Sample records for c-terminal coiled-coil motif

  1. 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:


    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

  2. Plasmodium vivax antigen discovery based on alpha-helical coiled coil protein motif

    DEFF Research Database (Denmark)

    Céspedes, Nora; Habel, Catherine; Lopez-Perez, Mary


    Protein α-helical coiled coil structures that elicit antibody responses, which block critical functions of medically important microorganisms, represent a means for vaccine development. By using bioinformatics algorithms, a total of 50 antigens with α-helical coiled coil motifs orthologous...

  3. Hinderin, a five-domains protein including coiled-coil motifs that binds to SMC3

    Directory of Open Access Journals (Sweden)

    Ghiselli Giancarlo


    Full Text Available Abstract Background The structural maintenance of chromosome proteins SMC1 and SMC3 play an important role in the maintenance of chromosomal integrity by preventing the premature separation of the sister chromatids at the onset of anaphase. The two proteins are constitutive components of the multimeric complex cohesin and form dimers by interacting at their central globular regions. Results In order to identify proteins that by binding to SMC3 may interfere with the protein dimerization process, a human cDNA library was screened by the yeast two-hybrid system by using the hinge region of SMC3 as bait. This has lead to the identification of Hinderin, a novel five domains protein including two coiled-coil motifs and sharing a strikingly structural similarity to the SMC family of proteins. Hinderin is ubiquitously expressed in human tissues. Orthologue forms of the protein are present in other vertebrates but not in lower organisms. A mapping of the interaction sites revealed that the N- and C-terminal globular domains mediate the binding of Hinderin to SMC3. Hinderin/SMC3 complexes could be recovered by immunoprecipitation from cell lysates using an anti-SMC3 antibody, thus demonstrating that the two proteins interact in vivo. On the contrary, Hinderin did not interact with SMC1. In vivo the rate of SMC1/SMC3 interaction was decreased by the ectopic expression of Hinderin. Conclusions Hinderin is a novel binding partner of SMC3. Based on its ability to modulate SMC1/SMC3 interaction we postulate that Hinderin affects the availability of SMC3 to engage in the formation of multimeric protein complexes.

  4. Structural model of the TRPP2/PKD1 C-terminal coiled-coil complex produced by a combined computational and experimental approach. (United States)

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


    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.

  5. Plasmodium vivax antigen discovery based on alpha-helical coiled coil protein motif.

    Directory of Open Access Journals (Sweden)

    Nora Céspedes

    Full Text Available Protein α-helical coiled coil structures that elicit antibody responses, which block critical functions of medically important microorganisms, represent a means for vaccine development. By using bioinformatics algorithms, a total of 50 antigens with α-helical coiled coil motifs orthologous to Plasmodium falciparum were identified in the P. vivax genome. The peptides identified in silico were chemically synthesized; circular dichroism studies indicated partial or high α-helical content. Antigenicity was evaluated using human sera samples from malaria-endemic areas of Colombia and Papua New Guinea. Eight of these fragments were selected and used to assess immunogenicity in BALB/c mice. ELISA assays indicated strong reactivity of serum samples from individuals residing in malaria-endemic regions and sera of immunized mice, with the α-helical coiled coil structures. In addition, ex vivo production of IFN-γ by murine mononuclear cells confirmed the immunogenicity of these structures and the presence of T-cell epitopes in the peptide sequences. Moreover, sera of mice immunized with four of the eight antigens recognized native proteins on blood-stage P. vivax parasites, and antigenic cross-reactivity with three of the peptides was observed when reacted with both the P. falciparum orthologous fragments and whole parasites. Results here point to the α-helical coiled coil peptides as possible P. vivax malaria vaccine candidates as were observed for P. falciparum. Fragments selected here warrant further study in humans and non-human primate models to assess their protective efficacy as single components or assembled as hybrid linear epitopes.

  6. Control of Recombination Directionality by the Listeria Phage A118 Protein Gp44 and the Coiled-Coil Motif of Its Serine Integrase. (United States)

    Mandali, Sridhar; Gupta, Kushol; Dawson, Anthony R; Van Duyne, Gregory D; Johnson, Reid C


    The serine integrase of phage A118 catalyzes integrative recombination between attP on the phage and a specific attB locus on the chromosome of Listeria monocytogenes , but it is unable to promote excisive recombination between the hybrid attL and attR sites found on the integrated prophage without assistance by a recombination directionality factor (RDF). We have identified and characterized the phage-encoded RDF Gp44, which activates the A118 integrase for excision and inhibits integration. Gp44 binds to the C-terminal DNA binding domain of integrase, and we have localized the primary binding site to be within the mobile coiled-coil (CC) motif but distinct from the distal tip of the CC that is required for recombination. This interaction is sufficient to inhibit integration, but a second interaction involving the N-terminal end of Gp44 is also required to activate excision. We provide evidence that these two contacts modulate the trajectory of the CC motifs as they extend out from the integrase core in a manner dependent upon the identities of the four att sites. Our results support a model whereby Gp44 shapes the Int-bound complexes to control which att sites can synapse and recombine. IMPORTANCE Serine integrases mediate directional recombination between bacteriophage and bacterial chromosomes. These highly regulated site-specific recombination reactions are integral to the life cycle of temperate phage and, in the case of Listeria monocytogenes lysogenized by A118 family phage, are an essential virulence determinant. Serine integrases are also utilized as tools for genetic engineering and synthetic biology because of their exquisite unidirectional control of the DNA exchange reaction. Here, we identify and characterize the recombination directionality factor (RDF) that activates excision and inhibits integration reactions by the phage A118 integrase. We provide evidence that the A118 RDF binds to and modulates the trajectory of the long coiled-coil motif that

  7. Conformational consequences of cooperative binding of a coiled-coil peptide motif to poly(N-(2-hydroxypropyl) methacrylamide) HPMA copolymers. (United States)

    Griffiths, Peter C; Paul, Alison; Apostolovic, Bojana; Klok, Harm-Anton; de Luca, Edoardo; King, Stephen M; Heenan, Richard K


    Small-angle neutron scattering and pulsed-gradient spin-echo NMR have been used to examine the solution conformation of a series of water soluble poly(N-(2-hydroxypropyl) methacrylamide) P(HPMA) co-polymer drug delivery vehicles incorporating a coiled-coil peptide motif as a novel pH sensitive non-covalent linker. The conformation of the HPMA homopolymer is well-described by a Gaussian coil model and changing pH from pH 7 to pH 5 has little effect on the solution conformation, as quantified via the radius of gyration. Copolymerisation with 5-10mol% of the K3 peptide bearing methacrylate monomer (K3-MA), gave a series of copolymers that exhibited an increase in radius of gyration at both pH's, despite being typically 30% lower in molecular weight, indicating that the K3-MA causes a perturbation (expansion) of the copolymer conformation. Subsequent addition of an equimolar amount of the complementary peptide E3 makes little further difference to the conformation, indicative of the intimate binding (coiled-coil motif) between the two peptides. Again, the effects of pH are small. Only the addition of a large aromatic structure such as methotrexate causes a further perturbation of the structure - the hydrophobic interaction between the MTX units causes a significant collapse of the polymer coil. These findings further elaborate the understanding of those factors that determine the solution conformation of novel polymer therapeutics. Copyright © 2011 Elsevier B.V. All rights reserved.

  8. 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* (United States)

    Lee, Eunhee; Hayes, David B.; Langsetmo, Knut; Sundberg, Eric J.; Tao, Terence C.


    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

  9. 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


    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.

  10. Programming protein self assembly with coiled coils

    Energy Technology Data Exchange (ETDEWEB)

    Dietz, Hendrik; Bornschloegl, Thomas; Heym, Roland; Koenig, Frauke; Rief, Matthias [Physik Department E22, Technische Universitaet Muenchen, James-Franck-Strasse 1, 85748 Garching (Germany)


    The controlled assembly of protein domains into supramolecular structures will be an important prerequisite for the use of functional proteins in future nanotechnology applications. Coiled coils are multimerization motifs whose dimerization properties can be programmed by amino acid sequence. Here, we report programmed supramolecular self-assembly of protein molecules using coiled coils and directly demonstrate its potential on the single molecule level by AFM force spectroscopy. We flanked two different model proteins, Ig27 from human cardiac titin and green fluorescent protein (GFP), by coiled coil binding partners and studied the capability of these elementary building blocks to self-assemble into linear chains. Simple sterical constraints are shown to control the assembly process, providing evidence that many proteins can be assembled with this method. An application for this technique is the design of polyproteins for single molecule force spectroscopy with an integrated force-calibration standard.

  11. CCHMM_PROF: a HMM-based coiled-coil predictor with evolutionary information

    DEFF Research Database (Denmark)

    Bartoli, Lisa; Fariselli, Piero; Krogh, Anders


    MOTIVATION: The widespread coiled-coil structural motif in proteins is known to mediate a variety of biological interactions. Recognizing a coiled-coil containing sequence and locating its coiled-coil domains are key steps towards the determination of the protein structure and function. Different......:// approximately lisa/coiled-coils. The predictor is freely available at CONTACT:

  12. Meiosis specific coiled-coil proteins in Shizosaccharomyces pombe

    Directory of Open Access Journals (Sweden)

    Okuzaki Daisuke


    Full Text Available Abstract Many meiosis-specific proteins in Schizosaccharomyces pombe contain coiled-coil motifs which play essential roles for meiotic progression. For example, the coiled-coil motifs present in Meu13 and Mcp7 are required for their function as a putative recombinase cofactor complex during meiotic recombination. Mcp6/Hrs1 and Mcp5/Num1 control horsetail chromosome movement by astral microtubule organization and anchoring dynein respectively. Dhc1 and Ssm4 are also required for horsetail chromosome movement. It is clear from these examples that the coiled-coil motif in these proteins plays an important role during the progression of cells through meiosis. However, there are still many unanswered questions on how these proteins operate. In this paper, we briefly review recent studies on the meiotic coiled-coil proteins in Sz. pombe.

  13. Biomaterials Made from Coiled-Coil Peptides. (United States)

    Conticello, Vincent; Hughes, Spencer; Modlin, Charles

    The development of biomaterials designed for specific applications is an important objective in personalized medicine. While the breadth and prominence of biomaterials have increased exponentially over the past decades, critical challenges remain to be addressed, particularly in the development of biomaterials that exhibit highly specific functions. These functional properties are often encoded within the molecular structure of the component molecules. Proteins, as a consequence of their structural specificity, represent useful substrates for the construction of functional biomaterials through rational design. This chapter provides an in-depth survey of biomaterials constructed from coiled-coils, one of the best-understood protein structural motifs. We discuss the utility of this structurally diverse and functionally tunable class of proteins for the creation of novel biomaterials. This discussion illustrates the progress that has been made in the development of coiled-coil biomaterials by showcasing studies that bridge the gap between the academic science and potential technological impact.

  14. Polymer cancerostatics targeted with an antibody fragment bound via a coiled coil motif: in vivo therapeutic efficacy against murine BCL1 leukemia

    Czech Academy of Sciences Publication Activity Database

    Pechar, Michal; Pola, Robert; Janoušková, Olga; Sieglová, Irena; Král, Vlastimil; Fábry, Milan; Tomalová, Barbora; Kovář, Marek


    Roč. 18, č. 1 (2018), s. 1-11, č. článku 1700173. ISSN 1616-5187 R&D Projects: GA MŠk(CZ) LO1507; GA MŠk(CZ) LQ1604; GA MZd(CZ) NV16-28594A; GA ČR(CZ) GA16-17207S; GA ČR GA13-12885S Institutional support: RVO:61389013 ; RVO:68378050 ; RVO:61388971 Keywords : cancer therapy * coiled coil * drug targeting Subject RIV: CD - Macromolecular Chemistry; CD - Macromolecular Chemistry (UMG-J); EE - Microbiology, Virology (MBU-M) OBOR OECD: Polymer science; Polymer science (UMG-J); Microbiology (MBU-M) Impact factor: 3.238, year: 2016

  15. High-resolution structures of a heterochiral coiled coil. (United States)

    Mortenson, David E; Steinkruger, Jay D; Kreitler, Dale F; Perroni, Dominic V; Sorenson, Gregory P; Huang, Lijun; Mittal, Ritesh; Yun, Hyun Gi; Travis, Benjamin R; Mahanthappa, Mahesh K; Forest, Katrina T; Gellman, Samuel H


    Interactions between polypeptide chains containing amino acid residues with opposite absolute configurations have long been a source of interest and speculation, but there is very little structural information for such heterochiral associations. The need to address this lacuna has grown in recent years because of increasing interest in the use of peptides generated from d amino acids (d peptides) as specific ligands for natural proteins, e.g., to inhibit deleterious protein-protein interactions. Coiled-coil interactions, between or among α-helices, represent the most common tertiary and quaternary packing motif in proteins. Heterochiral coiled-coil interactions were predicted over 50 years ago by Crick, and limited experimental data obtained in solution suggest that such interactions can indeed occur. To address the dearth of atomic-level structural characterization of heterochiral helix pairings, we report two independent crystal structures that elucidate coiled-coil packing between l- and d-peptide helices. Both structures resulted from racemic crystallization of a peptide corresponding to the transmembrane segment of the influenza M2 protein. Networks of canonical knobs-into-holes side-chain packing interactions are observed at each helical interface. However, the underlying patterns for these heterochiral coiled coils seem to deviate from the heptad sequence repeat that is characteristic of most homochiral analogs, with an apparent preference for a hendecad repeat pattern.

  16. Coiled-Coil Design: Updated and Upgraded. (United States)

    Woolfson, Derek N


    α-Helical coiled coils are ubiquitous protein-folding and protein-interaction domains in which two or more α-helical chains come together to form bundles. Through a combination of bioinformatics analysis of many thousands of natural coiled-coil sequences and structures, plus empirical protein engineering and design studies, there is now a deep understanding of the sequence-to-structure relationships for this class of protein architecture. This has led to considerable success in rational design and what might be termed in biro de novo design of simple coiled coils, which include homo- and hetero-meric parallel dimers, trimers and tetramers. In turn, these provide a toolkit for directing the assembly of both natural proteins and more complex designs in protein engineering, materials science and synthetic biology. Moving on, the increased and improved use of computational design is allowing access to coiled-coil structures that are rare or even not observed in nature, for example α-helical barrels, which comprise five or more α-helices and have central channels into which different functions may be ported. This chapter reviews all of these advances, outlining improvements in our knowledge of the fundamentals of coiled-coil folding and assembly, and highlighting new coiled coil-based materials and applications that this new understanding is opening up. Despite considerable progress, however, challenges remain in coiled-coil design, and the next decade promises to be as productive and exciting as the last.

  17. Crystal structure of a coiled-coil domain from human ROCK I.

    Directory of Open Access Journals (Sweden)

    Daqi Tu

    Full Text Available The small GTPase Rho and one of its targets, Rho-associated kinase (ROCK, participate in a variety of actin-based cellular processes including smooth muscle contraction, cell migration, and stress fiber formation. The ROCK protein consists of an N-terminal kinase domain, a central coiled-coil domain containing a Rho binding site, and a C-terminal pleckstrin homology domain. Here we present the crystal structure of a large section of the central coiled-coil domain of human ROCK I (amino acids 535-700. The structure forms a parallel α-helical coiled-coil dimer that is structurally similar to tropomyosin, an actin filament binding protein. There is an unusual discontinuity in the coiled-coil; three charged residues (E613, R617 and D620 are positioned at what is normally the hydrophobic core of coiled-coil packing. We speculate that this conserved irregularity could function as a hinge that allows ROCK to adopt its autoinhibited conformation.

  18. Coiled coil in the stalk region of ncd motor protein is nonlocally sustained. (United States)

    Ito, Mie; Morii, Hisayuki; Shimizu, Takashi; Tanokura, Masaru


    The dimeric structure of kinesin superfamily proteins plays an important role in their motile functions and characteristics. In this study, the coiled-coil-forming property of the stalk region (192-346) of Drosophila ncd, a C-terminal kinesin motor protein, was investigated by synthesizing various peptide fragments. The alpha helicity of a set of 46-residue peptides spanning the stalk region appeared too low to form a coiled-coil dimer, probably because of insufficient continuity of the hydrophobic residues at (a and d) core positions in amphipathic heptad repeats. On the other hand, several peptides with leucine residues introduced at core positions or with extensional sequences with high alpha helicity had an advantage in coiled-coil formation. When we analyzed the thermal and urea-induced unfolding of these dimeric peptides, we identified four domains having a relatively high potential to form coiled coils. Among them, three domains on the C-terminal side of the stalk region, i.e., (252-272), (276-330), and (336-346), were in the same heptad frame, although these potential coiled-coil domains were not self-sustaining individually. This is in sharp contrast to the fragment of human kinesin, (332-369), which has an extremely high tendency toward coiled-coil formation. One of the possible triggers for coiled-coil formation of the ncd stalk region may be the interaction between the motor domain and the C-terminal part of the stalk as previously revealed by X-ray crystallography. The residues, S331 and R335, seem to act as a breaking point for alpha-helix continuity. This would make the region (336-346), as the head-stalk joint, more flexible such as seen with a plus-end-directed kinesin, if this region had no interaction with the motor domain. These characteristic differences between ncd and kinesin suggest that the nonlocally sustained coiled coil of ncd is one of the factors important for minus-end-directed motility.

  19. Immune responses to coiled coil supramolecular biomaterials. (United States)

    Rudra, Jai S; Tripathi, Pulak K; Hildeman, David A; Jung, Jangwook P; Collier, Joel H


    Self-assembly has been increasingly utilized in recent years to create peptide-based biomaterials for 3D cell culture, tissue engineering, and regenerative medicine, but the molecular determinants of these materials' immunogenicity have remained largely unexplored. In this study, a set of molecules that self-assembled through coiled coil oligomerization was designed and synthesized, and immune responses against them were investigated in mice. Experimental groups spanned a range of oligomerization behaviors and included a peptide from the coiled coil region of mouse fibrin that did not form supramolecular structures, an engineered version of this peptide that formed coiled coil bundles, and a peptide-PEG-peptide triblock bioconjugate that formed coiled coil multimers and supramolecular aggregates. In mice, the native peptide and engineered peptide did not produce any detectable antibody response, and none of the materials elicited detectable peptide-specific T cell responses, as evidenced by the absence of IL-2 and interferon-gamma in cultures of peptide-challenged splenocytes or draining lymph node cells. However, specific antibody responses were elevated in mice injected with the multimerizing peptide-PEG-peptide. Minimal changes in secondary structure were observed between the engineered peptide and the triblock peptide-PEG-peptide, making it possible that the triblock's multimerization was responsible for this antibody response. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

  20. α-Helical coiled-coil peptide materials for biomedical applications. (United States)

    Wu, Yaoying; Collier, Joel H


    Self-assembling coiled coils, which occur commonly in native proteins, have received significant interest for the design of new biomaterials-based medical therapies. Considerable effort over recent years has led to a detailed understanding of the self-assembly process of coiled coils, and a diverse collection of strategies have been developed for designing functional materials using this motif. The ability to engineer the interface between coiled coils allows one to achieve variously connected components, leading to precisely defined structures such as nanofibers, nanotubes, nanoparticles, networks, gels, and combinations of these. Currently these materials are being developed for a range of biotechnological and medical applications, including drug delivery systems for controlled release, targeted nanomaterials, 'drug-free' therapeutics, vaccine delivery systems, and others. WIREs Nanomed Nanobiotechnol 2017, 9:e1424. doi: 10.1002/wnan.1424 For further resources related to this article, please visit the WIREs website. © 2016 Wiley Periodicals, Inc.

  1. Computational characterization of parallel dimeric and trimeric coiled-coils using effective amino acid indices. (United States)

    Li, Chen; Wang, Xiao-Feng; Chen, Zhen; Zhang, Ziding; Song, Jiangning


    The coiled-coil, which consists of two or more α-helices winding around each other, is a ubiquitous and the most frequently observed protein-protein interaction motif in nature. The coiled-coil is known for its straightforward heptad repeat pattern and can be readily recognized based on protein primary sequences, exhibiting a variety of oligomer states and topologies. Due to the stable interaction formed between their α-helices, coiled-coils have been under close scrutiny to design novel protein structures for potential applications in the fields of material science, synthetic biology and medicine. However, their broader application requires an in-depth and systematic analysis of the sequence-to-structure relationship of coiled-coil folding and oligomeric formation. In this article, we propose a new oligomerization state predictor, termed as RFCoil, which exploits the most useful and non-redundant amino acid indices combined with the machine learning algorithm - random forest (RF) - to predict the oligomeric states of coiled-coil regions. Benchmarking experiments show that RFCoil achieves an AUC (area under the ROC curve) of 0.849 on the 10-fold cross-validation test using the training dataset and 0.855 on the independent test using the validation dataset, respectively. Performance comparison results indicate that RFCoil outperforms the four existing predictors LOGICOIL, PrOCoil, SCORER 2.0 and Multicoil2. Furthermore, we extract a number of predominant rules from the trained RF model that underlie the oligomeric formation. We also present two case studies to illustrate the applicability of the extracted rules to the prediction of coiled-coil oligomerization state. The RFCoil web server, source codes and datasets are freely available for academic users at

  2. 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


    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

  3. Structural mapping of the coiled-coil domain of a bacterial condensin and comparative analyses across all domains of life suggest conserved features of SMC proteins. (United States)

    Waldman, Vincent M; Stanage, Tyler H; Mims, Alexandra; Norden, Ian S; Oakley, Martha G


    The structural maintenance of chromosomes (SMC) proteins form the cores of multisubunit complexes that are required for the segregation and global organization of chromosomes in all domains of life. These proteins share a common domain structure in which N- and C- terminal regions pack against one another to form a globular ATPase domain. This "head" domain is connected to a central, globular, "hinge" or dimerization domain by a long, antiparallel coiled coil. To date, most efforts for structural characterization of SMC proteins have focused on the globular domains. Recently, however, we developed a method to map interstrand interactions in the 50-nm coiled-coil domain of MukB, the divergent SMC protein found in γ-proteobacteria. Here, we apply that technique to map the structure of the Bacillus subtilis SMC (BsSMC) coiled-coil domain. We find that, in contrast to the relatively complicated coiled-coil domain of MukB, the BsSMC domain is nearly continuous, with only two detectable coiled-coil interruptions. Near the middle of the domain is a break in coiled-coil structure in which there are three more residues on the C-terminal strand than on the N-terminal strand. Close to the head domain, there is a second break with a significantly longer insertion on the same strand. These results provide an experience base that allows an informed interpretation of the output of coiled-coil prediction algorithms for this family of proteins. A comparison of such predictions suggests that these coiled-coil deviations are highly conserved across SMC types in a wide variety of organisms, including humans. © 2015 Wiley Periodicals, Inc.

  4. A periodic table of coiled-coil protein structures. (United States)

    Moutevelis, Efrosini; Woolfson, Derek N


    Coiled coils are protein structure domains with two or more alpha-helices packed together via interlacing of side chains known as knob-into-hole packing. We analysed and classified a large set of coiled-coil structures using a combination of automated and manual methods. This led to a systematic classification that we termed a "periodic table of coiled coils," which we have made available at In this table, coiled-coil assemblies are arranged in columns with increasing numbers of alpha-helices and in rows of increased complexity. The table provides a framework for understanding possibilities in and limits on coiled-coil structures and a basis for future prediction, engineering and design studies.

  5. Analysis of alpha-helical coiled coils with the program TWISTER reveals a structural mechanism for stutter compensation. (United States)

    Strelkov, Sergei V; Burkhard, Peter


    Alpha-helical coiled coils represent a widespread protein structure motif distinguished by a seven-residue periodicity of apolar residues in the primary sequence. A characteristic "knobs-into-holes" packing of these residues into a hydrophobic core results in a superhelical, usually left-handed, rope of two or more alpha-helices. Such a geometry can be parameterized. For this purpose, a new computer program, TWISTER, was developed. With the three-dimensional coordinates as input, TWISTER uses an original algorithm to determine the local coiled-coil parameters as a function of residue number. In addition, heptad positions are assigned based on structural criteria. It is known that frequently encountered discontinuities in the heptad repeat, such as stutters and skips, can be tolerated within a continuous coiled coil but result in a local distortion of its geometry. This was explored in detail with the help of TWISTER for several two- and three-stranded coiled coils. Depending on the particular protein, stutters were found to be compensated locally by an unwinding of the superhelix, alpha-helical unwinding, or both. In the first case, there is often a local switch from a left-handed to a right-handed superhelix. In general, the geometrical distortion is confined to about two alpha-helical turns at either side of the stutter. Furthermore, stutters result in a local increase of the coiled-coil radius. (c) 2002 Elsevier Science (USA).

  6. Type I macrophage scavenger receptor contains α-helical and collagen-like coiled coils (United States)

    Kodama, Tatsuhiko; Freeman, Mason; Rohrer, Lucia; Zabrecky, James; Matsudaira, Paul; Krieger, Monty


    The macrophage scavenger receptor is a trimeric membrane glycoprotein with unusual ligand-binding properties which has been implicated in the development of atherosclerosis. The trimeric structure of the bovine type I scavenger receptor, deduced by complementary DNA cloning, contains three extracellular C-terminal cysteine-rich domains connected to the transmembrane domain by a long fibrous stalk. This stalk structure, composed of an a-helical coiled coil and a collagen-like triple helix, has not previously been observed in an integral membrane protein.

  7. Coiled-coil domain-dependent homodimerization of intracellular barley immune receptors defines a minimal functional module for triggering cell death

    NARCIS (Netherlands)

    Maekawa, T.; Cheng, W.; Spiridon, L.N.; Töller, A.; Lukasik, E.; Saijo, Y.; Liu, P.; Shen, Q.H.; Micluta, M.A.; Somssich, I.E.; Takken, F.L.W.; Petrescu, A.J.; Chai, J.; Schulze-Lefert, P.


    Plants and animals have evolved structurally related innate immune sensors, designated NLRs, to detect intracellular nonself molecules. NLRs are modular, consisting of N-terminal coiled-coil (CC) or TOLL/interleukin-1 receptor (TIR) domains, a central nucleotide-binding (NB) domain, and C-terminal

  8. Coiled-coil forming peptides for the induction of silver nanoparticles

    Energy Technology Data Exchange (ETDEWEB)

    Božič Abram, Sabina [Department of Synthetic Biology and Immunology, National Institute of Chemistry, Hajdrihova 19, 1000 Ljubljana (Slovenia); Graduate School of Biomedicine, University of Ljubljana, Ljubljana 1000 (Slovenia); Aupič, Jana [Department of Synthetic Biology and Immunology, National Institute of Chemistry, Hajdrihova 19, 1000 Ljubljana (Slovenia); Doctoral Programme in Chemical Sciences, Faculty of Chemistry and Chemical Technology, University of Ljubljana, Ljubljana 1000 (Slovenia); Dražić, Goran [Laboratory for Materials Chemistry, National Institute of Chemistry, Hajdrihova 19, 1000 Ljubljana (Slovenia); Gradišar, Helena [Department of Synthetic Biology and Immunology, National Institute of Chemistry, Hajdrihova 19, 1000 Ljubljana (Slovenia); EN-FIST, Centre of Excellence, Trg Osvobodilne fronte 13, Ljubljana 1000 (Slovenia); Jerala, Roman, E-mail: [Department of Synthetic Biology and Immunology, National Institute of Chemistry, Hajdrihova 19, 1000 Ljubljana (Slovenia); EN-FIST, Centre of Excellence, Trg Osvobodilne fronte 13, Ljubljana 1000 (Slovenia)


    Biopolymers with defined sequence patterns offer an attractive alternative for the formation of silver nanoparticle (AgNP). A set of coiled-coil dimer forming peptides was tested for their AgNP formation ability. Seventeen of those peptides mediated the formation of AgNPs in aqueous solution at neutral pH, while the formation of a coiled-coil dimer inhibited the nanoparticle generation. A QSAR regression model on the relationship between sequence and function suggests that in this peptide type the patterns KXQQ and KXEE are favorable, whereas Ala residues appear to have an inhibitory effect. UV–VIS spectra of the obtained nanoparticles gave a peak at around 420 nm, typical for AgNPs in the size range around 40 nm, which was confirmed by dynamic light scattering and transmission electron microscopy. Peptide-induced AgNPs exhibited good antibacterial activity, even after a 15 min contact time, while they had low toxicity to human cells at the same concentrations. These results show that our designed peptides generate AgNPs with antibacterial activity at mild conditions and might be used for antibacterial coatings. - Highlights: • 17 of the 30 tested coiled-coil forming peptides induce AgNP formation. • Coiled-coil dimer formation suppresses AgNP generation of individual peptides. • Size of the peptide-induced silver nanoparticles is around 40 nm. • QSAR analysis points to the importance of KXQQ and KXEE motifs for AgNP generation. • Peptide-induced silver nanoparticles exhibit antibacterial activity.

  9. Structure and Misfolding of the Flexible Tripartite Coiled-Coil Domain of Glaucoma-Associated Myocilin

    Energy Technology Data Exchange (ETDEWEB)

    Hill, Shannon E.; Nguyen, Elaine; Donegan, Rebecca K.; Patterson-Orazem, Athéna C.; Hazel, Anthony; Gumbart, James C.; Lieberman, Raquel L.


    Glaucoma-associated myocilin is a member of the olfactomedins, a protein family involved in neuronal development and human diseases. Molecular studies of the myocilin N-terminal coiled coil demonstrate a unique tripartite architecture: a Y-shaped parallel dimer-of-dimers with distinct tetramer and dimer regions. The structure of the dimeric C-terminal 7-heptad repeats elucidates an unexpected repeat pattern involving inter-strand stabilization by oppositely charged residues. Molecular dynamics simulations reveal an alternate accessible conformation in which the terminal inter-strand disulfide limits the extent of unfolding and results in a kinked configuration. By inference, full-length myocilin is also branched, with two pairs of C-terminal olfactomedin domains. Selected variants within the N-terminal region alter the apparent quaternary structure of myocilin but do so without compromising stability or causing aggregation. In addition to increasing our structural knowledge of naturally occurring extracellular coiled coils and biomedically important olfactomedins, this work broadens the scope of protein misfolding in the pathogenesis of myocilin-associated glaucoma.

  10. Structure and Misfolding of the Flexible Tripartite Coiled-Coil Domain of Glaucoma-Associated Myocilin. (United States)

    Hill, Shannon E; Nguyen, Elaine; Donegan, Rebecca K; Patterson-Orazem, Athéna C; Hazel, Anthony; Gumbart, James C; Lieberman, Raquel L


    Glaucoma-associated myocilin is a member of the olfactomedins, a protein family involved in neuronal development and human diseases. Molecular studies of the myocilin N-terminal coiled coil demonstrate a unique tripartite architecture: a Y-shaped parallel dimer-of-dimers with distinct tetramer and dimer regions. The structure of the dimeric C-terminal 7-heptad repeats elucidates an unexpected repeat pattern involving inter-strand stabilization by oppositely charged residues. Molecular dynamics simulations reveal an alternate accessible conformation in which the terminal inter-strand disulfide limits the extent of unfolding and results in a kinked configuration. By inference, full-length myocilin is also branched, with two pairs of C-terminal olfactomedin domains. Selected variants within the N-terminal region alter the apparent quaternary structure of myocilin but do so without compromising stability or causing aggregation. In addition to increasing our structural knowledge of naturally occurring extracellular coiled coils and biomedically important olfactomedins, this work broadens the scope of protein misfolding in the pathogenesis of myocilin-associated glaucoma. Copyright © 2017 Elsevier Ltd. All rights reserved.

