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Sample records for n-ethylmaleimide-sensitive factor-attachment protein

  1. Soluble N-ethylmaleimide-sensitive factor attachment protein receptors required during Trypanosoma cruzi parasitophorous vacuole development.

    Science.gov (United States)

    Cueto, Juan Agustín; Vanrell, María Cristina; Salassa, Betiana Nebaí; Nola, Sébastien; Galli, Thierry; Colombo, María Isabel; Romano, Patricia Silvia

    2017-06-01

    Trypanosoma cruzi, the etiologic agent of Chagas disease, is an obligate intracellular parasite that exploits different host vesicular pathways to invade the target cells. Vesicular and target soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are key proteins of the intracellular membrane fusion machinery. During the early times of T. cruzi infection, several vesicles are attracted to the parasite contact sites in the plasma membrane. Fusion of these vesicles promotes the formation of the parasitic vacuole and parasite entry. In this work, we study the requirement and the nature of SNAREs involved in the fusion events that take place during T. cruzi infection. Our results show that inhibition of N-ethylmaleimide-sensitive factor protein, a protein required for SNARE complex disassembly, impairs T. cruzi infection. Both TI-VAMP/VAMP7 and cellubrevin/VAMP3, two v-SNAREs of the endocytic and exocytic pathways, are specifically recruited to the parasitophorous vacuole membrane in a synchronized manner but, although VAMP3 is acquired earlier than VAMP7, impairment of VAMP3 by tetanus neurotoxin fails to reduce T. cruzi infection. In contrast, reduction of VAMP7 activity by expression of VAMP7's longin domain, depletion by small interfering RNA or knockout, significantly decreases T. cruzi infection susceptibility as a result of a minor acquisition of lysosomal components to the parasitic vacuole. In addition, overexpression of the VAMP7 partner Vti1b increases the infection, whereas expression of a KIF5 kinesin mutant reduces VAMP7 recruitment to vacuole and, concomitantly, T. cruzi infection. Altogether, these data support a key role of TI-VAMP/VAMP7 in the fusion events that culminate in the T. cruzi parasitophorous vacuole development. © 2016 John Wiley & Sons Ltd.

  2. The Central Polybasic Region of the Soluble SNARE (Soluble N-Ethylmaleimide-sensitive Factor Attachment Protein Receptor) Vam7 Affects Binding to Phosphatidylinositol 3-Phosphate by the PX (Phox Homology) Domain.

    Science.gov (United States)

    Miner, Gregory E; Starr, Matthew L; Hurst, Logan R; Sparks, Robert P; Padolina, Mark; Fratti, Rutilio A

    2016-08-19

    The yeast vacuole requires four SNAREs to trigger membrane fusion including the soluble Qc-SNARE Vam7. The N-terminal PX domain of Vam7 binds to the lipid phosphatidylinositol 3-phosphate (PI3P) and the tethering complex HOPS (homotypic fusion and vacuole protein sorting complex), whereas the C-terminal SNARE motif forms SNARE complexes. Vam7 also contains an uncharacterized middle domain that is predicted to be a coiled-coil domain with multiple helices. One helix contains a polybasic region (PBR) composed of Arg-164, Arg-168, Lys-172, Lys-175, Arg-179, and Lys-186. Polybasic regions are often associated with nonspecific binding to acidic phospholipids including phosphoinositides. Although the PX (phox homology) domain alone binds PI3P, we theorized that the Vam7 PBR could bind to additional acidic phospholipids enriched at fusion sites. Mutating each of the basic residues in the PBR to an alanine (Vam7-6A) led to attenuated vacuole fusion. The defective fusion of Vam7-6A was due in part to inefficient association with its cognate SNAREs and HOPS, yet the overall vacuole association of Vam7-6A was similar to wild type. Experiments testing the binding of Vam7 to specific signaling lipids showed that mutating the PBR to alanines augmented binding to PI3P. The increased binding to PI3P by Vam7-6A likely contributed to the observed wild type levels of vacuole association, whereas protein-protein interactions were diminished. PI3P binding was inhibited when the PX domain mutant Y42A was introduced into Vam7-6A to make Vam7-7A. Thus the Vam7 PBR affects PI3P binding by the PX domain and in turn affects binding to SNAREs and HOPS to support efficient fusion. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  3. The Q-soluble N-Ethylmaleimide-sensitive Factor Attachment Protein Receptor (Q-SNARE) SNAP-47 Regulates Trafficking of Selected Vesicle-associated Membrane Proteins (VAMPs)*

    Science.gov (United States)

    Kuster, Aurelia; Nola, Sebastien; Dingli, Florent; Vacca, Barbara; Gauchy, Christian; Beaujouan, Jean-Claude; Nunez, Marcela; Moncion, Thomas; Loew, Damarys; Formstecher, Etienne; Galli, Thierry; Proux-Gillardeaux, Veronique

