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Sample records for binding proteins control

  1. Plant RNA binding proteins for control of RNA virus infection

    OpenAIRE

    Huh, Sung Un; Paek, Kyung-Hee

    2013-01-01

    Plant RNA viruses have effective strategies to infect host plants through either direct or indirect interactions with various host proteins, thus suppressing the host immune system. When plant RNA viruses enter host cells exposed RNAs of viruses are recognized by the host immune system through processes such as siRNA-dependent silencing. Interestingly, some host RNA binding proteins have been involved in the inhibition of RNA virus replication, movement, and translation through RNA-specific b...

  2. Control of a neuronal morphology program by an RNA-binding zinc finger protein, Unkempt.

    Science.gov (United States)

    Murn, Jernej; Zarnack, Kathi; Yang, Yawei J; Durak, Omer; Murphy, Elisabeth A; Cheloufi, Sihem; Gonzalez, Dilenny M; Teplova, Marianna; Curk, Tomaž; Zuber, Johannes; Patel, Dinshaw J; Ule, Jernej; Luscombe, Nicholas M; Tsai, Li-Huei; Walsh, Christopher A; Shi, Yang

    2015-03-01

    Cellular morphology is an essential determinant of cellular function in all kingdoms of life, yet little is known about how cell shape is controlled. Here we describe a molecular program that controls the early morphology of neurons through a metazoan-specific zinc finger protein, Unkempt. Depletion of Unkempt in mouse embryos disrupts the shape of migrating neurons, while ectopic expression confers neuronal-like morphology to cells of different nonneuronal lineages. We found that Unkempt is a sequence-specific RNA-binding protein and identified its precise binding sites within coding regions of mRNAs linked to protein metabolism and trafficking. RNA binding is required for Unkempt-induced remodeling of cellular shape and is directly coupled to a reduced production of the encoded proteins. These findings link post-transcriptional regulation of gene expression with cellular shape and have general implications for the development and disease of multicellular organisms. PMID:25737280

  3. RNA Binding Proteins that Control Human Papillomavirus Gene Expression.

    OpenAIRE

    Naoko Kajitani; Stefan Schwartz

    2015-01-01

    The human papillomavirus (HPV) life cycle is strictly linked to the differentiation program of the infected mucosal epithelial cell. In the basal and lower levels of the epithelium, early genes coding for pro-mitotic proteins and viral replication factors are expressed, while terminal cell differentiation is required for activation of late gene expression and production of viral particles at the very top of the epithelium. Such productive infections are normally cleared within 18–24 months. I...

  4. Steric control of CO binding in a totally synthetic heme protein model

    OpenAIRE

    Busch, Daryle H.; Zimmer, L. Lawrence; Grzybowski, Joseph J.; Olszanski, Dennis J.; Jackels, Susan C.; Callahan, Robert C.; Christoph, Gary G.

    1981-01-01

    A family of totally synthetic models for the carbon monoxide adducts of heme proteins has been synthesized and applied to the elucidation of the role of steric effects in the relative detoxification of carbon monoxide. The complexes are designed such that a sheltered void of controllable dimensions encompasses the CO binding site. Systematic variations in the available space for the iron-bound CO produce a wide range of equilibrium binding constants (KCO). An x-ray structure determination of ...

  5. Characterization of engineered actin binding proteins that control filament assembly and structure.

    Directory of Open Access Journals (Sweden)

    Crista M Brawley

    Full Text Available BACKGROUND: Eukaryotic cells strictly regulate the structure and assembly of their actin filament networks in response to various stimuli. The actin binding proteins that control filament assembly are therefore attractive targets for those who wish to reorganize actin filaments and reengineer the cytoskeleton. Unfortunately, the naturally occurring actin binding proteins include only a limited set of pointed-end cappers, or proteins that will block polymerization from the slow-growing end of actin filaments. Of the few that are known, most are part of large multimeric complexes that are challenging to manipulate. METHODOLOGY/PRINCIPAL FINDINGS: We describe here the use of phage display mutagenesis to generate of a new class of binding protein that can be targeted to the pointed-end of actin. These proteins, called synthetic antigen binders (sABs, are based on an antibody-like scaffold where sequence diversity is introduced into the binding loops using a novel "reduced genetic code" phage display library. We describe effective strategies to select and screen for sABs that ensure the generated sABs bind to the pointed-end surface of actin exclusively. CONCLUSIONS/SIGNIFICANCE: From our set of pointed-end binders, we identify three sABs with particularly useful properties to systematically probe actin dynamics: one protein that caps the pointed end, a second that crosslinks actin filaments, and a third that severs actin filaments and promotes disassembly.

  6. Gemin5: A Multitasking RNA-Binding Protein Involved in Translation Control

    Directory of Open Access Journals (Sweden)

    David Piñeiro

    2015-04-01

    Full Text Available Gemin5 is a RNA-binding protein (RBP that was first identified as a peripheral component of the survival of motor neurons (SMN complex. This predominantly cytoplasmic protein recognises the small nuclear RNAs (snRNAs through its WD repeat domains, allowing assembly of the SMN complex into small nuclear ribonucleoproteins (snRNPs. Additionally, the amino-terminal end of the protein has been reported to possess cap-binding capacity and to interact with the eukaryotic initiation factor 4E (eIF4E. Gemin5 was also shown to downregulate translation, to be a substrate of the picornavirus L protease and to interact with viral internal ribosome entry site (IRES elements via a bipartite non-canonical RNA-binding site located at its carboxy-terminal end. These features link Gemin5 with translation control events. Thus, beyond its role in snRNPs biogenesis, Gemin5 appears to be a multitasking protein cooperating in various RNA-guided processes. In this review, we will summarise current knowledge of Gemin5 functions. We will discuss the involvement of the protein on translation control and propose a model to explain how the proteolysis fragments of this RBP in picornavirus-infected cells could modulate protein synthesis.

  7. The alternative complement pathway control protein H binds to immune complexes and serves their detection

    International Nuclear Information System (INIS)

    During solubilization of immune complexes C3b becomes fixed to the immunoglobulin part and serves as a receptor for the alternative complement pathway control protein H. The H-C3b immune complex interaction can be made detectable using 4% polyethyleneglycol to separate free from bound 125I-H. Tetanus toxoid (Te)/anti-Te complexes kept soluble with fresh serum and containing 125 IU of specific antibody bound 18% of 125I-H; when fresh serum was chelated with 10 mM EDTA, 125I-H binding was only 5%. On sucrose density gradients, the H-binding material sedimented in the range of 12 to 30 S. In 36 serum samples from rheumatoid arthritis (RA) patients and in 12 serum samples from patients with systemic lupus erythematosus (SLE), 125I-H binding was significantly elevated to 9.5 +/- 4.7% (mean +/- 1 SD) and 13.3 +/- 5.6%, respectively, while 125I-H binding by 36 normal human sera was 4 +/- 2%. RA samples (17/36, 47%) and SLE samples (9/12, 75%) had H-binding values increased by more than 2 SD above the normal mean. The serum samples were also assessed for conglutinin- and C1q-binding activities; a significant correlation between H and C1q binding was observed (P less than 0.001); there was no correlation between H and conglutinin binding. Although binding to immune complexes through its interaction with C3b, H clearly detects a population of complexes other than conglutinin, thus expanding the possibilities of further characterizing pathological complexes

  8. Binding of SGTA to Rpn13 selectively modulates protein quality control.

    Science.gov (United States)

    Leznicki, Pawel; Korac-Prlic, Jelena; Kliza, Katarzyna; Husnjak, Koraljka; Nyathi, Yvonne; Dikic, Ivan; High, Stephen

    2015-09-01

    Rpn13 is an intrinsic ubiquitin receptor of the 26S proteasome regulatory subunit that facilitates substrate capture prior to degradation. Here we show that the C-terminal region of Rpn13 binds to the tetratricopeptide repeat (TPR) domain of SGTA, a cytosolic factor implicated in the quality control of mislocalised membrane proteins (MLPs). The overexpression of SGTA results in a substantial increase in steady-state MLP levels, consistent with an effect on proteasomal degradation. However, this effect is strongly dependent upon the interaction of SGTA with the proteasomal component Rpn13. Hence, overexpression of the SGTA-binding region of Rpn13 or point mutations within the SGTA TPR domain both inhibit SGTA binding to the proteasome and substantially reduce MLP levels. These findings suggest that SGTA can regulate the access of MLPs to the proteolytic core of the proteasome, implying that a protein quality control cycle that involves SGTA and the BAG6 complex can operate at the 19S regulatory particle. We speculate that the binding of SGTA to Rpn13 enables specific polypeptides to escape proteasomal degradation and/or selectively modulates substrate degradation. PMID:26169395

  9. Specific interactions between DNA and regulatory protein controlled by ligand-binding: Ab initio molecular simulation

    Energy Technology Data Exchange (ETDEWEB)

    Matsushita, Y., E-mail: kurita@cs.tut.ac.jp; Murakawa, T., E-mail: kurita@cs.tut.ac.jp; Shimamura, K., E-mail: kurita@cs.tut.ac.jp; Oishi, M., E-mail: kurita@cs.tut.ac.jp; Ohyama, T., E-mail: kurita@cs.tut.ac.jp; Kurita, N., E-mail: kurita@cs.tut.ac.jp [Department of Computer Science and Engineering, Toyohashi University of Technology, Tempaku-cho, Toyohashi, Aichi, 441-8580 (Japan)

    2015-02-27

    The catabolite activator protein (CAP) is one of the regulatory proteins controlling the transcription mechanism of gene. Biochemical experiments elucidated that the complex of CAP with cyclic AMP (cAMP) is indispensable for controlling the mechanism, while previous molecular simulations for the monomer of CAP+cAMP complex revealed the specific interactions between CAP and cAMP. However, the effect of cAMP-binding to CAP on the specific interactions between CAP and DNA is not elucidated at atomic and electronic levels. We here considered the ternary complex of CAP, cAMP and DNA in solvating water molecules and investigated the specific interactions between them at atomic and electronic levels using ab initio molecular simulations based on classical molecular dynamics and ab initio fragment molecular orbital methods. The results highlight the important amino acid residues of CAP for the interactions between CAP and cAMP and between CAP and DNA.

  10. Specific interactions between DNA and regulatory protein controlled by ligand-binding: Ab initio molecular simulation

    International Nuclear Information System (INIS)

    The catabolite activator protein (CAP) is one of the regulatory proteins controlling the transcription mechanism of gene. Biochemical experiments elucidated that the complex of CAP with cyclic AMP (cAMP) is indispensable for controlling the mechanism, while previous molecular simulations for the monomer of CAP+cAMP complex revealed the specific interactions between CAP and cAMP. However, the effect of cAMP-binding to CAP on the specific interactions between CAP and DNA is not elucidated at atomic and electronic levels. We here considered the ternary complex of CAP, cAMP and DNA in solvating water molecules and investigated the specific interactions between them at atomic and electronic levels using ab initio molecular simulations based on classical molecular dynamics and ab initio fragment molecular orbital methods. The results highlight the important amino acid residues of CAP for the interactions between CAP and cAMP and between CAP and DNA

  11. Posttranscriptional control of the hypoxic response by RNA-binding proteins and microRNAs

    Directory of Open Access Journals (Sweden)

    Myriam eGorospe

    2011-07-01

    Full Text Available Mammalian gene expression patterns change profoundly in response to low oxygen levels. These changes in gene expression programs are strongly influenced by post-transcriptional mechanisms mediated by mRNA-binding factors: RNA-binding proteins (RBPs and microRNAs (miRNAs. Here, we review the RBPs and miRNAs that modulate mRNA turnover and translation in response to hypoxic challenge. RBPs such as HuR (human antigen R, PTB (polypyrimidine tract-binding protein, heterogeneous nuclear ribonucleoproteins (hnRNPs, tristetraprolin, nucleolin, iron-response element binding proteins (IRPs, and cytoplasmic polyadenylation-element-binding proteins (CPEBs, selectively bind to numerous hypoxia-regulated transcripts and play a major role in establishing hypoxic gene expression patterns. MiRNAs including miR-210, miR-373, and miR-21 associate with hypoxia-regulated transcripts and further modulate the levels of the encoded proteins to implement the hypoxic gene expression profile. We discuss the potent regulation of hypoxic gene expression by RBPs and miRNAs and their integrated actions in the cellular hypoxic response.

  12. Control of synaptic plasticity and memory via suppression of poly(A)-binding protein.

    Science.gov (United States)

    Khoutorsky, Arkady; Yanagiya, Akiko; Gkogkas, Christos G; Fabian, Marc R; Prager-Khoutorsky, Masha; Cao, Ruifeng; Gamache, Karine; Bouthiette, Frederic; Parsyan, Armen; Sorge, Robert E; Mogil, Jeffrey S; Nader, Karim; Lacaille, Jean-Claude; Sonenberg, Nahum

    2013-04-24

    Control of protein synthesis is critical for synaptic plasticity and memory formation. However, the molecular mechanisms linking neuronal activity to activation of mRNA translation are not fully understood. Here, we report that the translational repressor poly(A)-binding protein (PABP)-interacting protein 2A (PAIP2A), an inhibitor of PABP, is rapidly proteolyzed by calpains in stimulated neurons and following training for contextual memory. Paip2a knockout mice exhibit a lowered threshold for the induction of sustained long-term potentiation and an enhancement of long-term memory after weak training. Translation of CaMKIIα mRNA is enhanced in Paip2a⁻/⁻ slices upon tetanic stimulation and in the hippocampus of Paip2a⁻/⁻ mice following contextual fear learning. We demonstrate that activity-dependent degradation of PAIP2A relieves translational inhibition of memory-related genes through PABP reactivation and conclude that PAIP2A is a pivotal translational regulator of synaptic plasticity and memory. PMID:23622065

  13. Control of silicification by genetically engineered fusion proteins: silk-silica binding peptides.

    Science.gov (United States)

    Zhou, Shun; Huang, Wenwen; Belton, David J; Simmons, Leo O; Perry, Carole C; Wang, Xiaoqin; Kaplan, David L

    2015-03-01

    In the present study, an artificial spider silk gene, 6mer, derived from the consensus sequence of Nephila clavipes dragline silk gene, was fused with different silica-binding peptides (SiBPs), A1, A3 and R5, to study the impact of the fusion protein sequence chemistry on silica formation and the ability to generate a silk-silica composite in two different bioinspired silicification systems: solution-solution and solution-solid. Condensed silica nanoscale particles (600-800 nm) were formed in the presence of the recombinant silk and chimeras, which were smaller than those formed by 15mer-SiBP chimeras, revealing that the molecular weight of the silk domain correlated to the sizes of the condensed silica particles in the solution system. In addition, the chimeras (6mer-A1/A3/R5) produced smaller condensed silica particles than the control (6mer), revealing that the silica particle size formed in the solution system is controlled by the size of protein assemblies in solution. In the solution-solid interface system, silicification reactions were performed on the surface of films fabricated from the recombinant silk proteins and chimeras and then treated to induce β-sheet formation. A higher density of condensed silica formed on the films containing the lowest β-sheet content while the films with the highest β-sheet content precipitated the lowest density of silica, revealing an inverse correlation between the β-sheet secondary structure and the silica content formed on the films. Intriguingly, the 6mer-A3 showed the highest rate of silica condensation but the lowest density of silica deposition on the films, compared with 6mer-A1 and -R5, revealing antagonistic crosstalk between the silk and the SiBP domains in terms of protein assembly. These findings offer a path forward in the tailoring of biopolymer-silica composites for biomaterial related needs. PMID:25462851

  14. Combinatorial Control of mRNA Fates by RNA-Binding Proteins and Non-Coding RNAs

    Directory of Open Access Journals (Sweden)

    Valentina Iadevaia

    2015-09-01

    Full Text Available Post-transcriptional control of gene expression is mediated by RNA-binding proteins (RBPs and small non-coding RNAs (e.g., microRNAs that bind to distinct elements in their mRNA targets. Here, we review recent examples describing the synergistic and/or antagonistic effects mediated by RBPs and miRNAs to determine the localisation, stability and translation of mRNAs in mammalian cells. From these studies, it is becoming increasingly apparent that dynamic rearrangements of RNA-protein complexes could have profound implications in human cancer, in synaptic plasticity, and in cellular differentiation.

  15. Regulation of transcription attenuation and translation initiation by allosteric control of an RNA-binding protein: the Bacillus subtilis TRAP protein.

    Science.gov (United States)

    Babitzke, Paul

    2004-04-01

    Tryptophan allosterically controls the 11-subunit trp RNA-binding attenuation protein (TRAP) of Bacillus subtilis. When activated by tryptophan, TRAP binds to multiple trinucleotide repeats in target transcripts. TRAP is responsible for the decision to terminate transcription in the leader region of the trpEDCFBA operon or to allow transcription to proceed into the structural genes. TRAP also regulates translation of trpE by promoting formation of an RNA structure that prevents ribosome binding. In addition, bound TRAP regulates translation initiation of pabA, trpP and ycbK by directly blocking ribosome binding. The anti-TRAP protein inhibits TRAP activity by competing with RNA for the RNA binding surface of TRAP. PMID:15063849

  16. Control of silicification by genetically engineered fusion proteins: Silk–silica binding peptides

    OpenAIRE

    Zhou, Shun; Huang, Wenwen; Belton, David J.; Simmons, Leo O.; Perry, Carole C.; Wang, Xiaoqin; Kaplan, David L.

    2014-01-01

    In the present study, an artificial spider silk gene, 6mer, derived from the consensus sequence of Nephila clavipes dragline silk gene, was fused with different silica-binding peptides (SiBPs), A1, A3 and R5, to study the impact of the fusion protein sequence chemistry on silica formation and the ability to generate a silk–silica composite in two different bioinspired silicification systems: solution–solution and solution– solid. Condensed silica nanoscale particles (600–800 nm) were formed i...

  17. HIV-1 TAR RNA-binding proteins control TAT activation of translation in Xenopus oocytes.

    Science.gov (United States)

    Braddock, M; Powell, R; Blanchard, A D; Kingsman, A J; Kingsman, S M

    1993-01-01

    Human immunodeficiency virus (HIV-1) gene expression is activated by the viral TAT protein that interacts with an RNA sequence, TAR, located at the 5' end of all viral mRNAs. TAT functions primarily as a transcriptional activator in mammalian cells. However, in Xenopus oocytes TAT functions primarily as a translational activator. TAR is an RNA structure comprising a partially base-paired stem, a tripyrimidine bulge in the upper stem, and an unpaired six-nucleotide loop. In vitro, TAT binds directly to the bulge with no requirement for the loop. In vivo, however, mutations in the loop abolish TAT activation of transcription and translation, implying a requirement for TAR-binding cellular factors. We now provide genetic evidence for the presence of two TAR-specific cellular factors in Xenopus oocytes. These factors display independent and mutually exclusive interactions with either the loop or the bulge region of TAR. Furthermore, by using in vivo RNA competition assays we show that the cellular factors regulate the accessibility of the TAT binding site. The fact that Xenopus oocytes contain factors that specifically interact with a human viral RNA sequence might indicate that the TAT/TAR interaction is subverting a conserved pathway in the cell. PMID:8422967

  18. Control of a neuronal morphology program by an RNA-binding zinc finger protein, Unkempt

    OpenAIRE

    Murn, Jernej; Zarnack, Kathi; Yang, Yawei J.; Durak, Omer; Murphy, Elisabeth A.; Cheloufi, Sihem; Gonzalez, Dilenny M.; Teplova, Marianna; Curk, Tomaž; Zuber, Johannes; Patel, Dinshaw J.; Ule, Jernej; Luscombe, Nicholas M.; Tsai, Li-Huei; Walsh, Christopher A.

    2015-01-01

    Cellular morphology is an essential determinant of cellular function in all kingdoms of life, yet little is known about how cell shape is controlled. Here we describe a molecular program that controls the early morphology of neurons through a metazoan-specific zinc finger protein, Unkempt. Depletion of Unkempt in mouse embryos disrupts the shape of migrating neurons, while ectopic expression confers neuronal-like morphology to cells of different nonneuronal lineages. We found that Unkempt is ...

  19. Competitive binding of viral E2 protein and mammalian core-binding factor to transcriptional control sequences of human papillomavirus type 8 and bovine papillomavirus type 1.

    OpenAIRE

    Schmidt, H. M.; Steger, G; Pfister, H

    1997-01-01

    The promoter P7535 of human papillomavirus type 8 and the promoter P7185 of bovine papillomavirus type 1 are negatively regulated by viral E2 proteins via the promoter proximal binding sites P2 and BS1, respectively. Mutations of these E2 binding sites can reduce basal promoter activity. This suggests binding of a transcription-stimulating factor and may indicate that repression by E2 is due to competitive binding of viral and cellular proteins. A computer search revealed putative binding sit...

  20. Organic crystal-binding peptides: morphology control and one-pot formation of protein-displaying organic crystals

    Science.gov (United States)

    Niide, Teppei; Ozawa, Kyohei; Nakazawa, Hikaru; Oliveira, Daniel; Kasai, Hitoshi; Onodera, Mari; Asano, Ryutaro; Kumagai, Izumi; Umetsu, Mitsuo

    2015-11-01

    Crystalline assemblies of fluorescent molecules have different functional properties than the constituent monomers, as well as unique optical characteristics that depend on the structure, size, and morphological homogeneity of the crystal particles. In this study, we selected peptides with affinity for the surface of perylene crystal particles by exposing a peptide-displaying phage library in aqueous solution to perylene crystals, eluting the surface-bound phages by means of acidic desorption or liquid-liquid extraction, and amplifying the obtained phages in Escherichia coli. One of the perylene-binding peptides, PeryBPb1: VQHNTKYSVVIR, selected by this biopanning procedure induced perylene molecules to form homogenous planar crystal nanoparticles by means of a poor solvent method, and fusion of the peptide to a fluorescent protein enabled one-pot formation of protein-immobilized crystalline nanoparticles. The nanoparticles were well-dispersed in aqueous solution, and Förster resonance energy transfer from the perylene crystals to the fluorescent protein was observed. Our results show that the crystal-binding peptide could be used for simultaneous control of perylene crystal morphology and dispersion and protein immobilization on the crystals.Crystalline assemblies of fluorescent molecules have different functional properties than the constituent monomers, as well as unique optical characteristics that depend on the structure, size, and morphological homogeneity of the crystal particles. In this study, we selected peptides with affinity for the surface of perylene crystal particles by exposing a peptide-displaying phage library in aqueous solution to perylene crystals, eluting the surface-bound phages by means of acidic desorption or liquid-liquid extraction, and amplifying the obtained phages in Escherichia coli. One of the perylene-binding peptides, PeryBPb1: VQHNTKYSVVIR, selected by this biopanning procedure induced perylene molecules to form homogenous planar

  1. Crystal and solution studies of the "Plus-C" odorant-binding protein 48 from Anopheles gambiae: control of binding specificity through three-dimensional domain swapping.

    Science.gov (United States)

    Tsitsanou, Katerina E; Drakou, Christina E; Thireou, Trias; Vitlin Gruber, Anna; Kythreoti, Georgia; Azem, Abdussalam; Fessas, Dimitrios; Eliopoulos, Elias; Iatrou, Kostas; Zographos, Spyros E

    2013-11-15

    Much physiological and behavioral evidence has been provided suggesting that insect odorant-binding proteins (OBPs) are indispensable for odorant recognition and thus are appealing targets for structure-based discovery and design of novel host-seeking disruptors. Despite the fact that more than 60 putative OBP-encoding genes have been identified in the malaria vector Anopheles gambiae, the crystal structures of only six of them are known. It is therefore clear that OBP structure determination constitutes the bottleneck for structure-based approaches to mosquito repellent/attractant discovery. Here, we describe the three-dimensional structure of an A. gambiae "Plus-C" group OBP (AgamOBP48), which exhibits the second highest expression levels in female antennae. This structure represents the first example of a three-dimensional domain-swapped dimer in dipteran species. A combined binding site is formed at the dimer interface by equal contribution of each monomer. Structural comparisons with the monomeric AgamOBP47 revealed that the major structural difference between the two Plus-C proteins localizes in their N- and C-terminal regions, and their concerted conformational change may account for monomer-swapped dimer conversion and furthermore the formation of novel binding pockets. Using a combination of gel filtration chromatography, differential scanning calorimetry, and analytical ultracentrifugation, we demonstrate the AgamOBP48 dimerization in solution. Eventually, molecular modeling calculations were used to predict the binding mode of the most potent synthetic ligand of AgamOBP48 known so far, discovered by ligand- and structure-based virtual screening. The structure-aided identification of multiple OBP binders represents a powerful tool to be employed in the effort to control transmission of the vector-borne diseases. PMID:24097978

  2. Vitamin D-binding protein controls T cell responses to vitamin D

    DEFF Research Database (Denmark)

    Kongsbak, Martin; von Essen, Marina Rode; Levring, Trine Bøegh;

    2014-01-01

    BACKGROUND: In vitro studies have shown that the active form of vitamin D3, 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3), can regulate differentiation of CD4+ T cells by inhibiting Th1 and Th17 cell differentiation and promoting Th2 and Treg cell differentiation. However, the serum concentration of 1...... activated T cells express the 25(OH)D-1α-hydroxylase CYP27B1 that converts 25(OH)D3 to 1,25(OH)2D3, it is still controversial whether activated T cells have the capacity to produce sufficient amounts of 1,25(OH)2D3 to affect vitamin D-responsive genes. Furthermore, it is not known how the vitamin D......-binding protein (DBP) found in high concentrations in serum affects T cell responses to 25(OH)D3. RESULTS: We found that activated T cells express CYP27B1 and have the capacity to produce sufficient 1,25(OH)2D3 to affect vitamin D-responsive genes when cultured with physiological concentrations of 25(OH)D3 in...

  3. The RNA binding protein CsrA controls c-di-GMP metabolism by directly regulating the expression of GGDEF proteins

    OpenAIRE

    Jonas, Kristina; Edwards, Adrianne N.; Simm, Roger; Romeo, Tony; Römling, Ute; Melefors, Öjar

    2008-01-01

    The carbon storage regulator CsrA is an RNA binding protein that controls carbon metabolism, biofilm formation and motility in various eubacteria. Nevertheless, in Escherichia coli only five target mRNAs have been shown to be directly regulated by CsrA at the post-transcriptional level. Here we identified two new direct targets for CsrA, ycdT and ydeH, both of which encode proteins with GGDEF domains. A csrA mutation caused mRNA levels of ycdT and ydeH to increase more than 10-fold. RNA mobil...

  4. BINDING ISOTHERMS SURFACTANT-PROTEINS

    OpenAIRE

    Elena Irina Moater; Cristiana Radulescu; Ionica Ionita

    2011-01-01

    The interactions between surfactants and proteins shows some similarities with interactions between surfactants and polymers, but the hydrophobic amphoteric nature of proteins and their secondary and tertiary structure components make them different from conventional polymer systems. Many studies from the past about surfactant - proteins bonding used the dialysis techniques. Other techniques used to determine the binding isotherm, included ultrafiltration, ultracentrifugation, potentiometry, ...

  5. Protein Dynamics in an RNA Binding Protein

    Science.gov (United States)

    Hall, Kathleen

    2006-03-01

    Using ^15N NMR relaxation measurements, analyzed with the Lipari-Szabo formalism, we have found that the human U1A RNA binding protein has ps-ns motions in those loops that make contact with RNA. Specific mutations can alter the extent and pattern of motions, and those proteins inevitably lose RNA binding affinity. Proteins with enhanced mobility of loops and termini presumably lose affinity due to increased conformational sampling by those parts of the protein that interact directly with RNA. There is an entropic penalty associated with locking down those elements upon RNA binding, in addition to a loss of binding efficiency caused by the increased number of conformations adopted by the protein. However, in addition to local conformational heterogeneity, analysis of molecular dynamics trajectories by Reorientational Eigenmode Dynamics reveals that loops of the wild type protein undergo correlated motions that link distal sites across the binding surface. Mutations that disrupt correlated motions result in weaker RNA binding, implying that there is a network of interactions across the surface of the protein. (KBH was a Postdoctoral Fellow with Al Redfield from 1985-1990). This work was supported by the NIH (to KBH) and NSF (SAS).

  6. Grafting of protein-protein binding sites

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    A strategy for grafting protein-protein binding sites is described. Firstly, key interaction residues at the interface of ligand protein to be grafted are identified and suitable positions in scaffold protein for grafting these key residues are sought. Secondly, the scaffold proteins are superposed onto the ligand protein based on the corresponding Ca and Cb atoms. The complementarity between the scaffold protein and the receptor protein is evaluated and only matches with high score are accepted. The relative position between scaffold and receptor proteins is adjusted so that the interface has a reasonable packing density. Then the scaffold protein is mutated to corresponding residues in ligand protein at each candidate position. And the residues having bad steric contacts with the receptor proteins, or buried charged residues not involved in the formation of any salt bridge are mutated. Finally, the mutated scaffold protein in complex with receptor protein is co-minimized by Charmm. In addition, we deduce a scoring function to evaluate the affinity between mutated scaffold protein and receptor protein by statistical analysis of rigid binding data sets.

  7. RNA binding protein Pub1p regulates glycerol production and stress tolerance by controlling Gpd1p activity during winemaking.

    Science.gov (United States)

    Orozco, Helena; Sepúlveda, Ana; Picazo, Cecilia; Matallana, Emilia; Aranda, Agustín

    2016-06-01

    Glycerol is a key yeast metabolite in winemaking because it contributes to improve the organoleptic properties of wine. It is also a cellular protective molecule that enhances the tolerance of yeasts to osmotic stress and promotes longevity. Thus, its production increases by genetic manipulation, which is of biotechnological and basic interest. Glycerol is produced by diverting glycolytic glyceraldehyde-3-phosphate through the action of glycerol-3-phosphate dehydrogenase (coded by genes GPD1 and GPD2). Here, we demonstrate that RNA-binding protein Pub1p regulates glycerol production by controlling Gpd1p activity. Its deletion does not alter GPD1 mRNA levels, but protein levels and enzymatic activity increase, which explains the higher intracellular glycerol concentration and greater tolerance to osmotic stress of the pub1∆ mutant. PUB1 deletion also enhances the activity of nicotinamidase, a longevity-promoting enzyme. Both enzymatic activities are partially located in peroxisomes, and we detected peroxisome formation during wine fermentation. The role of Pub1p in life span control depends on nutrient conditions and is related with the TOR pathway, and a major connection between RNA metabolism and the nutrient signaling response is established. PMID:26846624

  8. Novel control of cardiac myofilament response to calcium by S-glutahionylation at specific sites of myosin binding protein C

    Directory of Open Access Journals (Sweden)

    R.JohnSolaro

    2013-11-01

    Full Text Available Our previous studies demonstrated a relation between glutathionylation of cardiac myosin binding protein C and diastolic dysfunction in a hypertensive mouse model stressed by treatment with salt, deoxycorticosterone acetate, and unilateral nephrectomy. Although these results strongly indicated an important role for S-glutathionylation of myosin binding protein C as a modifier of myofilament function, indirect effects of other post-translational modifications may have occurred. Moreover, we did not determine the sites of thiol modification by glutathionylation. To address these issues, we developed an in vitro method to mimic the in situ S-glutathionylation of myofilament proteins and determined direct functional effects and sites of oxidative modification employing Western blotting and mass spectrometry. We induced glutathionylation in vitro by treatment of isolated myofibrils and detergent extracted fiber bundles (skinned fibers with oxidized glutathione (GSSG. Immuno-blotting results revealed increased glutathionylation with GSSG treatment of a protein band around 140 kDa. Using tandem mass spectrometry, we identified the 140 kDa band as cardiac myosin binding protein C and determined the sites of glutathionylation to be at cysteines 655, 479, and 627. Determination of the relation between Ca2+-activation of myofibrillar acto-myosin ATPase rate demonstrated an increased Ca2+-sensitivity induced by the S-glutathionylation. Force generating skinned fiber bundles also showed an increase in Ca-sensitivity when treated with oxidized glutathione, which was reversed with the reducing agent, dithiothreitol. Our data demonstrate that a specific and direct effect of S-glutathionylation of myosin binding protein C is a significant increase in myofilament Ca2+-sensitivity. Our data also provide new insights into the functional significance of oxidative modification of myosin binding protein C and the potential role of domains not previously considered to

  9. Probing protein phosphatase substrate binding

    DEFF Research Database (Denmark)

    Højlys-Larsen, Kim B.; Sørensen, Kasper Kildegaard; Jensen, Knud Jørgen; Gammeltoft, Steen

    2012-01-01

    Proteomics and high throughput analysis for systems biology can benefit significantly from solid-phase chemical tools for affinity pull-down of proteins from complex mixtures. Here we report the application of solid-phase synthesis of phosphopeptides for pull-down and analysis of the affinity...... profile of the integrin-linked kinase associated phosphatase (ILKAP), a member of the protein phosphatase 2C (PP2C) family. Phosphatases can potentially dephosphorylate these phosphopeptide substrates but, interestingly, performing the binding studies at 4 °C allowed efficient binding to phosphopeptides......, without the need for phosphopeptide mimics or phosphatase inhibitors. As no proven ILKAP substrates were available, we selected phosphopeptide substrates among known PP2Cδ substrates including the protein kinases: p38, ATM, Chk1, Chk2 and RSK2 and synthesized directly on PEGA solid supports through a BAL...

  10. Structural analysis of DNA binding by C.Csp231I, a member of a novel class of R-M controller proteins regulating gene expression

    Energy Technology Data Exchange (ETDEWEB)

    Shevtsov, M. B.; Streeter, S. D.; Thresh, S.-J.; Swiderska, A.; McGeehan, J. E.; Kneale, G. G., E-mail: geoff.kneale@port.ac.uk [University of Portsmouth, Portsmouth PO1 2DY (United Kingdom)

    2015-02-01

    The structure of the new class of controller proteins (exemplified by C.Csp231I) in complex with its 21 bp DNA-recognition sequence is presented, and the molecular basis of sequence recognition in this class of proteins is discussed. An unusual extended spacer between the dimer binding sites suggests a novel interaction between the two C-protein dimers. In a wide variety of bacterial restriction–modification systems, a regulatory ‘controller’ protein (or C-protein) is required for effective transcription of its own gene and for transcription of the endonuclease gene found on the same operon. We have recently turned our attention to a new class of controller proteins (exemplified by C.Csp231I) that have quite novel features, including a much larger DNA-binding site with an 18 bp (∼60 Å) spacer between the two palindromic DNA-binding sequences and a very different recognition sequence from the canonical GACT/AGTC. Using X-ray crystallography, the structure of the protein in complex with its 21 bp DNA-recognition sequence was solved to 1.8 Å resolution, and the molecular basis of sequence recognition in this class of proteins was elucidated. An unusual aspect of the promoter sequence is the extended spacer between the dimer binding sites, suggesting a novel interaction between the two C-protein dimers when bound to both recognition sites correctly spaced on the DNA. A U-bend model is proposed for this tetrameric complex, based on the results of gel-mobility assays, hydrodynamic analysis and the observation of key contacts at the interface between dimers in the crystal.

  11. Structural analysis of DNA binding by C.Csp231I, a member of a novel class of R-M controller proteins regulating gene expression

    International Nuclear Information System (INIS)

    The structure of the new class of controller proteins (exemplified by C.Csp231I) in complex with its 21 bp DNA-recognition sequence is presented, and the molecular basis of sequence recognition in this class of proteins is discussed. An unusual extended spacer between the dimer binding sites suggests a novel interaction between the two C-protein dimers. In a wide variety of bacterial restriction–modification systems, a regulatory ‘controller’ protein (or C-protein) is required for effective transcription of its own gene and for transcription of the endonuclease gene found on the same operon. We have recently turned our attention to a new class of controller proteins (exemplified by C.Csp231I) that have quite novel features, including a much larger DNA-binding site with an 18 bp (∼60 Å) spacer between the two palindromic DNA-binding sequences and a very different recognition sequence from the canonical GACT/AGTC. Using X-ray crystallography, the structure of the protein in complex with its 21 bp DNA-recognition sequence was solved to 1.8 Å resolution, and the molecular basis of sequence recognition in this class of proteins was elucidated. An unusual aspect of the promoter sequence is the extended spacer between the dimer binding sites, suggesting a novel interaction between the two C-protein dimers when bound to both recognition sites correctly spaced on the DNA. A U-bend model is proposed for this tetrameric complex, based on the results of gel-mobility assays, hydrodynamic analysis and the observation of key contacts at the interface between dimers in the crystal

  12. Control of the level of unusual estrogen-binding protein in rat liver by sex steroids and the pituitary

    Energy Technology Data Exchange (ETDEWEB)

    Smirnova, O.V.; Rozen, V.B.; Vishnyakova, T.G.

    1986-02-01

    This paper studies the role of sex steriods and the pituitary in regulation of the unusual estrogen-binding protein (UEBP) level in male rat liver. The concentration of E/sub 2/-binding sites of UEBP in the liver cytosol was determined by measuring binding of a minimal addition of 2,4,6,7-tritium-E/sub 2/, with specific radioactivity of 98-100 Ci/mmole. Data on the effect of hypophysectomy on the UEBP level in the liver of different groups of rats are presented. The presence of comparable quantities of E/sub 2/ and androgens in rats of both sexes is evidence of the existence of a fine mechanism of combined regulation of the UEBP concentration under natural conditions that reflect changes in the absolute E/sub 2/ or androgen levels or in the ratio between them.

  13. Control of the level of unusual estrogen-binding protein in rat liver by sex steroids and the pituitary

    International Nuclear Information System (INIS)

    This paper studies the role of sex steriods and the pituitary in regulation of the unusual estrogen-binding protein (UEBP) level in male rat liver. The concentration of E2-binding sites of UEBP in the liver cytosol was determined by measuring binding of a minimal addition of 2,4,6,7-tritium-E2, with specific radioactivity of 98-100 Ci/mmole. Data on the effect of hypophysectomy on the UEBP level in the liver of different groups of rats are presented. The presence of comparable quantities of E2 and androgens in rats of both sexes is evidence of the existence of a fine mechanism of combined regulation of the UEBP concentration under natural conditions that reflect changes in the absolute E2 or androgen levels or in the ratio between them

  14. The fission yeast RNA binding protein Mmi1 regulates meiotic genes by controlling intron specific splicing and polyadenylation coupled RNA turnover.

    Directory of Open Access Journals (Sweden)

    Huei-Mei Chen

    Full Text Available The polyA tails of mRNAs are monitored by the exosome as a quality control mechanism. We find that fission yeast, Schizosaccharomyces pombe, adopts this RNA quality control mechanism to regulate a group of 30 or more meiotic genes at the level of both splicing and RNA turnover. In vegetative cells the RNA binding protein Mmi1 binds to the primary transcripts of these genes. We find the novel motif U(U/C/GAAAC highly over-represented in targets of Mmi1. Mmi1 can specifically regulate the splicing of particular introns in a transcript: it inhibits the splicing of introns that are in the vicinity of putative Mmi1 binding sites, while allowing the splicing of other introns that are far from such sites. In addition, binding of Mmi1, particularly near the 3' end, alters 3' processing to promote extremely long polyA tails of up to a kilobase. The hyperadenylated transcripts are then targeted for degradation by the nuclear exonuclease Rrp6. The nuclear polyA binding protein Pab2 assists this hyperadenylation-mediated RNA decay. Rrp6 also targets other hyperadenylated transcripts, which become hyperadenylated in an unknown, but Mmi1-independent way. Thus, hyperadenylation may be a general signal for RNA degradation. In addition, binding of Mmi1 can affect the efficiency of 3' cleavage. Inactivation of Mmi1 in meiosis allows meiotic expression, through splicing and RNA stabilization, of at least 29 target genes, which are apparently constitutively transcribed.

  15. Control of Neuronal Excitability by Calcium Binding Proteins: A New Mathematical Model for Striatal Fast-Spiking Interneurons

    OpenAIRE

    Don Patrick Bischop

    2012-01-01

    Calcium binding proteins, such as parvalbumin (PV), are abundantly expressed in distinctive patterns in the central nervous system but their physiological function remains poorly under-stood. Notably, at the level of the striatum, where PV is only expressed in the fast-spiking (FS) interneurons. FS interneurons form an inhibitory network modulating the output of the striatum by synchronizing medium-sized spiny neurons (MSN). So far the existing conductance-based computational models for FS ne...

  16. Control of neuronal excitability by calcium binding proteins : a new mathematical model for striatal fast-spiking interneurons

    OpenAIRE

    Don Patrick Bischop

    2012-01-01

    Calcium binding proteins, such as parvalbumin (PV), are abundantly expressed in distinctive patterns in the central nervous system but their physiological function remains poorly understood. Notably, at the level of the striatum, where PV is only expressed in the fast spiking (FS) interneurons. FS interneurons form an inhibitory network modulating the output of the striatum by synchronizing medium-sized spiny neurons (MSN). So far the existing conductance-based computational models for FS n...

  17. Structure of the apo form of the catabolite control protein A (CcpA) from Bacillus megaterium with a DNA-binding domain

    International Nuclear Information System (INIS)

    Crystal structure analysis of the apo form of catabolite control protein A reveals the three-helix bundle of the DNA-binding domain. In the crystal packing, this domain interacts with the binding site for the corepressor protein. Crystal structure determination of catabolite control protein A (CcpA) at 2.6 Å resolution reveals for the first time the structure of a full-length apo-form LacI-GalR family repressor protein. In the crystal structures of these transcription regulators, the three-helix bundle of the DNA-binding domain has only been observed in cognate DNA complexes; it has not been observed in other crystal structures owing to its mobility. In the crystal packing of apo-CcpA, the protein–protein contacts between the N-terminal three-helix bundle and the core domain consisted of interactions between the homodimers that were similar to those between the corepressor protein HPr and the CcpA N-subdomain in the ternary DNA complex. In contrast to the DNA complex, the apo-CcpA structure reveals large subdomain movements in the core, resulting in a complete loss of contacts between the N-subdomains of the homodimer

  18. Erythropoietin binding protein from mammalian serum

    Energy Technology Data Exchange (ETDEWEB)

    Clemons, G.K.

    1997-04-29

    Purified mammalian erythropoietin binding-protein is disclosed, and its isolation, identification, characterization, purification, and immunoassay are described. The erythropoietin binding protein can be used for regulation of erythropoiesis by regulating levels and half-life of erythropoietin. A diagnostic kit for determination of level of erythropoietin binding protein is also described. 11 figs.

  19. Erythropoietin binding protein from mammalian serum

    Energy Technology Data Exchange (ETDEWEB)

    Clemons, Gisela K. (Berkeley, CA)

    1997-01-01

    Purified mammalian erythropoietin binding-protein is disclosed, and its isolation, identification, characterization, purification, and immunoassay are described. The erythropoietin binding protein can be used for regulation of erythropoiesis by regulating levels and half-life of erythropoietin. A diagnostic kit for determination of level of erythropoietin binding protein is also described.

  20. The multicopy sRNA LhrC controls expression of the oligopeptide-binding protein OppA in Listeria monocytogenes.

    Science.gov (United States)

    Sievers, Susanne; Lund, Anja; Menendez-Gil, Pilar; Nielsen, Aaraby; Storm Mollerup, Maria; Lambert Nielsen, Stine; Buch Larsson, Pernille; Borch-Jensen, Jonas; Johansson, Jörgen; Kallipolitis, Birgitte Haahr

    2015-01-01

    Listeria monocytogenes is the causative agent of the foodborne disease listeriosis. During infection, L. monocytogenes produces an array of non-coding RNAs, including the multicopy sRNA LhrC. These five, nearly identical sRNAs are highly induced in response to cell envelope stress and target the virulence adhesin lapB at the post-transcriptional level. Here, we demonstrate that LhrC controls expression of additional genes encoding cell envelope-associated proteins with virulence function. Using transcriptomics and proteomics, we identified a set of genes affected by LhrC in response to cell envelope stress. Three targets were significantly down-regulated by LhrC at both the RNA and protein level: lmo2349, tcsA and oppA. All three genes encode membrane-associated proteins: A putative substrate binding protein of an amino acid ABC transporter (Lmo2349); the CD4+ T cell-stimulating antigen TcsA, and the oligopeptide binding protein OppA, of which the latter 2 are required for full virulence of L. monocytogenes. For OppA, we show that LhrC acts by direct base paring to the ribosome binding site of the oppA mRNA, leading to an impediment of its translation and a decreased mRNA level. The sRNA-mRNA interaction depends on 2 of 3 CU-rich regions in LhrC allowing binding of 2 oppA mRNAs to a single LhrC molecule. Finally, we found that LhrC contributes to infection in macrophage-like cells. These findings demonstrate a central role for LhrC in controlling the level of OppA and other virulence-associated cell envelope proteins in response to cell envelope stress. PMID:26176322

  1. Advances on Plant Pathogenic Mycotoxin Binding Proteins

    Institute of Scientific and Technical Information of China (English)

    WANG Chao-hua; DONG Jin-gao

    2002-01-01

    Toxin-binding protein is one of the key subjects in plant pathogenic mycotoxin research. In this paper, new advances in toxin-binding proteins of 10 kinds of plant pathogenic mycotoxins belonging to Helminthosporium ,Alternaria ,Fusicoccum ,Verticillium were reviewed, especially the techniques and methods of toxin-binding proteins of HS-toxin, HV-toxin, HMT-toxin, HC-toxin. It was proposed that the isotope-labeling technique and immunological chemistry technique should be combined together in research of toxin-binding protein, which will be significant to study the molecular recognition mechanism between host and pathogenic fungus.

  2. Spatial control of translation repression and polarized growth by conserved NDR kinase Orb6 and RNA-binding protein Sts5

    Science.gov (United States)

    Nuñez, Illyce; Rodriguez Pino, Marbelys; Wiley, David J; Das, Maitreyi E; Chen, Chuan; Goshima, Tetsuya; Kume, Kazunori; Hirata, Dai; Toda, Takashi; Verde, Fulvia

    2016-01-01

    RNA-binding proteins contribute to the formation of ribonucleoprotein (RNP) granules by phase transition, but regulatory mechanisms are not fully understood. Conserved fission yeast NDR (Nuclear Dbf2-Related) kinase Orb6 governs cell morphogenesis in part by spatially controlling Cdc42 GTPase. Here we describe a novel, independent function for Orb6 kinase in negatively regulating the recruitment of RNA-binding protein Sts5 into RNPs to promote polarized cell growth. We find that Orb6 kinase inhibits Sts5 recruitment into granules, its association with processing (P) bodies, and degradation of Sts5-bound mRNAs by promoting Sts5 interaction with 14-3-3 protein Rad24. Many Sts5-bound mRNAs encode essential factors for polarized cell growth, and Orb6 kinase spatially and temporally controls the extent of Sts5 granule formation. Disruption of this control system affects cell morphology and alters the pattern of polarized cell growth, revealing a role for Orb6 kinase in the spatial control of translational repression that enables normal cell morphogenesis. DOI: http://dx.doi.org/10.7554/eLife.14216.001 PMID:27474797

  3. Control of neuronal excitability by calcium binding proteins: a new mathematical model for striatal fast-spiking interneurons.

    Science.gov (United States)

    Bischop, D P; Orduz, D; Lambot, L; Schiffmann, S N; Gall, D

    2012-01-01

    Calcium binding proteins, such as parvalbumin (PV), are abundantly expressed in distinctive patterns in the central nervous system but their physiological function remains poorly understood. Notably, at the level of the striatum, where PV is only expressed in the fast-spiking (FS) interneurons. FS interneurons form an inhibitory network modulating the output of the striatum by synchronizing medium-sized spiny neurons (MSN). So far the existing conductance-based computational models for FS neurons did not allow the study of the coupling between PV concentration and electrical activity. In the present paper, we propose a new mathematical model for the striatal FS interneurons that includes apamin-sensitive small conductance Ca(2+)-dependent K(+) channels (SK) and the presence of a calcium buffer. Our results show that a variation in the concentration of PV can modulate substantially the intrinsic excitability of the FS interneurons and therefore may be involved in the information processing at the striatal level. PMID:22787441

  4. Control of neuronal excitability by calcium binding proteins : a new mathematical model for striatal fast-spiking interneurons.

    Directory of Open Access Journals (Sweden)

    Don Patrick Bischop

    2012-07-01

    Full Text Available Calcium binding proteins, such as parvalbumin, are abundantly expressed in very distinctive patterns in the central nervous system but their physiological function remains poorly understood. Notably, at the level of the striatum, parvalbumin is only expressed in the fast spiking (FS interneurons, which form a inhibitory network modulating the output of the striatum by synchronizing medium-sized spiny neurons (MSN. So far the existing conductance-based computational models for FS neurons did not allow the study of the the coupling between parvalbumin concentration and electrical activity. In the present paper, we propose a new mathematical model for the striatal FS interneurons that includes apamin-sensitive small conductance \\ca -dependent \\kk channels (SK and takes into account the presence of a calcium buffer. Our results demonstrate that a variation in the concentration of parvalbumin can modulate substantially the intrinsic excitability of the FS interneurons and therefore may be involved in the information processing at the striatal level.

  5. Retinoid-binding proteins: similar protein architectures bind similar ligands via completely different ways.

    Directory of Open Access Journals (Sweden)

    Yu-Ru Zhang

    Full Text Available BACKGROUND: Retinoids are a class of compounds that are chemically related to vitamin A, which is an essential nutrient that plays a key role in vision, cell growth and differentiation. In vivo, retinoids must bind with specific proteins to perform their necessary functions. Plasma retinol-binding protein (RBP and epididymal retinoic acid binding protein (ERABP carry retinoids in bodily fluids, while cellular retinol-binding proteins (CRBPs and cellular retinoic acid-binding proteins (CRABPs carry retinoids within cells. Interestingly, although all of these transport proteins possess similar structures, the modes of binding for the different retinoid ligands with their carrier proteins are different. METHODOLOGY/PRINCIPAL FINDINGS: In this work, we analyzed the various retinoid transport mechanisms using structure and sequence comparisons, binding site analyses and molecular dynamics simulations. Our results show that in the same family of proteins and subcellular location, the orientation of a retinoid molecule within a binding protein is same, whereas when different families of proteins are considered, the orientation of the bound retinoid is completely different. In addition, none of the amino acid residues involved in ligand binding is conserved between the transport proteins. However, for each specific binding protein, the amino acids involved in the ligand binding are conserved. The results of this study allow us to propose a possible transport model for retinoids. CONCLUSIONS/SIGNIFICANCE: Our results reveal the differences in the binding modes between the different retinoid-binding proteins.

  6. FBXL5-mediated degradation of single-stranded DNA-binding protein hSSB1 controls DNA damage response.

    Science.gov (United States)

    Chen, Zhi-Wei; Liu, Bin; Tang, Nai-Wang; Xu, Yun-Hua; Ye, Xiang-Yun; Li, Zi-Ming; Niu, Xiao-Min; Shen, Sheng-Ping; Lu, Shun; Xu, Ling

    2014-10-01

    Human single-strand (ss) DNA binding proteins 1 (hSSB1) has been shown to participate in DNA damage response and maintenance of genome stability by regulating the initiation of ATM-dependent signaling. ATM phosphorylates hSSB1 and prevents hSSB1 from ubiquitin-proteasome-mediated degradation. However, the E3 ligase that targets hSSB1 for destruction is still unknown. Here, we report that hSSB1 is the bona fide substrate for an Fbxl5-containing SCF (Skp1-Cul1-F box) E3 ligase. Fbxl5 interacts with and targets hSSB1 for ubiquitination and degradation, which could be prevented by ATM-mediated hSSB1 T117 phosphorylation. Furthermore, cells overexpression of Fbxl5 abrogated the cellular response to DSBs, including activation of ATM and phosphorylation of ATM targets and exhibited increased radiosensitivity, chemosensitivity and defective checkpoint activation after genotoxic stress stimuli. Moreover, the protein levels of hSSB1 and Fbxl5 showed an inverse correlation in lung cancer cells lines and clinical lung cancer samples. Therefore, Fbxl5 may negatively modulate hSSB1 to regulate DNA damage response, implicating Fbxl5 as a novel, promising therapeutic target for lung cancers. PMID:25249620

  7. Penicillin-Binding Protein Imaging Probes

    OpenAIRE

    Kocaoglu, Ozden; Carlson, Erin E.

    2013-01-01

    Penicillin-binding proteins (PBPs) are membrane-associated proteins involved in the biosynthesis of peptidoglycan (PG), the main component of bacterial cell walls. These proteins were discovered and named for their affinity to bind the β-lactam antibiotic penicillin. The importance of the PBPs has long been appreciated; however, the apparent functional redundancy of the ~5–15 proteins that most bacteria possess makes determination of their individual roles difficult. Existing techniques to st...

  8. Calmodulin Binding Proteins and Alzheimer's Disease.

    Science.gov (United States)

    O'Day, Danton H; Eshak, Kristeen; Myre, Michael A

    2015-01-01

    The small, calcium-sensor protein, calmodulin, is ubiquitously expressed and central to cell function in all cell types. Here the literature linking calmodulin to Alzheimer's disease is reviewed. Several experimentally-verified calmodulin-binding proteins are involved in the formation of amyloid-β plaques including amyloid-β protein precursor, β-secretase, presenilin-1, and ADAM10. Many others possess potential calmodulin-binding domains that remain to be verified. Three calmodulin binding proteins are associated with the formation of neurofibrillary tangles: two kinases (CaMKII, CDK5) and one protein phosphatase (PP2B or calcineurin). Many of the genes recently identified by genome wide association studies and other studies encode proteins that contain putative calmodulin-binding domains but only a couple (e.g., APOE, BIN1) have been experimentally confirmed as calmodulin binding proteins. At least two receptors involved in calcium metabolism and linked to Alzheimer's disease (mAchR; NMDAR) have also been identified as calmodulin-binding proteins. In addition to this, many proteins that are involved in other cellular events intimately associated with Alzheimer's disease including calcium channel function, cholesterol metabolism, neuroinflammation, endocytosis, cell cycle events, and apoptosis have been tentatively or experimentally verified as calmodulin binding proteins. The use of calmodulin as a potential biomarker and as a therapeutic target is discussed. PMID:25812852

  9. Impact of Circulating Vitamin D Binding Protein Levels on the Association Between 25-Hydroxyvitamin D and Pancreatic Cancer Risk: A Nested Case-Control Study

    OpenAIRE

    Weinstein, Stephanie J.; Stolzenberg-Solomon, Rachael Z; Kopp, William; Rager, Helen; Virtamo, Jarmo; Albanes, Demetrius

    2012-01-01

    High concentrations of circulating 25-hydroxyvitamin D [25(OH)D] have been associated with elevated pancreatic cancer risk. As this is contrary to an expected inverse association between vitamin D status and cancer, we examined whether vitamin D binding protein (DBP), the primary carrier of vitamin D compounds in circulation, plays a role in this relationship. Prediagnostic serum DBP and 25(OH)D were studied in relation to risk of pancreatic cancer in a nested case-control study of 234 pancre...

  10. Mercury-binding proteins of Mytilus edulis

    Energy Technology Data Exchange (ETDEWEB)

    Roesijadi, G.; Morris, J. E.; Calabrese, A.

    1981-11-01

    Mytilus edulis possesses low molecular weight, mercury-binding proteins. The predominant protein isolated from gill tissue is enriched in cysteinyl residues (8%) and possesses an amino acid composition similar to cadmium-binding proteins of mussels and oysters. Continuous exposure of mussels to 5 ..mu..g/l mercury results in spillover of mercury from these proteins to high molecular weight proteins. Antibodies to these proteins have been isolated, and development of immunoassays is presently underway. Preliminary studies to determine whether exposure of adult mussels to mercury will result in induction of mercury-binding proteins in offspring suggest that such proteins occur in larvae although additional studies are indicated for a conclusive demonstration.

  11. Computational Prediction of RNA-Binding Proteins and Binding Sites

    Directory of Open Access Journals (Sweden)

    Jingna Si

    2015-11-01

    Full Text Available Proteins and RNA interaction have vital roles in many cellular processes such as protein synthesis, sequence encoding, RNA transfer, and gene regulation at the transcriptional and post-transcriptional levels. Approximately 6%–8% of all proteins are RNA-binding proteins (RBPs. Distinguishing these RBPs or their binding residues is a major aim of structural biology. Previously, a number of experimental methods were developed for the determination of protein–RNA interactions. However, these experimental methods are expensive, time-consuming, and labor-intensive. Alternatively, researchers have developed many computational approaches to predict RBPs and protein–RNA binding sites, by combining various machine learning methods and abundant sequence and/or structural features. There are three kinds of computational approaches, which are prediction from protein sequence, prediction from protein structure, and protein-RNA docking. In this paper, we review all existing studies of predictions of RNA-binding sites and RBPs and complexes, including data sets used in different approaches, sequence and structural features used in several predictors, prediction method classifications, performance comparisons, evaluation methods, and future directions.

  12. Identification of a Ubiquitin-Binding Structure in the S-Locus F-Box Protein Controlling S-RNase-Based Self-Incompatibility

    Institute of Scientific and Technical Information of China (English)

    Guang Chen; Bin Zhang; Lijing Liu; Qun Li; Yu'e Zhang; Qi Xie; Yongbiao Xue

    2012-01-01

    In flowering plants,self-incompatibility (SI) serves as an important intraspecific reproductive barrier to promote outbreeding.In species from the Solanaceae,Plantaginaceae and Rosaceae,S-RNase and SLF (S-locus F-box) proteins have been shown to control the female and male specificity of SI,respectively.However,little is known about structure features of the SLF protein apart from its conserved F-box domain.Here we show that the SLF C-terminal region possesses a novel ubiquitin-binding domain (UBD) structure conserved among the SLF protein family.By using an ex vivo system of Nicotiana benthamiana,we found that the UBD mediates the SLF protein turnover by the ubiquitin-proteasome pathway.Furthermore,we detected that the SLF protein was directly involved in S-RNase degradation.Taken together,our results provide a novel insight into the SLF structure and highlight a potential role of SLF protein stability and degradation in S-RNase-based self-incompatibility.

  13. Radiation damage to DNA-binding proteins

    International Nuclear Information System (INIS)

    The DNA-binding properties of proteins are strongly affected upon irradiation. The tetrameric lactose repressor (a dimer of dimers) losses its ability to bind operator DNA as soon as at least two damages per protomer of each dimer occur. The monomeric MC1 protein losses its ability to bind DNA in two steps : i) at low doses only the specific binding is abolished, whereas the non-specific one is still possible; ii) at high doses all binding vanishes. Moreover, the DNA bending induced by MC1 binding is less pronounced for a protein that underwent the low dose irradiation. When the entire DNA-protein complexes are irradiated, the observed disruption of the complexes is mainly due to the damage of the proteins and not to that of DNA. The doses necessary for complex disruption are higher than those inactivating the free protein. This difference, larger for MC1 than for lactose repressor, is due to the protection of the protein by the bound DNA. The oxidation of the protein side chains that are accessible to the radiation-induced hydroxyl radicals seems to represent the inactivating damage

  14. Alcohol Binding to the Odorant Binding Protein LUSH: Multiple Factors Affecting Binding Affinities

    OpenAIRE

    Ader, Lauren; Jones, David N. M.; Lin, Hai

    2010-01-01

    Density function theory (DFT) calculations have been carried out to investigate the binding of alcohols to the odorant binding protein LUSH from Drosophila melanogaster. LUSH is one of the few proteins known to bind to ethanol at physiologically relevant concentrations and where high-resolution structural information is available for the protein bound to alcohol at these concentrations. The structures of the LUSH–alcohol complexes identify a set of specific hydrogen-bonding interactions as cr...

  15. Control of translation and miRNA-dependent repression by a novel poly(A) binding protein, hnRNP-Q.

    Science.gov (United States)

    Svitkin, Yuri V; Yanagiya, Akiko; Karetnikov, Alexey E; Alain, Tommy; Fabian, Marc R; Khoutorsky, Arkady; Perreault, Sandra; Topisirovic, Ivan; Sonenberg, Nahum

    2013-01-01

    Translation control often operates via remodeling of messenger ribonucleoprotein particles. The poly(A) binding protein (PABP) simultaneously interacts with the 3' poly(A) tail of the mRNA and the eukaryotic translation initiation factor 4G (eIF4G) to stimulate translation. PABP also promotes miRNA-dependent deadenylation and translational repression of target mRNAs. We demonstrate that isoform 2 of the mouse heterogeneous nuclear protein Q (hnRNP-Q2/SYNCRIP) binds poly(A) by default when PABP binding is inhibited. In addition, hnRNP-Q2 competes with PABP for binding to poly(A) in vitro. Depleting hnRNP-Q2 from translation extracts stimulates cap-dependent and IRES-mediated translation that is dependent on the PABP/poly(A) complex. Adding recombinant hnRNP-Q2 to the extracts inhibited translation in a poly(A) tail-dependent manner. The displacement of PABP from the poly(A) tail by hnRNP-Q2 impaired the association of eIF4E with the 5' m(7)G cap structure of mRNA, resulting in the inhibition of 48S and 80S ribosome initiation complex formation. In mouse fibroblasts, silencing of hnRNP-Q2 stimulated translation. In addition, hnRNP-Q2 impeded let-7a miRNA-mediated deadenylation and repression of target mRNAs, which require PABP. Thus, by competing with PABP, hnRNP-Q2 plays important roles in the regulation of global translation and miRNA-mediated repression of specific mRNAs. PMID:23700384

  16. Co-ordinate control of synthesis of mitochondrial and non-mitochondrial hemoproteins: a binding site for the HAP1 (CYP1) protein in the UAS region of the yeast catalase T gene (CTT1).

    OpenAIRE

    Winkler, H.; Adam, G.; Mattes, E; Schanz, M.; Hartig, A; Ruis, H

    1988-01-01

    Control of expression of the Saccharomyces cerevisiae CTT1 (catalase T) gene by the HAP1 (CYP1) gene, a mediator of heme control of mitochondrial cytochromes, was studied. Expression of a CTT1-lacZ fusion in a hap1 mutant showed that the CTT1 promoter is under HAP1 control. As demonstrated by a gel retardation assay, the HAP1 protein binds to a heme control region of the CTT1 gene. This binding in vitro is stimulated by hemin. The HAP1-binding sequence was localized by using DNA fragments spa...

  17. Calcineurin homologous protein: a multifunctional Ca2+-binding protein family

    OpenAIRE

    Di Sole, Francesca; Vadnagara, Komal; MOE, ORSON W.; Babich, Victor

    2012-01-01

    The calcineurin homologous protein (CHP) belongs to an evolutionarily conserved Ca2+-binding protein subfamily. The CHP subfamily is composed of CHP1, CHP2, and CHP3, which in vertebrates share significant homology at the protein level with each other and between other Ca2+-binding proteins. The CHP structure consists of two globular domains containing from one to four EF-hand structural motifs (calcium-binding regions composed of two helixes, E and F, joined by a loop), the myristoylation, a...

  18. Abnormal SDS-PAGE migration of cytosolic proteins can identify domains and mechanisms that control surfactant binding

    OpenAIRE

    Shi, Yunhua; Mowery, Richard A; Ashley, Jonathan; Hentz, Michelle; Ramirez, Alejandro J; Bilgicer, Basar; Slunt-Brown, Hilda; David R Borchelt; Bryan F Shaw

    2012-01-01

    The amino acid substitution or post-translational modification of a cytosolic protein can cause unpredictable changes to its electrophoretic mobility during SDS-PAGE. This type of “gel shifting” has perplexed biochemists and biologists for decades. We identify a mechanism for “gel shifting” that predominates among a set of ALS (amyotrophic lateral sclerosis) mutant hSOD1 (superoxide dismutase) proteins, post-translationally modified hSOD1 proteins, and homologous SOD1 proteins from different ...

  19. Megalin binds and mediates cellular internalization of folate binding protein

    DEFF Research Database (Denmark)

    Birn, Henrik; Zhai, Xiaoyue; Holm, Jan;

    2005-01-01

    to express high levels of megalin, is inhibitable by excess unlabeled FBP and by receptor associated protein, a known inhibitor of binding to megalin. Immortalized rat yolk sac cells, representing an established model for studying megalin-mediated uptake, reveal (125)I-labeled FBP uptake which is...

  20. Affinity purification of proteins binding to GST fusion proteins.

    Science.gov (United States)

    Swaffield, J C; Johnston, S A

    2001-05-01

    This unit describes the use of proteins fused to glutathione-S-transferase (GST fusion proteins) to affinity purify other proteins, a technique also known as GST pulldown purification. The describes a strategy in which a GST fusion protein is bound to agarose affinity beads and the complex is then used to assay the binding of a specific test protein that has been labeled with [35S]methionine by in vitro translation. However, this method can be adapted for use with other types of fusion proteins; for example, His6, biotin tags, or maltose-binding protein fusions (MBP), and these may offer particular advantages. A describes preparation of an E. coli extract that is added to the reaction mixture with purified test protein to reduce nonspecific binding. PMID:18265191

  1. CREB-binding protein controls response to cocaine by acetylating histones at the fosB promoter in the mouse striatum

    OpenAIRE

    Levine, Amir A.; Guan, Zhonghui; Barco, Angel; Xu, Shiqin; Kandel, Eric R.; Schwartz, James H.

    2005-01-01

    Remodeling chromatin is essential for cAMP-regulated gene expression, necessary not only for development but also for memory storage and other enduring mental states. Histone acetylation and deacetylation mediate long-lasting forms of synaptic plasticity in Aplysia as well as cognition in mice. Here, we show that histone acetylation by the cAMP-response element binding protein (CREB)-binding protein (CBP) mediates sensitivity to cocaine by regulating expression of the fosB gene and its splice...

  2. The N-terminal Peptide of Mammalian GTP Cyclohydrolase I Is an Autoinhibitory Control Element and Contributes to Binding the Allosteric Regulatory Protein GFRP*

    Science.gov (United States)

    Higgins, Christina E.; Gross, Steven S.

    2011-01-01

    GTP cyclohydrolase I (GTPCH) is the rate-limiting enzyme for biosynthesis of tetrahydrobiopterin (BH4), an obligate cofactor for NO synthases and aromatic amino acid hydroxylases. BH4 can limit its own synthesis by triggering decameric GTPCH to assemble in an inhibitory complex with two GTPCH feedback regulatory protein (GFRP) pentamers. Subsequent phenylalanine binding to the GTPCH·GFRP inhibitory complex converts it to a stimulatory complex. An N-terminal inhibitory peptide in GTPCH may also contribute to autoregulation of GTPCH activity, but mechanisms are undefined. To characterize potential regulatory actions of the N-terminal peptide in rat GTPCH, we expressed, purified, and characterized a truncation mutant, devoid of 45 N-terminal amino acids (Δ45-GTPCH) and contrasted its catalytic and GFRP binding properties to wild type GTPCH (wt-GTPCH). Contrary to prior reports, we show that GFRP binds wt-GTPCH in the absence of any small molecule effector, resulting in allosteric stimulation of GTPCH activity: a 20% increase in Vmax, 50% decrease in KmGTP, and increase in Hill coefficient to 1.6, from 1.0. These features of GFRP-stimulated wt-GTPCH activity were phenocopied by Δ45-GTPCH in the absence of bound GFRP. Addition of GFRP to Δ45-GTPCH failed to elicit complex formation or a substantial further increase in GTPCH catalytic activity. Expression of Δ45-GTPCH in HEK-293 cells elicited 3-fold greater BH4 accumulation than an equivalent of wt-GTPCH. Together, results indicate that the N-terminal peptide exerts autoinhibitory control over rat GTPCH and is required for GFRP binding on its own. Displacement of the autoinhibitory peptide provides a molecular mechanism for physiological up-regulation of GTPCH activity. PMID:21163945

  3. The N-terminal peptide of mammalian GTP cyclohydrolase I is an autoinhibitory control element and contributes to binding the allosteric regulatory protein GFRP.

    Science.gov (United States)

    Higgins, Christina E; Gross, Steven S

    2011-04-01

    GTP cyclohydrolase I (GTPCH) is the rate-limiting enzyme for biosynthesis of tetrahydrobiopterin (BH4), an obligate cofactor for NO synthases and aromatic amino acid hydroxylases. BH4 can limit its own synthesis by triggering decameric GTPCH to assemble in an inhibitory complex with two GTPCH feedback regulatory protein (GFRP) pentamers. Subsequent phenylalanine binding to the GTPCH·GFRP inhibitory complex converts it to a stimulatory complex. An N-terminal inhibitory peptide in GTPCH may also contribute to autoregulation of GTPCH activity, but mechanisms are undefined. To characterize potential regulatory actions of the N-terminal peptide in rat GTPCH, we expressed, purified, and characterized a truncation mutant, devoid of 45 N-terminal amino acids (Δ45-GTPCH) and contrasted its catalytic and GFRP binding properties to wild type GTPCH (wt-GTPCH). Contrary to prior reports, we show that GFRP binds wt-GTPCH in the absence of any small molecule effector, resulting in allosteric stimulation of GTPCH activity: a 20% increase in Vmax, 50% decrease in KmGTP, and increase in Hill coefficient to 1.6, from 1.0. These features of GFRP-stimulated wt-GTPCH activity were phenocopied by Δ45-GTPCH in the absence of bound GFRP. Addition of GFRP to Δ45-GTPCH failed to elicit complex formation or a substantial further increase in GTPCH catalytic activity. Expression of Δ45-GTPCH in HEK-293 cells elicited 3-fold greater BH4 accumulation than an equivalent of wt-GTPCH. Together, results indicate that the N-terminal peptide exerts autoinhibitory control over rat GTPCH and is required for GFRP binding on its own. Displacement of the autoinhibitory peptide provides a molecular mechanism for physiological up-regulation of GTPCH activity. PMID:21163945

  4. Treponema pallidum Fibronectin-Binding Proteins

    OpenAIRE

    Cameron, Caroline E.; Brown, Elizabeth L.; Kuroiwa, Janelle M. Y.; Schnapp, Lynn M.; Brouwer, Nathan L.

    2004-01-01

    Putative adhesins were predicted by computer analysis of the Treponema pallidum genome. Two treponemal proteins, Tp0155 and Tp0483, demonstrated specific attachment to fibronectin, blocked bacterial adherence to fibronectin-coated slides, and supported attachment of fibronectin-producing mammalian cells. These results suggest Tp0155 and Tp0483 are fibronectin-binding proteins mediating T. pallidum-host interactions.