  11. Structural basis for cargo binding and autoinhibition of Bicaudal-D1 by a parallel coiled-coil with homotypic registry

    Energy Technology Data Exchange (ETDEWEB)

    Terawaki, Shin-ichi, E-mail: [Graduate School of Science and Technology, Gunma University, 1-5-1 Tenjin-cho, Kiryu, Gunma 376-8515 (Japan); SPring-8 Center, RIKEN, 1-1-1 Koto, Sayo-cho, Sayo-gun, Hyogo 679-5148 (Japan); Yoshikane, Asuka [Graduate School of Science and Technology, Gunma University, 1-5-1 Tenjin-cho, Kiryu, Gunma 376-8515 (Japan); SPring-8 Center, RIKEN, 1-1-1 Koto, Sayo-cho, Sayo-gun, Hyogo 679-5148 (Japan); Higuchi, Yoshiki [Department of Life Science, Graduate School of Life Science, University of Hyogo, 3-2-1 Koto, Kamigori-cho, Ako-gun, Hyogo 678-1297 (Japan); Department of Picobiology, Graduate School of Life Science, University of Hyogo, 3-2-1 Koto, Kamigori-cho, Ako-gun, Hyogo 678-1297 (Japan); SPring-8 Center, RIKEN, 1-1-1 Koto, Sayo-cho, Sayo-gun, Hyogo 679-5148 (Japan); Wakamatsu, Kaori [Graduate School of Science and Technology, Gunma University, 1-5-1 Tenjin-cho, Kiryu, Gunma 376-8515 (Japan); SPring-8 Center, RIKEN, 1-1-1 Koto, Sayo-cho, Sayo-gun, Hyogo 679-5148 (Japan)


    Bicaudal-D1 (BICD1) is an α-helical coiled-coil protein mediating the attachment of specific cargo to cytoplasmic dynein. It plays an essential role in minus end-directed intracellular transport along microtubules. The third C-terminal coiled-coil region of BICD1 (BICD1 CC3) has an important role in cargo sorting, including intracellular vesicles associating with the small GTPase Rab6 and the nuclear pore complex Ran binding protein 2 (RanBP2), and inhibiting the association with cytoplasmic dynein by binding to the first N-terminal coiled-coil region (CC1). The crystal structure of BICD1 CC3 revealed a parallel homodimeric coiled-coil with asymmetry and complementary knobs-into-holes interactions, differing from Drosophila BicD CC3. Furthermore, our binding study indicated that BICD1 CC3 possesses a binding surface for two distinct cargos, Rab6 and RanBP2, and that the CC1-binding site overlaps with the Rab6-binding site. These findings suggest a molecular basis for cargo recognition and autoinhibition of BICD proteins during dynein-dependent intracellular retrograde transport. - Highlights: • BICD1 CC3 is a parallel homodimeric coiled-coil with axial asymmetry. • The coiled-coil packing of BICD1 CC3 is adapted to the equivalent heptad position. • BICD1 CC3 has distinct binding sites for two classes of cargo, Rab6 and RanBP2. • The CC1-binding site of BICD1 CC3 overlaps with the Rab6-binding site.

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

    Directory of Open Access Journals (Sweden)

    Matthew R Harter


    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.

  13. Evidence of α-helical coiled coils and β-sheets in hornet silk. (United States)

    Kameda, Tsunenori; Nemoto, Takashi; Ogawa, Tetsuya; Tosaka, Masatoshi; Kurata, Hiroki; Schaper, Andreas K


    α-Helical coiled coil and β-sheet complexes are essential structural building elements of silk proteins produced by different species of the Hymenoptera. Beside X-ray scattering at wide and small angles we applied cryo-electron diffraction and microscopy to demonstrate the presence and the details of such structures in silk of the giant hornet Vespa mandarinia japonica. Our studies on the assembly of the fibrous silk proteins and their internal organization in relation to the primary chain structure suggest a 172 Å pitch supercoil consisting of four intertwined alanine-rich α-helical strands. The axial periodicity may adopt even multiples of the pitch value. Coiled coil motifs form the largest portion of the hornet silk structure and are aligned nearly parallel to the cocoon fiber axis in the same way as the membrane-like parts of the cocoon are molecularly orientated in the spinning direction. Supercoils were found to be associated with β-crystals, predominantly localized in the l-serine-rich chain sequences terminating each of the four predominant silk proteins. Such β-sheet blocks are considered resulting from transformation of random coil molecular sequences due to the action of elongational forces during the spinning process. Copyright © 2013 Elsevier Inc. All rights reserved.

  14. Electrostatic determinants of stability in parallel 3-stranded coiled coils. (United States)

    Roy, Liton; Case, Martin A


    The optimal positioning of salt-bridging interactions in a parallel alpha-helical homotrimeric coiled coil has been explored in a metal ion-assembled polypeptide trimer of 60 residues; arginine-glutamate pairs are more stabilizing than the corresponding lysine-glutamate pairs, and optimal stabilization is obtained with positively charged arginine residues at the c positions of the alpha-helical heptad and negatively charged glutamate residues at the e positions.

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

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


    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

  16. 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

    Directory of Open Access Journals (Sweden)

    Chattopadhyay Santanu


    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.

  17. The structure of the GemC1 coiled coil and its interaction with the Geminin family of coiled-coil proteins

    Energy Technology Data Exchange (ETDEWEB)

    Caillat, Christophe; Fish, Alexander [The Netherlands Cancer Institute, 1066 CX Amsterdam (Netherlands); Pefani, Dafni-Eleftheria; Taraviras, Stavros; Lygerou, Zoi [University of Patras, 26505 Rio, Patras (Greece); Perrakis, Anastassis, E-mail: [The Netherlands Cancer Institute, 1066 CX Amsterdam (Netherlands)


    The GemC1 coiled-coil structure has subtle differences compared with its homologues Geminin and Idas. Co-expression experiments in cells and biophysical stability analysis of the Geminin-family coiled coils suggest that the GemC1 coiled coil alone is unstable. GemC1, together with Idas and Geminin, an important regulator of DNA-replication licensing and differentiation decisions, constitute a superfamily sharing a homologous central coiled-coil domain. To better understand this family of proteins, the crystal structure of a GemC1 coiled-coil domain variant engineered for better solubility was determined to 2.2 Å resolution. GemC1 shows a less typical coiled coil compared with the Geminin homodimer and the Geminin–Idas heterodimer structures. It is also shown that both in vitro and in cells GemC1 interacts with Geminin through its coiled-coil domain, forming a heterodimer that is more stable that the GemC1 homodimer. Comparative analysis of the thermal stability of all of the possible superfamily complexes, using circular dichroism to follow the unfolding of the entire helix of the coiled coil, or intrinsic tryptophan fluorescence of a unique conserved N-terminal tryptophan, shows that the unfolding of the coiled coil is likely to take place from the C-terminus towards the N-terminus. It is also shown that homodimers show a single-state unfolding, while heterodimers show a two-state unfolding, suggesting that the dimer first falls apart and the helices then unfold according to the stability of each protein. The findings argue that Geminin-family members form homodimers and heterodimers between them, and this ability is likely to be important for modulating their function in cycling and differentiating cells.

  18. Platinum (IV) coiled coil nanotubes selectively kill human glioblastoma cells. (United States)

    Thanasupawat, Thatchawan; Bergen, Hugo; Hombach-Klonisch, Sabine; Krcek, Jerry; Ghavami, Saeid; Del Bigio, Marc R; Krawitz, Sherry; Stelmack, Gerald; Halayko, Andrew; McDougall, Matthew; Meier, Markus; Stetefeld, Jörg; Klonisch, Thomas


    Malignant glioma are often fatal and pose a significant therapeutic challenge. Here we have employed α-helical right handed coiled coils (RHCC) which self-assemble into tetrameric nanotubes that stably associate with platinum (Pt) (IV) compound. This Pt(IV)-RHCC complex showed superior in vitro and in vivo toxicity in human malignant glioma cells at up to 5 fold lower platinum concentrations when compared to free Pt(IV). Pt(IV)-RHCC nanotubes activated multiple cell death pathways in GB cells without affecting astrocytes in vitro or causing damage to normal mouse brain. This Pt(IV)-RHCC nanotubes may serve as a promising new therapeutic tool for low dose Pt(IV) prodrug application for highly efficient and selective treatment of human brain tumors. The prognosis of malignant glioma remains poor despite medical advances. Platinum, one of the chemotherapeutic agents used, has significant systemic side effects. In this article, the authors employed α-helical right handed coiled coil (RHCC) protein nanotubes as a carrier for cisplatin. It was shown that the new compound achieved higher tumor kill rate but lower toxicity to normal cells and thus may hold promise to be a highly efficient treatment for the future. Copyright © 2015 Elsevier Inc. All rights reserved.

  19. Localization and trafficking of an isoform of the AtPRA1 family to the Golgi apparatus depend on both N- and C-terminal sequence motifs. (United States)

    Jung, Chan Jin; Lee, Myoung Hui; Min, Myung Ki; Hwang, Inhwan


    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.

  20. ELKS controls the pool of readily releasable vesicles at excitatory synapses through its N-terminal coiled-coil domains. (United States)

    Held, Richard G; Liu, Changliang; Kaeser, Pascal S


    In a presynaptic nerve terminal, synaptic strength is determined by the pool of readily releasable vesicles (RRP) and the probability of release (P) of each RRP vesicle. These parameters are controlled at the active zone and vary across synapses, but how such synapse specific control is achieved is not understood. ELKS proteins are enriched at vertebrate active zones and enhance P at inhibitory hippocampal synapses, but ELKS functions at excitatory synapses are not known. Studying conditional knockout mice for ELKS, we find that ELKS enhances the RRP at excitatory synapses without affecting P. Surprisingly, ELKS C-terminal sequences, which interact with RIM, are dispensable for RRP enhancement. Instead, the N-terminal ELKS coiled-coil domains that bind to Liprin-α and Bassoon are necessary to control RRP. Thus, ELKS removal has differential, synapse-specific effects on RRP and P, and our findings establish important roles for ELKS N-terminal domains in synaptic vesicle priming.

  1. Growth Factor Identity Is Encoded by Discrete Coiled-Coil Rotamers in the EGFR Juxtamembrane Region. (United States)

    Doerner, Amy; Scheck, Rebecca; Schepartz, Alanna


    Binding of transforming growth factor α (TGF-α) to the epidermal growth factor receptor (EGFR) extracellular domain is encoded through the formation of a unique antiparallel coiled coil within the juxtamembrane segment. This new coiled coil is an "inside-out" version of the coiled coil formed in the presence of epidermal growth factor (EGF). A third, intermediary coiled-coil interface is formed in the juxtamembrane region when EGFR is stimulated with betacellulin. The seven growth factors that activate EGFR in mammalian systems (EGF, TGF-α, epigen, epiregulin, betacellulin, heparin-binding EGF, and amphiregulin) fall into distinct categories in which the structure of the coiled coil induced within the juxtamembrane region correlates with cell state. The observation that coiled-coil state tracks with the downstream signaling profiles for each ligand provides evidence for growth factor functional selectivity by EGFR. Encoding growth factor identity in alternative coiled-coil rotamers provides a simple and elegant method for communicating chemical information across the plasma membrane. Copyright © 2015 Elsevier Ltd. All rights reserved.

  2. The basic amino acids in the coiled-coil domain of CIN85 regulate its interaction with c-Cbl and phosphatidic acid during epidermal growth factor receptor (EGFR) endocytosis. (United States)

    Zheng, Xiudan; Zhang, Jing; Liao, Kan


    During EGFR internalization CIN85 bridges EGFR-Cbl complex, endocytic machinery and fusible membrane through the interactions of CIN85 with c-Cbl, endophilins and phosphatidic acid. These protein-protein and protein-lipid interactions are mediated or regulated by the positively charged C-terminal coiled-coil domain of CIN85. However, the details of CIN85-lipid interaction remain unknown. The present study suggested a possible electric interaction between the negative charge of phosphatidic acid and the positive charge of basic amino acids in coiled-coil domain. Mutations of the basic amino acids in the coiled-coil domain, especially K645, K646, R648 and R650, into neutral amino acid alanine completely blocked the interaction of CIN85 with c-Cbl or phosphatidic acid. However, they did not affect CIN85-endophilin interaction. In addition, CIN85 was found to associate with the internalized EGFR endosomes. It interacted with several ESCRT (Endosomal Sorting Complex Required for Transport) component proteins for ESCRT assembly on endosomal membrane. Mutations in the coiled-coil domain (deletion of the coiled-coil domain or point mutations of the basic amino acids) dissociated CIN85 from endosomes. These mutants bound the ESCRT components in cytoplasm to prevent them from assembly on endosomal membrane and inhibited EGFR sorting for degradation. As an adaptor protein, CIN85 interacts with variety of partners through several domains. The positive charges of basic amino acids in the coiled-coil domain are not only involved in the interaction with phosphatidic acid, but also regulate the interaction of CIN85 with c-Cbl. CIN85 also interacts with ESCRT components for protein sorting in endosomes. These CIN85-protein and CIN85-lipid interactions enable CIN85 to link EGFR-Cbl endocytic complex with fusible membrane during EGFR endocytosis and subsequently to facilitate ESCRT formation on endosomal membrane for EGFR sorting and degradation.

  3. 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.


    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.

  4. NMR Structure of the C-Terminal Transmembrane Domain of the HDL Receptor, SR-BI, and a Functionally Relevant Leucine Zipper Motif. (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


    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.

  5. Transport vesicle tethering at the trans Golgi network: coiled coil proteins in action

    Directory of Open Access Journals (Sweden)

    Pak-yan Patricia Cheung


    Full Text Available The Golgi complex is decorated with so-called Golgin proteins that share a common feature: a large proportion of their amino acid sequences are predicted to form coiled-coil structures. The possible presence of extensive coiled coils implies that these proteins are highly elongated molecules that can extend a significant distance from the Golgi surface. This property would help them to capture or trap inbound transport vesicles and to tether Golgi mini-stacks together. This review will summarize our current understanding of coiled coil tethers that are needed for the receipt of transport vesicles at the trans Golgi network. How do long tethering proteins actually catch vesicles? Golgi-associated, coiled coil tethers contain numerous binding sites for small GTPases, SNARE proteins, and vesicle coat proteins. How are these interactions coordinated and are any or all of them important for the tethering process? Progress towards understanding these questions and remaining, unresolved mysteries will be discussed.

  6. Multicoil2: predicting coiled coils and their oligomerization states from sequence in the twilight zone.

    Directory of Open Access Journals (Sweden)

    Jason Trigg

    Full Text Available The alpha-helical coiled coil can adopt a variety of topologies, among the most common of which are parallel and antiparallel dimers and trimers. We present Multicoil2, an algorithm that predicts both the location and oligomerization state (two versus three helices of coiled coils in protein sequences. Multicoil2 combines the pairwise correlations of the previous Multicoil method with the flexibility of Hidden Markov Models (HMMs in a Markov Random Field (MRF. The resulting algorithm integrates sequence features, including pairwise interactions, through multinomial logistic regression to devise an optimized scoring function for distinguishing dimer, trimer and non-coiled-coil oligomerization states; this scoring function is used to produce Markov Random Field potentials that incorporate pairwise correlations localized in sequence. Multicoil2 significantly improves both coiled-coil detection and dimer versus trimer state prediction over the original Multicoil algorithm retrained on a newly-constructed database of coiled-coil sequences. The new database, comprised of 2,105 sequences containing 124,088 residues, includes reliable structural annotations based on experimental data in the literature. Notably, the enhanced performance of Multicoil2 is evident when tested in stringent leave-family-out cross-validation on the new database, reflecting expected performance on challenging new prediction targets that have minimal sequence similarity to known coiled-coil families. The Multicoil2 program and training database are available for download from

  7. Reversible and irreversible coiled coils in the stalk domain of ncd motor protein. (United States)

    Makino, Tsukasa; Morii, Hisayuki; Shimizu, Takashi; Arisaka, Fumio; Kato, Yusuke; Nagata, Koji; Tanokura, Masaru


    Ncd is a microtubule minus end-directed motor protein from Drosophila, a member of the kinesin-14 family, and an essential protein in mitosis and meiosis. Full-length ncd exists as a dimer via the formation of an alpha-helical coiled coil in its central stalk domain (P192-R346), which is thought to be one of the key regions for its motility. In our previous studies, however, none of the various synthetic polypeptide fragments (up to 46 residues) from the stalk domain formed a coiled coil. Herein, we have investigated the structural properties of the full-length ncd stalk domain using recombinant polypeptides together with shorter segments. These new fragments did form coiled coils as verified by far-UV circular dichroism (CD) spectroscopy and analytical ultracentrifugation, suggesting that a certain length of polypeptide would be required for dimer formation. Moreover, deletion mapping revealed that the cooperativity among the neighboring subdomains in the stalk domain is required for formation of the coiled coil. Interestingly, the intact stalk domain segments showed three-state transition in thermal unfolding measurements with CD, indicating the presence of two regions: (i) a coiled-coil region (P227-R306) that exhibits reversible denaturation at a lower temperature (20-30 degrees C) and (ii) a more rigid coiled-coil region (T307-E334) that exhibits irreversible denaturation at a high temperature (ca. 60 degrees C). These results imply that the N-terminal region of the stalk domain might be able to adopt both a coiled-coil conformation and a dissociated one, which might be relevant to the functions of ncd.

  8. 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. (United States)

    Manfra, Ornella; Van Craenenbroeck, Kathleen; Skieterska, Kamila; Frimurer, Thomas; Schwartz, Thue W; Levy, Finn Olav; Andressen, Kjetil Wessel


    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.

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

    Directory of Open Access Journals (Sweden)

    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.

  10. pH sensitive coiled coils: a strategy for enhanced liposomal drug delivery (United States)

    Reja, Rahi M.; Khan, Mohsina; Singh, Sumeet K.; Misra, Rajkumar; Shiras, Anjali; Gopi, Hosahudya N.


    Stimuli responsive controlled release from liposome based vesicles is a promising strategy for the site specific delivery of drugs. Herein, we report the design of pH sensitive coiled coils and their incorporation into the liposome as triggers for the controlled release of encapsulated drugs. The designed coiled coil peptides with the incorporation of environment sensitive fluorescent amino acids were found to be stable at physiological pH and unstructured while changing the pH of the environment to either acidic or basic. This pH dependent conformational switch of the coiled-coil polypeptides was exploited as triggers for the enhanced release of the encapsulated drug molecules from liposomes. The SEM, DLS and TEM analysis revealed the uniform morphology of the peptide liposome hybrid vesicles. Further, the drug encapsulated liposome internalization experiments with cancer cells revealed the enhanced release and accumulation of drugs in the acidic lysosomal compartments in comparison with liposomes without coiled coils.Stimuli responsive controlled release from liposome based vesicles is a promising strategy for the site specific delivery of drugs. Herein, we report the design of pH sensitive coiled coils and their incorporation into the liposome as triggers for the controlled release of encapsulated drugs. The designed coiled coil peptides with the incorporation of environment sensitive fluorescent amino acids were found to be stable at physiological pH and unstructured while changing the pH of the environment to either acidic or basic. This pH dependent conformational switch of the coiled-coil polypeptides was exploited as triggers for the enhanced release of the encapsulated drug molecules from liposomes. The SEM, DLS and TEM analysis revealed the uniform morphology of the peptide liposome hybrid vesicles. Further, the drug encapsulated liposome internalization experiments with cancer cells revealed the enhanced release and accumulation of drugs in the acidic

  11. The Structures of Coiled-Coil Domains from Type III Secretion System Translocators Reveal Homology to Pore-Forming Toxins

    Energy Technology Data Exchange (ETDEWEB)

    Barta, Michael L.; Dickenson, Nicholas E.; Patil, Mrinalini; Keightley, Andrew; Wyckoff, Gerald J.; Picking, William D.; Picking, Wendy L.; Geisbrecht, Brian V. (UMKC); (OKLU)


    Many pathogenic Gram-negative bacteria utilize type III secretion systems (T3SSs) to alter the normal functions of target cells. Shigella flexneri uses its T3SS to invade human intestinal cells to cause bacillary dysentery (shigellosis) that is responsible for over one million deaths per year. The Shigella type III secretion apparatus is composed of a basal body spanning both bacterial membranes and an exposed oligomeric needle. Host altering effectors are secreted through this energized unidirectional conduit to promote bacterial invasion. The active needle tip complex of S. flexneri is composed of a tip protein, IpaD, and two pore-forming translocators, IpaB and IpaC. While the atomic structure of IpaD has been elucidated and studied, structural data on the hydrophobic translocators from the T3SS family remain elusive. We present here the crystal structures of a protease-stable fragment identified within the N-terminal regions of IpaB from S. flexneri and SipB from Salmonella enterica serovar Typhimurium determined at 2.1 {angstrom} and 2.8 {angstrom} limiting resolution, respectively. These newly identified domains are composed of extended-length (114 {angstrom} in IpaB and 71 {angstrom} in SipB) coiled-coil motifs that display a high degree of structural homology to one another despite the fact that they share only 21% sequence identity. Further structural comparisons also reveal substantial similarity to the coiled-coil regions of pore-forming proteins from other Gram-negative pathogens, notably, colicin Ia. This suggests that these mechanistically separate and functionally distinct membrane-targeting proteins may have diverged from a common ancestor during the course of pathogen-specific evolutionary events.

  12. Allosteric effects in coiled-coil proteins folding and lanthanide-ion binding. (United States)

    Samiappan, Manickasundaram; Alasibi, Samaa; Cohen-Luria, Rivka; Shanzer, Abraham; Ashkenasy, Gonen


    Peptide sequences modified with lanthanide-chelating groups at their N-termini, or at their lysine side chains, were synthesized, and new Ln(III) complexes were characterized. We show that partial folding of the conjugates to form trimer coiled coil structures induces coordination of lanthanides to the ligand, which in turn further stabilizes the 3D structure.

  13. Folding Topology of a Short Coiled-Coil Peptide Structure Templated by an Oligonucleotide Triplex

    DEFF Research Database (Denmark)

    Lou, Chenguang; Christensen, Niels Johan; Martos Maldonado, Manuel Cristo


    The rational design of a well-defined protein-like tertiary structure formed by small peptide building blocks is still a formidable challenge. By using peptide-oligonucleotide conjugates (POC) as building blocks, we present the self-assembly of miniature coiled-coil α-helical peptides guided...

  14. Antiparallel Four-Stranded Coiled Coil Specified by a 3-3-1 Hyrdrophobic Heptad Repeat

    Energy Technology Data Exchange (ETDEWEB)

    Deng,Y.; Liu, J.; Zheng, Q.; Eliezer, D.; Kallenbach, N.; Lu, M.


    Coiled-coil sequences in proteins commonly share a seven-amino acid repeat with nonpolar side chains at the first (a) and fourth (d) positions. We investigate here the role of a 3-3-1 hydrophobic repeat containing nonpolar amino acids at the a, d, and g positions in determining the structures of coiled coils using mutants of the GCN4 leucine zipper dimerization domain. When three charged residues at the g positions in the parental sequence are replaced by nonpolar alanine or valine side chains, stable four-helix structures result. The X-ray crystal structures of the tetramers reveal antiparallel, four-stranded coiled coils in which the a, d, and g side chains interlock in a combination of knobs-into-knobs and knobs-into-holes packing. Interfacial interactions in a coiled coil can therefore be prescribed by hydrophobic-polar patterns beyond the canonical 3-4 heptad repeat. The results suggest that the conserved, charged residues at the g positions in the GCN4 leucine zipper can impart a negative design element to disfavor thermodynamically more stable, antiparallel tetramers.

  15. Golgi coiled-coil proteins contain multiple binding sites for Rab family G proteins

    NARCIS (Netherlands)

    Sinka, Rita; Gillingham, Alison K.; Kondylis, Vangelis; Munro, Sean


    Vesicles and other carriers destined for the Golgi apparatus must be guided to the correct cisternae. Golgins, long coiled-coil proteins that localize to particular Golgi subdomains via their C termini, are candidate regulators of vesicle sorting. In this study, we report that the GRIP domain

  16. Insights into the coiled-coil organization of the Hendra virus phosphoprotein from combined biochemical and SAXS studies. (United States)

    Beltrandi, Matilde; Blocquel, David; Erales, Jenny; Barbier, Pascale; Cavalli, Andrea; Longhi, Sonia


    Nipah and Hendra viruses are recently emerged paramyxoviruses belonging to the Henipavirus genus. The Henipavirus phosphoprotein (P) consists of a large intrinsically disordered domain and a C-terminal domain (PCT) containing alternating disordered and ordered regions. Among these latter is the P multimerization domain (PMD). Using biochemical, analytical ultracentrifugation and small-angle X-ray scattering (SAXS) studies, we show that Hendra virus (HeV) PMD forms an elongated coiled-coil homotrimer in solution, in agreement with our previous findings on Nipah virus (NiV) PMD. However, the orientation of the N-terminal region differs from that observed in solution for NiV PMD, consistent with the ability of this region to adopt different conformations. SAXS studies provided evidence for a trimeric organization also in the case of PCT, thus extending and strengthening our findings on PMD. The present results are discussed in light of conflicting reports in the literature pointing to a tetrameric organization of paramyxoviral P proteins. Copyright © 2014 Elsevier Inc. All rights reserved.

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

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


    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.

  18. 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)


    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.

  19. Design of beta-domain swapping, alpha/beta-protein, environmentally sensitive coiled coil and peptide functionalized titania materials (United States)

    Nagarkar, Radhika P.


    The objective of this dissertation is to apply rational peptide design to fabricate nanomaterials via self-assembly. This has been demonstrated in structurally diverse systems with an aim of deciphering the underlying principles governing how sequence affects the peptide's ability to adopt a specific secondary structure and ultimate material properties that are realized from the association of these secondary structural elements. Several amyloidogenic proteins have been shown to self-assemble into fibrils using a mechanism known as domain swapping. Here, discreet units of secondary structure are exchanged among discreet proteins during self-assembly to form extended networks with precise three dimensional organization. The possibility of using these mechanisms to design peptides capable of controlled assembly and fibril formation leading to materials with targeted properties is explored. By altering the placement of a beta-turn sequence that varies the size and location of the exchanged strand, twisting, non-twisting and laminated fibrillar nanostructures are obtained. Hydrogels prepared from these strand swapping beta-hairpins have varied rheological properties due to differences in their fibrillar nanostructures. In a second distinct design, alpha/beta-proteins are used to prepare environmentally sensitive hydrogels. Here, multiple distinct motifs for structural integrity and dynamic response within a single self-assembling peptide allow the amyloid-like fibrils formed to controllably alter their nano-topography in response to an external stimulus such as temperature. The development of these self-assembling alpha/beta-protein motifs also necessitated the design of pH sensitive antiparallel coiled coils. Exploring the basic principles responsible for pH dependent conformational changes in coiled coils can lead to new insights in the control of protein structure and function. Lastly, this dissertation discusses the interface between biomolecules and inorganic

  20. d-Cysteine Ligands Control Metal Geometries within De Novo Designed Three-Stranded Coiled Coils

    DEFF Research Database (Denmark)

    Ruckthong, Leela; Peacock, Anna F.A.; Pascoe, Cherilyn E.


    Although metal ion binding to naturally occurring l-amino acid proteins is well documented, understanding the impact of the opposite chirality (d-)amino acids on the structure and stereochemistry of metals is in its infancy. We examine the effect of a d-configuration cysteine within a designed l......-amino acid three-stranded coiled coil in order to enforce a precise coordination number on a metal center. The d chirality does not alter the native fold, but the side-chain re-orientation modifies the sterics of the metal binding pocket. l-Cys side chains within the coiled-coil structure have previously...... been shown to rotate substantially from their preferred positions in the apo structure to create a binding site for a tetra-coordinate metal ion. However, here we show by X-ray crystallography that d-Cys side chains are preorganized within a suitable geometry to bind such a ligand. This is confirmed...

  1. Application of Coiled Coil Peptides in Liposomal Anticancer Drug Delivery Using a Zebrafish Xenograft Model. (United States)

    Yang, Jian; Shimada, Yasuhito; Olsthoorn, René C L; Snaar-Jagalska, B Ewa; Spaink, Herman P; Kros, Alexander


    The complementary coiled coil forming peptides E4 [(EIAALEK)4] and K4 [(KIAALKE)4] are known to trigger liposomal membrane fusion when tethered to lipid vesicles in the form of lipopeptides. In this study, we examined whether these coiled coil forming peptides can be used for drug delivery applications. First, we prepared E4 peptide modified liposomes containing the far-red fluorescent dye TO-PRO-3 iodide (E4-Lipo-TP3) and confirmed that E4-liposomes could deliver TP3 into HeLa cells expressing K4 peptide on the membrane (HeLa-K) under cell culture conditions in a selective manner. Next, we prepared doxorubicin-containing E4-liposomes (E4-Lipo-DOX) and confirmed that E4-liposomes could also deliver DOX into HeLa-K cells. Moreover, E4-Lipo-DOX showed enhanced cytotoxicity toward HeLa-K cells compared to free doxorubicin. To prove the suitability of E4/K4 coiled coil formation for in vivo drug delivery, we injected E4-Lipo-TP3 or E4-Lipo-DOX into zebrafish xenografts of HeLa-K. As a result, E4-liposomes delivered TP3 to the implanted HeLa-K cells, and E4-Lipo-DOX could suppress cancer proliferation in the xenograft when compared to nontargeted conditions (i.e., zebrafish xenograft with free DOX injection). These data demonstrate that coiled coil formation enables drug selectivity and efficacy in vivo. It is envisaged that these findings are a step forward toward biorthogonal targeting systems as a tool for clinical drug delivery.

  2. CCBuilder 2.0: Powerful and accessible coiled-coil modeling. (United States)

    Wood, Christopher W; Woolfson, Derek N


    The increased availability of user-friendly and accessible computational tools for biomolecular modeling would expand the reach and application of biomolecular engineering and design. For protein modeling, one key challenge is to reduce the complexities of 3D protein folds to sets of parametric equations that nonetheless capture the salient features of these structures accurately. At present, this is possible for a subset of proteins, namely, repeat proteins. The α-helical coiled coil provides one such example, which represents ≈ 3-5% of all known protein-encoding regions of DNA. Coiled coils are bundles of α helices that can be described by a small set of structural parameters. Here we describe how this parametric description can be implemented in an easy-to-use web application, called CCBuilder 2.0, for modeling and optimizing both α-helical coiled coils and polyproline-based collagen triple helices. This has many applications from providing models to aid molecular replacement for X-ray crystallography, in silico model building and engineering of natural and designed protein assemblies, and through to the creation of completely de novo "dark matter" protein structures. CCBuilder 2.0 is available as a web-based application, the code for which is open-source and can be downloaded freely. We have created CCBuilder 2.0, an easy to use web-based application that can model structures for a whole class of proteins, the α-helical coiled coil, which is estimated to account for 3-5% of all proteins in nature. CCBuilder 2.0 will be of use to a large number of protein scientists engaged in fundamental studies, such as protein structure determination, through to more-applied research including designing and engineering novel proteins that have potential applications in biotechnology. © 2017 The Authors Protein Science published by Wiley Periodicals, Inc. on behalf of The Protein Society.