    2015-01-01

    SNAREs constitute the core machinery of intracellular membrane fusion, but vesicular SNAREs localize to specific compartments via largely unknown mechanisms. Here, we identified an interaction between VAMP7 and SNAP-47 using a proteomics approach. We found that SNAP-47 mainly localized to cytoplasm, the endoplasmic reticulum (ER), and ERGIC and could also shuttle between the cytoplasm and the nucleus. SNAP-47 preferentially interacted with the trans-Golgi network VAMP4 and post-Golgi VAMP7 and -8. SNAP-47 also interacted with ER and Golgi syntaxin 5 and with syntaxin 1 in the absence of Munc18a, when syntaxin 1 is retained in the ER. A C-terminally truncated SNAP-47 was impaired in interaction with VAMPs and affected their subcellular distribution. SNAP-47 silencing further shifted the subcellular localization of VAMP4 from the Golgi apparatus to the ER. WT and mutant SNAP-47 overexpression impaired VAMP7 exocytic activity. We conclude that SNAP-47 plays a role in the proper localization and function of a subset of VAMPs likely via regulation of their transport through the early secretory pathway. PMID:26359495

  4. The Q-soluble N-Ethylmaleimide-sensitive Factor Attachment Protein Receptor (Q-SNARE) SNAP-47 Regulates Trafficking of Selected Vesicle-associated Membrane Proteins (VAMPs).

    Science.gov (United States)

    Kuster, Aurelia; Nola, Sebastien; Dingli, Florent; Vacca, Barbara; Gauchy, Christian; Beaujouan, Jean-Claude; Nunez, Marcela; Moncion, Thomas; Loew, Damarys; Formstecher, Etienne; Galli, Thierry; Proux-Gillardeaux, Veronique

    2015-11-20

    SNAREs constitute the core machinery of intracellular membrane fusion, but vesicular SNAREs localize to specific compartments via largely unknown mechanisms. Here, we identified an interaction between VAMP7 and SNAP-47 using a proteomics approach. We found that SNAP-47 mainly localized to cytoplasm, the endoplasmic reticulum (ER), and ERGIC and could also shuttle between the cytoplasm and the nucleus. SNAP-47 preferentially interacted with the trans-Golgi network VAMP4 and post-Golgi VAMP7 and -8. SNAP-47 also interacted with ER and Golgi syntaxin 5 and with syntaxin 1 in the absence of Munc18a, when syntaxin 1 is retained in the ER. A C-terminally truncated SNAP-47 was impaired in interaction with VAMPs and affected their subcellular distribution. SNAP-47 silencing further shifted the subcellular localization of VAMP4 from the Golgi apparatus to the ER. WT and mutant SNAP-47 overexpression impaired VAMP7 exocytic activity. We conclude that SNAP-47 plays a role in the proper localization and function of a subset of VAMPs likely via regulation of their transport through the early secretory pathway. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. A library of 7TM receptor C-terminal tails - Interactions with the proposed post-endocytic sorting proteins ERM-binding phosphoprotein 50 (EBP50), N-ethylmaleimide-sensitive factor (NSF), sorting nexin 1 (SNX1), and G protein-coupled receptor-associated sorting protein (GASP)

    DEFF Research Database (Denmark)

    Heydorn, A.; Sondergaard, B.P.; Ersbøll, Bjarne Kjær

    2004-01-01

    sequestration through interactions, mainly with the C-terminal intracellular tails of the receptors. A library of tails from 59 representative members of the super family of seven-transmembrane receptors was probed as glutathione S-transferase fusion proteins for interactions with four different adaptor...... only a single receptor tail, i.e. the beta(2)-adrenergic receptor, whereas N-ethylmaleimide-sensitive factor bound 11 of the tail-fusion proteins. Of the two proteins proposed to target receptors for lysosomal degradation, sorting nexin 1 (SNX1) bound 10 and the C-terminal domain of G protein......-coupled receptor-associated sorting protein bound 23 of the 59 tail proteins. Surface plasmon resonance analysis of the binding kinetics of selected hits from the glutathione S-transferase pull-down experiments, i.e. the tails of the virally encoded receptor US28 and the delta-opioid receptor, confirmed...

  6. The N-ethylmaleimide-sensitive factor and dysbindin interact to modulate synaptic plasticity.

    Science.gov (United States)

    Gokhale, Avanti; Mullin, Ariana P; Zlatic, Stephanie A; Easley, Charles A; Merritt, Megan E; Raj, Nisha; Larimore, Jennifer; Gordon, David E; Peden, Andrew A; Sanyal, Subhabrata; Faundez, Victor

    2015-05-13

    Dysbindin is a schizophrenia susceptibility factor and subunit of the biogenesis of lysosome-related organelles complex 1 (BLOC-1) required for lysosome-related organelle biogenesis, and in neurons, synaptic vesicle assembly, neurotransmission, and plasticity. Protein networks, or interactomes, downstream of dysbindin/BLOC-1 remain partially explored despite their potential to illuminate neurodevelopmental disorder mechanisms. Here, we conducted a proteome-wide search for polypeptides whose cellular content is sensitive to dysbindin/BLOC-1 loss of function. We identified components of the vesicle fusion machinery as factors downregulated in dysbindin/BLOC-1 deficiency in neuroectodermal cells and iPSC-derived human neurons, among them the N-ethylmaleimide-sensitive factor (NSF). Human dysbindin/BLOC-1 coprecipitates with NSF and vice versa, and both proteins colocalized in a Drosophila model synapse. To test the hypothesis that NSF and dysbindin/BLOC-1 participate in a pathway-regulating synaptic function, we examined the role for NSF in dysbindin/BLOC-1-dependent synaptic homeostatic plasticity in Drosophila. As previously described, we found that mutations in dysbindin precluded homeostatic synaptic plasticity elicited by acute blockage of postsynaptic receptors. This dysbindin mutant phenotype is fully rescued by presynaptic expression of either dysbindin or Drosophila NSF. However, neither reduction of NSF alone or in combination with dysbindin haploinsufficiency impaired homeostatic synaptic plasticity. Our results demonstrate that dysbindin/BLOC-1 expression defects result in altered cellular content of proteins of the vesicle fusion apparatus and therefore influence synaptic plasticity. Copyright © 2015 the authors 0270-6474/15/357643-11$15.00/0.