  5. Palmitoylation controls the dynamics of budding-yeast heterochromatin via the telomere-binding protein Rif1

    OpenAIRE

    Park, Sookhee; Patterson, Erin E.; Cobb, Jenel; Audhya, Anjon; Gartenberg, Marc R.; Fox, Catherine A.

    2011-01-01

    The posttranslational addition of palmitate to cysteines occurs ubiquitously in eukaryotic cells, where it functions in anchoring target proteins to membranes and in vesicular trafficking. Here we show that the Saccharomyces cerevisiae palmitoyltransferase Pfa4 enhanced heterochromatin formation at the cryptic mating-type loci HMR and HML via Rif1, a telomere regulatory protein. Acylated Rif1 was detected in extracts from wild-type but not pfa4Δ mutant cells. In a pfa4Δ mutant, Rif1-GFP dispe...

  6. Pentatricopeptide repeats: Modular blocks for building RNA-binding proteins

    OpenAIRE

    Filipovska, Aleksandra; Rackham, Oliver

    2013-01-01

    Pentatricopeptide repeat (PPR) proteins control diverse aspects of RNA metabolism across the eukaryotic domain. Recent computational and structural studies have provided new insights into how they recognize RNA, and show that the recognition is sequence-specific and modular. The modular code for RNA-binding by PPR proteins holds great promise for the engineering of new tools to target RNA and identifying RNAs bound by natural PPR proteins.

  7. Regulating a Post-Transcriptional Regulator: Protein Phosphorylation, Degradation and Translational Blockage in Control of the Trypanosome Stress-Response RNA-Binding Protein ZC3H11.

    Directory of Open Access Journals (Sweden)

    Igor Minia

    2016-03-01

    Full Text Available The life cycle of the mammalian pathogen Trypanosoma brucei involves commuting between two markedly different environments: the homeothermic mammalian host and the poikilothermic invertebrate vector. The ability to resist temperature and other stresses is essential for trypanosome survival. Trypanosome gene expression is mainly post-transcriptional, but must nevertheless be adjusted in response to environmental cues, including host-specific physical and chemical stresses. We investigate here the control of ZC3H11, a CCCH zinc finger protein which stabilizes stress response mRNAs. ZC3H11 protein levels increase at least 10-fold when trypanosomes are stressed by heat shock, proteasome inhibitors, ethanol, arsenite, and low doses of puromycin, but not by various other stresses. We found that increases in protein stability and translation efficiency both contribute to ZC3H11 accumulation. ZC3H11 is an in vitro substrate for casein kinase 1 isoform 2 (CK1.2, and results from CK1.2 depletion and other experiments suggest that phosphorylation of ZC3H11 can promote its instability in vivo. Results from sucrose density centrifugation indicate that under normal culture conditions translation initiation on the ZC3H11 mRNA is repressed, but after suitable stresses the ZC3H11 mRNA moves to heavy polysomes. The ZC3H11 3'-UTR is sufficient for translation suppression and a region of 71 nucleotides is required for the regulation. Since the control works in both bloodstream forms, where ZC3H11 translation is repressed at 37°C, and in procyclic forms, where ZC3H11 translation is activated at 37°C, we predict that this regulatory RNA sequence is targeted by repressive trans acting factor that is released upon stress.

  8. Regulating a Post-Transcriptional Regulator: Protein Phosphorylation, Degradation and Translational Blockage in Control of the Trypanosome Stress-Response RNA-Binding Protein ZC3H11.

    Science.gov (United States)

    Minia, Igor; Clayton, Christine

    2016-03-01

    The life cycle of the mammalian pathogen Trypanosoma brucei involves commuting between two markedly different environments: the homeothermic mammalian host and the poikilothermic invertebrate vector. The ability to resist temperature and other stresses is essential for trypanosome survival. Trypanosome gene expression is mainly post-transcriptional, but must nevertheless be adjusted in response to environmental cues, including host-specific physical and chemical stresses. We investigate here the control of ZC3H11, a CCCH zinc finger protein which stabilizes stress response mRNAs. ZC3H11 protein levels increase at least 10-fold when trypanosomes are stressed by heat shock, proteasome inhibitors, ethanol, arsenite, and low doses of puromycin, but not by various other stresses. We found that increases in protein stability and translation efficiency both contribute to ZC3H11 accumulation. ZC3H11 is an in vitro substrate for casein kinase 1 isoform 2 (CK1.2), and results from CK1.2 depletion and other experiments suggest that phosphorylation of ZC3H11 can promote its instability in vivo. Results from sucrose density centrifugation indicate that under normal culture conditions translation initiation on the ZC3H11 mRNA is repressed, but after suitable stresses the ZC3H11 mRNA moves to heavy polysomes. The ZC3H11 3'-UTR is sufficient for translation suppression and a region of 71 nucleotides is required for the regulation. Since the control works in both bloodstream forms, where ZC3H11 translation is repressed at 37°C, and in procyclic forms, where ZC3H11 translation is activated at 37°C, we predict that this regulatory RNA sequence is targeted by repressive trans acting factor that is released upon stress. PMID:27002830

  9. Regulating a Post-Transcriptional Regulator: Protein Phosphorylation, Degradation and Translational Blockage in Control of the Trypanosome Stress-Response RNA-Binding Protein ZC3H11

    Science.gov (United States)

    Minia, Igor; Clayton, Christine

    2016-01-01

    The life cycle of the mammalian pathogen Trypanosoma brucei involves commuting between two markedly different environments: the homeothermic mammalian host and the poikilothermic invertebrate vector. The ability to resist temperature and other stresses is essential for trypanosome survival. Trypanosome gene expression is mainly post-transcriptional, but must nevertheless be adjusted in response to environmental cues, including host-specific physical and chemical stresses. We investigate here the control of ZC3H11, a CCCH zinc finger protein which stabilizes stress response mRNAs. ZC3H11 protein levels increase at least 10-fold when trypanosomes are stressed by heat shock, proteasome inhibitors, ethanol, arsenite, and low doses of puromycin, but not by various other stresses. We found that increases in protein stability and translation efficiency both contribute to ZC3H11 accumulation. ZC3H11 is an in vitro substrate for casein kinase 1 isoform 2 (CK1.2), and results from CK1.2 depletion and other experiments suggest that phosphorylation of ZC3H11 can promote its instability in vivo. Results from sucrose density centrifugation indicate that under normal culture conditions translation initiation on the ZC3H11 mRNA is repressed, but after suitable stresses the ZC3H11 mRNA moves to heavy polysomes. The ZC3H11 3'-UTR is sufficient for translation suppression and a region of 71 nucleotides is required for the regulation. Since the control works in both bloodstream forms, where ZC3H11 translation is repressed at 37°C, and in procyclic forms, where ZC3H11 translation is activated at 37°C, we predict that this regulatory RNA sequence is targeted by repressive trans acting factor that is released upon stress. PMID:27002830

  10. Liver Fatty Acid Binding Protein and Obesity

    OpenAIRE

    Atshaves, B.P.; Martin, G G; Hostetler, H.A.; McIntosh, A.L.; Kier, A B; Schroeder, F.

    2010-01-01

    While low levels of unesterified long chain fatty acids (LCFAs) are normal metabolic intermediates of dietary and endogenous fat, LCFAs are also potent regulators of key receptors/enzymes, and at high levels become toxic detergents within the cell. Elevated levels of LCFAs are associated with diabetes, obesity, and metabolic syndrome. Consequently, mammals evolved fatty acid binding proteins (FABPs) that bind/sequester these potentially toxic free fatty acids in the cytosol and present them f...

  11. Nickel binding sites in histone proteins

    OpenAIRE

    Zoroddu, Maria Antonietta; Peana, Massimiliano Francesco; Solinas, Costantino; Medici, Serenella

    2012-01-01

    Nickel compounds are well known as human carcinogens, though the molecular events that are responsible for this are not well understood. It has been proposed that a crucial element in the mechanism of carcinogenesis is the binding of Ni(II) ions within the cell nucleus. It is known that DNA polymer binds Ni(II) only weakly, leaving the proteins of the cell nucleus as the likely Ni(II) targets. Being histone proteins the most abundant among them, they can be considered the primary sites fo...

  12. Ice-Binding Proteins and Their Function.

    Science.gov (United States)

    Bar Dolev, Maya; Braslavsky, Ido; Davies, Peter L

    2016-06-01

    Ice-binding proteins (IBPs) are a diverse class of proteins that assist organism survival in the presence of ice in cold climates. They have different origins in many organisms, including bacteria, fungi, algae, diatoms, plants, insects, and fish. This review covers the gamut of IBP structures and functions and the common features they use to bind ice. We discuss mechanisms by which IBPs adsorb to ice and interfere with its growth, evidence for their irreversible association with ice, and methods for enhancing the activity of IBPs. The applications of IBPs in the food industry, in cryopreservation, and in other technologies are vast, and we chart out some possibilities. PMID:27145844

  13. WBC27, an Adenosine Tri-phosphate-binding Cassette Protein, Controls Pollen Wall Formation and Patterning in Arabidopsis

    Institute of Scientific and Technical Information of China (English)

    Xiao-Ying Dou; Ke-Zhen Yang; Yi Zhang; Wei Wang; Xiao-Lei Liu; Li-Qun Chen; Xue-Qin Zhang; De Ye

    2011-01-01

    In flowering plants, the exine components are derived from tapetum. Despite its importance to sexual plant reproduction, little is known about the translocation of exine materials from tapetum to developing microspores. Here we report functional characterization of the arabidopsis WBC27 gene. WBC27 encodes an adenosine tri-phosphate binding cassette (ABC) transporter and is expressed preferentially in tapetum. Mutation of WBC27 disrupted the exine formation. The wbc27 mutant microspores began to degenerate once released from tetrads and most of the microspores collapsed at the uninucleate stage. Only a small number of wbc27-1 microspores could develop into tricellular pollen grains. These survival pollen grains lacked exine and germinated in the anther before anthesis. All of these results suggest that the ABC transporter, WBC27 plays important roles in the formation of arabidopsis exine, possibly by translocation of lipidic precursors of sporopollenin from tapetum to developing microspores.

  14. Predicting the binding patterns of hub proteins: a study using yeast protein interaction networks.

    Directory of Open Access Journals (Sweden)

    Carson M Andorf

    Full Text Available BACKGROUND: Protein-protein interactions are critical to elucidating the role played by individual proteins in important biological pathways. Of particular interest are hub proteins that can interact with large numbers of partners and often play essential roles in cellular control. Depending on the number of binding sites, protein hubs can be classified at a structural level as singlish-interface hubs (SIH with one or two binding sites, or multiple-interface hubs (MIH with three or more binding sites. In terms of kinetics, hub proteins can be classified as date hubs (i.e., interact with different partners at different times or locations or party hubs (i.e., simultaneously interact with multiple partners. METHODOLOGY: Our approach works in 3 phases: Phase I classifies if a protein is likely to bind with another protein. Phase II determines if a protein-binding (PB protein is a hub. Phase III classifies PB proteins as singlish-interface versus multiple-interface hubs and date versus party hubs. At each stage, we use sequence-based predictors trained using several standard machine learning techniques. CONCLUSIONS: Our method is able to predict whether a protein is a protein-binding protein with an accuracy of 94% and a correlation coefficient of 0.87; identify hubs from non-hubs with 100% accuracy for 30% of the data; distinguish date hubs/party hubs with 69% accuracy and area under ROC curve of 0.68; and SIH/MIH with 89% accuracy and area under ROC curve of 0.84. Because our method is based on sequence information alone, it can be used even in settings where reliable protein-protein interaction data or structures of protein-protein complexes are unavailable to obtain useful insights into the functional and evolutionary characteristics of proteins and their interactions. AVAILABILITY: We provide a web server for our three-phase approach: http://hybsvm.gdcb.iastate.edu.

  15. Antibodies against the calcium-binding protein

    International Nuclear Information System (INIS)

    Plant microsomes contain a protein clearly related to a calcium-binding protein, calsequestrin, originally found in the sarcoplasmic reticulum of muscle cells, responsible for the rapid release and uptake of Ca2+ within the cells. The location and role of calsequestrin in plant cells is unknown. To generate monoclonal antibodies specific to plant calsequestrin, mice were immunized with a microsomal fraction from cultured cells of Streptanthus tortuosus (Brassicaceae). Two clones cross-reacted with one protein band with a molecular weight equal to that of calsequestrin (57 kilodaltons) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. This band is able to bind 45Ca2+ and can be recognized by a polyclonal antibody against the canine cardiac muscle calsequestrin. Rabbit skeletal muscle calsequestrin cross-reacted with the plant monoclonal antibodies. The plant monoclonal antibodies generated here are specific to calsequestrin protein

  16. Signal transduction by guanine nucleotide binding proteins.

    Science.gov (United States)

    Spiegel, A M

    1987-01-01

    High affinity binding of guanine nucleotides and the ability to hydrolyze bound GTP to GDP are characteristics of an extended family of intracellular proteins. Subsets of this family include cytosolic initiation and elongation factors involved in protein synthesis, and cytoskeletal proteins such as tubulin (Hughes, S.M. (1983) FEBS Lett. 164, 1-8). A distinct subset of guanine nucleotide binding proteins is membrane-associated; members of this subset include the ras gene products (Ellis, R.W. et al. (1981) Nature 292, 506-511) and the heterotrimeric G-proteins (also termed N-proteins) (Gilman, A.G. (1984) Cell 36, 577-579). Substantial evidence indicates that G-proteins act as signal transducers by coupling receptors (R) to effectors (E). A similar function has been suggested but not proven for the ras gene products. Known G-proteins include Gs and Gi, the G-proteins associated with stimulation and inhibition, respectively, of adenylate cyclase; transducin (TD), the G-protein coupling rhodopsin to cGMP phosphodiesterase in rod photoreceptors (Bitensky, M.W. et al. (1981) Curr. Top. Membr. Transp. 15, 237-271; Stryer, L. (1986) Annu. Rev. Neurosci. 9, 87-119), and Go, a G-protein of unknown function that is highly abundant in brain (Sternweis, P.C. and Robishaw, J.D. (1984) J. Biol. Chem. 259, 13806-13813; Neer, E.J. et al. (1984) J. Biol. Chem. 259, 14222-14229). G-proteins also participate in other signal transduction pathways, notably that involving phosphoinositide breakdown. In this review, I highlight recent progress in our understanding of the structure, function, and diversity of G-proteins. PMID:2435586

  17. ALG-2, a multifunctional calcium binding protein?

    DEFF Research Database (Denmark)

    Tarabykina, Svetlana; Mollerup, Jens; Winding Gojkovic, P.;

    2004-01-01

    ALG-2 was originally discovered as a pro-apoptotic protein in a genetic screen. Due to its ability to bind calcium with high affinity it was postulated to provide a link between the known effect of calcium in programmed cell death and the molecular death execution machinery. This review article...

  18. Control of mRNA stability contributes to low levels of nuclear poly(A) binding protein 1 (PABPN1) in skeletal muscle

    OpenAIRE

    Apponi, Luciano H.; Corbett, Anita H.; Pavlath, Grace K.

    2013-01-01

    Background The nuclear poly(A) binding protein 1 (PABPN1) is a ubiquitously expressed protein that plays critical roles at multiple steps in post-transcriptional regulation of gene expression. Short expansions of the polyalanine tract in the N-terminus of PABPN1 lead to oculopharyngeal muscular dystrophy (OPMD), which is an adult onset disease characterized by eyelid drooping, difficulty in swallowing, and weakness in the proximal limb muscles. Why alanine-expanded PABPN1 leads to muscle-spec...

  19. Rrp6p controls mRNA poly(A) tail length and its decoration with poly(A) binding proteins.

    Science.gov (United States)

    Schmid, Manfred; Poulsen, Mathias Bach; Olszewski, Pawel; Pelechano, Vicent; Saguez, Cyril; Gupta, Ishaan; Steinmetz, Lars M; Moore, Claire; Jensen, Torben Heick

    2012-07-27

    Poly(A) (pA) tail binding proteins (PABPs) control mRNA polyadenylation, stability, and translation. In a purified system, S. cerevisiae PABPs, Pab1p and Nab2p, are individually sufficient to provide normal pA tail length. However, it is unknown how this occurs in more complex environments. Here we find that the nuclear exosome subunit Rrp6p counteracts the in vitro and in vivo extension of mature pA tails by the noncanonical pA polymerase Trf4p. Moreover, PABP loading onto nascent pA tails is controlled by Rrp6p; while Pab1p is the major PABP, Nab2p only associates in the absence of Rrp6p. This is because Rrp6p can interact with Nab2p and displace it from pA tails, potentially leading to RNA turnover, as evidenced for certain pre-mRNAs. We suggest that a nuclear mRNP surveillance step involves targeting of Rrp6p by Nab2p-bound pA-tailed RNPs and that pre-mRNA abundance is regulated at this level. PMID:22683267

  20. Notch2 controls prolactin and insulin-like growth factor binding protein-1 expression in decidualizing human stromal cells of early pregnancy.

    Directory of Open Access Journals (Sweden)

    Gerlinde R Otti

    Full Text Available Decidualization, the transformation of the human uterine mucosa into the endometrium of pregnancy, is critical for successful implantation and embryonic development. However, key regulatory factors controlling differentiation of uterine stromal cells into hormone-secreting decidual cells have not been fully elucidated. Hence, we herein investigated the role of the Notch signaling pathway in human decidual stromal cells (HDSC isolated from early pregnancy samples. Immunofluorescence of first trimester decidual tissues revealed expression of Notch2 receptor and its putative, membrane-anchored interaction partners Jagged1, Delta-like (DLL 1 and DLL4 in stromal cells whereas other Notch receptors and ligands were absent from these cells. During in vitro differentiation with estrogen/progesterone (E2P4 and/or cyclic adenosine monophosphate (cAMP HDSC constitutively expressed Notch2 and weakly downregulated Jagged1 mRNA and protein, measured by quantitative PCR (qPCR and Western blotting, respectively. However, increased levels of DLL1 and DLL4 were observed in the decidualizing cultures. Transfection of a Notch luciferase reporter and qPCR of the Notch target gene hairy and enhancer of split 1 (HES1 revealed an induction of canonical Notch activity during in vitro differentiation. In contrast, treatment of HDSC with a chemical Notch/γ-secretase inhibitor decreased cAMP/E2P4-stimulated Notch luciferase activity, HES1 transcript levels and mRNA expression of the decidual marker genes prolactin (PRL and insulin-like growth factor binding protein 1 (IGFBP1. Similarly, siRNA-mediated gene silencing or antibody-mediated blocking of Notch2 diminished HES1, PRL and IGFBP1 mRNA levels as well as secreted PRL protein. In summary, the data suggest that canonical, Notch2-dependent signaling plays a role in human decidualization.

  1. Identification of Enhancer Binding Proteins Important for Myxococcus xanthus Development▿

    OpenAIRE

    Giglio, Krista M.; Eisenstatt, Jessica; Garza, Anthony G.

    2009-01-01

    Enhancer binding proteins (EBPs) control the temporal expression of fruiting body development-associated genes in Myxococcus xanthus. Eleven previously uncharacterized EBP genes were inactivated. Six EBP gene mutations produced minor but reproducible defects in fruiting body development. One EBP gene mutation that affected A-motility produced strong developmental defects.

  2. Odorant-binding proteins in insects.

    Science.gov (United States)

    Zhou, Jing-Jiang

    2010-01-01

    Our understanding of the molecular and biochemical mechanisms that mediate chemoreception in insects has been greatly improved after the discovery of olfactory and taste receptor proteins. However, after 50 years of the discovery of first insect sex pheromone from the silkmoth Bombyx mori, it is still unclear how hydrophobic compounds reach the dendrites of sensory neurons in vivo across aqueous space and interact with the sensory receptors. The presence of soluble polypeptides in high concentration in the lymph of chemosensilla still poses unanswered questions. More than two decades after their discovery and despite the wealth of structural and biochemical information available, the physiological function of odorant-binding proteins (OBPs) is not well understood. Here, I review the structural properties of different subclasses of insect OBPs and their binding to pheromones and other small ligands. Finally, I discuss current ideas and models on the role of such proteins in insect chemoreception. PMID:20831949

  3. Quantifying drug-protein binding in vivo

    International Nuclear Information System (INIS)

    Accelerator mass spectrometry (AMS) provides precise quantitation of isotope labeled compounds that are bound to biological macromolecules such as DNA or proteins. The sensitivity is high enough to allow for sub-pharmacological (''micro-'') dosing to determine macromolecular targets without inducing toxicities or altering the system under study, whether it is healthy or diseased. We demonstrated an application of AMS in quantifying the physiologic effects of one dosed chemical compound upon the binding level of another compound in vivo at sub-toxic doses [4].We are using tissues left from this study to develop protocols for quantifying specific binding to isolated and identified proteins. We also developed a new technique to quantify nanogram to milligram amounts of isolated protein at precisions that are comparable to those for quantifying the bound compound by AMS

  4. Guanine Nucleotide-Binding Proteins of the G(12) Family Shape Immune Functions by Controlling CD4(+) T Cell Adhesiveness and Motility

    NARCIS (Netherlands)

    S. Herroeder; P. Reichardt; A. Sassmann; B. Zimmermann; D. Jaeneke; J. Hoeckner; M.W. Hollmann; K.D. Fischer; S. Vogt; R. Grosse; N. Hogg; M. Gunzer; S. Offermanns; N. Wettschureck

    2009-01-01

    Integrin-mediated adhesion plays a central role in T cell trafficking and activation. Genetic inactivation of the guanine nucleotide-binding (G) protein alpha-subunits G alpha(12) and G alpha(13) resulted in an increased activity of integrin leukocyte-function-antigen-1 in murine CD4(+) T cells. The

  5. Brain hyaluronan binding protein inhibits tumor growth

    Institute of Scientific and Technical Information of China (English)

    高锋; 曹曼林; 王蕾

    2004-01-01

    Background Great efforts have been made to search for the angiogenic inhibitors in avascular tissues. Several proteins isolated from cartilage have been proved to have anti-angiogenic or anti-tumour effects. Because cartilage contains a great amount of hyaluronic acid (HA) oligosaccharides and abundant HA binding proteins (HABP), therefore, we speculated that HABP might be one of the factors regulating vascularization in cartilage or anti-angiogenesis in tumours. The purpose of this research was to evaluale the effects of hyaluronan binding protein on inhibiting tumour growth both in vivo and vitro. Methods A unique protein termed human brain hyaluronan (HA) binding protein (b-HABP) was cloned from human brain cDNA library. MDA-435 human breast cancer cell line was chosen as a transfectant. The in vitro underlying mechanisms were investigated by determining the possibilities of MDA-435/b-HABP colony formation on soft agar, the effects of the transfectant on the proliferation of endothelial cells and the expression levels of caspase 3 and FasL from MDA-435/b-HABP. The in vivo study included tumour growth on the chorioallantoic membrane (CAM) of chicken embryos and nude mice. Results Colony formation assay revealed that the colonies formed by MDA-435/b-HABP were greatly reduced compared to mock transfectants. The conditioned media from MDA-435/b-HABP inhibited the growth of endothelial cells in culture. Caspase 3 and FasL expressions were induced by MDA-435/b-HABP. The size of tumours of MDA-435/b-HABP in both CAM and nude mice was much smaller than that of MDA-435 alone. Conclusions Human brain hyaluronan binding protein (b-HABP) may represent a new kind of naturally existing anti-tumour substance. This brain-derived glycoprotein may block tumour growth by inducing apoptosis of cancer cells or by decreasing angiogenesis in tumour tissue via inhibiting proliferation of endothelial cells.

  6. A structural classification of substrate-binding proteins

    NARCIS (Netherlands)

    Berntsson, Ronnie P. -A.; Smits, Sander H. J.; Schmitt, Lutz; Slotboom, Dirk-Jan; Poolman, Bert; Rydström, Jan

    2010-01-01

    Substrate-binding proteins (SBP) are associated with a wide variety of protein complexes. The proteins are part of ATP-binding cassette transporters for substrate uptake, ion gradient driven transporters, DNA-binding proteins, as well as channels and receptors from both pro-and eukaryotes. A wealth

  7. The human enhancer-binding protein Gata3 binds to several T-cell receptor regulatory elements.

    OpenAIRE

    Marine, J; Winoto, A

    1991-01-01

    The tissue-specific developmental regulation of the alpha, beta, gamma and delta T-cell antigen receptor (TCR) genes is controlled by the corresponding distinct enhancers and their enhancer-binding proteins. To find a common TCR regulatory element, we have studied the ability of the newly described enhancer-binding protein Gata3 to bind to the sequence motif (A/T)GATA(G/A) shared between enhancer elements of all four TCR genes. Gata3 was shown in the chicken to be an enhancer-binding protein ...

  8. The Actin Binding Protein Adseverin Regulates Osteoclastogenesis

    OpenAIRE

    Hassanpour, Siavash; Jiang, Hongwei; Wang, Yongqiang; Kuiper, Johannes W. P.; Glogauer, Michael

    2014-01-01

    Adseverin (Ads), a member of the Gelsolin superfamily of actin binding proteins, regulates the actin cytoskeleton architecture by severing and capping existing filamentous actin (F-actin) strands and nucleating the assembly of new F-actin filaments. Ads has been implicated in cellular secretion, exocytosis and has also been shown to regulate chondrogenesis and megakaryoblastic leukemia cell differentiation. Here we report for the first time that Ads is involved in regulating osteoclastogenesi...

  9. Where metal ions bind in proteins.

    OpenAIRE

    Yamashita, M M; Wesson, L.; Eisenman, G.; Eisenberg, D.

    1990-01-01

    The environments of metal ions (Li+, Na+, K+, Ag+, Cs+, Mg2+, Ca2+, Mn2+, Cu2+, Zn2+) in proteins and other metal-host molecules have been examined. Regardless of the metal and its precise pattern of ligation to the protein, there is a common qualitative feature to the binding site: the metal is ligated by a shell of hydrophilic atomic groups (containing oxygen, nitrogen, or sulfur atoms) and this hydrophilic shell is embedded within a larger shell of hydrophobic atomic groups (containing car...

  10. The N-terminal Peptide of Mammalian GTP Cyclohydrolase I Is an Autoinhibitory Control Element and Contributes to Binding the Allosteric Regulatory Protein GFRP*

    OpenAIRE

    Higgins, Christina E.; Gross, Steven S.

    2010-01-01

    GTP cyclohydrolase I (GTPCH) is the rate-limiting enzyme for biosynthesis of tetrahydrobiopterin (BH4), an obligate cofactor for NO synthases and aromatic amino acid hydroxylases. BH4 can limit its own synthesis by triggering decameric GTPCH to assemble in an inhibitory complex with two GTPCH feedback regulatory protein (GFRP) pentamers. Subsequent phenylalanine binding to the GTPCH·GFRP inhibitory complex converts it to a stimulatory complex. An N-terminal inhibitory peptide in GTPCH may als...

  11. Association Study of Mannose-Binding Lectin Levels and Genetic Variants in Lectin Pathway Proteins with Susceptibility to Age-Related Macular Degeneration: A Case-Control Study

    OpenAIRE

    Osthoff, Michael; Dean, Melinda M.; Baird, Paul N.; Richardson, Andrea J.; Daniell, Mark; Guymer, Robyn H.; Eisen, Damon P

    2015-01-01

    Background In age-related macular degeneration (AMD) the complement system is thought to be activated by chronic oxidative damage with genetic variants identified in the alternative pathway as susceptibility factors. However, the involvement of the lectin pathway of complement, a key mediator of oxidative damage, is controversial. This study investigated whether mannose-binding lectin (MBL) levels and genetic variants in lectin pathway proteins, are associated with the predisposition to and s...

  12. DNA and RNA Quadruplex-Binding Proteins

    Directory of Open Access Journals (Sweden)

    Václav Brázda

    2014-09-01

    Full Text Available Four-stranded DNA structures were structurally characterized in vitro by NMR, X-ray and Circular Dichroism spectroscopy in detail. Among the different types of quadruplexes (i-Motifs, minor groove quadruplexes, G-quadruplexes, etc., the best described are G-quadruplexes which are featured by Hoogsteen base-paring. Sequences with the potential to form quadruplexes are widely present in genome of all organisms. They are found often in repetitive sequences such as telomeric ones, and also in promoter regions and 5' non-coding sequences. Recently, many proteins with binding affinity to G-quadruplexes have been identified. One of the initially portrayed G-rich regions, the human telomeric sequence (TTAGGGn, is recognized by many proteins which can modulate telomerase activity. Sequences with the potential to form G-quadruplexes are often located in promoter regions of various oncogenes. The NHE III1 region of the c-MYC promoter has been shown to interact with nucleolin protein as well as other G-quadruplex-binding proteins. A number of G-rich sequences are also present in promoter region of estrogen receptor alpha. In addition to DNA quadruplexes, RNA quadruplexes, which are critical in translational regulation, have also been predicted and observed. For example, the RNA quadruplex formation in telomere-repeat-containing RNA is involved in interaction with TRF2 (telomere repeat binding factor 2 and plays key role in telomere regulation. All these fundamental examples suggest the importance of quadruplex structures in cell processes and their understanding may provide better insight into aging and disease development.

  13. A homeodomain protein binds to. gamma. -globin gene regulatory sequences

    Energy Technology Data Exchange (ETDEWEB)

    Lavelle, D.; Ducksworth, J.; Eves, E.; Gomes, G.; Keller, M.; Heller, P.; DeSimone, J. (Univ. of Illinois, Chicago (United States) Veterans Administration Westside Medical Center, Chicago, IL (United States))

    1991-08-15

    Developmental regulation of {gamma}-globin gene expression probably occurs through developmental-stage-specific trans-acting factors able to promote the interaction of enhancer elements located in the far upstream locus control region with regulatory elements in the {gamma} gene promoters and 3{prime}{sup A}{gamma} enhancer located in close proximity to the genes. The authors have detected a nuclear protein in K562 and baboon fetal bone marrow nuclear extracts capable of binding to A+T-rich sequences in the locus control region, {gamma} gene promoter, and 3{prime} {sup A}{gamma} enhancer. SDS/polyacrylamide gel analysis of the purified K562 binding activity revealed a single protein of 87 kDa. A K562 cDNA clone was isolated encoding a {beta}-galactosidase fusion protein with a DNA binding specificity identical to that of the K562/fetal bone marrow nuclear protein. The cDNA clone encodes a homeodomain homologous to the Drosophila antennapedia protein.

  14. Comparative study of methyl-CpG-binding domain proteins

    Directory of Open Access Journals (Sweden)

    Ropers H Hilger

    2003-01-01

    Full Text Available Abstract Background Methylation at CpG dinucleotides in genomic DNA is a fundamental epigenetic mechanism of gene expression control in vertebrates. Proteins with a methyl-CpG-binding domain (MBD can bind to single methylated CpGs and most of them are involved in transcription control. So far, five vertebrate MBD proteins have been described as MBD family members: MBD1, MBD2, MBD3, MBD4 and MECP2. Results We performed database searches for new proteins containing an MBD and identified six amino acid sequences which are different from the previously described ones. Here we present a comparison of their MBD sequences, additional protein motifs and the expression of the encoding genes. A calculated unrooted dendrogram indicates the existence of at least four different groups of MBDs within these proteins. Two of these polypeptides, KIAA1461 and KIAA1887, were only present as predicted amino acid sequences based on a partial human cDNA. We investigated their expression by Northern blot analysis and found transcripts of ~8 kb and ~5 kb respectively, in all eight normal tissues studied. Conclusions Eleven polypeptides with a MBD could be identified in mouse and man. The analysis of protein domains suggests a role in transcriptional regulation for most of them. The knowledge of additional existing MBD proteins and their expression pattern is important in the context of Rett syndrome.

  15. Protein and ligand adaptation in a retinoic acid binding protein.

    OpenAIRE

    Pattanayek, R.; Newcomer, M E

    1999-01-01

    A retinoic acid binding protein isolated from the lumen of the rat epididymis (ERABP) is a member of the lipocalin superfamily. ERABP binds both the all-trans and 9-cis isomers of retinoic acid, as well as the synthetic retinoid (E)-4-[2-(5,6,7,8)-tetrahydro-5,5,8,8-tetramethyl-2 napthalenyl-1 propenyl]-benzoic acid (TTNPB), a structural analog of all-trans retinoic acid. The structure of ERABP with a mixture of all-trans and 9-cis retinoic acid has previously been reported. To elucidate any ...

  16. Dissection of the Critical Binding Determinants of Cellular Retinoic Acid Binding Protein II by Mutagenesis and Fluorescence Binding Assay

    OpenAIRE

    Vasileiou, Chrysoula; Lee, Kin Sing Stephen; Crist, Rachael M.; Vaezeslami, Soheila; Goins, Sarah M.; Geiger, James H.; Borhan, Babak

    2009-01-01

    The binding of retinoic acid to mutants of Cellular Retinoic Acid Binding Protein II (CRABPII) was evaluated to better understand the importance of the direct protein/ligand interactions. The important role of Arg111 for the correct structure and function of the protein was verified and other residues that directly affect retinoic acid binding have been identified. Furthermore, retinoic acid binding to CRABPII mutants that lack all previously identified interacting amino acids was rescued by ...

  17. Landscape of protein-small ligand binding modes.

    Science.gov (United States)

    Kasahara, Kota; Kinoshita, Kengo

    2016-09-01

    Elucidating the mechanisms of specific small-molecule (ligand) recognition by proteins is a long-standing conundrum. While the structures of these molecules, proteins and ligands, have been extensively studied, protein-ligand interactions, or binding modes, have not been comprehensively analyzed. Although methods for assessing similarities of binding site structures have been extensively developed, the methods for the computational treatment of binding modes have not been well established. Here, we developed a computational method for encoding the information about binding modes as graphs, and assessing their similarities. An all-against-all comparison of 20,040 protein-ligand complexes provided the landscape of the protein-ligand binding modes and its relationships with protein- and chemical spaces. While similar proteins in the same SCOP Family tend to bind relatively similar ligands with similar binding modes, the correlation between ligand and binding similarities was not very high (R(2)  = 0.443). We found many pairs with novel relationships, in which two evolutionally distant proteins recognize dissimilar ligands by similar binding modes (757,474 pairs out of 200,790,780 pairs were categorized into this relationship, in our dataset). In addition, there were an abundance of pairs of homologous proteins binding to similar ligands with different binding modes (68,217 pairs). Our results showed that many interesting relationships between protein-ligand complexes are still hidden in the structure database, and our new method for assessing binding mode similarities is effective to find them. PMID:27327045

  18. Interaction of the bovine papillomavirus type 1 E2 transcriptional control protein with the viral enhancer: purification of the DNA-binding domain and analysis of its contact points with DNA.

    OpenAIRE

    Moskaluk, C A; Bastia, D

    1988-01-01

    The E2 gene of bovine papillomavirus type 1 positively and negatively regulates the transcriptional enhancer located in the long control region of the viral genome. The DNA-binding domain of the E2 gene product was suspected to interact with the DNA sequence motif ACCN6GGT. We have shown that the carboxy-terminal 126 amino acids of the E2 protein constitute the DNA-binding domain. In this paper we described the expression of the E2 carboxy terminus in Escherichia coli and its subsequent purif...

  19. Measuring Binding Affinity of Protein-Ligand Interaction Using Spectrophotometry: Binding of Neutral Red to Riboflavin-Binding Protein

    Science.gov (United States)

    Chenprakhon, Pirom; Sucharitakul, Jeerus; Panijpan, Bhinyo; Chaiyen, Pimchai

    2010-01-01

    The dissociation constant, K[subscript d], of the binding of riboflavin-binding protein (RP) with neutral red (NR) can be determined by titrating RP to a fixed concentration of NR. Upon adding RP to the NR solution, the maximum absorption peak of NR shifts to 545 nm from 450 nm for the free NR. The change of the absorption can be used to determine…

  20. Alternative polyadenylation and RNA-binding proteins.

    Science.gov (United States)

    Erson-Bensan, Ayse Elif

    2016-08-01

    Our understanding of the extent of microRNA-based gene regulation has expanded in an impressive pace over the past decade. Now, we are beginning to better appreciate the role of 3'-UTR (untranslated region) cis-elements which harbor not only microRNA but also RNA-binding protein (RBP) binding sites that have significant effect on the stability and translational rate of mRNAs. To add further complexity, alternative polyadenylation (APA) emerges as a widespread mechanism to regulate gene expression by producing shorter or longer mRNA isoforms that differ in the length of their 3'-UTRs or even coding sequences. Resulting shorter mRNA isoforms generally lack cis-elements where trans-acting factors bind, and hence are differentially regulated compared with the longer isoforms. This review focuses on the RBPs involved in APA regulation and their action mechanisms on APA-generated isoforms. A better understanding of the complex interactions between APA and RBPs is promising for mechanistic and clinical implications including biomarker discovery and new therapeutic approaches. PMID:27208003

  1. Comparison of the Folding Mechanism of Highly Homologous Proteins in the Lipid-binding Protein Family

    Science.gov (United States)

    The folding mechanism of two closely related proteins in the intracellular lipid binding protein family, human bile acid binding protein (hBABP) and rat bile acid binding protein (rBABP) were examined. These proteins are 77% identical (93% similar) in sequence Both of these singl...

  2. Determining Membrane Protein-Lipid Binding Thermodynamics Using Native Mass Spectrometry.

    Science.gov (United States)

    Cong, Xiao; Liu, Yang; Liu, Wen; Liang, Xiaowen; Russell, David H; Laganowsky, Arthur

    2016-04-01

    Membrane proteins are embedded in the biological membrane where the chemically diverse lipid environment can modulate their structure and function. However, the thermodynamics governing the molecular recognition and interaction of lipids with membrane proteins is poorly understood. Here, we report a method using native mass spectrometry (MS), to determine thermodynamics of individual ligand binding events to proteins. Unlike conventional methods, native MS can resolve individual ligand binding events and, coupled with an apparatus to control the temperature, determine binding thermodynamic parameters, such as for protein-lipid interactions. We validated our approach using three soluble protein-ligand systems (maltose binding protein, lysozyme, and nitrogen regulatory protein) and obtained similar results to those using isothermal titration calorimetry and surface plasmon resonance. We also determined for the first time the thermodynamics of individual lipid binding to the ammonia channel (AmtB), an integral membrane protein from Escherichia coli. Remarkably, we observed distinct thermodynamic signatures for the binding of different lipids and entropy-enthalpy compensation for binding lipids of variable chain length. Additionally, using a mutant form of AmtB that abolishes a specific phosphatidylglycerol (PG) binding site, we observed distinct changes in the thermodynamic signatures for binding PG, implying these signatures can identify key residues involved in specific lipid binding and potentially differentiate between specific lipid binding sites. PMID:27015007

  3. Isolation of a Thiamine-binding Protein from Rice Germ and Distribution of Similar Proteins.

    Science.gov (United States)

    Shimizu, M; Yoshida, T; Toda, T; Iwashima, A; Mitsunaga, T

    1996-01-01

    A thiamine-binding protein was purified from rice germ (Oryza sativa L.) by extraction, salting-out with ammonium sulfate, and column chromatography. From the results of molecular mass, Kd and Bmax values for thiamine-binding, binding specificity for thiamine phosphates and analog, the protein was suggested to be identical to the thiamine-binding protein in rice bran. The thiamine-binding protein w as more efficiently purified from rice germ than from rice bran. The protein was rich in glutamic acid (and/or glutamine) and glycine. The protein did not show immunological similarity to thiamine-binding proteins in buckwheat and sesame seeds. However proteins similar to the thiamine-binding protein from rice germ existed in gramineous seeds. They were suggested to have thiamine-binding activity and to be of the same molecular mass as the thiamine-binding protein. PMID:27299548

  4. Isolation and characterizations of oxalate-binding proteins in the kidney

    International Nuclear Information System (INIS)

    Highlights: ► The first large-scale characterizations of oxalate-binding kidney proteins. ► The recently developed oxalate-conjugated EAH Sepharose 4B beads were applied. ► 38 forms of 26 unique oxalate-binding kidney proteins were identified. ► 25/26 (96%) of identified proteins had “L-x(3,5)-R-x(2)-[AGILPV]” domain. -- Abstract: Oxalate-binding proteins are thought to serve as potential modulators of kidney stone formation. However, only few oxalate-binding proteins have been identified from previous studies. Our present study, therefore, aimed for large-scale identification of oxalate-binding proteins in porcine kidney using an oxalate-affinity column containing oxalate-conjugated EAH Sepharose 4B beads for purification followed by two-dimensional gel electrophoresis (2-DE) to resolve the recovered proteins. Comparing with those obtained from the controlled column containing uncoupled EAH-Sepharose 4B (to subtract the background of non-specific bindings), a total of 38 protein spots were defined as oxalate-binding proteins. These protein spots were successfully identified by quadrupole time-of-flight mass spectrometry (MS) and/or tandem MS (MS/MS) as 26 unique proteins, including several nuclear proteins, mitochondrial proteins, oxidative stress regulatory proteins, metabolic enzymes and others. Identification of oxalate-binding domain using the PRATT tool revealed “L-x(3,5)-R-x(2)-[AGILPV]” as a functional domain responsible for oxalate-binding in 25 of 26 (96%) unique identified proteins. We report herein, for the first time, large-scale identification and characterizations of oxalate-binding proteins in the kidney. The presence of positively charged arginine residue in the middle of this functional domain suggested its significance for binding to the negatively charged oxalate. These data will enhance future stone research, particularly on stone modulators.

  5. Identification of Treponema pallidum penicillin-binding proteins.

    OpenAIRE

    Cunningham, T M; Miller, J N; Lovett, M A

    1987-01-01

    Penicillin-binding proteins of 180, 89, 80, 68, 61, 41, and 38 kilodaltons were identified in Treponema pallidum (Nichols) by their covalent binding of [35S]benzylpenicillin. Penicillin-binding proteins are localized in the plasma membranes of many bacterial species and may serve as useful markers for determining plasma membrane intactness in T. pallidum fractionation studies.

  6. Novel RNA-binding properties of the MTG chromatin regulatory proteins

    Directory of Open Access Journals (Sweden)

    Sacchi Nicoletta

    2008-10-01

    Full Text Available Abstract Background The myeloid translocation gene (MTG proteins are non-DNA-binding transcriptional regulators capable of interacting with chromatin modifying proteins. As a consequence of leukemia-associated chromosomal translocations, two of the MTG proteins, MTG8 and MTG16, are fused to the DNA-binding domain of AML1, a transcriptional activator crucial for hematopoiesis. The AML1-MTG fusion proteins, as the wild type MTGs, display four conserved homology regions (NHR1-4 related to the Drosophila nervy protein. Structural protein analyses led us to test the hypothesis that specific MTG domains may mediate RNA binding. Results By using an RNA-binding assay based on synthetic RNA homopolymers and a panel of MTG deletion mutants, here we show that all the MTG proteins can bind RNA. The RNA-binding properties can be traced to two regions: the Zinc finger domains in the NHR4, which mediate Zinc-dependent RNA binding, and a novel short basic region (SBR upstream of the NHR2, which mediates Zinc-independent RNA binding. The two AML1-MTG fusion proteins, retaining both the Zinc fingers domains and the SBR, also display RNA-binding properties. Conclusion Evidence has been accumulating that RNA plays a role in transcriptional control. Both wild type MTGs and chimeric AML1-MTG proteins display in vitro RNA-binding properties, thus opening new perspectives on the possible involvement of an RNA component in MTG-mediated chromatin regulation.

  7. Histidine switch controlling pH-dependent protein folding and DNA binding in a transcription factor at the core of synthetic network devices.

    Science.gov (United States)

    Deochand, D K; Perera, I C; Crochet, R B; Gilbert, N C; Newcomer, M E; Grove, A

    2016-07-19

    Therapeutic strategies have been reported that depend on synthetic network devices in which a urate-sensing transcriptional regulator detects pathological levels of urate and triggers production or release of urate oxidase. The transcription factor involved, HucR, is a member of the multiple antibiotic resistance (MarR) protein family. We show that protonation of stacked histidine residues at the pivot point of long helices that form the scaffold of the dimer interface leads to reversible formation of a molten globule state and significantly attenuated DNA binding at physiological temperatures. We also show that binding of urate to symmetrical sites in each protein lobe is communicated via the dimer interface. This is the first demonstration of regulation of a MarR family transcription factor by pH-dependent interconversion between a molten globule and a compact folded state. Our data further suggest that HucR may be utilized in synthetic devices that depend on detection of pH changes. PMID:27282811

  8. The Cobalamin-binding Protein in Zebrafish is an Intermediate Between the Three Cobalamin-binding Proteins in Human

    OpenAIRE

    Greibe, Eva Holm; Fedosov, Sergey; Nexø, Ebba

    2012-01-01

    In humans, three soluble extracellular cobalamin-binding proteins; transcobalamin (TC), intrinsic factor (IF), and haptocorrin (HC), are involved in the uptake and transport of cobalamin. In this study, we investigate a cobalamin-binding protein from zebrafish (Danio rerio) and summarize current knowledge concerning the phylogenetic evolution of kindred proteins. We identified a cobalamin binding capacity in zebrafish protein extracts (8.2 pmol/fish) and ambient water (13.5 pmol/fish) associa...

  9. Glycan masking of Plasmodium vivax Duffy Binding Protein for probing protein binding function and vaccine development.

    Directory of Open Access Journals (Sweden)

    Sowmya Sampath

    Full Text Available Glycan masking is an emerging vaccine design strategy to focus antibody responses to specific epitopes, but it has mostly been evaluated on the already heavily glycosylated HIV gp120 envelope glycoprotein. Here this approach was used to investigate the binding interaction of Plasmodium vivax Duffy Binding Protein (PvDBP and the Duffy Antigen Receptor for Chemokines (DARC and to evaluate if glycan-masked PvDBPII immunogens would focus the antibody response on key interaction surfaces. Four variants of PVDBPII were generated and probed for function and immunogenicity. Whereas two PvDBPII glycosylation variants with increased glycan surface coverage distant from predicted interaction sites had equivalent binding activity to wild-type protein, one of them elicited slightly better DARC-binding-inhibitory activity than wild-type immunogen. Conversely, the addition of an N-glycosylation site adjacent to a predicted PvDBP interaction site both abolished its interaction with DARC and resulted in weaker inhibitory antibody responses. PvDBP is composed of three subdomains and is thought to function as a dimer; a meta-analysis of published PvDBP mutants and the new DBPII glycosylation variants indicates that critical DARC binding residues are concentrated at the dimer interface and along a relatively flat surface spanning portions of two subdomains. Our findings suggest that DARC-binding-inhibitory antibody epitope(s lie close to the predicted DARC interaction site, and that addition of N-glycan sites distant from this site may augment inhibitory antibodies. Thus, glycan resurfacing is an attractive and feasible tool to investigate protein structure-function, and glycan-masked PvDBPII immunogens might contribute to P. vivax vaccine development.

  10. STRUCTURAL FEATURES OF PLANT CHITINASES AND CHITIN-BINDING PROTEINS

    NARCIS (Netherlands)

    BEINTEMA, JJ

    1994-01-01

    Structural features of plant chitinases and chitin-binding proteins are discussed. Many of these proteins consist of multiple domains,of which the chitin-binding hevein domain is a predominant one. X-ray and NMR structures of representatives of the major classes of these proteins are available now,

  11. Cobalamin and folate binding proteins in human tumour tissue.

    OpenAIRE

    Sheppard, K; Bradbury, D A; Davies, J. M.; Ryrie, D. R.

    1984-01-01

    The serum of an 84 year old man with disseminated carcinoma was found to contain extremely high concentrations of cobalamin and of a cobalamin binding protein with trans-cobalamin I characteristics. Tumour tissue samples obtained at necropsy contained considerably higher concentrations of cobalamin binding protein (R-binder) than normal tissues. Tumour tissues also contained increased concentrations of specific folate binding protein. In all tissues studied a close correlation existed between...

  12. Minimalistic predictor of protein binding energy: contribution of solvation factor to protein binding.

    Science.gov (United States)

    Choi, Jeong-Mo; Serohijos, Adrian W R; Murphy, Sean; Lucarelli, Dennis; Lofranco, Leo L; Feldman, Andrew; Shakhnovich, Eugene I

    2015-02-17

    It has long been known that solvation plays an important role in protein-protein interactions. Here, we use a minimalistic solvation-based model for predicting protein binding energy to estimate quantitatively the contribution of the solvation factor in protein binding. The factor is described by a simple linear combination of buried surface areas according to amino-acid types. Even without structural optimization, our minimalistic model demonstrates a predictive power comparable to more complex methods, making the proposed approach the basis for high throughput applications. Application of the model to a proteomic database shows that receptor-substrate complexes involved in signaling have lower affinities than enzyme-inhibitor and antibody-antigen complexes, and they differ by chemical compositions on interfaces. Also, we found that protein complexes with components that come from the same genes generally have lower affinities than complexes formed by proteins from different genes, but in this case the difference originates from different interface areas. The model was implemented in the software PYTHON, and the source code can be found on the Shakhnovich group webpage: http://faculty.chemistry.harvard.edu/shakhnovich/software. PMID:25692584

  13. Diagnosis of Non-ST-Elevation Acute Coronary Syndrome by the Measurement of Heart-Type Fatty Acid Binding Protein in Serum: A Prospective Case Control Study

    Directory of Open Access Journals (Sweden)

    Priscilla Abraham Chandran

    2014-01-01

    Full Text Available A prospective case control study was undertaken to evaluate the diagnostic performance of serum heart-type fatty acid binding protein (HFABP in comparison to cardiac TnT and TnI in 33 patients admitted with chest pain, diagnosed as NSTE-ACS (non ST elevation acute coronary syndrome and 22 healthy controls. Area under the receiver operating curve (AUC was highest for H-FABP (AUC 0.79; 95% CI 0.66–0.89 versus cTnI (AUC 0.73; 95% CI 0.59–0.84 and cTnT (AUC 0.71; 95% CI 0.57–0.83. The H-FABP level above 6.5 ng/mL showed 56.7% (CI 37.4–74.5 sensitivity, 0.5 (95% CI 0.3–0.7 negative likelihood ratio (−LR, 100% (CI 84.6–100.0 specificity, and 100% (CI 79.4–100.0 positive predictive value (PPV, 62.9% (CI 44.9–78.5 negative predictive value (NPV. cTnI level above 0.009 μg/L had 40% (CI 22.7–59.4 sensitivity, 0.6 (95% CI 0.4–0.8 −LR, 100% (CI 84.6–100.0 specificity, 100% (CI 73.5–100.0 PPV, and 55% (CI 38.5–70.7 NPV. cTnT showed 46.7% (CI 28.3–65.7 sensitivity, 0.5 (95% CI 0.4–0.7 −LR, 100% (CI 84.6–100.0 specificity, 100% (CI 76.8–100.0 PPV, and 57.9% (CI 40.8–73.7 NPV at level above 9 μg/L. +LR were 12.5 (95% CI 1.8–86.8, 1.7 (95% CI 1.0–3.0, and 1.2 (95% CI 0.8–1.9 for H-FABP, cTnI, and cTnT respectively. In conclusion measurement of H-FABP is a valuable tool in the early diagnosis of patients with chest pain (6–8 hrs and seems to be a preferred biomarker in the differential diagnosis of NSTE-ACS. More studies are needed to determine whether serum H-FABP further improves diagnostic performance.

  14. Solution Structure and Backbone Dynamics of Human Liver Fatty Acid Binding Protein: Fatty Acid Binding Revisited

    OpenAIRE

    Cai, Jun; Lücke, Christian; Chen, Zhongjing; Qiao, Ye; Klimtchuk, Elena; Hamilton, James A.

    2012-01-01

    Liver fatty acid binding protein (L-FABP), a cytosolic protein most abundant in liver, is associated with intracellular transport of fatty acids, nuclear signaling, and regulation of intracellular lipolysis. Among the members of the intracellular lipid binding protein family, L-FABP is of particular interest as it can i), bind two fatty acid molecules simultaneously and ii), accommodate a variety of bulkier physiological ligands such as bilirubin and fatty acyl CoA. To better understand the p...

  15. RNA-Binding Proteins in Trichomonas vaginalis: Atypical Multifunctional Proteins

    Directory of Open Access Journals (Sweden)

    Elisa E. Figueroa-Angulo

    2015-11-01

    Full Text Available Iron homeostasis is highly regulated in vertebrates through a regulatory system mediated by RNA-protein interactions between the iron regulatory proteins (IRPs that interact with an iron responsive element (IRE located in certain mRNAs, dubbed the IRE-IRP regulatory system. Trichomonas vaginalis, the causal agent of trichomoniasis, presents high iron dependency to regulate its growth, metabolism, and virulence properties. Although T. vaginalis lacks IRPs or proteins with aconitase activity, possesses gene expression mechanisms of iron regulation at the transcriptional and posttranscriptional levels. However, only one gene with iron regulation at the transcriptional level has been described. Recently, our research group described an iron posttranscriptional regulatory mechanism in the T. vaginalis tvcp4 and tvcp12 cysteine proteinase mRNAs. The tvcp4 and tvcp12 mRNAs have a stem-loop structure in the 5'-coding region or in the 3'-UTR, respectively that interacts with T. vaginalis multifunctional proteins HSP70, α-Actinin, and Actin under iron starvation condition, causing translation inhibition or mRNA stabilization similar to the previously characterized IRE-IRP system in eukaryotes. Herein, we summarize recent progress and shed some light on atypical RNA-binding proteins that may participate in the iron posttranscriptional regulation in T. vaginalis.

  16. Inhibition of tristetraprolin deadenylation by poly(A) binding protein

    OpenAIRE

    Rowlett, Robert M.; Chrestensen, Carol A.; Schroeder, Melanie J.; Harp, Mary G.; Pelo, Jared W.; Shabanowitz, Jeffery; DeRose, Robert; Hunt, Donald F.; Sturgill, Thomas W.; Worthington, Mark T.

    2008-01-01

    Tristetraprolin (TTP) is the prototype for a family of RNA binding proteins that bind the tumor necrosis factor (TNF) messenger RNA AU-rich element (ARE), causing deadenylation of the TNF poly(A) tail, RNA decay, and silencing of TNF protein production. Using mass spectrometry sequencing we identified poly(A) binding proteins-1 and -4 (PABP1 and PABP4) in high abundance and good protein coverage from TTP immunoprecipitates. PABP1 significantly enhanced TNF ARE binding by RNA EMSA and prevente...

  17. Protein-binding RNA aptamers affect molecular interactions distantly from their binding sites.

    Directory of Open Access Journals (Sweden)

    Daniel M Dupont

    Full Text Available Nucleic acid aptamer selection is a powerful strategy for the development of regulatory agents for molecular intervention. Accordingly, aptamers have proven their diligence in the intervention with serine protease activities, which play important roles in physiology and pathophysiology. Nonetheless, there are only a few studies on the molecular basis underlying aptamer-protease interactions and the associated mechanisms of inhibition. In the present study, we use site-directed mutagenesis to delineate the binding sites of two 2´-fluoropyrimidine RNA aptamers (upanap-12 and upanap-126 with therapeutic potential, both binding to the serine protease urokinase-type plasminogen activator (uPA. We determine the subsequent impact of aptamer binding on the well-established molecular interactions (plasmin, PAI-1, uPAR, and LRP-1A controlling uPA activities. One of the aptamers (upanap-126 binds to the area around the C-terminal α-helix in pro-uPA, while the other aptamer (upanap-12 binds to both the β-hairpin of the growth factor domain and the kringle domain of uPA. Based on the mapping studies, combined with data from small-angle X-ray scattering analysis, we construct a model for the upanap-12:pro-uPA complex. The results suggest and highlight that the size and shape of an aptamer as well as the domain organization of a multi-domain protein such as uPA, may provide the basis for extensive sterical interference with protein ligand interactions considered distant from the aptamer binding site.

  18. Bacterial periplasmic sialic acid-binding proteins exhibit a conserved binding site

    Energy Technology Data Exchange (ETDEWEB)

    Gangi Setty, Thanuja [Institute for Stem Cell Biology and Regenerative Medicine, NCBS Campus, GKVK Post, Bangalore, Karnataka 560 065 (India); Cho, Christine [Carver College of Medicine, University of Iowa, Iowa City, IA 52242-1109 (United States); Govindappa, Sowmya [Institute for Stem Cell Biology and Regenerative Medicine, NCBS Campus, GKVK Post, Bangalore, Karnataka 560 065 (India); Apicella, Michael A. [Carver College of Medicine, University of Iowa, Iowa City, IA 52242-1109 (United States); Ramaswamy, S., E-mail: ramas@instem.res.in [Institute for Stem Cell Biology and Regenerative Medicine, NCBS Campus, GKVK Post, Bangalore, Karnataka 560 065 (India)

    2014-07-01

    Structure–function studies of sialic acid-binding proteins from F. nucleatum, P. multocida, V. cholerae and H. influenzae reveal a conserved network of hydrogen bonds involved in conformational change on ligand binding. Sialic acids are a family of related nine-carbon sugar acids that play important roles in both eukaryotes and prokaryotes. These sialic acids are incorporated/decorated onto lipooligosaccharides as terminal sugars in multiple bacteria to evade the host immune system. Many pathogenic bacteria scavenge sialic acids from their host and use them for molecular mimicry. The first step of this process is the transport of sialic acid to the cytoplasm, which often takes place using a tripartite ATP-independent transport system consisting of a periplasmic binding protein and a membrane transporter. In this paper, the structural characterization of periplasmic binding proteins from the pathogenic bacteria Fusobacterium nucleatum, Pasteurella multocida and Vibrio cholerae and their thermodynamic characterization are reported. The binding affinities of several mutations in the Neu5Ac binding site of the Haemophilus influenzae protein are also reported. The structure and the thermodynamics of the binding of sugars suggest that all of these proteins have a very well conserved binding pocket and similar binding affinities. A significant conformational change occurs when these proteins bind the sugar. While the C1 carboxylate has been identified as the primary binding site, a second conserved hydrogen-bonding network is involved in the initiation and stabilization of the conformational states.

  19. Bacterial periplasmic sialic acid-binding proteins exhibit a conserved binding site

    International Nuclear Information System (INIS)

    Structure–function studies of sialic acid-binding proteins from F. nucleatum, P. multocida, V. cholerae and H. influenzae reveal a conserved network of hydrogen bonds involved in conformational change on ligand binding. Sialic acids are a family of related nine-carbon sugar acids that play important roles in both eukaryotes and prokaryotes. These sialic acids are incorporated/decorated onto lipooligosaccharides as terminal sugars in multiple bacteria to evade the host immune system. Many pathogenic bacteria scavenge sialic acids from their host and use them for molecular mimicry. The first step of this process is the transport of sialic acid to the cytoplasm, which often takes place using a tripartite ATP-independent transport system consisting of a periplasmic binding protein and a membrane transporter. In this paper, the structural characterization of periplasmic binding proteins from the pathogenic bacteria Fusobacterium nucleatum, Pasteurella multocida and Vibrio cholerae and their thermodynamic characterization are reported. The binding affinities of several mutations in the Neu5Ac binding site of the Haemophilus influenzae protein are also reported. The structure and the thermodynamics of the binding of sugars suggest that all of these proteins have a very well conserved binding pocket and similar binding affinities. A significant conformational change occurs when these proteins bind the sugar. While the C1 carboxylate has been identified as the primary binding site, a second conserved hydrogen-bonding network is involved in the initiation and stabilization of the conformational states

  20. Rapid determination of thyroxine binding proteins of human serum

    Directory of Open Access Journals (Sweden)

    Arima,Terukatsu

    1976-02-01

    Full Text Available A simple method is described for determing thyroxine binding proteins in human serum by electrophoresis at pH 8.6, using cellulose acetate membrane as the supporting medium. The procedure had high reliability in sera of normal subjects, pregnant women and patients with decreased thyroxine binding capacity of thyroxine binding globulin.

  1. Escherichia coli cell division protein FtsZ is a guanine nucleotide binding protein.

    OpenAIRE

    Mukherjee, A; Dai, K; Lutkenhaus, J

    1993-01-01

    FtsZ is an essential cell division protein in Escherichia coli that forms a ring structure at the division site under cell cycle control. The dynamic nature of the FtsZ ring suggests possible similarities to eukaryotic filament forming proteins such as tubulin. In this study we have determined that FtsZ is a GTP/GDP binding protein with GTPase activity. A short segment of FtsZ is homologous to a segment in tubulin believed to be involved in the interaction between tubulin and guanine nucleoti...

  2. Thermodynamics of ligand binding to acyl-coenzyme A binding protein studied by titration calorimetry

    DEFF Research Database (Denmark)

    Færgeman, Nils J.; Sigurskjold, B W; Kragelund, B B;

    1996-01-01

    Ligand binding to recombinant bovine acyl-CoA binding protein (ACBP) was examined using isothermal microcalorimetry. Microcalorimetric measurements confirm that the binding affinity of acyl-CoA esters for ACBP is strongly dependent on the length of the acyl chain with a clear preference for acyl-...

  3. Calmodulin Binding Proteins and Alzheimer’s Disease

    Science.gov (United States)

    O’Day, Danton H.; Eshak, Kristeen; Myre, Michael A.

    2015-01-01

    Abstract The small, calcium-sensor protein, calmodulin, is ubiquitously expressed and central to cell function in all cell types. Here the literature linking calmodulin to Alzheimer’s disease is reviewed. Several experimentally-verified calmodulin-binding proteins are involved in the formation of amyloid-β plaques including amyloid-β protein precursor, β-secretase, presenilin-1, and ADAM10. Many others possess potential calmodulin-binding domains that remain to be verified. Three calmodulin binding proteins are associated with the formation of neurofibrillary tangles: two kinases (CaMKII, CDK5) and one protein phosphatase (PP2B or calcineurin). Many of the genes recently identified by genome wide association studies and other studies encode proteins that contain putative calmodulin-binding domains but only a couple (e.g., APOE, BIN1) have been experimentally confirmed as calmodulin binding proteins. At least two receptors involved in calcium metabolism and linked to Alzheimer’s disease (mAchR; NMDAR) have also been identified as calmodulin-binding proteins. In addition to this, many proteins that are involved in other cellular events intimately associated with Alzheimer’s disease including calcium channel function, cholesterol metabolism, neuroinflammation, endocytosis, cell cycle events, and apoptosis have been tentatively or experimentally verified as calmodulin binding proteins. The use of calmodulin as a potential biomarker and as a therapeutic target is discussed. PMID:25812852

  4. Local Unfolding of Fatty Acid Binding Protein to Allow Ligand Entry for Binding.

    Science.gov (United States)

    Xiao, Tianshu; Fan, Jing-Song; Zhou, Hu; Lin, Qingsong; Yang, Daiwen

    2016-06-01

    Fatty acid binding proteins are responsible for the transportation of fatty acids in biology. Despite intensive studies, the molecular mechanism of fatty acid entry to and exit from the protein cavity is still unclear. Here a cap-closed variant of human intestinal fatty acid binding protein was generated by mutagenesis, in which the helical cap is locked to the β-barrel by a disulfide linkage. Structure determination shows that this variant adopts a closed conformation, but still uptakes fatty acids. Stopped-flow experiments indicate that a rate-limiting step exists before the ligand association and this step corresponds to the conversion of the closed form to the open one. NMR relaxation dispersion and H-D exchange data demonstrate the presence of two excited states: one is native-like, but the other adopts a locally unfolded structure. Local unfolding of helix 2 generates an opening for ligands to enter the protein cavity, and thus controls the ligand association rate. PMID:27105780

  5. SCOWLP classification: Structural comparison and analysis of protein binding regions

    Directory of Open Access Journals (Sweden)

    Anders Gerd

    2008-01-01

    Full Text Available Abstract Background Detailed information about protein interactions is critical for our understanding of the principles governing protein recognition mechanisms. The structures of many proteins have been experimentally determined in complex with different ligands bound either in the same or different binding regions. Thus, the structural interactome requires the development of tools to classify protein binding regions. A proper classification may provide a general view of the regions that a protein uses to bind others and also facilitate a detailed comparative analysis of the interacting information for specific protein binding regions at atomic level. Such classification might be of potential use for deciphering protein interaction networks, understanding protein function, rational engineering and design. Description Protein binding regions (PBRs might be ideally described as well-defined separated regions that share no interacting residues one another. However, PBRs are often irregular, discontinuous and can share a wide range of interacting residues among them. The criteria to define an individual binding region can be often arbitrary and may differ from other binding regions within a protein family. Therefore, the rational behind protein interface classification should aim to fulfil the requirements of the analysis to be performed. We extract detailed interaction information of protein domains, peptides and interfacial solvent from the SCOWLP database and we classify the PBRs of each domain family. For this purpose, we define a similarity index based on the overlapping of interacting residues mapped in pair-wise structural alignments. We perform our classification with agglomerative hierarchical clustering using the complete-linkage method. Our classification is calculated at different similarity cut-offs to allow flexibility in the analysis of PBRs, feature especially interesting for those protein families with conflictive binding regions

  6. Sequence and structural features of binding site residues in protein-protein complexes: comparison with protein-nucleic acid complexes

    OpenAIRE

    Selvaraj S; Jayaram B; Saranya N; Gromiha M; Fukui Kazuhiko

    2011-01-01

    Abstract Background Protein-protein interactions are important for several cellular processes. Understanding the mechanism of protein-protein recognition and predicting the binding sites in protein-protein complexes are long standing goals in molecular and computational biology. Methods We have developed an energy based approach for identifying the binding site residues in protein–protein complexes. The binding site residues have been analyzed with sequence and structure based parameters such...

  7. Clinical relevance of drug binding to plasma proteins

    Science.gov (United States)

    Ascenzi, Paolo; Fanali, Gabriella; Fasano, Mauro; Pallottini, Valentina; Trezza, Viviana

    2014-12-01

    Binding to plasma proteins highly influences drug efficacy, distribution, and disposition. Serum albumin, the most abundant protein in plasma, is a monomeric multi-domain macromolecule that displays an extraordinary ligand binding capacity, providing a depot and carrier for many endogenous and exogenous compounds, such as fatty acids and most acidic drugs. α-1-Acid glycoprotein, the second main plasma protein, is a glycoprotein physiologically involved in the acute phase reaction and is the main carrier for basic and neutral drugs. High- and low-density lipoproteins play a limited role in drug binding and are natural drug delivery system only for few lipophilic drugs or lipid-based formulations. Several factors influence drug binding to plasma proteins, such as pathological conditions, concurrent administration of drugs, sex, and age. Any of these factors, in turn, influences drug efficacy and toxicity. Here, biochemical, biomedical, and biotechnological aspects of drug binding to plasma proteins are reviewed.

  8. Maintaining cholesterol homeostasis:Sterol regulatory element-binding proteins

    Institute of Scientific and Technical Information of China (English)

    Lutz W. Weber; Meinrad Boll; Andreas Stampfl

    2004-01-01

    The molecular mechanism of how hepatocytes maintain cholesterol homeostasis has become much more transparent with the discovery of sterol regulatory element binding proteins (SREBPs) in recent years. These membrane proteins are members of the basic helix-loop-helix-leucine zipper (bHLHZip) family of transcription factors. They activate the expression of at least 30 genes involved in the synthesis of cholesterol and lipids. SREBPs are synthesized as precursor proteins in the endoplasmic reticulum (ER), where they form a complex with another protein, SREBP cleavage activating protein (SCAP).The SCAP molecule contains a sterol sensory domain. In the presence of high cellular sterol concentrations SCAP confines SREBP to the ER. With low cellular concentrations, SCAP escorts SREBP to activation in the Golgi. There, SREBP undergoes two proteolytic cleavage steps to release the mature, biologically active transcription factor, nuclear SREBP (nSREBP). nSREBP translocates to the nucleus and binds to sterol response elements (SRE) in the promoter/enhancer regions of target genes. Additional transcription factors are required to activate transcription of these genes. Three different SREBPs are known, SREBPs-1a, -1c and -2. SREBP-1a and -1c are isoforms produced from a single gene by alternate splicing. SREBP-2is encoded by a different gene and does not display any isoforms. It appears that SREBPs alone, in the sequence described above, can exert complete control over cholesterol synthesis, whereas many additional factors (hormones,cytokines, etc.) are required for complete control of lipid metabolism. Medicinal manipulation of the SREBP/SCAP system is expected to prove highly beneficial in the management of cholesterol-related disease.

  9. The actin binding protein adseverin regulates osteoclastogenesis.

    Science.gov (United States)

    Hassanpour, Siavash; Jiang, Hongwei; Wang, Yongqiang; Kuiper, Johannes W P; Glogauer, Michael

    2014-01-01

    Adseverin (Ads), a member of the Gelsolin superfamily of actin binding proteins, regulates the actin cytoskeleton architecture by severing and capping existing filamentous actin (F-actin) strands and nucleating the assembly of new F-actin filaments. Ads has been implicated in cellular secretion, exocytosis and has also been shown to regulate chondrogenesis and megakaryoblastic leukemia cell differentiation. Here we report for the first time that Ads is involved in regulating osteoclastogenesis (OCG). Ads is induced during OCG downstream of RANK-ligand (RANKL) stimulation and is highly expressed in mature osteoclasts. The D5 isoform of Ads is not involved in regulating OCG, as its expression is not induced in response to RANKL. Three clonal Ads knockdown RAW264.7 (RAW) macrophage cell lines with varying degrees of Ads expression and OCG deficiency were generated. The most drastic OCG defect was noted in the clonal cell line with the greatest degree of Ads knockdown as indicated by a lack of TRAcP staining and multinucleation. RNAi mediated knockdown of Ads in osteoclast precursors resulted in distinct morphological changes characterized by altered F-actin distribution and increased filopodia formation. Ads knockdown precursor cells experienced enhanced migration while fusion of knockdown precursors cells was limited. Transient reintroduction of de novo Ads back into the knockdown system was capable of rescuing TRAcP expression but not osteoclast multinucleation most likely due to the transient nature of Ads expression. This preliminary study allows us to conclude that Ads is a RANKL induced early regulator of OCG with a potential role in pre-osteoclast differentiation and fusion. PMID:25275604

  10. The actin binding protein adseverin regulates osteoclastogenesis.

    Directory of Open Access Journals (Sweden)

    Siavash Hassanpour

    Full Text Available Adseverin (Ads, a member of the Gelsolin superfamily of actin binding proteins, regulates the actin cytoskeleton architecture by severing and capping existing filamentous actin (F-actin strands and nucleating the assembly of new F-actin filaments. Ads has been implicated in cellular secretion, exocytosis and has also been shown to regulate chondrogenesis and megakaryoblastic leukemia cell differentiation. Here we report for the first time that Ads is involved in regulating osteoclastogenesis (OCG. Ads is induced during OCG downstream of RANK-ligand (RANKL stimulation and is highly expressed in mature osteoclasts. The D5 isoform of Ads is not involved in regulating OCG, as its expression is not induced in response to RANKL. Three clonal Ads knockdown RAW264.7 (RAW macrophage cell lines with varying degrees of Ads expression and OCG deficiency were generated. The most drastic OCG defect was noted in the clonal cell line with the greatest degree of Ads knockdown as indicated by a lack of TRAcP staining and multinucleation. RNAi mediated knockdown of Ads in osteoclast precursors resulted in distinct morphological changes characterized by altered F-actin distribution and increased filopodia formation. Ads knockdown precursor cells experienced enhanced migration while fusion of knockdown precursors cells was limited. Transient reintroduction of de novo Ads back into the knockdown system was capable of rescuing TRAcP expression but not osteoclast multinucleation most likely due to the transient nature of Ads expression. This preliminary study allows us to conclude that Ads is a RANKL induced early regulator of OCG with a potential role in pre-osteoclast differentiation and fusion.

