acid-Rich Extension in Its SDS Resistance, Thermostability, and Activity.

    Science.gov (United States)

    Hu, Hang; Chen, Kaixiang; Li, Lulu; Long, Liangkun; Ding, Shaojun

    2017-04-28

    A neutral xylanase (CcXyn) was identified from Coprinus cinereus. It has a single GH10 catalytic domain with a basic amino acid-rich extension (PVRRK) at the C-terminus. In this study, the wild-type (CcXyn) and C-terminus-truncated xylanase (CcXyn-Δ5C) were heterologously expressed in Pichia pastoris and their characteristics were comparatively analyzed with aims to examine the effect of this extension on the enzyme function. The circular dichorism analysis indicated that both enzymes in general had a similar structure, but CcXyn-Δ5C contained less α-helices (42.9%) and more random coil contents (35.5%) than CcXyn (47.0% and 32.8%, respectively). Both enzymes had the same pH (7.0) and temperature (45°C) optima, and similar substrate specificity on different xylans. They all hydrolyzed beechwood xylan primarily to xylobiose and xylotriose. The amounts of xylobiose and xylotriose accounted for 91.5% and 92.2% (w/w) of total xylooligosaccharides (XOS) generated from beechwood by CcXyn and CcXyn-Δ5C, respectively. However, truncation of the C-terminal 5-amino-acids extension significantly improved the thermostability, SDS resistance, and pH stability at pH 6.0-9.0. Furthermore, CcXyn-Δ5C exhibited a much lower Km value than CcXyn (0.27 mg/ml vs 0.83 mg/ml), and therefore, the catalytic efficiency of CcXyn-Δ5C was 2.4-times higher than that of CcXyn. These properties make CcXyn-Δ5C a good model for the structure-function study of (α/β)8-barrel-folded enzymes and a promising candidate for various applications, especially in the detergent industry and XOS production.

  1. Structures of pseudechetoxin and pseudecin, two snake-venom cysteine-rich secretory proteins that target cyclic nucleotide-gated ion channels: implications for movement of the C-terminal cysteine-rich domain

    Energy Technology Data Exchange (ETDEWEB)

    Suzuki, Nobuhiro [Department of Applied Biochemistry, University of Tsukuba, Tsukuba, Ibaraki 305-8572 (Japan); Department of Biochemistry, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602 (Japan); Yamazaki, Yasuo [Department of Biochemistry, Meiji Pharmaceutical University, Kiyose, Tokyo 204-8588 (Japan); Brown, R. Lane [Neurological Science Institute, Oregon Health and Science University, Beaverton, Oregon 97006 (United States); Fujimoto, Zui [Department of Biochemistry, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602 (Japan); Morita, Takashi, E-mail: tmorita@my-pharm.ac.jp [Department of Biochemistry, Meiji Pharmaceutical University, Kiyose, Tokyo 204-8588 (Japan); Mizuno, Hiroshi, E-mail: tmorita@my-pharm.ac.jp [Department of Biochemistry, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602 (Japan); VALWAY Technology Center, NEC Soft Ltd, Koto-ku, Tokyo 136-8627 (Japan); Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology, Central 6, Tsukuba, Ibaraki 305-8566 (Japan); Department of Applied Biochemistry, University of Tsukuba, Tsukuba, Ibaraki 305-8572 (Japan)

    2008-10-01

    The structures of pseudechetoxin and pseudecin suggest that both proteins bind to cyclic nucleotide-gated ion channels in a manner in which the concave surface occludes the pore entrance. Cyclic nucleotide-gated (CNG) ion channels play pivotal roles in sensory transduction by retinal photoreceptors and olfactory neurons. The elapid snake toxins pseudechetoxin (PsTx) and pseudecin (Pdc) are the only known protein blockers of CNG channels. These toxins belong to a cysteine-rich secretory protein (CRISP) family containing an N-terminal pathogenesis-related proteins of group 1 (PR-1) domain and a C-terminal cysteine-rich domain (CRD). PsTx and Pdc are highly homologous proteins, but their blocking affinities on CNG channels are different: PsTx blocks both the olfactory and retinal channels with ∼15–30-fold higher affinity than Pdc. To gain further insights into their structure and function, the crystal structures of PsTx, Pdc and Zn{sup 2+}-bound Pdc were determined. The structures revealed that most of the amino-acid-residue differences between PsTx and Pdc are located around the concave surface formed between the PR-1 domain and the CRD, suggesting that the concave surface is functionally important for CNG-channel binding and inhibition. A structural comparison in the presence and absence of Zn{sup 2+} ion demonstrated that the concave surface can open and close owing to movement of the CRD upon Zn{sup 2+} binding. The data suggest that PsTx and Pdc occlude the pore entrance and that the dynamic motion of the concave surface facilitates interaction with the CNG channels.

  2. The C-terminal 20 Amino Acids of Drosophila Topoisomerase 2 Are Required for Binding to a BRCA1 C Terminus (BRCT) Domain-containing Protein, Mus101, and Fidelity of DNA Segregation*

    Science.gov (United States)