  7. Developmental and Diurnal Expression of the Synaptosomal-Associated Protein 25 (Snap25) in the Rat Pineal Gland

    DEFF Research Database (Denmark)

    Karlsen, Anna S; Rath, Martin Fredensborg; Rohde, Kristian

    2013-01-01

    Snap25 (synaptosomal-associated protein) is a 25 kDa protein, belonging to the SNARE-family (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) of proteins, essential for synaptic and secretory vesicle exocytosis. Snap25 has by immunohistochemistry been demonstrated in the rat...

  8. N-Ethylmaleimide-Sensitive Factor b (nsfb) Is Required for Normal Pigmentation of the Zebrafish Retinal Pigment Epithelium.

    Science.gov (United States)

    Hanovice, Nicholas J; Daly, Christina M S; Gross, Jeffrey M

    2015-11-01

    Despite the number of albinism-causing mutations identified in human patients and animal models, there remain a significant number of cases for which no mutation has been identified, suggesting that our understanding of melanogenesis is incomplete. Previously, we identified two oculocutaneous albinism mutations in zebrafish, au13 and au18. Here, we sought to identify the mutated loci and determine how the affected proteins contribute to normal pigmentation of the retinal pigment epithelium (RPE). Complementation analyses revealed that au13 and au18 belonged to a single complementation group, suggesting that they affected the same locus. Whole-genome sequencing and single nucleotide polymorphism (SNP) analysis was performed to identify putative mutations, which were confirmed by cDNA sequencing and mRNA rescue. Transmission electron microscopy (TEM) and image quantification were used to identify the cellular basis of hypopigmentation. Whole-genome sequencing and SNP mapping identified a nonsense mutation in the N-ethylmaleimide-sensitive factor b (nsfb) gene in au18 mutants. Complementary DNA sequencing confirmed the presence of the mutation (C893T), which truncates the nsfb protein by roughly two-thirds (Y297X). No coding sequence mutations were identified in au13, but quantitative PCR revealed a significant decrease in nsfb expression, and nsfb mRNA injection rescued the hypopigmentation phenotype, suggesting a regulatory mutation. In situ hybridization revealed that nsfb is broadly expressed during embryonic development, including in the RPE. Transmission electron microscopy analyses indicated that average melanosome density and maturity were significantly decreased in nsfb mutants. au18 and au13 contain mutations in nsfb, which encodes a protein that is required for the maturation of melanosomes in zebrafish RPE.

  9. The role of the N-D1 linker of the N-ethylmaleimide-sensitive factor in the SNARE disassembly.

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    Cui-Cui Liu

    Full Text Available N-ethylmaleimide-sensitive factor (NSF is a member of the type II AAA+ (ATPase associated with various cellular activities family. It plays a critical role in intracellular membrane trafficking by disassembling soluble NSF attachment protein receptor (SNARE complexes. Each NSF protomer consists of an N-terminal domain (N domain followed by two AAA ATPase domains (D1 and D2 in tandem. The N domain is required for SNARE/α-SNAP binding and the D1 domain accounts for the majority of ATP hydrolysis. Little is known about the role of the N-D1 linker in the NSF function. This study presents detailed mutagenesis analyses of NSF N-D1 linker, dissecting its role in the SNARE disassembly, the SNARE/α-SNAP complex binding, the basal ATPase activity and the SNARE/α-SNAP stimulated ATPase activity. Our results show that the N-terminal region of the N-D1 linker associated mutants cause severe defect in SNARE complex disassembly, but little effects on the SNARE/α-SNAP complex binding, the basal and the SNARE/α-SNAP stimulated ATPase activity, suggesting this region may be involved in the motion transmission from D1 to N domain. Mutating the residues in middle and C-terminal region of the N-D1 linker increases the basal ATPase activity, indicating it may play a role in autoinhibiting NSF activity until it encounters SNARE/α-SNAP complex substrate. Moreover, mutations at the C-terminal sequence GIGG exhibit completely abolished or severely reduced activities of the substrate binding, suggesting that the flexibility of N-D1 linker is critical for the movement of the N domain that is required for the substrate binding. Taken together, these data suggest that the whole N-D1 linker is critical for the biological function of NSF to disassemble SNARE complex substrate with different regions responsible for different roles.