  11. Concentration-dependent Cu(II) binding to prion protein

    Science.gov (United States)

    Hodak, Miroslav; Lu, Wenchang; Bernholc, Jerry

    2008-03-01

    The prion protein plays a causative role in several neurodegenerative diseases, including mad cow disease in cattle and Creutzfeldt-Jakob disease in humans. The normal function of the prion protein is unknown, but it has been linked to its ability to bind copper ions. Experimental evidence suggests that copper can be bound in three distinct modes depending on its concentration, but only one of those binding modes has been fully characterized experimentally. Using a newly developed hybrid DFT/DFT method [1], which combines Kohn-Sham DFT with orbital-free DFT, we have examined all the binding modes and obtained their detailed binding geometries and copper ion binding energies. Our results also provide explanation for experiments, which have found that when the copper concentration increases the copper binding mode changes, surprisingly, from a stronger to a weaker one. Overall, our results indicate that prion protein can function as a copper buffer. 1. Hodak, Lu, Bernholc, JCP, in press.

  12. Pumilio Puf domain RNA-binding proteins in Arabidopsis

    OpenAIRE

    Abbasi, Nazia; Park, Youn-Il; Choi, Sang-Bong

    2011-01-01

    Pumilio proteins are a class of RNA-binding proteins harboring Puf domains (or PUM-HD; Pumilio-Homology Domain), named after the founding members, Pumilio (from Drosophila melanogaster) and FBF (Fem-3 mRNA-Binding Factor from Caenorhabditis elegans). The domains contain multiple tandem repeats each of which recognizes one RNA base and is comprised of 35–39 amino acids. Puf domain proteins have been reported in organisms ranging from single-celled yeast to higher multicellular eukaryotes, such...

  13. Grafting odorant binding proteins on diamond bio-MEMS

    OpenAIRE

    Manai, Raafa; Scorsone, E.; Rousseau, L.; Ghassemi, F.; Possas Abreu, M.; Lissorgues, G.; Tremillon, N.; Ginisty, H; Arnault, J.-C.; Tuccori, E.; Bernabei, M.; Cali, K.; Persaud, K C; Bergonzo, P.

    2014-01-01

    Odorant binding proteins (OBPs) are small soluble proteins found in olfactory systems that are capable of binding several types of odorant molecules. Cantilevers based on polycrystalline diamond surfaces are very promising as chemical transducers. Here two methods were investigated for chemically grafting porcine OBPs on polycrystalline diamond surfaces for biosensor development. The first approach resulted in random orientation of the immobilized proteins over the surface. The second approac...

  14. The clinical significance of fatty acid binding proteins

    OpenAIRE

    Barbara Choromańska; Piotr Myśliwiec; Jacek Dadan; Hady Razak Hady; Adrian Chabowski

    2011-01-01

    Excessive levels of free fatty acids are toxic to cells. The human body has evolved a defense mechanism in the form of small cytoplasmic proteins called fatty acid binding proteins (FABPs) that bind long-chain fatty acids (LCFA), and then refer them to appropriate intracellular disposal sites (oxidation in mitochondria and peroxisomes or storage in the endoplasmic reticulum). So far, nine types of these proteins have been described, and their name refers to the place in which they were first ...

  15. Cooperative binding modes of Cu(II) in prion protein

    Science.gov (United States)

    Hodak, Miroslav; Chisnell, Robin; Lu, Wenchang; Bernholc, Jerry

    2007-03-01

    The misfolding of the prion protein, PrP, is responsible for a group of neurodegenerative diseases including mad cow disease and Creutzfeldt-Jakob disease. It is known that the PrP can efficiently bind copper ions; four high-affinity binding sites located in the octarepeat region of PrP are now well known. Recent experiments suggest that at low copper concentrations new binding modes, in which one copper ion is shared between two or more binding sites, are possible. Using our hybrid Thomas-Fermi/DFT computational scheme, which is well suited for simulations of biomolecules in solution, we investigate the geometries and energetics of two, three and four binding sites cooperatively binding one copper ion. These geometries are then used as inputs for classical molecular dynamics simulations. We find that copper binding affects the secondary structure of the PrP and that it stabilizes the unstructured (unfolded) part of the protein.

  16. Thermodynamic parameters of the binding of retinol to binding proteins and to membranes

    International Nuclear Information System (INIS)

    Retinol (vitamin A alcohol) is a hydrophobic compound and distributes in vivo mainly between binding proteins and cellular membranes. To better clarify the nature of the interactions of retinol with these phases which have a high affinity for it, the thermodynamic parameters of these interactions were studied. The temperature-dependence profiles of the binding of retinol to bovine retinol binding protein, bovine serum albumin, unilamellar vesicles of dioleoylphosphatidylcholine, and plasma membranes from rat liver were determined. It was found that binding of retinol to retinol binding protein is characterized by a large increase in entropy and no change in enthalpy. Binding to albumin is driven by enthalpy and is accompanied by a decrease in entropy. Partitioning of retinal into unilamellar vesicles and into plasma membranes is stabilized both by enthalpic and by entropic components. The implications of these finding are discussed

  17. Sequence and structural features of binding site residues in protein-protein complexes: comparison with protein-nucleic acid complexes

    Directory of Open Access Journals (Sweden)

    Selvaraj S

    2011-10-01

    Full Text Available Abstract Background Protein-protein interactions are important for several cellular processes. Understanding the mechanism of protein-protein recognition and predicting the binding sites in protein-protein complexes are long standing goals in molecular and computational biology. Methods We have developed an energy based approach for identifying the binding site residues in protein–protein complexes. The binding site residues have been analyzed with sequence and structure based parameters such as binding propensity, neighboring residues in the vicinity of binding sites, conservation score and conformational switching. Results We observed that the binding propensities of amino acid residues are specific for protein-protein complexes. Further, typical dipeptides and tripeptides showed high preference for binding, which is unique to protein-protein complexes. Most of the binding site residues are highly conserved among homologous sequences. Our analysis showed that 7% of residues changed their conformations upon protein-protein complex formation and it is 9.2% and 6.6% in the binding and non-binding sites, respectively. Specifically, the residues Glu, Lys, Leu and Ser changed their conformation from coil to helix/strand and from helix to coil/strand. Leu, Ser, Thr and Val prefer to change their conformation from strand to coil/helix. Conclusions The results obtained in this study will be helpful for understanding and predicting the binding sites in protein-protein complexes.

  18. Stereoselective binding of chiral drugs to plasma proteins

    Institute of Scientific and Technical Information of China (English)

    Qi SHEN; Lu WANG; Hui ZHOU; Hui-di JIANG; Lu-shan YU; Su ZENG

    2013-01-01

    Chiral drugs show distinct biochemical and pharmacological behaviors in the human body.The binding of chiral drugs to plasma proteins usually exhibits stereoselectivity,which has a far-reaching influence on their pharmacological activities and pharmacokinetic profiles.In this review,the stereoselective binding of chiral drugs to human serum albumin (HSA),α1-acid glycoprotein (AGP)and lipoprotein,three most important proteins in human plasma,are detailed.Furthermore,the application of AGP variants and recombinant fragments of HSA for studying enantiomer binding properties is also discussed.Apart from the stereoselectivity of enantiomer-protein binding,enantiomer-enantiomer interactions that may induce allosteric effects are also described.Additionally,the techniques and methods used to determine drug-protein binding parameters are briefly reviewed.

  19. The Pumilio protein binds RNA through a conserved domain that defines a new class of RNA-binding proteins.

    Science.gov (United States)

    Zamore, P D; Williamson, J R; Lehmann, R

    1997-01-01

    Translation of hunchback(mat) (hb[mat]) mRNA must be repressed in the posterior of the pre-blastoderm Drosophila embryo to permit formation of abdominal segments. This translational repression requires two copies of the Nanos Response Element (NRE), a 16-nt sequence in the hb[mat] 3' untranslated region. Translational repression also requires the action of two proteins: Pumilio (PUM), a sequence-specific RNA-binding protein; and Nanos, a protein that determines the location of repression. Binding of PUM to the NRE is thought to target hb(mat) mRNA for repression. Here, we show the RNA-binding domain of PUM to be an evolutionarily conserved, 334-amino acid region at the carboxy-terminus of the approximately 158-kDa PUM protein. This contiguous region of PUM retains the RNA-binding specificity of full-length PUM protein. Proteins with sequences homologous to the PUM RNA-binding domain are found in animals, plants, and fungi. The high degree of sequence conservation of the PUM RNA-binding domain in other far-flung species suggests that the domain is an ancient protein motif, and we show that conservation of sequence reflects conservation of function: that is, the homologous region from a human protein binds RNA with sequence specificity related to but distinct from Drosophila PUM. PMID:9404893

  20. Guardian of Genetic Messenger-RNA-Binding Proteins

    Directory of Open Access Journals (Sweden)

    Antje Anji

    2016-01-01

    Full Text Available RNA in cells is always associated with RNA-binding proteins that regulate all aspects of RNA metabolism including RNA splicing, export from the nucleus, RNA localization, mRNA turn-over as well as translation. Given their diverse functions, cells express a variety of RNA-binding proteins, which play important roles in the pathologies of a number of diseases. In this review we focus on the effect of alcohol on different RNA-binding proteins and their possible contribution to alcohol-related disorders, and discuss the role of these proteins in the development of neurological diseases and cancer. We further discuss the conventional methods and newer techniques that are employed to identify RNA-binding proteins.

  1. Convergent evolution among immunoglobulin G-binding bacterial proteins.

    OpenAIRE

    Frick, I M; Wikström, M.; Forsén, S.; Drakenberg, T; Gomi, H.; Sjöbring, U; Björck, L

    1992-01-01

    Protein G, a bacterial cell-wall protein with high affinity for the constant region of IgG (IgGFc) antibodies, contains homologous repeats responsible for the interaction with IgGFc. A synthetic peptide corresponding to an 11-amino acid-long sequence in the COOH-terminal region of the repeats was found to bind to IgGFc and block the interaction with protein G. Moreover, two other IgGFc-binding bacterial proteins (proteins A and H), which do not contain any sequences homologous to the peptide,...

  2. A computational method for the analysis and prediction of protein:phosphopeptide-binding sites

    OpenAIRE

    Joughin, Brian A.; Tidor, Bruce; Yaffe, Michael B.

    2005-01-01

    Phosphopeptide-binding domains, including the FHA, SH2, WW, WD40, MH2, and Polo-box domains, as well as the 14-3-3 proteins, exert control functions in important processes such as cell growth, division, differentiation, and apoptosis. Structures and mechanisms of phosphopeptide binding are generally diverse, revealing few general principles. A computational method for analysis of phosphopeptide-binding domains was therefore developed to elucidate the physical and chemical nature of phosphopep...

  3. Technical advance: identification of plant actin-binding proteins by F-actin affinity chromatography

    Science.gov (United States)

    Hu, S.; Brady, S. R.; Kovar, D. R.; Staiger, C. J.; Clark, G. B.; Roux, S. J.; Muday, G. K.

    2000-01-01

    Proteins that interact with the actin cytoskeleton often modulate the dynamics or organization of the cytoskeleton or use the cytoskeleton to control their localization. In plants, very few actin-binding proteins have been identified and most are thought to modulate cytoskeleton function. To identify actin-binding proteins that are unique to plants, the development of new biochemical procedures will be critical. Affinity columns using actin monomers (globular actin, G-actin) or actin filaments (filamentous actin, F-actin) have been used to identify actin-binding proteins from a wide variety of organisms. Monomeric actin from zucchini (Cucurbita pepo L.) hypocotyl tissue was purified to electrophoretic homogeneity and shown to be native and competent for polymerization to actin filaments. G-actin, F-actin and bovine serum albumin affinity columns were prepared and used to separate samples enriched in either soluble or membrane-associated actin-binding proteins. Extracts of soluble actin-binding proteins yield distinct patterns when eluted from the G-actin and F-actin columns, respectively, leading to the identification of a putative F-actin-binding protein of approximately 40 kDa. When plasma membrane-associated proteins were applied to these columns, two abundant polypeptides eluted selectively from the F-actin column and cross-reacted with antiserum against pea annexins. Additionally, a protein that binds auxin transport inhibitors, the naphthylphthalamic acid binding protein, which has been previously suggested to associate with the actin cytoskeleton, was eluted in a single peak from the F-actin column. These experiments provide a new approach that may help to identify novel actin-binding proteins from plants.

  4. Characterization of a cocaine binding protein in human placenta

    International Nuclear Information System (INIS)

    [3H]-Cocaine binding sites are identified in human placental villus tissue plasma membranes. These binding sites are associated with a protein and show saturable and specific binding of [3H]-cocaine with a high affinity site of 170 fmole/mg protein. The binding is lost with pretreatment with trypsin or heat. The membrane bound protein is solubilized with the detergent 3-(3-cholamidopropyl)dimethyl-ammonio-1-propane sulphonate (CHAPS) with retention of its saturable and specific binding of [3H]-cocaine. The detergent-protein complex migrates on a sepharose CL-6B gel chromatography column as a protein with an apparent molecular weight of 75,900. The protein has an S20,w value of 5.1. The binding of this protein to norcocaine, pseudococaine, nomifensine, imipramine, desipramine, amphetamine and dopamine indicates that it shares some, but not all, the properties of the brain cocaine receptor. The physiologic significance of this protein in human placenta is currently unclear

  5. Electrochemistry of heparin binding to tau protein on Au surfaces

    International Nuclear Information System (INIS)

    Highlights: • Anionic heparin binds tau protein film on Au • N-terminal of tau protein is critical for heparin binding • Negatively charged heparin binds positively charged tau domains • Heparin binding to tau increases charge transfer resistance - ABSTRACT: The tau protein is a neurodegenerative disease biomarker. The in vitro aggregation of tau is triggered by electrostatic charge imbalance induced by an anionic inducing agent, such as heparin. The binding of the tau-heparin complex is based on electrostatic interactions, but the exact binding mode of heparin to the tau protein has not been fully identified. In this work, the effects of the tau protein orientation on gold (Au) electrode to heparin were explored by the cyclic voltammetry and electrochemical impedance spectroscopy. To modulate the accessibility of N-terminal of the tau to heparin, the tau films on Au surfaces were fabricated in two ways: immobilization of tau via the N-terminal of tau protein (N-tau-Au) or by the Cys291/Cys322 residues, located in the R-repeat domains of the tau protein (Cys-tau-Au). The sulfur-Au bonding was characterized by X-ray photoelectron spectroscopy. The charge transfer resistance was measured for N-tau-Au and Cys-tau-Au as a function of heparin concentration. The heparin concentration range was varied from 0.2 pM to 216 μM with the optimal binding concentration at 21 nM (the highest charge transfer resistance value). The heparin binding to tau films was investigated in the presence of [Fe(CN)6]3−/4− or benzoquinone redox probes. The tau-heparin binding was greater for the Cys-tau-Au surface over N-tau-Au, indicating specific tau domains may be required for optimal heparin binding

  6. Acyl-CoA-binding protein/diazepam-binding inhibitor gene and pseudogenes

    DEFF Research Database (Denmark)

    Mandrup, S; Hummel, R; Ravn, S;

    1992-01-01

    Acyl-CoA-binding protein (ACBP) is a 10 kDa protein isolated from bovine liver by virtue of its ability to bind and induce the synthesis of medium-chain acyl-CoA esters. Surprisingly, it turned out to be identical to a protein named diazepam-binding Inhibitor (DBI) claimed to be an endogenous...... remarkable correspondence between the structural modules of ACBP/DBI as determined by 1H nuclear magnetic resonance spectroscopy and the exon-intron architecture of the ACBP/DBI gene. Detailed analyses of transcription of the ACBP/DBI gene in brain and liver were performed to map transcription initiation...

  7. Mutations in the RNA Binding Domain of Stem-Loop Binding Protein Define Separable Requirements for RNA Binding and for Histone Pre-mRNA Processing

    OpenAIRE

    Dominski, Zbigniew; Erkmann, Judith A.; Greenland, John A.; Marzluff, William F

    2001-01-01

    Expression of replication-dependent histone genes at the posttranscriptional level is controlled by stem-loop binding protein (SLBP). One function of SLBP is to bind the stem-loop structure in the 3′ untranslated region of histone pre-mRNAs and facilitate 3′ end processing. Interaction of SLBP with the stem-loop is mediated by the centrally located RNA binding domain (RBD). Here we identify several highly conserved amino acids in the RBD mutation of which results in complete or substantial lo...

  8. Prediction of heme binding residues from protein sequences with integrative sequence profiles

    OpenAIRE

    2012-01-01

    Background The heme-protein interactions are essential for various biological processes such as electron transfer, catalysis, signal transduction and the control of gene expression. The knowledge of heme binding residues can provide crucial clues to understand these activities and aid in functional annotation, however, insufficient work has been done on the research of heme binding residues from protein sequence information. Methods We propose a sequence-based approach for accurate prediction...

  9. Niobium Uptake and Release by Bacterial Ferric Ion Binding Protein

    Directory of Open Access Journals (Sweden)

    Yanbo Shi

    2010-01-01

    Full Text Available Ferric ion binding proteins (Fbps transport FeIII across the periplasm and are vital for the virulence of many Gram negative bacteria. Iron(III is tightly bound in a hinged binding cleft with octahedral coordination geometry involving binding to protein side chains (including tyrosinate residues together with a synergistic anion such as phosphate. Niobium compounds are of interest for their potential biological activity, which has been little explored. We have studied the binding of cyclopentadienyl and nitrilotriacetato NbV complexes to the Fbp from Neisseria gonorrhoeae by UV-vis spectroscopy, chromatography, ICP-OES, mass spectrometry, and Nb K-edge X-ray absorption spectroscopy. These data suggest that NbV binds strongly to Fbp and that a dinuclear NbV centre can be readily accommodated in the interdomain binding cleft. The possibility of designing niobium-based antibiotics which block iron uptake by pathogenic bacteria is discussed.

  10. PTPRT regulates the interaction of Syntaxin-binding protein 1 with Syntaxin 1 through dephosphorylation of specific tyrosine residue

    International Nuclear Information System (INIS)

    Highlights: •PTPRT is a brain-specific, expressed, protein tyrosine phosphatase. •PTPRT regulated the interaction of Syntaxin-binding protein 1 with Syntaxin 1. •PTPRT dephosphorylated the specific tyrosine residue of Syntaxin-binding protein 1. •Dephosphorylation of Syntaxin-binding protein 1 enhanced the interaction with Syntaxin 1. •PTPRT appears to regulate the fusion of synaptic vesicle through dephosphorylation. -- Abstract: PTPRT (protein tyrosine phosphatase receptor T), a brain-specific tyrosine phosphatase, has been found to regulate synaptic formation and development of hippocampal neurons, but its regulation mechanism is not yet fully understood. Here, Syntaxin-binding protein 1, a key component of synaptic vesicle fusion machinery, was identified as a possible interaction partner and an endogenous substrate of PTPRT. PTPRT interacted with Syntaxin-binding protein 1 in rat synaptosome, and co-localized with Syntaxin-binding protein 1 in cultured hippocampal neurons. PTPRT dephosphorylated tyrosine 145 located around the linker between domain 1 and 2 of Syntaxin-binding protein 1. Syntaxin-binding protein 1 directly binds to Syntaxin 1, a t-SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) protein, and plays a role as catalysts of SNARE complex formation. Syntaxin-binding protein 1 mutant mimicking non-phosphorylation (Y145F) enhanced the interaction with Syntaxin 1 compared to wild type, and therefore, dephosphorylation of Syntaxin-binding protein 1 appeared to be important for SNARE-complex formation. In conclusion, PTPRT could regulate the interaction of Syntaxin-binding protein 1 with Syntaxin 1, and as a result, the synaptic vesicle fusion appeared to be controlled through dephosphorylation of Syntaxin-binding protein 1

  11. Suppression of cellular transformation by poly (A binding protein interacting protein 2 (Paip2.

    Directory of Open Access Journals (Sweden)

    Amy B Rosenfeld

    Full Text Available Controlling translation is crucial for the homeostasis of a cell. Its deregulation can facilitate the development and progression of many diseases including cancer. Poly (A binding protein interacting protein 2 (Paip2 inhibits efficient initiation of translation by impairing formation of the necessary closed loop of mRNA. The over production of Paip2 in the presence of a constitutively active form of hRas(V12 can reduce colony formation in a semi-solid matrix and focus formation on a cell monolayer. The ability of Paip2 to bind to Pabp is required to suppress the transformed phenotype mediated by hRas(V12. These observations indicate that Paip2 is able to function as a tumor suppressor.

  12. Roles of RNA-Binding Proteins in DNA Damage Response.

    Science.gov (United States)

    Kai, Mihoko

    2016-01-01

    Living cells experience DNA damage as a result of replication errors and oxidative metabolism, exposure to environmental agents (e.g., ultraviolet light, ionizing radiation (IR)), and radiation therapies and chemotherapies for cancer treatments. Accumulation of DNA damage can lead to multiple diseases such as neurodegenerative disorders, cancers, immune deficiencies, infertility, and also aging. Cells have evolved elaborate mechanisms to deal with DNA damage. Networks of DNA damage response (DDR) pathways are coordinated to detect and repair DNA damage, regulate cell cycle and transcription, and determine the cell fate. Upstream factors of DNA damage checkpoints and repair, "sensor" proteins, detect DNA damage and send the signals to downstream factors in order to maintain genomic integrity. Unexpectedly, we have discovered that an RNA-processing factor is involved in DNA repair processes. We have identified a gene that contributes to glioblastoma multiforme (GBM)'s treatment resistance and recurrence. This gene, RBM14, is known to function in transcription and RNA splicing. RBM14 is also required for maintaining the stem-like state of GBM spheres, and it controls the DNA-PK-dependent non-homologous end-joining (NHEJ) pathway by interacting with KU80. RBM14 is a RNA-binding protein (RBP) with low complexity domains, called intrinsically disordered proteins (IDPs), and it also physically interacts with PARP1. Furthermore, RBM14 is recruited to DNA double-strand breaks (DSBs) in a poly(ADP-ribose) (PAR)-dependent manner (unpublished data). DNA-dependent PARP1 (poly-(ADP) ribose polymerase 1) makes key contributions in the DNA damage response (DDR) network. RBM14 therefore plays an important role in a PARP-dependent DSB repair process. Most recently, it was shown that the other RBPs with intrinsically disordered domains are recruited to DNA damage sites in a PAR-dependent manner, and that these RBPs form liquid compartments (also known as "liquid-demixing"). Among the

  13. Identification of Actin-Binding Proteins from Maize Pollen

    Energy Technology Data Exchange (ETDEWEB)

    Staiger, C.J.

    2004-01-13

    Specific Aims--The goal of this project was to gain an understanding of how actin filament organization and dynamics are controlled in flowering plants. Specifically, we proposed to identify unique proteins with novel functions by investigating biochemical strategies for the isolation and characterization of actin-binding proteins (ABPs). In particular, our hunt was designed to identify capping proteins and nucleation factors. The specific aims included: (1) to use F-actin affinity chromatography (FAAC) as a general strategy to isolate pollen ABPs (2) to produce polyclonal antisera and perform subcellular localization in pollen tubes (3) to isolate cDNA clones for the most promising ABPs (4) to further purify and characterize ABP interactions with actin in vitro. Summary of Progress By employing affinity chromatography on F-actin or DNase I columns, we have identified at least two novel ABPs from pollen, PrABP80 (gelsolin-like) and ZmABP30, We have also cloned and expressed recombinant protein, as well as generated polyclonal antisera, for 6 interesting ABPs from Arabidopsis (fimbrin AtFIM1, capping protein a/b (AtCP), adenylyl cyclase-associated protein (AtCAP), AtCapG & AtVLN1). We performed quantitative analyses of the biochemical properties for two of these previously uncharacterized ABPs (fimbrin and capping protein). Our studies provide the first evidence for fimbrin activity in plants, demonstrate the existence of barbed-end capping factors and a gelsolin-like severing activity, and provide the quantitative data necessary to establish and test models of F-actin organization and dynamics in plant cells.

  14. Studies of the silencing of Baculovirus DNA binding protein

    NARCIS (Netherlands)

    Quadt, I.; Lent, van J.W.M.; Knebel-Morsdorf, D.

    2007-01-01

    Baculovirus DNA binding protein (DBP) binds preferentially single-stranded DNA in vitro and colocalizes with viral DNA replication sites. Here, its putative role as viral replication factor has been addressed by RNA interference. Silencing of DBP in Autographa californica multiple nucleopolyhedrovir

  15. Somatomedin-1 binding protein-3: insulin-like growth factor-1 binding protein-3, insulin-like growth factor-1 carrier protein.

    Science.gov (United States)

    2003-01-01

    planned to move to Avecia's larger facility with a capacity of 10 000 litres. Somatomedin-1 binding protein-3 was originally licenced to Welfide for Japan. On October 1 2001, Welfide Corporation merged with Mitsubishi-Tokyo Pharmaceuticals to form Mitsubishi Pharma Corporation. The new company is a subsidiary of Mitsubishi Chemical. In April 2003 Insmed initiated a named patient programme in Europe, that will make available somatomedin-1 binding protein-3 for the treatment of growth hormone insensitivity syndrome (GHIS)--Laron syndrome. The treatment of patients was initiated in Scandinavia, with authorisation pending in several other European countries. Somatomedin-1 binding protein-3 will be made available to those GHIS patients who, in the opinion of their doctor, may benefit from IGF-1 therapy. At precommercial scale quantities, the drug will be available on a limited basis. Safety data generated from the named patient programme will be used to support marketing applications in 2004. A phase II dose-ranging study in children with GHIS was completed at Saint Bartholomew's and the Royal London School of Medicine, London, UK. A single dose of somatomedin-1 binding protein-3 delivered the same amount of IGF-1 as two daily injections of unbound IGF-1. There were no adverse events reported. GHIS is a genetic condition in which patients do not produce adequate quantities of IGF because of a failure to respond to the growth hormone signal. This results in a slower growth rate and short stature. Insmed has acquired an exclusive licence to Pharmacia's regulatory filings concerning yeast-derived IGF-1. These filings were used by Pharmacia to receive marketing approvals in several European countries and also in the investigational New Drug Application with the US FDA. This licence will facilitate the development of SomatoKine for the treatment of children with GHIS. In January 2003, Insmed announced positive results from a double-blind, placebo-controlled, dose-ranging study of

  16. Expected and unexpected features of protein-binding RNA aptamers

    DEFF Research Database (Denmark)

    Bjerregaard, Nils; Andreasen, Peter A; Dupont, Daniel M

    2016-01-01

    RNA molecules with high affinity to specific proteins can be isolated from libraries of up to 10(16) different RNA sequences by systematic evolution of ligands by exponential enrichment (SELEX). These so-called protein-binding RNA aptamers are often interesting, e.g., as modulators of protein...... function for therapeutic use, for probing the conformations of proteins, for studies of basic aspects of nucleic acid-protein interactions, etc. Studies on the interactions between RNA aptamers and proteins display a number of expected and unexpected features, including the chemical nature of the...... interacting RNA-protein surfaces, the conformation of protein-bound aptamer versus free aptamer, the conformation of aptamer-bound protein versus free protein, and the effects of aptamers on protein function. Here, we review current insights into the details of RNA aptamer-protein interactions. For further...

  17. The interrelationship between ligand binding and self-association of the folate binding protein

    DEFF Research Database (Denmark)

    Holm, Jan; Schou, Christian; Babol, Linnea N.;

    2011-01-01

    The folate binding protein (FBP) regulates homeostasis and intracellular trafficking of folic acid, a vitamin of decisive importance in cell division and growth. We analyzed whether interrelationship between ligand binding and self-association of FBP plays a significant role in the physiology of...

  18. Natural ligand binding and transfer from liver fatty acid binding protein (LFABP) to membranes.

    Science.gov (United States)

    De Gerónimo, Eduardo; Hagan, Robert M; Wilton, David C; Córsico, Betina

    2010-09-01

    Liver fatty acid-binding protein (LFABP) is distinctive among fatty acid-binding proteins because it binds more than one molecule of long-chain fatty acid and a variety of diverse ligands. Also, the transfer of fluorescent fatty acid analogues to model membranes under physiological ionic strength follows a different mechanism compared to most of the members of this family of intracellular lipid binding proteins. Tryptophan insertion mutants sensitive to ligand binding have allowed us to directly measure the binding affinity, ligand partitioning and transfer to model membranes of natural ligands. Binding of fatty acids shows a cooperative mechanism, while acyl-CoAs binding presents a hyperbolic behavior. Saturated fatty acids seem to have a stronger partition to protein vs. membranes, compared to unsaturated fatty acids. Natural ligand transfer rates are more than 200-fold higher compared to fluorescently-labeled analogues. Interestingly, oleoyl-CoA presents a markedly different transfer behavior compared to the rest of the ligands tested, probably indicating the possibility of specific targeting of ligands to different metabolic fates. PMID:20541621

  19. Autoinhibition of Mint1 adaptor protein regulates amyloid precursor protein binding and processing

    OpenAIRE

    Matos, Maria F.; Xu, Yibin; Dulubova, Irina; Otwinowski, Zbyszek; Richardson, John M.; Tomchick, Diana R.; Rizo, Josep; Ho, Angela

    2012-01-01

    Mint adaptor proteins bind to the amyloid precursor protein (APP) and regulate APP processing associated with Alzheimer’s disease; however, the molecular mechanisms underlying Mint regulation in APP binding and processing remain unclear. Biochemical, biophysical, and cellular experiments now show that the Mint1 phosphotyrosine binding (PTB) domain that binds to APP is intramolecularly inhibited by the adjacent C-terminal linker region. The crystal structure of a C-terminally extended Mint1 PT...

  20. RNAcontext: a new method for learning the sequence and structure binding preferences of RNA-binding proteins.

    Directory of Open Access Journals (Sweden)

    Hilal Kazan

    Full Text Available Metazoan genomes encode hundreds of RNA-binding proteins (RBPs. These proteins regulate post-transcriptional gene expression and have critical roles in numerous cellular processes including mRNA splicing, export, stability and translation. Despite their ubiquity and importance, the binding preferences for most RBPs are not well characterized. In vitro and in vivo studies, using affinity selection-based approaches, have successfully identified RNA sequence associated with specific RBPs; however, it is difficult to infer RBP sequence and structural preferences without specifically designed motif finding methods. In this study, we introduce a new motif-finding method, RNAcontext, designed to elucidate RBP-specific sequence and structural preferences with greater accuracy than existing approaches. We evaluated RNAcontext on recently published in vitro and in vivo RNA affinity selected data and demonstrate that RNAcontext identifies known binding preferences for several control proteins including HuR, PTB, and Vts1p and predicts new RNA structure preferences for SF2/ASF, RBM4, FUSIP1 and SLM2. The predicted preferences for SF2/ASF are consistent with its recently reported in vivo binding sites. RNAcontext is an accurate and efficient motif finding method ideally suited for using large-scale RNA-binding affinity datasets to determine the relative binding preferences of RBPs for a wide range of RNA sequences and structures.

  1. Surface expression of GABAA receptors is transcriptionally controlled by the interplay of cAMP-response element-binding protein and its binding partner inducible cAMP early repressor.

    Science.gov (United States)

    Hu, Yinghui; Lund, Ingrid V; Gravielle, Maria C; Farb, David H; Brooks-Kayal, Amy R; Russek, Shelley J

    2008-04-01

    The regulated expression of type A gamma-aminobutyric acid (GABA) receptor (GABA(A)R) subunit genes plays a critical role in neuronal maturation and synaptogenesis. It is also associated with a variety of neurological diseases. Changes in GABA(A) receptor alpha1 subunit gene (GABRA1) expression have been reported in animal models of epilepsy, alcohol abuse, withdrawal, and stress. Understanding the genetic mechanism behind such changes in alpha subunit expression will lead to a better understanding of the role that signal transduction plays in control over GABA(A)R function and brings with it the promise of providing new therapeutic tools for the prevention or cure of a variety of neurological disorders. Here we show that activation of protein kinase C increases alpha1 subunit levels via phosphorylation of CREB (pCREB) that is bound to the GABRA1 promoter (GABRA1p). In contrast, activation of protein kinase A decreases levels of alpha1 even in the presence of pCREB. Decrease of alpha1 is dependent upon the inducible cAMP early repressor (ICER) as directly demonstrated by ICER-induced down-regulation of endogenous alpha1-containing GABA(A)Rs at the cell surface of cortical neurons. Taken together with the fact that there are less alpha1gamma2-containing GABA(A)Rs in neurons after protein kinase A stimulation and that activation of endogenous dopamine receptors down-regulates alpha1 subunit mRNA levels subsequent to induction of ICER, our studies identify a transcriptional mechanism for regulating the cell surface expression of alpha1-containing GABA(A)Rs that is dependent upon the formation of CREB heterodimers. PMID:18180303

  2. Binding of fluorescent lanthanides to rat liver mitochondrial membranes and calcium ion-binding proteins.

    Science.gov (United States)

    Mikkelsen, R B; Wallach, D F

    1976-05-21

    (1) Tb3+ binding to mitochondrial membranes can be monitored by enhanced ion fluorescence at 545 nm with excitation at 285 nm. At low protein concentrations (less than 30 mug/ml) no inner filter effects are observed. (2) This binding is localized at the external surface of the inner membrane and is unaffected by inhibitors of respiration or oxidative phosphorylation. (3) A soluble Ca2+ binding protein isolated according to Lehninger, A.L. ((1971) Biochem. Biophys. Res. Commun. 42, 312-317) also binds Tb3+ with enhanced ion fluorescence upon excitation at 285 nm. The excitation spectrum of the isolated protein and of the intact mitochondria are indicative of an aromatic amino acid at the cation binding site. (4) Further characterization of the Tb3+-protein interaction revealed that there is more than one binding site per protein molecule and that these sites are clustered (less than 20 A). Neuraminidase treatment or organic solvent extraction of the protein did not affect fluorescent Tb3+ binding. (5) pH dependency studies of Tb3+ binding to the isolated protein or intact mitochondria demonstrated the importance of an ionizable group of pK greater than 6. At pH less than 7.5 the amount of Tb3+ bound to the isolated protein decreased with increase in pH as monitored by Tb3+ fluorescence. With intact mitochondria the opposite occurred with a large increase in Tb3+ fluorescence at higher pH. This increase was not observed when the mitochondria were preincubated with antimycin A and rotenone. PMID:6061

  3. Identification of lectin-binding proteins in Chlamydia species.

    OpenAIRE

    Swanson, A F; Kuo, C. C.

    1990-01-01

    Lectin-binding proteins of chlamydiae were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. All three Chlamydia species tested expressed two proteins when whole-elementary-body lysates were reacted with the biotinylated lectin Dolichos biflorus agglutinin. The protein with a molecular mass of 18 kilodaltons (kDa) responded strongly compared with a higher-molecular-mass protein that varied from 27 to 32 kDa with each chlamydia strain tested. Among six l...

  4. Protein kinase A binds and activates heat shock factor 1.

    Directory of Open Access Journals (Sweden)

    Ayesha Murshid

    Full Text Available BACKGROUND: Many inducible transcription factors are regulated through batteries of posttranslational modifications that couple their activity to inducing stimuli. We have studied such regulation of Heat Shock Factor 1 (HSF1, a key protein in control of the heat shock response, and a participant in carcinogenisis, neurological health and aging. As the mechanisms involved in the intracellular regulation of HSF1 in good health and its dysregulation in disease are still incomplete we are investigating the role of posttranslational modifications in such regulation. METHODOLOGY/PRINCIPAL FINDINGS: In a proteomic study of HSF1 binding partners, we have discovered its association with the pleiotropic protein kinase A (PKA. HSF1 binds avidly to the catalytic subunit of PKA, (PKAcα and becomes phosphorylated on a novel serine phosphorylation site within its central regulatory domain (serine 320 or S320, both in vitro and in vivo. Intracellular PKAcα levels and phosphorylation of HSF1 at S320 were both required for HSF1 to be localized to the nucleus, bind to response elements in the promoter of an HSF1 target gene (hsp70.1 and activate hsp70.1 after stress. Reduction in PKAcα levels by small hairpin RNA led to HSF1 exclusion from the nucleus, its exodus from the hsp70.1 promoter and decreased hsp70.1 transcription. Likewise, null mutation of HSF1 at S320 by alanine substitution for serine led to an HSF1 species excluded from the nucleus and deficient in hsp70.1 activation. CONCLUSIONS: These findings of PKA regulation of HSF1 through S320 phosphorylation add to our knowledge of the signaling networks converging on this factor and may contribute to elucidating its complex roles in the stress response and understanding HSF1 dysregulation in disease.

  5. Optimizing a coarse-grained model for the recognition of protein-protein binding

    OpenAIRE

    Emperador, Agustí; Orozco, Modesto

    2015-01-01

    We are optimizing a force-field to be used with our coarsegrained protein model for the recognition of protein -protein binding. We have found that, apart from ranking correctly the ligand-receptor conformations generated in a protein-protein docking algorithm, our model is able to distinguish binding (experimental structure) from nonbinding (false positive) conformations for many complexes. This suggests us that the model could have a good performance in complete cross-d...

  6. Statistical-mechanical lattice models for protein-DNA binding in chromatin

    International Nuclear Information System (INIS)

    Statistical-mechanical lattice models for protein-DNA binding are well established as a method to describe complex ligand binding equilibria measured in vitro with purified DNA and protein components. Recently, a new field of applications has opened up for this approach since it has become possible to experimentally quantify genome-wide protein occupancies in relation to the DNA sequence. In particular, the organization of the eukaryotic genome by histone proteins into a nucleoprotein complex termed chromatin has been recognized as a key parameter that controls the access of transcription factors to the DNA sequence. New approaches have to be developed to derive statistical-mechanical lattice descriptions of chromatin-associated protein-DNA interactions. Here, we present the theoretical framework for lattice models of histone-DNA interactions in chromatin and investigate the (competitive) DNA binding of other chromosomal proteins and transcription factors. The results have a number of applications for quantitative models for the regulation of gene expression.

  7. SH2 Domains Serve as Lipid-Binding Modules for pTyr-Signaling Proteins.

    Science.gov (United States)

    Park, Mi-Jeong; Sheng, Ren; Silkov, Antonina; Jung, Da-Jung; Wang, Zhi-Gang; Xin, Yao; Kim, Hyunjin; Thiagarajan-Rosenkranz, Pallavi; Song, Seohyeon; Yoon, Youngdae; Nam, Wonhee; Kim, Ilshin; Kim, Eui; Lee, Dong-Gyu; Chen, Yong; Singaram, Indira; Wang, Li; Jang, Myoung Ho; Hwang, Cheol-Sang; Honig, Barry; Ryu, Sungho; Lorieau, Justin; Kim, You-Me; Cho, Wonhwa

    2016-04-01

    The Src-homology 2 (SH2) domain is a protein interaction domain that directs myriad phosphotyrosine (pY)-signaling pathways. Genome-wide screening of human SH2 domains reveals that ∼90% of SH2 domains bind plasma membrane lipids and many have high phosphoinositide specificity. They bind lipids using surface cationic patches separate from pY-binding pockets, thus binding lipids and the pY motif independently. The patches form grooves for specific lipid headgroup recognition or flat surfaces for non-specific membrane binding and both types of interaction are important for cellular function and regulation of SH2 domain-containing proteins. Cellular studies with ZAP70 showed that multiple lipids bind its C-terminal SH2 domain in a spatiotemporally specific manner and thereby exert exquisite spatiotemporal control over its protein binding and signaling activities in T cells. Collectively, this study reveals how lipids control SH2 domain-mediated cellular protein-protein interaction networks and suggest a new strategy for therapeutic modulation of pY-signaling pathways. PMID:27052731

  8. PRELIMINARY STUDY OF EXTRACTABLE PROTEIN BINDING USING MALEIC ANHYDRIDE COPOLYMER

    Institute of Scientific and Technical Information of China (English)

    Thirawan Nipithakul; Ladawan Watthanachote; Nanticha Kalapat

    2012-01-01

    A preliminary study of using maleic anhydride copolymer for protein binding has been carried out.The polymeric films were prepared by compression of the purified resin and annealing the film to induce efficient back formation of the anhydride groups.The properties of the film surface were analyzed by attenuated total reflection Fourier transforms infrared spectroscopy and water contact angle measurements.The protein content was determined by Bradford assay.To obtain optimum conditions,immersion time for protein binding was examined.Results revealed that proteins can be successfully immobilized onto the film surface via covalent linkage.The efficiency of the covalent binding of the extractable protein to maleic anhydride-polyethylene film was estimated at 69.87 μtg/cm2,although the film had low anhydride content (3%) on the surface.

  9. Binding of CCAAT displacement protein CDP to adenovirus packaging sequences.

    Science.gov (United States)

    Erturk, Ece; Ostapchuk, Philomena; Wells, Susanne I; Yang, Jihong; Gregg, Keqin; Nepveu, Alain; Dudley, Jaquelin P; Hearing, Patrick

    2003-06-01

    Adenovirus (Ad) type 5 DNA packaging is initiated in a polar fashion from the left end of the genome. The packaging process is dependent upon the cis-acting packaging domain located between nucleotides 194 and 380. Seven A/T-rich repeats have been identified within this domain that direct packaging. A1, A2, A5, and A6 are the most important repeats functionally and share a bipartite sequence motif. Several lines of evidence suggest that there is a limiting trans-acting factor(s) that plays a role in packaging. Two cellular activities that bind to minimal packaging domains in vitro have been previously identified. These binding activities are P complex, an uncharacterized protein(s), and chicken ovalbumin upstream promoter transcription factor (COUP-TF). In this work, we report that a third cellular protein, octamer-1 protein (Oct-1), binds to minimal packaging domains. In vitro binding analyses and in vivo packaging assays were used to examine the relevance of these DNA binding activities to Ad DNA packaging. The results of these experiments reveal that COUP-TF and Oct-1 binding does not play a functional role in Ad packaging, whereas P-complex binding directly correlates with packaging function. We demonstrate that P complex contains the cellular protein CCAAT displacement protein (CDP) and that full-length CDP is found in purified virus particles. In addition to cellular factors, previous evidence indicates that viral factors play a role in the initiation of viral DNA packaging. We propose that CDP, in conjunction with one or more viral proteins, binds to the packaging sequences of Ad to initiate the encapsidation process. PMID:12743282

  10. Characterization of cap binding proteins associated with the nucleus

    International Nuclear Information System (INIS)

    Eucaryotic mRNAs a carry 7-methylguanosine triphosphate residue (called cap structure) at their 5' terminus. The cap plays an important role in RNA recognition. Cap binding proteins (CBP) of HeLa cells were identified by photoaffinity labelling using the cap analogue γ-(32P)-(4-(benzoyl-phenyl)methylamido)-7-methylguanosine-5'-triphosphate (BP-m7GTP). Photoreaction of this cap analogue with HeLa cell initiation factors resulted in specific labelling of two polypeptides of Msub(r) 37000 and 26000. The latter was also labelled in crude initiation factors prepared from reticulocytes and is identical to the cap binding protein CBP I previously identified. These cap binding proteins were also affinity labelled in poliovirus infected cell extracts. Photoaffinity reaction with BP-m7GTP of whole HeLa cell homogenate showed three additional polypeptides with Msub(r) 120000, 89000 and 80000. These cap binding proteins were found to be associated with the nucleus and are therefore referred to as nuclear cap binding proteins, i.e. NCBP 1, NCBP 2 and NCBP 3. They were also present in splicing extracts. Photoaffinity labelling in these nuclear extracts was differentially inhibited by various cap analogues and capped mRNAs. Affinity chromatography on immobilized globin mRNA led to a partial separation of the three nuclear cap binding proteins. Chromatography on m7GTP-Sepharose resulted in a specific binding of NCBP 3. The different behaviour of the cap binding proteins suggests that they are functionally distinct and that they might be involved in different processes requiring cap recognition. (Author)

  11. High-throughput analysis of protein-DNA binding affinity.

    Science.gov (United States)

    Franco-Zorrilla, José M; Solano, Roberto

    2014-01-01

    Sequence-specific protein-DNA interactions mediate most regulatory processes underlying gene expression, such as transcriptional regulation by transcription factors (TFs) or chromatin organization. Current knowledge about DNA-binding specificities of TFs is based mostly on low- to medium-throughput methodologies that are time-consuming and often fail to identify DNA motifs recognized by a TF with lower affinity but retaining biological relevance. The use of protein-binding microarrays (PBMs) offers a high-throughput alternative for the identification of protein-DNA specificities. PBM consists in an array of pseudorandomized DNA sequences that are optimized to include all the possible 10- or 11-mer DNA sequences, allowing the determination of binding specificities of most eukaryotic TFs. PBMs that can be synthesized by several manufacturing companies as single-stranded DNA are converted into double-stranded in a simple primer extension reaction. The protein of interest fused to an epitope tag is then incubated onto the PBM, and specific DNA-protein complexes are revealed in a series of immunological reactions coupled to a fluorophore. After scanning and quantifying PBMs, specific DNA motifs recognized by the protein are identified with ready-to-use scripts, generating comprehensive but accessible information about the DNA-binding specificity of the protein. This chapter describes detailed procedures for preparation of double-stranded PBMs, incubation with recombinant protein, and detection of protein-DNA complexes. Finally, we outline some cues for evaluating the biological role of DNA motifs obtained in vitro. PMID:24057393

  12. High-Fidelity DNA Sensing by Protein Binding Fluctuations

    CERN Document Server

    Tlusty, Tsvi; Libchaber, Albert; 10.1103/PhysRevLett.93.258103

    2010-01-01

    One of the major functions of RecA protein in the cell is to bind single-stranded DNA exposed upon damage, thereby triggering the SOS repair response.We present fluorescence anisotropy measurements at the binding onset, showing enhanced DNA length discrimination induced by adenosine triphosphate consumption. Our model explains the observed DNA length sensing as an outcome of out-of equilibrium binding fluctuations, reminiscent of microtubule dynamic instability. The cascade architecture of the binding fluctuations is a generalization of the kinetic proofreading mechanism. Enhancement of precision by an irreversible multistage pathway is a possible design principle in the noisy biological environment.

  13. Ubiquitin-binding proteins: similar, but different

    DEFF Research Database (Denmark)

    Andersen, Katrine M; Hofmann, Kay; Hartmann-Petersen, Rasmus

    2005-01-01

    ubiquitin conjugation to endoplasmic reticulum degradation), UEV [ubiquitin E2 (ubiquitin-conjugating enzyme) variant] and NZF (nuclear protein localization gene 4 zinc finger) domain-containing proteins appear to have more specialized functions. Here we discuss functional and structural properties of...

  14. Pulmonary surfactant protein A (SP-A) specifically binds dipalmitoylphosphatidylcholine

    International Nuclear Information System (INIS)

    Phospholipids are the major components of pulmonary surfactant. Dipalmitoylphosphatidylcholine is believed to be especially essential for the surfactant function of reducing the surface tension at the air-liquid interface. Surfactant protein A (SP-A) with a reduced denatured molecular mass of 26-38 kDa, characterized by a collagen-like structure and N-linked glycosylation, interacts strongly with a mixture of surfactant-like phospholipids. In the present study the direct binding of SP-A to phospholipids on a thin layer chromatogram was visualized using 125I-SP-A as a probe, so that the phospholipid specificities of SP-A binding and the structural requirements of SP-A and phospholipids for the binding could be examined. Although 125I-SP-A bound phosphatidylcholine and sphingomyeline, it was especially strong in binding dipalmitoylphosphatidylcholine, but failed to bind phosphatidylglycerol, phosphatidylinositol, phosphatidylethanolamine, and phosphatidylserine. Labeled SP-A also exhibited strong binding to distearoylphosphatidylcholine, but weak binding to dimyristoyl-, 1-palmitoyl-2-linoleoyl-, and dilinoleoylphosphatidylcholine. Unlabeled SP-A readily competed with labeled SP-A for phospholipid binding. SP-A strongly bound dipalmitoylglycerol produced by phospholipase C treatment of dipalmitoylphosphatidylcholine, but not palmitic acid. This protein also failed to bind lysophosphatidylcholine produced by phospholipase A2 treatment of dipalmitoylphosphatidylcholine. 125I-SP-A shows almost no binding to dipalmitoylphosphatidylglycerol and dipalmitoylphosphatidylethanolamine. The addition of 10 mM EGTA into the binding buffer reduced much of the 125I-SP-A binding to phospholipids. Excess deglycosylated SP-A competed with labeled SP-A for binding to dipalmitoylphosphatidylcholine, but the excess collagenase-resistant fragment of SP-A failed

  15. Lipid A binding proteins in macrophages detected by ligand blotting

    International Nuclear Information System (INIS)

    Endotoxin (LPS) stimulates a variety of eukaryotic cells. These actions are involved in the pathogenesis of Gram-negative septicemia. The site of action of the LPS toxic moiety, lipid A (LA), is unclear. Their laboratory has previously identified a bioactive LA precursor lipid IV/sub A/, which can be enzymatically labeled with 32P/sub i/ (109 dpm/nmole) and purified (99%). They now show that this ligand binds to specific proteins immobilized on nitrocellulose (NC) from LPS-sensitive RAW 264.7 cultured macrophages. NC blots were incubated with [32P]-IV/sub A/ in a buffer containing BSA, NaCl, polyethylene glycol, and azide. Binding was assessed using autoradiography or scintillation counting. Dot blot binding of the radioligand was inhibited by excess cold IV/sub A/, LA, or ReLPS but not by phosphatidylcholine, cardiolipin, phosphatidylinositol, or phosphatidic acid. Binding was trypsin-sensitive and dependent on protein concentration. Particulate macrophage proteins were subjected to SDS-PAGE and then electroblotted onto NC. Several discrete binding proteins were observed. Identical treatment of fetal bovine serum or molecular weight standards revealed no detectable binding. By avoiding high nonspecific binding of intact membranes, this ligand blotting assay may be useful in elucidating the molecular actions of LPS

  16. Mining the characteristic interaction patterns on protein-protein binding interfaces.

    Science.gov (United States)

    Li, Yan; Liu, Zhihai; Han, Li; Li, Chengke; Wang, Renxiao

    2013-09-23

    Protein-protein interactions are observed in various biological processes. They are important for understanding the underlying molecular mechanisms and can be potential targets for developing small-molecule regulators of such processes. Previous studies suggest that certain residues on protein-protein binding interfaces are "hot spots". As an extension to this concept, we have developed a residue-based method to identify the characteristic interaction patterns (CIPs) on protein-protein binding interfaces, in which each pattern is a cluster of four contacting residues. Systematic analysis was conducted on a nonredundant set of 1,222 protein-protein binding interfaces selected out of the entire Protein Data Bank. Favored interaction patterns across different protein-protein binding interfaces were retrieved by considering both geometrical and chemical conservations. As demonstrated on two test tests, our method was able to predict hot spot residues on protein-protein binding interfaces with good recall scores and acceptable precision scores. By analyzing the function annotations and the evolutionary tree of the protein-protein complexes in our data set, we also observed that protein-protein interfaces sharing common characteristic interaction patterns are normally associated with identical or similar biological functions. PMID:23930922

  17. The cobalamin-binding protein in zebrafish is an intermediate between the three cobalamin-binding proteins in human.

    Directory of Open Access Journals (Sweden)

    Eva Greibe

    Full Text Available In humans, three soluble extracellular cobalamin-binding proteins; transcobalamin (TC, intrinsic factor (IF, and haptocorrin (HC, are involved in the uptake and transport of cobalamin. In this study, we investigate a cobalamin-binding protein from zebrafish (Danio rerio and summarize current knowledge concerning the phylogenetic evolution of kindred proteins. We identified a cobalamin binding capacity in zebrafish protein extracts (8.2 pmol/fish and ambient water (13.5 pmol/fish associated with a single protein. The protein showed resistance toward degradation by trypsin and chymotrypsin (like human IF, but unlike human HC and TC. The cobalamin analogue, cobinamide, bound weaker to the zebrafish cobalamin binder than to human HC, but stronger than to human TC and IF. Affinity for another analogue, adenosyl-pseudo-cobalamin was low compared with human HC and TC, but high compared with human IF. The absorbance spectrum of the purified protein in complex with hydroxo-cobalamin resembled those of human HC and IF, but not TC. We searched available databases to further explore the phylogenies of the three cobalamin-binding proteins in higher vertebrates. Apparently, TC-like proteins are the oldest evolutionary derivatives followed by IF and HC (the latter being present only in reptiles and most but not all mammals. Our findings suggest that the only cobalamin-binding protein in zebrafish is an intermediate between the three human cobalamin binders. These findings support the hypothesis about a common ancestral gene for all cobalamin-binding proteins in higher vertebrates.

  18. Analysis of the ligand binding properties of recombinant bovine liver-type fatty acid binding protein

    DEFF Research Database (Denmark)

    Rolf, B; Oudenampsen-Krüger, E; Börchers, T;

    1995-01-01

    The coding part of the cDNA for bovine liver-type fatty acid binding protein (L-FABP) has been amplified by RT-PCR, cloned and used for the construction of an Escherichia coli (E. coli) expression system. The recombinant protein made up to 25% of the soluble E. coli proteins and could be isolated...... by a simple two step protocol combining ion exchange chromatography and gel filtration. Dissociation constants for binding of oleic acid, arachidonic acid, oleoyl-CoA, lysophosphatidic acid and the peroxisomal proliferator bezafibrate to L-FABP have been determined by titration calorimetry. All ligands were...... bound in a 2:1 stoichiometry, the dissociation constants for the first ligand bound were all in the micro molar range. Oleic acid was bound with the highest affinity and a Kd of 0.26 microM. Furthermore, binding of cholesterol to L-FABP was investigated with the Lipidex assay, a liposome binding assay...

  19. Glycosylation status of vitamin D binding protein in cancer patients.

    Science.gov (United States)

    Rehder, Douglas S; Nelson, Randall W; Borges, Chad R

    2009-10-01

    On the basis of the results of activity studies, previous reports have suggested that vitamin D binding protein (DBP) is significantly or even completely deglycosylated in cancer patients, eliminating the molecular precursor of the immunologically important Gc macrophage activating factor (GcMAF), a glycosidase-derived product of DBP. The purpose of this investigation was to directly determine the relative degree of O-linked trisaccharide glycosylation of serum-derived DBP in human breast, colorectal, pancreatic, and prostate cancer patients. Results obtained by electrospray ionization-based mass spectrometric immunoassay showed that there was no significant depletion of DBP trisaccharide glycosylation in the 56 cancer patients examined relative to healthy controls. These results suggest that alternative hypotheses regarding the molecular and/or structural origins of GcMAF must be considered to explain the relative inability of cancer patient serum to activate macrophages. PMID:19642159

  20. Rapid identification of DNA-binding proteins by mass spectrometry

    DEFF Research Database (Denmark)

    Nordhoff, E; Krogsdam, A M; Jorgensen, H F;

    1999-01-01

    We report a protocol for the rapid identification of DNA-binding proteins. Immobilized DNA probes harboring a specific sequence motif are incubated with cell or nuclear extract. Proteins are analyzed directly off the solid support by matrix-assisted laser desorption/ionization time-of-flight mass...

  1. Solid-binding Proteins for Modification of Inorganic Substrates

    Science.gov (United States)

    Coyle, Brandon Laurence

    Robust and simple strategies to directly functionalize graphene- and diamond-based nanostructures with proteins are of considerable interest for biologically driven manufacturing, biosensing and bioimaging. In this work, we identify a new set of carbon binding peptides that vary in overall hydrophobicity and charge, and engineer two of these sequences (Car9 and Car15) within the framework of various proteins to exploit their binding ability. In addition, we conducted a detailed analysis of the mechanisms that underpin the interaction of the fusion proteins with carbon and silicon surfaces. Through these insights, we were able to develop proteins suitable for dispersing graphene flakes and carbon nanotubes in aqueous solutions, while retaining protein activity. Additionally, our investigation into the mechanisms of adhesion for our carbon binding peptides inspired a cheap, disposable protein purification system that is more than 10x cheaper than commonly used His-tag protein purification. Our results emphasize the importance of understanding both bulk and molecular recognition events when exploiting the adhesive properties of solid-binding peptides and proteins in technological applications.

  2. Zeatin-binding proteins in barley leaves

    International Nuclear Information System (INIS)

    Highly labelled tritium-zeatin was used in the work to clarify for the first time a protein factor that is present in cytokinin-sensitive vegetative organs of plants (barley leaves) and which possesses the properties of a cytokinin receptor. Aliquots of tritium-zeatin were mixed with a solution of protein and incubated for several hours in buffer. Following incubation, protein was precipitated by ammonium sulfate at 90% of saturation, and radioactivity of the precipitate was checked in a dioxane scintillator with an efficiency of about 35%. It is shown that the characteristics of interaction of the clarified specific protein sites with cytokinins in regard to a number of criteria correspond to the characteristics expected of receptors of these phytohormones

  3. Zeatin-binding proteins in barley leaves

    Energy Technology Data Exchange (ETDEWEB)

    Romanov, G.A.; Kulaeva, O.N.; Taryan, V.Y.

    1986-01-01

    Highly labelled tritium-zeatin was used in the work to clarify for the first time a protein factor that is present in cytokinin-sensitive vegetative organs of plants (barley leaves) and which possesses the properties of a cytokinin receptor. Aliquots of tritium-zeatin were mixed with a solution of protein and incubated for several hours in buffer. Following incubation, protein was precipitated by ammonium sulfate at 90% of saturation, and radioactivity of the precipitate was checked in a dioxane scintillator with an efficiency of about 35%. It is shown that the characteristics of interaction of the clarified specific protein sites with cytokinins in regard to a number of criteria correspond to the characteristics expected of receptors of these phytohormones.

  4. Relating the shape of protein binding sites to binding affinity profiles: is there an association?

    Directory of Open Access Journals (Sweden)

    Bitter István

    2010-10-01

    Full Text Available Abstract Background Various pattern-based methods exist that use in vitro or in silico affinity profiles for classification and functional examination of proteins. Nevertheless, the connection between the protein affinity profiles and the structural characteristics of the binding sites is still unclear. Our aim was to investigate the association between virtual drug screening results (calculated binding free energy values and the geometry of protein binding sites. Molecular Affinity Fingerprints (MAFs were determined for 154 proteins based on their molecular docking energy results for 1,255 FDA-approved drugs. Protein binding site geometries were characterized by 420 PocketPicker descriptors. The basic underlying component structure of MAFs and binding site geometries, respectively, were examined by principal component analysis; association between principal components extracted from these two sets of variables was then investigated by canonical correlation and redundancy analyses. Results PCA analysis of the MAF variables provided 30 factors which explained 71.4% of the total variance of the energy values while 13 factors were obtained from the PocketPicker descriptors which cumulatively explained 94.1% of the total variance. Canonical correlation analysis resulted in 3 statistically significant canonical factor pairs with correlation values of 0.87, 0.84 and 0.77, respectively. Redundancy analysis indicated that PocketPicker descriptor factors explain 6.9% of the variance of the MAF factor set while MAF factors explain 15.9% of the total variance of PocketPicker descriptor factors. Based on the salient structures of the factor pairs, we identified a clear-cut association between the shape and bulkiness of the drug molecules and the protein binding site descriptors. Conclusions This is the first study to investigate complex multivariate associations between affinity profiles and the geometric properties of protein binding sites. We found that

  5. Theoretical studies of binding of mannose-binding protein to monosaccharides

    Science.gov (United States)

    Aida-Hyugaji, Sachiko; Takano, Keiko; Takada, Toshikazu; Hosoya, Haruo; Kojima, Naoya; Mizuochi, Tsuguo; Inoue, Yasushi

    2004-11-01

    Binding properties of mannose-binding protein (MBP) to monosaccharides are discussed based on ab initio molecular orbital calculations for cluster models constructed. The calculated binding energies indicate that MBP has an affinity for N-acetyl- D-glucosamine, D-mannose, L-fucose, and D-glucose rather than D-galactose and N-acetyl- D-galactosamine, which is consistent with the biochemical experimental results. Electrostatic potential surfaces at the binding site of four monosaccharides having binding properties matched well with that of MBP. A vacant frontier orbital was found to be localized around the binding site of MBP, suggesting that MBP-monosaccharide interaction may occur through electrostatic and orbital interactions.

  6. Cholesterol-binding viral proteins in virus entry and morphogenesis.

    Science.gov (United States)

    Schroeder, Cornelia

    2010-01-01

    Up to now less than a handful of viral cholesterol-binding proteins have been characterized, in HIV, influenza virus and Semliki Forest virus. These are proteins with roles in virus entry or morphogenesis. In the case of the HIV fusion protein gp41 cholesterol binding is attributed to a cholesterol recognition consensus (CRAC) motif in a flexible domain of the ectodomain preceding the trans-membrane segment. This specific CRAC sequence mediates gp41 binding to a cholesterol affinity column. Mutations in this motif arrest virus fusion at the hemifusion stage and modify the ability of the isolated CRAC peptide to induce segregation of cholesterol in artificial membranes.Influenza A virus M2 protein co-purifies with cholesterol. Its proton translocation activity, responsible for virus uncoating, is not cholesterol-dependent, and the transmembrane channel appears too short for integral raft insertion. Cholesterol binding may be mediated by CRAC motifs in the flexible post-TM domain, which harbours three determinants of binding to membrane rafts. Mutation of the CRAC motif of the WSN strain attenuates virulence for mice. Its affinity to the raft-non-raft interface is predicted to target M2 protein to the periphery of lipid raft microdomains, the sites of virus assembly. Its influence on the morphology of budding virus implicates M2 as factor in virus fission at the raft boundary. Moreover, M2 is an essential factor in sorting the segmented genome into virus particles, indicating that M2 also has a role in priming the outgrowth of virus buds.SFV E1 protein is the first viral type-II fusion protein demonstrated to directly bind cholesterol when the fusion peptide loop locks into the target membrane. Cholesterol binding is modulated by another, proximal loop, which is also important during virus budding and as a host range determinant, as shown by mutational studies. PMID:20213541

  7. Detergent activation of the binding protein in the folate radioassay

    International Nuclear Information System (INIS)

    A minor cow's whey protein associated with β-lactoglobulin is used as binding protein in the competitive radioassay for serum and erythrocyte folate. Seeking to optimize the assay, we tested the performance of binder solutions of increasing purity. The folate binding protein was isolated from cow's whey by means of CM-Sepharose CL-6B cation-exchange chromatography, and further purified on a methotrexate-AH-Sepharose 4B affinity matrix. In contrast to β-lactoglobulin, the purified protein did not bind folate unless the detergents cetyltrimethylammonium (10 mmol/Ll) or Triton X-100 (1 g/L) were present. Such detergent activation was not needed in the presence of serum. There seems to be a striking analogy between these phenomena and the well-known reactivation of certain purified membrane-derived enzymes by surfactants

  8. Drug Promiscuity in PDB: Protein Binding Site Similarity Is Key.

    Directory of Open Access Journals (Sweden)

    V Joachim Haupt

    Full Text Available Drug repositioning applies established drugs to new disease indications with increasing success. A pre-requisite for drug repurposing is drug promiscuity (polypharmacology - a drug's ability to bind to several targets. There is a long standing debate on the reasons for drug promiscuity. Based on large compound screens, hydrophobicity and molecular weight have been suggested as key reasons. However, the results are sometimes contradictory and leave space for further analysis. Protein structures offer a structural dimension to explain promiscuity: Can a drug bind multiple targets because the drug is flexible or because the targets are structurally similar or even share similar binding sites? We present a systematic study of drug promiscuity based on structural data of PDB target proteins with a set of 164 promiscuous drugs. We show that there is no correlation between the degree of promiscuity and ligand properties such as hydrophobicity or molecular weight but a weak correlation to conformational flexibility. However, we do find a correlation between promiscuity and structural similarity as well as binding site similarity of protein targets. In particular, 71% of the drugs have at least two targets with similar binding sites. In order to overcome issues in detection of remotely similar binding sites, we employed a score for binding site similarity: LigandRMSD measures the similarity of the aligned ligands and uncovers remote local similarities in proteins. It can be applied to arbitrary structural binding site alignments. Three representative examples, namely the anti-cancer drug methotrexate, the natural product quercetin and the anti-diabetic drug acarbose are discussed in detail. Our findings suggest that global structural and binding site similarity play a more important role to explain the observed drug promiscuity in the PDB than physicochemical drug properties like hydrophobicity or molecular weight. Additionally, we find ligand

  9. Structural Basis for Antagonism by Suramin of Heparin Binding to Vaccinia Complement Protein

    Energy Technology Data Exchange (ETDEWEB)

    Ganesh, Vannakambadi K.; Muthuvel, Suresh Kumar; Smith, Scott A.; Kotwal, Girish J.; Murthy, Krishna H.M. (U. of Cape Town); (UAB); (U. of Louisville)

    2010-07-19

    Suramin is a competitive inhibitor of heparin binding to many proteins, including viral envelope proteins, protein tyrosine phosphatases, and fibroblast growth factors (FGFs). It has been clinically evaluated as a potential therapeutic in treatment of cancers caused by unregulated angiogenesis, triggered by FGFs. Although it has shown clinical promise in treatment of several cancers, suramin has many undesirable side effects. There is currently no experimental structure that reveals the molecular interactions responsible for suramin inhibition of heparin binding, which could be of potential use in structure-assisted design of improved analogues of suramin. We report the structure of suramin, in complex with the heparin-binding site of vaccinia virus complement control protein (VCP), which interacts with heparin in a geometrically similar manner to many FGFs. The larger than anticipated flexibility of suramin manifested in this structure, and other details of VCP-suramin interactions, might provide useful structural information for interpreting interactions of suramin with many proteins.

  10. Protein-protein binding affinities calculated using the LIE method

    OpenAIRE

    Andberg, Tor Arne Heim

    2011-01-01

    Absolute binding free energies for the third domain of the turkey ovomucoid inhibitor in complex with Streptomyces griseus proteinase B and porcine pancreatic elastase has been calculated using the linear interaction energy method.

  11. The Role Stress Granules and RNA Binding Proteins in Neurodegeneration

    OpenAIRE

    Vanderweyde, Tara; Youmans, Katie; Liu-Yesucevitz, Liqun; Wolozin, Benjamin

    2013-01-01

    The eukaryotic stress response involves translational suppression of non-housekeeping proteins and the sequestration of unnecessary mRNA transcripts into stress granules (SGs). This process is dependent on mRNA binding proteins (RBPs) that interact with capped mRNA transcripts through RNA recognition motifs, and exhibit reversible aggregation through hydrophobic poly-glycine domains, some of which are homologous to yeast prion proteins. The activity and aggregation of RBPs appears to be impor...

  12. Convolutional neural network architectures for predicting DNA–protein binding

    Science.gov (United States)

    Zeng, Haoyang; Edwards, Matthew D.; Liu, Ge; Gifford, David K.

    2016-01-01

    Motivation: Convolutional neural networks (CNN) have outperformed conventional methods in modeling the sequence specificity of DNA–protein binding. Yet inappropriate CNN architectures can yield poorer performance than simpler models. Thus an in-depth understanding of how to match CNN architecture to a given task is needed to fully harness the power of CNNs for computational biology applications. Results: We present a systematic exploration of CNN architectures for predicting DNA sequence binding using a large compendium of transcription factor datasets. We identify the best-performing architectures by varying CNN width, depth and pooling designs. We find that adding convolutional kernels to a network is important for motif-based tasks. We show the benefits of CNNs in learning rich higher-order sequence features, such as secondary motifs and local sequence context, by comparing network performance on multiple modeling tasks ranging in difficulty. We also demonstrate how careful construction of sequence benchmark datasets, using approaches that control potentially confounding effects like positional or motif strength bias, is critical in making fair comparisons between competing methods. We explore how to establish the sufficiency of training data for these learning tasks, and we have created a flexible cloud-based framework that permits the rapid exploration of alternative neural network architectures for problems in computational biology. Availability and Implementation: All the models analyzed are available at http://cnn.csail.mit.edu. Contact: gifford@mit.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:27307608

  13. Profiling Protein Kinases and Other ATP Binding Proteins in Arabidopsis Using Acyl-ATP Probes*

    OpenAIRE

    Villamor, J. G.; Kaschani, F.; Colby, T; Oeljeklaus, J.; Zhao, D; Kaiser, M.; Patricelli, M. P.; R. A. L. van der Hoorn

    2013-01-01

    Many protein activities are driven by ATP binding and hydrolysis. Here, we explore the ATP binding proteome of the model plant Arabidopsis thaliana using acyl-ATP (AcATP)1 probes. These probes target ATP binding sites and covalently label lysine residues in the ATP binding pocket. Gel-based profiling using biotinylated AcATP showed that labeling is dependent on pH and divalent ions and can be competed by nucleotides. The vast majority of these AcATP-labeled proteins are known ATP binding prot...