  3. Historical review: another 50th anniversary--new periodicities in coiled coils. (United States)

    Gruber, Markus; Lupas, Andrei N


    In 1953, Francis Crick and Linus Pauling both proposed models of supercoiled alpha helices ('coiled coils') for the structure of keratin. These were the first attempts at modelling the tertiary structure of a protein. Crick emphasized the packing mode of the side-chains ('knobs-into-holes'), which required a periodicity of seven residues over two helical turns (7/2) and a supercoil in the opposite sense of the constituent helices. By contrast, Pauling envisaged a broader set of periodicities (4/1, 7/2, 18/5, 15/4, 11/3) and supercoils of both senses. Crick's model became canonical and the 'heptad repeat' essentially synonymous with coiled coils, but 50 years later new crystal structures and protein sequences show that the less common periodicities envisaged by Pauling also occur in coiled coils, adding a variant packing mode ('knobs-to-knobs') to the standard model. Pauling's laboratory notebooks suggest that he searched unsuccessfully for this packing mode in 1953.

  4. Coiled-Coil Proteins Facilitated the Functional Expansion of the Centrosome (United States)

    Kuhn, Michael; Hyman, Anthony A.; Beyer, Andreas


    Repurposing existing proteins for new cellular functions is recognized as a main mechanism of evolutionary innovation, but its role in organelle evolution is unclear. Here, we explore the mechanisms that led to the evolution of the centrosome, an ancestral eukaryotic organelle that expanded its functional repertoire through the course of evolution. We developed a refined sequence alignment technique that is more sensitive to coiled coil proteins, which are abundant in the centrosome. For proteins with high coiled-coil content, our algorithm identified 17% more reciprocal best hits than BLAST. Analyzing 108 eukaryotic genomes, we traced the evolutionary history of centrosome proteins. In order to assess how these proteins formed the centrosome and adopted new functions, we computationally emulated evolution by iteratively removing the most recently evolved proteins from the centrosomal protein interaction network. Coiled-coil proteins that first appeared in the animal–fungi ancestor act as scaffolds and recruit ancestral eukaryotic proteins such as kinases and phosphatases to the centrosome. This process created a signaling hub that is crucial for multicellular development. Our results demonstrate how ancient proteins can be co-opted to different cellular localizations, thereby becoming involved in novel functions. PMID:24901223

  5. Regulation of c-Fes Tyrosine Kinase and Biological Activities by N-Terminal Coiled-Coil Oligomerization Domains (United States)

    Cheng, Haiyun; Rogers, Jim A.; Dunham, Nancy A.; Smithgall, Thomas E.


    The cytoplasmic protein-tyrosine kinase Fes has been implicated in cytokine signal transduction, hematopoiesis, and embryonic development. Previous work from our laboratory has shown that active Fes exists as a large oligomeric complex in vitro. However, when Fes is expressed in mammalian cells, its kinase activity is tightly repressed. The Fes unique N-terminal sequence has two regions with strong homology to coiled-coil-forming domains often found in oligomeric proteins. Here we show that disruption or deletion of the first coiled-coil domain upregulates Fes tyrosine kinase and transforming activities in Rat-2 fibroblasts and enhances Fes differentiation-inducing activity in myeloid leukemia cells. Conversely, expression of a Fes truncation mutant consisting only of the unique N-terminal domain interfered with Rat-2 fibroblast transformation by an activated Fes mutant, suggesting that oligomerization is essential for Fes activation in vivo. Coexpression with the Fes N-terminal region did not affect the transforming activity of v-Src in Rat-2 cells, arguing against a nonspecific suppressive effect. Taken together, these findings suggest a model in which Fes activation may involve coiled-coil-mediated interconversion of monomeric and oligomeric forms of the kinase. Mutation of the first coiled-coil domain may activate Fes by disturbing intramolecular coiled-coil interaction, allowing for oligomerization via the second coiled-coil domain. Deletion of the second coiled-coil domain blocks fibroblast transformation by an activated form of c-Fes, consistent with this model. These results provide the first evidence for regulation of a nonreceptor protein-tyrosine kinase by coiled-coil domains. PMID:10567558

  6. Coiled-coil interactions modulate multimerization, mitochondrial binding and kinase activity of myotonic dystrophy protein kinase splice isoforms.

    NARCIS (Netherlands)

    Herpen, R.E.M.A. van; Tjeertes, J.V.; Mulders, S.A.M.; Oude Ophuis, R.J.A.; Wieringa, B.; Wansink, D.G.


    The myotonic dystrophy protein kinase polypeptide repertoire in mice and humans consists of six different splice isoforms that vary in the nature of their C-terminal tails and in the presence or absence of an internal Val-Ser-Gly-Gly-Gly motif. Here, we demonstrate that myotonic dystrophy protein

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

    Directory of Open Access Journals (Sweden)

    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.

  8. The heterotrimeric laminin coiled-coil domain exerts anti-adhesive effects and induces a pro-invasive phenotype.

    Directory of Open Access Journals (Sweden)

    Patricia Santos-Valle

    Full Text Available Laminins are large heterotrimeric cross-shaped extracellular matrix glycoproteins with terminal globular domains and a coiled-coil region through which the three chains are assembled and covalently linked. Laminins are key components of basement membranes, and they serve as attachment sites for cell adhesion, migration and proliferation. In this work, we produced a recombinant fragment comprising the entire laminin coiled-coil of the α1-, β1-, and γ1-chains that assemble into a stable heterotrimeric coiled-coil structure independently of the rest of the molecule. This domain was biologically active and not only failed to serve as a substrate for cell attachment, spreading and focal adhesion formation but also inhibited cell adhesion to laminin when added to cells in a soluble form at the time of seeding. Furthermore, gene array expression profiling in cells cultured in the presence of the laminin coiled-coil domain revealed up-regulation of genes involved in cell motility and invasion. These findings were confirmed by real-time quantitative PCR and zymography assays. In conclusion, this study shows for the first time that the laminin coiled-coil domain displays anti-adhesive functions and has potential implications for cell migration during matrix remodeling.

  9. Inherited germline TP53 mutation encodes a protein with an aberrant C-terminal motif in a case of pediatric adrenocortical tumor. (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


    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.

  10. An autoinhibited coiled-coil design strategy for split-protein protease sensors. (United States)

    Shekhawat, Sujan S; Porter, Jason R; Sriprasad, Akshay; Ghosh, Indraneel


    Proteases are widely studied as they are integral players in cell-cycle control and apoptosis. We report a new approach for the design of a family of genetically encoded turn-on protease biosensors. In our design, an autoinhibited coiled-coil switch is turned on upon proteolytic cleavage, which results in the complementation of split-protein reporters. Utilizing this new autoinhibition design paradigm, we present the rational construction and optimization of three generations of protease biosensors, with the final design providing a 1000-fold increase in bioluminescent signal upon addition of the TEV protease. We demonstrate the generality of the approach utilizing two different split-protein reporters, firefly luciferase and beta-lactamase, while also testing our design in the context of a therapeutically relevant protease, caspase-3. Finally, we present a dual protease sensor geometry that allows for the use of these turn-on sensors as potential AND logic gates. Thus, these studies potentially provide a new method for the design and implementation of genetically encoded turn-on protease sensors while also providing a general autoinhibited coiled-coil strategy for controlling the activity of fragmented proteins.

  11. C-terminal domains implicated in the functional surface expression of potassium channels (United States)

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


    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

  12. Antibody engineering using phage display with a coiled-coil heterodimeric Fv antibody fragment.

    Directory of Open Access Journals (Sweden)

    Xinwei Wang

    Full Text Available A Fab-like antibody binding unit, ccFv, in which a pair of heterodimeric coiled-coil domains was fused to V(H and V(L for Fv stabilization, was constructed for an anti-VEGF antibody. The anti-VEGF ccFv showed the same binding affinity as scFv but significantly improved stability and phage display level. Furthermore, phage display libraries in the ccFv format were constructed for humanization and affinity maturation of the anti-VEGF antibody. A panel of V(H frameworks and V(H-CDR3 variants, with a significant improvement in affinity and expressibility in both E. coli and yeast systems, was isolated from the ccFv phage libraries. These results demonstrate the potential application of the ccFv antibody format in antibody engineering.

  13. Self-assembly of designed coiled coil peptides studied by small-angle X-ray scattering and analytical ultracentrifugation

    DEFF Research Database (Denmark)

    Malik, Leila; Nygaard, Jesper; Christensen, Niels Johan


    , they are promising tools for the construction of nanomaterials. Small-angle X-ray scattering (SAXS) has emerged as a new biophysical technique for elucidation of protein topology. Here, we describe a systematic study of the self-assembly of a small ensemble of coiled coil sequences using SAXS and analytical...

  14. Synchrotron radiation circular dichroism spectroscopy-defined structure of the C-terminal domain of NaChBac and its role in channel assembly (United States)

    Powl, Andrew M.; O’Reilly, Andrias O.; Miles, Andrew J.; Wallace, B. A.


    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

  15. Rescue of vitrified-warmed bovine oocytes with rho-associated coiled-coil kinase inhibitor. (United States)

    Hwang, In-Sul; Hara, Hiromasa; Chung, Hak-Jae; Hirabayashi, Masumi; Hochi, Shinichi


    Cryotolerance of matured bovine oocytes is not fully practical even though a promising vitrification procedure with a ultrarapid cooling rate was applied. The present study was conducted to investigate whether recovery culture of vitrified-warmed bovine oocytes with an inhibitor (Y-27632) of Rho-associated coiled-coil kinase (ROCK) can improve the developmental potential after in vitro fertilization (IVF) and in vitro culture. Immediately after warming, almost all oocytes appeared to be morphological normal. Treatment of the postwarming oocytes with 10 μM Y-27632 for 2 h resulted in the significantly higher oocyte survival rate before IVF as well as higher cleavage rate and blastocyst formation rate. Quality analysis of the resultant blastocysts in terms of total cell number and apoptotic cell ratio also showed the positive effect of the Y-27632 treatment. Time-dependent change in mitochondrial activity of the vitrified-warmed oocytes was not influenced by ROCK inhibition during the period of recovery culture. However, the ability of ooplasm to support single-aster formation was improved by the ROCK inhibition. Thus, inhibition of ROCK activity in vitrified-warmed bovine oocytes during a short-term recovery culture can lead to higher developmental competence, probably due to decreased apoptosis and normalized function of the microtubule-organizing center.

  16. Transforming the Energy Landscape of a Coiled-Coil Peptide via Point Mutations. (United States)

    Röder, Konstantin; Wales, David J


    We analyze the effect of point mutations on the energy landscape of a coiled-coil peptide, GCN4-pLI, where the native state is a parallel tetrameric configuration formed from two identical dimers. Experimentally, a single mutation, E20S, supports both antiparallel and parallel structures. Here, we analyze the potential energy landscapes of the dimeric units for the parent sequence and four mutants, namely E20S, E20A, E20P, and E20G. Despite sharing characteristic funnels containing the parallel and antiparallel structures, the point mutations change some parts of the landscape quite dramatically, and we predict new intermediate structures and characterize the associated heat capacities. For the mutants we predict that kinked intermediate structures facilitate the transition between parallel and antiparallel morphologies, in contrast to the parent sequence. Furthermore, we predict a change from a multifunnel energy landscape in the E20S mutant to a landscape dominated by an underlying single funnel in the parent sequence, with accompanying heat capacity signatures. Our results imply that changes in the landscape due to mutations might provide useful tools for functional protein design.

  17. A coiled-coil protein is required for coordination of karyokinesis and cytokinesis in Toxoplasma gondii. (United States)

    Courjol, Flavie; Gissot, Mathieu


    Toxoplasma gondii is a unicellular eukaryotic pathogen that belongs to the Apicomplexa phylum, which encompasses some of the deadliest pathogens of medical and veterinary importance. The centrosome is key to the organization and coordination of the cell cycle and division of apicomplexan parasites. The T. gondii centrosome possesses a particular bipartite structure (outer and inner core). One of the main roles of the centrosome is to ensure proper coordination of karyokinesis. However, how these two events are coordinated is still unknown in T. gondii, for which the centrosome components are poorly described. To gain more insights into the biology and the composition of the T. gondii centrosome, we characterized a protein that resides at the interface of the outer and inner core centrosome. TgCep530 is a large coil-coiled protein with an essential role in the survival of the parasite. Depletion of this protein leads to the accumulation of parasites lacking nuclei and disruption of the normal cell cycle. Lack of TgCep530 results in a discoordination between the nuclear cycle and the budding cycle that yields fully formed parasites without nuclei. TgCep530 has a crucial role in the coordination of karyokinesis and cytokinesis. This article is protected by copyright. All rights reserved.

  18. Midbody Targeting of the ESCRT Machinery by a Noncanonical Coiled Coil in CEP55

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Hyung Ho; Elia, Natalie; Ghirlando, Rodolfo; Lippincott-Schwartz, Jennifer; Hurley, James H. (NIH)


    The ESCRT (endosomal sorting complex required for transport) machinery is required for the scission of membrane necks in processes including the budding of HIV-1 and cytokinesis. An essential step in cytokinesis is recruitment of the ESCRT-I complex and the ESCRT-associated protein ALIX to the midbody (the structure that tethers two daughter cells) by the protein CEP55. Biochemical experiments show that peptides from ALIX and the ESCRT-I subunit TSG101 compete for binding to the ESCRT and ALIX-binding region (EABR) of CEP55. We solved the crystal structure of EABR bound to an ALIX peptide at a resolution of 2.0 angstroms. The structure shows that EABR forms an aberrant dimeric parallel coiled coil. Bulky and charged residues at the interface of the two central heptad repeats create asymmetry and a single binding site for an ALIX or TSG101 peptide. Both ALIX and ESCRT-I are required for cytokinesis, which suggests that multiple CEP55 dimers are required for function.

  19. Coiled coil rich proteins (Ccrp influence molecular pathogenicity of Helicobacter pylori.

    Directory of Open Access Journals (Sweden)

    Sarah Schätzle

    Full Text Available Pathogenicity of the human pathogen Helicobacter pylori relies on its capacity to adapt to a hostile environment and to escape the host response. Although there have been great advances in our understanding of the bacterial cytoskeleton, major gaps remain in our knowledge of its contribution to virulence. In this study we have explored the influence of coiled coil rich proteins (Ccrp cytoskeletal elements on pathogenicity factors of H. pylori. Deletion of any of the ccrp resulted in a strongly decreased activity of the main pathogenicity factor urease. We further investigated their role using in vitro co-culture experiments with the human gastric adenocarcinoma cell line AGS modeling H. pylori - host cell interactions. Intriguingly, host cell showed only a weak "scattering/hummingbird" phenotype, in which host cells are transformed from a uniform polygonal shape into a severely elongated state characterized by the formation of needle-like projections, after co-incubation with any ccrp deletion mutant. Furthermore, co-incubation with the ccrp59 mutant resulted in reduced type IV secretion system associated activities, e.g. IL-8 production and CagA translocation/phosphorylation. Thus, in addition to their role in maintaining the helical cell shape of H. pylori Ccrp proteins influence many cellular processes and are thereby crucial for the virulence of this human pathogen.

  20. Rational design of helical nanotubes from self-assembly of coiled-coil lock washers. (United States)

    Xu, Chunfu; Liu, Rui; Mehta, Anil K; Guerrero-Ferreira, Ricardo C; Wright, Elizabeth R; Dunin-Horkawicz, Stanislaw; Morris, Kyle; Serpell, Louise C; Zuo, Xiaobing; Wall, Joseph S; Conticello, Vincent P


    Design of a structurally defined helical assembly is described that involves recoding of the amino acid sequence of peptide GCN4-pAA. In solution and the crystalline state, GCN4-pAA adopts a 7-helix bundle structure that resembles a supramolecular lock washer. Structurally informed mutagenesis of the sequence of GCN4-pAA afforded peptide 7HSAP1, which undergoes self-association into a nanotube via noncovalent interactions between complementary interfaces of the coiled-coil lock-washer structures. Biophysical measurements conducted in solution and the solid state over multiple length scales of structural hierarchy are consistent with self-assembly of nanotube structures derived from 7-helix bundle subunits. The dimensions of the supramolecular assemblies are similar to those observed in the crystal structure of GCN4-pAA. Fluorescence studies of the interaction of 7HSAP1 with the solvatochromic fluorophore PRODAN indicated that the nanotubes could encapsulate shape-appropriate small molecules with high binding affinity.

  1. Rice Cellulose SynthaseA8 Plant-Conserved Region Is a Coiled-Coil at the Catalytic Core Entrance

    Energy Technology Data Exchange (ETDEWEB)

    Rushton, Phillip S.; Olek, Anna T.; Makowski, Lee; Badger, John; Steussy, C. Nicklaus; Carpita, Nicholas C.; Stauffacher, Cynthia V. (NEU); (Purdue)


    The crystallographic structure of a rice (Oryza sativa) cellulose synthase, OsCesA8, plant-conserved region (P-CR), one of two unique domains in the catalytic domain of plant CesAs, was solved to 2.4 Å resolution. Two antiparallel α-helices form a coiled-coil domain linked by a large extended connector loop containing a conserved trio of aromatic residues. The P-CR structure was fit into a molecular envelope for the P-CR domain derived from small-angle X-ray scattering data. The P-CR structure and molecular envelope, combined with a homology-based chain trace of the CesA8 catalytic core, were modeled into a previously determined CesA8 small-angle X-ray scattering molecular envelope to produce a detailed topological model of the CesA8 catalytic domain. The predicted position for the P-CR domain from the molecular docking models places the P-CR connector loop into a hydrophobic pocket of the catalytic core, with the coiled-coil aligned near the entrance of the substrate UDP-glucose into the active site. In this configuration, the P-CR coiled-coil alone is unlikely to regulate substrate access to the active site, but it could interact with other domains of CesA, accessory proteins, or other CesA catalytic domains to control substrate delivery.

  2. Balance between Coiled-Coil Stability and Dynamics Regulates Activity of BvgS Sensor Kinase in Bordetella

    Directory of Open Access Journals (Sweden)

    E. Lesne


    Full Text Available The two-component system BvgAS controls the expression of the virulence regulon of Bordetella pertussis. BvgS is a prototype of bacterial sensor kinases with extracytoplasmic Venus flytrap perception domains. Following its transmembrane segment, BvgS harbors a cytoplasmic Per-Arnt-Sim (PAS domain and then a predicted 2-helix coiled coil that precede the dimerization-histidine-phosphotransfer domain of the kinase. BvgS homologs have a similar domain organization, or they harbor only a predicted coiled coil between the transmembrane and the dimerization-histidine-phosphotransfer domains. Here, we show that the 2-helix coiled coil of BvgS regulates the enzymatic activity in a mechanical manner. Its marginally stable hydrophobic interface enables a switch between a state of great rotational dynamics in the kinase mode and a more rigid conformation in the phosphatase mode in response to signal perception by the periplasmic domains. We further show that the activity of BvgS is controlled in the same manner if its PAS domain is replaced with the natural α-helical sequences of PAS-less homologs. Clamshell motions of the Venus flytrap domains trigger the shift of the coiled coil’s dynamics. Thus, we have uncovered a general mechanism of regulation for the BvgS family of Venus flytrap-containing two-component sensor kinases.

  3. The Rsv3 Locus Conferring Resistance to Soybean Mosaic Virus is Associated with a Cluster of Coiled-Coil Nucleotide-Binding Leucine-Rich Repeat Genes

    National Research Council Canada - National Science Library

    Suh, Su Jeoung; Bowman, Brian C; Jeong, Namhee; Yang, Kiwoung; Kastl, Christin; Tolin, Sue A; Maroof, M.A. Saghai; Jeong, Soon-Chun


    ...), has been characterized by examination of the soybean genome sequence. The 154 kbp interval encompassing contains a family of closely related coiled-coil nucleotide-binding leucine-rich repeat (CC-NB-LRR) genes...

  4. The Golgi-associated long coiled-coil protein NECC1 participates in the control of the regulated secretory pathway in PC12 cells

    National Research Council Canada - National Science Library

    Cruz-García, David; Díaz-Ruiz, Alberto; Rabanal-Ruiz, Yoana; Peinado, Juan R; Gracia-Navarro, Francisco; Castaño, Justo P; Montero-Hadjadje, Maité; Tonon, Marie-Christine; Vaudry, Hubert; Anouar, Youssef; Vázquez-Martínez, Rafael; Malagón, María M


    .... In the present study, we characterize the intracellular distribution as well as the biochemical and functional properties of a novel long coiled-coil protein present in neuroendocrine tissues, NECC1...

  5. Alanine zipper-like coiled-coil domains are necessary for homotypic dimerization of plant GAGA-factors in the nucleus and nucleolus.

    Directory of Open Access Journals (Sweden)

    Dierk Wanke

    Full Text Available GAGA-motif binding proteins control transcriptional activation or repression of homeotic genes. Interestingly, there are no sequence similarities between animal and plant proteins. Plant BBR/BPC-proteins can be classified into two distinct groups: Previous studies have elaborated on group I members only and so little is known about group II proteins. Here, we focused on the initial characterization of AtBPC6, a group II protein from Arabidopsis thaliana. Comparison of orthologous BBR/BPC sequences disclosed two conserved signatures besides the DNA binding domain. A first peptide signature is essential and sufficient to target AtBPC6-GFP to the nucleus and nucleolus. A second domain is predicted to form a zipper-like coiled-coil structure. This novel type of domain is similar to Leucine zippers, but contains invariant alanine residues with a heptad spacing of 7 amino acids. By yeast-2-hybrid and BiFC-assays we could show that this Alanine zipper domain is essential for homotypic dimerization of group II proteins in vivo. Interhelical salt bridges and charge-stabilized hydrogen bonds between acidic and basic residues of the two monomers are predicted to form an interaction domain, which does not follow the classical knobs-into-holes zipper model. FRET-FLIM analysis of GFP/RFP-hybrid fusion proteins validates the formation of parallel dimers in planta. Sequence comparison uncovered that this type of domain is not restricted to BBR/BPC proteins, but is found in all kingdoms.

  6. Novel Anti-Nicotine Vaccine Using a Trimeric Coiled-Coil Hapten Carrier.

    Directory of Open Access Journals (Sweden)

    Keith D Miller

    Full Text Available Tobacco addiction represents one of the largest public health problems in the world and is the leading cause of cancer and heart disease, resulting in millions of deaths a year. Vaccines for smoking cessation have shown considerable promise in preclinical models, although functional antibody responses induced in humans are only modestly effective in preventing nicotine entry into the brain. The challenge in generating serum antibodies with a large nicotine binding capacity is made difficult by the fact that this drug is non-immunogenic and must be conjugated as a hapten to a protein carrier. To circumvent the limitations of traditional carriers like keyhole limpet hemocyanin (KLH, we have synthesized a short trimeric coiled-coil peptide (TCC that creates a series of B and T cell epitopes with uniform stoichiometry and high density. Here we compared the relative activities of a TCC-nic vaccine and two control KLH-nic vaccines using Alum as an adjuvant or GLA-SE, which contains a synthetic TLR4 agonist formulated in a stable oil-in-water emulsion. The results showed that the TCC's high hapten density correlated with a better immune response in mice as measured by anti-nicotine Ab titer, affinity, and specificity, and was responsible for a reduction in anti-carrier immunogenicity. The Ab responses achieved with this synthetic vaccine resulted in a nicotine binding capacity in serum that could prevent >90% of a nicotine dose equivalent to three smoked cigarettes (0.05 mg/kg from reaching the brain.

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


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


    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. Structure-function evolution of the Transforming acidic coiled coil genes revealed by analysis of phylogenetically diverse organisms

    Directory of Open Access Journals (Sweden)

    DiMatteo Anthony


    Full Text Available Abstract Background Examination of ancient gene families can provide an insight into how the evolution of gene structure can relate to function. Functional homologs of the evolutionarily conserved transforming acidic coiled coil (TACC gene family are present in organisms from yeast to man. However, correlations between functional interactions and the evolution of these proteins have yet to be determined. Results We have performed an extensive database analysis to determine the genomic and cDNA sequences of the TACCs from phylogenetically diverse organisms. This analysis has determined the phylogenetic relationship of the TACC proteins to other coiled coil proteins, the resolution of the placement of the rabbit TACC4 as the orthologue of human TACC3, and RHAMM as a distinct family of coiled coil proteins. We have also extended the analysis of the TACCs to the interaction databases of C. elegans and D. melanogaster to identify potentially novel TACC interactions. The validity of this modeling was confirmed independently by the demonstration of direct binding of human TACC2 to the nuclear hormone receptor RXRβ. Conclusion The data so far suggest that the ancestral TACC protein played a role in centrosomal/mitotic spindle dynamics. TACC proteins were then recruited to complexes involved in protein translation, RNA processing and transcription by interactions with specific bridging proteins. However, during evolution, the TACC proteins have now acquired the ability to directly interact with components of these complexes (such as the LSm proteins, nuclear hormone receptors, GAS41, and transcription factors. This suggests that the function of the TACC proteins may have evolved from performing assembly or coordination functions in the centrosome to include a more intimate role in the functional evolution of chromatin remodeling, transcriptional and posttranscriptional complexes in the cell.

  9. Oncogenic TPM3-ALK activation requires dimerization through the coiled-coil structure of TPM3

    Energy Technology Data Exchange (ETDEWEB)

    Amano, Yosuke; Ishikawa, Rie; Sakatani, Toshio [Department of Respiratory Medicine, The University of Tokyo Hospital, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655 (Japan); Ichinose, Junji [Department of Cardiothoracic Surgery, The University of Tokyo Hospital, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655 (Japan); Sunohara, Mitsuhiro; Watanabe, Kousuke; Kage, Hidenori [Department of Respiratory Medicine, The University of Tokyo Hospital, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655 (Japan); Nakajima, Jun [Department of Cardiothoracic Surgery, The University of Tokyo Hospital, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655 (Japan); Nagase, Takahide; Ohishi, Nobuya [Department of Respiratory Medicine, The University of Tokyo Hospital, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655 (Japan); Takai, Daiya, E-mail: [Department of Respiratory Medicine, The University of Tokyo Hospital, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655 (Japan); Department of Clinical Laboratory, The University of Tokyo Hospital, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655 (Japan)


    Inflammatory myofibroblastic tumor (IMT) is a mesenchymal tumor that can arise from anywhere in the body. Anaplastic lymphoma kinase (ALK) gene rearrangements, most often resulting in the tropomyosin 3 (TPM3)-ALK fusion gene, are the main causes of IMT. However, the mechanism of malignant transformation in IMT has yet to be elucidated. The purpose of this study was to clarify the role of the TPM3 region in the transformation of IMT via TPM3-ALK. Lentivirus vectors containing a TPM3-ALK fusion gene lacking various lengths of TPM3 were constructed and expressed in HEK293T and NIH3T3 cell lines. Focus formation assay revealed loss of contact inhibition in NIH3T3 cells transfected with full-length TPM3-ALK, but not with ALK alone. Blue-native polyacrylamide gel electrophoresis (BN-PAGE) revealed that TPM3-ALK dimerization increased in proportion to the length of TPM3. Western blot showed phosphorylation of ALK, ERK1/2, and STAT3 in HEK293T cells transfected with TPM3-ALK. Thus, the coiled-coil structure of TPM3 contributes to the transforming ability of the TPM3-ALK fusion protein, and longer TPM3 region leads to higher dimer formation. - Highlights: • TPM3-ALK fusion protein dimerizes through the coiled-coil structure of TPM3. • Longer coiled-coil structure of TPM3 leads to higher TPM3-ALK dimer formation. • Presence of TPM3-ALK dimer leads to ALK, STAT3, and ERK1/2 phosphorylation. • Presence of TPM3-ALK leads to loss of contact inhibition. • BN-PAGE is a simple technique for visualizing oncogenic dimerization.

  10. Crystal structure of tetranectin, a trimeric plasminogen-binding protein with an alpha-helical coiled coil

    DEFF Research Database (Denmark)

    Nielsen, B B; Kastrup, J S; Rasmussen, H


    Tetranectin is a plasminogen kringle 4-binding protein. The crystal structure has been determined at 2.8 A resolution using molecular replacement. Human tetranectin is a homotrimer forming a triple alpha-helical coiled coil. Each monomer consists of a carbohydrate recognition domain (CRD) connected...... to a long alpha-helix. Tetranectin has been classified in a distinct group of the C-type lectin superfamily but has structural similarity to the proteins in the group of collectins. Tetranectin has three intramolecular disulfide bridges. Two of these are conserved in the C-type lectin superfamily, whereas...

  11. Caracterización celular y molecular de la proteina neuroendocrine long coiled-coil 2 (NECC2)


    Díaz-Ruiz, Alberto


    Como se mencionó en la introducción del presente trabajo, estudios previos en nuestro grupo de investigación permitieron la identificación de una nueva proteína long coiled-coil específica de vertebrados a partir de un análisis genómico diferencial sobre dos subtipos de células endocrinas, las células melanotropas, que muestran fenotipos opuestos de hipo e hipersecreción hormonal (Gonzalez de Aguilar et al., 1997; Peinado et al., 2002; Cruz-Garcia et al., 2007). Esta proteína fue denominada N...

  12. Design considerations in coiled-coil fusion constructs for the structural determination of a problematic region of the human cardiac myosin rod

    Energy Technology Data Exchange (ETDEWEB)

    Andreas, Michael P.; Ajay, Gautam; Gellings, Jaclyn A.; Rayment, Ivan


    X-ray structural determination of segments of the myosin rod has proved difficult because of the strong salt-dependent aggregation properties and repeating pattern of charges on the surface of the coiled-coil that lead to the formation of paracrystals. This problem has been resolved in part through the use of globular assembly domains that improve protein folding and prevent aggregation. The primary consideration now in designing coiled-coil fusion constructs for myosin is deciding where to truncate the coiled-coil and which amino acid residues to include from the folding domain. This is especially important for myosin that contains numerous regions of low predicted coiled-coil propensity. Here we describe the strategy adopted to determine the structure of the region that extends from Arg1677 – Leu1797 that included two areas that do not show a strong sequence signature of a conventional left-handed coiled coil or canonical heptad repeat. This demonstrates again that, with careful choice of fusion constructs, overlapping structures exhibit very similar conformations for the myosin rod fragments in the canonical regions. However, conformational variability is seen around Leu1706 which is a hot spot for cardiomyopathy mutations suggesting that this might be important for function.

  13. Self-assembly of coiled coil peptides into nanoparticles vs 2-d plates: effects of assembly pathway (United States)

    Kim, Kyunghee; Pochan, Darrin

    Molecular solution assembly, or self-assembly, is a process by which ordered nanostructures or patterns are formed by non-covalent interactions during assembly. Biomimicry, the use of bioinspired molecules or biologically relevant materials, is an important area of self-assembly research with peptides serving a critical role as molecular tools. The morphology of peptide assemblies can be controlled by adjusting solution conditions such as the concentration of peptides, the temperature, and pH. Herein, spherical nanostructures, which have potential for creating an encapsulation system, are formed by self-assembly when coiled coil peptides are combined in solution. These peptides are homotrimeric and heterodimeric coiled-coil bundles and the homotrimer is connected with each of heterodimer through their external surfaces via disulfide bonds. The resultant covalent constructs could co-assemble into complementary trimeric hubs, respectively. The two peptide constructs are directly mixed and assembled in solution in order to produce either spherical particles or 2-d plates depending on the solution conditions and kinetic pathway of assembly. In particular, structural changes of the self-assembled peptides are explored by control of the thermal history of the assembly solution.