  10. A library of 7TM receptor C-terminal tails. Interactions with the proposed post-endocytic sorting proteins ERM-binding phosphoprotein 50 (EBP50), N-ethylmaleimide-sensitive factor (NSF), sorting nexin 1 (SNX1), and G protein-coupled receptor-associated sorting protein (GASP)

    DEFF Research Database (Denmark)

    Heydorn, Arne; Søndergaard, Birgitte P; Ersbøll, Bjarne

    2004-01-01

    Adaptor and scaffolding proteins determine the cellular targeting, the spatial, and thereby the functional association of G protein-coupled seven-transmembrane receptors with co-receptors, transducers, and downstream effectors and the adaptors determine post-signaling events such as receptor...... sequestration through interactions, mainly with the C-terminal intracellular tails of the receptors. A library of tails from 59 representative members of the super family of seven-transmembrane receptors was probed as glutathione S-transferase fusion proteins for interactions with four different adaptor...... that the tail library provides useful information on the general importance of certain adaptor proteins, for example, in this case, ruling out EBP50 as being a broad spectrum-recycling adaptor....

  11. Munc18-1 and Munc18-2 Proteins Modulate beta-Cell Ca2+ Sensitivity and Kinetics of Insulin Exocytosis Differently

    OpenAIRE

    Mandic, Slavena A.; Skelin, Masa; Johansson, Jenny U.; Rupnik, Marjan S.; Berggren, Per-Olof; Bark, Christina

    2011-01-01

    Fast neurotransmission and slower hormone release share the same core fusion machinery consisting of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins. In evoked neurotransmission, interactions between SNAREs and the Munc18-1 protein, a member of the Sec1/Munc18 (SM) protein family, are essential for exocytosis, whereas other SM proteins are dispensable. To address if the exclusivity of Munc18-1 demonstrated in neuroexocytosis also applied to fast insulin ...

  12. The maintenance of long-term memory in the hippocampus depends on the interaction between N-ethylmaleimide-sensitive factor and GluA2.

    Science.gov (United States)

    Migues, Paola Virginia; Hardt, Oliver; Finnie, Peter; Wang, Yu Wang; Nader, Karim

    2014-09-01

    The maintenance of established memories has recently been shown to involve the stabilization of GluA2-containing AMPA receptors (GluA2/AMPARs) at postsynaptic membranes. Previous studies have suggested that N-ethylmaleimide-sensitive factor (NSF) regulates the stabilization of AMPARs at the synaptic membrane. We therefore disrupted the interaction between GluA2 and NSF in the dorsal hippocampus and examined its effect on the maintenance of object location and contextual fear memory. We used two interference peptides, pep2m and pepR845A, that have been shown to block the binding of NSF to GluA2 and reduce GluA2 synaptic content. Either peptide disrupted consolidated memory, and these effects persisted for at least 5 or 28 days after peptide administration. Following peptide administration and long-term memory disruption, rats were able to acquire new memories. Memory acquisition or consolidation was not impaired when pepR845A was given immediately before the training sessions. Blocking GluA2 endocytosis with the peptide GluA23Y prevented the memory impairment effect of pepR845A. Taken together, our results indicate that the persistence of long-term memory depends on the maintenance of a steady-state level of synaptic GluA2/AMPARs, which requires the interaction of NSF with GluA2. © 2014 Wiley Periodicals, Inc.

  13. α-SNAP Interferes with the Zippering of the SNARE Protein Membrane Fusion Machinery

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    Park, Yongsoo; Vennekate, Wensi; Yavuz, Halenur; Preobraschenski, Julia; Hernandez, Javier M.; Riedel, Dietmar; Walla, Peter Jomo; Jahn, Reinhard

    2014-01-01

    Neuronal exocytosis is mediated by soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins. Before fusion, SNARE proteins form complexes bridging the membrane followed by assembly toward the C-terminal membrane anchors, thus initiating membrane fusion. After fusion, the SNARE complex is disassembled by the AAA-ATPase N-ethylmaleimide-sensitive factor that requires the cofactor α-SNAP to first bind to the assembled SNARE complex. Using chromaffin granules and liposomes we now show that α-SNAP on its own interferes with the zippering of membrane-anchored SNARE complexes midway through the zippering reaction, arresting SNAREs in a partially assembled trans-complex and preventing fusion. Intriguingly, the interference does not result in an inhibitory effect on synaptic vesicles, suggesting that membrane properties also influence the final outcome of α-SNAP interference with SNARE zippering. We suggest that binding of α-SNAP to the SNARE complex affects the ability of the SNARE complex to harness energy or transmit force to the membrane. PMID:24778182

  14. Exocytosis proteins as novel targets for diabetes prevention and/or remediation?

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    Aslamy, Arianne; Thurmond, Debbie C

    2017-05-01

    Diabetes remains one of the leading causes of morbidity and mortality worldwide, affecting an estimated 422 million adults. In the US, it is predicted that one in every three children born as of 2000 will suffer from diabetes in their lifetime. Type 2 diabetes results from combinatorial defects in pancreatic β-cell glucose-stimulated insulin secretion and in peripheral glucose uptake. Both processes, insulin secretion and glucose uptake, are mediated by exocytosis proteins, SNARE (soluble N -ethylmaleimide-sensitive factor attachment protein receptor) complexes, Sec1/Munc18 (SM), and double C2-domain protein B (DOC2B). Increasing evidence links deficiencies in these exocytosis proteins to diabetes in rodents and humans. Given this, emerging studies aimed at restoring and/or enhancing cellular levels of certain exocytosis proteins point to promising outcomes in maintaining functional β-cell mass and enhancing insulin sensitivity. In doing so, new evidence also shows that enhancing exocytosis protein levels may promote health span and longevity and may also harbor anti-cancer and anti-Alzheimer's disease capabilities. Herein, we present a comprehensive review of the described capabilities of certain exocytosis proteins and how these might be targeted for improving metabolic dysregulation. Copyright © 2017 the American Physiological Society.