  14. Natural history of S-adenosylmethionine-binding proteins

    Directory of Open Access Journals (Sweden)

    Mushegian Arcady R

    2005-10-01

    Full Text Available Abstract Background S-adenosylmethionine is a source of diverse chemical groups used in biosynthesis and modification of virtually every class of biomolecules. The most notable reaction requiring S-adenosylmethionine, transfer of methyl group, is performed by a large class of enzymes, S-adenosylmethionine-dependent methyltransferases, which have been the focus of considerable structure-function studies. Evolutionary trajectories of these enzymes, and especially of other classes of S-adenosylmethionine-binding proteins, nevertheless, remain poorly understood. We addressed this issue by computational comparison of sequences and structures of various S-adenosylmethionine-binding proteins. Results Two widespread folds, Rossmann fold and TIM barrel, have been repeatedly used in evolution for diverse types of S-adenosylmethionine conversion. There were also cases of recruitment of other relatively common folds for S-adenosylmethionine binding. Several classes of proteins have unique unrelated folds, specialized for just one type of chemistry and unified by the theme of internal domain duplications. In several cases, functional divergence is evident, when evolutionarily related enzymes have changed the mode of binding and the type of chemical transformation of S-adenosylmethionine. There are also instances of functional convergence, when biochemically similar processes are performed by drastically different classes of S-adenosylmethionine-binding proteins. Comparison of remote sequence similarities and analysis of phyletic patterns suggests that the last universal common ancestor of cellular life had between 10 and 20 S-adenosylmethionine-binding proteins from at least 5 fold classes, providing for S-adenosylmethionine formation, polyamine biosynthesis, and methylation of several substrates, including nucleic acids and peptide chain release factor. Conclusion We have observed several novel relationships between families that were not known to be

  15. Quantitative analysis of EGR proteins binding to DNA: assessing additivity in both the binding site and the protein

    Directory of Open Access Journals (Sweden)

    Stormo Gary D

    2005-07-01

    Full Text Available Abstract Background Recognition codes for protein-DNA interactions typically assume that the interacting positions contribute additively to the binding energy. While this is known to not be precisely true, an additive model over the DNA positions can be a good approximation, at least for some proteins. Much less information is available about whether the protein positions contribute additively to the interaction. Results Using EGR zinc finger proteins, we measure the binding affinity of six different variants of the protein to each of six different variants of the consensus binding site. Both the protein and binding site variants include single and double mutations that allow us to assess how well additive models can account for the data. For each protein and DNA alone we find that additive models are good approximations, but over the combined set of data there are context effects that limit their accuracy. However, a small modification to the purely additive model, with only three additional parameters, improves the fit significantly. Conclusion The additive model holds very well for every DNA site and every protein included in this study, but clear context dependence in the interactions was detected. A simple modification to the independent model provides a better fit to the complete data.

  16. Thermodynamics of tryptophan-mediated activation of the trp RNA-binding attenuation protein.

    Science.gov (United States)

    McElroy, Craig A; Manfredo, Amanda; Gollnick, Paul; Foster, Mark P

    2006-06-27

    The trp RNA-binding attenuation protein (TRAP) functions in many bacilli to control the expression of the tryptophan biosynthesis genes. Transcription of the trp operon is controlled by TRAP through an attenuation mechanism, in which competition between two alternative secondary-structural elements in the 5' leader sequence of the nascent mRNA is influenced by tryptophan-dependent binding of TRAP to the RNA. Previously, NMR studies of the undecamer (11-mer) suggested that tryptophan-dependent control of RNA binding by TRAP is accomplished through ligand-induced changes in protein dynamics. We now present further insights into this ligand-coupled event from hydrogen/deuterium (H/D) exchange analysis, differential scanning calorimetry (DSC), and isothermal titration calorimetry (ITC). Scanning calorimetry showed tryptophan dissociation to be independent of global protein unfolding, while analysis of the temperature dependence of the binding enthalpy by ITC revealed a negative heat capacity change larger than expected from surface burial, a hallmark of binding-coupled processes. Analysis of this excess heat capacity change using parameters derived from protein folding studies corresponds to the ordering of 17-24 residues per monomer of TRAP upon tryptophan binding. This result is in agreement with qualitative analysis of residue-specific broadening observed in TROSY NMR spectra of the 91 kDa oligomer. Implications for the mechanism of ligand-mediated TRAP activation through a shift in a preexisting conformational equilibrium and an induced-fit conformational change are discussed. PMID:16784236

  17. Identification and characterization of the RNA binding surface of the pentatricopeptide repeat protein

    OpenAIRE

    Kobayashi, Keiko; Kawabata, Masuyo; Hisano, Keizo; Kazama, Tomohiko; Matsuoka, Ken; Sugita, Mamoru; Nakamura, Takahiro

    2011-01-01

    The expressions of chloroplast and mitochondria genes are tightly controlled by numerous nuclear-encoded proteins, mainly at the post-transcriptional level. Recent analyses have identified a large, plant-specific family of pentatricopeptide repeat (PPR) motif-containing proteins that are exclusively involved in RNA metabolism of organelle genes via sequence-specific RNA binding. A tandem array of PPR motifs within the protein is believed to facilitate the RNA interaction, although little is k...

  18. Increased cortical expression of FK506 binding protein-51 in HIV-associated neurocognitive disorders

    OpenAIRE

    Soontornniyomkij, Virawudh; Everall, Ian P.; Moore, David J.; Gouaux, Ben; Tatro, Erick T.; Gospodarev, Vadim; Masliah, Eliezer; Yin, Nicole S.; Vinters, Harry V.; Achim, Cristian L.

    2012-01-01

    FK506 binding protein (FKBP)-51 and FKBP52 act as molecular chaperones to control glucocorticoid receptor (GR) sensitivity. Dysregulation of proteins involved in GR-mediated signaling can lead to maladaptive stress response and aging-related cognitive decline. As HIV infection is related to chronic stress, we hypothesized that altered cortical expression of these proteins was associated with HIV-associated neurocognitive disorders (HAND). We used quantitative immunohistochemistry to assess ex...

  19. A Genetic Screen Identifies Putative Targets and Binding Partners of CREB-Binding Protein in the Developing Drosophila Eye

    OpenAIRE

    Anderson, Jason; Bhandari, Rohan; Kumar, Justin P.

    2005-01-01

    Drosophila CREB-binding protein (dCBP) is a very large multidomain protein, which belongs to the CBP/p300 family of proteins that were first identified by their ability to bind the CREB transcription factor and the adenoviral protein E1. Since then CBP has been shown to bind to >100 additional proteins and functions in a multitude of different developmental contexts. Among other activities, CBP is known to influence development by remodeling chromatin, by serving as a transcriptional coactiva...

  20. Tetrapyrrole binding affinity of the murine and human p22HBP heme-binding proteins.

    Science.gov (United States)

    Micaelo, Nuno M; Macedo, Anjos L; Goodfellow, Brian J; Félix, Vítor

    2010-11-01

    We present the first systematic molecular modeling study of the binding properties of murine (mHBP) and human (hHBP) p22HBP protein (heme-binding protein) with four tetrapyrrole ring systems belonging to the heme biosynthetic pathway: iron protoporphyrin IX (HEMIN), protoporphyrin IX (PPIX), coproporphyrin III (CPIII), coproporphyrin I (CPI). The relative binding affinities predicted by our computational study were found to be similar to those observed experimentally, providing a first rational structural analysis of the molecular recognition mechanism, by p22HBP, toward a number of different tetrapyrrole ligands. To probe the structure of these p22HBP protein complexes, docking, molecular dynamics and MM-PBSA methodologies supported by experimental NMR ring current shift data have been employed. The tetrapyrroles studied were found to bind murine p22HBP with the following binding affinity order: HEMIN> PPIX> CPIII> CPI, which ranged from -22.2 to -6.1 kcal/mol. In general, the protein-tetrapyrrole complexes are stabilized by non-bonded interactions between the tetrapyrrole propionate groups and basic residues of the protein, and by the preferential solvation of the complex compared to the unbound components. PMID:20800521

  1. A poxvirus protein that binds to and inactivates IL-18, and inhibits NK cell response.

    Science.gov (United States)

    Born, T L; Morrison, L A; Esteban, D J; VandenBos, T; Thebeau, L G; Chen, N; Spriggs, M K; Sims, J E; Buller, R M

    2000-03-15

    IL-18 induces IFN-gamma and NK cell cytotoxicity, making it a logical target for viral antagonism of host defense. We demonstrate that the ectromelia poxvirus p13 protein, bearing homology to the mammalian IL-18 binding protein, binds IL-18, and inhibits its activity in vitro. Binding of IL-18 to the viral p13 protein was compared with binding to the cellular IL-18R. The dissociation constant of p13 for murine IL-18 is 5 nM, compared with 0.2 nM for the cellular receptor heterodimer. Mice infected with a p13 deletion mutant of ectromelia virus had elevated cytotoxicity for YAC-1 tumor cell targets compared with control animals. Additionally, the p13 deletion mutant virus exhibited decreased levels of infectivity. Our data suggest that inactivation of IL-18, and subsequent impairment of NK cell cytotoxicity, may be one mechanism by which ectromelia evades the host immune response. PMID:10706717

  2. Characterization of adenosine binding proteins in human placental membranes

    International Nuclear Information System (INIS)

    We have characterized two adenosine binding proteins in human placenta. In membranes, one site is detected with [3H] -N-ethylcarboxamidoadenosine ([3H]NECA). This site is similar to the adenosine A2 receptor. We call this site the adenosine A2-like binding site. In detergent extracts, the second site is detected and has the characteristics of an adenosine A1 receptor. The soluble adenosine A2-like binding site cannot be detected without a rapid assay. Binding to the adenosine A1 receptor with [3H]-2-chloroadenosine and [3H]NECA is time dependent, saturable, and reversible. Equilibrium displacement analysis with adenosine agonists reveals an A1 specificity: 2-chloroadenosine > R-phenylisopropyladenosine > 5'-N-ethylcarboxamidoadenosine. The antagonist potency order is 1,3-diethyl-8-phenylxanthine > isobutylmethylxanthine > theophylline. Competition analysis of membranes with the A,-selective ligands [3H]-cyclohexyladenosine [3H] cylopentylxanthine revealed adenosine A1 agonist and antagonist potency orders. We have purified the adenosine A2-like binding site. The adenosine A2-like binding site is an ubiquitous major cellular protein. It is glycosylated, highly asymmetric, and acidic. The native protein is an homodimer with a subunit molecular mass of 98 kDa. The sedimentation coefficient and partial specific volume of the binding complex are 6.9 s and 0.698 ml/g, respectively. The Stokes' radius is 70 Angstrom. The native molecular mass of the detergent-protein complex is 230 kDa. The adenosine A2-like binding site has an agonist potency order of 5'-N-ethylcarboxamidoadenosine > 2-chloroadenosine >> R-phenylisopropyladenosine and an antagonist potency order of isobutylmethylxanthine > theophylline >> 1,3-diethyl-8-phenylxanthine

  3. Characterization of adenosine binding proteins in human placental membranes

    Energy Technology Data Exchange (ETDEWEB)

    Hutchison, K.A.

    1989-01-01

    We have characterized two adenosine binding proteins in human placenta. In membranes, one site is detected with ({sup 3}H) -N-ethylcarboxamidoadenosine (({sup 3}H)NECA). This site is similar to the adenosine A{sub 2} receptor. We call this site the adenosine A{sub 2}-like binding site. In detergent extracts, the second site is detected and has the characteristics of an adenosine A{sub 1} receptor. The soluble adenosine A{sub 2}-like binding site cannot be detected without a rapid assay. Binding to the adenosine A{sub 1} receptor with ({sup 3}H)-2-chloroadenosine and ({sup 3}H)NECA is time dependent, saturable, and reversible. Equilibrium displacement analysis with adenosine agonists reveals an A{sub 1} specificity: 2-chloroadenosine > R-phenylisopropyladenosine > 5{prime}-N-ethylcarboxamidoadenosine. The antagonist potency order is 1,3-diethyl-8-phenylxanthine > isobutylmethylxanthine > theophylline. Competition analysis of membranes with the A,-selective ligands ({sup 3}H)-cyclohexyladenosine ({sup 3}H) cylopentylxanthine revealed adenosine A{sub 1} agonist and antagonist potency orders. We have purified the adenosine A{sub 2}-like binding site. The adenosine A{sub 2}-like binding site is an ubiquitous major cellular protein. It is glycosylated, highly asymmetric, and acidic. The native protein is an homodimer with a subunit molecular mass of 98 kDa. The sedimentation coefficient and partial specific volume of the binding complex are 6.9 s and 0.698 ml/g, respectively. The Stokes' radius is 70 {Angstrom}. The native molecular mass of the detergent-protein complex is 230 kDa. The adenosine A{sub 2}-like binding site has an agonist potency order of 5'-N-ethylcarboxamidoadenosine > 2-chloroadenosine >> R-phenylisopropyladenosine and an antagonist potency order of isobutylmethylxanthine > theophylline >> 1,3-diethyl-8-phenylxanthine.

  4. Identification of albumin-binding proteins in capillary endothelial cells

    OpenAIRE

    1988-01-01

    Isolated fat tissue microvessels and lung, whose capillary endothelia express in situ specific binding sites for albumin, were homogenized and subjected to SDS-gel electrophoresis and electroblotting. The nitrocellulose strips were incubated with either albumin-gold (Alb-Au) and directly visualized, or with [125I]albumin (monomeric or polymeric) and autoradiographed. The extracts of both microvascular endothelium and the lung express albumin-binding proteins (ABPs) represented by two pairs of...

  5. Yeast TATA-binding protein TFIID binds to TATA elements with both consensus and nonconsensus DNA sequences.

    OpenAIRE

    S. Hahn; Buratowski, S.; Sharp, P A; Guarente, L

    1989-01-01

    The DNA binding properties of the yeast TATA element-binding protein TFIID were investigated. The affinity (apparent equilibrium dissociation constant) of TFIID for the adenovirus major late promoter consensus TATA element is 2 x 10(-9) M, a value similar to the affinity of gene-specific regulatory proteins for their binding sites. TFIID binding is highly specific and recognizes nonspecific sites with approximately 10(5)-fold lower affinity. Despite this specificity, TFIID also binds with hig...

  6. Differential plasma protein binding to metal oxide nanoparticles

    International Nuclear Information System (INIS)

    Nanoparticles rapidly interact with the proteins present in biological fluids, such as blood. The proteins that are adsorbed onto the surface potentially dictate the biokinetics of the nanomaterials and their fate in vivo. Using nanoparticles with different sizes and surface characteristics, studies have reported the effects of physicochemical properties on the composition of adsorbed plasma proteins. However, to date, few studies have been conducted focusing on the nanoparticles that are commonly exposed to the general public, such as the metal oxides. Using previously established ultracentrifugation approaches, two-dimensional gel electrophoresis and mass spectrometry, the current study investigated the binding of human plasma proteins to commercially available titanium dioxide, silicon dioxide and zinc oxide nanoparticles. We found that, despite these particles having similar surface charges in buffer, they bound different plasma proteins. For TiO2, the shape of the nanoparticles was also an important determinant of protein binding. Agglomeration in water was observed for all of the nanoparticles and both TiO2 and ZnO further agglomerated in biological media. This led to an increase in the amount and number of different proteins bound to these nanoparticles. Proteins with important biological functions were identified, including immunoglobulins, lipoproteins, acute-phase proteins and proteins involved in complement pathways and coagulation. These results provide important insights into which human plasma proteins bind to particular metal oxide nanoparticles. Because protein absorption to nanoparticles may determine their interaction with cells and tissues in vivo, understanding how and why plasma proteins are adsorbed to these particles may be important for understanding their biological responses.

  7. The liver fatty acid binding protein--comparison of cavity properties of intracellular lipid-binding proteins.

    Science.gov (United States)

    Thompson, J; Ory, J; Reese-Wagoner, A; Banaszak, L

    1999-02-01

    The crystal and solution structures of all of the intracellular lipid binding proteins (iLBPs) reveal a common beta-barrel framework with only small local perturbations. All existing evidence points to the binding cavity and a poorly delimited 'portal' region as defining the function of each family member. The importance of local structure within the cavity appears to be its influence on binding affinity and specificity for the lipid. The portal region appears to be involved in the regulation of ligand exchange. Within the iLBP family, liver fatty acid binding protein or LFABP, has the unique property of binding two fatty acids within its internalized binding cavity rather than the commonly observed stoichiometry of one. Furthermore, LFABP will bind hydrophobic molecules larger than the ligands which will associate with other iLBPs. The crystal structure of LFABP contains two bound oleate molecules and provides the explanation for its unusual stoichiometry. One of the bound fatty acids is completely internalized and has its carboxylate interacting with an arginine and two serines. The second oleate represents an entirely new binding mode with the carboxylate on the surface of LFABP. The two oleates also interact with each other. Because of this interaction and its inner location, it appears the first oleate must be present before the second more external molecule is bound. PMID:10331654

  8. Binding dynamics of single-stranded DNA binding proteins to fluctuating bubbles in breathing DNA

    International Nuclear Information System (INIS)

    We investigate the dynamics of a single local denaturation zone in a DNA molecule, a so-called DNA bubble, in the presence of single-stranded DNA binding proteins (SSBs). In particular, we develop a dynamical description of the process in terms of a two-dimensional master equation for the time evolution of the probability distribution of having a bubble of size m with n bound SSBs, for the case when m and n are the slowest variables in the system. We derive explicit expressions for the equilibrium statistical weights for a given m and n, which depend on the statistical weight u associated with breaking a base-pair interaction, the loop closure exponent c, the cooperativity parameter σ0, the SSB size λ, and binding strength κ. These statistical weights determine, through the detailed balance condition, the transfer coefficient in the master equation. For the case of slow and fast binding dynamics the problem can be reduced to one-dimensional master equations. In the latter case, we perform explicitly the adiabatic elimination of the fast variable n. Furthermore, we find that for the case that the loop closure is neglected and the binding dynamics is vanishing (but with arbitrary σ0) the eigenvalues and the eigenvectors of the master equation can be obtained analytically, using an orthogonal polynomial approach. We solve the general case numerically (i.e., including SSB binding and the loop closure) as a function of statistical weight u, binding protein size λ, and binding strength κ, and compare to the fast and slow binding limits. In particular, we find that the presence of SSBs in general increases the relaxation time, compared to the case when no binding proteins are present. By tuning the parameters, we can drive the system from regular bubble fluctuation in the absence of SSBs to full denaturation, reflecting experimental and in vivo situations

  9. DNA-based control of protein activity.

    Science.gov (United States)

    Engelen, W; Janssen, B M G; Merkx, M

    2016-03-01

    DNA has emerged as a highly versatile construction material for nanometer-sized structures and sophisticated molecular machines and circuits. The successful application of nucleic acid based systems greatly relies on their ability to autonomously sense and act on their environment. In this feature article, the development of DNA-based strategies to dynamically control protein activity via oligonucleotide triggers is discussed. Depending on the desired application, protein activity can be controlled by directly conjugating them to an oligonucleotide handle, or expressing them as a fusion protein with DNA binding motifs. To control proteins without modifying them chemically or genetically, multivalent ligands and aptamers that reversibly inhibit their function provide valuable tools to regulate proteins in a noncovalent manner. The goal of this feature article is to give an overview of strategies developed to control protein activity via oligonucleotide-based triggers, as well as hurdles yet to be taken to obtain fully autonomous systems that interrogate, process and act on their environments by means of DNA-based protein control. PMID:26812623

  10. Holo- And Apo- Structures of Bacterial Periplasmic Heme Binding Proteins

    Energy Technology Data Exchange (ETDEWEB)

    Ho, W.W.; Li, H.; Eakanunkul, S.; Tong, Y.; Wilks, A.; Guo, M.; Poulos, T.L.

    2009-06-01

    An essential component of heme transport in Gram-negative bacterial pathogens is the periplasmic protein that shuttles heme between outer and inner membranes. We have solved the first crystal structures of two such proteins, ShuT from Shigella dysenteriae and PhuT from Pseudomonas aeruginosa. Both share a common architecture typical of Class III periplasmic binding proteins. The heme binds in a narrow cleft between the N- and C-terminal binding domains and is coordinated by a Tyr residue. A comparison of the heme-free (apo) and -bound (holo) structures indicates little change in structure other than minor alterations in the heme pocket and movement of the Tyr heme ligand from an 'in' position where it can coordinate the heme iron to an 'out' orientation where it points away from the heme pocket. The detailed architecture of the heme pocket is quite different in ShuT and PhuT. Although Arg{sup 228} in PhuT H-bonds with a heme propionate, in ShuT a peptide loop partially takes up the space occupied by Arg{sup 228}, and there is no Lys or Arg H-bonding with the heme propionates. A comparison of PhuT/ShuT with the vitamin B{sub 12}-binding protein BtuF and the hydroxamic-type siderophore-binding protein FhuD, the only two other structurally characterized Class III periplasmic binding proteins, demonstrates that PhuT/ShuT more closely resembles BtuF, which reflects the closer similarity in ligands, heme and B{sub 12}, compared with ligands for FhuD, a peptide siderophore.

  11. The plasma protein binding of HIDA

    International Nuclear Information System (INIS)

    By using Sephadex gel column chromatography to separate substances into their various components according to molecular weight, we have investigated the effect of incubating several brands of HIDA in plasma, in vitro. The results show that such incubation has no effect on either dimethyl HIDA, or diethyl HIDA, but that in the case of para-butyl HIDA, incubation in plasma increases the Rf value to that of HSA (human serum albumin). This indicates that para-butyl HIDA becomes bound to plasma proteins, in contrast to both dimethyl HIDA and diethyl HIDA. (orig.)

  12. Role of SUMO in RNF4-mediated Promyelocytic Leukemia Protein (PML) Degradation: Sumoylation of pml and phospho-switch control of its sumo binding domain dissected in living cells*

    OpenAIRE

    Percherancier, Yann; Germain-Desprez, Delphine; Galisson, Frédéric; Mascle, Xavier H.; Dianoux, Laurent; Estephan, Patricia; Chelbi-Alix, Mounira K.; Aubry, Muriel

    2009-01-01

    Promyelocytic leukemia protein (PML) is a tumor suppressor acting as the organizer of subnuclear structures called PML nuclear bodies (NBs). Both covalent modification of PML by the small ubiquitin-like modifier (SUMO) and non-covalent binding of SUMO to the PML SUMO binding domain (SBD) are necessary for PML NB formation and maturation. PML sumoylation and proteasome-dependent degradation induced by the E3 ubiquitin ligase, RNF4, are enhanced by the acute promyelocytic leukemia therapeutic a...

  13. Characterization of a calmodulin binding protein kinase from Arabidopsis thalian

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    A full-length calmodulin binding protein kinase cDNA, AtCBK1, from Arabidopsis has been isolated by screening of an Arabidopsis cDNA library and by 5′-RACE. Northern blot and in situ hybridization indicated that the expression of AtCBK1 was more abundant in the vascular bundles and the meristems than in other tissues. The phylogenetic analyses reveal that AtCBK1 is different from animal CaMKs and it falls into CRK subgroup, indicating that they may come from different ancestors. The result suggests that AtCBK1 encodes a CaM-binding serine/threonine protein kinase.

  14. Detection of Fibronectin-Binding Proteins in Clostridium perfringens.

    Directory of Open Access Journals (Sweden)

    Yokoyama,Masako

    2006-12-01

    Full Text Available Clostridium perfringens is an anaerobic spore-forming pathogen of humans and animals. C. perfringens type A strains, 13, CPN50, and NCTC8237, isolated from human gas gangrene, bound specifically to human fi bronectin (Fn. The trypsin-treatment of the bacterial cells significantly reduced the Fn-binding. A ligand blotting analysis of all three C. perfringens strains revealed that 5 protein bands of 34 kDa, 29 kDa, 26 kDa, 17 kDa, and 12 kDa specifically bound to biotinylated Fn. These results suggest that C. perfringens possesses certain Fn-binding proteins on the cell surface.

  15. Binding-regulated click ligation for selective detection of proteins.

    Science.gov (United States)

    Cao, Ya; Han, Peng; Wang, Zhuxin; Chen, Weiwei; Shu, Yongqian; Xiang, Yang

    2016-04-15

    Herein, a binding-regulated click ligation (BRCL) strategy for endowing selective detection of proteins is developed with the incorporation of small-molecule ligand and clickable DNA probes. The fundamental principle underlying the strategy is the regulating capability of specific protein-ligand binding against the ligation between clickable DNA probes, which could efficiently combine the detection of particular protein with enormous DNA-based sensing technologies. In this work, the feasibly of the BRCL strategy is first verified through agarose gel electrophoresis and electrochemical impedance spectroscopy measurements, and then confirmed by transferring it to a nanomaterial-assisted fluorescence assay. Significantly, the BRCL strategy-based assay is able to respond to target protein with desirable selectivity, attributing to the specific recognition between small-molecule ligand and its target. Further experiments validate the general applicability of the sensing method by tailoring the ligand toward different proteins (i.e., avidin and folate receptor), and demonstrate its usability in complex biological samples. To our knowledge, this work pioneers the practice of click chemistry in probing specific small-molecule ligand-protein binding, and therefore may pave a new way for selective detection of proteins. PMID:26599478

  16. The RNA-binding protein repertoire of Arabidopsis thaliana

    KAUST Repository

    Marondedze, Claudius

    2016-07-11

    RNA-binding proteins (RBPs) have essential roles in determining the fate of RNA from synthesis to decay and have been studied on a protein-by-protein basis, or computationally based on a number of well-characterised RNA-binding domains. Recently, high-throughput methods enabled the capture of mammalian RNA-binding proteomes. To gain insight into the role of Arabidopsis thaliana RBPs at the systems level, we have employed interactome capture techniques using cells from different ecotypes grown in cultures and leaves. In vivo UV-crosslinking of RNA to RBPs, oligo(dT) capture and mass spectrometry yielded 1,145 different proteins including 550 RBPs that either belong to the functional category ‘RNA-binding’, have known RNA-binding domains or have orthologs identified in mammals, C. elegans, or S. cerevisiae in addition to 595 novel candidate RBPs. We noted specific subsets of RBPs in cultured cells and leaves and a comparison of Arabidopsis, mammalian, C. elegans, and S. cerevisiae RBPs reveals a common set of proteins with a role in intermediate metabolism, as well as distinct differences suggesting that RBPs are also species and tissue specific. This study provides a foundation for studies that will advance our understanding of the biological significance of RBPs in plant developmental and stimulus specific responses.

  17. All-Purpose Containers? Lipid-Binding Protein - Drug Interactions.

    Directory of Open Access Journals (Sweden)

    Tiziana Beringhelli

    Full Text Available The combined use of in vitro (19F-NMR and in silico (molecular docking procedures demonstrates the affinity of a number of human calycins (lipid-binding proteins from ileum, liver, heart, adipose tissue and epidermis, and retinol-binding protein from intestine for different drugs (mainly steroids and vastatins. Comparative evaluations on the complexes outline some of the features relevant for interaction (non-polar character of the drugs; amino acids and water molecules in the protein calyx most often involved in binding. Dissociation constants (Ki for drugs typically lie in the same range as Ki for natural ligands; in most instances (different proteins and docking conditions, vastatins are the strongest interactors, with atorvastatin ranking top in half of the cases. The affinity of some calycins for some of the vastatins is in the order of magnitude of the drug Cmax after systemic administration in humans. The possible biological implications of this feature are discussed in connection with drug delivery parameters (route of administration, binding to carrier proteins, distribution to, and accumulation in, human tissues.

  18. Drug-drug plasma protein binding interactions of ivacaftor.

    Science.gov (United States)

    Schneider, Elena K; Huang, Johnny X; Carbone, Vincenzo; Baker, Mark; Azad, Mohammad A K; Cooper, Matthew A; Li, Jian; Velkov, Tony

    2015-06-01

    Ivacaftor is a novel cystic fibrosis (CF) transmembrane conductance regulator (CFTR) potentiator that improves the pulmonary function for patients with CF bearing a G551D CFTR-protein mutation. Because ivacaftor is highly bound (>97%) to plasma proteins, there is the strong possibility that co-administered CF drugs may compete for the same plasma protein binding sites and impact the free drug concentration. This, in turn, could lead to drastic changes in the in vivo efficacy of ivacaftor and therapeutic outcomes. This biochemical study compares the binding affinity of ivacaftor and co-administered CF drugs for human serum albumin (HSA) and α1 -acid glycoprotein (AGP) using surface plasmon resonance and fluorimetric binding assays that measure the displacement of site-selective probes. Because of their ability to strongly compete for the ivacaftor binding sites on HSA and AGP, drug-drug interactions between ivacaftor are to be expected with ducosate, montelukast, ibuprofen, dicloxacillin, omeprazole, and loratadine. The significance of these plasma protein drug-drug interactions is also interpreted in terms of molecular docking simulations. This in vitro study provides valuable insights into the plasma protein drug-drug interactions of ivacaftor with co-administered CF drugs. The data may prove useful in future clinical trials for a staggered treatment that aims to maximize the effective free drug concentration and clinical efficacy of ivacaftor. PMID:25707701

  19. Fatty Acid- and Retinoid-binding Proteins Have Distinct Binding Pockets for the Two Types of Cargo*

    OpenAIRE

    Jordanova, Rositsa; Groves, Matthew R.; Kostova, Elena; Woltersdorf, Christian; Liebau, Eva; Tucker, Paul A.

    2009-01-01

    Parasitic nematodes cause serious diseases in humans, animals, and plants. They have limited lipid metabolism and are reliant on lipid-binding proteins to acquire these metabolites from their hosts. Several structurally novel families of lipid-binding proteins in nematodes have been described, including the fatty acid- and retinoid-binding protein family (FAR). In Caenorhabditis elegans, used as a model for studying parasitic nematodes, eight C. elegans FAR proteins have been described. The c...

  20. Nutritional Control of mRNA Stability Is Mediated by a Conserved AU-rich Element That Binds the Cytoplasmic Shuttling Protein HuR*

    OpenAIRE

    Yaman, Ibrahim; Fernandez, James; Sarkar, Bedabrata; Schneider, Robert J.; Snider, Martin D.; Nagy, Laura E.; Hatzoglou, Maria

    2002-01-01

    The cationic amino acid transporter, Cat-1, is a high affinity transporter of the essential amino acids, arginine and lysine. Expression of the cat-1 gene increases during nutritional stress as part of the adaptive response to starvation. Amino acid limitation induces coordinate increases in stability and translation of the cat-1 mRNA, at a time when global protein synthesis decreases. It is shown here that increased cat-1 mRNA stability requires an 11 nucleotide AU-rich element within the di...

  1. Drug bioactivation, covalent binding to target proteins and toxicity relevance.

    Science.gov (United States)

    Zhou, Shufeng; Chan, Eli; Duan, Wei; Huang, Min; Chen, Yu-Zong

    2005-01-01

    A number of therapeutic drugs with different structures and mechanisms of action have been reported to undergo metabolic activation by Phase I or Phase II drug-metabolizing enzymes. The bioactivation gives rise to reactive metabolites/intermediates, which readily confer covalent binding to various target proteins by nucleophilic substitution and/or Schiff's base mechanism. These drugs include analgesics (e.g., acetaminophen), antibacterial agents (e.g., sulfonamides and macrolide antibiotics), anticancer drugs (e.g., irinotecan), antiepileptic drugs (e.g., carbamazepine), anti-HIV agents (e.g., ritonavir), antipsychotics (e.g., clozapine), cardiovascular drugs (e.g., procainamide and hydralazine), immunosupressants (e.g., cyclosporine A), inhalational anesthetics (e.g., halothane), nonsteroidal anti-inflammatory drugs (NSAIDSs) (e.g., diclofenac), and steroids and their receptor modulators (e.g., estrogens and tamoxifen). Some herbal and dietary constituents are also bioactivated to reactive metabolites capable of binding covalently and inactivating cytochrome P450s (CYPs). A number of important target proteins of drugs have been identified by mass spectrometric techniques and proteomic approaches. The covalent binding and formation of drug-protein adducts are generally considered to be related to drug toxicity, and selective protein covalent binding by drug metabolites may lead to selective organ toxicity. However, the mechanisms involved in the protein adduct-induced toxicity are largely undefined, although it has been suggested that drug-protein adducts may cause toxicity either through impairing physiological functions of the modified proteins or through immune-mediated mechanisms. In addition, mechanism-based inhibition of CYPs may result in toxic drug-drug interactions. The clinical consequences of drug bioactivation and covalent binding to proteins are unpredictable, depending on many factors that are associated with the administered drugs and patients

  2. Protein kinase Cbeta mediates hepatic induction of sterol-regulatory element binding protein-1c by insulin

    OpenAIRE

    Yamamoto, Takashi; Watanabe, Kazuhisa; Inoue, Noriyuki; Nakagawa, Yoshimi; Ishigaki, Naomi; Matsuzaka, Takashi; Takeuchi, Yoshinori; Kobayashi, Kazuto; Yatoh, Shigeru; Takahashi, Akimitsu; Suzuki, Hiroaki; Yahagi, Naoya; Gotoda, Takanari; Yamada, Nobuhiro; Shimano, Hitoshi

    2010-01-01

    Sterol-regulatory element binding protein-1c (SREBP-1c) is a transcription factor that controls lipogenesis in the liver. Hepatic SREBP-1c is nutritionally regulated, and its sustained activation causes hepatic steatosis and insulin resistance. Although regulation of SREBP-1c is known to occur at the transcriptional level, the precise mechanism by which insulin signaling activates SREBP-1c promoter remains to be elucidated. Here we show that protein kinase C beta (PKCbeta) is a key mediator o...

  3. Free enthalpies of replacing water molecules in protein binding pockets.

    Science.gov (United States)

    Riniker, Sereina; Barandun, Luzi J; Diederich, François; Krämer, Oliver; Steffen, Andreas; van Gunsteren, Wilfred F

    2012-12-01

    Water molecules in the binding pocket of a protein and their role in ligand binding have increasingly raised interest in recent years. Displacement of such water molecules by ligand atoms can be either favourable or unfavourable for ligand binding depending on the change in free enthalpy. In this study, we investigate the displacement of water molecules by an apolar probe in the binding pocket of two proteins, cyclin-dependent kinase 2 and tRNA-guanine transglycosylase, using the method of enveloping distribution sampling (EDS) to obtain free enthalpy differences. In both cases, a ligand core is placed inside the respective pocket and the remaining water molecules are converted to apolar probes, both individually and in pairs. The free enthalpy difference between a water molecule and a CH(3) group at the same location in the pocket in comparison to their presence in bulk solution calculated from EDS molecular dynamics simulations corresponds to the binding free enthalpy of CH(3) at this location. From the free enthalpy difference and the enthalpy difference, the entropic contribution of the displacement can be obtained too. The overlay of the resulting occupancy volumes of the water molecules with crystal structures of analogous ligands shows qualitative correlation between experimentally measured inhibition constants and the calculated free enthalpy differences. Thus, such an EDS analysis of the water molecules in the binding pocket may give valuable insight for potency optimization in drug design. PMID:23247390

  4. The distribution of ligand-binding pockets around protein-protein interfaces suggests a general mechanism for pocket formation

    OpenAIRE

    Gao, Mu; Skolnick, Jeffrey

    2012-01-01

    Protein-protein and protein-ligand interactions are ubiquitous in a biological cell. Here, we report a comprehensive study of the distribution of protein-ligand interaction sites, namely ligand-binding pockets, around protein-protein interfaces where protein-protein interactions occur. We inspected a representative set of 1,611 representative protein-protein complexes and identified pockets with a potential for binding small molecule ligands. The majority of these pockets are within a 6 Å dis...

  5. Pumilio Puf domain RNA-binding proteins in Arabidopsis.

    Science.gov (United States)

    Abbasi, Nazia; Park, Youn-Il; Choi, Sang-Bong

    2011-03-01

    Pumilio proteins are a class of RNA-binding proteins harboring Puf domains (or PUM-HD; Pumilio-Homology Domain), named after the founding members, Pumilio (from Drosophila melanogaster) and FBF (Fem-3 mRNA-Binding Factor from Caenorhabditis elegans). The domains contain multiple tandem repeats each of which recognizes one RNA base and is comprised of 35-39 amino acids. Puf domain proteins have been reported in organisms ranging from single-celled yeast to higher multicellular eukaryotes, such as humans and plants. In yeast and animals, they are involved in a variety of posttranscriptional RNA metabolism including RNA decay, RNA transport, rRNA processing and translational repression. However, their roles in plants are largely unknown. Recently, we have characterized the first member of the Puf family of RNA-binding proteins, APUM23, in Arabidopsis. Here, we discuss and summarize the diverse roles and targets of Puf proteins previously reported in other organisms and then highlight the potential regulatory roles of Puf proteins in Arabidopsis, using our recent study as an example. PMID:21350339

  6. Calcium-binding ability of soy protein hydrolysates

    Institute of Scientific and Technical Information of China (English)

    Xiao Lan Bao; Mei Song; Jing Zhang; Yang Chen; Shun Tang Guo

    2007-01-01

    This present study investigated the ability of various soy protein hydrolysates (SPHs) in binding calcium. It was demonstrated that the amount of Ca-bound depended greatly on the SPHs obtained using different proteases, which included: neutrase,flavourzyme, protease M and pepsin. The maximum level of Ca-bound (66.9 mg/g) occurred when protease M was used to hydrolyze soy protein. Peptide fragments exhibiting high Ca-binding capacity had molecular weights of either 14.4 or 8-9 kDa. The level of Ca-bound increased linearly with the increment of carboxyl content in SPHs, and further deamidation on SPHs from protease M improved Ca-binding of the hydrolysate.

  7. Characterization of the comparative drug binding to intra- (liver fatty acid binding protein) and extra- (human serum albumin) cellular proteins.

    Science.gov (United States)

    Rowland, Andrew; Hallifax, David; Nussio, Matthew R; Shapter, Joseph G; Mackenzie, Peter I; Brian Houston, J; Knights, Kathleen M; Miners, John O

    2015-01-01

    1. This study compared the extent, affinity, and kinetics of drug binding to human serum albumin (HSA) and liver fatty acid binding protein (LFABP) using ultrafiltration and surface plasmon resonance (SPR). 2. Binding of basic and neutral drugs to both HSA and LFABP was typically negligible. Binding of acidic drugs ranged from minor (fu > 0.8) to extensive (fu LFABP was observed for the acidic drugs torsemide and sulfinpyrazone, and for β-estradiol (a polar, neutral compound). 3. The extent of binding of acidic drugs to HSA was up to 40% greater than binding to LFABP. SPR experiments demonstrated comparable kinetics and affinity for the binding of representative acidic drugs (naproxen, sulfinpyrazone, and torsemide) to HSA and LFABP. 4. Simulations based on in vitro kinetic constants derived from SPR experiments and a rapid equilibrium model were undertaken to examine the impact of binding characteristics on compartmental drug distribution. Simulations provided mechanistic confirmation that equilibration of intracellular unbound drug with the extracellular unbound drug is attained rapidly in the absence of active transport mechanisms for drugs bound moderately or extensively to HSA and LFABP. PMID:25801059

  8. The Role of Microtubule End Binding (EB) Proteins in Ciliogenesis

    DEFF Research Database (Denmark)

    Schrøder, Jacob Morville

    biflagellate green alga Chlamydomonas (Pedersen et al., 2003), and is required for ciliogenesis in mouse fibroblasts (Schroder et al., 2007). However, the exact mechanism(s) involved and roles of the two additional mammalian members of the end binding (EB) protein family, EB2 and EB3, in ciliogenesis are...

  9. Genetic noise control via protein oligomerization

    Energy Technology Data Exchange (ETDEWEB)

    Ghim, C; Almaas, E

    2008-06-12

    Gene expression in a cell entails random reaction events occurring over disparate time scales. Thus, molecular noise that often results in phenotypic and population-dynamic consequences sets a fundamental limit to biochemical signaling. While there have been numerous studies correlating the architecture of cellular reaction networks with noise tolerance, only a limited effort has been made to understand the dynamical role of protein-protein associations. We have developed a fully stochastic model for the positive feedback control of a single gene, as well as a pair of genes (toggle switch), integrating quantitative results from previous in vivo and in vitro studies. In particular, we explicitly account for the fast protein binding-unbinding kinetics, RNA polymerases, and the promoter/operator sequences of DNA. We find that the overall noise-level is reduced and the frequency content of the noise is dramatically shifted to the physiologically irrelevant high-frequency regime in the presence of protein dimerization. This is independent of the choice of monomer or dimer as transcription factor and persists throughout the multiple model topologies considered. For the toggle switch, we additionally find that the presence of a protein dimer, either homodimer or heterodimer, may significantly reduce its intrinsic switching rate. Hence, the dimer promotes the robust function of bistable switches by preventing the uninduced (induced) state from randomly being induced (uninduced). The specific binding between regulatory proteins provides a buffer that may prevent the propagation of fluctuations in genetic activity. The capacity of the buffer is a non-monotonic function of association-dissociation rates. Since the protein oligomerization per se does not require extra protein components to be expressed, it provides a basis for the rapid control of intrinsic or extrinsic noise. The stabilization of phenotypically important toggle switches, and nested positive feedback loops in

  10. Monomeric Yeast Frataxin is an Iron-Binding Protein

    Energy Technology Data Exchange (ETDEWEB)

    Cook,J.; Bencze, K.; Jankovic, A.; Crater, A.; Busch, C.; Bradley, P.; Stemmler, A.; Spaller, M.; Stemmler, T.

    2006-01-01

    Friedreich's ataxia, an autosomal cardio- and neurodegenerative disorder that affects 1 in 50 000 humans, is caused by decreased levels of the protein frataxin. Although frataxin is nuclear-encoded, it is targeted to the mitochondrial matrix and necessary for proper regulation of cellular iron homeostasis. Frataxin is required for the cellular production of both heme and iron-sulfur (Fe-S) clusters. Monomeric frataxin binds with high affinity to ferrochelatase, the enzyme involved in iron insertion into porphyrin during heme production. Monomeric frataxin also binds to Isu, the scaffold protein required for assembly of Fe-S cluster intermediates. These processes (heme and Fe-S cluster assembly) share requirements for iron, suggesting that monomeric frataxin might function as the common iron donor. To provide a molecular basis to better understand frataxin's function, we have characterized the binding properties and metal-site structure of ferrous iron bound to monomeric yeast frataxin. Yeast frataxin is stable as an iron-loaded monomer, and the protein can bind two ferrous iron atoms with micromolar binding affinity. Frataxin amino acids affected by the presence of iron are localized within conserved acidic patches located on the surfaces of both helix-1 and strand-1. Under anaerobic conditions, bound metal is stable in the high-spin ferrous state. The metal-ligand coordination geometry of both metal-binding sites is consistent with a six-coordinate iron-(oxygen/nitrogen) based ligand geometry, surely constructed in part from carboxylate and possibly imidazole side chains coming from residues within these conserved acidic patches on the protein. On the basis of our results, we have developed a model for how we believe yeast frataxin interacts with iron.

  11. Monomeric Yeast Frataxin is an Iron-Binding Protein

    International Nuclear Information System (INIS)

    Friedreich's ataxia, an autosomal cardio- and neurodegenerative disorder that affects 1 in 50 000 humans, is caused by decreased levels of the protein frataxin. Although frataxin is nuclear-encoded, it is targeted to the mitochondrial matrix and necessary for proper regulation of cellular iron homeostasis. Frataxin is required for the cellular production of both heme and iron-sulfur (Fe-S) clusters. Monomeric frataxin binds with high affinity to ferrochelatase, the enzyme involved in iron insertion into porphyrin during heme production. Monomeric frataxin also binds to Isu, the scaffold protein required for assembly of Fe-S cluster intermediates. These processes (heme and Fe-S cluster assembly) share requirements for iron, suggesting that monomeric frataxin might function as the common iron donor. To provide a molecular basis to better understand frataxin's function, we have characterized the binding properties and metal-site structure of ferrous iron bound to monomeric yeast frataxin. Yeast frataxin is stable as an iron-loaded monomer, and the protein can bind two ferrous iron atoms with micromolar binding affinity. Frataxin amino acids affected by the presence of iron are localized within conserved acidic patches located on the surfaces of both helix-1 and strand-1. Under anaerobic conditions, bound metal is stable in the high-spin ferrous state. The metal-ligand coordination geometry of both metal-binding sites is consistent with a six-coordinate iron-(oxygen/nitrogen) based ligand geometry, surely constructed in part from carboxylate and possibly imidazole side chains coming from residues within these conserved acidic patches on the protein. On the basis of our results, we have developed a model for how we believe yeast frataxin interacts with iron

  12. The RNA binding domain of Pumilio antagonizes poly-adenosine binding protein and accelerates deadenylation

    OpenAIRE

    Weidmann, Chase A.; Raynard, Nathan A.; Blewett, Nathan H.; Van Etten, Jamie; Goldstrohm, Aaron C.

    2014-01-01

    This article analyzes the mechanism by which Pumilio represses the translation of its targets. The results show, rather surprisingly, that promotion of deadenylation is not required for expression. Instead, Pumilio interacts with poly(A) binding protein and somehow interferes with its activity.

  13. Cooperative binding of copper(I) to the metal binding domains in Menkes disease protein

    DEFF Research Database (Denmark)

    Jensen, P Y; Bonander, N; Møller, L B;

    1999-01-01

    We have optimised the overexpression and purification of the N-terminal end of the Menkes disease protein expressed in Escherichia coli, containing one, two and six metal binding domains (MBD), respectively. The domain(s) have been characterised using circular dichroism (CD) and fluorescence spec...

  14. The RNA binding domain of Pumilio antagonizes poly-adenosine binding protein and accelerates deadenylation.

    Science.gov (United States)

    Weidmann, Chase A; Raynard, Nathan A; Blewett, Nathan H; Van Etten, Jamie; Goldstrohm, Aaron C

    2014-08-01

    PUF proteins are potent repressors that serve important roles in stem cell maintenance, neurological processes, and embryonic development. These functions are driven by PUF protein recognition of specific binding sites within the 3' untranslated regions of target mRNAs. In this study, we investigated mechanisms of repression by the founding PUF, Drosophila Pumilio, and its human orthologs. Here, we evaluated a previously proposed model wherein the Pumilio RNA binding domain (RBD) binds Argonaute, which in turn blocks the translational activity of the eukaryotic elongation factor 1A. Surprisingly, we found that Argonautes are not necessary for repression elicited by Drosophila and human PUFs in vivo. A second model proposed that the RBD of Pumilio represses by recruiting deadenylases to shorten the mRNA's polyadenosine tail. Indeed, the RBD binds to the Pop2 deadenylase and accelerates deadenylation; however, this activity is not crucial for regulation. Rather, we determined that the poly(A) is necessary for repression by the RBD. Our results reveal that poly(A)-dependent repression by the RBD requires the poly(A) binding protein, pAbp. Furthermore, we show that repression by the human PUM2 RBD requires the pAbp ortholog, PABPC1. Pumilio associates with pAbp but does not disrupt binding of pAbp to the mRNA. Taken together, our data support a model wherein the Pumilio RBD antagonizes the ability of pAbp to promote translation. Thus, the conserved function of the PUF RBD is to bind specific mRNAs, antagonize pAbp function, and promote deadenylation. PMID:24942623

  15. A deep learning framework for modeling structural features of RNA-binding protein targets.

    Science.gov (United States)

    Zhang, Sai; Zhou, Jingtian; Hu, Hailin; Gong, Haipeng; Chen, Ligong; Cheng, Chao; Zeng, Jianyang

    2016-02-29

    RNA-binding proteins (RBPs) play important roles in the post-transcriptional control of RNAs. Identifying RBP binding sites and characterizing RBP binding preferences are key steps toward understanding the basic mechanisms of the post-transcriptional gene regulation. Though numerous computational methods have been developed for modeling RBP binding preferences, discovering a complete structural representation of the RBP targets by integrating their available structural features in all three dimensions is still a challenging task. In this paper, we develop a general and flexible deep learning framework for modeling structural binding preferences and predicting binding sites of RBPs, which takes (predicted) RNA tertiary structural information into account for the first time. Our framework constructs a unified representation that characterizes the structural specificities of RBP targets in all three dimensions, which can be further used to predict novel candidate binding sites and discover potential binding motifs. Through testing on the real CLIP-seq datasets, we have demonstrated that our deep learning framework can automatically extract effective hidden structural features from the encoded raw sequence and structural profiles, and predict accurate RBP binding sites. In addition, we have conducted the first study to show that integrating the additional RNA tertiary structural features can improve the model performance in predicting RBP binding sites, especially for the polypyrimidine tract-binding protein (PTB), which also provides a new evidence to support the view that RBPs may own specific tertiary structural binding preferences. In particular, the tests on the internal ribosome entry site (IRES) segments yield satisfiable results with experimental support from the literature and further demonstrate the necessity of incorporating RNA tertiary structural information into the prediction model. The source code of our approach can be found in https

  16. In vivo threonine phosphorylation of immunoglobulin binding protein (BiP) maps to its protein binding domain

    OpenAIRE

    Gaut, James R.

    1997-01-01

    lmmunoglobin binding protein (BiP) molecules exist as both monomers and oligomers and phosphorylated BiP is restricted to the oligomeric pool. Modified BiP is not bound to proteins such as immunoglobulin heavy chain and consequently, may constitute an inactive form. Unlike earlier analysis of mammalian BiP isolated by two-dimensional gel electrophoresis, results here demonstrated that immunoprecipitated BiP displayed predominantly threonine phosphorylation with only a trace of detectable phos...

  17. Engineering periplasmic ligand binding proteins as glucose nanosensors

    Directory of Open Access Journals (Sweden)

    Constance J. Jeffery

    2011-01-01

    Full Text Available Diabetes affects over 100 million people worldwide. Better methods for monitoring blood glucose levels are needed for improving disease management. Several labs have previously made glucose nanosensors by modifying members of the periplasmic ligand binding protein superfamily. This minireview summarizes recent developments in constructing new versions of these proteins that are responsive within the physiological range of blood glucose levels, employ new reporter groups, and/or are more robust. These experiments are important steps in the development of novel proteins that have the characteristics needed for an implantable glucose nanosensor for diabetes management: specificity for glucose, rapid response, sensitivity within the physiological range of glucose concentrations, reproducibility, and robustness.

  18. Predicting protein ligand binding motions with the conformation explorer

    Directory of Open Access Journals (Sweden)

    Flores Samuel C

    2011-10-01

    Full Text Available Abstract Background Knowledge of the structure of proteins bound to known or potential ligands is crucial for biological understanding and drug design. Often the 3D structure of the protein is available in some conformation, but binding the ligand of interest may involve a large scale conformational change which is difficult to predict with existing methods. Results We describe how to generate ligand binding conformations of proteins that move by hinge bending, the largest class of motions. First, we predict the location of the hinge between domains. Second, we apply an Euler rotation to one of the domains about the hinge point. Third, we compute a short-time dynamical trajectory using Molecular Dynamics to equilibrate the protein and ligand and correct unnatural atomic positions. Fourth, we score the generated structures using a novel fitness function which favors closed or holo structures. By iterating the second through fourth steps we systematically minimize the fitness function, thus predicting the conformational change required for small ligand binding for five well studied proteins. Conclusions We demonstrate that the method in most cases successfully predicts the holo conformation given only an apo structure.

  19. Treponema pallidum receptor binding proteins interact with fibronectin

    Energy Technology Data Exchange (ETDEWEB)

    Peterson, K.M.; Baseman, J.B.; Alderete, J.F.

    1983-06-01

    Analysis of plasma proteins avidly bound to T. pallidum surfaces revealed the ability of T. pallidum to acquire numerous host macromolecules. No acquisition was evident by the avirulent spirochete, T. phagedenis biotype Reiter. Western blotting technology using hyperimmune antifibronectin serum as a probe revealed the ability of virulent treponemes to avidly bind fibronectin from a complex medium such as plasma. The specificity of the tiplike adherence of motile T. pallidum to fibronectin-coated glass surfaces and to fibronectin on HEp-2 cells was reinforced by the observation that pretreatment of coverslips or cell monolayers with monospecific antiserum against fibronectin substantially reduced T. pallidum attachment. The stoichiometric binding of T. pallidum to fibronectin-coated coverslips and the inability of unlabeled or /sup 35/S-radiolabeled treponemes to interact with glass surfaces treated with other plasma proteins further established the specific nature of the interaction between virulent T. pallidum and fibronectin. The avid association between three outer envelope proteins of T. pallidum and fibronectin was also demonstrated. These treponemal surface proteins have been previously identified as putative receptor-binding proteins responsible for T. pallidum parasitism of host cells. The data suggest that surface fibronectin mediates tip-oriented attachment of T. pallidum to host cells via a receptor-ligand mechanism of recognition.

  20. Treponema pallidum receptor binding proteins interact with fibronectin

    International Nuclear Information System (INIS)

    Analysis of plasma proteins avidly bound to T. pallidum surfaces revealed the ability of T. pallidum to acquire numerous host macromolecules. No acquisition was evident by the avirulent spirochete, T. phagedenis biotype Reiter. Western blotting technology using hyperimmune antifibronectin serum as a probe revealed the ability of virulent treponemes to avidly bind fibronectin from a complex medium such as plasma. The specificity of the tiplike adherence of motile T. pallidum to fibronectin-coated glass surfaces and to fibronectin on HEp-2 cells was reinforced by the observation that pretreatment of coverslips or cell monolayers with monospecific antiserum against fibronectin substantially reduced T. pallidum attachment. The stoichiometric binding of T. pallidum to fibronectin-coated coverslips and the inability of unlabeled or 35S-radiolabeled treponemes to interact with glass surfaces treated with other plasma proteins further established the specific nature of the interaction between virulent T. pallidum and fibronectin. The avid association between three outer envelope proteins of T. pallidum and fibronectin was also demonstrated. These treponemal surface proteins have been previously identified as putative receptor-binding proteins responsible for T. pallidum parasitism of host cells. The data suggest that surface fibronectin mediates tip-oriented attachment of T. pallidum to host cells via a receptor-ligand mechanism of recognition

  1. Characterization of flavonoid-protein interactions using fluorescence spectroscopy: Binding of pelargonidin to dairy proteins.

    Science.gov (United States)

    Arroyo-Maya, Izlia J; Campos-Terán, José; Hernández-Arana, Andrés; McClements, David Julian

    2016-12-15

    In this study, the interaction between the flavonoid pelargonidin and dairy proteins: β-lactoglobulin (β-LG), whey protein (WPI), and caseinate (CAS) was investigated. Fluorescence experiments demonstrated that pelargonidin quenched milk proteins fluorescence strongly. However, the protein secondary structure was not significantly affected by pelargonidin, as judged from far-UV circular dichroism. Analysis of fluorescence data indicated that pelargonidin-induced quenching does not arise from a dynamical mechanism, but instead is due to protein-ligand binding. Therefore, quenching data were analyzed using the model of independent binding sites. Both β-LG and CAS, but not WPI, showed hyperbolic binding isotherms indicating that these proteins firmly bound pelargonidin at both pH 7.0 and 3.0 (binding constants ca. 1.0×10(5) at 25.0°C). To investigate the underlying thermodynamics, binding constants were determined at 25.0, 35.0, and 45.0°C. These results pointed to binding processes that depend on the structural conformation of the milk proteins. PMID:27451201

  2. Streptococcal IgA-binding proteins bind in the Calpha 2-Calpha 3 interdomain region and inhibit binding of IgA to human CD89.

    Science.gov (United States)

    Pleass, R J; Areschoug, T; Lindahl, G; Woof, J M

    2001-03-16

    Certain pathogenic bacteria express surface proteins that bind to the Fc part of human IgA or IgG. These bacterial proteins are important as immunochemical tools and model systems, but their biological function is still unclear. Here, we describe studies of three streptococcal proteins that bind IgA: the Sir22 and Arp4 proteins of Streptococcus pyogenes and the unrelated beta protein of group B streptococcus. Analysis of IgA domain swap and point mutants indicated that two loops at the Calpha2/Calpha3 domain interface are critical for binding of the streptococcal proteins. This region is also used in binding the human IgA receptor CD89, an important mediator of IgA effector function. In agreement with this finding, the three IgA-binding proteins and a 50-residue IgA-binding peptide derived from Sir22 blocked the ability of IgA to bind CD89. Further, the Arp4 protein inhibited the ability of IgA to trigger a neutrophil respiratory burst via CD89. Thus, we have identified residues on IgA-Fc that play a key role in binding of different streptococcal IgA-binding proteins, and we have identified a mechanism by which a bacterial IgA-binding protein may interfere with IgA effector function. PMID:11096107

  3. The Cobalamin-binding Protein in Zebrafish is an Intermediate Between the Three Cobalamin-binding Proteins in Human

    DEFF Research Database (Denmark)

    Greibe, Eva Holm; Fedosov, Sergey; Nexø, Ebba

    2012-01-01

    the oldest evolutionary derivatives followed by IF and HC (the latter being present only in reptiles and most but not all mammals). Our findings suggest that the only cobalamin-binding protein in zebrafish is an intermediate between the three human cobalamin binders. These findings support the...

  4. Prediction of the key binding site of odorant-binding protein of Holotrichia oblita Faldermann (Coleoptera: Scarabaeida).

    Science.gov (United States)

    Zhuang, X; Wang, Q; Wang, B; Zhong, T; Cao, Y; Li, K; Yin, J

    2014-06-01

    The scarab beetle Holotrichia oblita Faldermann (Coleoptera: Scarabaeidae) is a predominant underground pest in the northern parts of China, and its larvae (grubs) cause great economic losses because of its wide range of host plants and covert habitats. Environmentally friendly strategies for controlling adults would have novel and broad potential applications. One potential pest management measure is the regulation of olfactory chemoreception to control target insect pests. In the process of olfactory recognition, odorant-binding proteins (OBPs) are believed to carry hydrophobic odorants from the environment to the surface of olfactory receptor neurons. To obtain a better understanding of the relationship between OBP structures and their ligands, homology modelling and molecular docking have been conducted on the interaction between HoblOBP1 and hexyl benzoate in the present study. Based on the results, site-directed mutagenesis and binding experiments were combined to describe the binding sites of HoblOBP1 and to explore its ligand-binding mechanism. After homology modelling of HoblOBP1, it was found that the three-dimensional structure of HoblOBP1 consists of six α-helices and three disulphide bridges that connect the helices, and the hydrophobic pockets are both composed of five helices. Based on the docking study, we found that van der Waals interactions and hydrophobic interactions are both important in the bonding between HoblOBP1 and hexyl benzoate. Intramolecular residues formed the hydrogen bonds in the C terminus of the protein and the bonds are crucial for the ligand-binding specificity. Finally, MET48, ILE80 and TYR111 are binding sites predicted for HoblOBP1. Using site-directed mutagenesis and fluorescence assays, it was found that ligands could not be recognized by mutant of Tyr111. A possible explanation is that the compound could not be recognized by the mutant, and remains in the binding cavity because of the loss of the intramolecular

  5. Polyamine binding to proteins in oat and Petunia protoplasts

    Science.gov (United States)

    Mizrahi, Y.; Applewhite, P. B.; Galston, A. W.

    1989-01-01

    Previous work (A Apelbaum et al. [1988] Plant Physiol 88: 996-998) has demonstrated binding of labeled spermidine (Spd) to a developmentally regulated 18 kilodalton protein in tobacco tissue cultures derived from thin surface layer explants. To assess the general importance of such Spd-protein complexes, we attempted bulk isolation from protoplasts of Petunia and oat (Avena sativa). In Petunia, as in tobacco, fed radioactive Spd is bound to protein, but in oat, Spd is first converted to 1,3,-diaminopropane (DAP), probably by polyamine oxidase action. In oat, binding of DAP to protein depends on age of donor leaf and conditions of illumination and temperature, and the extraction of the DAP-protein complex depends upon buffer and pH. The yield of the DAP-protein complex was maximized by extraction of frozen-thawed protoplasts with a pH 8.8 carbonate buffer containing SDS. Its molecular size, based on Sephacryl column fractionation of ammonium sulfate precipitated material, exceeded 45 kilodaltons. Bound Spd or DAP can be released from their complexes by the action of Pronase, but not DNAse, RNAse, or strong salt solutions, indicating covalent attachment to protein.

  6. Controlled Activation of Protein Rotational Dynamics Using Smart Hydrogel Tethering

    Energy Technology Data Exchange (ETDEWEB)

    Beech, Brenda M.; Xiong, Yijia; Boschek, Curt B.; Baird, Cheryl L.; Bigelow, Diana J.; Mcateer, Kathleen; Squier, Thomas C.

    2014-09-05

    Stimulus-responsive hydrogel materials that stabilize and control protein dynamics have the potential to enable a range of applications to take advantage of the inherent specificity and catalytic efficiencies of proteins. Here we describe the modular construction of a hydrogel using an engineered calmodulin (CaM) within a polyethylene glycol (PEG) matrix that involves the reversible tethering of proteins through an engineered CaM-binding sequence. For these measurements, maltose binding protein (MBP) was isotopically labeled with [13C] and [15N], permitting dynamic structural measurements using TROSY-HSQC NMR spectroscopy. Upon initial formation of hydrogels protein dynamics are suppressed, with concomitant increases in protein stability. Relaxation of the hydrogel matrix following transient heating results in the activation of protein dynamics and restoration of substrate-induced large-amplitude domain motions necessary for substrate binding.

  7. Prediction of DNA-binding specificity in zinc finger proteins

    Indian Academy of Sciences (India)

    Sumedha Roy; Shayoni Dutta; Kanika Khanna; Shruti Singla; Durai Sundar

    2012-07-01

    Zinc finger proteins interact via their individual fingers to three base pair subsites on the target DNA. The four key residue positions −1, 2, 3 and 6 on the alpha-helix of the zinc fingers have hydrogen bond interactions with the DNA. Mutating these key residues enables generation of a plethora of combinatorial possibilities that can bind to any DNA stretch of interest. Exploiting the binding specificity and affinity of the interaction between the zinc fingers and the respective DNA can help to generate engineered zinc fingers for therapeutic purposes involving genome targeting. Exploring the structure–function relationships of the existing zinc finger–DNA complexes can aid in predicting the probable zinc fingers that could bind to any target DNA. Computational tools ease the prediction of such engineered zinc fingers by effectively utilizing information from the available experimental data. A study of literature reveals many approaches for predicting DNA-binding specificity in zinc finger proteins. However, an alternative approach that looks into the physico-chemical properties of these complexes would do away with the difficulties of designing unbiased zinc fingers with the desired affinity and specificity. We present a physico-chemical approach that exploits the relative strengths of hydrogen bonding between the target DNA and all combinatorially possible zinc fingers to select the most optimum zinc finger protein candidate.

  8. Insulin-like growth factor binding proteins: a structural perspective

    Directory of Open Access Journals (Sweden)

    Briony eForbes

    2012-03-01

    Full Text Available Insulin-like growth factor binding proteins (IGFBP-1 to -6 bind insulin-like growth factors-I and -II (IGF-I and IGF-II with high affinity. These binding proteins maintain IGFs in the circulation and direct them to target tissues, where they promote cell growth, proliferation, differentiation and survival via the type 1 IGF receptor (IGF-1R. IGFBPs also interact with many other molecules, which not only influence their modulation of IGF action but also mediate IGF-independent activities that influence processes such as cell migration and apoptosis by influencing gene transcription.IGFBPs-1 to -6 are structurally similar proteins consisting of three distinct domains, N-terminal, Linker and C-terminal. There have been major advances in our understanding of IGFBP structure in the last decade and a half. While there is still no structure of an intact IGFBP to date, several structures of individual N- and C-domains have been solved. The structure of a complex of N-BP-4:IGF-I:C-BP-4 has also been solved, providing a detailed picture of the structural features of the IGF binding site and the mechanism of binding. Structural studies have also identified features important for interaction with extracellular matrix components and integrins. This review summarises structural studies reported so far and highlights features important for binding not only IGF but also other partners. It also highlights future directions in which structural studies will add to our knowledge of the role played by the IGFBP family in normal growth and development, as well as in disease.

  9. Stable Isotope Labeling Strategy for Protein-Ligand Binding Analysis in Multi-Component Protein Mixtures

    Science.gov (United States)

    DeArmond, Patrick D.; West, Graham M.; Huang, Hai-Tsang; Fitzgerald, Michael C.

    2011-03-01

    Described here is a stable isotope labeling protocol that can be used with a chemical modification- and mass spectrometry-based protein-ligand binding assay for detecting and quantifying both the direct and indirect binding events that result from protein-ligand binding interactions. The protocol utilizes an H{2/16}O2 and H{2/18}O2 labeling strategy to evaluate the chemical denaturant dependence of methionine oxidation in proteins both in the presence and absence of a target ligand. The differential denaturant dependence to the oxidation reactions performed in the presence and absence of ligand provides a measure of the protein stability changes that occur as a result of direct interactions of proteins with the target ligand and/or as a result of indirect interactions involving other protein-ligand interactions that are either induced or disrupted by the ligand. The described protocol utilizes the 18O/16O ratio in the oxidized protein samples to quantify the ligand-induced protein stability changes. The ratio is determined using the isotopic distributions observed for the methionine-containing peptides used for protein identification in the LC-MS-based proteomics readout. The strategy is applied to a multi-component protein mixture in this proof-of-principle experiment, which was designed to evaluate the technique's ability to detect and quantify the direct binding interaction between cyclosporin A and cyclophilin A and to detect the indirect binding interaction between cyclosporin A and calcineurin (i.e., the protein-protein interaction between cyclophilin A and calcineurin that is induced by cyclosporin A binding to cyclophilin A).

  10. Myb-binding protein 1A (MYBBP1A is essential for early embryonic development, controls cell cycle and mitosis, and acts as a tumor suppressor.

    Directory of Open Access Journals (Sweden)

    Silvia Mori

    Full Text Available MYBBP1A is a predominantly nucleolar transcriptional regulator involved in rDNA synthesis and p53 activation via acetylation. However little further information is available as to its function. Here we report that MYBBP1A is developmentally essential in the mouse prior to blastocyst formation. In cell culture, down-regulation of MYBBP1A decreases the growth rate of wild type mouse embryonic stem cells, mouse embryo fibroblasts (MEFs and of human HeLa cells, where it also promotes apoptosis. HeLa cells either arrest at G2/M or undergo delayed and anomalous mitosis. At mitosis, MYBBP1A is localized to a parachromosomal region and gene-expression profiling shows that its down-regulation affects genes controlling chromosomal segregation and cell cycle. However, MYBBP1A down-regulation increases the growth rate of the immortalized NIH3T3 cells. Such Mybbp1a down-regulated NIH3T3 cells are more susceptible to Ras-induced transformation and cause more potent Ras-driven tumors. We conclude that MYBBP1A is an essential gene with novel roles at the pre-mitotic level and potential tumor suppressor activity.

  11. [Penicillin-binding proteins of various strains of Lactobacillus].

    Science.gov (United States)

    Griaznova, N S; Subbotina, N A; Beliavskaia, I V; Taisova, A S; Afonin, V I; Tiurin, M V; Shenderov, B A; Sazykina, Iu O; Navashin, S M

    1990-02-01

    Sensitivity of different species of Lactobacillus i.e. L. casei, L. plantarum, L. acidophillus, L. buchneri, L. jugurti and others to penicillins and cephalosporins of various generations was studied. Penicillin binding proteins (PBPs) of the Lactobacillus species were specified. It was shown that the number of PBPs depended on the Lactobacillus species. L. casei had the least number of PBPs (4) and L. brevis had the highest number of PBPs (11). Competition of 14C-benzylpenicillin with ampicillin, cefotaxime, ceftizoxime and cefoperazone for binding to separate PBPs in three strains of different Lactobacillus species was investigated. PMID:2110806

  12. Photoaffinity labelling of high affinity dopamine binding proteins

    Energy Technology Data Exchange (ETDEWEB)

    Ross, G.M.; McCarry, B.E.; Mishra, R.K.

    1986-03-01

    A photoactive analogue of the dopamine agonist 2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronapthalene (ADTN) has been synthesized and used to photoaffinity label dopamine binding proteins prepared from bovine caudate nucleus. N-(3-)N'-4-azidobenzamidol)-aminopropyl)-aminopropyl)-ADTN (AzB-AP-ADTN) was incubated with caudate membranes and irradiated with UV light. Membranes were then repeatedly washed by centrifugation to remove excess photolabel. A binding assay, using (/sup 3/H)-SCH 23390 (a D/sub 1/ specific antagonist), was then performed to evaluate the loss of receptor density in the photolyzed preparation. AzB-AP-ADTN irreversibly blocked (/sup 3/H)-SCH 23390 binding in a dose-dependent manner. Scatchard analysis revealed a decrease in the B/sub max/, with no significant change in the K/sub d/, of (/sup 3/H)-SCH 23390 binding. Compounds which compete for D/sub 1/ receptor binding (such as dopamine, SKF 38393 or apomorphine), proteted the SCH 23390 binding site from inactivation. This data would suggest that the novel photoaffinity ligand, AzB-AP-ADTN, can covalently label the D/sub 1/ (adenylate cyclase linked) dopamine receptor.

  13. Deoxyribonucleic-binding homeobox proteins are augmented in human cancer

    DEFF Research Database (Denmark)

    Wewer, U M; Mercurio, A M; Chung, S Y;

    1990-01-01

    Homeobox genes encode sequence-specific DNA-binding proteins that are involved in the regulation of gene expression during embryonic development. In this study, we examined the expression of homeobox proteins in human cancer. Antiserum was obtained against a synthetic peptide derived from the...... isolated and used to elicit a rabbit antiserum. In immunostaining, both antisera reacted with the nuclei of cultured tumor cells. In tissue sections of human carcinoma, nuclear immunoreactivity was observed in the tumor cells in 40 of 42 cases examined. Adjacent normal epithelial tissue obtained from the...... presence of the homeobox transcript in human carcinoma was documented by in situ hybridization and RNase protection mapping. These results demonstrate that human cancer is associated with the expression of homeobox proteins. Such homeobox proteins, as well as other regulatory proteins, could be involved in...