  14. Two coiled-coil domains of Chlamydia trachomatis IncA affect membrane fusion events during infection.

    Directory of Open Access Journals (Sweden)

    Erik Ronzone

    Full Text Available Chlamydia trachomatis replicates in a parasitophorous membrane-bound compartment called an inclusion. The inclusions corrupt host vesicle trafficking networks to avoid the degradative endolysosomal pathway but promote fusion with each other in order to sustain higher bacterial loads in a process known as homotypic fusion. The Chlamydia protein IncA (Inclusion protein A appears to play central roles in both these processes as it participates to homotypic fusion and inhibits endocytic SNARE-mediated membrane fusion. How IncA selectively inhibits or activates membrane fusion remains poorly understood. In this study, we analyzed the spatial and molecular determinants of IncA's fusogenic and inhibitory functions. Using a cell-free membrane fusion assay, we found that inhibition of SNARE-mediated fusion requires IncA to be on the same membrane as the endocytic SNARE proteins. IncA displays two coiled-coil domains showing high homology with SNARE proteins. Domain swap and deletion experiments revealed that although both these domains are capable of independently inhibiting SNARE-mediated fusion, these two coiled-coil domains cooperate in mediating IncA multimerization and homotypic membrane interaction. Our results support the hypothesis that Chlamydia employs SNARE-like virulence factors that positively and negatively affect membrane fusion and promote infection.

  15. Crystal structure of cytomegalovirus IE1 protein reveals targeting of TRIM family member PML via coiled-coil interactions.

    Directory of Open Access Journals (Sweden)

    Myriam Scherer


    Full Text Available PML nuclear bodies (PML-NBs are enigmatic structures of the cell nucleus that act as key mediators of intrinsic immunity against viral pathogens. PML itself is a member of the E3-ligase TRIM family of proteins that regulates a variety of innate immune signaling pathways. Consequently, viruses have evolved effector proteins to modify PML-NBs; however, little is known concerning structure-function relationships of viral antagonists. The herpesvirus human cytomegalovirus (HCMV expresses the abundant immediate-early protein IE1 that colocalizes with PML-NBs and induces their dispersal, which correlates with the antagonization of NB-mediated intrinsic immunity. Here, we delineate the molecular basis for this antagonization by presenting the first crystal structure for the evolutionary conserved primate cytomegalovirus IE1 proteins. We show that IE1 consists of a globular core (IE1CORE flanked by intrinsically disordered regions. The 2.3 Å crystal structure of IE1CORE displays an all α-helical, femur-shaped fold, which lacks overall fold similarity with known protein structures, but shares secondary structure features recently observed in the coiled-coil domain of TRIM proteins. Yeast two-hybrid and coimmunoprecipitation experiments demonstrate that IE1CORE binds efficiently to the TRIM family member PML, and is able to induce PML deSUMOylation. Intriguingly, this results in the release of NB-associated proteins into the nucleoplasm, but not of PML itself. Importantly, we show that PML deSUMOylation by IE1CORE is sufficient to antagonize PML-NB-instituted intrinsic immunity. Moreover, co-immunoprecipitation experiments demonstrate that IE1CORE binds via the coiled-coil domain to PML and also interacts with TRIM5α We propose that IE1CORE sequesters PML and possibly other TRIM family members via structural mimicry using an extended binding surface formed by the coiled-coil region. This mode of interaction might render the antagonizing activity less

  16. Optimizing the Photocontrol of bZIP Coiled Coils with Azobenzene Crosslinkers: Role of the Crosslinking Site. (United States)

    Ali, Ahmed M; Forbes, Matthew W; Woolley, G Andrew


    DNA binding by bZIP-type coiled-coil proteins can be inhibited by dominant negative versions of the proteins in which the N-terminal basic region is replaced by an acidic extension. Photocontrol of bZIP function can be achieved by introducing intramolecular azobenzene-based crosslinkers into dominant negatives. We show that the largest degree of photocontrol is achieved when the crosslinker is introduced into the zipper region of the dominant negative between Cys residues placed at f sites in the heptad segment showing the highest intrinsic helical propensity. The overall affinity of the dominant negative can then be tuned by varying the length of the acidic extension. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. The coiled-coil protein VIG1 is essential for tethering vacuoles to mitochondria during vacuole inheritance of Cyanidioschyzon merolae. (United States)

    Fujiwara, Takayuki; Kuroiwa, Haruko; Yagisawa, Fumi; Ohnuma, Mio; Yoshida, Yamato; Yoshida, Masaki; Nishida, Keiji; Misumi, Osami; Watanabe, Satoru; Tanaka, Kan; Kuroiwa, Tsuneyoshi


    Vacuoles/lysosomes function in endocytosis and in storage and digestion of metabolites. These organelles are inherited by the daughter cells in eukaryotes. However, the mechanisms of this inheritance are poorly understood because the cells contain multiple vacuoles that behave randomly. The primitive red alga Cyanidioschyzon merolae has a minimum set of organelles. Here, we show that C. merolae contains about four vacuoles that are distributed equally between the daughter cells by binding to dividing mitochondria. Binding is mediated by VIG1, a 30-kD coiled-coil protein identified by microarray analyses and immunological assays. VIG1 appears on the surface of free vacuoles in the cytosol and then tethers the vacuoles to the mitochondria. The vacuoles are released from the mitochondrion in the daughter cells following VIG1 digestion. Suppression of VIG1 by antisense RNA disrupted the migration of vacuoles. Thus, VIG1 is essential for tethering vacuoles to mitochondria during vacuole inheritance in C. merolae.

  18. A novel cryptic binding motif, LRSKSRSFQVSDEQY, in the C-terminal fragment of MMP-3/7-cleaved osteopontin as a novel ligand for α9β1 integrin is involved in the anti-type II collagen antibody-induced arthritis.

    Directory of Open Access Journals (Sweden)

    Shigeyuki Kon

    Full Text Available Osteopontin (OPN is a multifunctional protein that has been linked to various intractable inflammatory diseases. One way by which OPN induces inflammation is the production of various functional fragments by enzyme cleavage. It has been well appreciated that OPN is cleaved by thrombin, and/or matrix metalloproteinase-3 and -7 (MMP-3/7. Although the function of thrombin-cleaved OPN is well characterized, little is known about the function of MMP-3/7-cleaved OPN. In this study, we found a novel motif, LRSKSRSFQVSDEQY, in the C-terminal fragment of MMP-3/7-cleaved mouse OPN binds to α9β1 integrin. Importantly, this novel motif is involved in the development of anti-type II collagen antibody-induced arthritis (CAIA. This study provides the first in vitro and in vivo evidence that OPN cleavage by MMP-3/7 is an important regulatory mechanism for CAIA.

  19. 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. (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


    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.

  20. 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. (United States)

    Singh, Varsha; Lin, Rong; Yang, Jianbo; Cha, Boyoung; Sarker, Rafiquel; Tse, Chung Ming; Donowitz, Mark


    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.

  1. Biogenesis of the Secretory Granule: Chromogranin a Coiled-Coil Structure Results in Unusual Physical Properties And Suggests a Mechanism for Granule Core Condensation

    Energy Technology Data Exchange (ETDEWEB)

    Mosley, C.A.; Taupenot, L.; Biswas, N.; Taulane, J.P.; Olson, N.H.; Vaingankar, S.M.; Wen, G.; Schork, N.J.; Ziegler, M.G.; Mahata, S.K.; O' Connor, D.T.


    The secretory pro-hormone chromogranin A (CHGA) is densely packed into storage granules along with catecholamines, playing a catalytic role in granule biogenesis. 3-Dimensional structural data on CHGA are lacking. We found a superfamily structural homology for CHGA in the tropomyosin family of alpha-helical coiled-coils, even in mid-molecule regions where primary sequence identity is only modest. The assignment was confirmed by an independent algorithm, suggesting approximately 6-7 such domains spanning CHGA. We provide additional physiochemical evidence (chromatographic, spectral, microscopic) consistent with this unusual structure. Alpha-helical secondary structure (at up to approximately 45%) was confirmed by circular dichroism. CHGA molecular mass was estimated by MALDI-TOF mass spectrometry at approximately 50 kDa and by denaturing gel filtration at approximately 50-61 kDa, while its native Stokes radius was approximately 84.8 A, as compared to an expected approximately 30 A; the increase gave rise to an apparent native molecular weight of approximately 578 kDa, also consistent with the extended conformation of a coiled-coil. Small-angle X-ray scattering (SAXS) on CHGA in solution best fit an elongated cylindrical conformation in the monodisperse region with a radius of gyration of the rod cross-section (Rt) of approximately 52 A, compatible with a coiled-coil in the hydrated, aqueous state, or a multimeric coiled-coil. Electron microscopy with negative staining revealed an extended, filamentous CHGA structure with a diameter of approximately 94 +/- 4.5 A. Extended, coiled-coil conformation is likely to permit protein 'packing' in the secretory granule at approximately 50% higher density than a globular/spherical conformation. Natural allelic variation in the catestatin region was predicted to disrupt the coiled-coil. Chromaffin granule ultrastructure revealed a approximately 108 +/- 6.3 A periodicity of electron density, suggesting nucleation of a

  2. Magnetic Field Alignment of PS-P4VP: a Non-Liquid Crystalline Coil-Coil Block Copolymer (United States)

    Rokhlenko, Yekaterina; Zhang, Kai; Larson, Steven; Gopalan, Padma; O'Hern, Corey; Osuji, Chinedum


    Magnetic fields provide the ability to control alignment of self-assembled soft materials such as block copolymers. Most prior work in this area has relied on the presence of ordered assemblies of anisotropic liquid crystalline species to ensure sufficient magnetic anisotropy to drive alignment. Recent experiments with poly(styrene-b-4-vinylpyridine), a non-liquid crystalline BCP, however, show field-induced alignment of a lamellar microstructure during cooling across the order-disorder transition. Using in situ x-ray scattering, we examine the roles of field strength and cooling rate on the alignment response of this low MW coil-coil BCP. Alignment is first observed at field strengths as low as 1 Tesla and improves markedly with both increasing field strength and slower cooling. We present a geometric argument to illustrate the origin of a finite, non-trivial magnetic susceptibility anisotropy for highly stretched surface-tethered polymer chains and corroborate this using coarse-grained molecular dynamics simulations. We rationalize the magnetic field response of the system in terms of the mobility afforded by the absence of entanglements, the intrinsic anisotropy resulting from the stretched polymer chains and sterically constrained conjugated rings, and the large grain size in these low molecular weight materials.

  3. The use of a P. falciparum specific coiled-coil domain to construct a self-assembling protein nanoparticle vaccine to prevent malaria. (United States)

    Karch, Christopher P; Doll, Tais A P F; Paulillo, Sara M; Nebie, Issa; Lanar, David E; Corradin, Giampietro; Burkhard, Peter


    The parasitic disease malaria remains a major global public health concern and no truly effective vaccine exists. One approach to the development of a malaria vaccine is to target the asexual blood stage that results in clinical symptoms. Most attempts have failed. New antigens such as P27A and P27 have emerged as potential new vaccine candidates. Multiple studies have demonstrated that antigens are more immunogenic and are better correlated with protection when presented on particulate delivery systems. One such particulate delivery system is the self-assembling protein nanoparticle (SAPN) that relies on coiled-coil domains of proteins to form stable nanoparticles. In the past we have used de novo designed amino acid domains to drive the formation of the coiled-coil scaffolds which present the antigenic epitopes on the particle surface. Here we use naturally occurring domains found in the tex1 protein to form the coiled-coil scaffolding of the nanoparticle. Thus, by engineering P27A and a new extended form of the coiled-coil domain P27 onto the N and C terminus of the SAPN protein monomer we have developed a particulate delivery system that effectively displays both antigens on a single particle that uses malaria tex1 sequences to form the nanoparticle scaffold. These particles are immunogenic in a murine model and induce immune responses similar to the ones observed in seropositive individuals in malaria endemic regions. We demonstrate that our P27/P27A-SAPNs induce an immune response akin to the one in seropositive individuals in Burkina Faso. Since P27 is highly conserved among different Plasmodium species, these novel SAPNs may even provide cross-protection between Plasmodium falciparum and Plasmodium vivax the two major human malaria pathogens. As the SAPNs are also easy to manufacture and store they can be delivered to the population in need without complication thus providing a low cost malaria vaccine.

  4. Study on the interaction between methyl jasmonate and the coiled-coil domain of rice blast resistance protein Pi36 by spectroscopic methods (United States)

    Liu, Xin Q.; Zhang, Dan; Zhang, Xiang M.; Wang, Chun T.; Liu, Xue Q.; Tan, Yan P.; Wu, Yun H.


    Interaction between the coiled-coil domain of rice blast resistance protein Pi36 and methyl-jasmonate (MeJA) was studied by fluorescence and UV-vis spectroscopic techniques. The quenching mechanism of fluorescence of MeJA by this domain was discussed to be a static quenching procedure. Fluorescence quenching was explored to measure the number of binding sites n and apparent binding constants K. The thermodynamics parameters ΔH, ΔG, ΔS were also calculated. The results indicate the binding reaction was not entropy-driven but enthalpy-driven, and hydrophobic binding played major role in the interaction. The binding sites of MeJA with the coiled-coil structural domain of rice blast resistance protein Pi36 were found to approach the microenvironment of both Tyr and Trp by the synchronous fluorescence spectrometry. The distance r between donor (the coiled-coil domain of rice blast resistance protein Pi36) and acceptor (MeJA) was obtained according to Förster theory of non-radioactive energy transfer.

  5. The Golgi-associated long coiled-coil protein NECC1 participates in the control of the regulated secretory pathway in PC12 cells. (United States)

    Cruz-García, David; Díaz-Ruiz, Alberto; Rabanal-Ruiz, Yoana; Peinado, Juan R; Gracia-Navarro, Francisco; Castaño, Justo P; Montero-Hadjadje, Maité; Tonon, Marie-Christine; Vaudry, Hubert; Anouar, Youssef; Vázquez-Martínez, Rafael; Malagón, María M


    Golgi-associated long coiled-coil proteins, often referred to as golgins, are involved in the maintenance of the structural organization of the Golgi apparatus and the regulation of membrane traffic events occurring in this organelle. Little information is available on the contribution of golgins to Golgi function in cells specialized in secretion such as endocrine cells or neurons. In the present study, we characterize the intracellular distribution as well as the biochemical and functional properties of a novel long coiled-coil protein present in neuroendocrine tissues, NECC1 (neuroendocrine long coiled-coil protein 1). The present study shows that NECC1 is a peripheral membrane protein displaying high stability to detergent extraction, which distributes across the Golgi apparatus in neuroendocrine cells. In addition, NECC1 partially localizes to post-Golgi carriers containing secretory cargo in PC12 cells. Overexpression of NECC1 resulted in the formation of juxtanuclear aggregates together with a slight fragmentation of the Golgi and a decrease in K+-stimulated hormone release. In contrast, NECC1 silencing did not alter Golgi architecture, but enhanced K+-stimulated hormone secretion in PC12 cells. In all, the results of the present study identify NECC1 as a novel component of the Golgi matrix and support a role for this protein as a negative modulator of the regulated trafficking of secretory cargo in neuroendocrine cells.

  6. 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

    DEFF Research Database (Denmark)

    Manfra, Ornella; Van Craenenbroeck, Kathleen; Skieterska, Kamila


    -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......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...

  7. Structural Comparisons of Apo- and Metalated Three-Stranded Coiled Coils Clarify Metal Binding Determinants in Thiolate Containing Designed Peptides

    Energy Technology Data Exchange (ETDEWEB)

    Chakraborty, Saumen; Touw, Debra S.; Peacock, Anna F.A.; Stuckey, Jeanne; Pecoraro, Vincent L. (Michigan)


    Over the past two decades, designed metallopeptides have held the promise for understanding a variety of fundamental questions in metallobiochemistry; however, these dreams have not yet been realized because of a lack of structural data to elaborate the protein scaffolds before metal complexation and the resultant metalated structures which ultimately exist. This is because there are few reports of structural characterization of such systems either in their metalated or nonmetalated forms and no examples where an apo structure and the corresponding metalated peptide assembly have both been defined by X-ray crystallography. Herein we present X-ray structures of two de novo designed parallel three-stranded coiled coils (designed using the heptad repeat (a {yields} g)) CSL9C (CS = Coil Ser) and CSL19C in their nonmetalated forms, determined to 1.36 and 2.15 {angstrom} resolutions, respectively. Leucines from either position 9 (a site) or 19 (d site) are replaced by cysteine to generate the constructs CSL9C and CSL19C, respectively, yielding thiol-rich pockets at the hydrophobic interior of these peptides, suitable to bind heavy metals such as As(III), Hg(II), Cd(II), and Pb(II). We use these structures to understand the inherent structural differences between a and d sites to clarify the basis of the observed differential spectroscopic behavior of metal binding in these types of peptides. Cys side chains of (CSL9C){sub 3} show alternate conformations and are partially preorganized for metal binding, whereas cysteines in (CSL19C){sub 3} are present as a single conformer. Zn(II) ions, which do not coordinate or influence Cys residues at the designed metal sites but are essential for forming X-ray quality crystals, are bound to His and Glu residues at the crystal packing interfaces of both structures. These 'apo' structures are used to clarify the changes in metal site organization between metalated As(CSL9C){sub 3} and to speculate on the differential basis of

  8. pH-Sensitive Coiled-Coil Peptide-Cross-Linked Hyaluronic Acid Nanogels: Synthesis and Targeted Intracellular Protein Delivery to CD44 Positive Cancer Cells. (United States)

    Ding, Lingling; Jiang, Yu; Zhang, Jian; Klok, Harm-Anton; Zhong, Zhiyuan


    The clinical translation of protein drugs that act intracellularly is limited by the absence of safe and efficient intracellular protein delivery vehicles. Here, pH-sensitive coiled-coil peptide-cross-linked hyaluronic acid nanogels (HA-cNGs) were designed and investigated for targeted intracellular protein delivery to CD44 overexpressing MCF-7 breast cancer cells. HA-cNGs were obtained with a small size of 176 nm from an equivalent mixture of hyaluronic acid conjugates with GY(EIAALEK) 3 GC (E3) and GY(KIAALKE) 3 GC (K3) peptides, respectively, at pH 7.4 by nanoprecipitation. Circular dichroism (CD) proved the formation of coiled-coil structures between E3 and K3 peptides at pH 7.4 while fast uncoiling at pH 5.0. HA-cNGs showed facile loading of cytochrome C (CC) and greatly accelerated CC release under mild acidic conditions (18.4%, 76.8%, and 91.4% protein release in 24 h at pH 7.4, 6.0, and 5.0, respectively). Confocal microscopy and flow cytometry displayed efficient internalization of CC-loaded HA-cNGs and effective endosomal escape of CC in MCF-7 cancer cells. Remarkably, HA-cNGs loaded with saporin, a ribosome inactivating protein, exhibited significantly enhanced apoptotic activity to MCF-7 cells with a low IC 50 of 12.2 nM. These coiled-coil peptide-cross-linked hyaluronic acid nanogels have appeared as a simple and multifunctional platform for efficient intracellular protein delivery.

  9. Three novel mutations in KIF21A highlight the importance of the third coiled-coil stalk domain in the etiology of CFEOM1

    Directory of Open Access Journals (Sweden)

    Gutowski Nicholas J


    Full Text Available Abstract Background Congenital fibrosis of the extraocular muscles types 1 and 3 (CFEOM1/CFEOM3 are autosomal dominant strabismus disorders that appear to result from maldevelopment of ocular nuclei and nerves. We previously reported that most individuals with CFEOM1 and rare individuals with CFEOM3 harbor heterozygous mutations in KIF21A. KIF21A encodes a kinesin motor involved in anterograde axonal transport, and the familial and de novo mutations reported to date predictably alter one of only a few KIF21A amino acids – three within the third coiled-coil region of the stalk and one in the distal motor domain, suggesting they result in altered KIF21A function. To further define the spectrum of KIF21A mutations in CFEOM we have now identified all CFEOM probands newly enrolled in our study and determined if they harbor mutations in KIF21A. Results Sixteen CFEOM1 and 29 CFEOM3 probands were studied. Three previously unreported de novo KIF21A mutations were identified in three CFEOM1 probands, all located in the same coiled-coil region of the stalk that contains all but one of the previously reported mutations. Eight additional CFEOM1 probands harbored three of the mutations previously reported in KIF21A; seven had one of the two most common mutations, while one harbored the mutation in the distal motor domain. No mutation was detected in 5 CFEOM1 or any CFEOM3 probands. Conclusion Analysis of sixteen CFEOM1 probands revealed three novel KIF21A mutations and confirmed three reported mutations, bringing the total number of reported KIF21A mutations in CFEOM1 to 11 mutations among 70 mutation positive probands. All three new mutations alter amino acids in heptad repeats within the third coiled-coil region of the KIF21A stalk, further highlighting the importance of alterations in this domain in the etiology of CFEOM1.

  10. The Coiled-Coil Protein VIG1 Is Essential for Tethering Vacuoles to Mitochondria during Vacuole Inheritance of Cyanidioschyzon merolae[C][W][OA (United States)

    Fujiwara, Takayuki; Kuroiwa, Haruko; Yagisawa, Fumi; Ohnuma, Mio; Yoshida, Yamato; Yoshida, Masaki; Nishida, Keiji; Misumi, Osami; Watanabe, Satoru; Tanaka, Kan; Kuroiwa, Tsuneyoshi


    Vacuoles/lysosomes function in endocytosis and in storage and digestion of metabolites. These organelles are inherited by the daughter cells in eukaryotes. However, the mechanisms of this inheritance are poorly understood because the cells contain multiple vacuoles that behave randomly. The primitive red alga Cyanidioschyzon merolae has a minimum set of organelles. Here, we show that C. merolae contains about four vacuoles that are distributed equally between the daughter cells by binding to dividing mitochondria. Binding is mediated by VIG1, a 30-kD coiled-coil protein identified by microarray analyses and immunological assays. VIG1 appears on the surface of free vacuoles in the cytosol and then tethers the vacuoles to the mitochondria. The vacuoles are released from the mitochondrion in the daughter cells following VIG1 digestion. Suppression of VIG1 by antisense RNA disrupted the migration of vacuoles. Thus, VIG1 is essential for tethering vacuoles to mitochondria during vacuole inheritance in C. merolae. PMID:20348431

  11. cDNA cloning of the basement membrane chondroitin sulfate proteoglycan core protein, bamacan: a five domain structure including coiled-coil motifs

    DEFF Research Database (Denmark)

    Wu, R R; Couchman, J R


    Basement membranes contain several proteoglycans, and those bearing heparan sulfate glycosaminoglycans such as perlecan and agrin usually predominate. Most mammalian basement membranes also contain chondroitin sulfate, and a core protein, bamacan, has been partially characterized. We have now....../translation product from a full-length bamacan cDNA. The unusual structure of this proteoglycan is indicative of specific functional roles in basement membrane physiology, commensurate with its distinct expression in development and changes in disease models....

  12. Structure of Shroom domain 2 reveals a three-segmented coiled-coil required for dimerization, Rock binding, and apical constriction. (United States)

    Mohan, Swarna; Rizaldy, Ryan; Das, Debamitra; Bauer, Robert J; Heroux, Annie; Trakselis, Michael A; Hildebrand, Jeffrey D; VanDemark, Andrew P


    Shroom (Shrm) proteins are essential regulators of cell shape and tissue morpho-logy during animal development that function by interacting directly with the coiled-coil region of Rho kinase (Rock). The Shrm-Rock interaction is sufficient to direct Rock subcellular localization and the subsequent assembly of contractile actomyosin networks in defined subcellular locales. However, it is unclear how the Shrm-Rock interaction is regulated at the molecular level. To begin investigating this issue, we present the structure of Shrm domain 2 (SD2), which mediates the interaction with Rock and is required for Shrm function. SD2 is a unique three-segmented dimer with internal symmetry, and we identify conserved residues on the surface and within the dimerization interface that are required for the Rock-Shrm interaction and Shrm activity in vivo. We further show that these residues are critical in both vertebrate and invertebrate Shroom proteins, indicating that the Shrm-Rock signaling module has been functionally and molecularly conserved. The structure and biochemical analysis of Shrm SD2 indicate that it is distinct from other Rock activators such as RhoA and establishes a new paradigm for the Rock-mediated assembly of contractile actomyosin networks.

  13. Control of Smc Coiled Coil Architecture by the ATPase Heads Facilitates Targeting to Chromosomal ParB/parS and Release onto Flanking DNA (United States)

    Minnen, Anita; Bürmann, Frank; Wilhelm, Larissa; Anchimiuk, Anna; Diebold-Durand, Marie-Laure; Gruber, Stephan


    Summary Smc/ScpAB promotes chromosome segregation in prokaryotes, presumably by compacting and resolving nascent sister chromosomes. The underlying mechanisms, however, are poorly understood. Here, we investigate the role of the Smc ATPase activity in the recruitment of Smc/ScpAB to the Bacillus subtilis chromosome. We demonstrate that targeting of Smc/ScpAB to ParB/parS loading sites is strictly dependent on engagement of Smc head domains and relies on an open organization of the Smc coiled coils. We find that dimerization of the Smc hinge domain stabilizes closed Smc rods and hinders head engagement as well as chromosomal targeting. Conversely, the ScpAB sub-complex promotes head engagement and Smc rod opening and thereby facilitates recruitment of Smc to parS sites. Upon ATP hydrolysis, Smc/ScpAB is released from loading sites and relocates within the chromosome—presumably through translocation along DNA double helices. Our findings define an intermediate state in the process of chromosome organization by Smc. PMID:26904953

  14. The Rsv3 Locus Conferring Resistance to Soybean Mosaic Virus is Associated with a Cluster of Coiled-Coil Nucleotide-Binding Leucine-Rich Repeat Genes

    Directory of Open Access Journals (Sweden)

    Su Jeoung Suh


    Full Text Available The (SMV resistance locus, , previously mapped between markers A519F/R and M3Satt in the soybean molecular linkage group B2 (chromosome 14, has been characterized by examination of the soybean genome sequence. The 154 kbp interval encompassing contains a family of closely related coiled-coil nucleotide-binding leucine-rich repeat (CC-NB-LRR genes. Tightly linked to this region are additional CC-NB-LRR genes and several leucine-rich repeat receptor-like kinase (LRR-RLK genes, thereby indicating that members of both multigene families constitute a heterogeneous cluster at the chromosomal region. To further confirm the sequence and genetic map concordance, we developed 16 markers from the genomic sequence including predicted CC-NB-LRR genes and their flanking sequences. Mapping of the resultant markers in three populations showed parallel alignment between the genetic and sequence maps in the -containing region. Phylogenetic analysis of five CC-NB-LRR genes including a pseudogene showed they were highly similar to each other and formed a subclade within a CC-NB-LRR gene clade with representatives from several plant families including legume species. These results demonstrate that the locus is associated with this cluster of CC-NB-LRR genes, thereby suggesting that the gene most likely encodes a member of this gene family. In addition, information from this study should facilitate marker-assisted selection and pyramiding of resistance genes.

  15. BECN2 interacts with ATG14 through a metastable coiled-coil to mediate autophagy: BECN2 CCD Structure and Interaction with ATG14

    Energy Technology Data Exchange (ETDEWEB)

    Su, Minfei; Li, Yue; Wyborny, Shane; Neau, David; Chakravarthy, Srinivas; Levine, Beth; Colbert, Christopher L.; Sinha, Sangita C. (NDSU); (IIT); (Cornell); (UTSMC)


    ATG14 binding to BECN/Beclin homologs is essential for autophagy, a critical catabolic homeostasis pathway. Here, we show that the α-helical, coiled-coil domain (CCD) of BECN2, a recently identified mammalian BECN1 paralog, forms an antiparallel, curved homodimer with seven pairs of nonideal packing interactions, while the BECN2 CCD and ATG14 CCD form a parallel, curved heterodimer stabilized by multiple, conserved polar interactions. Compared to BECN1, the BECN2 CCD forms a weaker homodimer, but binds more tightly to the ATG14 CCD. Mutation of nonideal BECN2 interface residues to more ideal pairs improves homodimer self-association and thermal stability. Unlike BECN1, all BECN2 CCD mutants bind ATG14, although more weakly than wild type. Thus, polar BECN2 CCD interface residues result in a metastable homodimer, facilitating dissociation, but enable better interactions with polar ATG14 residues stabilizing the BECN2:ATG14 heterodimer. These structure-based mechanistic differences in BECN1 and BECN2 homodimerization and heterodimerization likely dictate competitive ATG14 recruitment.

  16. Missense mutation in immunodeficient patients shows the multifunctional roles of coiled-coil domain 3 (CC3) in STIM1 activation. (United States)

    Maus, Mate; Jairaman, Amit; Stathopulos, Peter B; Muik, Martin; Fahrner, Marc; Weidinger, Carl; Benson, Melina; Fuchs, Sebastian; Ehl, Stephan; Romanin, Christoph; Ikura, Mitsuhiko; Prakriya, Murali; Feske, Stefan


    Store-operated Ca(2+) entry (SOCE) is a universal Ca(2+) influx pathway that is important for the function of many cell types. SOCE occurs upon depletion of endoplasmic reticulum (ER) Ca(2+) stores and relies on a complex molecular interplay between the plasma membrane (PM) Ca(2+) channel ORAI1 and the ER Ca(2+) sensor stromal interaction molecule (STIM) 1. Patients with null mutations in ORAI1 or STIM1 genes present with severe combined immunodeficiency (SCID)-like disease. Here, we describe the molecular mechanisms by which a loss-of-function STIM1 mutation (R429C) in human patients abolishes SOCE. R429 is located in the third coiled-coil (CC3) domain of the cytoplasmic C terminus of STIM1. Mutation of R429 destabilizes the CC3 structure and alters the conformation of the STIM1 C terminus, thereby releasing a polybasic domain that promotes STIM1 recruitment to ER-PM junctions. However, the mutation also impairs cytoplasmic STIM1 oligomerization and abolishes STIM1-ORAI1 interactions. Thus, despite its constitutive localization at ER-PM junctions, mutant STIM1 fails to activate SOCE. Our results demonstrate multifunctional roles of the CC3 domain in regulating intra- and intermolecular STIM1 interactions that control (i) transition of STIM1 from a quiescent to an active conformational state, (ii) cytoplasmic STIM1 oligomerization, and (iii) STIM1-ORAI1 binding required for ORAI1 activation.