  15. Discrete and continuous models of protein sorting in the Golgi

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    Gong, Haijun; Schwartz, Russell

    2009-03-01

    The Golgi apparatus plays an important role in processing and sorting proteins and lipids. Golgi compartments constantly exchange material with each other and with other cellular components, allowing them to maintain and reform distinct identities despite dramatic changes in structure and size during cell division, development and osmotic stress. We have developed two minimal models of membrane and protein exchange in the Golgi --- a discrete, stochastic model [1] and a continuous ordinary differential equation (ODE) model --- both based on two fundamental mechanisms: vesicle-coat-mediated selective concentration of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins during vesicle formation and SNARE-mediated selective fusion of vesicles. Both show similar ability to establish and maintain distinct identities over broad parameter ranges, but they diverge in extreme conditions where Golgi collapse and reassembly may be observed. By exploring where the models differ, we hope to better identify those features essential to minimal models of various Golgi behaviors. [1] H. Gong, D. Sengupta, A. D. Linstedt, R. Schwartz. Biophys J. 95: 1674-1688, 2008.

  16. Discrete, continuous, and stochastic models of protein sorting in the Golgi apparatus

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    Gong, Haijun; Guo, Yusong; Linstedt, Adam; Schwartz, Russell

    2010-01-01

    The Golgi apparatus plays a central role in processing and sorting proteins and lipids in eukaryotic cells. Golgi compartments constantly exchange material with each other and with other cellular components, allowing them to maintain and reform distinct identities despite dramatic changes in structure and size during cell division, development, and osmotic stress. We have developed three minimal models of membrane and protein exchange in the Golgi—a discrete, stochastic model, a continuous ordinary differential equation model, and a continuous stochastic differential equation model—each based on two fundamental mechanisms: vesicle-coat-mediated selective concentration of cargoes and soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins during vesicle formation and SNARE-mediated selective fusion of vesicles. By exploring where the models differ, we hope to discover whether the discrete, stochastic nature of vesicle-mediated transport is likely to have appreciable functional consequences for the Golgi. All three models show similar ability to restore and maintain distinct identities over broad parameter ranges. They diverge, however, in conditions corresponding to collapse and reassembly of the Golgi. The results suggest that a continuum model provides a good description of Golgi maintenance but that considering the discrete nature of vesicle-based traffic is important to understanding assembly and disassembly of the Golgi. Experimental analysis validates a prediction of the models that altering guanine nucleotide exchange factor expression levels will modulate Golgi size.

  17. Developmental and diurnal expression of the synaptosomal-associated protein 25 (Snap25) in the rat pineal gland.

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    Karlsen, Anna S; Rath, Martin F; Rohde, Kristian; Toft, Trine; Møller, Morten

    2013-06-01

    Snap25 (synaptosomal-associated protein) is a 25 kDa protein, belonging to the SNARE-family (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) of proteins, essential for synaptic and secretory vesicle exocytosis. Snap25 has by immunohistochemistry been demonstrated in the rat pineal gland but the biological importance of this is unknown. In this study, we demonstrate a high expression of mRNA encoding Snap25 in all parts of the rat pineal complex, the superficial-, and deep-pineal gland, as well as in the pineal stalk. Snap25 showed a low pineal expression during embryonic stages with a strong increase in expression levels just after birth. The expression showed no day/night variations. Neither removal of the sympathetic input to the pineal gland by superior cervical ganglionectomy nor bilateral decentralization of the superior cervical ganglia significantly affected the expression of Snap25 in the gland. The pineal expression levels of Snap25 were not changed following intraperitoneal injection of isoproterenol. The strong expression of Snap25 in the pineal gland suggests the presence of secretory granules and microvesicles in the rat pinealocyte supporting the concept of a vesicular release. At the transcriptional level, this Snap25-based release mechanism does not exhibit any diurnal rhythmicity and is regulated independently of the sympathetic nervous input to the gland.

  18. SNARE Requirements En Route to Exocytosis

    DEFF Research Database (Denmark)

    Mohrmann, Ralf; Sørensen, Jakob Balslev

    2012-01-01

    Although it has been known for almost two decades that the ternary complex of N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) constitutes the functional unit driving membrane fusion, our knowledge about the dynamical arrangement and organization of SNARE proteins and their......Although it has been known for almost two decades that the ternary complex of N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) constitutes the functional unit driving membrane fusion, our knowledge about the dynamical arrangement and organization of SNARE proteins...