  14. Competitive protein binding analysis for thyroxine using Sephadex column (Tetralute)

    International Nuclear Information System (INIS)

    The method of competitive protein binding analysis of thyroxine (T4) using Tetralute kit was evaluated. The net retention was decreased when the procedure of competition and separation was performed at a higher temperature but the final T4-I values were constant when the standard and test sera were treated identically. Coefficient of variation (C.V.) was 4% (within-assay) and 6% (between-assay) respectively. However, the T4-I values of pooled serum for quality control were slightly lower in earlier experiments in which correction factors (1.03--1.62 in 18 out of 21 assays) were necessary. T4-I values were determined by the Tetralute in 155 cases. They were as follows: 4.9+-0.8 μg/dl (euthyroid subjects), 6.4+-1.2 μg/dl (cord serum), 7.1+-1.1 μg/dl (pregnant women). 9.0+-3.6 μg/dl (trophoblastic disease), 13.3+-4.8 μg/dl (Graves' disease), 6.3+-1.6 μg/dl (Plummer's disease), 4-I values determined by Tetralute and Res-O-Mat T4 (r=0.96). Following oral administration of Telepaque the serum protein-bound iodine was markedly elevated, while the T4-I determined by Tetralute did not change. In vitro addition of diphenylhydantoin (500 μg/ml), salicylate (4 mg/ml) and phenobarbital (1 mg/ml) had no or little effect on T4 determination by Tetralute. A high concentration of benzbromarone (0.1 mg/ml) caused a higher value of T4-I determined by Tetralute when added to a TBG solution but there was only a slight increase when it was added to serum. (auth.)

  15. Poisson-Boltzmann Calculations of Nonspecific Salt Effects on Protein-Protein Binding Free Energies

    OpenAIRE

    Bertonati, Claudia; Honig, Barry; Alexov, Emil

    2007-01-01

    The salt dependence of the binding free energy of five protein-protein hetero-dimers and two homo-dimers/tetramers was calculated from numerical solutions to the Poisson-Boltzmann equation. Overall, the agreement with experimental values is very good. In all cases except one involving the highly charged lactoglobulin homo-dimer, increasing the salt concentration is found both experimentally and theoretically to decrease the binding affinity. To clarify the source of salt effects, the salt-dep...

  16. Isolation and characterization of a novel calmodulin-binding protein from potato

    Science.gov (United States)

    Reddy, Anireddy S N.; Day, Irene S.; Narasimhulu, S. B.; Safadi, Farida; Reddy, Vaka S.; Golovkin, Maxim; Harnly, Melissa J.

    2002-01-01

    Tuberization in potato is controlled by hormonal and environmental signals. Ca(2+), an important intracellular messenger, and calmodulin (CaM), one of the primary Ca(2+) sensors, have been implicated in controlling diverse cellular processes in plants including tuberization. The regulation of cellular processes by CaM involves its interaction with other proteins. To understand the role of Ca(2+)/CaM in tuberization, we have screened an expression library prepared from developing tubers with biotinylated CaM. This screening resulted in isolation of a cDNA encoding a novel CaM-binding protein (potato calmodulin-binding protein (PCBP)). Ca(2+)-dependent binding of the cDNA-encoded protein to CaM is confirmed by (35)S-labeled CaM. The full-length cDNA is 5 kb long and encodes a protein of 1309 amino acids. The deduced amino acid sequence showed significant similarity with a hypothetical protein from another plant, Arabidopsis. However, no homologs of PCBP are found in nonplant systems, suggesting that it is likely to be specific to plants. Using truncated versions of the protein and a synthetic peptide in CaM binding assays we mapped the CaM-binding region to a 20-amino acid stretch (residues 1216-1237). The bacterially expressed protein containing the CaM-binding domain interacted with three CaM isoforms (CaM2, CaM4, and CaM6). PCBP is encoded by a single gene and is expressed differentially in the tissues tested. The expression of CaM, PCBP, and another CaM-binding protein is similar in different tissues and organs. The predicted protein contained seven putative nuclear localization signals and several strong PEST motifs. Fusion of the N-terminal region of the protein containing six of the seven nuclear localization signals to the reporter gene beta-glucuronidase targeted the reporter gene to the nucleus, suggesting a nuclear role for PCBP.

  17. The Pumilio protein binds RNA through a conserved domain that defines a new class of RNA-binding proteins.

    OpenAIRE

    Zamore, P. D.; Williamson, J R; Lehmann, R.

    1997-01-01

    Translation of hunchback(mat) (hb[mat]) mRNA must be repressed in the posterior of the pre-blastoderm Drosophila embryo to permit formation of abdominal segments. This translational repression requires two copies of the Nanos Response Element (NRE), a 16-nt sequence in the hb[mat] 3' untranslated region. Translational repression also requires the action of two proteins: Pumilio (PUM), a sequence-specific RNA-binding protein; and Nanos, a protein that determines the location of repression. Bin...

  18. Detection of an endothelin-1-binding protein complex by low temperature SDS-PAGE

    International Nuclear Information System (INIS)

    We found that the complex of ET-1 and its binding protein was stable enough to be separated by SDS-PAGE when electrophoresis was run at a low temperature. Cross-linking was not necessary for the detection of 125I-ET-1 and its binding protein complex by autoradiography. This simple method could be used in qualitative (estimation of apparent molecular weight of ET-1 binding protein) and quantitative (determination of relative content of ET-binding protein) analysis of the ET-binding protein complex. ET-binding protein complexes of various animal species and organs were investigated by this method

  19. Phosphorylation of bovine interphotoreceptor retinoid-binding protein (IRBP)

    International Nuclear Information System (INIS)

    IRBP is the major soluble (glycolipo) protein of the interphotoreceptor matrix (IPM) and a putative intercellular retinoid-transport vehicle. The authors have now examined phosphorylation of proteins in a crude bovine IPM wash using γ-32P-ATP. SDS-polyacrylamide gel electrophoresis (PAGE) of IPM proteins showed several phosphorylated protein bands, one of them migrating in the same position as purified IRBP. When an aliquot of phosphorylated IPM proteins was incubated overnight with 3H-retinol and subjected to either size-exclusion or ion-exchange HPLC, a peak of 32P was observed in both cases which coincided with 3H-retinol binding and had a retention time identical to that of purified IRBP. When phosphorylated IPM was subjected to Con A Sepharose affinity chromatography and the 50mM methyl α-D-mannoside eluate chromatographed on ion-exchange HPLC, the 32P-peak was not present although a substantial amount of non-phosphorylated IRBP was recovered as assessed by SDS-PAGE and Western blotting. However, when the Con A Sepharose beads were dissolved in SDS and subjected to SDS-PAGE and Western blotting, a band of phosphorylated IRBP was observed, indicating that the phosphorylated IRBP was more tightly bound to the Con A Sepharose. The authors conclude that a fraction of IRBP can be phosphorylated by a yet to be characterized protein kinase and that the binding characteristics of IRBP are markedly altered by phosphorylation

  20. Crystal Structure of Human Retinoblastoma Binding Protein 9

    Energy Technology Data Exchange (ETDEWEB)

    Vorobiev, S.; Su, M; Seetharaman, J; Huang, Y; Chen, C; Maglaqui, M; Janjua, H; Montelione, G; Tong, L; et. al.

    2009-01-01

    As a step towards better integrating protein three-dimensional (3D) structural information in cancer systems biology, the Northeast Structural Genomics Consortium (NESG) (www.nesg.org) has constructed a Human Cancer Pathway Protein Interaction Network (HCPIN) by analysis of several classical cancer-associated signaling pathways and their physical protein-protein interactions. Many well-known cancer-associated proteins play central roles as hubs or bottlenecks in the HCPIN (http://nmr.cabm.rutgers.edu/hcpin). NESG has selected more than 1000 human proteins and protein domains from the HCPIN for sample production and 3D structure determination. The long-range goal of this effort is to provide a comprehensive 3D structure-function database for human cancer-associated proteins and protein complexes, in the context of their interaction networks. Human retinoblastoma binding protein 9 (RBBP9) is one of the HCPIN proteins targeted by NESG. RBBP9 was initially identified as the product of a new gene, Bog (for B5T over-expressed gene), in several transformed rat liver epithelial cell lines resistant to the growth-inhibitory effect of TGF-1 as well as in primary human liver tumors. RBBP9 contains the retinoblastoma (Rb) binding motif LxCxE in its sequence, and was shown to interact with Rb by yeast two-hybrid and coimmunoprecipitation experiments. Mutation of the Leu residue in this motif to Gln blocked the binding to Rb. RBBP9 can displace E2F1 from E2F1-Rb complexes, and over expression of RBBP9 overcomes TGF-1 induced growth arrest and results in transformation of rat liver epithelial cells leading to hepatoblastoma-like tumors in nude mice. RBBP9 may also play a role in cellular responses to chronic low dose radiation. A close homolog of RBBP9, sharing 93% amino acid sequence identity and also known as RBBP10, interacts with a protein with sua5-yciO-yrdC domains.

  1. Protein-binding RNA aptamers affect molecular interactions distantly from their binding sites

    DEFF Research Database (Denmark)

    Dupont, Daniel Miotto; Thuesen, Cathrine K; Bøtkjær, Kenneth A;

    2015-01-01

    Nucleic acid aptamer selection is a powerful strategy for the development of regulatory agents for molecular intervention. Accordingly, aptamers have proven their diligence in the intervention with serine protease activities, which play important roles in physiology and pathophysiology. Nonetheless......, there are only a few studies on the molecular basis underlying aptamer-protease interactions and the associated mechanisms of inhibition. In the present study, we use site-directed mutagenesis to delineate the binding sites of two 2´-fluoropyrimidine RNA aptamers (upanap-12 and upanap-126) with...... therapeutic potential, both binding to the serine protease urokinase-type plasminogen activator (uPA). We determine the subsequent impact of aptamer binding on the well-established molecular interactions (plasmin, PAI-1, uPAR, and LRP-1A) controlling uPA activities. One of the aptamers (upanap-126) binds to...

  2. Predicting the Impact of Missense Mutations on Protein-Protein Binding Affinity.

    Science.gov (United States)

    Li, Minghui; Petukh, Marharyta; Alexov, Emil; Panchenko, Anna R

    2014-04-01

    The crucial prerequisite for proper biological function is the protein's ability to establish highly selective interactions with macromolecular partners. A missense mutation that alters the protein binding affinity may cause significant perturbations or complete abolishment of the function, potentially leading to diseases. The availability of computational methods to evaluate the impact of mutations on protein-protein binding is critical for a wide range of biomedical applications. Here, we report an efficient computational approach for predicting the effect of single and multiple missense mutations on protein-protein binding affinity. It is based on a well-tested simulation protocol for structure minimization, modified MM-PBSA and statistical scoring energy functions with parameters optimized on experimental sets of several thousands of mutations. Our simulation protocol yields very good agreement between predicted and experimental values with Pearson correlation coefficients of 0.69 and 0.63 and root-mean-square errors of 1.20 and 1.90 kcal mol(-1) for single and multiple mutations, respectively. Compared with other available methods, our approach achieves high speed and prediction accuracy and can be applied to large datasets generated by modern genomics initiatives. In addition, we report a crucial role of water model and the polar solvation energy in estimating the changes in binding affinity. Our analysis also reveals that prediction accuracy and effect of mutations on binding strongly depends on the type of mutation and its location in a protein complex. PMID:24803870

  3. DNA binding protein identification by combining pseudo amino acid composition and profile-based protein representation

    Science.gov (United States)

    Liu, Bin; Wang, Shanyi; Wang, Xiaolong

    2015-10-01

    DNA-binding proteins play an important role in most cellular processes. Therefore, it is necessary to develop an efficient predictor for identifying DNA-binding proteins only based on the sequence information of proteins. The bottleneck for constructing a useful predictor is to find suitable features capturing the characteristics of DNA binding proteins. We applied PseAAC to DNA binding protein identification, and PseAAC was further improved by incorporating the evolutionary information by using profile-based protein representation. Finally, Combined with Support Vector Machines (SVMs), a predictor called iDNAPro-PseAAC was proposed. Experimental results on an updated benchmark dataset showed that iDNAPro-PseAAC outperformed some state-of-the-art approaches, and it can achieve stable performance on an independent dataset. By using an ensemble learning approach to incorporate more negative samples (non-DNA binding proteins) in the training process, the performance of iDNAPro-PseAAC was further improved. The web server of iDNAPro-PseAAC is available at http://bioinformatics.hitsz.edu.cn/iDNAPro-PseAAC/.

  4. The surface protein Shr of Streptococcus pyogenes binds heme and transfers it to the streptococcal heme-binding protein Shp

    OpenAIRE

    Lei Benfang; Liu Mengyao; Zhu Hui

    2008-01-01

    Abstract Background The heme acquisition machinery in Streptococcus pyogenes is believed to consist of the surface proteins, Shr and Shp, and heme-specific ATP-binding cassette transporter HtsABC. Shp has been shown to rapidly transfer its heme to the lipoprotein component, HtsA, of HtsABC. The function of Shr and the heme source of Shp have not been established. Results The objective of this study was to determine whether Shr binds heme and is a heme source of Shp. To achieve the objective, ...

  5. Human neutrophil calmodulin-binding proteins: identification of the calmodulin-dependent protein phosphatase

    International Nuclear Information System (INIS)

    The molecular events in linking neutrophil activation and ligand binding to specific membrane receptors are mediated in part by an increase in intracellular Ca2+. One mechanism by which Ca2+ may trigger neutrophil activation is through Ca2+/calmodulin (CaM)-regulated proteins and enzymes. To determine which Ca2+/CaM-regulated enzymes may be present in the neutrophil, they have used Western blotting techniques and 125I-CaM to identify neutrophil CaM-binding proteins. Eleven proteins with molecular weights ranging from 230K to 13.5K bound 125I-CaM in a Ca2+-dependent manner. One predominant region of 125I-Cam binding was to a 59K protein; a protein with an identical mobility was labeled by an antisera against brain CaM-dependent phosphatase. Ca2+-dependent phosphatase activity, which was inhibited by the CaM antagonist trifluoperazine, was detected in a neutrophil extract; a radioimmunoassay for the phosphatase indicated that it was present in the extract at approximately 0.2 μg/mg protein. Most of the CaM-binding proteins, including the 59K protein, were rapidly degraded upon lysis of the neutrophil. There was a close correlation between the degradation of the 59K protein and the loss of Ca2+-dependent phosphatase activity in the neutrophil extract. Thus, human neutrophils contain numerous CaM-binding proteins which are presumably Ca2+/calmodulin-regulated enzymes and proteins; the 59K protein is a CaM-dependent phosphatase

  6. Analysis of Copper-Binding Proteins in Rice Radicles Exposed to Excess Copper and Hydrogen Peroxide Stress.

    Science.gov (United States)

    Zhang, Hongxiao; Xia, Yan; Chen, Chen; Zhuang, Kai; Song, Yufeng; Shen, Zhenguo

    2016-01-01

    Copper (Cu) is an essential micronutrient for plants, but excess Cu can inactivate and disturb the protein function due to unavoidable binding to proteins at the cellular level. As a redox-active metal, Cu toxicity is mediated by the formation of reactive oxygen species (ROS). Cu-binding structural motifs may alleviate Cu-induced damage by decreasing free Cu(2+) activity in cytoplasm or scavenging ROS. The identification of Cu-binding proteins involved in the response of plants to Cu or ROS toxicity may increase our understanding the mechanisms of metal toxicity and tolerance in plants. This study investigated change of Cu-binding proteins in radicles of germinating rice seeds under excess Cu and oxidative stress using immobilized Cu(2+) affinity chromatography, two-dimensional electrophoresis, and mass spectra analysis. Quantitative image analysis revealed that 26 protein spots showed more than a 1.5-fold difference in abundances under Cu or H2O2 treatment compared to the control. The identified Cu-binding proteins were involved in anti-oxidative defense, stress response and detoxification, protein synthesis, protein modification, and metabolism regulation. The present results revealed that 17 out of 24 identified Cu-binding proteins have a similar response to low concentration Cu (20 μM Cu) and H2O2 stress, and 5 out of 24 were increased under low and high concentration Cu (100 μM Cu) but unaffected under H2O2 stress, which hint Cu ions can regulate Cu-binding proteins accumulation by H2O2 or no H2O2 pathway to cope with excess Cu in cell. The change pattern of these Cu-binding proteins and their function analysis warrant to further study the roles of Cu ions in these Cu-binding proteins of plant cells. PMID:27582750

  7. Mapping the Anopheles gambiae Odorant Binding Protein 1 (AgamOBP1) using modeling techniques, site directed mutagenesis, circular dichroism and ligand binding assays

    OpenAIRE

    Rusconi, B.; Maranhao, A.C.; Fuhrer, J P; Krotee, P.; Choi, S. H.; Grun, F; Thireou, T; Dimitratos, S.D.; Woods, D F; Marinotti, O.; Walter, M.F.; Eliopoulos, E.

    2012-01-01

    The major malaria vector in Sub-Saharan Africa is the Anopheles gambiae mosquito. This species is a key target of malaria control measures. Mosquitoes find humans primarily through olfaction, yet the molecular mechanisms associated with host-seeking behavior remain largely unknown. To further understand the functionality of A. gambiae odorant binding protein 1 (AgamOBP1), we combined in silico protein structure modeling and site-directed mutagenesis to generate 16 AgamOBP1 protein analogues c...

  8. Glycosylation status of vitamin D binding protein in cancer patients

    OpenAIRE

    Rehder, Douglas S.; Nelson, Randall W.; Borges, Chad R.

    2009-01-01

    On the basis of the results of activity studies, previous reports have suggested that vitamin D binding protein (DBP) is significantly or even completely deglycosylated in cancer patients, eliminating the molecular precursor of the immunologically important Gc macrophage activating factor (GcMAF), a glycosidase-derived product of DBP. The purpose of this investigation was to directly determine the relative degree of O-linked trisaccharide glycosylation of serum-derived DBP in human breast, co...

  9. Interactions of human mannose-binding protein with lipoteichoic acids.

    OpenAIRE

    Polotsky, V Y; Fischer, W; Ezekowitz, R A; Joiner, K A

    1996-01-01

    We explored the interaction of human recombinant mannose-binding protein and lipoteichoic acids (LTAs) by enzyme-linked immunosorbent assay. The best ligand was Micrococcus luteus lipomannan, followed by Enterococcus spp. LTA containing mono-, di-, and oligoglucosyl substituents. LTAs lacking terminal sugars (those of Streptococcus pyogenes and Staphylococcus aureus) or containing galactosyl substituents (those of Listeria spp. and Lactococcus spp.) were poor ligands. These results are consis...

  10. Vibrational Softening of a Protein on Ligand Binding

    Energy Technology Data Exchange (ETDEWEB)

    Balog, Erica [Semmelweis University, Budapest, Hungary; Perahia, David [Ecole Normale Superieure de Cachan, Cachan, France; Smith, Jeremy C [ORNL; Merzel, Franci [National Institute of Chemistry, Solvenia

    2011-01-01

    Neutron scattering experiments have demonstrated that binding of the cancer drug methotrexate softens the low-frequency vibrations of its target protein, dihydrofolate reductase (DHFR). Here, this softening is fully reproduced using atomic detail normal-mode analysis. Decomposition of the vibrational density of states demonstrates that the largest contributions arise from structural elements of DHFR critical to stability and function. Mode-projection analysis reveals an increase of the breathing-like character of the affected vibrational modes consistent with the experimentally observed increased adiabatic compressibility of the protein on complexation.

  11. Identifying Interactions that Determine Fragment Binding at Protein Hotspots.

    Science.gov (United States)

    Radoux, Chris J; Olsson, Tjelvar S G; Pitt, Will R; Groom, Colin R; Blundell, Tom L

    2016-05-12

    Locating a ligand-binding site is an important first step in structure-guided drug discovery, but current methods do little to suggest which interactions within a pocket are the most important for binding. Here we illustrate a method that samples atomic hotspots with simple molecular probes to produce fragment hotspot maps. These maps specifically highlight fragment-binding sites and their corresponding pharmacophores. For ligand-bound structures, they provide an intuitive visual guide within the binding site, directing medicinal chemists where to grow the molecule and alerting them to suboptimal interactions within the original hit. The fragment hotspot map calculation is validated using experimental binding positions of 21 fragments and subsequent lead molecules. The ligands are found in high scoring areas of the fragment hotspot maps, with fragment atoms having a median percentage rank of 97%. Protein kinase B and pantothenate synthetase are examined in detail. In each case, the fragment hotspot maps are able to rationalize a Free-Wilson analysis of SAR data from a fragment-based drug design project. PMID:27043011

  12. Characterization of auxin-binding proteins from zucchini plasma membrane

    Science.gov (United States)

    Hicks, G. R.; Rice, M. S.; Lomax, T. L.

    1993-01-01

    We have previously identified two auxin-binding polypeptides in plasma membrane (PM) preparations from zucchini (Cucurbita pepo L.) (Hicks et al. 1989, Proc. Natl. Acad. Sci. USA 86, 4948-4952). These polypeptides have molecular weights of 40 kDa and 42 kDa and label specifically with the photoaffinity auxin analog 5-N3-7-3H-IAA (azido-IAA). Azido-IAA permits both the covalent and radioactive tagging of auxin-binding proteins and has allowed us to characterize further the 40-kDa and 42-kDa polypeptides, including the nature of their attachment to the PM, their relationship to each other, and their potential function. The azido-IAA-labeled polypeptides remain in the pelleted membrane fraction following high-salt and detergent washes, which indicates a tight and possibly integral association with the PM. Two-dimensional electrophoresis of partially purified azido-IAA-labeled protein demonstrates that, in addition to the major isoforms of the 40-kDa and 42-kDa polypeptides, which possess isoelectric points (pIs) of 8.2 and 7.2, respectively, several less abundant isoforms that display unique pIs are apparent at both molecular masses. Tryptic and chymotryptic digestion of the auxin-binding proteins indicates that the 40-kDa and 42-kDa polypeptides are closely related or are modifications of the same polypeptide. Phase extraction with the nonionic detergent Triton X-114 results in partitioning of the azido-IAA-labeled polypeptides into the aqueous (hydrophilic) phase. This apparently paradoxical behavior is also exhibited by certain integral membrane proteins that aggregate to form channels. The results of gel filtration indicate that the auxin-binding proteins do indeed aggregate strongly and that the polypeptides associate to form a dimer or multimeric complex in vivo. These characteristics are consistent with the hypothesis that the 40-kDa and 42-kDa polypeptides are subunits of a multimeric integral membrane protein which has an auxin-binding site, and which may

  13. The expanding universe of ribonucleoproteins: of novel RNA-binding proteins and unconventional interactions.

    Science.gov (United States)

    Beckmann, Benedikt M; Castello, Alfredo; Medenbach, Jan

    2016-06-01

    Post-transcriptional regulation of gene expression plays a critical role in almost all cellular processes. Regulation occurs mostly by RNA-binding proteins (RBPs) that recognise RNA elements and form ribonucleoproteins (RNPs) to control RNA metabolism from synthesis to decay. Recently, the repertoire of RBPs was significantly expanded owing to methodological advances such as RNA interactome capture. The newly identified RNA binders are involved in diverse biological processes and belong to a broad spectrum of protein families, many of them exhibiting enzymatic activities. This suggests the existence of an extensive crosstalk between RNA biology and other, in principle unrelated, cell functions such as intermediary metabolism. Unexpectedly, hundreds of new RBPs do not contain identifiable RNA-binding domains (RBDs), raising the question of how they interact with RNA. Despite the many functions that have been attributed to RNA, our understanding of RNPs is still mostly governed by a rather protein-centric view, leading to the idea that proteins have evolved to bind to and regulate RNA and not vice versa. However, RNPs formed by an RNA-driven interaction mechanism (RNA-determined RNPs) are abundant and offer an alternative explanation for the surprising lack of classical RBDs in many RNA-interacting proteins. Moreover, RNAs can act as scaffolds to orchestrate and organise protein networks and directly control their activity, suggesting that nucleic acids might play an important regulatory role in many cellular processes, including metabolism. PMID:27165283

  14. The Roles of Monomeric GTP-Binding Proteins in Macroautophagy in Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Shu Yang

    2014-10-01

    Full Text Available Autophagy is a cellular degradation process that sequesters components into a double-membrane structure called the autophagosome, which then fuses with the lysosome or vacuole for hydrolysis and recycling of building blocks. Bulk phase autophagy, also known as macroautophagy, controlled by specific Atg proteins, can be triggered by a variety of stresses, including starvation. Because autophagy relies extensively on membrane traffic to form the membranous structures, factors that control membrane traffic are essential for autophagy. Among these factors, the monomeric GTP-binding proteins that cycle between active and inactive conformations form an important group. In this review, we summarize the functions of the monomeric GTP-binding proteins in autophagy, especially with reference to experiments in Saccharomyces cerevisiae.

  15. Architecture of the sugar binding sites in carbohydrate binding proteins--a computer modeling study.

    Science.gov (United States)

    Rao, V S; Lam, K; Qasba, P K

    1998-11-01

    Different sugars, Gal, GalNAc and Man were docked at the monosaccharide binding sites of Erythrina corallodenron (EcorL), peanut lectin (PNA), Lathyrus ochrus (LOLI), and pea lectin (PSL). To study the lectin-carbohydrate interactions, in the complexes, the hydroxymethyl group in Man and Gal favors, gg and gt conformations respectively, and is the dominant recognition determination. The monosaccharide binding site in lectins that are specific to Gal/GalNAc is wider due to the additional amino acid residues in loop D as compared to that in lectins specific to Man/Glc, and affects the hydrogen bonds of the sugar involving residues from loop D, but not its orientation in the binding site. The invariant amino acid residues Asp from loop A, and Asn and an aromatic residue (Phe or Tyr) in loop C provides the basic architecture to recognize the common features in C4 epimers. The invariant Gly in loop B together with one or two residues in the variable region of loop D/A holds the sugar tightly at both ends. Loss of any one of these hydrogen bonds leads to weak interaction. While the subtle variations in the sequence and conformation of peptide fragment that resulted due to the size and location of gaps present in amino acid sequence in the neighborhood of the sugar binding site of loop D/A seems to discriminate the binding of sugars which differ at C4 atom (galacto and gluco configurations). The variations at loop B are important in discriminating Gal and GalNAc binding. The present study thus provides a structural basis for the observed specificities of legume lectins which uses the same four invariant residues for binding. These studies also bring out the information that is important for the design/engineering of proteins with the desired carbohydrate specificity. PMID:9849627

  16. Enhanced resistance in Theobroma cacao against oomycete and fungal pathogens by secretion of phosphatidylinositol-3-phosphate-binding proteins.

    Science.gov (United States)

    Helliwell, Emily E; Vega-Arreguín, Julio; Shi, Zi; Bailey, Bryan; Xiao, Shunyuan; Maximova, Siela N; Tyler, Brett M; Guiltinan, Mark J

    2016-03-01

    The internalization of some oomycete and fungal pathogen effectors into host plant cells has been reported to be blocked by proteins that bind to the effectors' cell entry receptor, phosphatidylinositol-3-phosphate (PI3P). This finding suggested a novel strategy for disease control by engineering plants to secrete PI3P-binding proteins. In this study, we tested this strategy using the chocolate tree Theobroma cacao. Transient expression and secretion of four different PI3P-binding proteins in detached leaves of T. cacao greatly reduced infection by two oomycete pathogens, Phytophthora tropicalis and Phytophthora palmivora, which cause black pod disease. Lesion size and pathogen growth were reduced by up to 85%. Resistance was not conferred by proteins lacking a secretory leader, by proteins with mutations in their PI3P-binding site, or by a secreted PI4P-binding protein. Stably transformed, transgenic T. cacao plants expressing two different PI3P-binding proteins showed substantially enhanced resistance to both P. tropicalis and P. palmivora, as well as to the fungal pathogen Colletotrichum theobromicola. These results demonstrate that secretion of PI3P-binding proteins is an effective way to increase disease resistance in T. cacao, and potentially in other plants, against a broad spectrum of pathogens. PMID:26214158

  17. Development of computational methods for the prediction of protein structure, protein binding, and mutational effects using free energy calculations.

    OpenAIRE

    Becker, Caroline

    2014-01-01

    A molecular understanding of protein-protein or protein-ligand binding is of crucial importance for the design of proteins or ligands with defined binding characteristics. The comprehensive analysis of biomolecular binding and the coupled rational in silico design of protein-ligand interfaces requires both, accurate and computationally fast methods for the prediction of free energies. Accurate free energy methods usually involve atomistic molecular dynamics simulations that are computationall...

  18. SiteComp: a server for ligand binding site analysis in protein structures

    OpenAIRE

    Lin, Yingjie; Yoo, Seungyeul; Sanchez, Roberto

    2012-01-01

    Motivation: Computational characterization of ligand-binding sites in proteins provides preliminary information for functional annotation, protein design and ligand optimization. SiteComp implements binding site analysis for comparison of binding sites, evaluation of residue contribution to binding sites and identification of sub-sites with distinct molecular interaction properties.

  19. Promoter-distal RNA polymerase II binding discriminates active from inactive CCAAT/ enhancer-binding protein beta binding sites

    Science.gov (United States)

    Savic, Daniel; Roberts, Brian S.; Carleton, Julia B.; Partridge, E. Christopher; White, Michael A.; Cohen, Barak A.; Cooper, Gregory M.; Gertz, Jason; Myers, Richard M.

    2015-01-01

    Transcription factors (TFs) bind to thousands of DNA sequences in mammalian genomes, but most of these binding events appear to have no direct effect on gene expression. It is unclear why only a subset of TF bound sites are actively involved in transcriptional regulation. Moreover, the key genomic features that accurately discriminate between active and inactive TF binding events remain ambiguous. Recent studies have identified promoter-distal RNA polymerase II (RNAP2) binding at enhancer elements, suggesting that these interactions may serve as a marker for active regulatory sequences. Despite these correlative analyses, a thorough functional validation of these genomic co-occupancies is still lacking. To characterize the gene regulatory activity of DNA sequences underlying promoter-distal TF binding events that co-occur with RNAP2 and TF sites devoid of RNAP2 occupancy using a functional reporter assay, we performed cis-regulatory element sequencing (CRE-seq). We tested more than 1000 promoter-distal CCAAT/enhancer-binding protein beta (CEBPB)-bound sites in HepG2 and K562 cells, and found that CEBPB-bound sites co-occurring with RNAP2 were more likely to exhibit enhancer activity. CEBPB-bound sites further maintained substantial cell-type specificity, indicating that local DNA sequence can accurately convey cell-type–specific regulatory information. By comparing our CRE-seq results to a comprehensive set of genome annotations, we identified a variety of genomic features that are strong predictors of regulatory element activity and cell-type–specific activity. Collectively, our functional assay results indicate that RNAP2 occupancy can be used as a key genomic marker that can distinguish active from inactive TF bound sites. PMID:26486725

  20. Functional interactions between polypyrimidine tract binding protein and PRI peptide ligand containing proteins.

    Science.gov (United States)

    Coelho, Miguel B; Ascher, David B; Gooding, Clare; Lang, Emma; Maude, Hannah; Turner, David; Llorian, Miriam; Pires, Douglas E V; Attig, Jan; Smith, Christopher W J

    2016-08-15

    Polypyrimidine tract binding protein (PTBP1) is a heterogeneous nuclear ribonucleoprotein (hnRNP) that plays roles in most stages of the life-cycle of pre-mRNA and mRNAs in the nucleus and cytoplasm. PTBP1 has four RNA binding domains of the RNA recognition motif (RRM) family, each of which can bind to pyrimidine motifs. In addition, RRM2 can interact via its dorsal surface with proteins containing short peptide ligands known as PTB RRM2 interacting (PRI) motifs, originally found in the protein Raver1. Here we review our recent progress in understanding the interactions of PTB with RNA and with various proteins containing PRI ligands. PMID:27528752

  1. Bile salt recognition by human liver fatty acid binding protein.

    Science.gov (United States)

    Favretto, Filippo; Santambrogio, Carlo; D'Onofrio, Mariapina; Molinari, Henriette; Grandori, Rita; Assfalg, Michael

    2015-04-01

    Fatty acid binding proteins (FABPs) act as intracellular carriers of lipid molecules, and play a role in global metabolism regulation. Liver FABP (L-FABP) is prominent among FABPs for its wide ligand repertoire, which includes long-chain fatty acids as well as bile acids (BAs). In this work, we performed a detailed molecular- and atomic-level analysis of the interactions established by human L-FABP with nine BAs to understand the binding specificity for this important class of cholesterol-derived metabolites. Protein-ligand complex formation was monitored using heteronuclear NMR, steady-state fluorescence spectroscopy, and mass spectrometry. BAs were found to interact with L-FABP with dissociation constants in the narrow range of 0.6-7 μm; however, the diverse substitution patterns of the sterol nucleus and the presence of side-chain conjugation resulted in complexes endowed with various degrees of conformational heterogeneity. Trihydroxylated BAs formed monomeric complexes in which single ligand molecules occupied similar internal binding sites, based on chemical-shift perturbation data. Analysis of NMR line shapes upon progressive addition of taurocholate indicated that the binding mechanism departed from a simple binary association equilibrium, and instead involved intermediates along the binding path. The co-linear chemical shift behavior observed for L-FABP complexes with cholate derivatives added insight into conformational dynamics in the presence of ligands. The observed spectroscopic features of L-FABP/BA complexes, discussed in relation to ligand chemistry, suggest possible molecular determinants of recognition, with implications regarding intracellular BA transport. Our findings suggest that human L-FABP is a poorly selective, universal BA binder. PMID:25639618

  2. Interactome map uncovers phosphatidylserine transport by oxysterol-binding proteins.

    Science.gov (United States)

    Maeda, Kenji; Anand, Kanchan; Chiapparino, Antonella; Kumar, Arun; Poletto, Mattia; Kaksonen, Marko; Gavin, Anne-Claude

    2013-09-12

    The internal organization of eukaryotic cells into functionally specialized, membrane-delimited organelles of unique composition implies a need for active, regulated lipid transport. Phosphatidylserine (PS), for example, is synthesized in the endoplasmic reticulum and then preferentially associates--through mechanisms not fully elucidated--with the inner leaflet of the plasma membrane. Lipids can travel via transport vesicles. Alternatively, several protein families known as lipid-transfer proteins (LTPs) can extract a variety of specific lipids from biological membranes and transport them, within a hydrophobic pocket, through aqueous phases. Here we report the development of an integrated approach that combines protein fractionation and lipidomics to characterize the LTP-lipid complexes formed in vivo. We applied the procedure to 13 LTPs in the yeast Saccharomyces cerevisiae: the six Sec14 homology (Sfh) proteins and the seven oxysterol-binding homology (Osh) proteins. We found that Osh6 and Osh7 have an unexpected specificity for PS. In vivo, they participate in PS homeostasis and the transport of this lipid to the plasma membrane. The structure of Osh6 bound to PS reveals unique features that are conserved among other metazoan oxysterol-binding proteins (OSBPs) and are required for PS recognition. Our findings represent the first direct evidence, to our knowledge, for the non-vesicular transfer of PS from its site of biosynthesis (the endoplasmic reticulum) to its site of biological activity (the plasma membrane). We describe a new subfamily of OSBPs, including human ORP5 and ORP10, that transfer PS and propose new mechanisms of action for a protein family that is involved in several human pathologies such as cancer, dyslipidaemia and metabolic syndrome. PMID:23934110

  3. Arylfluorosulfates Inactivate Intracellular Lipid Binding Protein(s) through Chemoselective SuFEx Reaction with a Binding Site Tyr Residue.

    Science.gov (United States)

    Chen, Wentao; Dong, Jiajia; Plate, Lars; Mortenson, David E; Brighty, Gabriel J; Li, Suhua; Liu, Yu; Galmozzi, Andrea; Lee, Peter S; Hulce, Jonathan J; Cravatt, Benjamin F; Saez, Enrique; Powers, Evan T; Wilson, Ian A; Sharpless, K Barry; Kelly, Jeffery W

    2016-06-15

    Arylfluorosulfates have appeared only rarely in the literature and have not been explored as probes for covalent conjugation to proteins, possibly because they were assumed to possess high reactivity, as with other sulfur(VI) halides. However, we find that arylfluorosulfates become reactive only under certain circumstances, e.g., when fluoride displacement by a nucleophile is facilitated. Herein, we explore the reactivity of structurally simple arylfluorosulfates toward the proteome of human cells. We demonstrate that the protein reactivity of arylfluorosulfates is lower than that of the corresponding aryl sulfonyl fluorides, which are better characterized with regard to proteome reactivity. We discovered that simple hydrophobic arylfluorosulfates selectively react with a few members of the intracellular lipid binding protein (iLBP) family. A central function of iLBPs is to deliver small-molecule ligands to nuclear hormone receptors. Arylfluorosulfate probe 1 reacts with a conserved tyrosine residue in the ligand-binding site of a subset of iLBPs. Arylfluorosulfate probes 3 and 4, featuring a biphenyl core, very selectively and efficiently modify cellular retinoic acid binding protein 2 (CRABP2), both in vitro and in living cells. The X-ray crystal structure of the CRABP2-4 conjugate, when considered together with binding site mutagenesis experiments, provides insight into how CRABP2 might activate arylfluorosulfates toward site-specific reaction. Treatment of breast cancer cells with probe 4 attenuates nuclear hormone receptor activity mediated by retinoic acid, an endogenous client lipid of CRABP2. Our findings demonstrate that arylfluorosulfates can selectively target single iLBPs, making them useful for understanding iLBP function. PMID:27191344

  4. Vaccinia complement control protein: Multi-functional protein and a potential wonder drug

    Indian Academy of Sciences (India)

    Purushottam Jha; Girish J Kotwal

    2003-04-01

    Vaccinia virus complement control protein (VCP) was one of the first viral molecules demonstrated to have a role in blocking complement and hence in the evasion of host defense. Structurally it is very similar to the human C4b-BP and the other members of complement control protein. Functionally it is most similar to the CR1 protein. VCP blocks both major pathways of complement activation. The crystal structure of VCP was determined a little over a year ago and it is the only known structure of an intact and complete complement control protein. In addition to binding complement, VCP also binds to heparin. These two binding abilities can take place simultaneously and contribute to its many function and to its potential use in several inflammatory diseases, e.g. Alzheimer’s disease (AD), CNS injury, xenotransplantation, etc. making it a truly fascinating molecule and potential drug.

  5. Buffer Interference with Protein Dynamics: A Case Study on Human Liver Fatty Acid Binding Protein

    OpenAIRE

    Long, Dong; Yang, Daiwen

    2009-01-01

    Selection of suitable buffer types is often a crucial step for generating appropriate protein samples for NMR and x-ray crystallographic studies. Although the possible interaction between MES buffer (2-(N-morpholino)ethanesulfonic acid) and proteins has been discussed previously, the interaction is usually thought to have no significant effects on the structures of proteins. In this study, we demonstrate the direct, albeit weak, interaction between MES and human liver fatty acid binding prote...

  6. Shrimp arginine kinase being a binding protein of WSSV envelope protein VP31

    Science.gov (United States)

    Ma, Cuiyan; Gao, Qiang; Liang, Yan; Li, Chen; Liu, Chao; Huang, Jie

    2016-03-01

    Viral entry into the host is the earliest stage of infection in the viral life cycle in which attachment proteins play a key role. VP31 (WSV340/WSSV396), an envelope protein of white spot syndrome virus (WSSV), contains an Arg-Gly-Asp (RGD) peptide domain known as a cellular attachment site. At present, the process of VP31 interacting with shrimp host cells has not been explored. Therefore, the VP31 gene was cloned into pET30a (+), expressed in Escherichia coli strain BL21 and purified with immobilized metal ion affinity chromatography. Four gill cellular proteins of shrimp (Fenneropenaeus chinensis) were pulled down by an affinity column coupled with recombinant VP31 (rVP31), and the amino acid sequences were identified with MALDI-TOF/TOF mass spectrometry. Hemocyanin, beta-actin, arginine kinase (AK), and an unknown protein were suggested as the putative VP31 receptor proteins. SDS-PAGE showed that AK is the predominant binding protein of VP31. An i n vitro binding activity experiment indicated that recombinant AK's (rAK) binding activity with rVP31 is comparable to that with the same amount of WSSV. These results suggested that AK, as a member of the phosphagen kinase family, plays a role in WSSV infection. This is the first evidence showing that AK is a binding protein of VP31. Further studies on this topic will elucidate WSSV infection mechanism in the future.

  7. Conformation-controlled binding kinetics of antibodies

    Science.gov (United States)

    Galanti, Marta; Fanelli, Duccio; Piazza, Francesco

    2016-01-01

    Antibodies are large, extremely flexible molecules, whose internal dynamics is certainly key to their astounding ability to bind antigens of all sizes, from small hormones to giant viruses. In this paper, we build a shape-based coarse-grained model of IgG molecules and show that it can be used to generate 3D conformations in agreement with single-molecule Cryo-Electron Tomography data. Furthermore, we elaborate a theoretical model that can be solved exactly to compute the binding rate constant of a small antigen to an IgG in a prescribed 3D conformation. Our model shows that the antigen binding process is tightly related to the internal dynamics of the IgG. Our findings pave the way for further investigation of the subtle connection between the dynamics and the function of large, flexible multi-valent molecular machines.

  8. Interplay between binding affinity and kinetics in protein-protein interactions.

    Science.gov (United States)

    Cao, Huaiqing; Huang, Yongqi; Liu, Zhirong

    2016-07-01

    To clarify the interplay between the binding affinity and kinetics of protein-protein interactions, and the possible role of intrinsically disordered proteins in such interactions, molecular simulations were carried out on 20 protein complexes. With bias potential and reweighting techniques, the free energy profiles were obtained under physiological affinities, which showed that the bound-state valley is deep with a barrier height of 12 - 33 RT. From the dependence of the affinity on interface interactions, the entropic contribution to the binding affinity is approximated to be proportional to the interface area. The extracted dissociation rates based on the Arrhenius law correlate reasonably well with the experimental values (Pearson correlation coefficient R = 0.79). For each protein complex, a linear free energy relationship between binding affinity and the dissociation rate was confirmed, but the distribution of the slopes for intrinsically disordered proteins showed no essential difference with that observed for ordered proteins. A comparison with protein folding was also performed. Proteins 2016; 84:920-933. © 2016 Wiley Periodicals, Inc. PMID:27018856

  9. Robust transcriptome-wide discovery of RNA-binding protein binding sites with enhanced CLIP (eCLIP).

    Science.gov (United States)

    Van Nostrand, Eric L; Pratt, Gabriel A; Shishkin, Alexander A; Gelboin-Burkhart, Chelsea; Fang, Mark Y; Sundararaman, Balaji; Blue, Steven M; Nguyen, Thai B; Surka, Christine; Elkins, Keri; Stanton, Rebecca; Rigo, Frank; Guttman, Mitchell; Yeo, Gene W

    2016-06-01

    As RNA-binding proteins (RBPs) play essential roles in cellular physiology by interacting with target RNA molecules, binding site identification by UV crosslinking and immunoprecipitation (CLIP) of ribonucleoprotein complexes is critical to understanding RBP function. However, current CLIP protocols are technically demanding and yield low-complexity libraries with high experimental failure rates. We have developed an enhanced CLIP (eCLIP) protocol that decreases requisite amplification by ∼1,000-fold, decreasing discarded PCR duplicate reads by ∼60% while maintaining single-nucleotide binding resolution. By simplifying the generation of paired IgG and size-matched input controls, eCLIP improves specificity in the discovery of authentic binding sites. We generated 102 eCLIP experiments for 73 diverse RBPs in HepG2 and K562 cells (available at https://www.encodeproject.org), demonstrating that eCLIP enables large-scale and robust profiling, with amplification and sample requirements similar to those of ChIP-seq. eCLIP enables integrative analysis of diverse RBPs to reveal factor-specific profiles, common artifacts for CLIP and RNA-centric perspectives on RBP activity. PMID:27018577

  10. Computational analysis of HIV-1 protease protein binding pockets.

    Science.gov (United States)

    Ko, Gene M; Reddy, A Srinivas; Kumar, Sunil; Bailey, Barbara A; Garg, Rajni

    2010-10-25

    Mutations that arise in HIV-1 protease after exposure to various HIV-1 protease inhibitors have proved to be a difficult aspect in the treatment of HIV. Mutations in the binding pocket of the protease can prevent the protease inhibitor from binding to the protein effectively. In the present study, the crystal structures of 68 HIV-1 proteases complexed with one of the nine FDA approved protease inhibitors from the Protein Data Bank (PDB) were analyzed by (a) identifying the mutational changes with the aid of a developed mutation map and (b) correlating the structure of the binding pockets with the complexed inhibitors. The mutations of each crystal structure were identified by comparing the amino acid sequence of each structure against the HIV-1 wild-type strain HXB2. These mutations were visually presented in the form of a mutation map to analyze mutation patterns corresponding to each protease inhibitor. The crystal structure mutation patterns of each inhibitor (in vitro) were compared against the mutation patterns observed in in vivo data. The in vitro mutation patterns were found to be representative of most of the major in vivo mutations. We then performed a data mining analysis of the binding pockets from each crystal structure in terms of their chemical descriptors to identify important structural features of the HIV-1 protease protein with respect to the binding conformation of the HIV-1 protease inhibitors. Data mining analysis is performed using several classification techniques: Random Forest (RF), linear discriminant analysis (LDA), and logistic regression (LR). We developed two hybrid models, RF-LDA and RF-LR. Random Forest is used as a feature selection proxy, reducing the descriptor space to a few of the most relevant descriptors determined by the classifier. These descriptors are then used to develop the subsequent LDA, LR, and hierarchical classification models. Clustering analysis of the binding pockets using the selected descriptors used to

  11. Human pentraxin 3 binds to the complement regulator c4b-binding protein.

    Directory of Open Access Journals (Sweden)

    Anne Braunschweig

    Full Text Available The long pentraxin 3 (PTX3 is a soluble recognition molecule with multiple functions including innate immune defense against certain microbes and the clearance of apoptotic cells. PTX3 interacts with recognition molecules of the classical and lectin complement pathways and thus initiates complement activation. In addition, binding of PTX3 to the alternative complement pathway regulator factor H was shown. Here, we show that PTX3 binds to the classical and lectin pathway regulator C4b-binding protein (C4BP. A PTX3-binding site was identified within short consensus repeats 1-3 of the C4BP α-chain. PTX3 did not interfere with the cofactor activity of C4BP in the fluid phase and C4BP maintained its complement regulatory activity when bound to PTX3 on surfaces. While C4BP and factor H did not compete for PTX3 binding, the interaction of C4BP with PTX3 was inhibited by C1q and by L-ficolin. PTX3 bound to human fibroblast- and endothelial cell-derived extracellular matrices and recruited functionally active C4BP to these surfaces. Whereas PTX3 enhanced the activation of the classical/lectin pathway and caused enhanced C3 deposition on extracellular matrix, deposition of terminal pathway components and the generation of the inflammatory mediator C5a were not increased. Furthermore, PTX3 enhanced the binding of C4BP to late apoptotic cells, which resulted in an increased rate of inactivation of cell surface bound C4b and a reduction in the deposition of C5b-9. Thus, in addition to complement activators, PTX3 interacts with complement inhibitors including C4BP. This balanced interaction on extracellular matrix and on apoptotic cells may prevent excessive local complement activation that would otherwise lead to inflammation and host tissue damage.

  12. Liver fatty acid-binding protein binds monoacylglycerol in vitro and in mouse liver cytosol.

    Science.gov (United States)

    Lagakos, William S; Guan, Xudong; Ho, Shiu-Ying; Sawicki, Luciana Rodriguez; Corsico, Betina; Kodukula, Sarala; Murota, Kaeko; Stark, Ruth E; Storch, Judith

    2013-07-01

    Liver fatty acid-binding protein (LFABP; FABP1) is expressed both in liver and intestinal mucosa. Mice null for LFABP were recently shown to have altered metabolism of not only fatty acids but also monoacylglycerol, the two major products of dietary triacylglycerol hydrolysis (Lagakos, W. S., Gajda, A. M., Agellon, L., Binas, B., Choi, V., Mandap, B., Russnak, T., Zhou, Y. X., and Storch, J. (2011) Am. J. Physiol. Gastrointest. Liver Physiol. 300, G803-G814). Nevertheless, the binding and transport of monoacylglycerol (MG) by LFABP are uncertain, with conflicting reports in the literature as to whether this single chain amphiphile is in fact bound by LFABP. In the present studies, gel filtration chromatography of liver cytosol from LFABP(-/-) mice shows the absence of the low molecular weight peak of radiolabeled monoolein present in the fractions that contain LFABP in cytosol from wild type mice, indicating that LFABP binds sn-2 MG in vivo. Furthermore, solution-state NMR spectroscopy demonstrates two molecules of sn-2 monoolein bound in the LFABP binding pocket in positions similar to those found for oleate binding. Equilibrium binding affinities are ∼2-fold lower for MG compared with fatty acid. Finally, kinetic studies examining the transfer of a fluorescent MG analog show that the rate of transfer of MG is 7-fold faster from LFABP to phospholipid membranes than from membranes to membranes and occurs by an aqueous diffusion mechanism. These results provide strong support for monoacylglycerol as a physiological ligand for LFABP and further suggest that LFABP functions in the efficient intracellular transport of MG. PMID:23658011

  13. Liver Fatty Acid-binding Protein Binds Monoacylglycerol in Vitro and in Mouse Liver Cytosol*

    Science.gov (United States)

    Lagakos, William S.; Guan, Xudong; Ho, Shiu-Ying; Sawicki, Luciana Rodriguez; Corsico, Betina; Kodukula, Sarala; Murota, Kaeko; Stark, Ruth E.; Storch, Judith

    2013-01-01

    Liver fatty acid-binding protein (LFABP; FABP1) is expressed both in liver and intestinal mucosa. Mice null for LFABP were recently shown to have altered metabolism of not only fatty acids but also monoacylglycerol, the two major products of dietary triacylglycerol hydrolysis (Lagakos, W. S., Gajda, A. M., Agellon, L., Binas, B., Choi, V., Mandap, B., Russnak, T., Zhou, Y. X., and Storch, J. (2011) Am. J. Physiol. Gastrointest. Liver Physiol. 300, G803–G814). Nevertheless, the binding and transport of monoacylglycerol (MG) by LFABP are uncertain, with conflicting reports in the literature as to whether this single chain amphiphile is in fact bound by LFABP. In the present studies, gel filtration chromatography of liver cytosol from LFABP−/− mice shows the absence of the low molecular weight peak of radiolabeled monoolein present in the fractions that contain LFABP in cytosol from wild type mice, indicating that LFABP binds sn-2 MG in vivo. Furthermore, solution-state NMR spectroscopy demonstrates two molecules of sn-2 monoolein bound in the LFABP binding pocket in positions similar to those found for oleate binding. Equilibrium binding affinities are ∼2-fold lower for MG compared with fatty acid. Finally, kinetic studies examining the transfer of a fluorescent MG analog show that the rate of transfer of MG is 7-fold faster from LFABP to phospholipid membranes than from membranes to membranes and occurs by an aqueous diffusion mechanism. These results provide strong support for monoacylglycerol as a physiological ligand for LFABP and further suggest that LFABP functions in the efficient intracellular transport of MG. PMID:23658011

  14. Targeting Human Cancer by a Glycosaminoglycan Binding Malaria Protein

    DEFF Research Database (Denmark)

    Salanti, Ali; Clausen, Thomas M.; Agerbæk, Mette Ø.;

    2015-01-01

    Plasmodium falciparum engineer infected erythrocytes to present the malarial protein, VAR2CSA, which binds a distinct type chondroitin sulfate (CS) exclusively expressed in the placenta. Here, we show that the same CS modification is present on a high proportion of malignant cells and that it can...... be specifically targeted by recombinant VAR2CSA (rVAR2). In tumors, placental-like CS chains are linked to a limited repertoire of cancer-associated proteoglycans including CD44 and CSPG4. The rVAR2 protein localizes to tumors in vivo and rVAR2 fused to diphtheria toxin or conjugated to hemiasterlin compounds...... strongly inhibits in vivo tumor cell growth and metastasis. Our data demonstrate how an evolutionarily refined parasite-derived protein can be exploited to target a common, but complex, malignancy-associated glycosaminoglycan modification....

  15. Heart-type fatty-acid-binding protein (FABP3) is a lysophosphatidic acid-binding protein in human coronary artery endothelial cells

    Science.gov (United States)

    Tsukahara, Ryoko; Haniu, Hisao; Matsuda, Yoshikazu; Tsukahara, Tamotsu

    2014-01-01

    Fatty-acid-binding protein 3, muscle and heart (FABP3), also known as heart-type FABP, is a member of the family of intracellular lipid-binding proteins. It is a small cytoplasmic protein with a molecular mass of about 15 kDa. FABPs are known to be carrier proteins for transporting fatty acids and other lipophilic substances from the cytoplasm to the nucleus, where these lipids are released to a group of nuclear receptors such as peroxisome proliferator-activated receptors (PPARs). In this study, using lysophosphatidic acid (LPA)-coated agarose beads, we have identified FABP3 as an LPA carrier protein in human coronary artery endothelial cells (HCAECs). Administration of LPA to HCAECs resulted in a dose-dependent increase in PPARγ activation. Furthermore, the LPA-induced PPARγ activation was abolished when the FABP3 expression was reduced using small interfering RNA (siRNA). We further show that the nuclear fraction of control HCAECs contained a significant amount of exogenously added LPA, whereas FABP3 siRNA-transfected HCAECs had a decreased level of LPA in the nucleus. Taken together, these results suggest that FABP3 governs the transcriptional activities of LPA by targeting them to cognate PPARγ in the nucleus. PMID:25426414

  16. Heart-type fatty-acid-binding protein (FABP3 is a lysophosphatidic acid-binding protein in human coronary artery endothelial cells

    Directory of Open Access Journals (Sweden)

    Ryoko Tsukahara

    2014-01-01

    Full Text Available Fatty-acid-binding protein 3, muscle and heart (FABP3, also known as heart-type FABP, is a member of the family of intracellular lipid-binding proteins. It is a small cytoplasmic protein with a molecular mass of about 15 kDa. FABPs are known to be carrier proteins for transporting fatty acids and other lipophilic substances from the cytoplasm to the nucleus, where these lipids are released to a group of nuclear receptors such as peroxisome proliferator-activated receptors (PPARs. In this study, using lysophosphatidic acid (LPA-coated agarose beads, we have identified FABP3 as an LPA carrier protein in human coronary artery endothelial cells (HCAECs. Administration of LPA to HCAECs resulted in a dose-dependent increase in PPARγ activation. Furthermore, the LPA-induced PPARγ activation was abolished when the FABP3 expression was reduced using small interfering RNA (siRNA. We further show that the nuclear fraction of control HCAECs contained a significant amount of exogenously added LPA, whereas FABP3 siRNA-transfected HCAECs had a decreased level of LPA in the nucleus. Taken together, these results suggest that FABP3 governs the transcriptional activities of LPA by targeting them to cognate PPARγ in the nucleus.

  17. The clinical significance of fatty acid binding proteins

    Directory of Open Access Journals (Sweden)

    Barbara Choromańska

    2011-11-01

    Full Text Available Excessive levels of free fatty acids are toxic to cells. The human body has evolved a defense mechanism in the form of small cytoplasmic proteins called fatty acid binding proteins (FABPs that bind long-chain fatty acids (LCFA, and then refer them to appropriate intracellular disposal sites (oxidation in mitochondria and peroxisomes or storage in the endoplasmic reticulum. So far, nine types of these proteins have been described, and their name refers to the place in which they were first identified or where they can be found in the greatest concentration. The most important FABPs were isolated from the liver (L-FABP, heart (H-FABP, intestine (I-FABP, brain (B-FABP, epidermis (E-FABP and adipocytes (A-FABP. Determination of H-FABP is used in the diagnosis of myocardial infarction, and L-FABP in kidney lesions of different etiologies. It is postulated that FABPs play an important role in the pathogenesis of metabolic diseases. Elevated levels of A-FABP have been found in the pericardial fat tissue and were associated with cardiac dysfunction in obese people. A rise in A-FABP has been observed in patients with type II diabetes. I-FABP is known as a marker of cell damage in the small intestine. Increased concentration of B-FABP has been associated with human brain tumors such as glioblastoma and astrocytoma, as well as with neurodegenerative diseases (Alzheimer’s, Parkinson’s and other disorders of cognitive function. The aim of this work was to present current data on the clinical significance of fatty acid binding proteins.

  18. Cloud computing for protein-ligand binding site comparison.

    Science.gov (United States)

    Hung, Che-Lun; Hua, Guan-Jie

    2013-01-01

    The proteome-wide analysis of protein-ligand binding sites and their interactions with ligands is important in structure-based drug design and in understanding ligand cross reactivity and toxicity. The well-known and commonly used software, SMAP, has been designed for 3D ligand binding site comparison and similarity searching of a structural proteome. SMAP can also predict drug side effects and reassign existing drugs to new indications. However, the computing scale of SMAP is limited. We have developed a high availability, high performance system that expands the comparison scale of SMAP. This cloud computing service, called Cloud-PLBS, combines the SMAP and Hadoop frameworks and is deployed on a virtual cloud computing platform. To handle the vast amount of experimental data on protein-ligand binding site pairs, Cloud-PLBS exploits the MapReduce paradigm as a management and parallelizing tool. Cloud-PLBS provides a web portal and scalability through which biologists can address a wide range of computer-intensive questions in biology and drug discovery. PMID:23762824

  19. Serum Vitamin D, Vitamin D Binding Protein, and Risk of Colorectal Cancer

    OpenAIRE

    Anic, Gabriella M.; Weinstein, Stephanie J.; Mondul, Alison M.; Satu Männistö; Demetrius Albanes

    2014-01-01

    Background We previously reported a positive association between serum 25-hydroxyvitamin D (25(OH)D) and colorectal cancer risk. To further elucidate this association, we examined the molar ratio of 25(OH)D to vitamin D binding protein (DBP), the primary 25(OH)D transport protein, and whether DBP modified the association between 25(OH)D and colorectal cancer risk. Methods In a nested case-control study within the Alpha-Tocopherol, Beta-Carotene Cancer Prevention Study, controls were 1∶1 match...

  20. Arabidopsis chloroplast chaperonin 10 is a calmodulin-binding protein

    Science.gov (United States)

    Yang, T.; Poovaiah, B. W.

    2000-01-01

    Calcium regulates diverse cellular activities in plants through the action of calmodulin (CaM). By using (35)S-labeled CaM to screen an Arabidopsis seedling cDNA expression library, a cDNA designated as AtCh-CPN10 (Arabidopsis thaliana chloroplast chaperonin 10) was cloned. Chloroplast CPN10, a nuclear-encoded protein, is a functional homolog of E. coli GroES. It is believed that CPN60 and CPN10 are involved in the assembly of Rubisco, a key enzyme involved in the photosynthetic pathway. Northern analysis revealed that AtCh-CPN10 is highly expressed in green tissues. The recombinant AtCh-CPN10 binds to CaM in a calcium-dependent manner. Deletion mutants revealed that there is only one CaM-binding site in the last 31 amino acids of the AtCh-CPN10 at the C-terminal end. The CaM-binding region in AtCh-CPN10 has higher homology to other chloroplast CPN10s in comparison to GroES and mitochondrial CPN10s, suggesting that CaM may only bind to chloroplast CPN10s. Furthermore, the results also suggest that the calcium/CaM messenger system is involved in regulating Rubisco assembly in the chloroplast, thereby influencing photosynthesis. Copyright 2000 Academic Press.

  1. Integrating protein structures and precomputed genealogies in the Magnum database: Examples with cellular retinoid binding proteins

    Directory of Open Access Journals (Sweden)

    Bradley Michael E

    2006-02-01

    Full Text Available Abstract Background When accurate models for the divergent evolution of protein sequences are integrated with complementary biological information, such as folded protein structures, analyses of the combined data often lead to new hypotheses about molecular physiology. This represents an excellent example of how bioinformatics can be used to guide experimental research. However, progress in this direction has been slowed by the lack of a publicly available resource suitable for general use. Results The precomputed Magnum database offers a solution to this problem for ca. 1,800 full-length protein families with at least one crystal structure. The Magnum deliverables include 1 multiple sequence alignments, 2 mapping of alignment sites to crystal structure sites, 3 phylogenetic trees, 4 inferred ancestral sequences at internal tree nodes, and 5 amino acid replacements along tree branches. Comprehensive evaluations revealed that the automated procedures used to construct Magnum produced accurate models of how proteins divergently evolve, or genealogies, and correctly integrated these with the structural data. To demonstrate Magnum's capabilities, we asked for amino acid replacements requiring three nucleotide substitutions, located at internal protein structure sites, and occurring on short phylogenetic tree branches. In the cellular retinoid binding protein family a site that potentially modulates ligand binding affinity was discovered. Recruitment of cellular retinol binding protein to function as a lens crystallin in the diurnal gecko afforded another opportunity to showcase the predictive value of a browsable database containing branch replacement patterns integrated with protein structures. Conclusion We integrated two areas of protein science, evolution and structure, on a large scale and created a precomputed database, known as Magnum, which is the first freely available resource of its kind. Magnum provides evolutionary and structural

  2. In vitro binding of selenium by rat liver mitochondrial selenium-binding protein

    International Nuclear Information System (INIS)

    Last year the authors reported that upon freezing and thawing mitochondria from rats injected with [75Se]Na2SeO3 (75Se-selenite), a 75Se-binding protein (SeBP) was released. They have studied further in vitro labelling of SeBP. This matrix protein was labelled in vitro when lysed mitochondria (containing non-matrix material) were incubated with 75Se-selenite but not when matrix material alone was incubated with 75Se-selenite. Thus, there are one or more promoters of in vitro SeBP labelling in the non-matrix fraction. SeBP was also labelled in vitro when 75Se-selenite was added to matrix alone and dialyzed. Dialysis tubing, and not the dialysis process, promoted labelling by affecting SeBP and not by affecting 75Se-selenite. Labelling did not occur when matrix alone and 75Se-selenite were incubated (not dialyzed) in a glass test tube but did occur in a polystyrene test tube. They hypothesize that non-covalent interactions occur between SeBP and dialysis tubing or polystyrene that expose Se binding sites on the protein. A similar mechanism involving mitochondrial non-matrix material may function in vivo. Non-denaturing disc gel electrophoresis of partially purified SeBP labelled in vivo or in vitro suggested that the same protein was labelled in both conditions. Using in vitro binding techniques, SeBP was also found in sheep liver mitochondrial matrix. This supports the theory that SeBP is important in Se metabolism

  3. Functional zinc-binding motifs in enzymes and DNA-binding proteins.

    Science.gov (United States)

    Vallee, B L; Auld, D S

    1992-01-01

    Zinc is now known to be an integral component of a large number and variety of enzymes and proteins involved in virtually all aspects of metabolism, thus accounting for the fact that this element is essential for growth and development. The chemistry of zinc, superficially bland, in reality has turned out to be ideally appropriate and versatile for the unexpected development of multiple and unique chemical structures which biology has used for specific life processes. The present discussion will centre on those distinctive zinc-binding motifs that are critical both to enzyme function and the expression of the genetic message. X-Ray diffraction structure determination of 15 zinc enzymes belonging to IUB classes I-IV provide absolute standards of reference for the identity and nature of zinc ligands in their families. Three types of zinc enzyme binding motifs emerge through analysis of these: catalytic, coactive or cocatalytic, and structural. In contrast to zinc enzymes virtually all DNA-binding proteins contain multiple zinc atoms. With the availability of NMR and X-ray structure analyses three distinct motifs now emerge for those: zinc fingers, twists and clusters. PMID:1290939

  4. The Movable Type Method Applied to Protein-Ligand Binding

    Science.gov (United States)

    Zheng, Zheng; Ucisik, Melek N.; Merz, Kenneth M.

    2013-01-01

    Accurately computing the free energy for biological processes like protein folding or protein-ligand association remains a challenging problem. Both describing the complex intermolecular forces involved and sampling the requisite configuration space make understanding these processes innately difficult. Herein, we address the sampling problem using a novel methodology we term “movable type”. Conceptually it can be understood by analogy with the evolution of printing and, hence, the name movable type. For example, a common approach to the study of protein-ligand complexation involves taking a database of intact drug-like molecules and exhaustively docking them into a binding pocket. This is reminiscent of early woodblock printing where each page had to be laboriously created prior to printing a book. However, printing evolved to an approach where a database of symbols (letters, numerals, etc.) was created and then assembled using a movable type system, which allowed for the creation of all possible combinations of symbols on a given page, thereby, revolutionizing the dissemination of knowledge. Our movable type (MT) method involves the identification of all atom pairs seen in protein-ligand complexes and then creating two databases: one with their associated pairwise distant dependent energies and another associated with the probability of how these pairs can combine in terms of bonds, angles, dihedrals and non-bonded interactions. Combining these two databases coupled with the principles of statistical mechanics allows us to accurately estimate binding free energies as well as the pose of a ligand in a receptor. This method, by its mathematical construction, samples all of configuration space of a selected region (the protein active site here) in one shot without resorting to brute force sampling schemes involving Monte Carlo, genetic algorithms or molecular dynamics simulations making the methodology extremely efficient. Importantly, this method explores the

  5. Zinc ions bind to and inhibit activated protein C

    DEFF Research Database (Denmark)

    Zhu, Tianqing; Ubhayasekera, Wimal; Nickolaus, Noëlle;

    2010-01-01

    Zn2+ ions were found to efficiently inhibit activated protein C (APC), suggesting a potential regulatory function for such inhibition. APC activity assays employing a chromogenic peptide substrate demonstrated that the inhibition was reversible and the apparent K I was 13 +/- 2 microM. k cat was...... seven fold decreased whereas K M was unaffected in the presence of 10 microM Zn2+. The inhibitory effect of Zn2+ on APC activity was also observed when factor Va was used as a substrate in an assay coupled to a prothrombinase assay. The interaction of Zn2+ with APC was accompanied by a reversible...... fold enhanced, presumably due to the Ca2+-induced conformational change affecting the conformation of the Zn2+-binding site. The inhibition mechanism was non-competitive both in the absence and presence of Ca2+. Comparisons of sequences and structures suggested several possible sites for zinc binding...

  6. A unique bivalent binding and inhibition mechanism by the yatapoxvirus interleukin 18 binding protein.

    Directory of Open Access Journals (Sweden)

    Brian Krumm

    Full Text Available Interleukin 18 (IL18 is a cytokine that plays an important role in inflammation as well as host defense against microbes. Mammals encode a soluble inhibitor of IL18 termed IL18 binding protein (IL18BP that modulates IL18 activity through a negative feedback mechanism. Many poxviruses encode homologous IL18BPs, which contribute to virulence. Previous structural and functional studies on IL18 and IL18BPs revealed an essential binding hot spot involving a lysine on IL18 and two aromatic residues on IL18BPs. The aromatic residues are conserved among the very diverse mammalian and poxviruses IL18BPs with the notable exception of yatapoxvirus IL18BPs, which lack a critical phenylalanine residue. To understand the mechanism by which yatapoxvirus IL18BPs neutralize IL18, we solved the crystal structure of the Yaba-Like Disease Virus (YLDV IL18BP and IL18 complex at 1.75 Å resolution. YLDV-IL18BP forms a disulfide bonded homo-dimer engaging IL18 in a 2∶2 stoichiometry, in contrast to the 1∶1 complex of ectromelia virus (ECTV IL18BP and IL18. Disruption of the dimer interface resulted in a functional monomer, however with a 3-fold decrease in binding affinity. The overall architecture of the YLDV-IL18BP:IL18 complex is similar to that observed in the ECTV-IL18BP:IL18 complex, despite lacking the critical lysine-phenylalanine interaction. Through structural and mutagenesis studies, contact residues that are unique to the YLDV-IL18BP:IL18 binding interface were identified, including Q67, P116 of YLDV-IL18BP and Y1, S105 and D110 of IL18. Overall, our studies show that YLDV-IL18BP is unique among the diverse family of mammalian and poxvirus IL-18BPs in that it uses a bivalent binding mode and a unique set of interacting residues for binding IL18. However, despite this extensive divergence, YLDV-IL18BP binds to the same surface of IL18 used by other IL18BPs, suggesting that all IL18BPs use a conserved inhibitory mechanism by blocking a putative receptor-binding

  7. Intramitochondrial localization of universal minicircle sequence-binding protein, a trypanosomatid protein that binds kinetoplast minicircle replication origins.

    Science.gov (United States)

    Abu-Elneel, K; Robinson, D R; Drew, M E; Englund, P T; Shlomai, J

    2001-05-14

    Kinetoplast DNA (kDNA), the mitochondrial DNA of the trypanosomatid Crithidia fasciculata, is a unique structure containing 5,000 DNA minicircles topologically linked into a massive network. In vivo, the network is condensed into a disk-shaped structure. Replication of minicircles initiates at unique origins that are bound by universal minicircle sequence (UMS)-binding protein (UMSBP), a sequence-specific DNA-binding protein. This protein, encoded by a nuclear gene, localizes within the cell's single mitochondrion. Using immunofluorescence, we found that UMSBP localizes exclusively to two neighboring sites adjacent to the face of the kDNA disk nearest the cell's flagellum. This site is distinct from the two antipodal positions at the perimeter of the disk that is occupied by DNA polymerase beta, topoisomerase II, and a structure-specific endonuclease. Although we found constant steady-state levels of UMSBP mRNA and protein and a constant rate of UMSBP synthesis throughout the cell cycle, immunofluorescence indicated that UMSBP localization within the kinetoplast is not static. The intramitochondrial localization of UMSBP and other kDNA replication enzymes significantly clarifies our understanding of the process of kDNA replication. PMID:11352934

  8. Factors Affecting the Binding of a Recombinant Heavy Metal-Binding Domain (CXXC motif Protein to Heavy Metals

    Directory of Open Access Journals (Sweden)

    Kamala Boonyodying

    2012-06-01

    Full Text Available A number of heavy metal-binding proteins have been used to study bioremediation. CXXC motif, a metal binding domain containing Cys-X-X-Cys motif, has been identified in various organisms. These proteins are capable of binding various types of heavy metals. In this study, heavy metal binding domain (CXXC motif recombinant protein encoded from mcsA gene of S. aureus were cloned and overexpressed in Escherichia coli. The factors involved in the metal-binding activity were determined in order to analyze the potential of recombinant protein for bioremediation. A recombinant protein can be bound to Cd2+, Co2+, Cu2+ and Zn2+. The thermal stability of a recombinant protein was tested, and the results showed that the metal binding activity to Cu2+ and Zn2+ still exist after treating the protein at 85ºC for 30 min. The temperature and pH that affected the metal binding activity was tested and the results showed that recombinant protein was still bound to Cu2+ at 65ºC, whereas a pH of 3-7 did not affect the metal binding E. coli harboring a pRset with a heavy metal-binding domain CXXC motif increased the resistance of heavy metals against CuCl2 and CdCl2. This study shows that metal binding domain (CXXC motif recombinant protein can be effectively bound to various types of heavy metals and may be used as a potential tool for studying bioremediation.