  17. Mapping of the Neisseria meningitidis NadA cell-binding site: relevance of predicted {alpha}-helices in the NH2-terminal and dimeric coiled-coil regions. (United States)

    Tavano, Regina; Capecchi, Barbara; Montanari, Paolo; Franzoso, Susanna; Marin, Oriano; Sztukowska, Maryta; Cecchini, Paola; Segat, Daniela; Scarselli, Maria; Aricò, Beatrice; Papini, Emanuele


    NadA is a trimeric autotransporter protein of Neisseria meningitidis belonging to the group of oligomeric coiled-coil adhesins. It is implicated in the colonization of the human upper respiratory tract by hypervirulent serogroup B N. meningitidis strains and is part of a multiantigen anti-serogroup B vaccine. Structure prediction indicates that NadA is made by a COOH-terminal membrane anchor (also necessary for autotranslocation to the bacterial surface), an intermediate elongated coiled-coil-rich stalk, and an NH(2)-terminal region involved in cell interaction. Electron microscopy analysis and structure prediction suggest that the apical region of NadA forms a compact and globular domain. Deletion studies proved that the NH(2)-terminal sequence (residues 24 to 87) is necessary for cell adhesion. In this study, to better define the NadA cell binding site, we exploited (i) a panel of NadA mutants lacking sequences along the coiled-coil stalk and (ii) several oligoclonal rabbit antibodies, and their relative Fab fragments, directed to linear epitopes distributed along the NadA ectodomain. We identified two critical regions for the NadA-cell receptor interaction with Chang cells: the NH(2) globular head domain and the NH(2) dimeric intrachain coiled-coil α-helices stemming from the stalk. This raises the importance of different modules within the predicted NadA structure. The identification of linear epitopes involved in receptor binding that are able to induce interfering antibodies reinforces the importance of NadA as a vaccine antigen.

  18. Mapping of the Neisseria meningitidis NadA Cell-Binding Site: Relevance of Predicted α-Helices in the NH2-Terminal and Dimeric Coiled-Coil Regions▿ (United States)

    Tavano, Regina; Capecchi, Barbara; Montanari, Paolo; Franzoso, Susanna; Marin, Oriano; Sztukowska, Maryta; Cecchini, Paola; Segat, Daniela; Scarselli, Maria; Aricò, Beatrice; Papini, Emanuele


    NadA is a trimeric autotransporter protein of Neisseria meningitidis belonging to the group of oligomeric coiled-coil adhesins. It is implicated in the colonization of the human upper respiratory tract by hypervirulent serogroup B N. meningitidis strains and is part of a multiantigen anti-serogroup B vaccine. Structure prediction indicates that NadA is made by a COOH-terminal membrane anchor (also necessary for autotranslocation to the bacterial surface), an intermediate elongated coiled-coil-rich stalk, and an NH2-terminal region involved in cell interaction. Electron microscopy analysis and structure prediction suggest that the apical region of NadA forms a compact and globular domain. Deletion studies proved that the NH2-terminal sequence (residues 24 to 87) is necessary for cell adhesion. In this study, to better define the NadA cell binding site, we exploited (i) a panel of NadA mutants lacking sequences along the coiled-coil stalk and (ii) several oligoclonal rabbit antibodies, and their relative Fab fragments, directed to linear epitopes distributed along the NadA ectodomain. We identified two critical regions for the NadA-cell receptor interaction with Chang cells: the NH2 globular head domain and the NH2 dimeric intrachain coiled-coil α-helices stemming from the stalk. This raises the importance of different modules within the predicted NadA structure. The identification of linear epitopes involved in receptor binding that are able to induce interfering antibodies reinforces the importance of NadA as a vaccine antigen. PMID:20971901

  19. Cryopreservation of Brain Endothelial Cells Derived from Human Induced Pluripotent Stem Cells Is Enhanced by Rho-Associated Coiled Coil-Containing Kinase Inhibition. (United States)

    Wilson, Hannah K; Faubion, Madeline G; Hjortness, Michael K; Palecek, Sean P; Shusta, Eric V


    The blood-brain barrier (BBB) maintains brain homeostasis but also presents a major obstacle to brain drug delivery. Brain microvascular endothelial cells (BMECs) form the principal barrier and therefore represent the major cellular component of in vitro BBB models. Such models are often used for mechanistic studies of the BBB in health and disease and for drug screening. Recently, human induced pluripotent stem cells (iPSCs) have emerged as a new source for generating BMEC-like cells for use in in vitro human BBB studies. However, the inability to cryopreserve iPSC-BMECs has impeded implementation of this model by requiring a fresh differentiation to generate cells for each experiment. Cryopreservation of differentiated iPSC-BMECs would have a number of distinct advantages, including enabling production of larger scale lots, decreasing lead time to generate purified iPSC-BMEC cultures, and facilitating use of iPSC-BMECs in large-scale screening. In this study, we demonstrate that iPSC-BMECs can be successfully cryopreserved at multiple differentiation stages. Cryopreserved iPSC-BMECs retain high viability, express standard endothelial and BBB markers, and reach a high transendothelial electrical resistance (TEER) of ∼3000 Ω·cm2, equivalent to nonfrozen controls. Rho-associated coiled coil-containing kinase (ROCK) inhibitor Y-27632 substantially increased survival and attachment of cryopreserved iPSC-BMECs, as well as stabilized TEER above 800 Ω·cm2 out to 7 days post-thaw. Overall, cryopreservation will ease handling and storage of high-quality iPSC-BMECs, reducing a key barrier to greater implementation of these cells in modeling the human BBB.

  20. Klotho gene delivery ameliorates renal hypertrophy and fibrosis in streptozotocin-induced diabetic rats by suppressing the Rho-associated coiled-coil kinase signaling pathway. (United States)

    Deng, Minghong; Luo, Yumei; Li, Yunkui; Yang, Qiuchen; Deng, Xiaoqin; Wu, Ping; Ma, Houxun


    The present study aimed to investigate whether klotho gene delivery attenuated renal hypertrophy and fibrosis in streptozotocin-induced diabetic rats. A recombinant adeno-associated virus (rAAV) carrying mouse klotho full-length cDNA (rAAV.mKL), was constructed for in vivo investigation of klotho expression. Diabetes was induced in rats by a single tail vein injection of 60 mg/kg streptozotocin. Subsequently, the diabetic rats received an intravenous injection of rAAV.mKL, fluorescent protein (GFP) or phosphate-buffered saline (PBS). The Sprague-Dawley rat group received PBS and served as the control group. After 12 weeks, all the rats were sacrificed and ELISA, immunohistochemical and histological analyses, fluorescence microscopy, semi-quantitative reverse transcription-polymerase chain reaction and western blottin were performed. A single dose of rAAV.mKL was found to prevent the progression of renal hypertrophy and fibrosis for at least 12 weeks (duration of study). Klotho expression was suppressed in the diabetic rats, but was increased by rAAV.mKL delivery. rAAV.mKL significantly suppressed diabetes-induced renal hypertrophy and histopathological changes, reduced renal collagen fiber generation and decreased kidney hypertrophy index. In addition, rAAV.mKL decreased the protein expression levels of fibronectin and vimentin, while it downregulated the mRNA expression and activity of Rho-associated coiled-coil kinase (ROCK)I in the kidneys of the diabetic rats. These results indicated that klotho gene delivery ameliorated renal hypertrophy and fibrosis in diabetic rats, possibly by suppressing the ROCK signaling pathway. This may offer a novel approach for the long-term control and renoprotection of diabetes.

  1. CC2D2A, Encoding A Coiled-Coil and C2 Domain Protein, Causes Autosomal-Recessive Mental Retardation with Retinitis Pigmentosa (United States)

    Noor, Abdul; Windpassinger, Christian; Patel, Megha; Stachowiak, Beata; Mikhailov, Anna; Azam, Matloob; Irfan, Muhammad; Siddiqui, Zahid Kamal; Naeem, Farooq; Paterson, Andrew D.; Lutfullah, Muhammad; Vincent, John B.; Ayub, Muhammad


    Autosomal-recessive inheritance is believed to be relatively common in mental retardation (MR), although only four genes for nonsyndromic autosomal-recessive mental retardation (ARMR) have been reported. In this study, we ascertained a consanguineous Pakistani family with ARMR in four living individuals from three branches of the family, plus an additional affected individual later identified as a phenocopy. Retinitis pigmentosa was present in affected individuals, but no other features suggestive of a syndromic form of MR were found. We used Affymetrix 500K microarrays to perform homozygosity mapping and identified a homozygous and haploidentical region of 11.2 Mb on chromosome 4p15.33-p15.2. Linkage analysis across this region produced a maximum two-point LOD score of 3.59. We sequenced genes within the critical region and identified a homozygous splice-site mutation segregating in the family, within a coiled-coil and C2 domain-containing gene, CC2D2A. This mutation leads to the skipping of exon 19, resulting in a frameshift and a truncated protein lacking the C2 domain. Conservation analysis for CC2D2A suggests a functional domain near the C terminus as well as the C2 domain. Preliminary functional studies of CC2D2A suggest a possible role in Ca2+-dependent signal transduction. Identifying the function of CC2D2A, and a possible common pathway with CC2D1A, in correct neuronal development and functioning may help identify possible therapeutic targets for MR. PMID:18387594

  2. Structural analysis of intermolecular interactions in the kinesin adaptor complex fasciculation and elongation protein zeta 1/ short coiled-coil protein (FEZ1/SCOCO.

    Directory of Open Access Journals (Sweden)

    Marcos Rodrigo Alborghetti

    Full Text Available Cytoskeleton and protein trafficking processes, including vesicle transport to synapses, are key processes in neuronal differentiation and axon outgrowth. The human protein FEZ1 (fasciculation and elongation protein zeta 1 / UNC-76, in C. elegans, SCOCO (short coiled-coil protein / UNC-69 and kinesins (e.g. kinesin heavy chain / UNC116 are involved in these processes. Exploiting the feature of FEZ1 protein as a bivalent adapter of transport mediated by kinesins and FEZ1 protein interaction with SCOCO (proteins involved in the same path of axonal growth, we investigated the structural aspects of intermolecular interactions involved in this complex formation by NMR (Nuclear Magnetic Resonance, cross-linking coupled with mass spectrometry (MS, SAXS (Small Angle X-ray Scattering and molecular modelling. The topology of homodimerization was accessed through NMR (Nuclear Magnetic Resonance studies of the region involved in this process, corresponding to FEZ1 (92-194. Through studies involving the protein in its monomeric configuration (reduced and dimeric state, we propose that homodimerization occurs with FEZ1 chains oriented in an anti-parallel topology. We demonstrate that the interaction interface of FEZ1 and SCOCO defined by MS and computational modelling is in accordance with that previously demonstrated for UNC-76 and UNC-69. SAXS and literature data support a heterotetrameric complex model. These data provide details about the interaction interfaces probably involved in the transport machinery assembly and open perspectives to understand and interfere in this assembly and its involvement in neuronal differentiation and axon outgrowth.

  3. Complexes of neutralizing and non-neutralizing affinity matured Fabs with a mimetic of the internal trimeric coiled-coil of HIV-1 gp41.

    Directory of Open Access Journals (Sweden)

    Elena Gustchina

    Full Text Available A series of mini-antibodies (monovalent and bivalent Fabs targeting the conserved internal trimeric coiled-coil of the N-heptad repeat (N-HR of HIV-1 gp41 has been previously constructed and reported. Crystal structures of two closely related monovalent Fabs, one (Fab 8066 broadly neutralizing across a wide panel of HIV-1 subtype B and C viruses, and the other (Fab 8062 non-neutralizing, representing the extremes of this series, were previously solved as complexes with 5-Helix, a gp41 pre-hairpin intermediate mimetic. Binding of these Fabs to covalently stabilized chimeric trimers of N-peptides of HIV-1 gp41 (named (CCIZN363 or 3-H has now been investigated using X-ray crystallography, cryo-electron microscopy, and a variety of biophysical methods. Crystal structures of the complexes between 3-H and Fab 8066 and Fab 8062 were determined at 2.8 and 3.0 Å resolution, respectively. Although the structures of the complexes with the neutralizing Fab 8066 and its non-neutralizing counterpart Fab 8062 were generally similar, small differences between them could be correlated with the biological properties of these antibodies. The conformations of the corresponding CDRs of each antibody in the complexes with 3-H and 5-Helix are very similar. The adaptation to a different target upon complex formation is predominantly achieved by changes in the structure of the trimer of N-HR helices, as well as by adjustment of the orientation of the Fab molecule relative to the N-HR in the complex, via rigid-body movement. The structural data presented here indicate that binding of three Fabs 8062 with high affinity requires more significant changes in the structure of the N-HR trimer compared to binding of Fab 8066. A comparative analysis of the structures of Fabs complexed to different gp41 intermediate mimetics allows further evaluation of biological relevance for generation of neutralizing antibodies, as well as provides novel structural insights into immunogen

  4. A Rho-associated coiled-coil containing kinases (ROCK) inhibitor, Y-27632, enhances adhesion, viability and differentiation of human term placenta-derived trophoblasts in vitro. (United States)

    Motomura, Kenichiro; Okada, Naoko; Morita, Hideaki; Hara, Mariko; Tamari, Masato; Orimo, Keisuke; Matsuda, Go; Imadome, Ken-Ichi; Matsuda, Akio; Nagamatsu, Takeshi; Fujieda, Mikiya; Sago, Haruhiko; Saito, Hirohisa; Matsumoto, Kenji


    Although human term placenta-derived primary cytotrophoblasts (pCTBs) represent a good human syncytiotrophoblast (STB) model, in vitro culture of pCTBs is not always easily accomplished. Y-27632, a specific inhibitor of Rho-associated coiled-coil containing kinases (ROCK), reportedly prevented apoptosis and improved cell-to-substrate adhesion and culture stability of dissociated cultured human embryonic stem cells and human corneal endothelial cells. The Rho kinase pathway regulates various kinds of cell behavior, some of which are involved in pCTB adhesion and differentiation. In this study, we examined Y-27632's potential for enhancing pCTB adhesion, viability and differentiation. pCTBs were isolated from term, uncomplicated placentas by trypsin-DNase I-Dispase II treatment and purified by HLA class I-positive cell depletion. Purified pCTBs were cultured on uncoated plates in the presence of epidermal growth factor (10 ng/ml) and various concentrations of Y-27632. pCTB adhesion to the plates was evaluated by phase-contrast imaging, viability was measured by WST-8 assay, and differentiation was evaluated by immunofluorescence staining, expression of fusogenic genes and hCG-β production. Ras-related C3 botulinum toxin substrate 1 (Rac1; one of the effector proteins of the Rho family) and protein kinase A (PKA) involvement was evaluated by using their specific inhibitors, NSC-23766 and H-89. We found that Y-27632 treatment significantly enhanced pCTB adhesion to plates, viability, cell-to-cell fusion and hCG-β production, but showed no effects on pCTB proliferation or apoptosis. Furthermore, NSC-23766 and H-89 each blocked the effects of Y-27632, suggesting that Y-27632 significantly enhanced pCTB differentiation via Rac1 and PKA activation. Our findings suggest that Rac1 and PKA may be interactively involved in CTB differentiation, and addition of Y-27632 to cultures may be an effective method for creating a stable culture model for studying CTB and STB biology

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

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


    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. 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. (United States)

    Nakamura, Yoko; Hasebe, Ai; Takahashi, Kaneyoshi; Iijima, Masumi; Yoshimoto, Nobuo; Maturana, Andrés D; Ting, Kang; Kuroda, Shun'ichi; Niimi, Tomoaki


    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.

  7. 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* (United States)

    Nakamura, Yoko; Hasebe, Ai; Takahashi, Kaneyoshi; Iijima, Masumi; Yoshimoto, Nobuo; Maturana, Andrés D.; Ting, Kang; Kuroda, Shun'ichi; Niimi, Tomoaki


    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

  8. Screening for microsatellite instability identifies frequent 3'-untranslated region mutation of the RB1-inducible coiled-coil 1 gene in colon tumors.

    Directory of Open Access Journals (Sweden)

    Bogdan C Paun

    Full Text Available BACKGROUND: Coding region microsatellite instability (MSI results in loss of gene products and promotion of microsatellite-unstable (MSI-H carcinogenesis. Recent studies have indicated that MSI within 3'-untranslated regions (3'UTRs may post-transcriptionally dysregulate gene products. Within this context, we conducted a broad mutational survey of 42 short 3'UTR microsatellites (MSs in 45 MSI-H colorectal tumors and their corresponding normal colonic mucosae. METHODOLOGY/PRINCIPAL FINDINGS: In order to estimate the overall susceptibility of MSs to MSI in MSI-H tumors, the observed MSI frequency of each MS was correlated with its length, interspecies sequence conservation level, and distance from some genetic elements (i.e., stop codon, polyA signal, and microRNA binding sites. All MSs were stable in normal colonic mucosae. The MSI frequency at each MS in MSI-H tumors was independent of sequence conservation level and distance from other genetic elements. In contrast, MS length correlated significantly with MSI frequency in MSI-H tumors (r=0.86, p=7.2x10(-13. 3'UTR MSs demonstrated MSI frequencies in MSI-H tumors higher than the 99% upper limit predicted by MS length for RB1-inducible coiled-coil 1(RB1CC1, mutation frequency 68.4%, NUAK family SNF1-like kinase 1(NUAK1, 31.0%, and Rtf1, Paf1/RNA polymerase II complex component, homolog (RTF1, 25.0%. An in silico prediction of RNA structure alterations was conducted for these MSI events to gauge their likelihood of affecting post-transcriptional regulation. RB1CC1 mutant was predicted to lose a microRNA-accessible loop structure at a putative binding site for the tumor-suppressive microRNA, miR-138. In contrast, the predicted 3'UTR structural change was minimal for NUAK1- and RTF1 mutants. Notably, real-time quantitative RT-PCR analysis revealed significant RB1CC1 mRNA overexpression vs. normal colonic mucosae in MSI-H cancers manifesting RB1CC1 3'UTR MSI (9.0-fold; p = 3.6x10(-4. CONCLUSIONS: This

  9. Identification and solution structure of a highly conserved C-terminal domain within ORF1p required for retrotransposition of long interspersed nuclear element-1. (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


    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.

  10. NMR assignments of SPOC domain of the human transcriptional corepressor SHARP in complex with a C-terminal SMRT peptide. (United States)

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


    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.

  11. C-terminal Src kinase-mediated EPIYA phosphorylation of Pragmin creates a feed-forward C-terminal Src kinase activation loop that promotes cell motility. (United States)

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


    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.

  12. 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


    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

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


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


    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.

  14. Modules for C-terminal epitope tagging of Tetrahymena genes. (United States)

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


    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.

  15. 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 ...

  16. Protein kinase C and rho activated coiled coil protein kinase 2 (ROCK2 modulate Alzheimer's APP metabolism and phosphorylation of the Vps10-domain protein, SorL1

    Directory of Open Access Journals (Sweden)

    Ehrlich Michelle E


    Full Text Available Abstract Background Generation of the amyloid β (Aβ peptide of Alzheimer's disease (AD is differentially regulated through the intracellular trafficking of the amyloid β precursor protein (APP within the secretory and endocytic pathways. Protein kinase C (PKC and rho-activated coiled-coil kinases (ROCKs are two "third messenger" signaling molecules that control the relative utilization of these two pathways. Several members of the Vps family of receptors (Vps35, SorL1, SorCS1 play important roles in post-trans-Golgi network (TGN sorting and generation of APP derivatives, including Aβ at the TGN, endosome and the plasma membrane. We now report that Vps10-domain proteins are candidate substrates for PKC and/or ROCK2 and act as phospho-state-sensitive physiological effectors for post-TGN sorting of APP and its derivatives. Results Analysis of the SorL1 cytoplasmic tail revealed multiple consensus sites for phosphorylation by protein kinases. SorL1 was subsequently identified as a phosphoprotein, based on sensitivity of its electrophoretic migration pattern to calf intestine alkaline phosphatase and on its reaction with anti-phospho-serine antibodies. Activation of PKC resulted in increased shedding of the ectodomains of both APP and SorL1, and this was paralleled by an apparent increase in the level of the phosphorylated form of SorL1. ROCK2, the neuronal isoform of another protein kinase, was found to form complexes with SorL1, and both ROCK2 inhibition and ROCK2 knockdown enhanced generation of both soluble APP and Aβ. Conclusion These results highlight the potential importance of SorL1 in elucidating phospho-state sensitive mechanisms in the regulation of metabolism of APP and Aβ by PKC and ROCK2.

  17. 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


    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

  18. C-terminal Amidation of an Osteocalcin-derived Peptide Promotes Hydroxyapatite Crystallization* (United States)

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


    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

  19. C-terminal amidation of an osteocalcin-derived peptide promotes hydroxyapatite crystallization. (United States)

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


    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.

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

    NARCIS (Netherlands)

    Popović, S.


    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

  1. Crystal structure of the C-terminal globular domain of the third paralog of the Archaeoglobus fulgidus oligosaccharyltransferases. (United States)

    Matsumoto, Shunsuke; Shimada, Atsushi; Kohda, Daisuke


    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

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

    Directory of Open Access Journals (Sweden)

    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.

  3. C-terminal functionalization of nylon-3 polymers: effects of C-terminal groups on antibacterial and hemolytic activities. (United States)

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


    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.

  4. Strategies for the Analysis of Bam Recognition Motifs in Outer Membrane Proteins. (United States)

    Paramasivam, Nagarajan; Linke, Dirk


    Well-structured proteins interact with other proteins through surface-surface interactions. In such cases, the residues that form the interacting surface are not necessarily neighboring residues on the level of protein sequence. In contrast, unfolded or partially unfolded proteins can interact with other proteins through defined linear motifs. In the case of the β-barrel assembly machinery (BAM) in the outer membrane of Gram-negative bacteria, unfolded β-barrel proteins are recognized through a C-terminal linear motif, and are inserted into the membrane. While the exact mechanism of recognition is still under investigation, it has been shown that mutations in the recognition motif can partially or completely abolish membrane insertion. In this chapter, we demonstrate the workflow for motif discovery, motif extraction, and motif visualization on the example of the C-terminal motifs in transmembrane β-barrel proteins.

  5. Linear motif atlas for phosphorylation-dependent signaling

    DEFF Research Database (Denmark)

    Miller, Martin Lee; Jensen, LJ; Diella, F


    bind to them remains a challenge. NetPhorest is an atlas of consensus sequence motifs that covers 179 kinases and 104 phosphorylation-dependent binding domains [Src homology 2 (SH2), phosphotyrosine binding (PTB), BRCA1 C-terminal (BRCT), WW, and 14-3-3]. The atlas reveals new aspects of signaling...... sequence models of linear motifs. The atlas is available as a community resource (

  6. Physical association of GPR54 C-terminal with protein phosphatase 2A. (United States)

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


    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.

  7. Solution structure of the RecQ C-terminal domain of human Bloom syndrome protein. (United States)

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


    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.

  8. The in Silico Map-Based Cloning of Pi36, a Rice Coiled-Coil Nucleotide-Binding Site Leucine-Rich Repeat Gene That Confers Race-Specific Resistance to the Blast Fungus

    National Research Council Canada - National Science Library

    Liu, Xinqiong; Lin, Fei; Wang, Ling; Pan, Qinghua


    ...) gene content of the interval and hence for the identification of candidate gene(s) for Pi36. Three such sequences, which all had both a nucleotide-binding site and a leucine-rich repeat motif, were present...

  9. Evolutionarily conserved bias of amino-acid usage refines the definition of PDZ-binding motif

    Directory of Open Access Journals (Sweden)

    Launey Thomas


    Full Text Available Abstract Background The interactions between PDZ (PSD-95, Dlg, ZO-1 domains and PDZ-binding motifs play central roles in signal transductions within cells. Proteins with PDZ domains bind to PDZ-binding motifs almost exclusively when the motifs are located at the carboxyl (C- terminal ends of their binding partners. However, it remains little explored whether PDZ-binding motifs show any preferential location at the C-terminal ends of proteins, at genome-level. Results Here, we examined the distribution of the type-I (x-x-S/T-x-I/L/V or type-II (x-x-V-x-I/V PDZ-binding motifs in proteins encoded in the genomes of five different species (human, mouse, zebrafish, fruit fly and nematode. We first established that these PDZ-binding motifs are indeed preferentially present at their C-terminal ends. Moreover, we found specific amino acid (AA bias for the 'x' positions in the motifs at the C-terminal ends. In general, hydrophilic AAs were favored. Our genomics-based findings confirm and largely extend the results of previous interaction-based studies, allowing us to propose refined consensus sequences for all of the examined PDZ-binding motifs. An ontological analysis revealed that the refined motifs are functionally relevant since a large fraction of the proteins bearing the motif appear to be involved in signal transduction. Furthermore, co-precipitation experiments confirmed two new protein interactions predicted by our genomics-based approach. Finally, we show that influenza virus pathogenicity can be correlated with PDZ-binding motif, with high-virulence viral proteins bearing a refined PDZ-binding motif. Conclusions Our refined definition of PDZ-binding motifs should provide important clues for identifying functional PDZ-binding motifs and proteins involved in signal transduction.

  10. 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


    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

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

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


    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. Structural differences between C-terminal regions of tropomyosin isoforms

    Directory of Open Access Journals (Sweden)

    Małgorzata Śliwińska


    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.

  13. Hevea brasiliensis prohevein possesses a conserved C-terminal domain with amyloid-like properties in vitro. (United States)

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


    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.

  14. Identification of a novel binding site between HIV type 1 Nef C-terminal flexible loop and AP2 required for Nef-mediated CD4 downregulation. (United States)

    Jin, Yong-Jiu; Cai, Catherine Yi; Mezei, Mihaly; Ohlmeyer, Michael; Sanchez, Roberto; Burakoff, Steven J


    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.

  15. The C-terminal polyproline-containing region of ELMO contributes to an increase in the life-time of the ELMO-DOCK complex. (United States)

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


    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.

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

    NARCIS (Netherlands)

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


    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

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


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


    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.

  18. Cdc15 Phosphorylates the C-terminal Domain of RNA Polymerase II for Transcription during Mitosis. (United States)

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


    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.

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

    Directory of Open Access Journals (Sweden)

    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.

  20. MAS C-Terminal Tail Interacting Proteins Identified by Mass Spectrometry- Based Proteomic Approach. (United States)

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


    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.

  1. C-terminal in Sp1-like artificial zinc-finger proteins plays crucial roles in determining their DNA binding affinity. (United States)

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


    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.

  2. C-terminals in the mouse branchiomotor nuclei originate from the magnocellular reticular formation. (United States)

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


    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.

  3. Age-dependent loss of the C-terminal amino acid from alpha crystallin (United States)

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


    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.

  4. FastMotif: spectral sequence motif discovery

    National Research Council Canada - National Science Library

    Colombo, Nicoló; Vlassis, Nikos


    ... datasets produced by modern high-throughput sequencing technologies. We present FastMotif, a new motif discovery algorithm that is built on a recent machine learning technique referred to as Method of Moments...

  5. 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


    .... 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...

  6. 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

  7. NMR determines transient structure and dynamics in the disordered C-terminal domain of WASp interacting protein. (United States)

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


    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.

  8. The flexible C-terminal arm of the Lassa arenavirus Z-protein mediates interactions with multiple binding partners. (United States)

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


    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.

  9. Structural investigation of a C-terminal EphA2 receptor mutant: Does mutation affect the structure and interaction properties of the Sam domain? (United States)

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


    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.

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

    Directory of Open Access Journals (Sweden)

    Noonan James P


    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.

  11. Development of a cysteine-deprived and C-terminally truncated GLP-1 receptor. (United States)

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


    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.

  12. Diuretic activity of C-terminal group analogues of the insect kinins in Acheta domesticus. (United States)

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


    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.

  13. Updating the profile of C-terminal MECP2 deletions in Rett syndrome (United States)

    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


    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

  14. 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


    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.

  15. Computational and experimental studies of the interaction between phospho-peptides and the C-terminal domain of BRCA1 (United States)

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


    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.

  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


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

    Directory of Open Access Journals (Sweden)

    P.P. Moerman


    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.

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

    DEFF Research Database (Denmark)

    Davies, Michael Jonathan


    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...

  19. 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: [Manchester Interdisciplinary Biocentre, Faculty of Life Sciences, University of Manchester, 131 Princess Street, Manchester M1 7DN (United Kingdom)


    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.

  20. The C-terminal domain of Tetrahymena thermophila telomerase holoenzyme protein p65 induces multiple structural changes in telomerase RNA (United States)

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


    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

  1. Pengembangan Motif Batik Khas Bali

    Directory of Open Access Journals (Sweden)

    Irfa'ina Rohana Salma


    Full Text Available ABSTRAKIndustri batik berkembang pesat di Bali, namun motif-motif batiknya tidak mencerminkan identitas khas daerah. Oleh karena itu perlu diciptakan desain motif batik khas Bali yang sumber inspirasinya digali budaya dan alam Bali. Tujuan penelitian dan penciptaan seni ini adalah untuk menghasilkan motif batik yang mempunyai bentuk  unik dan karakteristik sehingga dapat mencerminkan budaya dan alam Bali. Metode yang digunakan yaitu pengumpulan data, perancangan motif, perwujudan menjadi batik, serta uji estetikanya. Dari penciptaan seni ini berhasil diciptakan 5 motif batik yaitu: (1 Motif Jepun Alit; (2 Motif Jepun Ageng; (3 Motif Sekar Jagad Bali; (4 Motif Teratai Banji; dan (5 Motif Poleng Biru. Berdasarkan hasil penilaian “Selera Estetika” diketahui bahwa motif yang paling banyak disukai adalah Motif Jepun Alit, Motif Sekar Jagad Bali,  dan Motif Teratai Banji. Kata kunci: Motif Jepun Alit, Motif Jepun Ageng, Motif Sekar Jagad Bali, Motif Teratai Banji, Motif Poleng Biru ABSTRACT Batik industry is growing rapidly in Bali, but its batik motifs do not reflect the typical regional identities. Therefore, it is necessary to create a distinctive design motif source of Bali excavated  from the repertoire of traditional Balinese arts and culture. The purpose of this research and its art creation is to produce batik motifs that have a unique shape and characteristics  to reflect the Balinese culture and natural surroundings. The method used by gathering and collecting data, designing motifs to  become the embodiment of batik. From the creation of this art had been created 5 motifs, namely: (1 Motif Jepun Alit; (2 Motif Jepun Ageng; (3 Motif Sekar Jagad Bali; (4 Motif Teratai Banji; and (5 Motif Poleng Biru. Based on the results of aesthetical assessment known that the most preferred motif are  Motif Jepun Alit, Motif Sekar Jagad Bali, and Motif Teratai Banji. Key words: Motif Jepun Alit, Motif Jepun Ageng, Motif Sekar Jagad Bali, Motif

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

    Directory of Open Access Journals (Sweden)

    Tukalo M. A.


    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.

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

    DEFF Research Database (Denmark)

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


    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....

  4. Mining Conditional Phosphorylation Motifs. (United States)

    Liu, Xiaoqing; Wu, Jun; Gong, Haipeng; Deng, Shengchun; He, Zengyou


    Phosphorylation motifs represent position-specific amino acid patterns around the phosphorylation sites in the set of phosphopeptides. Several algorithms have been proposed to uncover phosphorylation motifs, whereas the problem of efficiently discovering a set of significant motifs with sufficiently high coverage and non-redundancy still remains unsolved. Here we present a novel notion called conditional phosphorylation motifs. Through this new concept, the motifs whose over-expressiveness mainly benefits from its constituting parts can be filtered out effectively. To discover conditional phosphorylation motifs, we propose an algorithm called C-Motif for a non-redundant identification of significant phosphorylation motifs. C-Motif is implemented under the Apriori framework, and it tests the statistical significance together with the frequency of candidate motifs in a single stage. Experiments demonstrate that C-Motif outperforms some current algorithms such as MMFPh and Motif-All in terms of coverage and non-redundancy of the results and efficiency of the execution. The source code of C-Motif is available at: https://sourceforge. net/projects/cmotif/.