  19. 2BC Non-Structural Protein of Enterovirus A71 Interacts with SNARE Proteins to Trigger Autolysosome Formation

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    Jeffrey K. F. Lai

    2017-07-01

    Full Text Available Viruses have evolved unique strategies to evade or subvert autophagy machinery. Enterovirus A71 (EV-A71 induces autophagy during infection in vitro and in vivo. In this study, we report that EV-A71 triggers autolysosome formation during infection in human rhabdomyosarcoma (RD cells to facilitate its replication. Blocking autophagosome-lysosome fusion with chloroquine inhibited virus RNA replication, resulting in lower viral titres, viral RNA copies and viral proteins. Overexpression of the non-structural protein 2BC of EV-A71 induced autolysosome formation. Yeast 2-hybrid and co-affinity purification assays showed that 2BC physically and specifically interacted with a N-ethylmaleimide-sensitive factor attachment receptor (SNARE protein, syntaxin-17 (STX17. Co-immunoprecipitation assay further showed that 2BC binds to SNARE proteins, STX17 and synaptosome associated protein 29 (SNAP29. Transient knockdown of STX17, SNAP29, and microtubule-associated protein 1 light chain 3B (LC3B, crucial proteins in the fusion between autophagosomes and lysosomes as well as the lysosomal-associated membrane protein 1 (LAMP1 impaired production of infectious EV-A71 in RD cells. Collectively, these results demonstrate that the generation of autolysosomes triggered by the 2BC non-structural protein is important for EV-A71 replication, revealing a potential molecular pathway targeted by the virus to exploit autophagy. This study opens the possibility for the development of novel antivirals that specifically target 2BC to inhibit formation of autolysosomes during EV-A71 infection.

  20. Binary polypeptide system for permanent and oriented protein immobilization

    Directory of Open Access Journals (Sweden)

    Bailes Julian

    2010-05-01

    Full Text Available Abstract Background Many techniques in molecular biology, clinical diagnostics and biotechnology rely on binary affinity tags. The existing tags are based on either small molecules (e.g., biotin/streptavidin or glutathione/GST or peptide tags (FLAG, Myc, HA, Strep-tag and His-tag. Among these, the biotin-streptavidin system is most popular due to the nearly irreversible interaction of biotin with the tetrameric protein, streptavidin. The major drawback of the stable biotin-streptavidin system, however, is that neither of the two tags can be added to a protein of interest via recombinant means (except for the Strep-tag case leading to the requirement for chemical coupling. Results Here we report a new immobilization system which utilizes two monomeric polypeptides which self-assemble to produce non-covalent yet nearly irreversible complex which is stable in strong detergents, chaotropic agents, as well as in acids and alkali. Our system is based on the core region of the tetra-helical bundle known as the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex. This irreversible protein attachment system (IPAS uses either a shortened syntaxin helix and fused SNAP25-synaptobrevin or a fused syntaxin-synaptobrevin and SNAP25 allowing a two-component system suitable for recombinant protein tagging, capture and immobilization. We also show that IPAS is suitable for use with traditional beads and chromatography, planar surfaces and Biacore, gold nanoparticles and for protein-protein interaction in solution. Conclusions IPAS offers an alternative to chemical cross-linking, streptavidin-biotin system and to traditional peptide affinity tags and can be used for a wide range of applications in nanotechnology and molecular sciences.

  1. Binary polypeptide system for permanent and oriented protein immobilization.

    Science.gov (United States)

    Ferrari, Enrico; Darios, Frédéric; Zhang, Fan; Niranjan, Dhevahi; Bailes, Julian; Soloviev, Mikhail; Davletov, Bazbek

    2010-05-12

    Many techniques in molecular biology, clinical diagnostics and biotechnology rely on binary affinity tags. The existing tags are based on either small molecules (e.g., biotin/streptavidin or glutathione/GST) or peptide tags (FLAG, Myc, HA, Strep-tag and His-tag). Among these, the biotin-streptavidin system is most popular due to the nearly irreversible interaction of biotin with the tetrameric protein, streptavidin. The major drawback of the stable biotin-streptavidin system, however, is that neither of the two tags can be added to a protein of interest via recombinant means (except for the Strep-tag case) leading to the requirement for chemical coupling. Here we report a new immobilization system which utilizes two monomeric polypeptides which self-assemble to produce non-covalent yet nearly irreversible complex which is stable in strong detergents, chaotropic agents, as well as in acids and alkali. Our system is based on the core region of the tetra-helical bundle known as the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex. This irreversible protein attachment system (IPAS) uses either a shortened syntaxin helix and fused SNAP25-synaptobrevin or a fused syntaxin-synaptobrevin and SNAP25 allowing a two-component system suitable for recombinant protein tagging, capture and immobilization. We also show that IPAS is suitable for use with traditional beads and chromatography, planar surfaces and Biacore, gold nanoparticles and for protein-protein interaction in solution. IPAS offers an alternative to chemical cross-linking, streptavidin-biotin system and to traditional peptide affinity tags and can be used for a wide range of applications in nanotechnology and molecular sciences.

  2. Vesicle-associated Membrane Protein-2 (VAMP2) Mediates cAMP-stimulated Renin Release in Mouse Juxtaglomerular Cells*

    Science.gov (United States)

    Mendez, Mariela; Gross, Kenneth W.; Glenn, Sean T.; Garvin, Jeffrey L.; Carretero, Oscar A.