  9. Calcium ion binding properties and the effect of phosphorylation on the intrinsically disordered Starmaker protein.

    Science.gov (United States)

    Wojtas, Magdalena; Hołubowicz, Rafał; Poznar, Monika; Maciejewska, Marta; Ożyhar, Andrzej; Dobryszycki, Piotr

    2015-10-27

    Starmaker (Stm) is an intrinsically disordered protein (IDP) involved in otolith biomineralization in Danio rerio. Stm controls calcium carbonate crystal formation in vivo and in vitro. Phosphorylation of Stm affects its biomineralization properties. This study examined the effects of calcium ions and phosphorylation on the structure of Stm. We have shown that CK2 kinase phosphorylates 25 or 26 residues in Stm. Furthermore, we have demonstrated that Stm's affinity for calcium binding is dependent on its phosphorylation state. Phosphorylated Stm (StmP) has an estimated 30 ± 1 calcium binding sites per protein molecule with a dissociation constant (KD) of 61 ± 4 μM, while the unphosphorylated protein has 28 ± 3 sites and a KD of 210 ± 22 μM. Calcium ion binding induces a compaction of the Stm molecule, causing a significant decrease in its hydrodynamic radius and the formation of a secondary structure. The screening effect of Na(+) ions on calcium binding was also observed. Analysis of the hydrodynamic properties of Stm and StmP showed that Stm and StmP molecules adopt the structure of native coil-like proteins. PMID:26445027

  10. HCV NS5A abrogates p53 protein function by interfering with p53-DNA binding

    Institute of Scientific and Technical Information of China (English)

    Guo-Zhong Gong; Yong-Fang Jiang; Yan He; Li-Ying Lai; Ying-Hua Zhu; Xian-Shi Su

    2004-01-01

    AIM: To evaluate the inhibition effect of HCV NS5A on p53 transactivation on p21 promoter and explore its possible mechanism for influencing p53 function.METHODS: p53 function of transactivation on p21 promoter was studied with a luciferase reporter system in which the luciferase gene is driven by p21 promoter, and the p53-DNA binding ability was observed with the use of electrophoretic mobility-shift assay (EMSA). Lipofectin mediated p53 or HCV NS5A expression vectors were used to transfect hepatoma cell lines to observe whether HCV NS5A could abrogate the binding ability of p53 to its specific DNA sequence and p53 transactivation on p21 promoter.Western blot experiment was used for detection of HCV NS5A and p53 proteins expression.RESULTS: Relative luciferase activity driven by p21 promoter increased significantly in the presence of endogenous p53 protein. Compared to the control group, exogenous p53 protein also stimulated p21 promoter driven luciferase gene expression in a dose-dependent way. HCV NS5A protein gradually inhibited both endogenous and exogenous p53 transactivation on p21 promoter with increase of the dose of HCV NS5A expression plasmid. By the experiment of EMSA, we could find p53 binding to its specific DNA sequence and, when co-transfected with increased dose of HCV NS5A expression vector, the p53 binding affinity to its DNA gradually decreased and finally disappeared. Between the Huh 7 cells transfected with p53 expression vector alone or co-transfected with HCV NS5A expression vector, there was no difference in the p53 protein expression.CONCLUSION: HCV NS5A inhibits p53 transactivation on p21 promoter through abrogating p53 binding affinity to its specific DNA sequence. It does not affect p53 protein expression.

  11. RNA-binding protein Dnd1 inhibits microRNA access to target mRNA

    DEFF Research Database (Denmark)

    Kedde, Martijn; Strasser, Markus J; Boldajipour, Bijan;

    2007-01-01

    MicroRNAs (miRNAs) are inhibitors of gene expression capable of controlling processes in normal development and cancer. In mammals, miRNAs use a seed sequence of 6-8 nucleotides (nt) to associate with 3' untranslated regions (3'UTRs) of mRNAs and inhibit their expression. Intriguingly, occasionally...... evolutionary conserved RNA-binding protein (RBP), counteracts the function of several miRNAs in human cells and in primordial germ cells of zebrafish by binding mRNAs and prohibiting miRNAs from associating with their target sites. These effects of Dnd1 are mediated through uridine-rich regions present in the...

  12. The MTA family proteins as novel histone H3 binding proteins

    Directory of Open Access Journals (Sweden)

    Wu Meng

    2013-01-01

    Full Text Available Abstract Background The nucleosome remodeling and histone deacetylase complex (Mi2/NRD/NuRD/NURD has a broad role in regulation of transcription, DNA repair and cell cycle. Previous studies have revealed a specific interaction between NURD and histone H3N-terminal tail in vitro that is not observed for another HDAC1/2-containing complex, Sin3A. However, the subunit(s responsible for specific binding of H3 by NURD has not been defined. Results In this study, we show among several class I HDAC-containing corepressor complexes only NURD exhibits a substantial H3 tail-binding activity in vitro. We present the evidence that the MTA family proteins within the NURD complex interact directly with H3 tail. Extensive in vitro binding assays mapped the H3 tail-binding domain to the C-terminal region of MTA1 and MTA2. Significantly, although the MTA1 and MTA2 mutant proteins with deletion of the C-terminal H3 tail binding domain were assembled into the endogenous NURD complex when expressed in mammalian cells, the resulting NURD complexes were deficient in binding H3 tail in vitro, indicating that the MTA family proteins are required for the observed specific binding of H3 tail peptide by NURD in vitro. However, chromatin fractionation experiments show that the NURD complexes with impaired MTA1/2-H3 tail binding activity remained to be associated with chromatin in cells. Conclusions Together our study reveals a novel histone H3-binding activity for the MTA family proteins and provides evidence that the MTA family proteins mediate the in vitro specific binding of H3 tail peptide by NURD complex. However, multiple mechanisms are likely to contribute to the chromatin association of NURD complex in cells. Our finding also raises the possibility that the MTA family proteins may exert their diverse biological functions at least in part through their direct interaction with H3 tail.

  13. Using persistent homology and dynamical distances to analyze protein binding.

    Science.gov (United States)

    Kovacev-Nikolic, Violeta; Bubenik, Peter; Nikolić, Dragan; Heo, Giseon

    2016-03-01

    Persistent homology captures the evolution of topological features of a model as a parameter changes. The most commonly used summary statistics of persistent homology are the barcode and the persistence diagram. Another summary statistic, the persistence landscape, was recently introduced by Bubenik. It is a functional summary, so it is easy to calculate sample means and variances, and it is straightforward to construct various test statistics. Implementing a permutation test we detect conformational changes between closed and open forms of the maltose-binding protein, a large biomolecule consisting of 370 amino acid residues. Furthermore, persistence landscapes can be applied to machine learning methods. A hyperplane from a support vector machine shows the clear separation between the closed and open proteins conformations. Moreover, because our approach captures dynamical properties of the protein our results may help in identifying residues susceptible to ligand binding; we show that the majority of active site residues and allosteric pathway residues are located in the vicinity of the most persistent loop in the corresponding filtered Vietoris-Rips complex. This finding was not observed in the classical anisotropic network model. PMID:26812805

  14. Recognition of methylated DNA through methyl-CpG binding domain proteins

    DEFF Research Database (Denmark)

    Zou, Xueqing; Ma, Wen; Solov'yov, Ilia;

    2012-01-01

    DNA methylation is a key regulatory control route in epigenetics, involving gene silencing and chromosome inactivation. It has been recognized that methyl-CpG binding domain (MBD) proteins play an important role in interpreting the genetic information encoded by methylated DNA (mDNA). Although the...... function of MBD proteins has attracted considerable attention and is well characterized, the mechanism underlying mDNA recognition by MBD proteins is still poorly understood. In this article, we demonstrate that the methyl-CpG dinucleotides are recognized at the MBD-mDNA interface by two MBD arginines...... through an interplay of hydrogen bonding and cation-p interaction. Through molecular dynamics and quantum-chemistry calculations we investigate the methyl-cytosine recognition process and demonstrate that methylation enhances MBD-mDNA binding by increasing the hydrophobic interfacial area and by...

  15. Interaction of the anaphase-promoting complex/cyclosome and proteasome protein complexes with multiubiquitin chain-binding proteins

    DEFF Research Database (Denmark)

    Seeger, Michael; Hartmann-Petersen, Rasmus; Wilkinson, Caroline R M; Wallace, Mairi; Samejima, Itaru; Taylor, Martin S; Gordon, Colin

    2003-01-01

    Fission yeast Rhp23 and Pus1 represent two families of multiubiquitin chain-binding proteins that associate with the proteasome. We show that both proteins bind to different regions of the proteasome subunit Mts4. The binding site for Pus1 was mapped to a cluster of repetitive sequences also foun...

  16. DnaT is a PriC-binding protein.

    Science.gov (United States)

    Huang, Chien-Chih; Huang, Cheng-Yang

    2016-09-01

    DnaT and PriC are replication restart primosomal proteins required for re-initiating chromosomal DNA replication. DnaT is a component of the PriA-dependent primosome, while PriC belongs to the PriC-dependent primosome. Whether DnaT can interact with PriC is still unknown. In this study, we define a direct interaction between PriC, a key initiator protein in PriC-mediated DNA replication restart, and DnaT, a DnaB/C complex loader protein, from Klebsiella pneumoniae. In fluorescence titrations, PriC bound to single-stranded DNA with a binding-site size of approximately 9 nt. Gold nanoparticle assay showed that the solution of DnaT-PriC changed from red to purple, which indicated the protein-protein interactions due to gold nanoparticle aggregate. In addition, this DnaT-PriC complex could be co-purified by the heparin HP column. Surface plasmon resonance analysis showed that the Kd value of DnaT bound to PriC was 2.9 × 10(-8) M. These results constitute a pioneering study of the DnaT-PriC interaction and present a putative link between the two independent replication restart pathways, namely, PriA- and PriC-dependent primosome assemblies. Further research can directly focus on determining how DnaT binds to the PriC-SSB-DNA tricomplex and regulates the PriC-dependent replication restart. PMID:27387236

  17. Complex regulation of CREB-binding protein by homeodomain-interacting protein kinase 2

    KAUST Repository

    Kovács, Krisztián A.

    2015-11-01

    CREB-binding protein (CBP) and p300 are transcriptional coactivators involved in numerous biological processes that affect cell growth, transformation, differentiation, and development. In this study, we provide evidence of the involvement of homeodomain-interacting protein kinase 2 (HIPK2) in the regulation of CBP activity. We show that HIPK2 interacts with and phosphorylates several regions of CBP. We demonstrate that serines 2361, 2363, 2371, 2376, and 2381 are responsible for the HIPK2-induced mobility shift of CBP C-terminal activation domain. Moreover, we show that HIPK2 strongly potentiates the transcriptional activity of CBP. However, our data suggest that HIPK2 activates CBP mainly by counteracting the repressive action of cell cycle regulatory domain 1 (CRD1), located between amino acids 977 and 1076, independently of CBP phosphorylation. Our findings thus highlight a complex regulation of CBP activity by HIPK2, which might be relevant for the control of specific sets of target genes involved in cellular proliferation, differentiation and apoptosis. © 2015 Elsevier Inc.

  18. A cDNA encoding a pRB-binding protein with properties of the transcription factor E2F

    DEFF Research Database (Denmark)

    Helin, K; Lees, J A; Vidal, M;

    1992-01-01

    The retinoblastoma protein (pRB) plays an important role in the control of cell proliferation, apparently by binding to and regulating cellular transcription factors such as E2F. Here we describe the characterization of a cDNA clone that encodes a protein with properties of E2F. This clone, RBP3...

  19. Distinct binding and immunogenic properties of the gonococcal homologue of meningococcal factor h binding protein.

    Directory of Open Access Journals (Sweden)

    Ilse Jongerius

    Full Text Available Neisseria meningitidis is a leading cause of sepsis and meningitis. The bacterium recruits factor H (fH, a negative regulator of the complement system, to its surface via fH binding protein (fHbp, providing a mechanism to avoid complement-mediated killing. fHbp is an important antigen that elicits protective immunity against the meningococcus and has been divided into three different variant groups, V1, V2 and V3, or families A and B. However, immunisation with fHbp V1 does not result in cross-protection against V2 and V3 and vice versa. Furthermore, high affinity binding of fH could impair immune responses against fHbp. Here, we investigate a homologue of fHbp in Neisseria gonorrhoeae, designated as Gonococcal homologue of fHbp (Ghfp which we show is a promising vaccine candidate for N. meningitidis. We demonstrate that Gfhp is not expressed on the surface of the gonococcus and, despite its high level of identity with fHbp, does not bind fH. Substitution of only two amino acids in Ghfp is sufficient to confer fH binding, while the corresponding residues in V3 fHbp are essential for high affinity fH binding. Furthermore, immune responses against Ghfp recognise V1, V2 and V3 fHbps expressed by a range of clinical isolates, and have serum bactericidal activity against N. meningitidis expressing fHbps from all variant groups.

  20. Distinct binding and immunogenic properties of the gonococcal homologue of meningococcal factor h binding protein.

    Science.gov (United States)

    Jongerius, Ilse; Lavender, Hayley; Tan, Lionel; Ruivo, Nicola; Exley, Rachel M; Caesar, Joseph J E; Lea, Susan M; Johnson, Steven; Tang, Christoph M

    2013-01-01

    Neisseria meningitidis is a leading cause of sepsis and meningitis. The bacterium recruits factor H (fH), a negative regulator of the complement system, to its surface via fH binding protein (fHbp), providing a mechanism to avoid complement-mediated killing. fHbp is an important antigen that elicits protective immunity against the meningococcus and has been divided into three different variant groups, V1, V2 and V3, or families A and B. However, immunisation with fHbp V1 does not result in cross-protection against V2 and V3 and vice versa. Furthermore, high affinity binding of fH could impair immune responses against fHbp. Here, we investigate a homologue of fHbp in Neisseria gonorrhoeae, designated as Gonococcal homologue of fHbp (Ghfp) which we show is a promising vaccine candidate for N. meningitidis. We demonstrate that Gfhp is not expressed on the surface of the gonococcus and, despite its high level of identity with fHbp, does not bind fH. Substitution of only two amino acids in Ghfp is sufficient to confer fH binding, while the corresponding residues in V3 fHbp are essential for high affinity fH binding. Furthermore, immune responses against Ghfp recognise V1, V2 and V3 fHbps expressed by a range of clinical isolates, and have serum bactericidal activity against N. meningitidis expressing fHbps from all variant groups. PMID:23935503

  1. The Bacillus thuringiensis insecticidal toxin binds biotin-containing proteins.

    OpenAIRE

    C. Du; Nickerson, K W

    1996-01-01

    Brush border membrane vesicles from larvae of the tobacco hornworm, Manduca sexta, contain protein bands of 85 and 120 kDa which react directly with streptavidin conjugated to alkaline phosphatase. The binding could be prevented either by including 10 microM biotin in the reaction mixture or by prior incubation of the brush border membrane vesicles with an activated 60- to 65-kDa toxin from Bacillus thuringiensis HD-73. The ability of B. thuringiensis toxins to recognize biotin-containing pro...

  2. Screening of hepatocyte proteins binding to F protein of hepatitis C virus by yeast two-hybrid system

    Institute of Scientific and Technical Information of China (English)

    Yan-Ping Huang; Xue-Song Gao; Dong Ji; Shu-Mei Lin; Qing Shao; Jun Cheng; Shu-Lin Zhang; Lin Wang; Jiang Guo; Yan Liu; Yuan Yang; Li-Ying Zhang; Gui-Qin Bai

    2005-01-01

    AIM: To investigate the biological function of F protein by yeast two-hybrid system.METHODS: We constructed F protein bait plasmid by cloning the gene of F protein into pGBKT7, then recombinant plasmid DNA was transformed into yeast AH109 (a type). The transformed yeast AH109 was mated with yeast Y187 (α type) containing liver cDNA library plasmid in 2×YPDA medium. Diploid yeast was plated on synthetic dropout nutrient medium (SD/-Trp-Leu-HisAde) containing X-α-gal for selection and screening.After extracting and sequencing plasmids from positive (blue) colonies, we underwent sequence analysis by bioinformatics.RESULTS: Thirty-six colonies were selected and sequenced.Among them, 11 colonies were zymogen granule protein,5 colonies were zinc finger protein, 4 colonies were zinc-α-2-glycoprotein, 1 colony was sialyltransferase, 1 colony was complement control protein factor Ⅰ, 1 colony was vitronectin, and 2 colonies were new genes with unknown function.CONCLUSION: The yeast two-hybrid system is an effective method for identifying hepatocyte proteins interacting with F protein of hepatitis C virus. F protein may bind to different proteins.

  3. Structural and binding properties of two paralogous fatty acid binding proteins of Taenia solium metacestode.

    Directory of Open Access Journals (Sweden)

    Seon-Hee Kim

    Full Text Available BACKGROUND: Fatty acid (FA binding proteins (FABPs of helminths are implicated in acquisition and utilization of host-derived hydrophobic substances, as well as in signaling and cellular interactions. We previously demonstrated that secretory hydrophobic ligand binding proteins (HLBPs of Taenia solium metacestode (TsM, a causative agent of neurocysticercosis (NC, shuttle FAs in the surrounding host tissues and inwardly transport the FAs across the parasite syncytial membrane. However, the protein molecules responsible for the intracellular trafficking and assimilation of FAs have remained elusive. METHODOLOGY/PRINCIPAL FINDINGS: We isolated two novel TsMFABP genes (TsMFABP1 and TsMFABP2, which encoded 133- and 136-amino acid polypeptides with predicted molecular masses of 14.3 and 14.8 kDa, respectively. They shared 45% sequence identity with each other and 15-95% with other related-members. Homology modeling demonstrated a characteristic β-barrel composed of 10 anti-parallel β-strands and two α-helices. TsMFABP2 harbored two additional loops between β-strands two and three, and β-strands six and seven, respectively. TsMFABP1 was secreted into cyst fluid and surrounding environments, whereas TsMFABP2 was intracellularly confined. Partially purified native proteins migrated to 15 kDa with different isoelectric points of 9.2 (TsMFABP1 and 8.4 (TsMFABP2. Both native and recombinant proteins bound to 11-([5-dimethylaminonaphthalene-1-sulfonyl]aminoundecannoic acid, dansyl-DL-α-amino-caprylic acid, cis-parinaric acid and retinol, which were competitively inhibited by oleic acid. TsMFABP1 exhibited high affinity toward FA analogs. TsMFABPs showed weak binding activity to retinol, but TsMFABP2 showed relatively high affinity. Isolation of two distinct genes from an individual genome strongly suggested their paralogous nature. Abundant expression of TsMFABP1 and TsMFABP2 in the canal region of worm matched well with the histological distributions

  4. Molecular control of vertebrate iron homeostasis by iron regulatory proteins

    OpenAIRE

    Wallander, Michelle L.; Leibold, Elizabeth A.; Eisenstein, Richard S.

    2006-01-01

    Both deficiencies and excesses of iron represent major public health problems throughout the world. Understanding the cellular and organismal processes controlling iron homeostasis is critical for identifying iron-related diseases and in advancing the clinical treatments for such disorders of iron metabolism. Iron regulatory proteins (IRPs) 1 and 2 are key regulators of vertebrate iron metabolism. These RNA binding proteins post-transcriptionally control the stability or translation of mRNAs ...

  5. Unusual Heme Binding in the Bacterial Iron Response Regulator Protein (Irr): Spectral Characterization of Heme Binding to Heme Regulatory Motif

    OpenAIRE

    Ishikawa, Haruto; Nakagaki, Megumi; Bamba, Ai; Uchida, Takeshi; Hori, Hiroshi; O'Brian, Mark R.; Iwai, Kazuhiro; Ishimori, Koichiro

    2011-01-01

    We characterized heme binding in the bacterial iron response regulator (Irr) protein, which is a simple heme-regulated protein having a single “heme-regulatory motif”, HRM, and plays a key role in the iron homeostasis of a nitrogen fixing bacterium. The heme titration to wild-type and mutant Irr clearly showed that Irr has two heme binding sites: one of the heme binding sites is in the HRM, where 29Cys is the axial ligand, and the other one, the secondary heme binding site, is located outside...

  6. Protein-protein binding before and after photo-modification of albumin

    Science.gov (United States)

    Rozinek, Sarah C.; Glickman, Randolph D.; Thomas, Robert J.; Brancaleon, Lorenzo

    2016-03-01

    Bioeffects of directed-optical-energy encompass a wide range of applications. One aspect of photochemical interactions involves irradiating a photosensitizer with visible light in order to induce protein unfolding and consequent changes in function. In the past, irradiation of several dye-protein combinations has revealed effects on protein structure. Beta lactoglobulin, human serum albumin (HSA) and tubulin have all been photo-modified with meso-tetrakis(4- sulfonatophenyl)porphyrin (TSPP) bound, but only in the case of tubulin has binding caused a verified loss of biological function (loss of ability to form microtubules) as a result of this light-induced structural change. The current work questions if the photo-induced structural changes that occur to HSA, are sufficient to disable its biological function of binding to osteonectin. The albumin-binding protein, osteonectin, is about half the molecular weight of HSA, so the two proteins and their bound product can be separated and quantified by size exclusion high performance liquid chromatography. TSPP was first bound to HSA and irradiated, photo-modifying the structure of HSA. Then native HSA or photo-modified HSA (both with TSPP bound) were compared, to assess loss in HSA's innate binding ability as a result of light-induced structure modification.

  7. Farnesoid X Receptor Inhibits the Transcriptional Activity of Carbohydrate Response Element Binding Protein in Human Hepatocytes

    OpenAIRE

    Caron, Sandrine; Huaman Samanez, Carolina; Dehondt, Hélène; Ploton, Maheul; Briand, Olivier; Lien, Fleur; Dorchies, Emilie; Dumont, Julie; Postic, Catherine; Cariou, Bertrand; Lefebvre, Philippe; Staels, Bart

    2013-01-01

    The glucose-activated transcription factor carbohydrate response element binding protein (ChREBP) induces the expression of hepatic glycolytic and lipogenic genes. The farnesoid X receptor (FXR) is a nuclear bile acid receptor controlling bile acid, lipid, and glucose homeostasis. FXR negatively regulates hepatic glycolysis and lipogenesis in mouse liver. The aim of this study was to determine whether FXR regulates the transcriptional activity of ChREBP in human hepatocytes and to unravel the...

  8. Posttranscriptional Regulation of p53 and Its Targets by RNA-Binding Proteins

    OpenAIRE

    Zhang, Jin; Chen, Xinbin

    2008-01-01

    p53 tumor suppressor plays a pivotal role in maintaining genomic integrity and preventing cancer development. The importance of p53 in tumor suppression is illustrated by the observation that about 50% human tumor cells have a dysfunctional p53 pathway. Although it has been well accepted that the activity of p53 is mainly controlled through post-translational modifications, recent studies have revealed that posttranscriptional regulations of p53 by various RNA-binding proteins also play a cru...

  9. Relationship between serum adiopocyte fatty acid binding protein and atherosclerosis in chronic kidney disease

    Institute of Scientific and Technical Information of China (English)

    吴晶

    2014-01-01

    Objective To investigate the expression of serum adiopocyte fatty acid binding protein(A-FABP)in chronic kidney disease(CKD)and the role that A-FABP plays in CKD with atherosclerosis.Methods A total of 138 patients with CKD and 20 health control volunteers(HC)were involved in this study.The levels of serum AFABP,free fatty acid(FFA),interleukin-6(IL-6),

  10. Circulating 25-Hydroxyvitamin D, Vitamin D Binding Protein, and Risk of Prostate Cancer

    OpenAIRE

    Weinstein, Stephanie J.; Mondul, Alison M.; Kopp, William; Rager, Helen; Virtamo, Jarmo; Albanes, Demetrius

    2012-01-01

    We recently reported a significant positive association between 25-hydroxyvitamin D [25(OH)D], the accepted biomarker of vitamin D status, and prostate cancer risk. To further elucidate this association, we examined the influence of vitamin D binding protein (DBP), the primary transporter of vitamin D compounds in the circulation. Prediagnostic serum concentrations of DBP were assayed for 950 cases and 964 matched controls with existing 25(OH)D measurements within the Alpha-Tocopherol, Beta-C...

  11. Diagnostic value of lipopolysaccharide-binding protein and procalcitonin for sepsis diagnosis in forensic pathology

    OpenAIRE

    Augsburger, Marc; Iglesias, Katia; Bardy, Daniel; Mangin, Patrice; Palmiere, Cristian

    2016-01-01

    The aims of this study were twofold. The first was to investigate the diagnostic performance of two biochemical markers, procalcitonin (PCT) and lipopolysaccharide-binding protein (LBP), considering each individually and then combined, for the postmortem diagnosis of sepsis. We also tested the usefulness of pericardial fluid for postmortem LBP determination. Two study groups were formed, a sepsis-related fatalities group of 12 cases and a control group of 30 cases. Postmortem native CT scans,...

  12. Orthogonal matrix factorization enables integrative analysis of multiple RNA binding proteins

    OpenAIRE

    Stražar, Martin; Žitnik, Marinka; Zupan, Blaž; Ule, Jernej; Curk, Tomaž

    2016-01-01

    Motivation: RNA binding proteins (RBPs) play important roles in post-transcriptional control of gene expression, including splicing, transport, polyadenylation and RNA stability. To model protein–RNA interactions by considering all available sources of information, it is necessary to integrate the rapidly growing RBP experimental data with the latest genome annotation, gene function, RNA sequence and structure. Such integration is possible by matrix factorization, where current approaches hav...

  13. Genetic noise control via protein oligomerization

    Directory of Open Access Journals (Sweden)

    Almaas Eivind

    2008-11-01

    Full Text Available Abstract Background Gene expression in a cell entails random reaction events occurring over disparate time scales. Thus, molecular noise that often results in phenotypic and population-dynamic consequences sets a fundamental limit to biochemical signaling. While there have been numerous studies correlating the architecture of cellular reaction networks with noise tolerance, only a limited effort has been made to understand the dynamic role of protein-protein interactions. Results We have developed a fully stochastic model for the positive feedback control of a single gene, as well as a pair of genes (toggle switch, integrating quantitative results from previous in vivo and in vitro studies. In particular, we explicitly account for the fast binding-unbinding kinetics among proteins, RNA polymerases, and the promoter/operator sequences of DNA. We find that the overall noise-level is reduced and the frequency content of the noise is dramatically shifted to the physiologically irrelevant high-frequency regime in the presence of protein dimerization. This is independent of the choice of monomer or dimer as transcription factor and persists throughout the multiple model topologies considered. For the toggle switch, we additionally find that the presence of a protein dimer, either homodimer or heterodimer, may significantly reduce its random switching rate. Hence, the dimer promotes the robust function of bistable switches by preventing the uninduced (induced state from randomly being induced (uninduced. Conclusion The specific binding between regulatory proteins provides a buffer that may prevent the propagation of fluctuations in genetic activity. The capacity of the buffer is a non-monotonic function of association-dissociation rates. Since the protein oligomerization per se does not require extra protein components to be expressed, it provides a basis for the rapid control of intrinsic or extrinsic noise. The stabilization of regulatory circuits

  14. A sequence-based dynamic ensemble learning system for protein ligand-binding site prediction

    KAUST Repository

    Chen, Peng

    2015-12-03

    Background: Proteins have the fundamental ability to selectively bind to other molecules and perform specific functions through such interactions, such as protein-ligand binding. Accurate prediction of protein residues that physically bind to ligands is important for drug design and protein docking studies. Most of the successful protein-ligand binding predictions were based on known structures. However, structural information is not largely available in practice due to the huge gap between the number of known protein sequences and that of experimentally solved structures

  15. Periplasmic Binding Proteins in Thermophiles: Characterization and Potential Application of an Arginine-Binding Protein from Thermotoga maritima: A Brief Thermo-Story

    Directory of Open Access Journals (Sweden)

    Sabato D'Auria

    2013-02-01

    Full Text Available Arginine-binding protein from the extremophile Thermotoga maritima is a 27.7 kDa protein possessing the typical two-domain structure of the periplasmic binding proteins family. The protein is characterized by a very high specificity and affinity to bind to arginine, also at high temperatures. Due to its features, this protein could be taken into account as a potential candidate for the design of a biosensor for arginine. It is important to investigate the stability of proteins when they are used for biotechnological applications. In this article, we review the structural and functional features of an arginine-binding protein from the extremophile Thermotoga maritima with a particular eye on its potential biotechnological applications.

  16. Protein interactions and ligand binding: From protein subfamilies to functional specificity

    OpenAIRE

    Rausell, A.; de Juan, D.; Pazos, F; Valencia, A.

    2010-01-01

    The divergence accumulated during the evolution of protein families translates into their internal organization as subfamilies, and it is directly reflected in the characteristic patterns of differentially conserved residues. These specifically conserved positions in protein subfamilies are known as “specificity determining positions” (SDPs). Previous studies have limited their analysis to the study of the relationship between these positions and ligand-binding specificity, demonstrating sign...

  17. Paracetamol and cytarabine binding competition in high affinity binding sites of transporting protein

    Science.gov (United States)

    Sułkowska, A.; Bojko, B.; Równicka, J.; Sułkowski, W. W.

    2006-07-01

    Paracetamol (acetaminophen, AA) the most popular analgesic drug is commonly used in the treatment of pain in patients suffering from cancer. In our studies, we evaluated the competition in binding with serum albumin between paracetamol (AA) and cytarabine, antyleukemic drug (araC). The presence of one drug can alter the binding affinity of albumin towards the second one. Such interaction can result in changing of the free fraction of the one of these drugs in blood. Two spectroscopic methods were used to determine high affinity binding sites and the competition of the drugs. Basing on the change of the serum albumin fluorescence in the presence of either of the drugs the quenching ( KQ) constants for the araC-BSA and AA-BSA systems were calculated. Analysis of UV difference spectra allowed us to describe the changes in drug-protein complexes (araC-albumin and AA-albumin) induced by the presence of the second drug (AA and araC, respectively). The mechanism of competition between araC and AA has been proposed.

  18. Cobalamin binding proteins in patients with HIV infection

    International Nuclear Information System (INIS)

    P-Cobalamins have been reported to be decreased in patients with HIV infection. Because of this, we found it of interest to examine both cobalamin-saturated binding proteins (holo-transcobalamin, holo-TC and holo-haptocorrin, holo-HC) and cobalamin unsaturated binding proteins (apo-transcobalamin, apo-TC and apo-haptocorrin, apo-HC). The results are given as range and (median). Eighteen male HIV-infected patients with plasma cobalamins below 200 pmol/l were studied. We found low concentrations of holo-TC (37-88(47.5)pmol/l) and holo-HC (64-184(135.3)pmol/l). The concentration of apo-TC and apo-HC was increased (480-1730(1025)pmol/l; 70-800(235)pmol/l). It is concluded that, in HIV-infected patients, low plasma cobalamin does not reflect a low concentration of transcobalamin or haptocorrin. In 20 HIV-infected patients and 31 patients with malignant haematological diseases, the TC isopeptide patterns were determined. In the HIV group, an increased frequency of TC isopeptide X was found and the overall distribution of TC isopeptides was significantly different from the reference population (p<0.05). There was no difference between the group of patients with malignant haematological diseases and the reference group. (au)

  19. Properties of Folate Binding Protein Purified from Cow’s Milk

    Directory of Open Access Journals (Sweden)

    SUBANDRATE

    2012-09-01

    Full Text Available Folic acid played an important role in the metabolism of the body. To measure the serum folic acid levels could use the folate binding protein (FBP from cow’s milk with a technique analogous to ELISA. The aims of this study were to identify characteristics of FBP from cow’s milk and binding capacity of FBP to folic acid and to purify FBP from other whey protein passed through DEAE-cellulose chromatography column. Each of DEAE-cellulose peaks was passed in affinity chromatography column. FBP was released from affinity column with sodium acetate buffer pH 3.5. The purity of obtained FBP was demonstrated by a single spot in SDS-PAGE analysis and the estimated molecular weight of FBP was around 31 kDa. Our study indicated that 1 mol FBP bound 1 mol folic acid. Alkylation with iodoacetic acid decreased the binding capacity of FBP which suggested the presence of a–SH or imidazol group in its active site. The importance of disulfide bridge was proven by decreasing of folate binding capacity of FBP after -mercaptoethanol treatment. In contrary, the folate binding didn need Ca2+ ion, as indicated by EDTA test which gave the same result as control.

  20. Localization of Cellular Retinol-Binding Protein and Retinol-Binding Protein in Cells Comprising the Blood-Brain Barrier of Rat and Human

    Science.gov (United States)

    MacDonald, Paul N.; Bok, Dean; Ong, David E.

    1990-06-01

    Brain is not generally recognized as an organ that requiries vitamin A, perhaps because no obvious histologic lesions have been observed in severely vitamin A-deficient animals. However, brain tissue does contain cellular vitamin A-binding proteins and a nuclear receptor protein for retinoic acid. In the present study, immunohistochemical techniques were used to determine the cell-specific location of cellular retinol-binding protein in human and rat brain tissue. Cellular retinol-binding protein was localized specifically within the endothelial cells of the brain microvasculature and within the cuboidal epithelial cells of the choroid plexus, two primary sites of the mammalian blood-brain barrier. In addition, autoradiographic procedures demonstrated binding sites for serum retinol-binding protein in the choroidal epithelium. These observations suggest that a significant movement of retinol across the blood-brain barrier may occur.

  1. Localization of cellular retinol-binding protein and retinol-binding protein in cells comprising the blood-brain barrier of rat and human

    Energy Technology Data Exchange (ETDEWEB)

    MacDonald, P.N.; Ong, D.E. (Vanderbilt Univ., Nashville, TN (USA)); Bok, D. (Univ. of California, Los Angeles (USA))

    1990-06-01

    Brain is not generally recognized as an organ that requires vitamin A, perhaps because no obvious histologic lesions have been observed in severely vitamin A-deficient animals. However, brain tissue does contain cellular vitamin A-binding proteins and a nuclear receptor protein for retinoic acid. In the present study, immunohistochemical techniques were used to determine the cell-specific location of cellular retinol-binding protein in human and rat brain tissue. Cellular retinol-binding protein was localized specifically within the cuboidal epithelial cells of the choroid plexus, two primary sites of the mammalian blood-brain barrier. In addition, autoradiographic procedures demonstrated binding sites for serum retinol-binding protein in the choroidal epithelium. These observations suggest that a significant movement of retinol across the blood-brain barrier may occur.

  2. Plant Cytosolic Acyl-CoA-Binding Proteins.

    Science.gov (United States)

    Ye, Zi-Wei; Chye, Mee-Len

    2016-01-01

    A gene family encoding six members of acyl-CoA-binding proteins (ACBP) exists in Arabidopsis and they are designated as AtACBP1-AtACBP6. They have been observed to play pivotal roles in plant lipid metabolism, consistent to the abilities of recombinant AtACBP in binding different medium- and long-chain acyl-CoA esters in vitro. While AtACBP1 and AtACBP2 are membrane-associated proteins with ankyrin repeats and AtACBP3 contains a signaling peptide for targeting to the apoplast, AtACBP4, AtACBP5 and AtACBP6 represent the cytosolic forms in the AtACBP family. They were verified to be subcellularly localized in the cytosol using diverse experimental methods, including cell fractionation followed by western blot analysis, immunoelectron microscopy and confocal laser-scanning microscopy using autofluorescence-tagged fusions. AtACBP4 (73.2 kDa) and AtACBP5 (70.1 kDa) are the largest, while AtACBP6 (10.4 kDa) is the smallest. Their binding affinities to oleoyl-CoA esters suggested that they can potentially transfer oleoyl-CoA esters from the plastids to the endoplasmic reticulum, facilitating the subsequent biosynthesis of non-plastidial membrane lipids in Arabidopsis. Recent studies on ACBP, extended from a dicot (Arabidopsis) to a monocot, revealed that six ACBP are also encoded in rice (Oryza sativa). Interestingly, three small rice ACBP (OsACBP1, OsACBP2 and OsACBP3) are present in the cytosol in comparison to one (AtACBP6) in Arabidopsis. In this review, the combinatory and distinct roles of the cytosolic AtACBP are discussed, including their functions in pollen and seed development, light-dependent regulation and substrate affinities to acyl-CoA esters. PMID:26662549

  3. Electrophilicities and Protein Covalent Binding of Demethylation Metabolites of Colchicine.

    Science.gov (United States)

    Guo, Xiucai; Lin, Dongju; Li, Weiwei; Wang, Kai; Peng, Ying; Zheng, Jiang

    2016-03-21

    Colchicine, an alkaloid existing in plants of Liliaceous colchicum, has been widely used in the treatment of gout and familial Mediterranean fever. The administration of colchicine was found to cause liver injury in humans. The mechanisms of colchicine-induced liver toxicity remain unknown. The objectives of this study were to determine the electrophilicities of demethylation metabolites of colchicine and investigate the protein adductions derived from the reactive metabolites of colchicine. Four demethylated colchicine (1-, 2-, 3-, and 10-DMCs), namely, M1-M4, were detected in colchicine-fortified microsomal incubations. Four N-acetyl cysteine (NAC) conjugates (M5-M8) derived from colchicine were detected in the microsomes in the presence of NAC. M5 and M6 were derived from 10-DMC. M7 resulted from the reaction of 2-DMC or 3-DMC with NAC, and M8 originated from 10-DMC. Microsomal protein covalent binding was observed after exposure to colchicine. Two cysteine adducts (CA-1 and CA-2) derived from 10-DMC were found in proteolytically digested microsomal protein samples after incubation with colchicine. The findings allow us to define the chemical property of demethylation metabolites of colchicine and the interaction between protein and the reactive metabolites of colchicine generated in situ. PMID:26845511

  4. Tannin-binding salivary proteins in three captive rhinoceros species.

    Science.gov (United States)

    Clauss, Marcus; Gehrke, Janin; Hatt, Jean-Michel; Dierenfeld, Ellen S; Flach, Edmund J; Hermes, Robert; Castell, Johanna; Streich, W Juergen; Fickel, Joerns

    2005-01-01

    Tannin-binding salivary proteins (TBSP) are considered to be counter-defences acquired in the course of evolution by animals whose natural forage contains such tannins. As tannins mostly occur in browse material but not in grasses, it is assumed that grazers do not have a need for TBSP. Whereas it has been shown in several non-ungulate species that TBSP can be induced by dietary tannins, their presence or absence in ungulates has, so far, been shown to be a species-specific characteristic independent of dietary manipulations. We investigated saliva from three rhinoceros species from zoological gardens fed comparable, conventional zoo diets. As expected, saliva from white rhinoceroses (Ceratotherum simum, grazer) had lower tannin-binding capacities than that from black rhinoceroses (Diceros bicornis, browser). Surprisingly, however, Indian rhinoceroses (Rhinoceros unicornis), commonly regarded as grazers as well, displayed the highest tannin-binding capacities of the three species investigated. It is speculated that this discrepancy might be a result of an evolutionarily recent switch to a grass-dominated diet in Indian rhinoceroses, and that the black rhinoceros, which is closer related to the white rhinoceros than the Indian species, has evolved an inducible mechanism of TBSP production. In separate trials during which the tannin content of the diets of black rhinoceroses was increased by the addition of either tannic acid or quebracho, the tannin-binding capacity of black rhinoceros saliva was increased to levels within the same range as that of Indian rhinoceroses on the conventional diets. While induction trials in white and Indian rhinoceroses remain to be performed for a full understanding of salivary anti-tannin defence in rhinoceroses, these results are the first report of an induced salivary response to increased dietary tannin levels in an ungulate species. PMID:15664314

  5. XAS and Pulsed EPR Studies of the Copper Binding Site in Riboflavin Binding Protein

    Energy Technology Data Exchange (ETDEWEB)

    Smith,S.; Bencze, K.; Wasiukanis, K.; Benore-Parsons, T.; Stemmler, T.

    2008-01-01

    Riboflavin Binding Protein (RBP) binds copper in a 1:1 molar ratio, forming a distinct well-ordered type II site. The nature of this site has been examined using X-ray absorption and pulsed electron paramagnetic resonance (EPR) spectroscopies, revealing a four coordinate oxygen/nitrogen rich environment. On the basis of analysis of the Cambridge Structural Database, the average protein bound copper-ligand bond length of 1.96 Angstroms, obtained by extended x-ray absorption fine structure (EXAFS), is consistent with four coordinate Cu(I) and Cu(II) models that utilize mixed oxygen and nitrogen ligand distributions. These data suggest a CuO3N coordination state for copper bound to RBP. While pulsed EPR studies including hyperfine sublevel correlation spectroscopy and electron nuclear double resonance show clear spectroscopic evidence for a histidine bound to the copper, inclusion of a histidine in the EXAFS simulation did not lead to any significant improvement in the fit.

  6. Evolving Transcription Factor Binding Site Models From Protein Binding Microarray Data

    KAUST Repository

    Wong, Ka-Chun

    2016-02-02

    Protein binding microarray (PBM) is a high-throughput platform that can measure the DNA binding preference of a protein in a comprehensive and unbiased manner. In this paper, we describe the PBM motif model building problem. We apply several evolutionary computation methods and compare their performance with the interior point method, demonstrating their performance advantages. In addition, given the PBM domain knowledge, we propose and describe a novel method called kmerGA which makes domain-specific assumptions to exploit PBM data properties to build more accurate models than the other models built. The effectiveness and robustness of kmerGA is supported by comprehensive performance benchmarking on more than 200 datasets, time complexity analysis, convergence analysis, parameter analysis, and case studies. To demonstrate its utility further, kmerGA is applied to two real world applications: 1) PBM rotation testing and 2) ChIP-Seq peak sequence prediction. The results support the biological relevance of the models learned by kmerGA, and thus its real world applicability.

  7. Enterocyte Fatty Acid Binding Proteins (FABPs): Different Functions of Liver- and Intestinal- FABPs in the Intestine

    OpenAIRE

    Gajda, Angela M.; Storch, Judith

    2014-01-01

    Fatty acid binding proteins (FABP) are highly abundant cytosolic proteins that are expressed in most mammalian tissues. In the intestinal enterocyte, both Liver- (LFABP; FABP1) and Intestinal-fatty acid binding proteins (IFABP; FABP2) are expressed. These proteins display high affinity binding for long chain fatty acids (FA) and other hydrophobic ligands, thus they are believed to be involved with uptake and trafficking of lipids in the intestine. In vitro studies have identified differences ...

  8. Effect of Protein Binding on the Pharmacological Activity of Highly Bound Antibiotics▿

    OpenAIRE

    Schmidt, Stephan; Röck, Katharina; Sahre, Martina; Burkhardt, Olaf; Brunner, Martin; Lobmeyer, Maximilian T.; Derendorf, Hartmut

    2008-01-01

    During antibiotic drug development, media are frequently spiked with either serum/plasma or protein supplements to evaluate the effect of protein binding. Usually, previously reported serum or plasma protein binding values are applied in the analysis. The aim of this study was to evaluate this approach by experimentally measuring free, unbound concentrations for antibiotics with reportedly high protein binding and their corresponding antimicrobial activities in media containing commonly used ...

  9. Acquisition of heme iron by Neisseria meningitidis does not involve meningococcal transferrin-binding proteins.

    Science.gov (United States)

    Martel, N; Lee, B C

    1994-02-01

    Similarities in size between hemin-binding protein 1 (HmBP1) and transferrin-binding protein 1 (TBP1) of Neisseria meningitidis suggest that these proteins are functionally homologous. However, a meningococcal mutant lacking the transferrin-binding proteins retained the capacity to acquire iron from heme and hemoglobin. In immunoblots, hyperimmune polyclonal antiserum against TBP1 did not react with HmBP1. PMID:8300227

  10. Evolutionary Conservation and Diversification of Puf RNA Binding Proteins and Their mRNA Targets.

    Science.gov (United States)

    Hogan, Gregory J; Brown, Patrick O; Herschlag, Daniel

    2015-11-01

    chain (ETC) complex I as well as hundreds of other mRNAs with nonmitochondrial functions. The many concerted and conserved changes in the RNA targets of Puf proteins strongly support an extensive role of RNA binding proteins in coordinating gene expression, as originally proposed by Keene. Rewiring of Puf-coordinated mRNA targets and transcriptional control of the same genes occurred at different points in evolution, suggesting that there have been distinct adaptations via RNA binding proteins and transcription factors. The changes in Puf targets and in the Puf proteins indicate an integral involvement of RNA binding proteins and their RNA targets in the adaptation, reprogramming, and function of gene expression. PMID:26587879

  11. A single rainbow trout cobalamin-binding protein stands in for three human binders

    DEFF Research Database (Denmark)

    Greibe, Eva; Fedosov, Sergey; Sorensen, Boe S; Højrup, Peter; Poulsen, Steen Seier; Nexo, Ebba

    2012-01-01

    Cobalamin uptake and transport in mammals are mediated by three cobalamin-binding proteins: haptocorrin, intrinsic factor, and transcobalamin. The nature of cobalamin-binding proteins in lower vertebrates remains to be elucidated. The aim of this study was to characterize the cobalamin-binding pr...

  12. Binding of the human papillomavirus E1 origin-recognition protein is regulated through complex formation with the E2 enhancer-binding protein.

    OpenAIRE

    Frattini, M G; Laimins, L A

    1994-01-01

    The papillomavirus E1 and E2 proteins form heteromeric complexes and individually bind specific sequences within the viral origin of replication. The mechanism by which these proteins are recruited to the origin and the role of the E1/E2 complex in replication remain undefined. To examine the interplay of these replication proteins, we have analyzed the binding of human papillomavirus (HPV) type 31b E1 and E2 proteins to the origin of replication. Binding of E1 to the origin was increased by ...

  13. APPLICATION OF IMMUNOGLOBULIN-BINDING PROTEINS A, G, L IN THE AFFINITY CHROMATOGRAPHY

    OpenAIRE

    Sviatenko, О.; Gorbatiuk, O.; Vasylchenko, О.

    2014-01-01

    Proteins A, G and L are native or recombinant proteins of microbial origin that bind to mammalian immunoglobulins. Preferably recombinant variants of proteins A, G, L are used in biotechnology for affinity sorbents production. Сomparative characteristics of proteins A, G, L and affinity sorbents on the basis of them, advantages and disadvantages of these proteins application as ligands in the affinity chromatography are done. Analysis of proteins A, G, L properties is presented. Binding speci...

  14. Myristylation alters DNA-binding activity and transactivation of FBR (gag-fos) protein.

    OpenAIRE

    Kamata, N; Jotte, R M; Holt, J. T.

    1991-01-01

    FBR murine sarcoma virus (gag-fos) protein, a virally transduced Fos protein, exhibits decreased gene transactivation in comparison with the cellular Fos protein. Biochemical analysis suggests that myristylation of the virally encoded N-terminal gag region results in decreased DNA binding and transcriptional activation without affecting heterodimerization with Jun protein. These findings demonstrate that protein myristylation can modulate gene regulation by a DNA-binding protein.

  15. Differential dissociation micromethod for the investigation of binding of metandrostenolone (Nerobol) to plasma proteins

    Energy Technology Data Exchange (ETDEWEB)

    Bojadzsieva, M.; Kocsar, L. (Orszagos Frederic Joliot-Curie Sugarbiologiai es Sugaregeszseguegyi Kutato Intezet, Budapest (Hungary)); Kremmer, T. (Orszagos Onkologiai Intezet, Budapest (Hungary))

    1985-01-01

    A micromethod was developed to determine the binding of anabolic steroids to plasma proteins. The new procedure combines precipitation with ammonium sulphate and differential dissociation. The binding parameters (association constant, specific binding capacity) are calculated on the basis of dissociation curves of sup(3)H-metandrostenolone from the precipitated sexual binding globuline.

  16. Chimeric Plant Calcium/Calmodulin-Dependent Protein Kinase Gene with a Neural Visinin-Like Calcium-Binding Domain

    Science.gov (United States)

    Patil, Shameekumar; Takezawa, D.; Poovaiah, B. W.

    1995-01-01

    Calcium, a universal second messenger, regulates diverse cellular processes in eukaryotes. Ca-2(+) and Ca-2(+)/calmodulin-regulated protein phosphorylation play a pivotal role in amplifying and diversifying the action of Ca-2(+)- mediated signals. A chimeric Ca-2(+)/calmodulin-dependent protein kinase (CCaMK) gene with a visinin-like Ca-2(+)- binding domain was cloned and characterized from lily. The cDNA clone contains an open reading frame coding for a protein of 520 amino acids. The predicted structure of CCaMK contains a catalytic domain followed by two regulatory domains, a calmodulin-binding domain and a visinin-like Ca-2(+)-binding domain. The amino-terminal region of CCaMK contains all 11 conserved subdomains characteristic of serine/threonine protein kinases. The calmodulin-binding region of CCaMK has high homology (79%) to alpha subunit of mammalian Ca-2(+)/calmodulin-dependent protein kinase. The calmodulin-binding region is fused to a neural visinin-like domain that contains three Ca-2(+)-binding EF-hand motifs and a biotin-binding site. The Escherichia coli-expressed protein (approx. 56 kDa) binds calmodulin in a Ca-2(+)-dependent manner. Furthermore, Ca-45-binding assays revealed that CCaMK directly binds Ca-2(+). The CCaMK gene is preferentially expressed in developing anthers. Southern blot analysis revealed that CCaMK is encoded by a single gene. The structural features of the gene suggest that it has multiple regulatory controls and could play a unique role in Ca-2(+) signaling in plants.

  17. Human Rif1 protein binds aberrant telomeres and aligns along anaphase midzone microtubules

    OpenAIRE

    Xu, Lifeng; Blackburn, Elizabeth H.

    2004-01-01

    We identified and characterized a human orthologue of Rif1 protein, which in budding yeast interacts in vivo with the major duplex telomeric DNA binding protein Rap1p and negatively regulates telomere length. Depletion of hRif1 by RNA interference in human cancer cells impaired cell growth but had no detectable effect on telomere length, although hRif1 overexpression in S. cerevisiae interfered with telomere length control, in a manner specifically dependent on the presence of yeast Rif1p. No...

  18. Computational study of ligand binding in lipid transfer proteins: Structures, interfaces, and free energies of protein-lipid complexes

    OpenAIRE

    Fernandez Pacios, Luis; Gomez Casado, Cristina; Tordesillas Villuendas, Leticia; Palacín Gómez, Aranzazu; Sanchez-Monge Laguna De Rins, Maria Rosa; Díaz Perales, Araceli

    2012-01-01

    Plant nonspecific lipid transfer proteins (nsLTPs) bind a wide variety of lipids, which allows them to perform disparate functions. Recent reports on their multifunctionality in plant growth processes have posed new questions on the versatile binding abilities of these proteins. The lack of binding specificity has been customarily explained in qualitative terms on the basis of a supposed structural flexibility and nonspecificity of hydrophobic protein-ligand interactions. We present here a co...

  19. IRBP-like proteins in the eyes of six cephalopod species--immunochemical relationship to vertebrate interstitial retinol-binding protein (IRBP) and cephalopod retinal-binding protein.

    Science.gov (United States)

    Fong, S L; Lee, P G; Ozaki, K; Hara, R; Hara, T; Bridges, C D

    1988-01-01

    SDS polyacrylamide gel electrophoresis and immunoblotting were used to examine soluble proteins from the eyes of six species of cephalopods i.e. Lolliguncula brevis, Sepia officinalis, Octopus maya, Octopus bimaculoides, Rossia pacifica and Loligo opalescens. All species had a protein ("IRBP") with molecular weight virtually identical with vertebrate interstitial retinol-binding protein (IRBP) averaging 132,400 +/- 700 (n = 6). "IRBP" reacted on nitrocellulose blot transfers with rabbit antibovine IRBP and rabbit antifrog IRBP antibodies. Unlike vertebrate IRBP, cephalopod "IRBP" (from L. brevis) did not bind exogenous retinol or concanavalin A. The N-terminal amino acid appeared to be blocked in samples electroeluted from SDS gels. The antifrog IRBP antibodies also reacted with a series of proteins with molecular weights between 46,000 and 47,000, identified as retinal-binding protein (RALBP) with anti-RALBP antibodies. Anti-IRBP also reacted with pure RALBP prepared from Todarodes pacificus. Occasionally, anti-RALBP antibodies were seen to react weakly with "IRBP" in some cephalopods. We conclude that RALBP, cephalopod "IRBP" and vertebrate IRBP share a common but distant ancestry, and that a protein resembling IRBP appeared before the vertebrates diverged from the invertebrates. Both RALBP and IRBP appear to have analogous functions in shuttling retinoids between rhodopsin and the corresponding isomerizing system, retinochrome in the cephalopods and retinol isomerase in the vertebrates. The function of cephalopod "IRBP" is unknown. PMID:3195063

  20. Effects of ligand binding on the mechanical stability of protein GB1 studied by steered molecular dynamics simulation.

    Science.gov (United States)

    Su, Ji-Guo; Zhao, Shu-Xin; Wang, Xiao-Feng; Li, Chun-Hua; Li, Jing-Yuan

    2016-08-01

    Regulation of the mechanical properties of proteins plays an important role in many biological processes, and sheds light on the design of biomaterials comprised of protein. At present, strategies to regulate protein mechanical stability focus mainly on direct modulation of the force-bearing region of the protein. Interestingly, the mechanical stability of GB1 can be significantly enhanced by the binding of Fc fragments of human IgG antibody, where the binding site is distant from the force-bearing region of the protein. The mechanism of this long-range allosteric control of protein mechanics is still elusive. In this work, the impact of ligand binding on the mechanical stability of GB1 was investigated using steered molecular dynamics simulation, and a mechanism underlying the enhanced protein mechanical stability is proposed. We found that the external force causes deformation of both force-bearing region and ligand binding site. In other words, there is a long-range coupling between these two regions. The binding of ligand restricts the distortion of the binding site and reduces the deformation of the force-bearing region through a long-range allosteric communication, which thus improves the overall mechanical stability of the protein. The simulation results are very consistent with previous experimental observations. Our studies thus provide atomic-level insights into the mechanical unfolding process of GB1, and explain the impact of ligand binding on the mechanical properties of the protein through long-range allosteric regulation, which should facilitate effective modulation of protein mechanical properties. PMID:27444879

  1. Easy mammalian expression and crystallography of maltose-binding protein-fused human proteins.

    Science.gov (United States)

    Bokhove, Marcel; Sadat Al Hosseini, Hamed; Saito, Takako; Dioguardi, Elisa; Gegenschatz-Schmid, Katharina; Nishimura, Kaoru; Raj, Isha; de Sanctis, Daniele; Han, Ling; Jovine, Luca

    2016-04-01

    We present a strategy to obtain milligrams of highly post-translationally modified eukaryotic proteins, transiently expressed in mammalian cells as rigid or cleavable fusions with a mammalianized version of bacterial maltose-binding protein (mMBP). This variant was engineered to combine mutations that enhance MBP solubility and affinity purification, as well as provide crystal-packing interactions for increased crystallizability. Using this cell type-independent approach, we could increase the expression of secreted and intracellular human proteins up to 200-fold. By molecular replacement with MBP, we readily determined five novel high-resolution structures of rigid fusions of targets that otherwise defied crystallization. PMID:26850170

  2. Evaluating the binding efficiency of pheromone binding protein with its natural ligand using molecular docking and fluorescence analysis

    Science.gov (United States)

    Ilayaraja, Renganathan; Rajkumar, Ramalingam; Rajesh, Durairaj; Muralidharan, Arumugam Ramachandran; Padmanabhan, Parasuraman; Archunan, Govindaraju

    2014-06-01

    Chemosignals play a crucial role in social and sexual communication among inter- and intra-species. Chemical cues are bound with protein that is present in the pheromones irrespective of sex are commonly called as pheromone binding protein (PBP). In rats, the pheromone compounds are bound with low molecular lipocalin protein α2u-globulin (α2u). We reported farnesol is a natural endogenous ligand (compound) present in rat preputial gland as a bound volatile compound. In the present study, an attempt has been made through computational method to evaluating the binding efficiency of α2u with the natural ligand (farnesol) and standard fluorescent molecule (2-naphthol). The docking analysis revealed that the binding energy of farnesol and 2-naphthol was almost equal and likely to share some binding pocket of protein. Further, to extrapolate the results generated through computational approach, the α2u protein was purified and subjected to fluorescence titration and binding assay. The results showed that the farnesol is replaced by 2-naphthol with high hydrophobicity of TYR120 in binding sites of α2u providing an acceptable dissociation constant indicating the binding efficiency of α2u. The obtained results are in corroboration with the data made through computational approach.

  3. RNA binding specificity of hnRNP proteins: a subset bind to the 3' end of introns.

    OpenAIRE

    Swanson, M S; Dreyfuss, G

    1988-01-01

    The binding of hnRNP proteins to pre-mRNAs in nuclear extracts, and as isolated proteins, was studied by using monoclonal antibody immunopurification of hnRNP proteins bound to RNase T1-generated fragments. Several major hnRNP proteins, A1, C and D, bind specifically to the 3' end of introns within a region containing the conserved polypyrimidine stretch between the branch site and the 3' splice site. Mutations which alter the conserved 3' splice site dinucleotide AG strongly impair or abolis...

  4. Crystal structure of a novel type of odorant binding protein from Anopheles gambiae, belonging to the C+ class

    OpenAIRE

    Lagarde, Amandine; Spinelli, Silvia; Qiao, Huili; Tegoni, Mariella; Pelosi, Paolo; Cambillau, Christian

    2011-01-01

    Anopheles gambiae (Agam) relies on its olfactory system to target human prey, leading eventually to injection of Plasmodium falciparum, the malaria vector. Odorant-binding proteins (OBPs) are the first line of proteins involved in odorant recognition. They interact with olfactory receptors and thus constitute an interesting target for insect control. We undertook a large-scale study of proteins belonging to the olfactory system of Agam with the aim of preventing insect bites by designing stro...

  5. Crystal structure of a novel type of odorant binding protein from Anopheles gambiae, belonging to the C+ class

    OpenAIRE

    2011-01-01

    Abstract Anopheles gambiae (Agam) relies on its olfactory system to target human prey, leading eventually to injection of Plasmodium falciparum, the malaria vector. Odorant-binding proteins (OBPs) are the first line of proteins involved in odorant recognition. They interact with olfactory receptors and thus constitute an interesting target for insect control. We undertook a large-scale study of proteins belonging to the olfactory system of Agam with the aim of preventing insect bit...

  6. New Cysteine-Rich Ice-Binding Protein Secreted from Antarctic Microalga, Chloromonas sp.

    Science.gov (United States)

    Jung, Woongsic; Campbell, Robert L; Gwak, Yunho; Kim, Jong Im; Davies, Peter L; Jin, EonSeon

    2016-01-01

    Many microorganisms in Antarctica survive in the cold environment there by producing ice-binding proteins (IBPs) to control the growth of ice around them. An IBP from the Antarctic freshwater microalga, Chloromonas sp., was identified and characterized. The length of the Chloromonas sp. IBP (ChloroIBP) gene was 3.2 kb with 12 exons, and the molecular weight of the protein deduced from the ChloroIBP cDNA was 34.0 kDa. Expression of the ChloroIBP gene was up- and down-regulated by freezing and warming conditions, respectively. Western blot analysis revealed that native ChloroIBP was secreted into the culture medium. This protein has fifteen cysteines and is extensively disulfide bonded as shown by in-gel mobility shifts between oxidizing and reducing conditions. The open-reading frame of ChloroIBP was cloned and over-expressed in Escherichia coli to investigate the IBP's biochemical characteristics. Recombinant ChloroIBP produced as a fusion protein with thioredoxin was purified by affinity chromatography and formed single ice crystals of a dendritic shape with a thermal hysteresis activity of 0.4±0.02°C at a concentration of 5 mg/ml. In silico structural modeling indicated that the three-dimensional structure of ChloroIBP was that of a right-handed β-helix. Site-directed mutagenesis of ChloroIBP showed that a conserved region of six parallel T-X-T motifs on the β-2 face was the ice-binding region, as predicted from the model. In addition to disulfide bonding, hydrophobic interactions between inward-pointing residues on the β-1 and β-2 faces, in the region of ice-binding motifs, were crucial to maintaining the structural conformation of ice-binding site and the ice-binding activity of ChloroIBP. PMID:27097164

  7. Divergence of Pumilio/fem-3 mRNA Binding Factor (PUF) Protein Specificity through Variations in an RNA-binding Pocket*

    Science.gov (United States)

    Qiu, Chen; Kershner, Aaron; Wang, Yeming; Holley, Cynthia P.; Wilinski, Daniel; Keles, Sunduz; Kimble, Judith; Wickens, Marvin; Hall, Traci M. Tanaka

    2012-01-01

    mRNA control networks depend on recognition of specific RNA sequences. Pumilio-fem-3 mRNA binding factor (PUF) RNA-binding proteins achieve that specificity through variations on a conserved scaffold. Saccharomyces cerevisiae Puf3p achieves specificity through an additional binding pocket for a cytosine base upstream of the core RNA recognition site. Here we demonstrate that this chemically simple adaptation is prevalent and contributes to the diversity of RNA specificities among PUF proteins. Bioinformatics analysis shows that mRNAs associated with Caenorhabditis elegans fem-3 mRNA binding factor (FBF)-2 in vivo contain an upstream cytosine required for biological regulation. Crystal structures of FBF-2 and C. elegans PUF-6 reveal binding pockets structurally similar to that of Puf3p, whereas sequence alignments predict a pocket in PUF-11. For Puf3p, FBF-2, PUF-6, and PUF-11, the upstream pockets and a cytosine are required for maximal binding to RNA, but the quantitative impact on binding affinity varies. Furthermore, the position of the upstream cytosine relative to the core PUF recognition site can differ, which in the case of FBF-2 originally masked the identification of this consensus sequence feature. Importantly, other PUF proteins lack the pocket and so do not discriminate upstream bases. A structure-based alignment reveals that these proteins lack key residues that would contact the cytosine, and in some instances, they also present amino acid side chains that interfere with binding. Loss of the pocket requires only substitution of one serine, as appears to have occurred during the evolution of certain fungal species. PMID:22205700

  8. Divergence of Pumilio/fem-3 mRNA binding factor (PUF) protein specificity through variations in an RNA-binding pocket.

    Science.gov (United States)

    Qiu, Chen; Kershner, Aaron; Wang, Yeming; Holley, Cynthia P; Wilinski, Daniel; Keles, Sunduz; Kimble, Judith; Wickens, Marvin; Hall, Traci M Tanaka

    2012-02-24

    mRNA control networks depend on recognition of specific RNA sequences. Pumilio-fem-3 mRNA binding factor (PUF) RNA-binding proteins achieve that specificity through variations on a conserved scaffold. Saccharomyces cerevisiae Puf3p achieves specificity through an additional binding pocket for a cytosine base upstream of the core RNA recognition site. Here we demonstrate that this chemically simple adaptation is prevalent and contributes to the diversity of RNA specificities among PUF proteins. Bioinformatics analysis shows that mRNAs associated with Caenorhabditis elegans fem-3 mRNA binding factor (FBF)-2 in vivo contain an upstream cytosine required for biological regulation. Crystal structures of FBF-2 and C. elegans PUF-6 reveal binding pockets structurally similar to that of Puf3p, whereas sequence alignments predict a pocket in PUF-11. For Puf3p, FBF-2, PUF-6, and PUF-11, the upstream pockets and a cytosine are required for maximal binding to RNA, but the quantitative impact on binding affinity varies. Furthermore, the position of the upstream cytosine relative to the core PUF recognition site can differ, which in the case of FBF-2 originally masked the identification of this consensus sequence feature. Importantly, other PUF proteins lack the pocket and so do not discriminate upstream bases. A structure-based alignment reveals that these proteins lack key residues that would contact the cytosine, and in some instances, they also present amino acid side chains that interfere with binding. Loss of the pocket requires only substitution of one serine, as appears to have occurred during the evolution of certain fungal species. PMID:22205700

  9. Binding of a fluorescence reporter and a ligand to an odorant-binding protein of the yellow fever mosquito, Aedes aegypti

    Science.gov (United States)

    Leal, Gabriel M.; Leal, Walter S.

    2015-01-01

    Odorant-binding proteins (OBPs), also named pheromone-binding proteins when the odorant is a pheromone, are essential for insect olfaction. They solubilize odorants that reach the port of entry of the olfactory system, the pore tubules in antennae and other olfactory appendages. Then, OBPs transport these hydrophobic compounds through an aqueous sensillar lymph to receptors embedded on dendritic membranes of olfactory receptor neurons. Structures of OBPs from mosquito species have shed new light on the mechanism of transport, although there is considerable debate on how they deliver odorant to receptors. An OBP from the southern house mosquito, Culex quinquefasciatus, binds the hydrophobic moiety of a mosquito oviposition pheromone (MOP) on the edge of its binding cavity. Likewise, it has been demonstrated that the orthologous protein from the malaria mosquito binds the insect repellent DEET on a similar edge of its binding pocket. A high school research project was aimed at testing whether the orthologous protein from the yellow fever mosquito, AaegOBP1, binds DEET and other insect repellents, and MOP was used as a positive control. Binding assays using the fluorescence reporter N-phenyl-1-naphtylamine (NPN) were inconclusive. However, titration of NPN fluorescence emission in AaegOBP1 solution with MOP led to unexpected and intriguing results. Quenching was observed in the initial phase of titration, but addition of higher doses of MOP led to a stepwise increase in fluorescence emission coupled with a blue shift, which can be explained at least in part by formation of MOP micelles to house stray NPN molecules. PMID:25671088

  10. Calciomics:prediction and analysis of EF-hand calcium binding proteins by protein engineering

    Institute of Scientific and Technical Information of China (English)

    YANG; Jenny; Jie

    2010-01-01

    Ca2+ plays a pivotal role in the physiology and biochemistry of prokaryotic and mammalian organisms.Viruses also utilize the universal Ca2+ signal to create a specific cellular environment to achieve coexistence with the host,and to propagate.In this paper we first describe our development of a grafting approach to understand site-specific Ca2+ binding properties of EF-hand proteins with a helix-loop-helix Ca2+ binding motif,then summarize our prediction and identification of EF-hand Ca2+ binding sites on a genome-wide scale in bacteria and virus,and next report the application of the grafting approach to probe the metal binding capability of predicted EF-hand motifs within the streptococcal hemoprotein receptor(Shr) of Streptococcus pyrogenes and the nonstructural protein 1(nsP1) of Sindbis virus.When methods such as the grafting approach are developed in conjunction with prediction algorithms we are better able to probe continuous Ca2+-binding sites that have been previously underrepresented due to the limitation of conventional methodology.

  11. NRIP, a novel calmodulin binding protein, activates calcineurin to dephosphorylate human papillomavirus E2 protein.

    Science.gov (United States)

    Chang, Szu-Wei; Tsao, Yeou-Ping; Lin, Chia-Yi; Chen, Show-Li

    2011-07-01

    Previously, we found a gene named nuclear receptor interaction protein (NRIP) (or DCAF6 or IQWD1). We demonstrate that NRIP is a novel binding protein for human papillomavirus 16 (HPV-16) E2 protein. HPV-16 E2 and NRIP can directly associate into a complex in vivo and in vitro, and the N-terminal domain of NRIP interacts with the transactivation domain of HPV-16 E2. Only full-length NRIP can stabilize E2 protein and induce HPV gene expression, and NRIP silenced by two designed small interfering RNAs (siRNAs) decreases E2 protein levels and E2-driven gene expression. We found that NRIP can directly bind with calmodulin in the presence of calcium through its IQ domain, resulting in decreased E2 ubiquitination and increased E2 protein stability. Complex formation between NRIP and calcium/calmodulin activates the phosphatase calcineurin to dephosphorylate E2 and increase E2 protein stability. We present evidences for E2 phosphorylation in vivo and show that NRIP acts as a scaffold to recruit E2 and calcium/calmodulin to prevent polyubiquitination and degradation of E2, enhancing E2 stability and E2-driven gene expression. PMID:21543494

  12. Characterization of a small acyl-CoA-binding protein (ACBP) from Helianthus annuus L. and its binding affinities.

    Science.gov (United States)

    Aznar-Moreno, Jose A; Venegas-Calerón, Mónica; Du, Zhi-Yan; Garcés, Rafael; Tanner, Julian A; Chye, Mee-Len; Martínez-Force, Enrique; Salas, Joaquín J

    2016-05-01

    Acyl-CoA-binding proteins (ACBPs) bind to acyl-CoA esters and promote their interaction with other proteins, lipids and cell structures. Small class I ACBPs have been identified in different plants, such as Arabidopsis thaliana (AtACBP6), Brassica napus (BnACBP) and Oryza sativa (OsACBP1, OsACBP2, OsACBP3), and they are capable of binding to different acyl-CoA esters and phospholipids. Here we characterize HaACBP6, a class I ACBP expressed in sunflower (Helianthus annuus) tissues, studying the specificity of its corresponding recombinant HaACBP6 protein towards various acyl-CoA esters and phospholipids in vitro, particularly using isothermal titration calorimetry and protein phospholipid binding assays. This protein binds with high affinity to de novo synthetized derivatives palmitoly-CoA, stearoyl-CoA and oleoyl-CoA (Kd 0.29, 0.14 and 0.15 μM respectively). On the contrary, it showed lower affinity towards linoleoyl-CoA (Kd 5.6 μM). Moreover, rHaACBP6 binds to different phosphatidylcholine species (dipalmitoyl-PC, dioleoyl-PC and dilinoleoyl-PC), yet it displays no affinity towards other phospholipids like lyso-PC, phosphatidic acid and lysophosphatidic acid derivatives. In the light of these results, the possible involvement of this protein in sunflower oil synthesis is considered. PMID:26938582

  13. A Venom Gland Extracellular Chitin-Binding-Like Protein from Pupal Endoparasitoid Wasps, Pteromalus Puparum, Selectively Binds Chitin

    Directory of Open Access Journals (Sweden)

    Yu Zhu

    2015-11-01

    Full Text Available Chitin-binding proteins (CBPs are present in many species and they act in a variety of biological processes. We analyzed a Pteromalus puparum venom apparatus proteome and transcriptome and identified a partial gene encoding a possible CBP. Here, we report cloning a full-length cDNA of a sequence encoding a chitin-binding-like protein (PpCBP from P. puparum, a pupal endoparasitoid of Pieris rapae. The cDNA encoded a 96-amino-acid protein, including a secretory signal peptide and a chitin-binding peritrophin-A domain. Phylogenetic analysis of chitin binding domains (CBDs of cuticle proteins and peritrophic matrix proteins in selected insects revealed that the CBD of PpCBP clustered with the CBD of Nasonia vitripennis. The PpCBP is specifically expressed in the venom apparatus of P. puparum, mostly in the venom gland. PpCBP expression was highest at day one after adult eclosion and much lower for the following five days. We produced a recombinant PpCBP and binding assays showed the recombinant protein selectively binds chitin but not cellulose in vitro. We infer that PpCBP serves a structural role in the venom reservoir, or may be injected into the host to help wound healing of the host exoskeleton.