  5. 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


    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 ...

  6. 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.


    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,

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

    Directory of Open Access Journals (Sweden)

    Raats Jos MH


    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.

  8. Urinary uromodulin carries an intact ZP domain generated by a conserved C-terminal proteolytic cleavage. (United States)

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


    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.

  9. General inverse solid-phase synthesis method for C-terminally modified peptide mimetics. (United States)

    Sasubilli, Ramakrishna; Gutheil, William G


    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.

  10. 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.


    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

  11. 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.


    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...

  12. 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 ...

  13. A C-terminal Aldehyde Analog of the Insect Kinins Inhibits Diuresis in the Housefly (United States)


    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

  14. 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


    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.

  15. 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

  16. WXG100 Protein Superfamily Consists of Three Subfamilies and Exhibits an α-Helical C-Terminal Conserved Residue Pattern (United States)

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


    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

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


    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.

  18. CAPN3-mediated processing of C-terminal titin replaced by pathological cleavage in titinopathy. (United States)

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


    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:

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

    Directory of Open Access Journals (Sweden)

    Bo Mi Kim


    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.

  20. Design, synthesis, and evaluation of Trolox-conjugated amyloid-β C-terminal peptides for therapeutic intervention in an in vitro model of Alzheimer's disease. (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


    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.

  1. Solution structure of the Arabidopsis thaliana telomeric repeat-binding protein DNA binding domain: a new fold with an additional C-terminal helix. (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


    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.

  2. FastMotif: spectral sequence motif discovery. (United States)

    Colombo, Nicoló; Vlassis, Nikos


    Sequence discovery tools play a central role in several fields of computational biology. In the framework of Transcription Factor binding studies, most of the existing motif finding algorithms are computationally demanding, and they may not be able to support the increasingly large datasets produced by modern high-throughput sequencing technologies. We present FastMotif, a new motif discovery algorithm that is built on a recent machine learning technique referred to as Method of Moments. Based on spectral decompositions, our method is robust to model misspecifications and is not prone to locally optimal solutions. We obtain an algorithm that is extremely fast and designed for the analysis of big sequencing data. On HT-Selex data, FastMotif extracts motif profiles that match those computed by various state-of-the-art algorithms, but one order of magnitude faster. We provide a theoretical and numerical analysis of the algorithm's robustness and discuss its sensitivity with respect to the free parameters. The Matlab code of FastMotif is available from Supplementary data are available at Bioinformatics online. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail:

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

    Directory of Open Access Journals (Sweden)

    Lindblad Peter


    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

  4. Isolation of influenza virus A hemagglutinin C-terminal domain by hemagglutinin proteolysis in octylglucoside micelles. (United States)

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


    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.

  5. 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


    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...

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


    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.

  7. Structural and Functional Characterization of the C-terminal Transmembrane Region of NBCe1-A* (United States)

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


    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

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


    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


    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.

  9. Structure of the Reston ebolavirus VP30 C-terminal domain. (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


    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.

  10. Influenza A hemagglutinin C-terminal anchoring peptide: identification and mass spectrometric study. (United States)

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


    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.

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

    African Journals Online (AJOL)



    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 ...

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

    Directory of Open Access Journals (Sweden)

    Jonathan D. Steckbeck


    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.

  13. 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.


    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...

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

    Directory of Open Access Journals (Sweden)

    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.

  15. Bacteriophage endolysin Lyt μ1/6: characterization of the C-terminal binding domain. (United States)

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


    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.

  16. A C-terminal Membrane Anchor Affects the Interactions of Prion Proteins with Lipid Membranes* (United States)

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


    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

  17. A C-terminal membrane anchor affects the interactions of prion proteins with lipid membranes. (United States)

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


    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.

  18. Resonance assignments and secondary structure of apolipoprotein E C-terminal domain in DHPC micelles. (United States)

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


    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.

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

    Directory of Open Access Journals (Sweden)

    Fiona K Wong


    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.

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

    Energy Technology Data Exchange (ETDEWEB)

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


    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

  1. Structural features discriminate androgen receptor N/C terminal and coactivator interactions. (United States)

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


    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.

  2. Role of p54 RNA helicase activity and its C-terminal domain in translational repression, P-body localization and assembly. (United States)

    Minshall, Nicola; Kress, Michel; Weil, Dominique; Standart, Nancy


    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.

  3. A di-arginine motif contributes to the ER localization of the type I transmembrane ER oxidoreductase TMX4

    DEFF Research Database (Denmark)

    Roth, Doris; Lynes, Emily; Riemer, Jan


    expressed in melanoma cells. Unlike many type I membrane proteins, TMX4 lacks a typical C-terminal di-lysine retrieval signal. Instead, the cytoplasmic tail has a conserved di-arginine motif of the RXR type. We show that mutation of the RQR sequence in TMX4 to KQK interferes with ER localization...

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

    Directory of Open Access Journals (Sweden)

    Guillermo Salgado-Moran


    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.

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

    Directory of Open Access Journals (Sweden)

    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.

  6. C-Terminal Truncated α-Synuclein Fibrils Contain Strongly Twisted β-Sheets. (United States)

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


    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Raaij, Mark J. van, E-mail: [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)


    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.

  8. C-terminal tyrosine residues modulate the fusion activity of the Hendra virus fusion protein. (United States)

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


    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.

  9. Two Disease-Causing SNAP-25B Mutations Selectively Impair SNARE C-terminal Assembly. (United States)

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


    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.

  10. C-Terminal acetylene derivatized peptides via silyl-based alkyne immobilization. (United States)

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


    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.

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

    DEFF Research Database (Denmark)

    Schrøder, H D


    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....

  12. 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. (United States)

    Staudt, Catherine; Gilis, Florentine; Tevel, Virginie; Jadot, Michel; Boonen, Marielle


    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.

  13. Efficient exact motif discovery. (United States)

    Marschall, Tobias; Rahmann, Sven


    The motif discovery problem consists of finding over-represented patterns in a collection of biosequences. It is one of the classical sequence analysis problems, but still has not been satisfactorily solved in an exact and efficient manner. This is partly due to the large number of possibilities of defining the motif search space and the notion of over-representation. Even for well-defined formalizations, the problem is frequently solved in an ad hoc manner with heuristics that do not guarantee to find the best motif. We show how to solve the motif discovery problem (almost) exactly on a practically relevant space of IUPAC generalized string patterns, using the p-value with respect to an i.i.d. model or a Markov model as the measure of over-representation. In particular, (i) we use a highly accurate compound Poisson approximation for the null distribution of the number of motif occurrences. We show how to compute the exact clump size distribution using a recently introduced device called probabilistic arithmetic automaton (PAA). (ii) We define two p-value scores for over-representation, the first one based on the total number of motif occurrences, the second one based on the number of sequences in a collection with at least one occurrence. (iii) We describe an algorithm to discover the optimal pattern with respect to either of the scores. The method exploits monotonicity properties of the compound Poisson approximation and is by orders of magnitude faster than exhaustive enumeration of IUPAC strings (11.8 h compared with an extrapolated runtime of 4.8 years). (iv) We justify the use of the proposed scores for motif discovery by showing our method to outperform other motif discovery algorithms (e.g. MEME, Weeder) on benchmark datasets. We also propose new motifs on Mycobacterium tuberculosis. The method has been implemented in Java. It can be obtained from

  14. 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.

  15. Evaluation of the C-Terminal Fragment of Entamoeba histolytica Gal/GalNAc Lectin Intermediate Subunit as a Vaccine Candidate against Amebic Liver Abscess. (United States)

    Min, Xiangyang; Feng, Meng; Guan, Yue; Man, Suqin; Fu, Yongfeng; Cheng, Xunjia; Tachibana, Hiroshi


    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.

  16. 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. (United States)

    Halavaty, Andrei S; Moffat, Keith


    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.

  17. Differentiation of odontoblasts is negatively regulated by MEPE via its C-terminal fragment. (United States)

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


    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.

  18. Structure of the RecQ C-terminal domain of human Bloom syndrome protein. (United States)

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


    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.

  19. The spt5 C-terminal region recruits yeast 3' RNA cleavage factor I. (United States)

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


    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.

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


    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.

  1. Effect of C-terminal domain truncation of Thermus thermophilus trehalose synthase on its substrate specificity. (United States)

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


    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.

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

    Directory of Open Access Journals (Sweden)

    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.

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


    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.

  4. 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: [EMBL Hamburg Outstation, c/o DESY, Notkestrasse 85, D-22603 Hamburg (Germany)


    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.

  5. 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.

  6. 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.

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


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


    Tobacco contains different isoforms of chitinase (EC, 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...

  8. Structural and Regulatory Elements of HCV NS5B Polymerase – β-Loop and C-Terminal Tail – Are Required for Activity of Allosteric Thumb Site II Inhibitors (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


    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

  9. Requirement for the E1 Helicase C-Terminal Domain in Papillomavirus DNA Replication In Vivo. (United States)

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


    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

  10. Nucleation process of a fibril precursor in the C-terminal segment of amyloid-β. (United States)

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


    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.

  11. The C-terminal region of E1A: a molecular tool for cellular cartography. (United States)

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


    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.

  12. Requirement for the E1 Helicase C-Terminal Domain in Papillomavirus DNA Replication In Vivo (United States)

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


    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

  13. C-terminal region of Mad2 plays an important role during mitotic spindle checkpoint in fission yeast Schizosaccharomyces pombe. (United States)

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


    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.

  14. Loss of Smad4 function in pancreatic tumors: C-terminal truncation leads to decreased stability. (United States)

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


    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.

  15. PrPSc-Specific Antibody Reveals C-Terminal Conformational Differences between Prion Strains. (United States)

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


    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.

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


    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.

  17. Characteristic NH3 and CO losses from sodiated peptides C-terminated by glutamine residues. (United States)

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


    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.

  18. Visibility graph motifs

    CERN Document Server

    Iacovacci, Jacopo


    Visibility algorithms transform time series into graphs and encode dynamical information in their topology, paving the way for graph-theoretical time series analysis as well as building a bridge between nonlinear dynamics and network science. In this work we introduce and study the concept of visibility graph motifs, smaller substructures that appear with characteristic frequencies. We develop a theory to compute in an exact way the motif profiles associated to general classes of deterministic and stochastic dynamics. We find that this simple property is indeed a highly informative and computationally efficient feature capable to distinguish among different dynamics and robust against noise contamination. We finally confirm that it can be used in practice to perform unsupervised learning, by extracting motif profiles from experimental heart-rate series and being able, accordingly, to disentangle meditative from other relaxation states. Applications of this general theory include the automatic classification a...

  19. A vector system for efficient and economical switching of C-terminal epitope tags in Saccharomyces cerevisiae. (United States)

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


    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.

  20. The C-terminal region of alpha-crystallin: involvement in protection against heat-induced denaturation (United States)

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


    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.

  1. 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


    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...

  2. The effect of C-terminal amidation on the efficacy and selectivity of antimicrobial and anticancer peptides. (United States)

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


    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.

  3. The MHC motif viewer

    DEFF Research Database (Denmark)

    Rapin, Nicolas Philippe Jean-Pierre; Hoof, Ilka; Lund, Ole


    In vertebrates, the onset of cellular immune reactions is controlled by presentation of peptides in complex with major histocompatibility complex (MHC) molecules to T cell receptors. In humans, MHCs are called human leukocyte antigens (HLAs). Different MHC molecules present different subsets...... is hampered by the lack of tools for browsing and comparing specificity of these molecules. We have developed a Web server, MHC Motif Viewer, which allows the display of the binding motif for MHC class I proteins for human, chimpanzee, rhesus monkey, mouse, and swine, as well as HLA-DR protein sequences...

  4. The arginine residue within the C-terminal active core of Bombyx mori pheromone biosynthesis-activating neuropeptide (PBAN is essential for receptor binding and activation

    Directory of Open Access Journals (Sweden)

    Takeshi eKawai


    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.

  5. Identification of two pentatricopeptide repeat genes required for RNA editing and zinc binding by C-terminal cytidine deaminase-like domains. (United States)

    Hayes, Michael L; Giang, Karolyn; Berhane, Beniam; Mulligan, R Michael


    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.

  6. The C-terminal region of the non-structural protein 2B from Hepatitis A Virus demonstrates lipid-specific viroporin-like activity (United States)

    Shukla, Ashutosh; Dey, Debajit; Banerjee, Kamalika; Nain, Anshu; Banerjee, Manidipa


    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.

  7. C-Terminal Repeats of Clostridium difficile Toxin A Induce Production of Chemokine and Adhesion Molecules in Endothelial Cells and Promote Migration of Leukocytes▿ (United States)

    Yeh, Chiou-Yueh; Lin, Chun-Nan; Chang, Chuan-Fa; Lin, Chun-Hung; Lien, Huei-Ting; Chen, Jen-Yang; Chia, Jean-San


    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. C-Terminal 23 kDa polypeptide of soybean Gly m Bd 28 K is a potential allergen. (United States)

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


    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. MHC motif viewer

    DEFF Research Database (Denmark)

    Rapin, Nicolas Philippe Jean-Pierre; Hoof, Ilka; Lund, Ole


    viewer, that allows the display of the likely binding motif for all human class I proteins of the loci HLA A, B, C, and E and for MHC class I molecules from chimpanzee (Pan troglodytes), rhesus monkey (Macaca mulatta), and mouse (Mus musculus). Furthermore, it covers all HLA-DR protein sequences...

  10. Fingerprint motifs of phytases

    African Journals Online (AJOL)

    Fan CM


    Mar 6, 2013 ... unique sequences including 131 prokaryotic and 102 eukaryotic phytase sequences covered phytases from. 190 species including 131 bacterium sequences, 70 fungus sequences, 27 plant sequences, one animal sequence and four yeast sequences. For motif analysis, 54 sequences were randomly.

  11. [Personal motif in art]. (United States)

    Gerevich, József


    One of the basic questions of the art psychology is whether a personal motif is to be found behind works of art and if so, how openly or indirectly it appears in the work itself. Analysis of examples and documents from the fine arts and literature allow us to conclude that the personal motif that can be identified by the viewer through symbols, at times easily at others with more difficulty, gives an emotional plus to the artistic product. The personal motif may be found in traumatic experiences, in communication to the model or with other emotionally important persons (mourning, disappointment, revenge, hatred, rivalry, revolt etc.), in self-searching, or self-analysis. The emotions are expressed in artistic activity either directly or indirectly. The intention nourished by the artist's identity (Kunstwollen) may stand in the way of spontaneous self-expression, channelling it into hidden paths. Under the influence of certain circumstances, the artist may arouse in the viewer, consciously or unconsciously, an illusionary, misleading image of himself. An examination of the personal motif is one of the important research areas of art therapy.

  12. Sequence-specific high mobility group box factors recognize 10-12-base pair minor groove motifs

    DEFF Research Database (Denmark)

    van Beest, M; Dooijes, D; van De Wetering, M


    , 12, and 10 base pairs, respectively. Footprinting with a deletion mutant of Ste11 reveals a novel interaction between the 3' base pairs of the extended DNA motif and amino acids C-terminal to the HMG domain. The sequence-specific interaction of Ste11 with these 3' base pairs contributes significantly......Sequence-specific high mobility group (HMG) box factors bind and bend DNA via interactions in the minor groove. Three-dimensional NMR analyses have provided the structural basis for this interaction. The cognate HMG domain DNA motif is generally believed to span 6-8 bases. However, alignment...... of promoter elements controlled by the yeast genes ste11 and Rox1 has indicated strict conservation of a larger DNA motif. By site selection, we identify a highly specific 12-base pair motif for Ste11, AGAACAAAGAAA. Similarly, we show that Tcf1, MatMc, and Sox4 bind unique, highly specific DNA motifs of 12...

  13. [C-terminal lysosome targeting domain of CD63 modifies cellular localization of rabies virus glycoprotein]. (United States)

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


    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.

  14. 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: [Department of Biotechnology, College of Natural Sciences, Chosun University, Gwangju 501-759 (Korea, Republic of); Lee, Weontae, E-mail: [Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749 (Korea, Republic of)


    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

  15. 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.


    ) 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...

  16. 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


    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....

  17. The E3 Ligase CHIP: Insights into Its Structure and Regulation (United States)

    Paul, Indranil; Ghosh, Mrinal K.


    The carboxy-terminus of Hsc70 interacting protein (CHIP) is a cochaperone E3 ligase containing three tandem repeats of tetratricopeptide (TPR) motifs and a C-terminal U-box domain separated by a charged coiled-coil region. CHIP is known to function as a central quality control E3 ligase and regulates several proteins involved in a myriad of physiological and pathological processes. Recent studies have highlighted varied regulatory mechanisms operating on the activity of CHIP which is crucial for cellular homeostasis. In this review article, we give a concise account of our current knowledge on the biochemistry and regulation of CHIP. PMID:24868554

  18. ActiveMotif: Interactive motif discovery with human feedback. (United States)

    Younghoon Kim; Woonghee Lee; Keonwoo Kim


    Motif detection, which is to discover short patterns involved in many important biological processes, has been recently raised as an important task in bioinformatics. The traditional algorithms to find a sequence motif have been developed using machine learning only without involving the experience and domain knowledge of human experts effectively. In this paper, we propose an interactive motif discovery system by introducing a new learning algorithm, by generalizing a well-known statistical motif model, whose inference can be shepherded by human feedback.

  19. Functional stabilization of an RNA recognition motif by a noncanonical N-terminal expansion. (United States)

    Netter, Catharina; Weber, Gert; Benecke, Heike; Wahl, Markus C


    RNA recognition motifs (RRMs) constitute versatile macromolecular interaction platforms. They are found in many components of spliceosomes, in which they mediate RNA and protein interactions by diverse molecular strategies. The human U11/U12-65K protein of the minor spliceosome employs a C-terminal RRM to bind hairpin III of the U12 small nuclear RNA (snRNA). This interaction comprises one side of a molecular bridge between the U11 and U12 small nuclear ribonucleoprotein particles (snRNPs) and is reminiscent of the binding of the N-terminal RRMs in the major spliceosomal U1A and U2B'' proteins to hairpins in their cognate snRNAs. Here we show by mutagenesis and electrophoretic mobility shift assays that the beta-sheet surface and a neighboring loop of 65K C-terminal RRM are involved in RNA binding, as previously seen in canonical RRMs like the N-terminal RRMs of the U1A and U2B'' proteins. However, unlike U1A and U2B'', some 30 residues N-terminal of the 65K C-terminal RRM core are additionally required for stable U12 snRNA binding. The crystal structure of the expanded 65K C-terminal RRM revealed that the N-terminal tail adopts an alpha-helical conformation and wraps around the protein toward the face opposite the RNA-binding platform. Point mutations in this part of the protein had only minor effects on RNA affinity. Removal of the N-terminal extension significantly decreased the thermal stability of the 65K C-terminal RRM. These results demonstrate that the 65K C-terminal RRM is augmented by an N-terminal element that confers stability to the domain, and thereby facilitates stable RNA binding.

  20. Inherited germline TP53 mutation encodes a protein with an aberrant C-terminal motif in a case of pediatric adrenocortical tumor


    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.


    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-p...

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

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


    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. Characterization of AWAP IV, the C-terminal domain of the avian protein AWAK. (United States)

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


    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.

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


    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.

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

    Directory of Open Access Journals (Sweden)

    Sunny Li-Yun Chang


    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.

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

    Directory of Open Access Journals (Sweden)

    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.

  6. Impedance Analysis of Ovarian Cancer Cells upon Challenge with C-terminal Clostridium Perfringens Enterotoxin (United States)

    Gordon, Geoffrey; Lo, Chun-Min


    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.

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

    Directory of Open Access Journals (Sweden)

    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.

  8. 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


    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...

  9. 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


    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...

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

    Directory of Open Access Journals (Sweden)

    Merkx Maarten


    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. 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.


    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.

  12. 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.


    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...

  13. Topological generalizations of network motifs (United States)

    Kashtan, N.; Itzkovitz, S.; Milo, R.; Alon, U.


    Biological and technological networks contain patterns, termed network motifs, which occur far more often than in randomized networks. Network motifs were suggested to be elementary building blocks that carry out key functions in the network. It is of interest to understand how network motifs combine to form larger structures. To address this, we present a systematic approach to define “motif generalizations”: families of motifs of different sizes that share a common architectural theme. To define motif generalizations, we first define “roles” in a subgraph according to structural equivalence. For example, the feedforward loop triad—a motif in transcription, neuronal, and some electronic networks—has three roles: an input node, an output node, and an internal node. The roles are used to define possible generalizations of the motif. The feedforward loop can have three simple generalizations, based on replicating each of the three roles and their connections. We present algorithms for efficiently detecting motif generalizations. We find that the transcription networks of bacteria and yeast display only one of the three generalizations, the multi-output feedforward generalization. In contrast, the neuronal network of C. elegans mainly displays the multi-input generalization. Forward-logic electronic circuits display a multi-input, multi-output hybrid. Thus, networks which share a common motif can have very different generalizations of that motif. Using mathematical modeling, we describe the information processing functions of the different motif generalizations in transcription, neuronal, and electronic networks.

  14. Effect of copper variation in yeast hydrolysate on C-terminal lysine levels of a monoclonal antibody. (United States)

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


    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.

  15. Trypanosoma evansi: identification and characterization of a variant surface glycoprotein lacking cysteine residues in its C-terminal domain. (United States)

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


    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.

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


    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.

  17. Small epitope-linker modules for PCR-based C-terminal tagging in Saccharomyces cerevisiae. (United States)

    Funakoshi, Minoru; Hochstrasser, Mark


    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 (

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


    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.

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

    Directory of Open Access Journals (Sweden)

    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.

  20. Cooperative cold denaturation: the case of the C-terminal domain of ribosomal protein L9. (United States)

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


    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.

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

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


    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.

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

    Directory of Open Access Journals (Sweden)

    Adam Z. Blatt


    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.

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

    Directory of Open Access Journals (Sweden)

    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.

  4. 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.

  5. CD3 gamma contains a phosphoserine-dependent di-leucine motif involved in down-regulation of the T cell receptor

    DEFF Research Database (Denmark)

    Dietrich, J; Hou, X; Wegener, A M


    -regulation of the TCR. Furthermore, analysis of a series of CD3 gamma truncation mutants indicated that in addition to S126 phosphorylation a motif C-terminal of S126 was required for TCR down-regulation. Point mutation analyses confirmed this observation and demonstrated that a membrane-proximal di-leucine motif (L131......, indicating that the TCR was down-regulated by endocytosis via clathrin coated pits. Based on the present results and previously published observations on intracellular receptor sorting, a general model for intracellular sorting of receptors containing di-leucine- or tyrosine-based motifs is proposed....

  6. A phosphoserine/threonine-binding pocket in AGC kinases and PDK1 mediates activation by hydrophobic motif phosphorylation

    DEFF Research Database (Denmark)

    Frödin, Morten; Antal, Torben L; Dümmler, Bettina A


    The growth factor-activated AGC protein kinases RSK, S6K, PKB, MSK and SGK are activated by serine/threonine phosphorylation in the activation loop and in the hydrophobic motif, C-terminal to the kinase domain. In some of these kinases, phosphorylation of the hydrophobic motif creates a specific...... docking site that recruits and activates PDK1, which then phosphorylates the activation loop. Here, we discover a pocket in the kinase domain of PDK1 that recognizes the phosphoserine/phosphothreonine in the hydrophobic motif by identifying two oppositely positioned arginine and lysine residues that bind...... in which the phosphorylated hydrophobic motif and activation loop act on the alphaC-helix of the kinase structure to induce synergistic stimulation of catalytic activity. Sequence conservation suggests that this mechanism is a key feature in activation of >40 human AGC kinases....

  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. (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


    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 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.


    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.

  9. 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.


    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

  10. Ras GAP-related and C-terminal domain-dependent localization and tumorigenic activities of IQGAP1 in melanoma cells.

    Directory of Open Access Journals (Sweden)

    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.

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

    Directory of Open Access Journals (Sweden)

    Yen-Hua Huang


    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.

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


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


    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...

  13. Hemoglobin Cochin-Port-Royal: consequences of the replacement of the beta chain C-terminal by an arginine. (United States)

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


    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.

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


    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.

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

    Directory of Open Access Journals (Sweden)

    Stefania Trazzi


    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.

  16. The C-terminal Six Amino Acids of the FNT Channel FocA Are Required for Formate Translocation But Not Homopentamer Integrity. (United States)

    Hunger, Doreen; Röcker, Marie; Falke, Dörte; Lilie, Hauke; Sawers, R Gary


    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

  17. Novel protein domains and motifs in the marine planctomycete Rhodopirellula baltica. (United States)

    Studholme, David J; Fuerst, John A; Bateman, Alex


    The planctomycetes are a phylum of bacteria that have a unique cell compartmentalisation and yeast-like budding cell division and peptidoglycan-less proteinaceous cell walls. We wished to further our understanding of these unique organisms at the molecular level by searching for conserved amino acid sequence motifs and domains in the proteins encoded by Rhodopirellula baltica. Using BLAST and single-linkage clustering, we have discovered several new protein domains and sequence motifs in this planctomycete. R. baltica has multiple members of the newly discovered GEFGR protein family and the ASPIC C-terminal domain family, whilst most other organisms for which whole genome sequence is available have no more than one. Many of the domains and motifs appear to be restricted to the planctomycetes. It is possible that these protein domains and motifs may have been lost or replaced in other phyla, or they may have undergone multiple duplication events in the planctomycete lineage. One of the novel motifs probably represents a novel N-terminal export signal peptide. With their unique cell biology, it may be that the planctomycete cell compartmentalisation plan in particular needs special membrane transport mechanisms. The discovery of these new domains and motifs, many of which are associated with secretion and cell-surface functions, will help to stimulate experimental work and thus enhance further understanding of this fascinating group of organisms.

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


    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.

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

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


    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.

  20. 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.


    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.

  1. Insights into the oligomerization process of the C-terminal domain of human plasma membrane Ca²+-ATPase. (United States)

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


    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.

  2. The C-terminal part of microcin B is crucial for DNA gyrase inhibition and antibiotic uptake by sensitive cells. (United States)

    Shkundina, Irina; Serebryakova, Marina; Severinov, Konstantin


    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.

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


    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.

  4. Influence of C-terminal tail deletion on structure and stability of hyperthermophile Sulfolobus tokodaii RNase HI. (United States)

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


    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.

  5. Tandem duplications in the C-terminal domain of the mesotocin receptor exclusively identified among East Eurasian thrushes. (United States)

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


    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.

  6. 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


    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.

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

    Directory of Open Access Journals (Sweden)

    Merkel George


    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

  8. Structural alphabet motif discovery and a structural motif database. (United States)

    Ku, Shih-Yen; Hu, Yuh-Jyh


    This study proposes a general framework for structural motif discovery. The framework is based on a modular design in which the system components can be modified or replaced independently to increase its applicability to various studies. It is a two-stage approach that first converts protein 3D structures into structural alphabet sequences, and then applies a sequence motif-finding tool to these sequences to detect conserved motifs. We named the structural motif database we built the SA-Motifbase, which provides the structural information conserved at different hierarchical levels in SCOP. For each motif, SA-Motifbase presents its 3D view; alphabet letter preference; alphabet letter frequency distribution; and the significance. SA-Motifbase is available at Copyright © 2011 Elsevier Ltd. All rights reserved.

  9. Cornelia de Lange individuals with new and recurrent SMC1A mutations enhance delineation of mutation repertoire and phenotypic spectrum. (United States)

    Gervasini, Cristina; Russo, Silvia; Cereda, Anna; Parenti, Ilaria; Masciadri, Maura; Azzollini, Jacopo; Melis, Daniela; Aravena, Teresa; Doray, Bérénice; Ferrarini, Alessandra; Garavelli, Livia; Selicorni, Angelo; Larizza, Lidia


    We report on the clinical and molecular characterization of eight patients, one male and seven females, with clinical diagnosis of Cornelia de Lange syndrome (CdLS), who were found to carry distinct mutations of the SMC1A gene. Five of the eight mutations are novel, with two involving amino acid residues previously described as altered in a different way. The other three have been reported each in a single case. Comparison of pairs of individuals with the same mutation indicates only partial overlap of their clinical phenotypes. The following novel missense mutations, all affecting highly conserved amino acid residues, were found: p.R398G in the N-terminal coiled-coil domain, p.V651M in the C-terminal coiled-coil/hinge junction, p.R693G in the C-terminal coiled-coil, and p.N1166T and p.L1189F in the C-terminal ABC cassette. The latter is localized in the H-loop, and represents the first mutation involving a functional motif of SMC1A protein. The effect of the mutations on SMC1A protein function has been predicted using four bioinformatic tools. All mutations except p.V651M were scored as pathogenic by three or four of the tools. p.V651M was found in the only male individual of our cohort, who presented with the most severe phenotype. This raises the issue of gender effect when addressing mutation-phenotype correlation for genes such as SMC1A, which incompletely escapes X-inactivation. Our clinical and molecular findings expand the total number of characterized SMC1A-mutated patients (from 44 to 52) and the restricted repertoire of SMC1A mutations (from 29 to 34), contributing to the molecular and clinical signature of SMC1A-based CdLS. © 2013 Wiley Periodicals, Inc.

  10. Self-regulation of functional pathways by motifs inside the disordered tails of beta-catenin. (United States)

    Zhao, Bi; Xue, Bin


    Beta-catenin has two major functions: coordinating cell-cell adhesion by interacting with cadherin in cadherin junction formation pathway; and regulating gene expression through Wnt signaling pathway. Accomplishing these two functions requires synergistic action of various sequential regions of the same beta-Catenin molecule, including the N-terminal tail, the middle armadillo domain, and the C-terminal tail. Although the middle armadillo domain is the major functional unit of beta-Catenin, the involvement of tails in the regulation of interaction between beta-Catenin and its partners has been well observed. Nonetheless, the regulatory processes of both tails are still elusive. In addition, it is interesting to note that the three sequential regions have different structural features: The middle armadillo domain is structured, but both N- and C-terminal tails are disordered. This observation leads to another important question on the functions and mechanisms of disordered tails, which is also largely unknown. In this study, we focused on the characterization of sequential, structural, and functional features of the disordered tails of beta-Catenin. We identified multiple functional motifs and conserved sequence motifs in the disordered tails, discovered the correlation between cancer-associated mutations and functional motifs, explored the abundance of protein intrinsic disorder in the interactomes of beta-Catenin, and elaborated a working model on the regulatory roles of disordered tails in the functional pathways of beta-Catenin. Disordered tails of beta-Catenin contain multiple functional motifs. These motifs interact with each other and the armadillo domain of beta-catenin to regulate the function of beta-Catenin in both cadherin junction formation pathway and Wnt signaling pathway.

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

    Directory of Open Access Journals (Sweden)

    Britta Hansmann


    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.

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


    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.

  13. Silyl-based alkyne-modifying linker for the preparation of C-terminal acetylene-derivatized protected peptides. (United States)

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


    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.