    2011-01-01

    Renin is essential for blood pressure control. Renin is stored in granules in juxtaglomerular (JG) cells, located in the pole of the renal afferent arterioles. The second messenger cAMP stimulates renin release. However, it is unclear whether fusion and exocytosis of renin-containing granules is involved. In addition, the role of the fusion proteins, SNAREs (soluble N-ethylmaleimide-sensitive factor attachment proteins), in renin release from JG cells has not been studied. The vesicle SNARE proteins VAMP2 (vesicle associated membrane protein 2) and VAMP3 mediate cAMP-stimulated exocytosis in other endocrine cells. Thus, we hypothesized that VAMP2 and/or -3 mediate cAMP-stimulated renin release from JG cells. By fluorescence-activated cell sorting, we isolated JG cells expressing green fluorescent protein and compared the relative abundance of VAMP2/3 in JG cells versus total mouse kidney mRNA by quantitative PCR. We found that VAMP2 and VAMP3 mRNA are expressed and enriched in JG cells. Confocal imaging of primary cultures of JG cells showed that VAMP2 (but not VAMP3) co-localized with renin-containing granules. Cleavage of VAMP2 and VAMP3 with tetanus toxin blocked cAMP-stimulated renin release from JG cells by ∼50% and impaired cAMP-stimulated exocytosis by ∼50%, as monitored with FM1–43. Then we specifically knocked down VAMP2 or VAMP3 by adenoviral-mediated delivery of short hairpin silencing RNA. We found that silencing VAMP2 blocked cAMP-induced renin release by ∼50%. In contrast, silencing VAMP3 had no effect on basal or cAMP-stimulated renin release. We conclude that VAMP2 and VAMP3 are expressed in JG cells, but only VAMP2 is targeted to renin-containing granules and mediates the stimulatory effect of cAMP on renin exocytosis. PMID:21708949

  3. Role of the SNARE protein SNAP23 on cAMP-stimulated renin release in mouse juxtaglomerular cells

    Science.gov (United States)

    Gaisano, Herbert Y.

    2013-01-01

    Renin, the rate-limiting enzyme in the formation of angiotensin II, is synthesized and stored in granules in juxtaglomerular (JG) cells. Therefore, the controlled mechanism involved in renin release is essential for the regulation of blood pressure. Exocytosis of renin-containing granules is likely involved in renin release; a process stimulated by cAMP. We found that the “soluble NSF (N-ethylmaleimide-sensitive factor) attachment protein receptor” (SNARE) protein VAMP2 mediates cAMP-stimulated renin release and exocytosis in JG cells. To mediate exocytosis, VAMP2 must interact with a synaptosome-associated protein (SNAP). In the renal cortex, the isoform SNAP23 is abundantly expressed. We hypothesized that SNAP23 mediates cAMP-stimulated renin release from primary cultures of mouse JG cells. We found that SNAP23 protein is expressed and colocalized with renin-containing granules in primary cultures of mouse JG cell lysates. Thus, we then tested the involvement of SNAP23 in cAMP-stimulated renin release by transducing JG cells with a dominant-negative SNAP23 construct. In control JG cells transduced with a scrambled sequence, increasing cAMP stimulated renin release from 1.3 ± 0.3 to 5.3 ± 1.2% of renin content. In cells transduced with dominant-negative SNAP23, cAMP increased renin from 1.0 ± 0.1 to 3.0 ± 0.6% of renin content, a 50% blockade. Botulinum toxin E, which cleaves and inactivates SNAP23, reduced cAMP-stimulated renin release by 42 ± 17%. Finally, adenovirus-mediated silencing of SNAP23 significantly blocked cAMP-stimulated renin release by 50 ± 13%. We concluded that the SNARE protein SNAP23 mediates cAMP-stimulated renin release. These data show that renin release is a SNARE-dependent process. PMID:23269646

  4. The yeast vacuolar Rab GTPase Ypt7p has an activity beyond membrane recruitment of the homotypic fusion and protein sorting-Class C Vps complex.

    Science.gov (United States)

    Stroupe, Christopher

    2012-04-01

    A previous report described lipid mixing of reconstituted proteoliposomes made using lipid mixtures that mimic the composition of yeast vacuoles. This lipid mixing required SNARE {SNAP [soluble NSF (N-ethylmaleimide-sensitive factor)-attachment protein] receptor} proteins, Sec18p and Sec17p (yeast NSF and α-SNAP) and the HOPS (homotypic fusion and protein sorting)-Class C Vps (vacuole protein sorting) complex, but not the vacuolar Rab GTPase Ypt7p. The present study investigates the activity of Ypt7p in proteoliposome lipid mixing. Ypt7p is required for the lipid mixing of proteoliposomes lacking cardiolipin [1,3-bis-(sn-3'-phosphatidyl)-sn-glycerol]. Omission of other lipids with negatively charged and/or small head groups does not cause Ypt7p dependence for lipid mixing. Yeast vacuoles made from strains disrupted for CRD1 (cardiolipin synthase) fuse to the same extent as vacuoles from strains with functional CRD1. Disruption of CRD1 does not alter dependence on Rab GTPases for vacuole fusion. It has been proposed that the recruitment of the HOPS complex to membranes is the main function of Ypt7p. However, Ypt7p is still required for lipid mixing even when the concentration of HOPS complex in lipid-mixing reactions is adjusted such that cardiolipin-free proteoliposomes with or without Ypt7p bind to equal amounts of HOPS. Ypt7p therefore must stimulate membrane fusion by a mechanism that is in addition to recruitment of HOPS to the membrane. This is the first demonstration of such a stimulatory activity--that is, beyond bulk effector recruitment--for a Rab GTPase.