  14. A Venom Gland Extracellular Chitin-Binding-Like Protein from Pupal Endoparasitoid Wasps, Pteromalus Puparum, Selectively Binds Chitin.

    Science.gov (United States)

    Zhu, Yu; Ye, Xin-Hai; Liu, Yang; Yan, Zhi-Chao; Stanley, David; Ye, Gong-Yin; Fang, Qi

    2015-12-01

    Chitin-binding proteins (CBPs) are present in many species and they act in a variety of biological processes. We analyzed a Pteromalus puparum venom apparatus proteome and transcriptome and identified a partial gene encoding a possible CBP. Here, we report cloning a full-length cDNA of a sequence encoding a chitin-binding-like protein (PpCBP) from P. puparum, a pupal endoparasitoid of Pieris rapae. The cDNA encoded a 96-amino-acid protein, including a secretory signal peptide and a chitin-binding peritrophin-A domain. Phylogenetic analysis of chitin binding domains (CBDs) of cuticle proteins and peritrophic matrix proteins in selected insects revealed that the CBD of PpCBP clustered with the CBD of Nasonia vitripennis. The PpCBP is specifically expressed in the venom apparatus of P. puparum, mostly in the venom gland. PpCBP expression was highest at day one after adult eclosion and much lower for the following five days. We produced a recombinant PpCBP and binding assays showed the recombinant protein selectively binds chitin but not cellulose in vitro. We infer that PpCBP serves a structural role in the venom reservoir, or may be injected into the host to help wound healing of the host exoskeleton. PMID:26633500

  15. Binding of cationic surfactants to DNA, protein and DNA-protein mixtures.

    Science.gov (United States)

    Gani, S A; Chattoraj, D K; Mukherjee, D C

    1999-06-01

    Extent of binding (gamma 2(1)) of cationic surfactants cetyltrimethyl ammonium bromide (CTAB), myristyltrimethyl ammonium bromide (MTAB) and dodecyl trimethyl ammonium bromide (DTAB) to calf-thymus DNA, bovine serum albumin (BSA) and to their binary mixture respectively have been measured as function of bulk concentration of the surfactant by using equilibrium dialysis technique. Binding of CTAB has been studied at different pH, ionic strength (mu), temperature and biopolymer composition and with native and denatured states of the biopolymers. The chain-length of different long chain amines plays a significant role in the extent of binding under identical solution condition. The binding ratios for CTAB to collagen, gelatin, DNA-collagen and DNA-gelatin mixtures respectively have also been determined. The conformational structures of different biopolymers are observed to play significant role in macromolecular interactions between protein and DNA in the presence of CTAB. From the experimental values of the maximum binding ratio (gamma 2m) at the saturation level for each individual biopolymer, ideal values (gamma 2m)id have been theoretically calculated for binary mixtures of biopolymers using additivity rule. The protein-DNA-CTAB interaction in mixture has been explained in terms of the deviation (delta) of (gamma 2m) from (gamma 2m)id in the presence of a surfactant in bulk. The binding of surfactants to biopolymers and to their binary mixtures are compared more precisely in terms of the Gibbs' free energy decrease (-delta G degree) for the saturation of the binding sites in the biopolymers or biopolymer mixtures with the change of the bulk surfactant activity from zero to unity in the rational mole fraction scale. PMID:10650715

  16. Protein-ligand binding affinities from large-scale quantum mechanical simulations

    OpenAIRE

    Fox, Stephen J.

    2012-01-01

    The accurate prediction of protein-drug binding affinities is a major aim of computational drug optimisation and development. A quantitative measure of binding affinity is provided by the free energy of binding, and such calculations typically require extensive configurational sampling of entities such as proteins with thousands of atoms. Current binding free energy methods use force fields to perform the configurational sampling and to compute interaction energies. Due to the empirical natur...

  17. Protein and solvent dynamics of the water-soluble chlorophyll-binding protein (WSCP)

    International Nuclear Information System (INIS)

    This study presents quasielastic neutron scattering data of the water-soluble chlorophyll-binding protein (WSCP) and the corresponding buffer solution at room temperature. The contributions of protein and buffer solution to the overall scattering are carefully separated. Otherwise, the fast water dynamics dominating the buffer contribution is likely to mask the slow protein dynamics. In the case of WSCP, the protein scattering can be described by two contributions: first, internal protein dynamics represented by a diffusion in a sphere with an average radius of 2.7 Angstroms and secondly global (Brownian) diffusion of the WSCP macromolecule with an upper limit for the translational diffusion coefficient of 9.4*10-7 cm2/s. (authors)

  18. Characterization of the retinoblastoma binding proteins RBP1 and RBP2

    DEFF Research Database (Denmark)

    Fattaey, A R; Helin, K; Dembski, M S;

    1993-01-01

    The retinoblastoma gene product, pRB, regulates cell proliferation by binding to and inhibiting the activity of key growth promoting proteins. Several cellular proteins have been shown to bind directly to pRB and the genes encoding a number of them have been isolated. The protein product of one of...

  19. Inhibition of the vitamin B12 binding capacity of proteins by the hydrolysis product of cyclophosphamide

    International Nuclear Information System (INIS)

    The inhibitory effect of cyclophosphamide hydrolysis product (CPHP) on vitamin B12 binding ability to proteins has been established. The ester N-(2-chloroethyl)-N'-(3-phosphopropyl)-etheylenediamine hydrochloride is probably responsible, in vitro, for blocking the protein binding sites. Preincubation of proteins with vitamin B12 prevents the inhibitory effect of CPHP. (au)

  20. Functional interaction between Smad, CREB binding protein, and p68 RNA helicase

    International Nuclear Information System (INIS)

    The transforming growth factors β control a diversity of biological processes including cellular proliferation, differentiation, apoptosis, and extracellular matrix production, and are critical effectors of embryonic patterning and development, including that of the orofacial region. TGFβ superfamily members signal through specific cell surface receptors that phosphorylate the cytoplasmic Smad proteins, resulting in their translocation to the nucleus and interaction with promoters of TGFβ-responsive genes. Subsequent alterations in transcription are cell type-specific and dependent on recruitment to the Smad/transcription factor complex of coactivators, such as CBP and p300, or corepressors, such as c-ski and SnoN. Since the affinity of Smads for DNA is generally low, additional accessory proteins that facilitate Smad/DNA binding are required, and are often cell- and tissue-specific. In order to identify novel Smad 3 binding proteins in developing orofacial tissue, a yeast two hybrid assay was employed in which the MH2 domain of Smad 3 was used to screen an expression library derived from mouse embryonic orofacial tissue. The RNA helicase, p68, was identified as a unique Smad binding protein, and the specificity of the interaction was confirmed through various in vitro and in vivo assays. Co-expression of Smad 3 and a CBP-Gal4 DNA binding domain fusion protein in a Gal4-luciferase reporter assay resulted in increased TGFβ-stimulated reporter gene transcription. Moreover, co-expression of p68 RNA helicase along with Smad 3 and CBP-Gal4 resulted in synergistic activation of Gal4-luciferase reporter expression. Collectively, these data indicate that the RNA helicase, p68, can directly interact with Smad 3 resulting in formation of a transcriptionally active ternary complex containing Smad 3, p68, and CBP. This offers a means of enhancing TGFβ-mediated cellular responses in developing orofacial tissue

  1. Crystal Structure of Menin Reveals Binding Site for Mixed Lineage Leukemia (MLL) Protein

    Energy Technology Data Exchange (ETDEWEB)

    Murai, Marcelo J.; Chruszcz, Maksymilian; Reddy, Gireesh; Grembecka, Jolanta; Cierpicki, Tomasz (Michigan); (UV)

    2014-10-02

    Menin is a tumor suppressor protein that is encoded by the MEN1 (multiple endocrine neoplasia 1) gene and controls cell growth in endocrine tissues. Importantly, menin also serves as a critical oncogenic cofactor of MLL (mixed lineage leukemia) fusion proteins in acute leukemias. Direct association of menin with MLL fusion proteins is required for MLL fusion protein-mediated leukemogenesis in vivo, and this interaction has been validated as a new potential therapeutic target for development of novel anti-leukemia agents. Here, we report the first crystal structure of menin homolog from Nematostella vectensis. Due to a very high sequence similarity, the Nematostella menin is a close homolog of human menin, and these two proteins likely have very similar structures. Menin is predominantly an {alpha}-helical protein with the protein core comprising three tetratricopeptide motifs that are flanked by two {alpha}-helical bundles and covered by a {beta}-sheet motif. A very interesting feature of menin structure is the presence of a large central cavity that is highly conserved between Nematostella and human menin. By employing site-directed mutagenesis, we have demonstrated that this cavity constitutes the binding site for MLL. Our data provide a structural basis for understanding the role of menin as a tumor suppressor protein and as an oncogenic co-factor of MLL fusion proteins. It also provides essential structural information for development of inhibitors targeting the menin-MLL interaction as a novel therapeutic strategy in MLL-related leukemias.

  2. (11)C-PBR28 binding to translocator protein increases with progression of Alzheimer's disease.

    Science.gov (United States)

    Kreisl, William C; Lyoo, Chul Hyoung; Liow, Jeih-San; Wei, Monica; Snow, Joseph; Page, Emily; Jenko, Kimberly J; Morse, Cheryl L; Zoghbi, Sami S; Pike, Victor W; Turner, R Scott; Innis, Robert B

    2016-08-01

    This longitudinal study sought to determine whether the 18 kDa translocator protein (TSPO), a marker of neuroinflammation, increases over time in Alzheimer's disease. Positron emission tomography imaging with the TSPO radioligand (11)C-PBR28 was performed at baseline and after a median follow-up of 2.7 years in 14 amyloid-positive patients and 8 amyloid-negative controls. Patients had a greater increase in TSPO binding than controls in inferior parietal lobule, precuneus, occipital cortex, hippocampus, entorhinal cortex, and combined middle and inferior temporal cortex. TSPO binding in temporoparietal regions increased from 3.9% to 6.3% per annum in patients, but ranged from -0.5% to 1% per annum in controls. The change in TSPO binding correlated with cognitive worsening on clinical dementia rating scale-sum of boxes and reduced cortical volume. The annual rate of increased TSPO binding in temporoparietal regions was about 5-fold higher in patients with clinical progression (n = 9) compared with those who did not progress (n = 5). TSPO may serve as a biomarker of Alzheimer's progression and response to anti-inflammatory therapies. PMID:27318133

  3. Heterogeneity of binding subunits of the human 150K insulin-like growth factor binding protein.

    Science.gov (United States)

    Gelato, M C; Gaynes, L A; Greenstein, L A; Nissley, S P

    1990-04-01

    Models for the structure of the GH-dependent 150K insulin-like growth factor-binding protein (IGF-BP) complex include 1) a binding subunit of 40-60K mol wt associated with a larger nonbinding component, and 2) an oligomeric structure simply made up of six 25-28K monomeric IGF-BP complexes. To evaluate these alternative models we examined the IGF-binding characteristics and behavior on an SP-Sephadex ion exchange column of BP species identified by chemically cross-linking [125I]IGF-I and [125I]IGF-II. In addition, human serum was gel filtered on Sephadex G-200 in 0.05 M NH4HCO3, pH 8.0, and the 150K BP identified by binding of [125I]IGF-II to column fractions. When [125I]IGF-I or [125I]IGF-II was cross-linked to the 150K BP with disuccinimidyl suberate and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (10-15%) and autoradiography, four specifically labeled complexes of 20K, 24K, 33K, and 47K mol wt were identified. We examined the IGF-binding characteristics of these species by cross-linking [125I]IGF-I and [125I]IGF-II after incubation in the presence of increasing concentrations of unlabeled IGF-I or IGF-II. Formation of the 24K complex was inhibited more potently by IGF-II than IGF-I, whereas the relative potency of IGF-I vs. IGF-II for inhibition of the formation of the other complexes depended upon whether [125I]IGF-II or [125I]IGF-I was used. When the 150K BP complex generated from gel filtration on Sephadex G-200 was acid stripped, the only species seen with chemical cross-linking of either [125I]IGF-I or [125I]IGF-II was the 47K complex. By both conventional competitive binding studies and cross-linking [125I]IGF-I and [125I]IGF-II after incubation with increasing concentrations of unlabeled IGF-I or IGF-II, the formation of the 47K complex was usually more potently inhibited by IGF-I than IGF-II. When Cohn fraction IV extract was chromatographed on a SP-Sephadex column (pH 3) and cross-linking performed on the flow-through, the 47K

  4. Definition study for temperature control in advanced protein crystal growth

    Science.gov (United States)

    Nyce, Thomas A.; Rosenberger, Franz; Sowers, Jennifer W.; Monaco, Lisa A.

    1990-01-01

    Some of the technical requirements for an expedient application of temperature control to advanced protein crystal growth activities are defined. Lysozome was used to study the effects of temperature ramping and temperature gradients for nucleation/dissolution and consecutive growth of sizable crystals and, to determine a prototype temperature program. The solubility study was conducted using equine serum albumin (ESA) which is an extremely stable, clinically important protein due to its capability to bind and transport many different small ions and molecules.

  5. Cholesterol-lowering effect of rice bran protein containing bile acid-binding proteins.

    Science.gov (United States)

    Wang, Jilite; Shimada, Masaya; Kato, Yukina; Kusada, Mio; Nagaoka, Satoshi

    2015-01-01

    Dietary plant protein is well known to reduce serum cholesterol levels. Rice bran is a by-product of rice milling and is a good source of protein. The present study examined whether feeding rats a high-cholesterol diet containing 10% rice bran protein (RBP) for 10 d affected cholesterol metabolism. Rats fed dietary RBP had lower serum total cholesterol levels and increased excretion of fecal steroids, such as cholesterol and bile acids, than those fed dietary casein. In vitro assays showed that RBP strongly bound to taurocholate, and inhibited the micellar solubility of cholesterol, compared with casein. Moreover, the bile acid-binding proteins of the RBP were eluted by a chromatographic column conjugated with cholic acid, and one of them was identified as hypothetical protein OsJ_13801 (NCBI accession No. EAZ29742) using MALDI-TOF mass spectrometry analysis. These results suggest that the hypocholesterolemic action of the RBP may be caused by the bile acid-binding proteins. PMID:25374002

  6. A Column Free Protein Purification Procedure using E. coli Single ‐ Stranded DNA Binding Protein (SSB) as an Affinity Tag

    OpenAIRE

    Soffe, Mark

    2014-01-01

    SSBs are DNA binding proteins that are essential components of cells and play key roles in DNA replication, repair, and recombination. Here we utilize two biochemical properties associated with the E. coli SSB protein to develop a novel procedure to purify proteins using a resin-free strategy to combat the largest bottleneck in biochemical research— obtaining uncontaminated, single proteins from the total cellular contents. 1. E. coli SSB binds to single stranded DNA (ssDNA) with extremely...

  7. Interaction of rat hormone-sensitive lipase with adipocyte lipid-binding protein

    OpenAIRE

    Shen, Wen-Jun; Sridhar, Kunju; Bernlohr, David A.; Fredric B Kraemer

    1999-01-01

    Hormone-sensitive lipase (HSL) is a cytosolic neutral lipase that functions as the rate-limiting enzyme for the mobilization of free fatty acids in adipose tissue. By using the yeast two-hybrid system to examine the potential interaction of HSL with other cellular proteins, evidence is provided to demonstrate a direct interaction of HSL with adipocyte lipid-binding protein (ALBP), a member of the family of intracellular lipid-binding proteins that binds fatty acids, retinoids, and other hydro...

  8. A conformational analysis of Walker motif A [GXXXXGKT (S)] in nucleotide-binding and other proteins

    OpenAIRE

    Ramakrishnan, C; Dani, VS; Ramasarma, T

    2002-01-01

    The sequence GXXXXGKT/S, popularly known as Walker motif A, is widely believed to be the site for binding nucleotides in many proteins. Examination of the crystal structures in the Protein Data Bank showed that about half of the examples having these sequences do not bind or use nucleotides. Data analyses showed 92 different Walker sequences of the variable quartet (XXXX). Ramachandran angles in this segment revealed conformational similarity in the group of 45 proteins, known to bind or util...

  9. Isothermal Chemical Denaturation to Determine Binding Affinity of Small Molecules to G-Protein Coupled Receptors

    OpenAIRE

    Ross, Patrick; Weihofen, Wilhelm; Siu, Fai; Xie, Amy; Katakia, Hetal; Wright, S. Kirk; Hunt, Ian; Brown, Richard K; Freire, Ernesto

    2014-01-01

    The determination of accurate binding affinities is critical in drug discovery and development. Several techniques are available for characterizing the binding of small molecules to soluble proteins. The situation is different for integral membrane proteins. Isothermal chemical denaturation (ICD) has been shown to be a valuable biophysical method to determine in a direct and label-free fashion the binding of ligands to soluble proteins. In this communication, the application of isothermal che...

  10. Isotope-coded ATP Probe for Quantitative Affinity Profiling of ATP-binding Proteins

    OpenAIRE

    Xiao, Yongsheng; Guo, Lei; Wang, Yinsheng

    2013-01-01

    ATP-binding proteins play significant roles in numerous cellular processes. Here, we introduced a novel isotope-coded ATP-affinity probe (ICAP) as acylating agent to simultaneously enrich and incorporate isotope label to ATP-binding proteins. By taking advantage of the quantitative capability of this isotope-coded probe, we devised an affinity profiling strategy to comprehensively characterize ATP-protein interactions at the entire proteome scale. False-positive identification of ATP-binding ...

  11. The effects of GH and hormone replacement therapy on serum concentrations of mannan-binding lectin, surfactant protein D and vitamin D binding protein in Turner syndrome

    DEFF Research Database (Denmark)

    Gravholt, Claus Højbjerg; Leth-Larsen, Rikke; Lauridsen, Anna Lis;

    2004-01-01

    function. In the present study we examined whether GH or hormone replacement therapy (HRT) in Turner syndrome (TS) influence the serum concentrations of MBL and two other proteins partaking in the innate immune defence, surfactant protein D (SP-D) and vitamin D binding protein (DBP). DESIGN: Study 1: a...

  12. The TSG101 protein binds to connexins and is involved in connexin degradation

    International Nuclear Information System (INIS)

    Gap junctions mediate electrical and metabolic communication between cells in almost all tissues and are proposed to play important roles in cellular growth control, differentiation and embryonic development. Gap junctional communication and channel assembly were suggested to be regulated by interaction of connexins with different proteins including kinases and phosphatases. Here, we identified the tumor susceptibility gene 101 (TSG101) protein to bind to the carboxyterminal tail of connexin45 in a yeast two-hybrid protein interaction screen. Glutathione S-transferase pull down experiments and immunoprecipitation revealed that not only connexin45 but also connexin30.2, -36, and -43 carboxyterminal regions were associated with TSG101 protein in pull down analyses and that connexin31, -43 and -45 co-precipitate with endogenous TSG101 protein in lysates from HM1 embryonic stem cells. TSG101 has been shown to be involved in cell cycle control, transcriptional regulation and turnover of endocytosed proteins. Thus, we decided to study the functional role of this interaction. SiRNA mediated knock down of TSG101 in HM1 embryonic stem cells led to increased levels of connexin43 and -45, prolonged half life of these connexins and increased transfer of microinjected Lucifer yellow. Our results suggest that TSG101 is involved in the degradation of connexins via interaction with connexin proteins

  13. Split green fluorescent protein as a modular binding partner for protein crystallization

    International Nuclear Information System (INIS)

    A strategy using a new split green fluorescent protein (GFP) as a modular binding partner to form stable protein complexes with a target protein is presented. The modular split GFP may open the way to rapidly creating crystallization variants. A modular strategy for protein crystallization using split green fluorescent protein (GFP) as a crystallization partner is demonstrated. Insertion of a hairpin containing GFP β-strands 10 and 11 into a surface loop of a target protein provides two chain crossings between the target and the reconstituted GFP compared with the single connection afforded by terminal GFP fusions. This strategy was tested by inserting this hairpin into a loop of another fluorescent protein, sfCherry. The crystal structure of the sfCherry-GFP(10–11) hairpin in complex with GFP(1–9) was determined at a resolution of 2.6 Å. Analysis of the complex shows that the reconstituted GFP is attached to the target protein (sfCherry) in a structurally ordered way. This work opens the way to rapidly creating crystallization variants by reconstituting a target protein bearing the GFP(10–11) hairpin with a variety of GFP(1–9) mutants engineered for favorable crystallization

  14. Role of SUMO in RNF4-mediated promyelocytic leukemia protein (PML) degradation: sumoylation of PML and phospho-switch control of its SUMO binding domain dissected in living cells.

    Science.gov (United States)

    Percherancier, Yann; Germain-Desprez, Delphine; Galisson, Frédéric; Mascle, Xavier H; Dianoux, Laurent; Estephan, Patricia; Chelbi-Alix, Mounira K; Aubry, Muriel

    2009-06-12

    Promyelocytic leukemia protein (PML) is a tumor suppressor acting as the organizer of subnuclear structures called PML nuclear bodies (NBs). Both covalent modification of PML by the small ubiquitin-like modifier (SUMO) and non-covalent binding of SUMO to the PML SUMO binding domain (SBD) are necessary for PML NB formation and maturation. PML sumoylation and proteasome-dependent degradation induced by the E3 ubiquitin ligase, RNF4, are enhanced by the acute promyelocytic leukemia therapeutic agent, arsenic trioxide (As2O3). Here, we established a novel bioluminescence resonance energy transfer (BRET) assay to dissect and monitor PML/SUMO interactions dynamically in living cells upon addition of therapeutic agents. Using this sensitive and quantitative SUMO BRET assay that distinguishes PML sumoylation from SBD-mediated PML/SUMO non-covalent interactions, we probed the respective roles of covalent and non-covalent PML/SUMO interactions in PML degradation and interaction with RNF4. We found that, although dispensable for As2O3-enhanced PML sumoylation and RNF4 interaction, PML SBD core sequence was required for As2O3- and RNF4-induced PML degradation. As confirmed with a phosphomimetic mutant, phosphorylation of a stretch of serine residues, contained within PML SBD was needed for PML interaction with SUMO-modified protein partners and thus for NB maturation. However, mutation of these serine residues did not impair As2O3- and RNF4-induced PML degradation, contrasting with the known role of these phosphoserine residues for casein kinase 2-promoted PML degradation. Altogether, these data suggest a model whereby sumoylation- and SBD-dependent PML oligomerization within NBs is sufficient for RNF4-mediated PML degradation and does not require the phosphorylation-dependent association of PML with other sumoylated partners. PMID:19380586

  15. Stimulation of translation by human Unr requires cold shock domains 2 and 4, and correlates with poly(A) binding protein interaction

    OpenAIRE

    Swagat Ray; Anderson, Emma C.

    2016-01-01

    The RNA binding protein Unr, which contains five cold shock domains, has several specific roles in post-transcriptional control of gene expression. It can act as an activator or inhibitor of translation initiation, promote mRNA turnover, or stabilise mRNA. Its role depends on the mRNA and other proteins to which it binds, which includes cytoplasmic poly(A) binding protein 1 (PABP1). Since PABP1 binds to all polyadenylated mRNAs, and is involved in translation initiation by interaction with eu...

  16. Mapping the Anopheles gambiae odorant binding protein 1 (AgamOBP1) using modeling techniques, site directed mutagenesis, circular dichroism and ligand binding assays.

    Science.gov (United States)

    Rusconi, B; Maranhao, A C; Fuhrer, J P; Krotee, P; Choi, S H; Grun, F; Thireou, T; Dimitratos, S D; Woods, D F; Marinotti, O; Walter, M F; Eliopoulos, E

    2012-08-01

    The major malaria vector in Sub-Saharan Africa is the Anopheles gambiae mosquito. This species is a key target of malaria control measures. Mosquitoes find humans primarily through olfaction, yet the molecular mechanisms associated with host-seeking behavior remain largely unknown. To further understand the functionality of A. gambiae odorant binding protein 1 (AgamOBP1), we combined in silico protein structure modeling and site-directed mutagenesis to generate 16 AgamOBP1 protein analogues containing single point mutations of interest. Circular dichroism (CD) and ligand-binding assays provided data necessary to probe the effects of the point mutations on ligand binding and the overall structure of AgamOBP1. Far-UV CD spectra of mutated AgamOBP1 variants displayed both substantial decreases to ordered α-helix structure (up to22%) and increases to disordered α-helix structure(up to 15%) with only minimal changes in random coil (unordered) structure. In mutations Y54A, Y122A and W114Q, aromatic side chain removal from the binding site significantly reduced N-phenyl-1-naphthylamine binding. Several non-aromatic mutations (L15T, L19T, L58T, L58Y, M84Q, M84K, H111A, Y122A and L124T) elicited changes to protein conformation with subsequent effects on ligand binding. This study provides empirical evidence for the in silico predicted functions of specific amino acids in AgamOBP1 folding and ligand binding characteristics. PMID:22564768

  17. Identification of pheromone components and their binding affinity to the odorant binding protein CcapOBP83a-2 of the Mediterranean fruit fly, Ceratitis capitata

    Czech Academy of Sciences Publication Activity Database

    Siciliano, P.; He, X. L.; Woodcock, C.; Pickett, J. A.; Field, L. M.; Birkett, M. A.; Kalinová, Blanka; Gomulski, L. M.; Scolari, F.; Gasperi, G.; Malacrida, A. R.; Zhou, J. J.

    2014-01-01

    Roč. 48, May (2014), s. 51-62. ISSN 0965-1748 Institutional support: RVO:61388963 Keywords : medfly * Ceratitis capitata * olfaction * odorant binding protein * pheromone binding protein * pheromone * binding studies * protein expression * electroantennography * GC-EAG * fluorescence displacement Subject RIV: CE - Biochemistry Impact factor: 3.450, year: 2014

  18. Acute hantavirus infection induces galectin-3-binding protein.

    Science.gov (United States)

    Hepojoki, Jussi; Strandin, Tomas; Hetzel, Udo; Sironen, Tarja; Klingström, Jonas; Sane, Jussi; Mäkelä, Satu; Mustonen, Jukka; Meri, Seppo; Lundkvist, Ake; Vapalahti, Olli; Lankinen, Hilkka; Vaheri, Antti

    2014-11-01

    Hantaviruses are zoonotic viruses that cause life-threatening diseases when transmitted to humans. Severe hantavirus infection is manifested by impairment of renal function, pulmonary oedema and capillary leakage. Both innate and adaptive immune responses contribute to the pathogenesis, but the underlying mechanisms are not fully understood. Here, we showed that galectin-3-binding protein (Gal-3BP) was upregulated as a result of hantavirus infection both in vitro and in vivo. Gal-3BP is a secreted glycoprotein found in human serum, and increased Gal-3BP levels have been reported in chronic viral infections and in several types of cancer. Our in vitro experiments showed that, whilst Vero E6 cells (an African green monkey kidney cell line) constitutively expressed and secreted Gal-3BP, this protein was detected in primary human cells only as a result of hantavirus infection. Analysis of Gal-3BP levels in serum samples of cynomolgus macaques infected experimentally with hantavirus indicated that hantavirus infection induced Gal-3BP also in vivo. Finally, analysis of plasma samples collected from patients hospitalized because of acute hantavirus infection showed higher Gal-3BP levels during the acute than the convalescent phase. Furthermore, the Gal-3BP levels in patients with haemorrhagic fever with renal syndrome correlated with increased complement activation and with clinical variables reflecting the severity of acute hantavirus infection. PMID:25013204

  19. QM/MM Molecular Dynamics Studies of Metal Binding Proteins

    Directory of Open Access Journals (Sweden)

    Pietro Vidossich

    2014-07-01

    Full Text Available Mixed quantum-classical (quantum mechanical/molecular mechanical (QM/MM simulations have strongly contributed to providing insights into the understanding of several structural and mechanistic aspects of biological molecules. They played a particularly important role in metal binding proteins, where the electronic effects of transition metals have to be explicitly taken into account for the correct representation of the underlying biochemical process. In this review, after a brief description of the basic concepts of the QM/MM method, we provide an overview of its capabilities using selected examples taken from our work. Specifically, we will focus on heme peroxidases, metallo-β-lactamases, α-synuclein and ligase ribozymes to show how this approach is capable of describing the catalytic and/or structural role played by transition (Fe, Zn or Cu and main group (Mg metals. Applications will reveal how metal ions influence the formation and reduction of high redox intermediates in catalytic cycles and enhance drug metabolism, amyloidogenic aggregate formation and nucleic acid synthesis. In turn, it will become manifest that the protein frame directs and modulates the properties and reactivity of the metal ions.

  20. Serum protein inhibition of thyrotropin binding to human thyroid tissue

    International Nuclear Information System (INIS)

    We used a modificaton of the TSH radioreceptor assay to detect TSH-binding inhibition (TBI) activity in serum and serum fractions from normal subjects and patients with Graves' disease. TBI activity is present in normal IgG prepared by DEAE-Sephadex chromatography and in normal globulins prepared by precipitation at 1.6 M ammonium sulfate. Other normal serum proteins also had TBI activity when large concentrations were tested. Gel filtration chromatography and powder block electrophoresis were used to prepare fractions of normal and Graves' disease sera. In these fractions from normal serum, TBI activity was found in both γ-globulin and α-globulin-albumin fractions electrophoretically and in both 7S and 4S peaks from gel filtration. TBI activity from Graves' disease patients' sera was similarly distributed, but relatively more TBI accompanied the electrophoretic γ-globulins. Sepharose Protein-A and anti-IgG were used as immunoabsorbents to isolate and purify IgG from normal and Graves' disease sera. TBI activity in IgG was proportional to the IgG concentration, indicating that the TBI which migrates as a γ-globulin electrophoretically is an IgG and thus may possibly be an antibody. Inhibitory activity found in normal serum globulins and in the non-IgG fractions of both normal and abnormal sera seriously interferes with attempts to use the TSH radioreceptor assay to study the hypothesized anti-TSH receptor antibody in the serum of patients with Graves' disease

  1. Is vitamin D binding protein a novel predictor of labour?

    Directory of Open Access Journals (Sweden)

    Stella Liong

    Full Text Available Vitamin D binding protein (VDBP has previously been identified in the amniotic fluid and cervicovaginal fluid (CVF of pregnant women. The biological functions of VDBP include acting as a carrier protein for vitamin D metabolites, the clearance of actin that is released during tissue injury and the augmentation of the pro-inflammatory response. This longitudinal observational study was conducted on 221 healthy pregnant women who spontaneously laboured and delivered either at term or preterm. Serial CVF samples were collected and VDBP was measured by ELISA. Binary logistic regression analysis was performed to assess the utility of VDBP as a predictor of labour. VDBP in the CVF did not change between 20 and 35 weeks' gestation. VDBP measured in-labour was significantly increased 4.2 to 7.4-fold compared to 4-7, 8-14 and 15-28 days before labour (P<0.05. VDBP concentration was 4.3-fold significantly higher at 0-3 days compared to 15-28 days pre-labour (P<0.05. The efficacy of VDBP to predict spontaneous labour onset within 3 days provided a positive and negative predictive value of 82.8% and 95.3% respectively (area under receiver operator characteristic curve  = 0.974. This longitudinal study of pregnant women suggests that VDBP in the CVF may be a useful predictor of labour.

  2. A FRET-based method for monitoring septin polymerization and binding of septin-associated proteins.

    Science.gov (United States)

    Booth, E A; Thorner, J

    2016-01-01

    Much about septin function has been inferred from in vivo studies using mainly genetic methods, and much of what we know about septin organization has been obtained through examination of static structures in vitro primarily by electron microscopy. Deeper mechanistic insight requires real-time analysis of the dynamics of the assembly of septin-based structures and how other proteins associate with them. We describe here a Förster resonance energy transfer (FRET)-based approach for measuring in vitro the rate and extent of filament formation from septin complexes, binding of other proteins to septin structures, and the apparent affinities of these interactions. FRET is particularly well suited for interrogating protein-protein interactions, especially on a rapid timescale; the spectral change provides an unambiguous indication of whether two elements within the system under study are associating and serves as a molecular-level "ruler" because it is very sensitive to the separation between the donor and acceptor fluorophores over biologically relevant distances (≤10nm). The necessary procedures involve generation of appropriate cysteine-less and single cysteine-containing septin variants, expression and purification of the heterooctameric complexes containing them, efficient labeling of the purified complexes with desired fluorophores, fluorimetric measurement of FRET, and appropriate safeguards and controls in data acquisition and analysis. Our methods can be used to interrogate the effects of buffer conditions, small molecules, and septin-binding proteins on septin filament assembly or stability; determine the effect of alternative septin subunits, mutational alterations, or posttranslational modifications on assembly; and, delineate the location of septin-binding proteins. PMID:27473902

  3. Identification of novel viral receptors with cell line expressing viral receptor-binding protein.

    Science.gov (United States)

    Mei, Mei; Ye, Jianqiang; Qin, Aijian; Wang, Lin; Hu, Xuming; Qian, Kun; Shao, Hongxia

    2015-01-01

    The viral cell receptors and infection can be blocked by the expression of the viral receptor-binding protein. Thus, the viral cell receptor is an attractive target for anti-viral strategies, and the identification of viral cell receptor is critical for better understanding and controlling viral disease. As a model system for viral entry and anti-retroviral approaches, avian sarcoma/leukosis virus (ASLV, including the A-J ten subgroups) has been studied intensively and many milestone discoveries have been achieved based on work with ASLV. Here, we used a DF1 cell line expressed viral receptor-binding protein to efficiently identify chicken Annexin A2 (chANXA2) as a novel receptor for retrovirus ALV-J (avian leukosis virus subgroup J). Our data demonstrate that antibodies or siRNA to chANXA2 significantly inhibited ALV-J infection and replication, and over-expression of chANXA2 permitted the entry of ALV-J into its non-permissible cells. Our findings have not only identified chANXA2 as a novel biomarker for anti-ALV-J, but also demonstrated that cell lines with the expression of viral receptor-binding protein could be as efficient tools for isolating functional receptors to identify novel anti-viral targets. PMID:25604889

  4. Penicillin binding proteins as danger signals: meningococcal penicillin binding protein 2 activates dendritic cells through Toll-like receptor 4.

    Directory of Open Access Journals (Sweden)

    Marcelo Hill

    Full Text Available Neisseria meningitidis is a human pathogen responsible for life-threatening inflammatory diseases. Meningococcal penicillin-binding proteins (PBPs and particularly PBP2 are involved in bacterial resistance to β-lactams. Here we describe a novel function for PBP2 that activates human and mouse dendritic cells (DC in a time and dose-dependent manner. PBP2 induces MHC II (LOGEC50 = 4.7 µg/ml ± 0.1, CD80 (LOGEC50 = 4.88 µg/ml ± 0.15 and CD86 (LOGEC50 = 5.36 µg/ml ± 0.1. This effect was abolished when DCs were co-treated with anti-PBP2 antibodies. PBP2-treated DCs displayed enhanced immunogenic properties in vitro and in vivo. Furthermore, proteins co-purified with PBP2 showed no effect on DC maturation. We show through different in vivo and in vitro approaches that this effect is not due to endotoxin contamination. At the mechanistic level, PBP2 induces nuclear localization of p65 NF-kB of 70.7 ± 5.1% cells versus 12 ± 2.6% in untreated DCs and needs TLR4 expression to mature DCs. Immunoprecipitation and blocking experiments showed thatPBP2 binds TLR4. In conclusion, we describe a novel function of meningococcal PBP2 as a pathogen associated molecular pattern (PAMP at the host-pathogen interface that could be recognized by the immune system as a danger signal, promoting the development of immune responses.

  5. Somitogenesis clock-wave initiation requires differential decay and multiple binding sites for clock protein.

    Directory of Open Access Journals (Sweden)

    Mark Campanelli

    2010-04-01

    Full Text Available Somitogenesis is a process common to all vertebrate embryos in which repeated blocks of cells arise from the presomitic mesoderm (PSM to lay a foundational pattern for trunk and tail development. Somites form in the wake of passing waves of periodic gene expression that originate in the tailbud and sweep posteriorly across the PSM. Previous work has suggested that the waves result from a spatiotemporally graded control protein that affects the oscillation rate of clock-gene expression. With a minimally constructed mathematical model, we study the contribution of two control mechanisms to the initial formation of this gene-expression wave. We test four biologically motivated model scenarios with either one or two clock protein transcription binding sites, and with or without differential decay rates for clock protein monomers and dimers. We examine the sensitivity of wave formation with respect to multiple model parameters and robustness to heterogeneity in cell population. We find that only a model with both multiple binding sites and differential decay rates is able to reproduce experimentally observed waveforms. Our results show that the experimentally observed characteristics of somitogenesis wave initiation constrain the underlying genetic control mechanisms.

  6. Design of an actively controlled snow ski release binding.

    Science.gov (United States)

    Hull, M L; Allen, K W

    1981-08-01

    A new electronic ski binding has been designed which may better protect skiers from lower extremity injuries. A four-step procedure for developing binding release criteria aimed at preventing specific injuries is outlined. Using simplified biomechanical models, the release criteria for tibia fracture in both torsion and flexion are derived. A binding design which embodies the derived release criteria is described. The binding consists of three subsystems: 1) a dynamometer, 2) an analog computer controller, and 3) an electromechanical release mechanism. The strain gage dynamometer directly measures torsion and bending moments between the boot and ski. An analog computer controlled processes dynamometer signals. Dual release mode capability is achieved by parallel solution of differential equations which model the leg in both medial-lateral rotation and flexion. When the model solution reaches a critical value, the controller actuates the release mechanism. The release mechanism incorporates a unique closed circuit hydraulic system which rigidly locks the boot to the ski until release. Laboratory tests on a prototype confirm that the computer-controlled binding prevents inadvertent release under noninjurious high-magnitude, short-duration loads but releases before quasi-static loads reach injurious levels. PMID:7278190

  7. Gc protein (vitamin D-binding protein): Gc genotyping and GcMAF precursor activity.

    Science.gov (United States)

    Nagasawa, Hideko; Uto, Yoshihiro; Sasaki, Hideyuki; Okamura, Natsuko; Murakami, Aya; Kubo, Shinichi; Kirk, Kenneth L; Hori, Hitoshi

    2005-01-01

    The Gc protein (human group-specific component (Gc), a vitamin D-binding protein or Gc globulin), has important physiological functions that include involvement in vitamin D transport and storage, scavenging of extracellular G-actin, enhancement of the chemotactic activity of C5a for neutrophils in inflammation and macrophage activation (mediated by a GalNAc-modified Gc protein (GcMAF)). In this review, the structure and function of the Gc protein is focused on especially with regard to Gc genotyping and GcMAF precursor activity. A discussion of the research strategy "GcMAF as a target for drug discovery" is included, based on our own research. PMID:16302727

  8. Significance of lipopolysaccharide-binding protein (an acute phase protein) in monitoring critically ill patients

    OpenAIRE

    Prucha, Miroslav; Herold, Ivan; Zazula, Roman; Dubska, Ladislava; Dostal, Miroslav; Hildebrand, Thomas; Hyanek, Josef

    2003-01-01

    Introduction The present study was conducted to assess the value of serum concentration of lipopolysaccharide-binding protein (LBP) in patients with systemic inflammatory response syndrome (SIRS), sepsis and septic shock with respect to its ability to differentiate between infectious and noninfectious etiologies in SIRS and to predict prognosis. Methods This prospective cohort study was conducted in a multidisciplinary intensive care unit. Sixty-eight patients, admitted consecutively to the i...

  9. Identification of Pneumococcal Surface Protein A as a Lactoferrin-Binding Protein of Streptococcus pneumoniae

    OpenAIRE

    Hammerschmidt, Sven; Bethe, Gesina; H. Remane, Petra; Chhatwal, Gursharan S.

    1999-01-01

    Lactoferrin (Lf), an iron-sequestering glycoprotein, predominates in mucosal secretions, where the level of free extracellular iron (10−18 M) is not sufficient for bacterial growth. This represents a mechanism of resistance to bacterial infections by prevention of colonization of the host by pathogens. In this study we were able to show that Streptococcus pneumoniae specifically recognizes and binds the iron carrier protein human Lf (hLf). Pretreatment of pneumococci with proteases reduced hL...

  10. Biointerface: protein enhanced stem cells binding to implant surface.

    Science.gov (United States)

    Chrzanowski, W; Kondyurin, A; Lee, Jae Ho; Lord, Megan S; Bilek, M M M; Kim, Hae-Won

    2012-09-01

    The number of metallic implantable devices placed every year is estimated at 3.7 million. This number has been steadily increasing over last decades at a rate of around 8 %. In spite of the many successes of the devices the implantation of biomaterial into tissues almost universally leads to the development of an avascular sac, which consists of fibrous tissue around the device and walls off the implant from the body. This reaction can be detrimental to the function of implant, reduces its lifetime, and necessitates repeated surgery. Clearly, to reduce the number of revision surgeries and improve long-term implant function it is necessary to enhance device integration by modulating cell adhesion and function. In this paper we have demonstrated that it is possible to enhance stem cell attachment using engineered biointerfaces. To create this functional interface, samples were coated with polymer (as a precursor) and then ion implanted to create a reactive interface that aids the binding of biomolecules--fibronectin. Both AFM and XPS analyses confirmed the presence of protein layers on the samples. The amount of protein was significant greater for the ion implanted surfaces and was not disrupted upon washing with detergent, hence the formation of strong bonds with the interface was confirmed. While, for non ion implanted surfaces, a decrease of protein was observed after washing with detergent. Finally, the number of stem cells attached to the surface was enhanced for ion implanted surfaces. The studies presented confirm that the developed bionterface with immobilised fibronectin is an effective means to modulate stem cell attachment. PMID:22714559

  11. Regulation of RNA binding proteins in trypanosomatid protozoan parasites.

    Science.gov (United States)

    Romaniuk, María Albertina; Cervini, Gabriela; Cassola, Alejandro

    2016-02-26

    Posttranscriptional mechanisms have a critical role in the overall outcome of gene expression. These mechanisms are especially relevant in protozoa from the genus Trypanosoma, which is composed by death threatening parasites affecting people in Sub-saharan Africa or in the Americas. In these parasites the classic view of regulation of transcription initiation to modulate the products of a given gene cannot be applied. This is due to the presence of transcription start sites that give rise to long polycistronic units that need to be processed costranscriptionally by trans-splicing and polyadenylation to give mature monocistronic mRNAs. Posttranscriptional mechanisms such as mRNA degradation and translational repression are responsible for the final synthesis of the required protein products. In this context, RNA-binding proteins (RBPs) in trypanosomes have a relevant role as modulators of mRNA abundance and translational repression by associating to the 3' untranslated regions in mRNA. Many different RBPs have been proposed to modulate cohorts of mRNAs in trypanosomes. However, the current understanding of their functions lacks a dynamic view on the different steps at which these RBPs are regulated. Here, we discuss different evidences to propose regulatory events for different RBPs in these parasites. These events vary from regulated developmental expression, to biogenesis of cytoplasmic ribonucleoprotein complexes in the nucleus, and condensation of RBPs and mRNA into large cytoplasmic granules. Finally, we discuss how newly identified posttranslational modifications of RBPs and mRNA metabolism-related proteins could have an enormous impact on the modulation of mRNA abundance. To understand these modifications is especially relevant in these parasites due to the fact that the enzymes involved could be interesting targets for drug therapy. PMID:26981203

  12. RNA-binding protein hnRNPLL as a critical regulator of lymphocyte homeostasis and differentiation.

    Science.gov (United States)

    Chang, Xing

    2016-05-01

    RNA-binding proteins orchestrate posttranscriptional regulation of gene expression, such as messenger RNA (mRNA) splicing, RNA stability regulation, and translation regulation. Heterogeneous nuclear RNA-binding proteins (hnRNPs) refer to a collection of unrelated RNA-binding proteins predominantly located in the nucleus (Han et al. Biochem J 2010, 430:379-392). Although canonical functions of hnRNPs are to promote pre-mRNA splicing, they are involved in all the processes of RNA metabolism through recognizing specific cis-elements on RNA (Dreyfuss et al. Annu Rev Biochem 1993, 62:289-321; Huelga et al. Cell Rep 2012, 1:167-178; Krecic and Swanson. Curr Opin Cell Biol 1999, 11:363-371). Heterogeneous nuclear RNA-binding protein L like (hnRNPLL) is a tissue-specific hnRNP, which was identified as a regulator of CD45RA to CD45RO switching during memory T-cell development (Oberdoerffer et al. Science 2008, 321:686-691; Topp et al. RNA 2008, 14:2038-2049; Wu et al. Immunity 2008, 29:863-875). Since then, hnRNPLL has emerged as a critical regulator of lymphocyte homeostasis and terminal differentiation, controlling alternative splicing or expression of critical genes for the lymphocytes development (Wu et al. Immunity 2008, 29:863-875; Chang et al. Proc Natl Acad Sci USA 2015, 112:E1888-E1897). This review will summarize recent advances in understanding the functions of hnRNPLL, focusing on its biochemical functions and physiological roles in lymphocyte differentiation and homeostasis. WIREs RNA 2016, 7:295-302. doi: 10.1002/wrna.1335 For further resources related to this article, please visit the WIREs website. PMID:26821996

  13. Nucleomorphin. A novel, acidic, nuclear calmodulin-binding protein from dictyostelium that regulates nuclear number.

    Science.gov (United States)

    Myre, Michael A; O'Day, Danton H

    2002-05-31

    Probing of Dictyostelium discoideum cell extracts after SDS-PAGE using (35)S-recombinant calmodulin (CaM) as a probe has revealed approximately three-dozen Ca(2+)-dependent calmodulin binding proteins. Here, we report the molecular cloning, expression, and subcellular localization of a gene encoding a novel calmodulin-binding protein (CaMBP); we have called nucleomorphin, from D. discoideum. A lambdaZAP cDNA expression library of cells from multicellular development was screened using a recombinant calmodulin probe ((35)S-VU1-CaM). The open reading frame of 1119 nucleotides encodes a polypeptide of 340 amino acids with a calculated molecular mass of 38.7 kDa and is constitutively expressed throughout the Dictyostelium life cycle. Nucleomorphin contains a highly acidic glutamic/aspartic acid inverted repeat (DEED) with significant similarity to the conserved nucleoplasmin domain and a putative transmembrane domain in the carboxyl-terminal region. Southern blotting reveals that nucleomorphin exists as a single copy gene. Using gel overlay assays and CaM-agarose we show that bacterially expressed nucleomorphin binds to bovine CaM in a Ca(2+)-dependent manner. Amino-terminal fusion to the green fluorescence protein (GFP) showed that GFP-NumA localized to the nucleus as distinct arc-like patterns similar to heterochromatin regions. GFP-NumA lacking the acidic DEED repeat still showed arc-like accumulations at the nuclear periphery, but the number of nuclei in these cells was increased markedly compared with control cells. Cells expressing GFP-NumA lacking the transmembrane domain localized to the nuclear periphery but did not affect nuclear number or gross morphology. Nucleomorphin is the first nuclear CaMBP to be identified in Dictyostelium. Furthermore, these data present the first identification of a member of the nucleoplasmin family as a calmodulin-binding protein and suggest nucleomorphin has a role in nuclear structure in Dictyostelium. PMID:11919178

  14. Changes in GDP binding to brown adipose tissue mitochondria and the uncoupling protein

    International Nuclear Information System (INIS)

    Incubation in vitro of brown adipose tissue (BAT) mitochondria with divalent cations, spermine, or alkaline phosphatase led to a marked increase in the binding of [3H]GDP. The effect of Mg2+ appeared to be the most specific and led to the largest increase in GDP binding. A simplified method was developed for measuring GDP binding to purified uncoupling protein from rat BAT mitochondria. Application of this method indicates that uncoupling protein from cold-acclimated rats binds twice as much GDP as uncoupling protein from cold-acclimated rats that were briefly returned to thermoneutrality, paralleling changes in GDP binding to the mitochondria. Incubation of BAT mitochondria with Mg2+ led to a smaller increase in GDP binding to the subsequently purified uncoupling protein, suggesting that divalent cations may somehow participate in the regulation of the activity of the uncoupling protein

  15. Characterization of a zinc blotting technique: evidence that a retroviral gag protein binds zinc

    International Nuclear Information System (INIS)

    We have characterized a simple method that uses 65ZnCl2 to detect zinc-binding proteins that have been immobilized on nitrocellulose. Conditions have been identified that permit the detection of as little as 1 microgram of some zinc-binding proteins. The specificity of the binding is indicated by the ability of other divalent metal ions to compete with 65Zn(II) in this assay. We have used this technique to provide evidence that the nucleic acid-binding gag protein of retroviruses also binds zinc. This technique can be applied to biological mixtures of proteins and may be used in proteolytic mapping studies to identify protein fragments that have zinc-binding activity

  16. Rapid detection and purification of sequence specific DNA binding proteins using magnetic separation

    Directory of Open Access Journals (Sweden)

    TIJANA SAVIC

    2006-02-01

    Full Text Available In this paper, a method for the rapid identification and purification of sequence specific DNA binding proteins based on magnetic separation is presented. This method was applied to confirm the binding of the human recombinant USF1 protein to its putative binding site (E-box within the human SOX3 protomer. It has been shown that biotinylated DNA attached to streptavidin magnetic particles specifically binds the USF1 protein in the presence of competitor DNA. It has also been demonstrated that the protein could be successfully eluted from the beads, in high yield and with restored DNA binding activity. The advantage of these procedures is that they could be applied for the identification and purification of any high-affinity sequence-specific DNA binding protein with only minor modifications.

  17. Development of a protein microarray using sequence-specific DNA binding domain on DNA chip surface

    International Nuclear Information System (INIS)

    A protein microarray based on DNA microarray platform was developed to identify protein-protein interactions in vitro. The conventional DNA chip surface by 156-bp PCR product was prepared for a substrate of protein microarray. High-affinity sequence-specific DNA binding domain, GAL4 DNA binding domain, was introduced to the protein microarray as fusion partner of a target model protein, enhanced green fluorescent protein. The target protein was oriented immobilized directly on the DNA chip surface. Finally, monoclonal antibody of the target protein was used to identify the immobilized protein on the surface. This study shows that the conventional DNA chip can be used to make a protein microarray directly, and this novel protein microarray can be applicable as a tool for identifying protein-protein interactions

  18. Detection and properties of A-factor-binding protein from Streptomyces griseus

    International Nuclear Information System (INIS)

    The optically active form of tritium-labeled A-factor (2-isocapryloyl-3R-hydroxymethyl-gamma-butyrolactone), a pleiotropic autoregulator responsible for streptomycin production, streptomycin resistance, and sporulation in Streptomyces griseus, was chemically synthesized. By using the radioactive A-factor, a binding protein for A-factor was detected in the cytoplasmic fraction of this organism. The binding protein had an apparent molecular weight of approximately 26,000, as determined by gel filtration. Scatchard analysis suggested that A-factor bound the protein in the molar ratio of 1:1 with a binding constant, Kd, of 0.7 nM. The number of the binding protein was roughly estimated to be 37 per genome. The inducing material virginiae butanolide C (VB-C), which has a structure very similar to that of A-factor and is essential for virginiamycin production in Streptomyces virginiae, did not inhibit binding. In addition, no protein capable of specifically binding 3H-labeled VB-C was found in S. griseus. Together with the observation that VB-C had almost no biological activity on the restoration of streptomycin production or sporulation in an A-factor-deficient mutant of S. griseus, these results indicated that the binding protein had a strict ligand specificity. Examination for an A-factor-binding protein in Streptomyces coelicolor A3(2) and Streptomyces lividans showed the absence of any specifically binding protein

  19. Allosteric coupling from G protein to the agonist-binding pocket in GPCRs.

    Science.gov (United States)

    DeVree, Brian T; Mahoney, Jacob P; Vélez-Ruiz, Gisselle A; Rasmussen, Soren G F; Kuszak, Adam J; Edwald, Elin; Fung, Juan-Jose; Manglik, Aashish; Masureel, Matthieu; Du, Yang; Matt, Rachel A; Pardon, Els; Steyaert, Jan; Kobilka, Brian K; Sunahara, Roger K

    2016-07-01

    G-protein-coupled receptors (GPCRs) remain the primary conduit by which cells detect environmental stimuli and communicate with each other. Upon activation by extracellular agonists, these seven-transmembrane-domain-containing receptors interact with heterotrimeric G proteins to regulate downstream second messenger and/or protein kinase cascades. Crystallographic evidence from a prototypic GPCR, the β2-adrenergic receptor (β2AR), in complex with its cognate G protein, Gs, has provided a model for how agonist binding promotes conformational changes that propagate through the GPCR and into the nucleotide-binding pocket of the G protein α-subunit to catalyse GDP release, the key step required for GTP binding and activation of G proteins. The structure also offers hints about how G-protein binding may, in turn, allosterically influence ligand binding. Here we provide functional evidence that G-protein coupling to the β2AR stabilizes a ‘closed’ receptor conformation characterized by restricted access to and egress from the hormone-binding site. Surprisingly, the effects of G protein on the hormone-binding site can be observed in the absence of a bound agonist, where G-protein coupling driven by basal receptor activity impedes the association of agonists, partial agonists, antagonists and inverse agonists. The ability of bound ligands to dissociate from the receptor is also hindered, providing a structural explanation for the G-protein-mediated enhancement of agonist affinity, which has been observed for many GPCR–G-protein pairs. Our data also indicate that, in contrast to agonist binding alone, coupling of a G protein in the absence of an agonist stabilizes large structural changes in a GPCR. The effects of nucleotide-free G protein on ligand-binding kinetics are shared by other members of the superfamily of GPCRs, suggesting that a common mechanism may underlie G-protein-mediated enhancement of agonist affinity. PMID:27362234

  20. Antibody Responses in Patients with Staphylococcal Septicemia against Two Staphylococcus aureus Fibrinogen Binding Proteins: Clumping Factor and an Extracellular Fibrinogen Binding Protein

    OpenAIRE

    Colque-Navarro, Patricia; Palma, Marco; Söderquist, Bo; Flock, Jan-Ingmar; Möllby, Roland

    2000-01-01

    We analyzed the serum antibody responses against two Staphylococcus aureus fibrinogen binding proteins, the cell-bound clumping factor (Clf) and an extracellular fibrinogen binding protein (Efb). The material consisted of 105 consecutive serum samples from 41 patients suffering from S. aureus septicemia and 72 serum samples from healthy individuals. An enzyme-linked immunosorbent assay (ELISA) was developed. Healthy individuals showed variable levels of antibodies against the studied antigens...

  1. Secretion and Proteolysis of Heterologous Proteins Fused to the Escherichia coli Maltose Binding Protein in Pichia pastoris

    OpenAIRE

    Li, Zhiguo; Leung, Wilson; Yon, Amy; Nguyen, John; Perez, Vincent C.; Vu, Jane; Giang, William; Luong, Linda T.; Phan, Tracy; Salazar, Katherine A.; Gomez, Seth R.; Au, Colin; Xiang, Fan; Thomas, David W; Franz, Andreas H.

    2010-01-01

    The E. coli maltose binding protein (MBP) has been utilized as a translational fusion partner to improve the expression of foreign proteins made in E. coli. When located N-terminal to its cargo protein, MBP increases the solubility of intracellular proteins and improves the export of secreted proteins in bacterial systems. We initially explored whether MBP would have the same effect in the methylotrophic yeast Pichia pastoris, a popular eukaryotic host for heterologous protein expression. Whe...

  2. Standardization for cortisol determination in human blood by competitive protein-binding

    International Nuclear Information System (INIS)

    Standardization for determination of cortisol from human plasma (17-hydroxycorticosteroids) using competitive protein-binding method is presented. Activated carbon coated with dextrans is used for separation of the hormone-protein complexe and hormone labelled free

  3. Resistance to juvenile hormone and an insect growth regulator in Drosophila is associated with an altered cytosolic juvenile hormone-binding protein

    International Nuclear Information System (INIS)

    The Met mutant of Drosophila melanogaster is highly resistant to juvenile hormone III (JH III) or its chemical analog, methoprene, an insect growth regulator. Five major mechanisms of insecticide resistance were examined in Met and susceptible Met+ flies. These two strains showed only minor differences when penetration, excretion, tissue sequestration, or metabolism of [3H]JH III was measured. In contrast, when we examined JH III binding by a cytosolic binding protein from a JH target tissue, Met strains had a 10-fold lower binding affinity than did Met+ strains. Studies using deficiency-bearing chromosomes provide strong evidence that the Met locus controls the binding protein characteristics and may encode the protein. These studies indicate that resistance in Met flies results from reduced binding affinity of a cytosolic binding protein for JH III

  4. The presence of phosphate-binding protein in inner mitochondrial membrane

    Directory of Open Access Journals (Sweden)

    Hatase,Osamu

    1976-06-01

    Full Text Available Phosphate-binding protein(s was found in the inner mitochondrial membrane of calf heart by Sephadex G-200 and G-25 gel filtration. The binding activity was inhibited by N-ethylmaleimide and competed by a large amount of cold phosphate. The amount of phosphate bound to the fraction was 29 nmoles per mg of protein. Affinity chromatography with phosphate-bound Sepharose 4B confirmed the presence of phosphate-binding protein(s in the active fraction of mitochondrial membrane fractionated by gel filtration.

  5. Visualization of Iron-Binding Micelles in Acidic Recombinant Biomineralization Protein, MamC

    Energy Technology Data Exchange (ETDEWEB)

    Kashyap, Sanjay [Ames Laboratory; Woehl, Taylor [Ames Laboratory; Valverde-Tercedor, Carmen [University of Granada; Sanchez-Quesada, Miguel [University of Granada; Lopez, Concepcion Jimenez [University of Granada; Prozorov, Tanya [Ames Laboratory

    2014-03-07

    Biological macromolecules are utilized in low-temperature synthetic methods to exert precise control over nanoparticle nucleation and placement. They enable low-temperature formation of a variety of functional nanostructured materials with properties often not achieved via conventional synthetic techniques. Here we report on the in situ visualization of a novel acidic bacterial recombinant protein, MamC, commonly present in the magnetosome membrane of several magnetotactic bacteria, including Magnetococcus marinus, strain MC-1. Our findings provide an insight into the self-assembly of MamC and point to formation of the extended protein surface, which is assumed to play an important role in the formation of biotemplated inorganic nanoparticles. The self-organization of MamC is compared to the behavior of another acidic recombinant iron-binding protein, Mms6.

  6. Recombinant fusion protein of albumin-retinol binding protein inactivates stellate cells

    International Nuclear Information System (INIS)

    Highlights: ► We designed novel recombinant albumin-RBP fusion proteins. ► Expression of fusion proteins inactivates pancreatic stellate cells (PSCs). ► Fusion proteins are successfully internalized into and inactivate PSCs. ► RBP moiety mediates cell specific uptake of fusion protein. -- Abstract: Quiescent pancreatic- (PSCs) and hepatic- (HSCs) stellate cells store vitamin A (retinol) in lipid droplets via retinol binding protein (RBP) receptor and, when activated by profibrogenic stimuli, they transform into myofibroblast-like cells which play a key role in the fibrogenesis. Despite extensive investigations, there is, however, currently no appropriate therapy available for tissue fibrosis. We previously showed that the expression of albumin, composed of three homologous domains (I–III), inhibits stellate cell activation, which requires its high-affinity fatty acid-binding sites asymmetrically distributed in domain I and III. To attain stellate cell-specific uptake, albumin (domain I/III) was coupled to RBP; RBP-albumindomainIII (R-III) and albumindomainI-RBP-albuminIII (I-R-III). To assess the biological activity of fusion proteins, cultured PSCs were used. Like wild type albumin, expression of R-III or I-R-III in PSCs after passage 2 (activated PSCs) induced phenotypic reversal from activated to fat-storing cells. On the other hand, R-III and I-R-III, but not albumin, secreted from transfected 293 cells were successfully internalized into and inactivated PSCs. FPLC-purified R-III was found to be internalized into PSCs via caveolae-mediated endocytosis, and its efficient cellular uptake was also observed in HSCs and podocytes among several cell lines tested. Moreover, tissue distribution of intravenously injected R-III was closely similar to that of RBP. Therefore, our data suggest that albumin-RBP fusion protein comprises of stellate cell inactivation-inducing moiety and targeting moiety, which may lead to the development of effective anti

  7. Squid rhodopsin and GTP-binding protein crossreact with vertebrate photoreceptor enzymes.

    OpenAIRE

    Saibil, H R; Michel-Villaz, M

    1984-01-01

    The activation of photoreceptor GTP-binding protein by rhodopsin was studied in squid photoreceptors and in crossreactions between the squid and bovine proteins. Turbidity changes were observed in the far-red after photoexcitation of rhodopsin with brief flashes and were used to probe interactions between photoreceptor membrane suspensions and soluble protein extracts. Our findings are squid photoreceptors contain a GTP-binding protein detectable by light- and GTP-sensitive turbidity changes ...

  8. A FRET-based method for monitoring septin polymerization and binding of septin-associated proteins

    Science.gov (United States)

    Booth, E.A.; Thorner, J.

    2016-01-01

    Much about septin function has been inferred from in vivo studies using mainly genetic methods, and much of what we know about septin organization has been obtained through examination of static structures in vitro primarily by electron microscopy. Deeper mechanistic insight requires real-time analysis of the dynamics of the assembly of septin-based structures and how other proteins associate with them. We describe here a Förster resonance energy transfer (FRET)-based approach for measuring in vitro the rate and extent of filament formation from septin complexes, binding of other proteins to septin structures, and the apparent affinities of these interactions. FRET is particularly well suited for interrogating protein–protein interactions, especially on a rapid timescale; the spectral change provides an unambiguous indication of whether two elements within the system under study are associating and serves as a molecular-level “ruler” because it is very sensitive to the separation between the donor and acceptor fluorophores over biologically relevant distances (≤ 10 nm). The necessary procedures involve generation of appropriate cysteine-less and single cysteine-containing septin variants, expression and purification of the heterooctameric complexes containing them, efficient labeling of the purified complexes with desired fluorophores, fluorimetric measurement of FRET, and appropriate safeguards and controls in data acquisition and analysis. Our methods can be used to interrogate the effects of buffer conditions, small molecules, and septin-binding proteins on septin filament assembly or stability; determine the effect of alternative septin subunits, mutational alterations, or posttranslational modifications on assembly; and, delineate the location of septin-binding proteins. PMID:27473902

  9. Isolation and properties of the complex between the enhancer binding protein NIFA and the sensor NIFL.

    Science.gov (United States)

    Money, T; Jones, T; Dixon, R; Austin, S

    1999-08-01

    In Azotobacter vinelandii, activation of nif gene expression by the transcriptional regulatory enhancer binding protein NIFA is controlled by the sensor protein NIFL in response to changes in levels of oxygen and fixed nitrogen in vivo. NIFL is a novel redox-sensing flavoprotein which is also responsive to adenosine nucleotides in vitro. Inhibition of NIFA activity by NIFL requires stoichiometric amounts of the two proteins, implying that the mechanism of inhibition is by direct protein-protein interaction rather than by catalytic modification of the NIFA protein. The formation of the inhibitory complex between NIFL and NIFA may be regulated by the intracellular ATP/ADP ratio. We show that adenosine nucleotides promote complex formation between purified NIFA and NIFL in vitro, allowing isolation of the NIFL-NIFA complex. The complex can also be isolated from cell extracts containing coexpressed NIFL and NIFA in the presence of MgADP. Removal of the nucleotide causes dissociation of the complex. Experiments with truncated proteins demonstrate that the amino-terminal domain of NIFA and the C-terminal region of NIFL potentiate the ADP-dependent stimulation of NIFL-NIFA complex formation. PMID:10419940

  10. P1 plasmid replication: initiator sequestration is inadequate to explain control by initiator-binding sites.

    OpenAIRE

    S.K. Pal; Chattoraj, D K

    1988-01-01

    The unit-copy plasmid replicon mini-P1 consists of an origin, a gene for an initiator protein, RepA, and a control locus, incA. Both the origin and the incA locus contain repeat sequences that bind RepA. It has been proposed that the incA repeats control replication by sequestering the rate-limiting RepA initiator protein. Here we show that when the concentration of RepA was increased about fourfold beyond its normal physiological level from an inducible source in trans, the copy number of a ...

  11. Novel Prostate Specific Antigen plastic antibody designed with charged binding sites for an improved protein binding and its application in a biosensor of potentiometric transduction

    International Nuclear Information System (INIS)

    Graphical abstract: EF13-201, Novel Prostate Specific Antigen plastic antibody designed with charged binding sites for an improved protein binding and its application in a biosensor of potentiometric transduction. - Abstract: This work shows that the synthesis of protein plastic antibodies tailored with selected charged monomers around the binding site enhances protein binding. These charged receptor sites are placed over a neutral polymeric matrix, thus inducing a suitable orientation the protein reception to its site. This is confirmed by preparing control materials with neutral monomers and also with non-imprinted template. This concept has been applied here to Prostate Specific Antigen (PSA), the protein of choice for screening prostate cancer throughout the population, with serum levels >10 ng/mL pointing out a high probability of associated cancer. Protein Imprinted Materials with charged binding sites (C/PIM) have been produced by surface imprinting over graphene layers to which the protein was first covalently attached. Vinylbenzyl(trimethylammonium chloride) and vinyl benzoate were introduced as charged monomers labelling the binding site and were allowed to self-organize around the protein. The subsequent polymerization was made by radical polymerization of vinylbenzene. Neutral PIM (N/PIM) prepared without oriented charges and non imprinted materials (NIM) obtained without template were used as controls. These materials were used to develop simple and inexpensive potentiometric sensor for PSA. They were included as ionophores in plasticized PVC membranes, and tested over electrodes of solid or liquid conductive contacts, made of conductive carbon over a syringe or of inner reference solution over micropipette tips. The electrodes with charged monomers showed a more stable and sensitive response, with an average slope of -44.2 mV/decade and a detection limit of 5.8 × 10−11 mol/L (2 ng/mL). The corresponding non-imprinted sensors showed lower

  12. Expressing a bacterial mercuric ion binding protein in plant for phytoremediation of heavy metals.

    Science.gov (United States)

    Hsieh, Ju-Liang; Chen, Ching-Yi; Chiu, Meng-Hsuen; Chein, Mei-Fang; Chang, Jo-Shu; Endo, Ginro; Huang, Chieh-Chen

    2009-01-30

    A specific mercuric ion binding protein (MerP) originating from transposon TnMERI1 of Bacillus megaterium strain MB1 isolated from Minamata Bay displayed good adsorption capability for a variety of heavy metals. In this study, the Gram-positive MerP protein was expressed in transgenic Arabidopsis to create a model system for phytoremediation of heavy metals. Under control of an actin promoter, the transgenic Arabidpsis showed higher tolerance and accumulation capacity for mercury, cadium and lead when compared with the control plant. Results from confocal microscopy analysis also indicate that MerP was localized at the cell membrane and vesicles of plant cells. The developed transgenic plants possessing excellent metal-accumulative ability could have potential applications in decontamination of heavy metals. PMID:18538925

  13. Distinctive Binding of Avibactam to Penicillin-Binding Proteins of Gram-Negative and Gram-Positive Bacteria

    OpenAIRE

    Asli, Abdelhamid; Brouillette, Eric; Krause, Kevin M.; Nichols, Wright W.; Malouin, François

    2016-01-01

    Avibactam is a novel non-β-lactam β-lactamase inhibitor that covalently acylates a variety of β-lactamases, causing inhibition. Although avibactam presents limited antibacterial activity, its acylation ability toward bacterial penicillin-binding proteins (PBPs) was investigated. Staphylococcus aureus was of particular interest due to the reported β-lactamase activity of PBP4. The binding of avibactam to PBPs was measured by adding increasing concentrations to membrane preparations of a variet...