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


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


    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 ...

  15. 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:


    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

  16. Side-chain anchoring strategy for solid-phase synthesis of peptide acids with C-terminal cysteine. (United States)

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


    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

  17. EEVD motif of heat shock cognate protein 70 contributes to bacterial uptake by trophoblast giant cells

    Directory of Open Access Journals (Sweden)

    Kim Suk


    Full Text Available Abstract Background The uptake of abortion-inducing pathogens by trophoblast giant (TG cells is a key event in infectious abortion. However, little is known about phagocytic functions of TG cells against the pathogens. Here we show that heat shock cognate protein 70 (Hsc70 contributes to bacterial uptake by TG cells and the EEVD motif of Hsc70 plays an important role in this. Methods Brucella abortus and Listeria monocytogenes were used as the bacterial antigen in this study. Recombinant proteins containing tetratricopeptide repeat (TPR domains were constructed and confirmation of the binding capacity to Hsc70 was assessed by ELISA. The recombinant TPR proteins were used for investigation of the effect of TPR proteins on bacterial uptake by TG cells and on pregnancy in mice. Results The monoclonal antibody that inhibits bacterial uptake by TG cells reacted with the EEVD motif of Hsc70. Bacterial TPR proteins bound to the C-terminal of Hsc70 through its EEVD motif and this binding inhibited bacterial uptake by TG cells. Infectious abortion was also prevented by blocking the EEVD motif of Hsc70. Conclusions Our results demonstrate that surface located Hsc70 on TG cells mediates the uptake of pathogenic bacteria and proteins containing the TPR domain inhibit the function of Hsc70 by binding to its EEVD motif. These molecules may be useful in the development of methods for preventing infectious abortion.

  18. A conserved motif mediates both multimer formation and allosteric activation of phosphoglycerate mutase 5. (United States)

    Wilkins, Jordan M; McConnell, Cyrus; Tipton, Peter A; Hannink, Mark


    Phosphoglycerate mutase 5 (PGAM5) is an atypical mitochondrial Ser/Thr phosphatase that modulates mitochondrial dynamics and participates in both apoptotic and necrotic cell death. The mechanisms that regulate the phosphatase activity of PGAM5 are poorly understood. The C-terminal phosphoglycerate mutase domain of PGAM5 shares homology with the catalytic domains found in other members of the phosphoglycerate mutase family, including a conserved histidine that is absolutely required for catalytic activity. However, this conserved domain is not sufficient for maximal phosphatase activity. We have identified a highly conserved amino acid motif, WDXNWD, located within the unique N-terminal region, which is required for assembly of PGAM5 into large multimeric complexes. Alanine substitutions within the WDXNWD motif abolish the formation of multimeric complexes and markedly reduce phosphatase activity of PGAM5. A peptide containing the WDXNWD motif dissociates the multimeric complex and reduces but does not fully abolish phosphatase activity. Addition of the WDXNWD-containing peptide in trans to a mutant PGAM5 protein lacking the WDXNWD motif markedly increases phosphatase activity of the mutant protein. Our results are consistent with an intermolecular allosteric regulation mechanism for the phosphatase activity of PGAM5, in which the assembly of PGAM5 into multimeric complexes, mediated by the WDXNWD motif, results in maximal activation of phosphatase activity. Our results suggest the possibility of identifying small molecules that function as allosteric regulators of the phosphatase activity of PGAM5. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  19. 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.


    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...

  20. 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


    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.

  1. The C-terminal extension of human telomerase reverse transcriptase is necessary for high affinity binding to telomeric DNA. (United States)

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


    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.

  2. Function and control of RNA polymerase II C-terminal domain phosphorylation in vertebrate transcription and RNA processing. (United States)

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


    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.

  3. Contribution of chitinase A's C-terminal vacuolar sorting determinant to the study of soluble protein compartmentation. (United States)

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


    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. 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


    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.

  5. 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.

  6. Crystal structures reveal a thiol protease-like catalytic triad in the C-terminal region of Pasteurella multocida toxin. (United States)

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


    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.

  7. Production and characterization of N- and C-terminally truncated Mtx2: a mosquitocidal toxin from Bacillus sphaericus. (United States)

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


    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.

  8. PCSK9-mediated degradation of the LDL receptor generates a 17 kDa C-terminal LDL receptor fragment. (United States)

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


    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.

  9. An alternative outer membrane secretion mechanism for an autotransporter protein lacking a C-terminal stable core. (United States)

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


    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.

  10. An isoform of Arabidopsis myosin XI interacts with small GTPases in its C-terminal tail region (United States)

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


    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

  11. 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.

  12. 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.

  13. An Essential Role for the Glut1 PDZ-Binding Motif in Growth Factor Regulation of Glut1 Degradation and Trafficking


    Wieman, Heather L.; Horn, Sarah R.; Jacobs, Sarah R.; Altman, Brian J.; Kornbluth, Sally; Rathmell, Jeffrey C.


    Cell surface localization of the glucose transporter, Glut1, is a cytokine-controlled process essential to support the metabolism and survival of hematopoietic cells. Molecular mechanisms that regulate Glut1 trafficking, however, are not certain. Here we show a C-terminal PDZ-binding motif in Glut1 is critical to promote maximal cytokine-stimulated Glut1 cell surface localization and prevent Glut1 lysosomal degradation in the absence of growth factor. Disruption of this PDZ-binding sequence t...

  14. Structural and dynamic properties of the C-terminal region of the Escherichia coli RNA chaperone Hfq: integrative experimental and computational studies. (United States)

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


    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.

  15. Localization of Daucus carota NMCP1 to the nuclear periphery: the role of the N-terminal region and an NLS-linked sequence motif, RYNLRR, in the tail domain

    Directory of Open Access Journals (Sweden)

    Yuta eKimura


    Full Text Available Recent ultrastructural studies revealed that a structure similar to the vertebrate nuclear lamina exists in the nuclei of higher plants. However, plant genomes lack genes for lamins and intermediate-type filament proteins, and this suggests that plant-specific nuclear coiled-coil proteins make up the lamina-like structure in plants. NMCP1 is a protein, first identified in Daucus carota cells, that localizes exclusively to the nuclear periphery in interphase cells. It has a tripartite structure comprised of head, rod, and tail domains, and includes putative nuclear localization signal (NLS motifs. We identified the functional NLS of DcNMCP1 (carrot NMCP1 and determined the protein regions required for localizing to the nuclear periphery using EGFP-fused constructs transiently expressed in Apium graveolens epidermal cells. Transcription was driven under a CaMV35S promoter, and the genes were introduced into the epidermal cells by a DNA-coated microprojectile delivery system. Of the NLS motifs, KRRRK and RRHK in the tail domain were highly functional for nuclear localization. Addition of the N-terminal 141 amino acids from DcNMCP1 shifted the localization of a region including these NLSs from the entire nucleus to the nuclear periphery. Using this same construct, the replacement of amino acids in RRHK or its preceding sequence, YNL, with alanine residues abolished localization to the nuclear periphery, while replacement of KRRRK did not affect localization. The sequence R/Q/HYNLRR/H, including YNL and the first part of the sequence of RRHK, is evolutionarily conserved in a subclass of NMCP1 sequences from many plant species. These results show that NMCP1 localizes to the nuclear periphery by a combined action of a sequence composed of R/Q/HYNLRR/H, NLS, and the N-terminal region including the head and a portion of the rod domain, suggesting that more than one binding site is implicated in localization of NMCP1.

  16. A cluster refinement algorithm for motif discovery. (United States)

    Li, Gang; Chan, Tak-Ming; Leung, Kwong-Sak; Lee, Kin-Hong


    Finding Transcription Factor Binding Sites, i.e., motif discovery, is crucial for understanding the gene regulatory relationship. Motifs are weakly conserved and motif discovery is an NP-hard problem. We propose a new approach called Cluster Refinement Algorithm for Motif Discovery (CRMD). CRMD employs a flexible statistical motif model allowing a variable number of motifs and motif instances. CRMD first uses a novel entropy-based clustering to find complete and good starting candidate motifs from the DNA sequences. CRMD then employs an effective greedy refinement to search for optimal motifs from the candidate motifs. The refinement is fast, and it changes the number of motif instances based on the adaptive thresholds. The performance of CRMD is further enhanced if the problem has one occurrence of motif instance per sequence. Using an appropriate similarity test of motifs, CRMD is also able to find multiple motifs. CRMD has been tested extensively on synthetic and real data sets. The experimental results verify that CRMD usually outperforms four other state-of-the-art algorithms in terms of the qualities of the solutions with competitive computing time. It finds a good balance between finding true motif instances and screening false motif instances, and is robust on problems of various levels of difficulty.

  17. 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)


    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.

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

    Directory of Open Access Journals (Sweden)

    Jarábková Veronika


    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.

  19. α-Helical to β-Helical Conformation Change in the C-Terminal of the Mammalian Prion Protein (United States)

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


    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.

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

    Directory of Open Access Journals (Sweden)

    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.

  1. Transactivation activity and nucleocytoplasmic transport of β-catenin are independently regulated by its C-terminal end. (United States)

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


    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.

  2. Regulation of fibrinolysis by C-terminal lysines operates through plasminogen and plasmin but not tissue-type plasminogen activator. (United States)

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


    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.

  3. 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


    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.

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


    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.

  5. Unravelling daily human mobility motifs. (United States)

    Schneider, Christian M; Belik, Vitaly; Couronné, Thomas; Smoreda, Zbigniew; González, Marta C


    Human mobility is differentiated by time scales. While the mechanism for long time scales has been studied, the underlying mechanism on the daily scale is still unrevealed. Here, we uncover the mechanism responsible for the daily mobility patterns by analysing the temporal and spatial trajectories of thousands of persons as individual networks. Using the concept of motifs from network theory, we find only 17 unique networks are present in daily mobility and they follow simple rules. These networks, called here motifs, are sufficient to capture up to 90 per cent of the population in surveys and mobile phone datasets for different countries. Each individual exhibits a characteristic motif, which seems to be stable over several months. Consequently, daily human mobility can be reproduced by an analytically tractable framework for Markov chains by modelling periods of high-frequency trips followed by periods of lower activity as the key ingredient.

  6. 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. (United States)

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


    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.

  7. A glycine zipper motif mediates the formation of toxic β-amyloid oligomers in vitro and in vivo

    Directory of Open Access Journals (Sweden)

    Fan Emily Y


    Full Text Available Abstract Background The β-amyloid peptide (Aβ contains a Gly-XXX-Gly-XXX-Gly motif in its C-terminal region that has been proposed to form a "glycine zipper" that drives the formation of toxic Aβ oligomers. We have tested this hypothesis by examining the toxicity of Aβ variants containing substitutions in this motif using a neuronal cell line, primary neurons, and a transgenic C. elegans model. Results We found that a Gly37Leu substitution dramatically reduced Aβ toxicity in all models tested, as measured by cell dysfunction, cell death, synaptic alteration, or tau phosphorylation. We also demonstrated in multiple models that Aβ Gly37Leu is actually anti-toxic, thereby supporting the hypothesis that interference with glycine zipper formation blocks assembly of toxic Aβ oligomers. To test this model rigorously, we engineered second site substitutions in Aβ predicted by the glycine zipper model to compensate for the Gly37Leu substitution and expressed these in C. elegans. We show that these second site substitutions restore in vivo Aβtoxicity, further supporting the glycine zipper model. Conclusions Our structure/function studies support the view that the glycine zipper motif present in the C-terminal portion of Aβ plays an important role in the formation of toxic Aβ oligomers. Compounds designed to interfere specifically with formation of the glycine zipper could have therapeutic potential.

  8. Biological network motif detection and evaluation. (United States)

    Kim, Wooyoung; Li, Min; Wang, Jianxin; Pan, Yi


    Molecular level of biological data can be constructed into system level of data as biological networks. Network motifs are defined as over-represented small connected subgraphs in networks and they have been used for many biological applications. Since network motif discovery involves computationally challenging processes, previous algorithms have focused on computational efficiency. However, we believe that the biological quality of network motifs is also very important. We define biological network motifs as biologically significant subgraphs and traditional network motifs are differentiated as structural network motifs in this paper. We develop five algorithms, namely, EDGEGO-BNM, EDGEBETWEENNESS-BNM, NMF-BNM, NMFGO-BNM and VOLTAGE-BNM, for efficient detection of biological network motifs, and introduce several evaluation measures including motifs included in complex, motifs included in functional module and GO term clustering score in this paper. Experimental results show that EDGEGO-BNM and EDGEBETWEENNESS-BNM perform better than existing algorithms and all of our algorithms are applicable to find structural network motifs as well. We provide new approaches to finding network motifs in biological networks. Our algorithms efficiently detect biological network motifs and further improve existing algorithms to find high quality structural network motifs, which would be impossible using existing algorithms. The performances of the algorithms are compared based on our new evaluation measures in biological contexts. We believe that our work gives some guidelines of network motifs research for the biological networks.

  9. Biological network motif detection and evaluation

    Directory of Open Access Journals (Sweden)

    Kim Wooyoung


    Full Text Available Abstract Background Molecular level of biological data can be constructed into system level of data as biological networks. Network motifs are defined as over-represented small connected subgraphs in networks and they have been used for many biological applications. Since network motif discovery involves computationally challenging processes, previous algorithms have focused on computational efficiency. However, we believe that the biological quality of network motifs is also very important. Results We define biological network motifs as biologically significant subgraphs and traditional network motifs are differentiated as structural network motifs in this paper. We develop five algorithms, namely, EDGEGO-BNM, EDGEBETWEENNESS-BNM, NMF-BNM, NMFGO-BNM and VOLTAGE-BNM, for efficient detection of biological network motifs, and introduce several evaluation measures including motifs included in complex, motifs included in functional module and GO term clustering score in this paper. Experimental results show that EDGEGO-BNM and EDGEBETWEENNESS-BNM perform better than existing algorithms and all of our algorithms are applicable to find structural network motifs as well. Conclusion We provide new approaches to finding network motifs in biological networks. Our algorithms efficiently detect biological network motifs and further improve existing algorithms to find high quality structural network motifs, which would be impossible using existing algorithms. The performances of the algorithms are compared based on our new evaluation measures in biological contexts. We believe that our work gives some guidelines of network motifs research for the biological networks.

  10. The BsaHI restriction-modification system: Cloning, sequencing and analysis of conserved motifs

    Directory of Open Access Journals (Sweden)

    Roberts Richard J


    Full Text Available Abstract Background Restriction and modification enzymes typically recognise short DNA sequences of between two and eight bases in length. Understanding the mechanism of this recognition represents a significant challenge that we begin to address for the BsaHI restriction-modification system, which recognises the six base sequence GRCGYC. Results The DNA sequences of the genes for the BsaHI methyltransferase, bsaHIM, and restriction endonuclease, bsaHIR, have been determined (GenBank accession #EU386360, cloned and expressed in E. coli. Both the restriction endonuclease and methyltransferase enzymes share significant similarity with a group of 6 other enzymes comprising the restriction-modification systems HgiDI and HgiGI and the putative HindVP, NlaCORFDP, NpuORFC228P and SplZORFNP restriction-modification systems. A sequence alignment of these homologues shows that their amino acid sequences are largely conserved and highlights several motifs of interest. We target one such conserved motif, reading SPERRFD, at the C-terminal end of the bsaHIR gene. A mutational analysis of these amino acids indicates that the motif is crucial for enzymatic activity. Sequence alignment of the methyltransferase gene reveals a short motif within the target recognition domain that is conserved among enzymes recognising the same sequences. Thus, this motif may be used as a diagnostic tool to define the recognition sequences of the cytosine C5 methyltransferases. Conclusion We have cloned and sequenced the BsaHI restriction and modification enzymes. We have identified a region of the R. BsaHI enzyme that is crucial for its activity. Analysis of the amino acid sequence of the BsaHI methyltransferase enzyme led us to propose two new motifs that can be used in the diagnosis of the recognition sequence of the cytosine C5-methyltransferases.

  11. Motif signatures of transcribed enhancers

    KAUST Repository

    Kleftogiannis, Dimitrios


    In mammalian cells, transcribed enhancers (TrEn) play important roles in the initiation of gene expression and maintenance of gene expression levels in spatiotemporal manner. One of the most challenging questions in biology today is how the genomic characteristics of enhancers relate to enhancer activities. This is particularly critical, as several recent studies have linked enhancer sequence motifs to specific functional roles. To date, only a limited number of enhancer sequence characteristics have been investigated, leaving space for exploring the enhancers genomic code in a more systematic way. To address this problem, we developed a novel computational method, TELS, aimed at identifying predictive cell type/tissue specific motif signatures. We used TELS to compile a comprehensive catalog of motif signatures for all known TrEn identified by the FANTOM5 consortium across 112 human primary cells and tissues. Our results confirm that distinct cell type/tissue specific motif signatures characterize TrEn. These signatures allow discriminating successfully a) TrEn from random controls, proxy of non-enhancer activity, and b) cell type/tissue specific TrEn from enhancers expressed and transcribed in different cell types/tissues. TELS codes and datasets are publicly available at

  12. MEET: motif elements estimation toolkit. (United States)

    Pairó, Erola; Maynou, Joan; Vallverdú, Montserrat; Caminal, Pere; Marco, Santiago; Perera, Alexandre


    MEET is an R package that integrates a set of algorithms for the detection of transcription factor binding sites (TFBS). The MEET R package includes five motif searching algorithms: MEME/MAST(Multiple Expectation-Maximization for Motif Elicitation), Q-residuals, MDscan (Motif Discovery scan), ITEME (Information Theory Elements for Motif Estimation) and MATCH. In addition MEET allows the user to work with different alignment algorithms: MUSCLE (Multiple Sequence Comparison by Log-Expectation), ClustalW and MEME. The package can work in two modes, training and detection. The training mode allows the user to choose the best parameters of a detector. Once the parameters are chosen, the detection mode allows to analyze a genome looking for binding sites. Both modes can combine the different alignment and detection methods, offering multiple possibilities. Combining the alignments and the detection algorithms makes possible the comparison between detection models at the same level, without having to care about the differences produced during the alignment process. The MEET R package can be downloaded from tar.gz.

  13. The C-terminal Region and SUMOylation of Cockayne Syndrome Group B Protein Play Critical Roles in Transcription-coupled Nucleotide Excision Repair. (United States)

    Sin, Yooksil; Tanaka, Kiyoji; Saijo, Masafumi


    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.

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

    Sin, Yooksil; Tanaka, Kiyoji; Saijo, Masafumi


    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

  15. 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


    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

  16. Expression of IQ-motif genes in human cells and ASPM domain structure. (United States)

    Rhoads, Allen; Kenguele, Hilaire


    Genes encoding multiple IQ-motif proteins have been identified in the human genome and may be regulated by calmodulin (CaM). Three genes of unknown function, abnormal spindle-like primary microcephaly (ASPM), KIAA0036, and KIAA1023, were expressed strongly in nearly all transformed human cell lines and in a panel of 16 adult human tissues by reverse transcription polymerase chain reaction. However, ASPM gene expression was not detected in adult brain or skeletal muscle. To better understand function, the domain structure of ASPM was examined. Abnormal spindle-like primary (ASP) protein (abnormal spindle) of Drosophila spp, an orthologue of ASPM, is involved in mitosis, and mutations lead to abnormal spindles and inhibition of cytokinesis. Studies of ASP have indicated that a microtubule binding region exists on the N-terminal third of the protein. Reiterative searches of the protein database using PSI-BLAST identified a common putative microtubular binding domain of 240 residues designated as MTASP. This nearly "all alpha" domain occurs in >25 related proteins including ASP and ASPM. The major C-terminal region of MTASP is basic with conserved hydrophobic residues and terminates at a flanking actin binding (CH) domain. This region is somewhat similar to other microtubule binding proteins such as MAP1B, MAP2, and tau. Multiple IQ motifs and often a conserved C-terminal domain occur in the remaining sequence. The multidomain structure of ASPM suggests a role in the coordination of cell cycle events. The extensive expression of multiple IQ-motif genes and the absence of ASPM in nondividing adult brain and skeletal muscle also suggest a role in cell division.

  17. 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. (United States)

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


    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.

  18. Bio-molecular architects: a scaffold provided by the C-terminal domain of eukaryotic RNA polymerase II. (United States)

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


    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.

  19. 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


    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.

  20. 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


    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.

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

    Directory of Open Access Journals (Sweden)

    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.

  2. The Stability Enhancement of Nitrile Hydratase from Bordetella petrii by Swapping the C-terminal Domain of β subunit. (United States)

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


    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.

  3. 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: [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)


    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.

  4. Plasmids for C-terminal tagging in Saccharomyces cerevisiae that contain improved GFP proteins, Envy and Ivy. (United States)

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


    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.

  5. Aurora A mediates cross-talk between N- and C-terminal post-translational modifications of p53 (United States)

    Raines, Sally Anne; Milner, Jo


    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

  6. Probing the interaction of the p53 C-terminal domain to the histone demethylase LSD1. (United States)

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


    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.

  7. The identification of putative RNA polymerase II C-terminal domain associated proteins in red and green algae. (United States)

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


    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.

  8. Ubiquitin C-terminal hydrolase-L3 regulates EMT process and cancer metastasis in prostate cell lines. (United States)

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


    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.

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

    Directory of Open Access Journals (Sweden)

    Yan Zhang


    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.

  10. C-terminal diversity within the p53 family accounts for differences in DNA binding and transcriptional activity (United States)

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


    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

  11. Hepatitis B Virus Core Protein Phosphorylation Sites Affect Capsid Stability and Transient Exposure of the C-terminal Domain. (United States)

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


    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.

  12. Fructose-1,6-bisphosphate aldolase of Neisseria meningitidis binds human plasminogen via its C-terminal lysine residue. (United States)

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


    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.

  13. 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.


    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    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:


    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.

  15. 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


    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.

  16. 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.

  17. Tight intramolecular regulation of the human Upf1 helicase by its N- and C-terminal domains. (United States)

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


    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.

  18. Structure of the S1 subunit C-terminal domain from bat-derived coronavirus HKU5 spike protein. (United States)

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


    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.

  19. 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


    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.

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

    Directory of Open Access Journals (Sweden)

    Jiivittha Veno


    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.

  1. Directed Evolution of Recombinant C-Terminal Truncated Staphylococcus epidermidis Lipase AT2 for the Enhancement of Thermostability (United States)

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


    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

  2. Neuroprotective effects of inositol 1,4,5-trisphosphate receptor C-terminal fragment in a Huntington's disease mouse model. (United States)

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


    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.

  3. 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.

  4. 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)


    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.

  5. Motif discovery in ranked lists of sequences

    DEFF Research Database (Denmark)

    Nielsen, Morten Muhlig; Tataru, Paula; Madsen, Tobias


    . These features make Regmex well suited for a range of biological sequence analysis problems related to motif discovery, exemplified by microRNA seed enrichment, but also including enrichment problems involving complex motifs and combinations of motifs. We demonstrate a number of usage scenarios that take......Motif analysis has long been an important method to characterize biological functionality and the current growth of sequencing-based genomics experiments further extends its potential. These diverse experiments often generate sequence lists ranked by some functional property. There is therefore...... a growing need for motif analysis methods that can exploit this coupled data structure and be tailored for specific biological questions. Here, we present an exploratory motif analysis tool, Regmex (REGular expression Motif EXplorer), which offers several methods to evaluate the correlation of motifs...

  6. MODIS: an audio motif discovery software


    Catanese, Laurence; Souviraà-Labastie, Nathan; Qu, Bingqing; Campion, Sébastien; Gravier, Guillaume; Vincent, Emmanuel; Bimbot, Frédéric


    International audience; MODIS is a free speech and audio motif discovery software developed at IRISA Rennes. Motif discovery is the task of discovering and collecting occurrences of repeating patterns in the absence of prior knowledge, or training material. MODIS is based on a generic approach to mine repeating audio sequences, with tolerance to motif variability. The algorithm implementation allows to process large audio streams at a reasonable speed where motif discovery often requires huge...

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

  8. 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


    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...

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

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


    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.

  10. 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.


    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

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

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


    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.

  12. 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)


    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.

  13. 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


    .... 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...

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

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


    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.

  15. 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


    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.

  16. Dissection of influenza A virus M1 protein: pH-dependent oligomerization of N-terminal domain and dimerization of C-terminal domain. (United States)

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


    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.

  17. Hydrophobic benzyl amines as supports for liquid-phase C-terminal amidated peptide synthesis: application to the preparation of ABT-510. (United States)

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


    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.

  18. 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: [The University of York, York YO10 5DD (United Kingdom); Northwest University, Xi’an 710069 (China)


    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.

  19. An overview of the sequence features of N- and C-terminal segments of the human chemokine receptors. (United States)

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


    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

  20. 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.

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

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


    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.

  2. 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


    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.

  3. Functional Characterization of C-terminal Ryanodine Receptor 1 Variants Associated with Central Core Disease or Malignant Hyperthermia. (United States)

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


    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.

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

    Directory of Open Access Journals (Sweden)

    Ashlyn M Spring


    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

  5. Energetic role of the paddle motif in voltage gating of Shaker K(+) channels. (United States)

    Xu, Yanping; Ramu, Yajamana; Shin, Hyeon-Gyu; Yamakaze, Jayden; Lu, Zhe


    Voltage-gated ion channels underlie rapid electric signaling in excitable cells. Electrophysiological studies have established that the N-terminal half of the fourth transmembrane segment ((NT)S4) of these channels is the primary voltage sensor, whereas crystallographic studies have shown that (NT)S4 is not located within a proteinaceous pore. Rather, (NT)S4 and the C-terminal half of S3 ((CT)S3 or S3b) form a helix-turn-helix motif, termed the voltage-sensor paddle. This unexpected structural finding raises two fundamental questions: does the paddle motif also exist in voltage-gated channels in a biological membrane, and, if so, what is its function in voltage gating? Here, we provide evidence that the paddle motif exists in the open state of Drosophila Shaker voltage-gated K(+) channels expressed in Xenopus oocytes and that (CT)S3 acts as an extracellular hydrophobic 'stabilizer' for (NT)S4, thus biasing the gating chemical equilibrium toward the open state.

  6. Detecting correlations among functional-sequence motifs (United States)

    Pirino, Davide; Rigosa, Jacopo; Ledda, Alice; Ferretti, Luca


    Sequence motifs are words of nucleotides in DNA with biological functions, e.g., gene regulation. Identification of such words proceeds through rejection of Markov models on the expected motif frequency along the genome. Additional biological information can be extracted from the correlation structure among patterns of motif occurrences. In this paper a log-linear multivariate intensity Poisson model is estimated via expectation maximization on a set of motifs along the genome of E. coli K12. The proposed approach allows for excitatory as well as inhibitory interactions among motifs and between motifs and other genomic features like gene occurrences. Our findings confirm previous stylized facts about such types of interactions and shed new light on genome-maintenance functions of some particular motifs. We expect these methods to be applicable to a wider set of genomic features.

  7. Mutagenesis of tyrosine and di-leucine motifs in the HIV-1 envelope cytoplasmic domain results in a loss of Env-mediated fusion and infectivity

    Directory of Open Access Journals (Sweden)

    Claiborne Daniel T


    Full Text Available Abstract Background The gp41 component of the Human Immunodeficiency Virus (HIV envelope glycoprotein (Env contains a long cytoplasmic domain (CD with multiple highly conserved tyrosine (Y and dileucine (LL motifs. Studies suggest that the motifs distal to major endocytosis motif (Y712HRL, located at residues 712-715 of Env, may contribute to Env functionality in the viral life cycle. In order to examine the biological contribution of these motifs in the biosynthesis, transport, and function of Env, we constructed two panels of mutants in which the conserved Y- and LL-motifs were sequentially substituted by alternative residues, either in the presence or absence of Y712. Additional mutants targeting individual motifs were then constructed. Results All mutant Envs, when expressed in the absence of other viral proteins, maintained at least WT levels of Env surface staining by multiple antibodies. The Y712 mutation (Y712C contributed to at least a 4-fold increase in surface expression for all mutants containing this change. Sequential mutagenesis of the Y- and LL-motifs resulted in a generally progressive decrease in Env fusogenicity. However, additive mutation of dileucine and tyrosine motifs beyond the tyrosine at residue 768 resulted in the most dramatic effects on Env incorporation into virions, viral infectivity, and virus fusion with target cells. Conclusions From the studies reported here, we show that mutations of the Y- and LL-motifs, which effectively eliminate the amphipathic nature of the lytic peptide 2 (LLP2 domain or disrupt YW and LL motifs in a region spanning residues 795-803 (YWWNLLQYW, just C-terminal of LLP2, can dramatically interfere with biological functions of HIV-1 Env and abrogate virus replication. Because these mutant proteins are expressed at the cell surface, we conclude that tyrosine and di-leucine residues within the cytoplasmic domain of gp41 play critical roles in HIV-1 replication that are distinct from that of

  8. Kopi dan Kakao dalam Kreasi Motif Batik Khas Jember

    Directory of Open Access Journals (Sweden)

    Irfa'ina Rohana Salma


    Full Text Available ABSTRAK Batik Jember selama ini identik dengan motif daun tembakau. Visualisasi daun tembakau dalam motif Batik Jember cukup lemah, yaitu kurang berkarakter karena motif yang muncul adalah seperti gambar daun pada umumnya. Oleh karena itu perlu diciptakan desain motif batik khas Jember yang sumber inspirasinya digali dari kekayaan alam lainnya dari Jember yang mempunyai bentuk spesifik dan karakteristik sehingga identitas motif bisa didapatkan dengan lebih kuat. Hasil alam khas Jember tersebut adalah kopi dan kakao. Tujuan penciptaan seni ini adalah untuk menghasilkan motif batik  baru yang mempunyai ciri khas Jember. Metode yang digunakan yaitu pengumpulan data, pengamatan mendalam terhadap objek penciptaan, pengkajian sumber inspirasi, pembuatan desain motif, dan perwujudan menjadi batik. Dari penciptaan seni ini berhasil dikreasikan 6 (enam motif batik yaitu: (1 Motif Uwoh Kopi; (2 Motif Godong Kopi;  (3 Motif Ceplok Kakao; (4 Motif Kakao Raja; (5 Motif Kakao Biru; dan (6 Motif Wiji Mukti. Berdasarkan hasil penilaian “Selera Estetika” diketahui bahwa motif yang paling banyak disukai adalah Motif Uwoh Kopi dan Motif Kakao Raja. Kata kunci: Motif Woh Kopi, Motif Godong Kopi, Motif Ceplok Kakao, Motif Kakao Raja, Motif Kakao Biru, Motif Wiji Mukti ABSTRACTBatik Jember is synonymous with tobacco leaf motif. Tobacco leaf shape is quite weak in the visual appearance characterized as that motif emerges like a picture of leaves in general. Therefore, it is necessary to create a distinctive design motif extracted from other natural resources of Jember that have specific shapes and characteristics that can be obtained as the stronger motif identity. The typical natural resources from Jember are coffee and cocoa. The purpose of the creation of this art is to produce the unique, creative and innovative batik and have specific characteristics of Jember. The method used are data collection, observation of the object, reviewing inspiration sources

  9. Statistical tests to compare motif count exceptionalities

    Directory of Open Access Journals (Sweden)

    Vandewalle Vincent


    Full Text Available Abstract Background Finding over- or under-represented motifs in biological sequences is now a common task in genomics. Thanks to p-value calculation for motif counts, exceptional motifs are identified and represent candidate functional motifs. The present work addresses the related question of comparing the exceptionality of one motif in two different sequences. Just comparing the motif count p-values in each sequence is indeed not sufficient to decide if this motif is significantly more exceptional in one sequence compared to the other one. A statistical test is required. Results We develop and analyze two statistical tests, an exact binomial one and an asymptotic likelihood ratio test, to decide whether the exceptionality of a given motif is equivalent or significantly different in two sequences of interest. For that purpose, motif occurrences are modeled by Poisson processes, with a special care for overlapping motifs. Both tests can take the sequence compositions into account. As an illustration, we compare the octamer exceptionalities in the Escherichia coli K-12 backbone versus variable strain-specific loops. Conclusion The exact binomial test is particularly adapted for small counts. For large counts, we advise to use the likelihood ratio test which is asymptotic but strongly correlated with the exact binomial test and very simple to use.