    Chen, Yu-tsung Shane; Wu, Jianhong; Modrich, Paul; Hsieh, Tao-shih

    2016-01-01

    Eukaryotic topoisomerase 2 (Top2) and one of its interacting partners, topoisomerase IIβ binding protein 1 (TopBP1) are two proteins performing essential cellular functions. We mapped the interacting domains of these two proteins using co-immunoprecipitation and pulldown experiments with truncated or mutant Drosophila Top2 with various Ser-to-Ala substitutions. We discovered that the last 20 amino acids of Top2 represent the key region for binding with Mus101 (the Drosophila homolog of TopBP1) and that phosphorylation of Ser-1428 and Ser-1443 is important for Top2 to interact with the N terminus of Mus101, which contains the BRCT1/2 domains. The interaction between Mus101 and the Top2 C-terminal regulatory domain is phosphorylation-dependent because treatment with phosphatase abolishes their association in pulldown assays. The binding affinity of N-terminal Mus101 with a synthetic phosphorylated peptide spanning the last 25 amino acids of Top2 (with Ser(P)-1428 and Ser(P)-1443) was determined by surface plasmon resonance with a Kd of 0.57 μm. In an in vitro decatenation assay, Mus101 can specifically reduce the decatenation activity of Top2, and dephosphorylation of Top2 attenuates this response. Next, we endeavored to establish a cellular system for testing the biological function of Top2-Mus101 interaction. Top2-silenced S2 cells rescued by Top2Δ20, Top2 with 20 amino acids truncated from the C terminus, developed abnormally high chromosome numbers, which implies that Top2-Mus101 interaction is important for maintaining the fidelity of chromosome segregation during mitosis. PMID:27129233

  3. Neuropeptide VGF C-Terminal Peptide TLQP-62 Alleviates Lipopolysaccharide-Induced Memory Deficits and Anxiety-like and Depression-like Behaviors in Mice: The Role of BDNF/TrkB Signaling.

    Science.gov (United States)

    Li, Chenli; Li, Mengmeng; Yu, Hanjie; Shen, Xinbei; Wang, Jinting; Sun, Xin; Wang, Qinwen; Wang, Chuang

    2017-09-20

    Peripheral inflammatory responses affect central nervous system (CNS) function, manifesting in symptoms of memory deficits, depression, and anxiety. Previous studies have revealed that neuropeptide VGF (nonacronymic) C-terminal peptide TLQP-62 rapidly reinforces brain-derived neurotrophic factor (BDNF)/tropomyosin receptor kinase B (TrkB) signaling, regulating memory consolidation and antidepressant-like action. However, whether it is beneficial for lipopolysaccharide (LPS)-induced neuropsychiatric dysfunction in mice is unknown. Herein, we explored the involvement of BDNF/TrkB signaling and biochemical alterations in inflammatory or oxidative stress markers in the alleviating effects of TLQP-62 on LPS-induced neuropsychiatric dysfunction. The mice were treated with TLQP-62 (2 μg/side) via intracerebroventricular (i.c.v.) injection 1 h before LPS (0.5 mg/kg, i.p.) administration. Our results showed that a single treatment with LPS (0.5 mg/kg, i.p) is sufficient to produce recognition memory deficits (in the novel object recognition test), depression-like behavior (in the forced swim test and sucrose preference test), and anxiety-like behavior (in the elevated zero maze). However, pretreatment with TLQP-62 prevented LPS-induced behavioral dysfunction, neuroinflammatory, and oxidative responses. In addition, our results further demonstrated that a reduction in BDNF expression mediated by BDNF-shRNA lentivirus significantly blocked the effects of TLQP-62, suggesting the critical role of BDNF/TrkB signaling in the neuroprotective effects of TLQP-62 in the mice. In conclusion, TLQP-62 could be a therapeutic approach for neuropsychiatric disorders, which are closely associated with neuroinflammation and oxidative stress.

  4. Rabbit IgG directed to a synthetic C-terminal peptide of the major grass pollen allergen Lol p I inhibits human basophil histamine release induced by natural Lol p I.

    Science.gov (United States)

    van Ree, R; Aalberse, R C

    1995-03-01

    The potential role of allergen-specific IgG antibodies as 'blocking' antibodies in allergen-induced human basophil histamine release was investigated. This was studied in a model with the major grass pollen allergen Lol p I and polyclonal rabbit antisera directed against this allergen and against a synthetic peptide of its C terminus. When allergen and antibodies were allowed to preincubate, Lol p I induced histamine release was inhibited up to 85% by the antiserum against Lol p I. By omitting preincubation, and thereby more closely mimicking an in vivo situation, up to 55% inhibition was realized. This indicates that allergen-specific IgG can act as 'blocking' antibody without preincubation. Immunization of rabbits with a synthetic C-terminal peptide of Lol p I resulted in antibodies reactive with natural Lol p I. Despite their 100-fold lower avidity for Lol p I (as compared with antinatural Lol p I), these antibodies had the capacity to inhibit Lol p I induced histamine release for > 90% (up to 50% without preincubation). This indicates that it is possible to block histamine release induced by a major allergen with low-avidity IgG antibodies directed against a minor proportion of the allergen (25 amino acids). IgE antibodies from the donors studied were unreactive with this synthetic peptide, indicating that for blocking activity identical epitope specificity of IgE and IgG is not essential. This opens interesting perspectives for application of synthetic peptides in immunotherapy, distinct from their effects on T cell reactivity.

  5. The foetal pig pineal gland is richly innervated by nerve fibres containing catecholamine-synthesizing enzymes, neuropeptide Y (NPY) and C-terminal flanking peptide of NPY, but it does not secrete melatonin.

    Science.gov (United States)

    Bulc, Michał; Lewczuk, Bogdan; Prusik, Magdalena; Całka, Jarosław

    2013-05-01

    Innervation of the mammalian pineal gland during prenatal development is poorly recognized. Therefore, immunofluorescence studies of the pineals of 70- and 90-day-old foetuses of the domestic pig were performed using antibodies against tyrosine hydroxylase (TH), dopamine-β-hydroxylase (DβH), neuropeptide Y (NPY) and C-terminal flanking peptide of NPY (CPON). The investigated glands were supplied by numerous nerve fibres containing TH and DβH. The density of these fibres was higher in the distal and middle parts of the gland than in the proximal one. NPY and CPON were identified in the majority of DβH-positive fibres as well as in a small population of DβH-negative fibres local