  14. Engineering of binding affinity at metal ion binding sites for the stabilization of proteins: Subtilisin as a test case

    International Nuclear Information System (INIS)

    A weak Ca2+ binding site in the bacterial serine protease subtilisin BPN' was chosen as a model to explore the feasibility of stabilizing a protein by increasing the binding affinity at a metal ion binding site. The existence of this weak Ca2+ binding site was first discovered through a study of the rate of thermal inactivation of wild-type subtilisin BPN' at 65/degrees/C as a function of the free [Ca2+]. Increasing the [Ca2+] in the range of 0.10-100 mM caused a 100-fold decrease in the rate of thermal inactivation. The data were found to closely fit a theoretical titration curve for a single Ca2+ specific binding site with an apparent log K/sub a/ = 1.49. A series of refined X-ray crystal structures of subtilisin in the presence of 0.0, 25.0, and 40.0 mM CaCl2 has allowed a detailed structural characterization of this Ca2+ binding site. Negatively charged side chains were introduced in the vicinity of the bound Ca2+ by changing Pro 172 and Gly 131 to Asp residues through site-directed and random mutagenesis techniques, respectively. These changes were found to increase the affinity of the Ca2+ binding site by 3.4- and 2-fold, respectively, when compared with the wild-type protein. X-ray studies of these new variants of subtilisin revealed the carboxylate side chains to be 6.8 and 13.2 /Angstrom/, respectively, from the bound Ca2+. These distances and the degree of enhanced binding are consistent with simple electrostatic theory. Moreover, when both Asp changes were introduced together, the binding affinity for Ca2+ was found to be increased about 6-fold over that for the wild-type protein, suggesting an independent and nearly additive effect on the total electrostatic potential at this locus

  15. Determining protein adducts of fipexide: mass spectrometry based assay for confirming the involvement of its reactive metabolite in covalent binding.

    Science.gov (United States)

    Sleno, Lekha; Varesio, Emmanuel; Hopfgartner, Gérard

    2007-01-01

    Fipexide is a nootropic drug, withdrawn from the market due to its idiosyncratic drug reactions causing adverse effects in man. Previous work on its metabolites has identified several potential reactive metabolites which could be implicated in protein binding. Here, we investigated the formation of these metabolites in rat and human hepatocytes. Based on these results, the o-quinone of fipexide (FIP), formed via the demethylenation reaction through a catechol intermediate, was chosen for further investigation. Studies were then pursued in order to relate this metabolite to protein binding, and thus better understand potential mechanisms for the toxicity of the parent compound. An assay was developed for determining the fipexide catechol-cysteine adduct in the microsomal protein fractions following in vitro incubations. This method digests the entire protein fraction into amino acids, followed by the detection of the Cys-metabolite adduct by liquid chromatography/mass spectrometry (LC/MS). We have designed a strategy where drug metabolism taking place in microsomal incubations and involved in protein binding can be assessed after the proteins have been digested, with the detection of the specific amino acid adduct. In this study, the structure of the fipexide adduct was hypothesized using knowledge previously gained in glutathione and N-acetylcysteine trapping experiments. Acetaminophen was used as a positive control for detecting a drug metabolite-cysteine adduct by LC/MS. This approach has the potential to be applicable as a protein-binding assay in early drug discovery without the need for radioactive compounds. PMID:18022964

  16. Structure and thermodynamics of effector molecule binding to the nitrogen signal transduction PII protein GlnZ from Azospirillum brasilense.

    Science.gov (United States)

    Truan, Daphné; Bjelić, Saša; Li, Xiao-Dan; Winkler, Fritz K

    2014-07-29

    The trimeric PII signal transduction proteins regulate the function of a variety of target proteins predominantly involved in nitrogen metabolism. ATP, ADP and 2-oxoglutarate (2-OG) are key effector molecules influencing PII binding to targets. Studies of PII proteins have established that the 20-residue T-loop plays a central role in effector sensing and target binding. However, the specific effects of effector binding on T-loop conformation have remained poorly documented. We present eight crystal structures of the Azospirillum brasilense PII protein GlnZ, six of which are cocrystallized and liganded with ADP or ATP. We find that interaction with the diphosphate moiety of bound ADP constrains the N-terminal part of the T-loop in a characteristic way that is maintained in ADP-promoted complexes with target proteins. In contrast, the interactions with the triphosphate moiety in ATP complexes are much more variable and no single predominant interaction mode is apparent except for the ternary MgATP/2-OG complex. These conclusions can be extended to most investigated PII proteins of the GlnB/GlnK subfamily. Unlike reported for other PII proteins, microcalorimetry reveals no cooperativity between the three binding sites of GlnZ trimers for any of the three effectors under carefully controlled experimental conditions. PMID:24846646

  17. Protein nanoparticle interaction: A spectrophotometric approach for adsorption kinetics and binding studies

    Science.gov (United States)

    Vaishanav, Sandeep K.; Chandraker, Kumudini; Korram, Jyoti; Nagwanshi, Rekha; Ghosh, Kallol K.; Satnami, Manmohan L.

    2016-08-01

    Investigating the protein nanoparticle interaction is crucial to understand how to control the biological interactions of nanoparticles. In this work, Model protein Bovine serum albumin (BSA) was used to evaluate the process of protein adsorption to the gold nanoparticles (GNPs) surface. The binding of a model protein (BSA) to GNPs was investigated through fluorescence quenching measurements. The strong affinities of BSA for GNPs were confirmed by the high value of binding constant (Ks) which was calculated to be 2.2 × 1011 L/mol. In this consequence, we also investigated the adsorption behavior of BSA on GNPs surface via UV-Vis spectroscopy. The effect of various operational parameters such as pH, contact time, initial BSA concentration, and temperature on adsorption of BSA was investigated using batch adsorption experiments. Kinetics of adsorption was found to follow the pseudo-second order rate equation. The suitability of Freundlich and Langmuir adsorption models to the equilibrium data was investigated. The equilibrium adsorption was well described by the Freundlich isotherm model. The maximum adsorption capacity for BSA adsorbed on GNPs was 58.71 mg/g and equilibrium constant was 0.0058 calculated by the Langmuir model at 298 K and pH = 11.0. Thermodynamic parameters showed that the adsorption of BSA onto GNPs was feasible, spontaneous, and exothermic.

  18. Detection and characterization of heparin-binding proteins with a gel overlay procedure

    International Nuclear Information System (INIS)

    The binding of 125I-labeled derivatives of heparin has been used by several investigators to identify heparin-binding fragments of different heparin-binding proteins. In this report we utilize the procedure described by J.W. Smith and D.J. Knauer (1987, Anal. Biochem. 160, 105-114) to produce 125I-fluorescein-heparin. Using this derivative, we compare the use of gel overlay procedures with Western blot procedures for the detection of heparin-binding proteins following polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. We show that the gel overlay procedure is a relatively simple and sensitive method for visualizing heparin-binding proteins. In addition, we use the procedure to characterize the heparin-binding properties of heparin-binding growth factor 1 (acidic fibroblast growth factor) with synthetic peptide competitors and site-directed mutants of the growth factor

  19. Expression Pattern of Fatty Acid Binding Proteins in Celiac Disease Enteropathy

    Directory of Open Access Journals (Sweden)

    Natalia M. Bottasso Arias

    2015-01-01

    Full Text Available Celiac disease (CD is an immune-mediated enteropathy that develops in genetically susceptible individuals following exposure to dietary gluten. Severe changes at the intestinal mucosa observed in untreated CD patients are linked to changes in the level and in the pattern of expression of different genes. Fully differentiated epithelial cells express two isoforms of fatty acid binding proteins (FABPs: intestinal and liver, IFABP and LFABP, respectively. These proteins bind and transport long chain fatty acids and also have other important biological roles in signaling pathways, particularly those related to PPARγ and inflammatory processes. Herein, we analyze the serum levels of IFABP and characterize the expression of both FABPs at protein and mRNA level in small intestinal mucosa in severe enteropathy and normal tissue. As a result, we observed higher levels of circulating IFABP in untreated CD patients compared with controls and patients on gluten-free diet. In duodenal mucosa a differential FABPs expression pattern was observed with a reduction in mRNA levels compared to controls explained by the epithelium loss in severe enteropathy. In conclusion, we report changes in FABPs’ expression pattern in severe enteropathy. Consequently, there might be alterations in lipid metabolism and the inflammatory process in the small intestinal mucosa.

  20. Expression Pattern of Fatty Acid Binding Proteins in Celiac Disease Enteropathy.

    Science.gov (United States)

    Bottasso Arias, Natalia M; García, Marina; Bondar, Constanza; Guzman, Luciana; Redondo, Agustina; Chopita, Nestor; Córsico, Betina; Chirdo, Fernando G

    2015-01-01

    Celiac disease (CD) is an immune-mediated enteropathy that develops in genetically susceptible individuals following exposure to dietary gluten. Severe changes at the intestinal mucosa observed in untreated CD patients are linked to changes in the level and in the pattern of expression of different genes. Fully differentiated epithelial cells express two isoforms of fatty acid binding proteins (FABPs): intestinal and liver, IFABP and LFABP, respectively. These proteins bind and transport long chain fatty acids and also have other important biological roles in signaling pathways, particularly those related to PPARγ and inflammatory processes. Herein, we analyze the serum levels of IFABP and characterize the expression of both FABPs at protein and mRNA level in small intestinal mucosa in severe enteropathy and normal tissue. As a result, we observed higher levels of circulating IFABP in untreated CD patients compared with controls and patients on gluten-free diet. In duodenal mucosa a differential FABPs expression pattern was observed with a reduction in mRNA levels compared to controls explained by the epithelium loss in severe enteropathy. In conclusion, we report changes in FABPs' expression pattern in severe enteropathy. Consequently, there might be alterations in lipid metabolism and the inflammatory process in the small intestinal mucosa. PMID:26346822

  1. Data for proteomic analysis of ATP-binding proteins and kinase inhibitor target proteins using an ATP probe

    OpenAIRE

    Jun Adachi; Marina Kishida; Shio Watanabe; Yuuki Hashimoto; Kazuna Fukamizu; Takeshi Tomonaga

    2015-01-01

    Interactions between ATP and ATP-binding proteins (ATPome) are common and are required for most cellular processes. Thus, it is clearly important to identify and quantify these interactions for understanding basic cellular mechanisms and the pathogenesis of various diseases. We used an ATP competition assay (competition between ATP and acyl-ATP probes) that enabled us to distinguish specific ATP-binding proteins from non-specific proteins (Adachi et al., 2014) [1]. As a result, we identified ...

  2. How Similar Are Protein Folding and Protein Binding Nuclei? Examination of Vibrational Motions of Energy Hot Spots and Conserved Residues

    OpenAIRE

    Haliloglu, Turkan; Keskin, Ozlem; Ma, Buyong; Nussinov, Ruth

    2004-01-01

    The underlying physico-chemical principles of the interactions between domains in protein folding are similar to those between protein molecules in binding. Here we show that conserved residues and experimental hot spots at intermolecular binding interfaces overlap residues that vibrate with high frequencies. Similarly, conserved residues and hot spots are found in protein cores and are also observed to vibrate with high frequencies. In both cases, these residues contribute significantly to t...

  3. The correlation of vitamin D level and vitamin D-binding protein gene polymorphism in chronic obstructive pulmonary disease

    Institute of Scientific and Technical Information of China (English)

    李晓晨

    2014-01-01

    Objective To assess the correlation of serum 25-hydroxyvitamin D(25-OHD)levels with vitamin D-binding protein(the group-specific component,GC)gene polymorphism in chronic obstructive pulmonary disease(COPD).Methods In a cross-sectional case-control study,250 participants,including 116 COPD patients with smoking history and 134 healthy smokers,were in-

  4. Mechanism of Fluorescence and Conformational Changes of the Sarcoplasmic Calcium Binding Protein of the Sand Worm Nereis diversicolor upon Ca2+ or Mg2+ Binding

    OpenAIRE

    Sillen, Alain; Verheyden, Stefan; Delfosse, Lotte; Braem, Tania; Robben, Johan; Volckaert, Guido; Engelborghs, Yves

    2003-01-01

    The calcium-binding protein isolated from the sarcoplasm of the muscles of the sand worm Nereis diversicolor has four EF-hands and three active binding sites for Ca2+ or Mg2+. Nereis diversicolor sarcoplasmic calcium-binding protein contains three tryptophan residues at positions 4, 57, and 170, respectively. The Wt protein shows a very limited fluorescence increase upon binding of Ca2+ or Mg2+. Single-tryptophan-containing mutants were produced and purified. The fluorescence titrations of th...

  5. Zinc fingers, zinc clusters, and zinc twists in DNA-binding protein domains.

    OpenAIRE

    Vallee, B L; Coleman, J E; Auld, D S

    1991-01-01

    We now recognize three distinct motifs of DNA-binding zinc proteins: (i) zinc fingers, (ii) zinc clusters, and (iii) zinc twists. Until very recently, x-ray crystallographic or NMR three-dimensional structure analyses of DNA-binding zinc proteins have not been available to serve as standards of reference for the zinc binding sites of these families of proteins. Those of the DNA-binding domains of the fungal transcription factor GAL4 and the rat glucocorticoid receptor are the first to have be...

  6. Mycobacterial PE_PGRS Proteins Contain Calcium-Binding Motifs with Parallel β-roll Folds

    Institute of Scientific and Technical Information of China (English)

    Nandita; Bachhawat; Balvinder; Singh

    2007-01-01

    The PE_PGRS family of proteins unique to mycobacteria is demonstrated to con- rain multiple calcium-binding and glycine-rich sequence motifs GGXGXD/NXUX. This sequence repeat constitutes a calcium-binding parallel/3-roll or parallel β-helix structure and is found in RTX toxins secreted by many Gram-negative bacteria. It is predicted that the highly homologous PE_PGRS proteins containing multiple copies of the nona-peptide motif could fold into similar calcium-binding structures. The implication of the predicted calcium-binding property of PE_PGRS proteins in the Ught of macrophage-pathogen interaction and pathogenesis is presented.

  7. A family of insulin-like growth factor II mRNA-binding proteins represses translation in late development

    DEFF Research Database (Denmark)

    Nielsen, J; Christiansen, J; Lykke-Andersen, J;

    1999-01-01

    day 12.5 followed by a decline towards birth, and, similar to IGF-II, IMPs are especially expressed in developing epithelia, muscle, and placenta in both mouse and human embryos. The results imply that cytoplasmic 5' UTR-binding proteins control IGF-II biosynthesis during late mammalian development.......Insulin-like growth factor II (IGF-II) is a major fetal growth factor. The IGF-II gene generates multiple mRNAs with different 5' untranslated regions (5' UTRs) that are translated in a differential manner during development. We have identified a human family of three IGF-II mRNA-binding proteins...

  8. OB protein binds specifically to the choroid plexus of mice and rats.

    Science.gov (United States)

    Devos, R; Richards, J G; Campfield, L A; Tartaglia, L A; Guisez, Y; van der Heyden, J; Travernier, J; Plaetinck, G; Burn, P

    1996-05-28

    Binding studies were conducted to identify the anatomical location of brain target sites for OB protein, the ob gene product. 125I-labeled recombinant mouse OB protein or alkaline phosphatase-OB fusion proteins were used for in vitro and in vivo binding studies. Coronal brain sections or fresh tissue from lean, obese ob/ob, and obese db/db mice as well as lean and obese Zucker rats were probed to identify potential central OB protein-binding sites. We report here that recombinant OB protein binds specifically to the choroid plexus. The binding of OB protein (either radiolabeled or the alkaline phosphatase-OB fusion protein) and its displacement by unlabeled OB protein was similar in lean, obese ob/ob, and obese db/db mice as well as lean and obese Zucker rats. These findings suggest that OB protein binds with high affinity to a specific receptor in the choroid plexus. After binding to the choroid plexus receptor, OB protein may then be transported across the blood-brain barrier into the cerebrospinal fluid. Alternatively, binding of OB protein to a specific receptor in the choroid plexus may activate afferent neural inputs to the neural network that regulates feeding behavior and energy balance or may result in the clearance or degradation of OB protein. The identification of the choroid plexus as a brain binding site for OB protein will provide the basis for the construction of expression libraries and facilitate the rapid cloning of the choroid plexus OB receptor. PMID:8643634

  9. The human fatty acid-binding protein family: Evolutionary divergences and functions

    Directory of Open Access Journals (Sweden)

    Smathers Rebecca L

    2011-03-01

    Full Text Available Abstract Fatty acid-binding proteins (FABPs are members of the intracellular lipid-binding protein (iLBP family and are involved in reversibly binding intracellular hydrophobic ligands and trafficking them throughout cellular compartments, including the peroxisomes, mitochondria, endoplasmic reticulum and nucleus. FABPs are small, structurally conserved cytosolic proteins consisting of a water-filled, interior-binding pocket surrounded by ten anti-parallel beta sheets, forming a beta barrel. At the superior surface, two alpha-helices cap the pocket and are thought to regulate binding. FABPs have broad specificity, including the ability to bind long-chain (C16-C20 fatty acids, eicosanoids, bile salts and peroxisome proliferators. FABPs demonstrate strong evolutionary conservation and are present in a spectrum of species including Drosophila melanogaster, Caenorhabditis elegans, mouse and human. The human genome consists of nine putatively functional protein-coding FABP genes. The most recently identified family member, FABP12, has been less studied.

  10. Altered binding of 125I-labeled calmodulin to a 46.5-kilodalton protein in skin fibroblasts cultured from patients with cystic fibrosis

    International Nuclear Information System (INIS)

    The levels of calmodulin and calmodulin-binding proteins have been determined in cultured skin fibroblasts from patients with cystic fibrosis (CF) and age- and sex-matched controls. Calmodulin ranged from 0.20 to 0.76 microgram/mg protein; there was no difference between calmodulin concentration in fibroblasts from CF patients and controls. Calmodulin-binding proteins of 230, 212, 204, 164, 139, 70, 59, 46.5, and 41 kD were identified. A protein with a mobility identical to the 59-kD calmodulin-binding protein was labeled by antiserum against calmodulin-dependent phosphatase. Although Ca2+/calmodulin-dependent phosphatase activity was detected, there was no different in activity between control and CF fibroblasts or in the level of phosphatase protein as determined by radioimmunoassay. Lower amounts of 125I-calmodulin were bound to the 46.5-kD calmodulin-binding protein in CF fibroblasts as compared with controls. The 46.5-kD calmodulin-binding protein may be reduced in CF fibroblasts or its structure may be altered resulting in a reduced binding capacity and/or affinity for calmodulin and perhaps reflecting, either directly or indirectly, the genetic defect responsible for cystic fibrosis

  11. Ligand-Binding Properties of the Carboxyl-Terminal Repeat Domain of Streptococcus mutans Glucan-Binding Protein A

    OpenAIRE

    Haas, Wolfgang; Banas, Jeffrey A.

    2000-01-01

    Streptococcus mutans glucan-binding protein A (GbpA) has sequence similarity in its carboxyl-terminal domain with glucosyltransferases (GTFs), the enzymes responsible for catalyzing the synthesis of the glucans to which GbpA and GTFs can bind and which promote S. mutans attachment to and accumulation on the tooth surface. It was predicted that this C-terminal region, comprised of what have been termed YG repeats, represents the GbpA glucan-binding domain (GBD). In an effort to test this hypot...

  12. Species Differences in the Carbohydrate Binding Preferences of Surfactant Protein D

    DEFF Research Database (Denmark)

    Crouch, Erika C.; Smith, Kelly; McDonald, Barbara; Briner, David; Linders, Bruce; McDonald, Joseph; Holmskov, Uffe; Head, James; Hartshorn, Kevan

    2006-01-01

    Interactions of surfactant protein D (SP-D) with micro-organisms and organic antigens involve binding to the trimeric neck plus carbohydrate recognition domain (neck+CRD). In these studies, we compared the ligand binding of homologous human, rat, and mouse trimeric neck+CRD fusion proteins, each...

  13. FK506-binding protein from adult Asian citrus psyllid, Diaphorina citri (Hemiptera: Psyllidae)

    Science.gov (United States)

    We successfully identified a new member of the FK-binding proteins from the Asian citrus psyllid, Diaphorina citri (Hemiptera: Psyllidae). FK-binding proteins (FKBP) function in many critical pathways needed for psyllid survival. The full length mRNA transcript provides us a new genetic target for ...

  14. Acyl-CoA binding proteins; structural and functional conservation over 2000 MYA

    DEFF Research Database (Denmark)

    Faergeman, Nils J; Wadum, Majken; Feddersen, Søren;

    2007-01-01

    -CoA binding protein, ACBP, has been proposed to play a pivotal role in the intracellular trafficking and utilization of long-chain fatty acyl-CoA esters. Depletion of acyl-CoA binding protein in yeast results in aberrant organelle morphology incl. fragmented vacuoles, multi-layered plasma membranes and...

  15. Steady-State Fluorescence Anisotropy to Investigate Flavonoids Binding to Proteins

    Science.gov (United States)

    Ingersoll, Christine M.; Strollo, Christen M.

    2007-01-01

    The steady-state fluorescence anisotropy is employed to study the binding of protein of a model protein, human serum albumin, to a commonly used flavonoid, quercetin. The experiment describes the thermodynamics, as well as the biochemical interactions of such binding effectively.

  16. A plant DNA-binding protein that recognizes 5-methylcytosine residues.

    OpenAIRE

    Zhang, D. L.; Ehrlich, K C; Supakar, P C; Ehrlich, M

    1989-01-01

    A novel, 5-methylcytosine-specific, DNA-binding protein, DBP-m, has been identified in nuclear extracts of peas. DBP-m specifically recognizes 5-methylcytosine residues in DNA without appreciable DNA sequence specificity, unlike a mammalian DNA-binding protein (MDBP), which recognizes 5-methylcytosine residues but only in a related family of 14-base-pair sequences.

  17. Identification of procollagen promoter DNA-binding proteins: effects of dexamethasone

    International Nuclear Information System (INIS)

    Glucocorticoids selectively decrease procollagen synthesis by decreasing procollagen mRNA transcription. Dexamethasone coordinately decreased total cellular type I and type III procollagen mRNAs in mouse embryonic skin fibroblasts. Since sequence specific DNA-binding proteins are known to modulate eukaryotic gene expression the authors identified in mouse fibroblasts nuclear proteins which bind to types I and III procollagen promoter DNAs. Nuclear proteins were electrophoresed, blotted onto nitrocellulose and probed with 32P-end-labeled type I and type III procollagen promoter DNAs in the presence of equimolar amounts of 32P-end-labeled vector DNA. Differences in total DNA binding were noted by the densitometric scans of the nuclear proteins. Dexamethasone treatment enhanced total DNA binding. Increasing the NaCl concentration decreased the number of promoter DNA-binding proteins without altering the relative specificity for the promoter DNAs. Promoter DNA binding to nuclear proteins was also inhibited by increasing concentrations of E. coli DNA. The number of DNA-binding proteins was greater for type III procollagen promoter DNA. The effect of dexamethasone treatment on promoter DNA binding to nuclear proteins was determined

  18. Cytosolic fatty acid-binding proteins: subjects and tools in metabolic research

    International Nuclear Information System (INIS)

    Fatty acid-binding proteins (FABPs) are major targets for specific binding of fatty acids in vivo. They constitute a widely expressed family of genetically related, small cytosolic proteins which very likely mediate intracellular transport of free long chain fatty acids. Genetic inhibition of FABP expression in vivo should therefore provide a useful tool to investigate and engineer fatty acid metabolism. (orig.)

  19. CLONING AND CHARACTERIZATION OF A NUCLEAR, SITE-SPECIFIC SSDNA BINDING-PROTEIN

    NARCIS (Netherlands)

    SMIDT, MP; RUSSCHEN, B; SNIPPE, L; WIJNHOLDS, J; AB, G

    1995-01-01

    Estradiol inducible, liver-specific expression of the apoVLDL II gene is mediated through the estrogen receptor and a variety of other DNA-binding proteins. In the present study we report the cloning and characterisation of a single-strand DNA binding protein that interacts with the lower strand of

  20. Computational Exploration of a Protein Receptor Binding Space with Student Proposed Peptide Ligands

    Science.gov (United States)

    King, Matthew D.; Phillips, Paul; Turner, Matthew W.; Katz, Michael; Lew, Sarah; Bradburn, Sarah; Andersen, Tim; McDougal, Owen M.

    2016-01-01

    Computational molecular docking is a fast and effective "in silico" method for the analysis of binding between a protein receptor model and a ligand. The visualization and manipulation of protein to ligand binding in three-dimensional space represents a powerful tool in the biochemistry curriculum to enhance student learning. The…

  1. Viral Proteins That Bind Double-Stranded RNA: Countermeasures Against Host Antiviral Responses

    OpenAIRE

    Krug, Robert M.

    2014-01-01

    Several animal viruses encode proteins that bind double-stranded RNA (dsRNA) to counteract host dsRNA-dependent antiviral responses. This article discusses the structure and function of the dsRNA-binding proteins of influenza A virus and Ebola viruses (EBOVs).

  2. Structural Basis for a Ribofuranosyl Binding Protein: Insights into the Furanose Specific Transport

    Energy Technology Data Exchange (ETDEWEB)

    Bagaria, A.; Swaminathan, S.; Kumaran, D.; Burley, S. K.

    2011-04-01

    The ATP-binding cassette transporters (ABC-transporters) are members of one of the largest protein superfamilies, with representatives in all extant phyla. These integral membrane proteins utilize the energy of ATP hydrolysis to carry out certain biological processes, including translocation of various substrates across membranes and non-transport related processes such as translation of RNA and DNA repair. Typically, such transport systems in bacteria consist of an ATP binding component, a transmembrane permease, and a periplasmic receptor or binding protein. Soluble proteins found in the periplasm of gram-negative bacteria serve as the primary receptors for transport of many compounds, such as sugars, small peptides, and some ions. Ligand binding activates these periplasmic components, permitting recognition by the membrane spanning domain, which supports for transport and, in some cases, chemotaxis. Transport and chemotaxis processes appear to be independent of one another, and a few mutants of bifunctional periplasmic components reveal the absence of one or the other function. Previously published high-resolution X-ray structures of various periplasmic ligand binding proteins include Arabinose binding protein (ABP), Allose binding protein (ALBP), Glucose-galactose binding protein (GBP) and Ribose binding protein (RBP). Each of these proteins consists of two structurally similar domains connected by a three-stranded hinge region, with ligand buried between the domains. Upon ligand binding and release, various conformational changes have been observed. For RBP, open (apo) and closed (ligand bound) conformations have been reported and so for MBP. The closed/active form of the protein interacts with the integral membrane component of the system in both transport and chemotaxis. Herein, we report 1.9{angstrom} resolution X-ray structure of the R{sub f}BP periplasmic component of an ABC-type sugar transport system from Hahella chejuensis (UniProt Id Q2S7D2) bound to

  3. Structural Basis for a Ribofuranosyl Binding Protein: Insights into the Furanose Specific Transport

    Energy Technology Data Exchange (ETDEWEB)

    A Bagaria; D Kumaran; S Burley; S Swaminathan

    2011-12-31

    The APT-binding cassette transporters (ABC-transporters) are members of one of the largest protein superfamilies, with representatives in all extant phyla. These integral membrane proteins utilize the energy of ATP hydrolysis to carry out certain biological processes, including translocation of various substrates across membranes and nontransport related processes such as translation of RNA and DNA repair. typically, such transport systems in bacteria consist of an ATP binding component, a transmembrane permease, and a periplasmic receptor or binding protein. Soluble proteins found in the periplasm of gram-negative bacteria serve as the primary receptors for transport of many compounds, such as sugars, small peptides, and some ions. Ligand binding activates these periplasmic components, permitting recognition by the membrane spanning domain, which supports for transport, and, in some cases, chemotaxis. Transport and chemotaxis processes appear to be independent of one another, and a few mutants of bifunctional periplasmic components reveal the absence of one or the other function. Previously published high-resolution X-ray structures of various periplasmic ligand binding proteins include Arabinose binding protein (ABP), Allose binding protein (ALBP), Glucose-galactose binding protein (GBP), and Ribose binding protein (RBP). Each of these proteins consits of two structurally similar domains connected by a three-stranded hinge region, with ligand buried between the domains. Upon ligand binding and release, various conformational changes have been observed. For RBP, open (apo) and closed (ligand bound) conformations hafve been reported and so for MBP. The closed/active form of the protein interacts with the ingral membrane component of the system in both transport and chemotaxis. Herein, they report 1.9 {angstrom} resolution X-ray structure of the R{sub f}BP periplasmic component of an ABC-type sugar transport system from Hahella chejuensis (UniProt Id Q2S7D2) bound

  4. The expression of selenium-binding protein 1 is decreased in uterine leiomyoma

    Directory of Open Access Journals (Sweden)

    Quddus M Ruhul

    2010-12-01

    Full Text Available Abstract Background Selenium has been shown to inhibit cancer development and growth through the mediation of selenium-binding proteins. Decreased expression of selenium-binding protein 1 has been reported in cancers of the prostate, stomach, colon, and lungs. No information, however, is available concerning the roles of selenium-binding protein 1 in uterine leiomyoma. Methods Using Western Blot analysis and immunohistochemistry, we examined the expression of selenium-binding protein 1 in uterine leiomyoma and normal myometrium in 20 patients who had undergone hysterectomy for uterine leiomyoma. Results and Discussion The patient age ranged from 34 to 58 years with a mean of 44.3 years. Proliferative endometrium was seen in 8 patients, secretory endometrium in 7 patients, and atrophic endometrium in 5 patients. Two patients showed solitary leiomyoma, and eighteen patients revealed 2 to 5 tumors. Tumor size ranged from 1 to 15.5 cm with a mean of 4.3 cm. Both Western Blot analysis and immunohistochemistry showed a significant lower level of selenium-binding protein 1 in leiomyoma than in normal myometrium. Larger tumors had a tendency to show a lower level of selenium-binding protein 1 than smaller ones, but the difference did not reach a statistical significance. The expression of selenium-binding protein 1 was the same among patients with proliferative, secretory, and atrophic endometrium in either leiomyoma or normal myometrium. Also, we did not find a difference of selenium-binding protein 1 level between patients younger than 45 years and older patients in either leiomyoma or normal myometrium. Conclusions Decreased expression of selenium-binding protein 1 in uterine leiomyoma may indicate a role of the protein in tumorigenesis. Our findings may provide a basis for future studies concerning the molecular mechanisms of selenium-binding protein 1 in tumorigenesis as well as the possible use of selenium in prevention and treatment of uterine

  5. Clinical significance of urinary liver-type fatty acid binding protein at various stages of nephropathy

    OpenAIRE

    Viswanathan, V.; S. Sivakumar; Sekar, V; Umapathy, D.; Kumpatla, S.

    2015-01-01

    This cross-sectional study was to evaluate the levels of urinary liver-type fatty acid binding protein (u-LFABP pg/mg urine creatinine ratio) at different stages of diabetic nephropathy and to see its correlation with other clinical parameters in South Indian patients with type 2 diabetes mellitus (T2DM). A total of 65 (M: F; 42:23) T2DM subjects were divided into three groups, and were compared with 13 (M: F; 3:10) nondiabetic controls. The study groups were as follows: normoalbuminuric (n =...

  6. Structure, Function, and Evolution of Biogenic Amine-binding Proteins in Soft Ticks

    Energy Technology Data Exchange (ETDEWEB)

    Mans, Ben J.; Ribeiro, Jose M.C.; Andersen, John F. (NIH)

    2008-08-19

    Two highly abundant lipocalins, monomine and monotonin, have been isolated from the salivary gland of the soft tick Argas monolakensis and shown to bind histamine and 5-hydroxytryptamine (5-HT), respectively. The crystal structures of monomine and a paralog of monotonin were determined in the presence of ligands to compare the determinants of ligand binding. Both the structures and binding measurements indicate that the proteins have a single binding site rather than the two sites previously described for the female-specific histamine-binding protein (FS-HBP), the histamine-binding lipocalin of the tick Rhipicephalus appendiculatus. The binding sites of monomine and monotonin are similar to the lower, low affinity site of FS-HBP. The interaction of the protein with the aliphatic amine group of the ligand is very similar for the all of the proteins, whereas specificity is determined by interactions with the aromatic portion of the ligand. Interestingly, protein interaction with the imidazole ring of histamine differs significantly between the low affinity binding site of FS-HBP and monomine, suggesting that histamine binding has evolved independently in the two lineages. From the conserved features of these proteins, a tick lipocalin biogenic amine-binding motif could be derived that was used to predict biogenic amine-binding function in other tick lipocalins. Heterologous expression of genes from salivary gland libraries led to the discovery of biogenic amine-binding proteins in soft (Ornithodoros) and hard (Ixodes) tick genera. The data generated were used to reconstruct the most probable evolutionary pathway for the evolution of biogenic amine-binding in tick lipocalins.

  7. Regulated Pumilio-2 binding controls RINGO/Spy mRNA translation and CPEB activation

    Science.gov (United States)

    Padmanabhan, Kiran; Richter, Joel D.

    2006-01-01

    CPEB is a sequence-specific RNA-binding protein that controls the polyadenylation-induced translation of mos and cyclin B1 mRNAs in maturing Xenopus oocytes. CPEB activity requires not only the phosphorylation of S174, but also the synthesis of a heretofore-unknown upstream effector molecule. We show that the synthesis of RINGO/Spy, an atypical activator of cyclin-dependent kinases (cdks), is necessary for CPEB-directed polyadenylation. Deletion analysis and mRNA reporter assays show that a cis element in the RINGO/Spy 3′UTR is necessary for translational repression in immature (G2-arrested) oocytes. The repression is mediated by 3′UTR Pumilio-Binding Elements (PBEs), and by its binding protein Pumilio 2 (Pum2). Pum2 also interacts with the Xenopus homolog of human Deleted for Azoospermia-like (DAZL) and the embryonic poly(A)-binding protein (ePAB). Following the induction of maturation, Pum2 dissociates not only from RINGO/Spy mRNA, but from XDAZL and ePAB as well; as a consequence, RINGO/Spy mRNA is translated. These results demonstrate that a reversible Pum2 interaction controls RINGO/Spy mRNA translation and, as a result, CPEB-mediated cytoplasmic polyadenylation. PMID:16418484

  8. Arabidopsis Pumilio protein APUM5 suppresses Cucumber mosaic virus infection via direct binding of viral RNAs

    OpenAIRE

    Huh, Sung Un; Kim, Min Jung; Paek, Kyung-Hee

    2012-01-01

    Posttranscriptional/translational regulation of gene expression is mediated by diverse RNA binding proteins and plays an important role in development and defense processes. Among the RNA-binding proteins, the mammalian Pumilio RNA-binding family (Puf) acts as posttranscriptional and translational repressors. An Arabidopsis Puf mutant, apum5-D, was isolated during a T-DNA insertional mutant screen for mutants with reduced susceptibility to Cucumber mosaic virus (CMV) infection. Interestingly,...

  9. D-fructose-binding proteins in bull seminal plasma: Isolation and characterization

    Czech Academy of Sciences Publication Activity Database

    Liberda, J.; Kraus, Marek; Ryšlavá, H.; Vlasáková, M.; Jonáková, Věra; Tichá, M.

    2001-01-01

    Roč. 47, č. 4 (2001), s. 113-119. ISSN 0015-5500 R&D Projects: GA ČR GA303/99/0357; GA ČR GV524/96/K162 Institutional research plan: CEZ:AV0Z5052915 Keywords : bull seminal plasma * non-heparin-binding and heparin-binding proteins * D- fructose -binding proteins Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 0.519, year: 2001

  10. Metal ion interaction of an oligopeptide fragment representing the regulatory metal binding site of a CueR protein

    DEFF Research Database (Denmark)

    Jancsó, Attila; Szokolai, Hajnalka; Roszahegyi, Livia; Szunyogh, Daniel; Hemmingsen, Lars Bo Stegeager; Thulstrup, Peter Waaben; Larsen, Flemming Hofmann

    2013-01-01

    Metalloregulatory proteins of the MerR family are transcriptional activators that sense/control the concentration of various metal ions inside bacteria.1 The Cu+ efflux regulator CueR, similarly to other MerR proteins, possesses a short multiple Cys-containing metal binding loop close to the C......-terminus. CueR has a high selectivity for Cu+, Ag+ and Au+, but exhibits no transcriptional activity for the divalent ions Hg2+ and Zn2+.2 The two Cys- residues of the metal binding loop were shown to settle M+ ions into a linear coordination environment but other factors may also play a role in the recognition...... of cognate metal ions.2 Nevertheless, it is an interesting question whether the same sequence, when removed from the protein, shows a flexibility to adopt different coordination environments and may efficiently bind metal ions having preferences for larger coordination numbers....

  11. Controlled Immobilization Strategies to Probe Short Hyaluronan-Protein Interactions

    Science.gov (United States)

    Minsky, Burcu Baykal; Antoni, Christiane H.; Boehm, Heike

    2016-01-01

    Well-controlled grafting of small hyaluronan oligosaccharides (sHA) enables novel approaches to investigate biological processes such as angiogenesis, immune reactions and cancer metastasis. We develop two strategies for covalent attachment of sHA, a fast high-density adsorption and a two-layer system that allows tuning the density and mode of immobilization. We monitored the sHA adlayer formation and subsequent macromolecular interactions by label-free quartz crystal microbalance with dissipation (QCM-D). The modified surfaces are inert to unspecific protein adsorption, and yet retain the specific binding capacity of sHA. Thus they are an ideal tool to study the interactions of hyaluronan-binding proteins and short hyaluronan molecules as demonstrated by the specific recognition of LYVE-1 and aggrecan. Both hyaladherins recognize sHA and the binding is independent to the presence of the reducing end. PMID:26883791

  12. Controlled Immobilization Strategies to Probe Short Hyaluronan-Protein Interactions

    Science.gov (United States)

    Minsky, Burcu Baykal; Antoni, Christiane H.; Boehm, Heike

    2016-02-01

    Well-controlled grafting of small hyaluronan oligosaccharides (sHA) enables novel approaches to investigate biological processes such as angiogenesis, immune reactions and cancer metastasis. We develop two strategies for covalent attachment of sHA, a fast high-density adsorption and a two-layer system that allows tuning the density and mode of immobilization. We monitored the sHA adlayer formation and subsequent macromolecular interactions by label-free quartz crystal microbalance with dissipation (QCM-D). The modified surfaces are inert to unspecific protein adsorption, and yet retain the specific binding capacity of sHA. Thus they are an ideal tool to study the interactions of hyaluronan-binding proteins and short hyaluronan molecules as demonstrated by the specific recognition of LYVE-1 and aggrecan. Both hyaladherins recognize sHA and the binding is independent to the presence of the reducing end.

  13. Requirement for the eIF4E binding proteins for the synergistic down-regulation of protein synthesis by hypertonic conditions and mTOR inhibition

    OpenAIRE

    Clemens, Michael J.; Elia, Androulla; Morley, Simon J.

    2013-01-01

    The protein kinase mammalian target of rapamycin (mTOR) regulates the phosphorylation and activity of several proteins that have the potential to control translation, including p70S6 kinase and the eIF4E binding proteins 4E-BP1 and 4E-BP2. In spite of this, in exponentially growing cells overall protein synthesis is often resistant to mTOR inhibitors. We report here that sensitivity of wild-type mouse embryonic fibroblasts (MEFs) to mTOR inhibitors can be greatly increased when the cells are ...

  14. Local conformational fluctuations can modulate the coupling between proton binding and global structural transitions in proteins

    OpenAIRE

    Whitten, Steven T; García-Moreno E., Bertrand; Hilser, Vincent J.

    2005-01-01

    Local conformational fluctuations in proteins can affect the coupling between ligand binding and global structural transitions. This finding was established by monitoring quantitatively how the population distribution in the ensemble of microstates of staphylococcal nuclease was affected by proton binding. Analysis of acid unfolding and proton-binding data with an ensemble-based model suggests that local fluctuations: (i) can be effective modulators of ligand-binding affinities, (ii) are impo...

  15. A Novel Approach for Identifying the Heme—Binding Proteins from Mouse Tissues

    Institute of Scientific and Technical Information of China (English)

    XiaoleiLi; XiaoshanWang; KangZhao; ZhengfengZhou; CaifengZhao; RenYan; LiangLin; TingtingLei; JianningYin; RongWang; ZhongshengSun; ZuyuanXu; JingyueBao; XiugingZhang; XiaoliFeng; SiqiLiu

    2003-01-01

    Heme is a key cofactor in aerobic life,both in eukaryotes and prokaryotes.Because of the high reactivity of ferrous protoporphyrin IX,the reactions of heme in cells are often carried out through heme-protein complexes.Traditionally studies of hemebinding proteins have been approached on a case by case basis,thus there is a limited global view of the distribution of heme-binding proteins in different cells or tissues.The procedure described here is aimed at profiling heme-binding proteins in mouse tissues sequentially by 1)purification of heme-binding proteins by hemeagarose,an affinity chromatographic resin;2)isolation of heme-binding proteins by SDS-PAGE or two-dimensional electrophoresis;3)identification of heme-binding proteins by mass spectrometry.In five mouse tissues,over 600 protein spots were visualized on 2DE gel stained by Commassie blue and 154 proteins were identified by MALDI-TOF,in which most proteins belong to heme related.This methodology makes it possible to globally characterize the heme-binding proteins in a biological system.

  16. RNA binding properties of the US11 protein from four primate simplexviruses

    Directory of Open Access Journals (Sweden)

    Tohme Sarah

    2011-11-01

    Full Text Available Abstract Background The protein encoded by the Us11 gene of herpes simplex viruses is a dsRNA binding protein which inhibits protein kinase R activity, thereby preventing the interferon-induced shut down of protein synthesis following viral infection. Us11 protein is not essential for infectivity in vitro and in mice in herpes simplex virus type 1 (HSV1, however this virus has a second, and apparently more important, inhibitor of PKR activity, the γ134.5 protein. Recently sequenced simian simplexviruses SA8, HVP2 and B virus do not have an ORF corresponding to the γ134.5 protein, yet they have similar, or greater, infectivity as HSV1 and HSV2. Methods We have expressed the US11 proteins of the simplexviruses HSV1, HSV2, HVP2 and B virus and measured their abilities to bind dsRNA, in order to investigate possible differences that could complement the absence of the γ134.5 protein. We employed a filter binding technique that allows binding of the Us11 protein under condition of excess dsRNA substrate and therefore a measurement of the true Kd value of Us11-dsRNA binding. Results and Conclusions The results show a Kd of binding in the range of 0.89 nM to 1.82 nM, with no significant difference among the four Us11 proteins.

  17. A Novel Approach for Identifying the Heme-Binding Proteins from Mouse Tissues

    Institute of Scientific and Technical Information of China (English)

    Xiaolei Li; Rong Wang; Zhongsheng Sun; Zuyuan Xu; Jingyue Bao; Xiuqing Zhang; Xiaoli Feng; Siqi Liu; Xiaoshan Wang; Kang Zhao; Zhengfeng Zhou; Caifeng Zhao; Ren Yan; Liang Lin; Tingting Lei; Jianning Yin

    2003-01-01

    Heme is a key cofactor in aerobic life, both in eukaryotes and prokaryotes. Because of the high reactivity of ferrous protoporphyrin IX, the reactions of heme in cells are often carried out through heme-protein complexes. Traditionally studies of hemebinding proteins have been approached on a case by case basis, thus there is a limited global view of the distribution of heme-binding proteins in different cells or tissues. The procedure described here is aimed at profiling hemne-binding proteins in mouse tissues sequentially by 1) purification of heme-binding proteins by hemeagarose, an affinity chromatographic resin; 2) isolation of heme-binding proteins by SDS-PAGE or two-dimensional electrophoresis; 3) identification of heme-binding proteins by mass spectrometry. In five mouse tissues, over 600 protein spots were visualized on 2DE gel stained by Commassie blue and 154 proteins were identified by MALDI-TOF, in which most proteins belong to heme related. This methodology makes it possible to globally characterize the heme-binding proteins in a biological system.

  18. A recombinant triblock protein polymer with dispersant and binding properties for digital printing.

    Science.gov (United States)

    Qi, Min; O'Brien, John P; Yang, Jianjun

    2008-01-01

    A structured triblock protein was designed to explore the potential of engineered peptides to function as high-performance ink dispersants and binders. The protein consists of three functional elements, including a pigment binding domain, a hydrophilic linker, and a printing surface binding domain. To construct such a chimeric protein, a carbon black binding peptide, FHENWPS, and a cellulose binding peptide, THKTSTQRLLAA, were identified from phage display libraries through biopanning, based on their strong and specific binding affinities to carbon black and cellulose. They were used as carbon black and cellulose binding domains, respectively, in a recombinant triblock protein. A linker sequence, PTPTPTPTPTPTPTPTPTPTPTP, was adapted from endoglucanase A of the bacterium Cellulomonas fimi, as a small, rigid, and hydrophilic interdomain linker. When incorporated into the triblock structure between the carbon black and cellulose binding sequences, the linker sufficiently isolates these two elements and allows dual binding activity. The structured triblock protein was shown to disperse carbon black particles and attach it to paper surfaces. Thus, the utility of structured proteins having useful dispersant and binding properties for digital printing inks was demonstrated. PMID:17972282

  19. Theory on the mechanism of rapid binding of transcription factor proteins at specific-sites on DNA

    CERN Document Server

    Murugan, Rajamanickam

    2014-01-01

    We develop revised theoretical ideas on the mechanism by which the transcription factor proteins locate their specific binding sites on DNA faster than the three-dimensional (3D) diffusion controlled rate limit. We demonstrate that the 3D-diffusion controlled rate limit can be enhanced when the protein molecule reads several possible binding stretches of the template DNA via one-dimensional (1D) diffusion upon each 3D-diffusion mediated collision or nonspecific binding event. The overall enhancement of site-specific association rate is directly proportional to the maximum possible sliding length (LA, square root of (6Do/kr) where Do is the 1D-diffusion coefficient and kr is the dissociation rate constant associated with the nonspecific DNA-protein complex) associated with the 1D-diffusion of protein molecule along DNA. Upon considering several possible mechanisms we find that the DNA binding proteins can efficiently locate their cognate sites on DNA by switching across fast-moving, slow-moving and reading sta...

  20. Sequence similarity between the erythrocyte binding domain of the Plasmodium vivax Duffy binding protein and the V3 loop of HIV-1 strain MN reveals a functional heparin binding motif involved in binding to the Duffy antigen receptor for chemokines

    Directory of Open Access Journals (Sweden)

    Bolton Michael J

    2011-11-01

    Full Text Available Abstract Background The HIV surface glycoprotein gp120 (SU, gp120 and the Plasmodium vivax Duffy binding protein (PvDBP bind to chemokine receptors during infection and have a site of amino acid sequence similarity in their binding domains that often includes a heparin binding motif (HBM. Infection by either pathogen has been found to be inhibited by polyanions. Results Specific polyanions that inhibit HIV infection and bind to the V3 loop of X4 strains also inhibited DBP-mediated infection of erythrocytes and DBP binding to the Duffy Antigen Receptor for Chemokines (DARC. A peptide including the HBM of PvDBP had similar affinity for heparin as RANTES and V3 loop peptides, and could be specifically inhibited from heparin binding by the same polyanions that inhibit DBP binding to DARC. However, some V3 peptides can competitively inhibit RANTES binding to heparin, but not the PvDBP HBM peptide. Three other members of the DBP family have an HBM sequence that is necessary for erythrocyte binding, however only the protein which binds to DARC, the P. knowlesi alpha protein, is inhibited by heparin from binding to erythrocytes. Heparitinase digestion does not affect the binding of DBP to erythrocytes. Conclusion The HBMs of DBPs that bind to DARC have similar heparin binding affinities as some V3 loop peptides and chemokines, are responsible for specific sulfated polysaccharide inhibition of parasite binding and invasion of red blood cells, and are more likely to bind to negative charges on the receptor than cell surface glycosaminoglycans.

  1. A rapid and simple assay for growth hormone-binding protein activity in human plasma

    International Nuclear Information System (INIS)

    The newly discovered circulating growth hormone binding proteins dictate a re-evaluation of the state of GH in plasma in health and disease as the binding proteins are known to affect GH metabolism and action. We describe a rapid and simple GH-binding assay that allows determination of free and complexed plasma GH, as well as GH-binding protein activity as an index of GH-binding protein levels, with relative ease. The method is based on incubation of plasma with 125I-GH and separation of bound from free GH on small DEAE-cellulose columns; it can be used on a large scale for routine determinations. The results obtained by this method are comparable to those obtained with the previously used slow and more cumbersome gel filtration technique. Initial data obtained in normal subject and certain disease states show that the bound fraction of plasma GH is similar in men, women and children, is unaffected by pregnancy or acute infection, but is marginally decreased in liver cirrhosis. In acromegaly, binding protein activity also appears normal when allowance is made for partial saturation of the binding proteins by the high prevailing GH levels. The technique we describe should facilitate investigations of normal and abnormal regulation of the GH binding proteins. (author)

  2. Serotonin binding in vitro by releasable proteins from human blood platelets

    International Nuclear Information System (INIS)

    Among the substances released from human blood platelets are serotonin and various proteins. It was hypothesized that one of these proteins binds serotonin and that serotonin might be important to the protein's function or that the protein might be important to serotonin's function. Two platelet-specific proteins, platelet factor 4 (PF4) and β-thromboglobulin (βTG) were found to bind serotonin in vitro. Endogenous PF4 was isolated by serotonin-affinity chromatography and was identified by radioimmunoassay. Purified [125I] -PF4 and native PF4 bound to and eluted from a serotonin-affinity column similarly. Ultrafiltration of the homologous protein, βTG, with [14C]-serotonin demonstrated binding of about 8 moles serotonin per mole tetrameric βTG with a dissociation constant of about 4 X 10(sup-8) M. Equilibrium dialysis of PF4 with radiolabelled serotonin was attempted, but no binding constant values were obtained because serotonin apparently bound to the dialysis membrane. Since EDTA was one of the two agents that eluted PF4 from the serotonin-affinity gel, calcium binding by PF4 was investigated by equilibrium dialysis. Evidence was obtained for positively cooperative binding of calcium ions by PF4. It is concluded that PF4 and βTG bind serotonin in vitro, that they may also bind in vivo when platelets undergo release, and that the functions of serotonin, PF4 and βTG may be mediated in part by serotonin-protein associations

  3. Enhancement of rabbit protein S anticoagulant cofactor activity in vivo by modulation of the protein S C4B binding protein interaction.

    OpenAIRE

    Weinstein, R E; Walker, F. J.

    1990-01-01

    The carboxy-terminal region of protein S has been recently been observed to be involved in the interaction between protein S and C4b-binding protein (Walker, F. J. 1989. J. Biol. Chem. 264:17645-17658). A synthetic peptide, GVQLDLDEAI, corresponding to that region of protein S has been used to investigate the protein S/C4b-binding protein interaction in vitro and in vivo. Rabbit activated protein C possesses species-specific anticoagulant activity for which rabbit protein S functions as a cof...

  4. The Mouthparts Enriched Odorant Binding Protein 11 of the Alfalfa Plant Bug Adelphocoris lineolatus Displays a Preferential Binding Behavior to Host Plant Secondary Metabolites

    Science.gov (United States)

    Sun, Liang; Wei, Yu; Zhang, Dan-Dan; Ma, Xiao-Yu; Xiao, Yong; Zhang, Ya-Nan; Yang, Xian-Ming; Xiao, Qiang; Guo, Yu-Yuan; Zhang, Yong-Jun

    2016-01-01

    Odorant binding proteins (OBPs) are proposed to be directly required for odorant discrimination and represent potential interesting targets for pest control. In the notoriously agricultural pest Adelphocoris lineolatus, our previous functional investigation of highly expressed antennal OBPs clearly supported this viewpoint, whereas the findings of the current study by characterizing of AlinOBP11 rather indicated that OBP in hemipterous plant bugs might fulfill a different and tantalizing physiological role. The phylogenetic analysis uncovered that AlinOBP11 together with several homologous bug OBP proteins are potential orthologs, implying they could exhibit a conserved function. Next, the results of expression profiles solidly showed that AlinOBP11 was predominantly expressed at adult mouthparts, the most important gustatory organ of Hemiptera mirid bug. Finally, a rigorously selective binding profile was observed in the fluorescence competitive binding assay, in which recombinant AlinOBP11 displayed much stronger binding abilities to non-volatile secondary metabolite compounds than the volatile odorants. These results reflect that AlinOBP11, even its orthologous proteins across bug species, could be associated with a distinctively conserved physiological role such as a crucial carrier for non-volatiles host secondary metabolites in gustatory system. PMID:27313540

  5. The Mouthparts Enriched Odorant Binding Protein 11 of the Alfalfa Plant Bug Adelphocoris lineolatus Displays a Preferential Binding Behavior to Host Plant Secondary Metabolites.

    Science.gov (United States)

    Sun, Liang; Wei, Yu; Zhang, Dan-Dan; Ma, Xiao-Yu; Xiao, Yong; Zhang, Ya-Nan; Yang, Xian-Ming; Xiao, Qiang; Guo, Yu-Yuan; Zhang, Yong-Jun

    2016-01-01

    Odorant binding proteins (OBPs) are proposed to be directly required for odorant discrimination and represent potential interesting targets for pest control. In the notoriously agricultural pest Adelphocoris lineolatus, our previous functional investigation of highly expressed antennal OBPs clearly supported this viewpoint, whereas the findings of the current study by characterizing of AlinOBP11 rather indicated that OBP in hemipterous plant bugs might fulfill a different and tantalizing physiological role. The phylogenetic analysis uncovered that AlinOBP11 together with several homologous bug OBP proteins are potential orthologs, implying they could exhibit a conserved function. Next, the results of expression profiles solidly showed that AlinOBP11 was predominantly expressed at adult mouthparts, the most important gustatory organ of Hemiptera mirid bug. Finally, a rigorously selective binding profile was observed in the fluorescence competitive binding assay, in which recombinant AlinOBP11 displayed much stronger binding abilities to non-volatile secondary metabolite compounds than the volatile odorants. These results reflect that AlinOBP11, even its orthologous proteins across bug species, could be associated with a distinctively conserved physiological role such as a crucial carrier for non-volatiles host secondary metabolites in gustatory system. PMID:27313540

  6. Comprehensive proteomic analysis of interphase and mitotic 14-3-3-binding proteins.

    Science.gov (United States)

    Meek, Sarah E M; Lane, William S; Piwnica-Worms, Helen

    2004-07-30

    14-3-3 proteins regulate the cell division cycle and play a pivotal role in blocking cell cycle advancement after activation of the DNA replication and DNA damage checkpoints. Here we describe a global proteomics analysis to identify proteins that bind to 14-3-3s during interphase and mitosis. 14-3-3-binding proteins were purified from extracts of interphase and mitotic HeLa cells using specific peptide elution from 14-3-3 zeta affinity columns. Proteins that specifically bound and eluted from the affinity columns were identified by microcapillary high pressure liquid chromatography tandem mass spectrometry analysis. Several known and novel 14-3-3-interacting proteins were identified in this screen. Identified proteins are involved in cell cycle regulation, signaling, metabolism, protein synthesis, nucleic acid binding, chromatin structure, protein folding, proteolysis, nucleolar function, and nuclear transport as well as several other cellular processes. In some cases 14-3-3 binding was cell cycle-dependent, whereas in other cases the binding was shown to be cell cycle-independent. This study adds to the growing list of human 14-3-3-binding proteins and implicates a role for 14-3-3 proteins in a plethora of essential biological processes. PMID:15161933

  7. Putative hAPN receptor binding sites in SARS_CoV spike protein

    Institute of Scientific and Technical Information of China (English)

    YUXiao-Jing; LUOCheng; LinJian-Cheng; HAOPei; HEYou-Yu; GUOZong-Ming; QINLei; SUJiong; LIUBo-Shu; HUANGYin; NANPeng; LIChuan-Song; XIONGBin; LUOXiao-Min; ZHAOGuo-Ping; PEIGang; CHENKai-Xian; SHENXu; SHENJian-Hua; ZOUJian-Ping; HEWei-Zhong; SHITie-Liu; ZHONGYang; JIANGHua-Liang; LIYi-Xue

    2003-01-01

    AIM:To obtain the information of ligand-receptor binding between thd S protein of SARS_CoV and CD13, identify the possible interacting domains or motifs related to binding sites, and provide clues for studying the functions of SARS proteins and designing anti-SARS drugs and vaccines. METHODS: On the basis of comparative genomics, the homology search, phylogenetic analyses, and multi-sequence alignment were used to predict CD13 related interacting domains and binding sites sites in the S protein of SARS_CoV. Molecular modeling and docking simulation methods were employed to address the interaction feature between CD13 and S protein of SARS_CoV in validating the bioinformatics predictions. RESULTS:Possible binding sites in the SARS_CoV S protein to CD13 have been mapped out by using bioinformatics analysis tools. The binding for one protein-protein interaction pair (D757-R761 motif of the SARS_CoV S protein to P585-A653 domain of CD13) has been simulated by molecular modeling and docking simulation methods. CONCLUSION:CD13 may be a possible receptor of the SARS_CoV S protein which may be associated with the SARS infection. This study also provides a possible strategy for mapping the possible binding receptors of the proteins in a genome.

  8. Phenanthrene binding by humic acid–protein complexes as studied by passive dosing technique

    International Nuclear Information System (INIS)

    This work investigated the binding behavior of phenanthrene by humic acids (HA-2 and HA-5), proteins (bovine serum albumin (BSA)), lysozyme and pepsin), and their complexes using a passive dosing technique. All sorption isotherms were fitted well with Freundlich model and the binding capability followed an order of HA-5 > HA-2 > BSA > pepsin > lysozyme. In NaCl solution, phenanthrene binding to HA-BSA complexes was much higher than the sum of binding to individual HA and BSA, while there was no enhancement for HA-pepsin. Positively charged lysozyme slightly lowered phenanthrene binding on both HAs due to strong aggregation of HA-lysozyme complexes, leading to reduction in the number of binding sites. The binding enhancement by HA-BSA was observed under all tested ion species and ionic strengths. This enhancement can be explained by unfolding of protein, reduction of aggregate size and formation of HA-BSA complexes with favorable conformations for binding phenanthrene. Highlights: • Phenanthrene binding capability followed an order: HA-5>HA-2>BSA>pepsin>lysozyme. • Phenanthrene binding to HA-BSA was enhanced relative to individual HA and BSA. • Binding enhancement to HA-BSA was observed under all tested solution conditions. • The enhancement is related to BSA unfolding, size reduction and HA-BSA complexation. -- Phenanthrene binding to HA-BSA complexes is much higher than the sum to individual HA and BSA while there was no binding enhancement to HA-pepsin or HA-lysozyme

  9. Insulin-like growth factors, insulin-like growth factor-binding proteins, insulin-like growth factor-binding protein-3 protease, and growth hormone-binding protein in lipodystrophic human immunodeficiency virus-infected patients

    DEFF Research Database (Denmark)

    Haugaard, Steen B; Andersen, Ove; Hansen, Birgitte Rønde;

    2004-01-01

    Human immunodeficiency virus (HIV)-lipodystrophy is associated with impaired growth hormone (GH) secretion. It remains to be elucidated whether insulin-like growth factors (IGFs), IGF-binding proteins (IGFBPs), IGFBP-3 protease, and GH-binding protein (GHBP) are abnormal in HIV......-lipodystrophy. These parameters were measured in overnight fasting serum samples from 16 Caucasian males with HIV-lipodystrophy (LIPO) and 15 Caucasian HIV-infected males without lipodystrophy (NONLIPO) matched for age, weight, duration of HIV infection, and antiretroviral therapy. In LIPO, abdominal fat mass and insulin...

  10. Ephemeral protein binding to DNA shapes stable nuclear bodies and chromatin domains

    CERN Document Server

    Brackley, C A; Michieletto, D; Mouvet, F; Cook, P R; Marenduzzo, D

    2016-01-01

    Fluorescence microscopy reveals that the contents of many (membrane-free) nuclear "bodies" exchange rapidly with the soluble pool whilst the underlying structure persists; such observations await a satisfactory biophysical explanation. To shed light on this, we perform large-scale Brownian dynamics simulations of a chromatin fiber interacting with an ensemble of (multivalent) DNA-binding proteins; these proteins switch between two states -- active (binding) and inactive (non-binding). This system provides a model for any DNA-binding protein that can be modified post-translationally to change its affinity for DNA (e.g., like the phosphorylation of a transcription factor). Due to this out-of-equilibrium process, proteins spontaneously assemble into clusters of self-limiting size, as individual proteins in a cluster exchange with the soluble pool with kinetics like those seen in photo-bleaching experiments. This behavior contrasts sharply with that exhibited by "equilibrium", or non-switching, proteins that exis...

  11. Identification of CP12 as a Novel Calcium-Binding Protein in Chloroplasts

    Directory of Open Access Journals (Sweden)

    Agostinho Gomes Rocha

    2013-08-01

    Full Text Available Calcium plays an important role in the regulation of several chloroplast processes. However, very little is still understood about the calcium fluxes or calcium-binding proteins present in plastids. Indeed, classical EF-hand containing calcium-binding proteins appears to be mostly absent from plastids. In the present study we analyzed the stroma fraction of Arabidopsis chloroplasts for the presence of novel calcium-binding proteins using 2D-PAGE separation followed by calcium overlay assay. A small acidic protein was identified by mass spectrometry analyses as the chloroplast protein CP12 and the ability of CP12 to bind calcium was confirmed with recombinant proteins. CP12 plays an important role in the regulation of the Calvin-Benson-Bassham Cycle participating in the assembly of a supramolecular complex between phosphoribulokinase and glyceraldehyde 3-phosphate dehydrogenase, indicating that calcium signaling could play a role in regulating carbon fixation.

  12. An insulin-binding protein from the venom of a solitary wasp Eumenes pomiformis binds to apolipophorin III in lepidopteran hemolymph.

    Science.gov (United States)

    Baek, Ji Hyeong; Lee, Si Hyeock; Kim, Woe-Yeon; Kim, Min Gab

    2016-03-01

    EpIBP, an insulin-like peptide-binding protein, is a major protein component of the venom of a solitary hunting wasp, Eumenes pomiformis. To evaluate the bioactivity, bacteria-expressed EpIBP was injected into Spodoptera exigua larvae, resulting in a higher survival rate and reduced loss of body weight under starvation conditions than control larvae. EpIBP was found to interact with apolipophorin III (apoLp III), implying that EpIBP might function by altering the apoLp III-mediated metabolism of prey. PMID:26748153

  13. In vitro protein binding of liraglutide in human plasma determined by reiterated stepwise equilibrium dialysis

    Science.gov (United States)

    Plum, Anne; Jensen, Lisbeth Bjerring; Kristensen, Jesper Bøggild

    2013-01-01

    Liraglutide is a human glucagon-like peptide-1 (GLP-1) analogue approved for the treatment of type 2 diabetes. It is based on human GLP-1 with the addition of a 16-carbon fatty acid, which facilitates binding to plasma proteins, thus prolonging the elimination half-life and allowing once-daily administration. It has not been possible to quantify liraglutide protein binding by ultrafiltration (the usual method of choice), as the lipophilic molecule becomes trapped in the filter membrane. Therefore, the aim of this study was to develop a methodology that could determine the extent of liraglutide binding to plasma proteins in vitro. We report here the details of a novel reiterated stepwise equilibrium dialysis assay that has successfully been used to quantify liraglutide plasma protein binding. The assay allowed quantification of liraglutide binding to proteins in purified plasma protein solutions and human plasma samples and was effective at plasma dilutions as low as 5%. At a clinically relevant liraglutide concentration (104 pM), greater than 98.9% of liraglutide was bound to protein. Specific binding to human serum albumin and α1-acid glycoprotein was 99.4% and 99.3%, respectively. The novel methodology described herein could have an application in the quantification of plasma protein binding of other highly lipophilic drug molecules. PMID:23853127

  14. Label-free measuring and mapping of binding kinetics of membrane proteins in single living cells

    Science.gov (United States)

    Wang, Wei; Yang, Yunze; Wang, Shaopeng; Nagaraj, Vinay J.; Liu, Qiang; Wu, Jie; Tao, Nongjian

    2012-10-01

    Membrane proteins mediate a variety of cellular responses to extracellular signals. Although membrane proteins are studied intensively for their values as disease biomarkers and therapeutic targets, in situ investigation of the binding kinetics of membrane proteins with their ligands has been a challenge. Traditional approaches isolate membrane proteins and then study them ex situ, which does not reflect accurately their native structures and functions. We present a label-free plasmonic microscopy method to map the local binding kinetics of membrane proteins in their native environment. This analytical method can perform simultaneous plasmonic and fluorescence imaging, and thus make it possible to combine the strengths of both label-based and label-free techniques in one system. Using this method, we determined the distribution of membrane proteins on the surface of single cells and the local binding kinetic constants of different membrane proteins. Furthermore, we studied the polarization of the membrane proteins on the cell surface during chemotaxis.

  15. Hemoglobin binding activity and hemoglobin-binding protein of prevotella nigrescens

    OpenAIRE

    Miyashita M; Oishi S; Kiso A; Kikuchi Y; Ueda O; Hirai K; Shibata Y; Fujimura S

    2010-01-01

    Abstract Prevotella nigrescens, lacking siderophores was found to bind to the hemoproteins. The binding was observed also in the envelope which was prepared by sonication of the cell. The binding occurred in the pH-dependent manner; the binding was observed below neutral pHs of the incubation mixtures but only slightly observed in the neutral and alkaline pHs. Furthermore, hemoglobin bound to the envelope was dissociated at high pHs buffers. Maximum amounts of hemoglobin bound to 1 mg envelop...

  16. Elucidation of binding mechanism and identification of binding site for an anti HIV drug, stavudine on human blood proteins.

    Science.gov (United States)

    Sandhya, B; Hegde, Ashwini H; Seetharamappa, J

    2013-05-01

    The binding of stavudine (STV) to two human blood proteins [human hemoglobin (HHb) and human serum albumin (HSA)] was studied in vitro under simulated physiological conditions by spectroscopic methods viz., fluorescence, UV absorption, resonance light scattering, synchronous fluorescence, circular dichroism (CD) and three-dimensional fluorescence. The binding parameters of STV-blood protein were determined from fluorescence quenching studies. Stern-Volmer plots indicated the presence of static quenching mechanism in the interaction of STV with blood proteins. The values of n close to unity indicated that one molecule of STV bound to one molecule of blood protein. The binding process was found to be spontaneous. Analysis of thermodynamic parameters revealed the presence of hydrogen bond and van der Waals forces between protein and STV. Displacement experiments indicated the binding of STV to Sudlow's site I on HSA. Secondary structures of blood proteins have undergone changes upon interaction with STV as evident from the reduction of α-helices (from 46.11% in free HHb to 38.34% in STV-HHb, and from 66.44% in free HSA to 52.26% in STV-HSA). Further, the alterations in secondary structures of proteins in the presence of STV were confirmed by synchronous and 3D-fluorescence spectral data. The distance between the blood protein (donor) and acceptor (STV) was found to be 5.211 and 5.402 nm for STV-HHb and STV-HSA, respectively based on Föster's non-radiative energy transfer theory. Effect of some metal ions was also investigated. The fraction of STV bound to HSA was found to be 87.8%. PMID:23275205

  17. Expression of liver fatty acid binding protein in hepatocellular carcinoma.

    Science.gov (United States)

    Cho, Soo-Jin; Ferrell, Linda D; Gill, Ryan M

    2016-04-01

    Loss of expression of liver fatty acid binding protein (LFABP) by immunohistochemistry has been shown to be characteristic of a subset of hepatocellular adenomas (HCAs) in which HNF1A is inactivated. Transformation to hepatocellular carcinoma is thought to be a very rare phenomenon in the HNF1A-inactivated variant of HCA. However, we recently observed 2 cases at our institution, 1 definite hepatocellular carcinoma and 1 possible hepatocellular carcinoma, with loss of LFABP staining, raising the possibility that LFABP down-regulation may be associated with hepatocellular carcinogenesis. Our aim was to evaluate hepatocellular carcinomas arising in various backgrounds and with varying degrees of differentiation for loss of LFABP staining. Twenty total cases of hepatocellular carcinoma were examined. Thirteen cases arose in a background of cirrhosis due to hepatitis C (n = 8) or steatohepatitis (n = 5); 7 cases arose in a noncirrhotic background, with 2 cases arising within HNF1A-inactivated variant HCA and 2 cases arising within inflammatory variant HCA. Complete loss of expression of LFABP was seen in 6 of 20 cases, including 2 cases of hepatocellular carcinoma arising within HNF1A-inactivated variant HCA. Thus, loss of staining for LFABP appears to be common in hepatocellular carcinoma and may be seen in well-differentiated hepatocellular carcinoma. Therefore, LFABP loss should not be interpreted as evidence for hepatocellular adenoma over carcinoma, when other features support a diagnosis of hepatocellular carcinoma. The findings raise consideration for a role of HNF1A inactivation in hepatocellular carcinogenesis, particularly in less differentiated tumors. PMID:26997447

  18. Proteomic Screening Method for Phosphopeptide Motif Binding Proteins Using Peptide Libraries

    OpenAIRE

    Christofk, Heather R.; Wu, Ning; Cantley, Lewis C.; John M. Asara

    2011-01-01

    Phosphopeptide binding domains mediate the directed and localized assembly of protein complexes essential to intracellular kinase signaling. To identify phosphopeptide binding proteins, we developed a proteomic screening method using immobilized partially-degenerate phosphopeptide mixtures combined with SILAC and microcapillary LC/MS/MS. The method was used to identify proteins that specifically bound to phosphorylated peptide library affinity matrices, including pTyr, and the motifs pSer/pTh...

  19. Identification and characterization of amylase binding protein C (AbpC) from Streptococcus mitis NS51

    OpenAIRE

    Vorrasi, John; Chaudhuri, Biswendu; Haase, Elaine M.; Scannapieco, Frank A.

    2010-01-01

    A substantial proportion of the streptococcal species found in dental plaque biofilms are able to interact with the abundant salivary enzyme α-amylase. These streptococci produce proteins that specifically bind amylase. An important plaque species, Streptococcus mitis, secretes a 36-kDa amylase binding protein into the extracellular milieu. Proteins precipitated from S. mitis NS51 cell culture supernatant by the addition of purified salivary amylase were separated by SDS-PAGE, transferred to ...

  20. Computational analysis of protein-ligand binding : from single continuous trajectories to multiple parallel simulations

    OpenAIRE

    Thorsteinsdottir, Holmfridur B.

    2010-01-01

    The interaction of proteins with other proteins or small molecules is essential for biological functions. Understanding the molecular basis of protein-ligand binding is of a vast interest for drug discovery, and computational methods to estimate proteinligand binding are starting to play an increasingly important role. In order to apply atomistic computational methods to the drug discovery process it is necessary to have accurate three-dimensional structures of the target prote...