  10. An Algorithm for Motif Discovery with Iteration on Lengths of Motifs. (United States)

    Fan, Yetian; Wu, Wei; Yang, Jie; Yang, Wenyu; Liu, Rongrong


    Analysis of DNA sequence motifs is becoming increasingly important in the study of gene regulation, and the identification of motif in DNA sequences is a complex problem in computational biology. Motif discovery has attracted the attention of more and more researchers, and varieties of algorithms have been proposed. Most existing motif discovery algorithms fix the motif's length as one of the input parameters. In this paper, a novel method is proposed to identify the optimal length of the motif and the optimal motif with that length, through an iteration process on increasing length numbers. For each fixed length, a modified genetic algorithm (GA) is used for finding the optimal motif with that length. Three operators are used in the modified GA: Mutation that is similar to the one used in usual GA but is modified to avoid local optimum in our case, and Addition and Deletion that are proposed by us for the problem. A criterion is given for singling out the optimal length in the increasing motif's lengths. We call this method AMDILM (an algorithm for motif discovery with iteration on lengths of motifs). The experiments on simulated data and real biological data show that AMDILM can accurately identify the optimal motif length. Meanwhile, the optimal motifs discovered by AMDILM are consistent with the real ones and are similar with the motifs obtained by the three well-known methods: Gibbs Sampler, MEME and Weeder.

  11. rMotifGen: random motif generator for DNA and protein sequences

    Directory of Open Access Journals (Sweden)

    Hardin C Timothy


    Full Text Available Abstract Background Detection of short, subtle conserved motif regions within a set of related DNA or amino acid sequences can lead to discoveries about important regulatory domains such as transcription factor and DNA binding sites as well as conserved protein domains. In order to help assess motif detection algorithms on motifs with varying properties and levels of conservation, we have developed a computational tool, rMotifGen, with the sole purpose of generating a number of random DNA or protein sequences containing short sequence motifs. Each motif consensus can be user-defined, randomly generated, or created from a position-specific scoring matrix (PSSM. Insertions and mutations within these motifs are created according to user-defined parameters and substitution matrices. The resulting sequences can be helpful in mutational simulations and in testing the limits of motif detection algorithms. Results Two implementations of rMotifGen have been created, one providing a graphical user interface (GUI for random motif construction, and the other serving as a command line interface. The second implementation has the added advantages of platform independence and being able to be called in a batch mode. rMotifGen was used to construct sample sets of sequences containing DNA motifs and amino acid motifs that were then tested against the Gibbs sampler and MEME packages. Conclusion rMotifGen provides an efficient and convenient method for creating random DNA or amino acid sequences with a variable number of motifs, where the instance of each motif can be incorporated using a position-specific scoring matrix (PSSM or by creating an instance mutated from its corresponding consensus using an evolutionary model based on substitution matrices. rMotifGen is freely available at:

  12. Motif Tool Manager: a web-based framework for motif discovery. (United States)

    Phan, Vinhthuy; Furlotte, Nicholas A


    Motif Tool Manager is a web-based framework for comparing and combining different approaches to discover novel DNA motifs. It comes with a set of five well-known approaches to motif discovery. It provides an easy mechanism for adding new motif finding tools to the framework through a web-interface and a minimal setup of the tools on the server. Users can execute the tools through the web-based framework and compare results from such executions. The framework provides a basic mechanism for identifying the most similar motif candidates found by a majority of themotif finding tools.

  13. The C-terminal residue of phage Vp16 PDF, the smallest peptide deformylase, acts as an offset element locking the active conformation. (United States)

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


    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.

  14. 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


    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.

  15. 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:; Petosa, Carlo [European Molecular Biology Laboratory, Grenoble Outstation, BP 181, 38042 Grenoble CEDEX 9 (France)


    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°.

  16. 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


    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....

  17. C-terminally truncated constitutively active androgen receptor variants and their biologic and clinical significance in castration-resistant prostate cancer. (United States)

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


    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.

  18. The C-terminal domain of BinA is responsible for Bacillus sphaericus binary toxin BinA-BinB interaction. (United States)

    Limpanawat, Suweeraya; Promdonkoy, Boonhiang; Boonserm, Panadda


    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.

  19. 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


    ) 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...

  20. Epstein-Barr Virus Latent Membrane Protein 1 (LMP1) C-Terminal-Activating Region 3 Contributes to LMP1-Mediated Cellular Migration via Its Interaction with Ubc9 ▿ (United States)

    Bentz, Gretchen L.; Whitehurst, Christopher B.; Pagano, Joseph S.


    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

  1. Epstein-Barr virus latent membrane protein 1 (LMP1) C-terminal-activating region 3 contributes to LMP1-mediated cellular migration via its interaction with Ubc9. (United States)

    Bentz, Gretchen L; Whitehurst, Christopher B; Pagano, Joseph S


    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.

  2. Spontaneous adsorption of coiled-coil model peptides K and E to a mixed lipid bilayer. (United States)

    Pluhackova, Kristyna; Wassenaar, Tsjerk A; Kirsch, Sonja; Böckmann, Rainer A


    A molecular description of the lipid-protein interactions underlying the adsorption of proteins to membranes is crucial for understanding, for example, the specificity of adsorption or the binding strength of a protein to a bilayer, or for characterizing protein-induced changes of membrane properties. In this paper, we extend an automated in silico assay (DAFT) for binding studies and apply it to characterize the adsorption of the model fusion peptides E and K to a mixed phospholipid/cholesterol membrane using coarse-grained molecular dynamics simulations. In addition, we couple the coarse-grained protocol to reverse transformation to atomistic resolution, thereby allowing to study molecular interactions with high detail. The experimentally observed differential binding of the peptides E and K to membranes, as well as the increased binding affinity of helical over unstructered peptides, could be well reproduced using the polarizable Martini coarse-grained (CG) force field. Binding to neutral membranes is shown to be dominated by initial binding of the positively charged N-terminus to the phospholipid headgroup region, followed by membrane surface-aligned insertion of the peptide at the interface between the hydrophobic core of the membrane and its polar headgroup region. Both coarse-grained and atomistic simulations confirm a before hypothesized snorkeling of lysine side chains for the membrane-bound state of the peptide K. Cholesterol was found to be enriched in peptide vicinity, which is probably of importance for the mechanism of membrane fusion. The applied sequential multiscale method, using coarse-grained simulations for the slow adsorption process of peptides to membranes followed by backward transformation to atomistic detail and subsequent atomistic simulations of the preformed peptide-lipid complexes, is shown to be a versatile approach to study the interactions of peptides or proteins with biomembranes.

  3. Oligomeric coiled-coil adhesin YadA is a double-edged sword.

    Directory of Open Access Journals (Sweden)

    Salome Casutt-Meyer

    Full Text Available Yersinia adhesin A (YadA is an essential virulence factor for the food-borne pathogens Yersinia enterocolitica and Yersinia pseudotuberculosis. Surprisingly, it is a pseudogene in Yersinia pestis. Even more intriguing, the introduction of a functional yadA gene in Y. pestis EV76 was shown to correlate with a decrease in virulence in a mouse model. Here, we report that wild type (wt Y. enterocolitica E40, as well as YadA-deprived E40 induced the synthesis of neutrophil extracellular traps (NETs upon contact with neutrophils, but only YadA-expressing Y. enterocolitica adhered to NETs and were killed. As binding seemed to be a prerequisite for killing, we searched for YadA-binding substrates and detected the presence of collagen within NETs. E40 bacteria expressing V98D,N99A mutant YadA with a severely reduced ability to bind collagen were found to be more resistant to killing, suggesting that collagen binding contributes significantly to sensitivity to NETs. Wt Y. pestis EV76 were resistant to killing by NETs, while recombinant EV76 expressing YadA from either Y. pseudotuberculosis or Y. enterocolitica were sensitive to killing by NETs, outlining the importance of YadA for susceptibility to NET-dependent killing. Recombinant EV76 endowed with YadA from Y. enterocolitica were also less virulent for the mouse than wt EV76, as shown before. In addition, EV76 carrying wt YadA were less virulent for the mouse than EV76 expressing YadA(V₉₈D,N₉₉A. The observation that YadA makes Yersinia sensitive to NETs provides an explanation as for why evolution selected for the inactivation of yadA in the flea-borne Y. pestis and clarifies an old enigma. Since YadA imposes the same cost to the food-borne Yersinia but was nevertheless conserved by evolution, this observation also illustrates the duality of some virulence functions.

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


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


    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...

  5. Generation of the beta-amyloid peptide and the amyloid precursor protein C-terminal fragment gamma are potentiated by FE65L1. (United States)

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


    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.

  6. 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.

  7. N- and C-terminal truncations of a GH10 xylanase significantly increase its activity and thermostability but decrease its SDS resistance. (United States)

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


    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.

  8. 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)


    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.

  9. Fitness for synchronization of network motifs

    DEFF Research Database (Denmark)

    Vega, Y.M.; Vázquez-Prada, M.; Pacheco, A.F.


    We study the synchronization of Kuramoto's oscillators in small parts of networks known as motifs. We first report on the system dynamics for the case of a scale-free network and show the existence of a non-trivial critical point. We compute the probability that network motifs synchronize, and fi...... that the fitness for synchronization correlates well with motifs interconnectedness and structural complexity. Possible implications for present debates about network evolution in biological and other systems are discussed....

  10. Motif discovery using optimized suffix tries


    Prado Martínez, Sergio


    Motif discovery is a challenging problem from a computational point of view [5] [6]. Binding sites are better conserved in DNA because they have a biological function and are therefore under selective pressure. Motif discovery algorithms can help us detect them. To tackle our problem we design and implement an index structure and a motif discovery algorithm. In this thesis we will investigate memory and performance optimizations. En el present article es presenta una implementació d'un ...

  11. Binding of a truncated form of lecithin:retinol acyltransferase and its N- and C-terminal peptides to lipid monolayers. (United States)

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


    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.

  12. 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.

  13. iPreny-PseAAC: Identify C-terminal Cysteine Prenylation Sites in Proteins by Incorporating Two Tiers of Sequence Couplings into PseAAC. (United States)

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


    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, 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

  14. The AvrM effector from flax rust has a structured C-terminal domain and interacts directly with the M resistance protein. (United States)

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


    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.

  15. 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


    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.

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


    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.

  17. 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


    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.

  18. Structural determinants for high-affinity binding in a Nedd4 WW3* domain-Comm PY motif complex. (United States)

    Kanelis, Voula; Bruce, M Christine; Skrynnikov, Nikolai R; Rotin, Daniela; Forman-Kay, Julie D


    Interactions between the WW domains of Drosophila Nedd4 (dNedd4) and Commissureless (Comm) PY motifs promote axon crossing at the CNS midline and muscle synaptogenesis. Here we report the solution structure of the dNedd4 WW3* domain complexed to the second PY motif (227'TGLPSYDEALH237') of Comm. Unexpectedly, there are interactions between WW3* and ligand residues both N- and C-terminal to the PY motif. Residues Y232'-L236' form a helical turn, following the PPII helical PY motif. Mutagenesis and binding studies confirm the importance of these extensive contacts, not simultaneously observed in other WW domain complexes, and identify a variable loop in WW3* responsible for its high-affinity interaction. These studies expand our general understanding of the molecular determinants involved in WW domain-ligand recognition. In addition, they provide insights into the specific regulation of dNedd4-mediated ubiquitination of Comm and subsequent internalization of Comm or the Comm/Roundabout complex, critical for CNS and muscle development.

  19. 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)


    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

  20. PI(4P Promotes Phosphorylation and Conformational Change of Smoothened through Interaction with Its C-terminal Tail.

    Directory of Open Access Journals (Sweden)

    Kai Jiang


    Full Text Available In Hedgehog (Hh signaling, binding of Hh to the Patched-Interference Hh (Ptc-Ihog receptor complex relieves Ptc inhibition on Smoothened (Smo. A longstanding question is how Ptc inhibits Smo and how such inhibition is relieved by Hh stimulation. In this study, we found that Hh elevates production of phosphatidylinositol 4-phosphate (PI(4P. Increased levels of PI(4P promote, whereas decreased levels of PI(4P inhibit, Hh signaling activity. We further found that PI(4P directly binds Smo through an arginine motif, which then triggers Smo phosphorylation and activation. Moreover, we identified the pleckstrin homology (PH domain of G protein-coupled receptor kinase 2 (Gprk2 as an essential component for enriching PI(4P and facilitating Smo activation. PI(4P also binds mouse Smo (mSmo and promotes its phosphorylation and ciliary accumulation. Finally, Hh treatment increases the interaction between Smo and PI(4P but decreases the interaction between Ptc and PI(4P, indicating that, in addition to promoting PI(4P production, Hh regulates the pool of PI(4P associated with Ptc and Smo.

  1. The C-terminal sequence of RhoB directs protein degradation through an endo-lysosomal pathway.

    Directory of Open Access Journals (Sweden)

    Dolores Pérez-Sala

    Full Text Available BACKGROUND: Protein degradation is essential for cell homeostasis. Targeting of proteins for degradation is often achieved by specific protein sequences or posttranslational modifications such as ubiquitination. METHODOLOGY/PRINCIPAL FINDINGS: By using biochemical and genetic tools we have monitored the localization and degradation of endogenous and chimeric proteins in live primary cells by confocal microscopy and ultra-structural analysis. Here we identify an eight amino acid sequence from the C-terminus of the short-lived GTPase RhoB that directs the rapid degradation of both RhoB and chimeric proteins bearing this sequence through a lysosomal pathway. Elucidation of the RhoB degradation pathway unveils a mechanism dependent on protein isoprenylation and palmitoylation that involves sorting of the protein into multivesicular bodies, mediated by the ESCRT machinery. Moreover, RhoB sorting is regulated by late endosome specific lipid dynamics and is altered in human genetic lipid traffic disease. CONCLUSIONS/SIGNIFICANCE: Our findings characterize a short-lived cytosolic protein that is degraded through a lysosomal pathway. In addition, we define a novel motif for protein sorting and rapid degradation, which allows controlling protein levels by means of clinically used drugs.

  2. Structure of the C-terminal domain of AspA (antigen I/II-family protein from Streptococcus pyogenes

    Directory of Open Access Journals (Sweden)

    Michael Hall


    Full Text Available The pathogenic bacteria Streptococcus pyogenes can cause an array of diseases in humans, including moderate infections such as pharyngitis (strep throat as well as life threatening conditions such as necrotizing fasciitis and puerperal fever. The antigen I/II family proteins are cell wall anchored adhesin proteins found on the surfaces of most oral streptococci and are involved in host colonization and biofilm formation. In the present study we have determined the crystal structure of the C2–3-domain of the antigen I/II type protein AspA from S. pyogenes M type 28. The structure was solved to 1.8 Å resolution and shows that the C2–3-domain is comprised of two structurally similar DEv-IgG motifs, designated C2 and C3, both containing a stabilizing covalent isopeptide bond. Furthermore a metal binding site is identified, containing a bound calcium ion. Despite relatively low sequence identity, interestingly, the overall structure shares high similarity to the C2–3-domains of antigen I/II proteins from Streptococcus gordonii and Streptococcus mutans, although certain parts of the structure exhibit distinct features. In summary this work constitutes the first step in the full structure determination of the AspA protein from S. pyogenes.

  3. PI(4)P Promotes Phosphorylation and Conformational Change of Smoothened through Interaction with Its C-terminal Tail (United States)

    Zhang, Jie; Li, Xiang-An; Evers, B. Mark; Zhu, Haining; Jia, Jianhang


    In Hedgehog (Hh) signaling, binding of Hh to the Patched-Interference Hh (Ptc-Ihog) receptor complex relieves Ptc inhibition on Smoothened (Smo). A longstanding question is how Ptc inhibits Smo and how such inhibition is relieved by Hh stimulation. In this study, we found that Hh elevates production of phosphatidylinositol 4-phosphate (PI(4)P). Increased levels of PI(4)P promote, whereas decreased levels of PI(4)P inhibit, Hh signaling activity. We further found that PI(4)P directly binds Smo through an arginine motif, which then triggers Smo phosphorylation and activation. Moreover, we identified the pleckstrin homology (PH) domain of G protein-coupled receptor kinase 2 (Gprk2) as an essential component for enriching PI(4)P and facilitating Smo activation. PI(4)P also binds mouse Smo (mSmo) and promotes its phosphorylation and ciliary accumulation. Finally, Hh treatment increases the interaction between Smo and PI(4)P but decreases the interaction between Ptc and PI(4)P, indicating that, in addition to promoting PI(4)P production, Hh regulates the pool of PI(4)P associated with Ptc and Smo. PMID:26863604

  4. Triaspartate: a model system for conformationally flexible DDD motifs in proteins. (United States)

    Duitch, Laura; Toal, Siobhan; Measey, Thomas J; Schweitzer-Stenner, Reinhard


    Understanding the interactions that govern turn formation in the unfolded state of proteins is necessary for a complete picture of the role that these turns play in both normal protein folding and functionally relevant yet disordered linear motifs. It is still unclear, however, whether short peptides can adopt stable turn structures in aqueous environments in the absence of any nonlocal interactions. To explore the effect that nearest-neighbor interactions and the local peptide environment have on the turn-forming capability of individual amino acid residues in short peptides, we combined vibrational (IR, Raman, and VCD), UV-CD, and (1)H NMR spectroscopies in order to probe the conformational ensemble of the central aspartic acid residue of the triaspartate peptide (DDD). The study was motivated by the recently discovered turn propensities of aspartic acid in GDG (Hagarman; et al. Chem.-Eur. J. 2011, 17, 6789). We investigated the DDD peptide under both acidic and neutral conditions in order to elucidate the effect that side-chain protonation has on the conformational propensity of the central aspartic acid residue. Amide I' profiles were analyzed in terms of two-dimensional Gaussian distributions representing conformational subdistributions in Ramachandran space. Interestingly, our results show that while the protonated form of the DDD peptide samples various turn-like conformations similar to GDG, deprotonation of the peptide eliminates this propensity for turns, causing the fully ionized peptide to exclusively sample pPII and β-strand-like structures. To further explore the factors stabilizing these more extended conformations in fully ionized DDD, we analyzed the temperature dependence of both the UV-CD spectrum and the (3)J(H(N),H(α)) coupling constants of the two amide protons (N- and C-terminal) in terms of a simple two-state (pPII-β) thermodynamic model. Thus, we were able to obtain the enthalpic and entropic differences between the pPII and

  5. MSDmotif: exploring protein sites and motifs

    Directory of Open Access Journals (Sweden)

    Henrick Kim


    Full Text Available Abstract Background Protein structures have conserved features – motifs, which have a sufficient influence on the protein function. These motifs can be found in sequence as well as in 3D space. Understanding of these fragments is essential for 3D structure prediction, modelling and drug-design. The Protein Data Bank (PDB is the source of this information however present search tools have limited 3D options to integrate protein sequence with its 3D structure. Results We describe here a web application for querying the PDB for ligands, binding sites, small 3D structural and sequence motifs and the underlying database. Novel algorithms for chemical fragments, 3D motifs, ϕ/ψ sequences, super-secondary structure motifs and for small 3D structural motif associations searches are incorporated. The interface provides functionality for visualization, search criteria creation, sequence and 3D multiple alignment options. MSDmotif is an integrated system where a results page is also a search form. A set of motif statistics is available for analysis. This set includes molecule and motif binding statistics, distribution of motif sequences, occurrence of an amino-acid within a motif, correlation of amino-acids side-chain charges within a motif and Ramachandran plots for each residue. The binding statistics are presented in association with properties that include a ligand fragment library. Access is also provided through the distributed Annotation System (DAS protocol. An additional entry point facilitates XML requests with XML responses. Conclusion MSDmotif is unique by combining chemical, sequence and 3D data in a single search engine with a range of search and visualisation options. It provides multiple views of data found in the PDB archive for exploring protein structures.

  6. Assessment of composite motif discovery methods

    Directory of Open Access Journals (Sweden)

    Johansen Jostein


    Full Text Available Abstract Background Computational discovery of regulatory elements is an important area of bioinformatics research and more than a hundred motif discovery methods have been published. Traditionally, most of these methods have addressed the problem of single motif discovery – discovering binding motifs for individual transcription factors. In higher organisms, however, transcription factors usually act in combination with nearby bound factors to induce specific regulatory behaviours. Hence, recent focus has shifted from single motifs to the discovery of sets of motifs bound by multiple cooperating transcription factors, so called composite motifs or cis-regulatory modules. Given the large number and diversity of methods available, independent assessment of methods becomes important. Although there have been several benchmark studies of single motif discovery, no similar studies have previously been conducted concerning composite motif discovery. Results We have developed a benchmarking framework for composite motif discovery and used it to evaluate the performance of eight published module discovery tools. Benchmark datasets were constructed based on real genomic sequences containing experimentally verified regulatory modules, and the module discovery programs were asked to predict both the locations of these modules and to specify the single motifs involved. To aid the programs in their search, we provided position weight matrices corresponding to the binding motifs of the transcription factors involved. In addition, selections of decoy matrices were mixed with the genuine matrices on one dataset to test the response of programs to varying levels of noise. Conclusion Although some of the methods tested tended to score somewhat better than others overall, there were still large variations between individual datasets and no single method performed consistently better than the rest in all situations. The variation in performance on individual

  7. PMS: a panoptic motif search tool.

    Directory of Open Access Journals (Sweden)

    Hieu Dinh

    Full Text Available Identification of DNA/Protein motifs is a crucial problem for biologists. Computational techniques could be of great help in this identification. In this direction, many computational models for motifs have been proposed in the literature.One such important model is the (l,d motif model. In this paper we describe a motif search web tool that predominantly employs this motif model. This web tool exploits the state-of-the art algorithms for solving the (l,d motif search problem.The online tool has been helping scientists identify many unknown motifs. Many of our predictions have been successfully verified as well. We hope that this paper will expose this crucial tool to many more scientists.Project name: PMS--Panoptic Motif Search Tool. Project home page: or Licence: PMS tools will be readily available to any scientist wishing to use it for non-commercial purposes, without restrictions. The online tool is freely available without login.

  8. Plk4-dependent phosphorylation of STIL is required for centriole duplication

    Directory of Open Access Journals (Sweden)

    Anne-Sophie Kratz


    Full Text Available Duplication of centrioles, namely the formation of a procentriole next to the parental centriole, is regulated by the polo-like kinase Plk4. Only a few other proteins, including STIL (SCL/TAL1 interrupting locus, SIL and Sas-6, are required for the early step of centriole biogenesis. Following Plk4 activation, STIL and Sas-6 accumulate at the cartwheel structure at the initial stage of the centriole assembly process. Here, we show that STIL interacts with Plk4 in vivo. A STIL fragment harboring both the coiled-coil domain and the STAN motif shows the strongest binding affinity to Plk4. Furthermore, we find that STIL is phosphorylated by Plk4. We identified Plk4-specific phosphorylation sites within the C-terminal domain of STIL and show that phosphorylation of STIL by Plk4 is required to trigger centriole duplication.

  9. A myxobacterial S-motility protein dances with poles. (United States)

    Huitema, Edgar; Viollier, Patrick H


    Coordinated movement of packs of S-motile Myxococcus xanthus cells relies on extrusion and retraction of pili that are located at one cell pole. At regular intervals the pili switch their polar location and cells reverse direction. Recently, the FrzS S-motility protein was observed to localize predominantly to the piliated pole. In time, FrzS was redeployed to the opposite pole and its sequestration at the new site coincided with cell reversal. The C-terminal region of FrzS, a response regulator homolog, is rich in coiled-coil motifs and is required for dynamic localization and proper motility. These results raise the possibility that proper spatial control of FrzS has an important role in the regulation of cell reversal and S-motility.

  10. MotifNet: a web-server for network motif analysis. (United States)

    Smoly, Ilan Y; Lerman, Eugene; Ziv-Ukelson, Michal; Yeger-Lotem, Esti


    Network motifs are small topological patterns that recur in a network significantly more often than expected by chance. Their identification emerged as a powerful approach for uncovering the design principles underlying complex networks. However, available tools for network motif analysis typically require download and execution of computationally intensive software on a local computer. We present MotifNet, the first open-access web-server for network motif analysis. MotifNet allows researchers to analyze integrated networks, where nodes and edges may be labeled, and to search for motifs of up to eight nodes. The output motifs are presented graphically and the user can interactively filter them by their significance, number of instances, node and edge labels, and node identities, and view their instances. MotifNet also allows the user to distinguish between motifs that are centered on specific nodes and motifs that recur in distinct parts of the network. MotifNet is freely available at . The website was implemented using ReactJs and supports all major browsers. The server interface was implemented in Python with data stored on a MySQL database. or Supplementary data are available at Bioinformatics online.

  11. CompleteMOTIFs: DNA motif discovery platform for transcription factor binding experiments. (United States)

    Kuttippurathu, Lakshmi; Hsing, Michael; Liu, Yongchao; Schmidt, Bertil; Maskell, Douglas L; Lee, Kyungjoon; He, Aibin; Pu, William T; Kong, Sek Won


    CompleteMOTIFs (cMOTIFs) is an integrated web tool developed to facilitate systematic discovery of overrepresented transcription factor binding motifs from high-throughput chromatin immunoprecipitation experiments. Comprehensive annotations and Boolean logic operations on multiple peak locations enable users to focus on genomic regions of interest for de novo motif discovery using tools such as MEME, Weeder and ChIPMunk. The pipeline incorporates a scanning tool for known motifs from TRANSFAC and JASPAR databases, and performs an enrichment test using local or precalculated background models that significantly improve the motif scanning result. Furthermore, using the cMOTIFs pipeline, we demonstrated that multiple transcription factors could cooperatively bind to the upstream of important stem cell differentiation regulators.

  12. Improved benchmarks for computational motif discovery

    Directory of Open Access Journals (Sweden)

    Walseng Vegard


    Full Text Available Abstract Background An important step in annotation of sequenced genomes is the identification of transcription factor binding sites. More than a hundred different computational methods have been proposed, and it is difficult to make an informed choice. Therefore, robust assessment of motif discovery methods becomes important, both for validation of existing tools and for identification of promising directions for future research. Results We use a machine learning perspective to analyze collections of transcription factors with known binding sites. Algorithms are presented for finding position weight matrices (PWMs, IUPAC-type motifs and mismatch motifs with optimal discrimination of binding sites from remaining sequence. We show that for many data sets in a recently proposed benchmark suite for motif discovery, none of the common motif models can accurately discriminate the binding sites from remaining sequence. This may obscure the distinction between the potential performance of the motif discovery tool itself versus the intrinsic complexity of the problem we are trying to solve. Synthetic data sets may avoid this problem, but we show on some previously proposed benchmarks that there may be a strong bias towards a presupposed motif model. We also propose a new approach to benchmark data set construction. This approach is based on collections of binding site fragments that are ranked according to the optimal level of discrimination achieved with our algorithms. This allows us to select subsets with specific properties. We present one benchmark suite with data sets that allow good discrimination between positive and negative instances with the common motif models. These data sets are suitable for evaluating algorithms for motif discovery that rely on these models. We present another benchmark suite where PWM, IUPAC and mismatch motif models are not able to discriminate reliably between positive and negative instances. This suite could be used

  13. Combining phylogenetic footprinting with motif models incorporating intra-motif dependencies. (United States)

    Nettling, Martin; Treutler, Hendrik; Cerquides, Jesus; Grosse, Ivo


    Transcriptional gene regulation is a fundamental process in nature, and the experimental and computational investigation of DNA binding motifs and their binding sites is a prerequisite for elucidating this process. Approaches for de-novo motif discovery can be subdivided in phylogenetic footprinting that takes into account phylogenetic dependencies in aligned sequences of more than one species and non-phylogenetic approaches based on sequences from only one species that typically take into account intra-motif dependencies. It has been shown that modeling (i) phylogenetic dependencies as well as (ii) intra-motif dependencies separately improves de-novo motif discovery, but there is no approach capable of modeling both (i) and (ii) simultaneously. Here, we present an approach for de-novo motif discovery that combines phylogenetic footprinting with motif models capable of taking into account intra-motif dependencies. We study the degree of intra-motif dependencies inferred by this approach from ChIP-seq data of 35 transcription factors. We find that significant intra-motif dependencies of orders 1 and 2 are present in all 35 datasets and that intra-motif dependencies of order 2 are typically stronger than those of order 1. We also find that the presented approach improves the classification performance of phylogenetic footprinting in all 35 datasets and that incorporating intra-motif dependencies of order 2 yields a higher classification performance than incorporating such dependencies of only order 1. Combining phylogenetic footprinting with motif models incorporating intra-motif dependencies leads to an improved performance in the classification of transcription factor binding sites. This may advance our understanding of transcriptional gene regulation and its evolution.

  14. MotifLab: a tools and data integration workbench for motif discovery and regulatory sequence analysis. (United States)

    Klepper, Kjetil; Drabløs, Finn


    Traditional methods for computational motif discovery often suffer from poor performance. In particular, methods that search for sequence matches to known binding motifs tend to predict many non-functional binding sites because they fail to take into consideration the biological state of the cell. In recent years, genome-wide studies have generated a lot of data that has the potential to improve our ability to identify functional motifs and binding sites, such as information about chromatin accessibility and epigenetic states in different cell types. However, it is not always trivial to make use of this data in combination with existing motif discovery tools, especially for researchers who are not skilled in bioinformatics programming. Here we present MotifLab, a general workbench for analysing regulatory sequence regions and discovering transcription factor binding sites and cis-regulatory modules. MotifLab supports comprehensive motif discovery and analysis by allowing users to integrate several popular motif discovery tools as well as different kinds of additional information, including phylogenetic conservation, epigenetic marks, DNase hypersensitive sites, ChIP-Seq data, positional binding preferences of transcription factors, transcription factor interactions and gene expression. MotifLab offers several data-processing operations that can be used to create, manipulate and analyse data objects, and complete analysis workflows can be constructed and automatically executed within MotifLab, including graphical presentation of the results. We have developed MotifLab as a flexible workbench for motif analysis in a genomic context. The flexibility and effectiveness of this workbench has been demonstrated on selected test cases, in particular two previously published benchmark data sets for single motifs and modules, and a realistic example of genes responding to treatment with forskolin. MotifLab is freely available at

  15. 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

  16. 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


    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 groo