  5. GsSNAP33, a novel Glycine soja SNAP25-type protein gene: Improvement of plant salt and drought tolerances in transgenic Arabidopsis thaliana.

    Science.gov (United States)

    Nisa, Zaib-Un; Mallano, Ali Inayat; Yu, Yang; Chen, Chao; Duan, Xiangbo; Amanullah, Sikandar; Kousar, Abida; Baloch, Abdul Wahid; Sun, Xiaoli; Tabys, Dina; Zhu, Yanming

    2017-10-01

    The N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) superfamily, specifically the SNAP25-type proteins and t-SNAREs, have been proposed to regulate cellular processes and plant resistance mechanisms. However, little is known about the role of SNAP25-type proteins in combating abiotic stresses, specifically in wild soybean. In the current study, the isolation and functional characterization of the putative synaptosomal-associated SNAP25-type protein gene GsSNAP33 from wild soybean (Glycine soja) were performed. GsSNAP33 has a molecular weight of 33,311 Da and comprises 300 amino acid residues along with Qb-Qc SNARE domains. Multiple sequence alignment revealed the highest similarity of the GsSNAP33 protein to GmSNAP33 (91%), VrSNAP33 (89%), PvSNAP33 (86%) and AtSNAP33 (63%). Phylogenetic studies revealed the abundance of SNAP33 proteins mostly in dicotyledons. Quantitative real-time PCR assays confirmed that GsSNAP33 expression can be induced by salt, alkali, ABA and PEG treatments and that GsSNAP33 is highly expressed in the pods, seeds and roots of Glycine soja. Furthermore, the overexpression of the GsSNAP33 gene in WT Arabidopsis thaliana resulted in increased germination rates, greater root lengths, improved photosynthesis, lower electrolyte leakage, higher biomass production and up-regulated expression levels of various stress-responsive marker genes, including KINI, COR15A, P5Cs, RAB18, RD29A and COR47 in transgenic lines compared with those in WT lines. Subcellular localization studies revealed that the GsSNAP33-eGFP fusion protein was localized to the plasma membrane, while eGFP was distributed throughout whole cytoplasm of onion epidermal cells. Collectively, our findings suggest that GsSNAP33, a novel plasma membrane protein gene of Glycine soja, might be involved in improving plant responses to salt and drought stresses in Arabidopsis. Copyright © 2017. Published by Elsevier Masson SAS.

  6. Alternative splicing of the human gene SYBL1 modulates protein domain architecture of longin VAMP7/TI-VAMP, showing both non-SNARE and synaptobrevin-like isoforms

    Directory of Open Access Journals (Sweden)

    De Franceschi Nicola

    2011-05-01

    Full Text Available Abstract Background The control of intracellular vesicle trafficking is an ideal target to weigh the role of alternative splicing in shaping genomes to make cells. Alternative splicing has been reported for several Soluble N-ethylmaleimide-sensitive factor Attachment protein REceptors of the vesicle (v-SNAREs or of the target membrane (t-SNARES, which are crucial to intracellular membrane fusion and protein and lipid traffic in Eukaryotes. However, splicing has not yet been investigated in Longins, i.e. the most widespread v-SNAREs. Longins are essential in Eukaryotes and prototyped by VAMP7, Sec22b and Ykt6, sharing a conserved N-terminal Longin domain which regulates membrane fusion and subcellular targeting. Human VAMP7/TI-VAMP, encoded by gene SYBL1, is involved in multiple cell pathways, including control of neurite outgrowth. Results Alternative splicing of SYBL1 by exon skipping events results in the production of a number of VAMP7 isoforms. In-frame or frameshift coding sequence modifications modulate domain architecture of VAMP7 isoforms, which can lack whole domains or domain fragments and show variant or extra domains. Intriguingly, two main types of VAMP7 isoforms either share the inhibitory Longin domain and lack the fusion-promoting SNARE motif, or vice versa. Expression analysis in different tissues and cell lines, quantitative real time RT-PCR and confocal microscopy analysis of fluorescent protein-tagged isoforms demonstrate that VAMP7 variants have different tissue specificities and subcellular localizations. Moreover, design and use of isoform-specific antibodies provided preliminary evidence for the existence of splice variants at the protein level. Conclusions Previous evidence on VAMP7 suggests inhibitory functions for the Longin domain and fusion/growth promoting activity for the Δ-longin molecule. Thus, non-SNARE isoforms with Longin domain and non-longin SNARE isoforms might have somehow opposite regulatory functions

  7. R-SNARE ykt6 resides in membrane-associated protease-resistant protein particles and modulates cell cycle progression when over-expressed.

    Science.gov (United States)

    Thayanidhi, Nandhakumar; Liang, Yingjian; Hasegawa, Haruki; Nycz, Deborah C; Oorschot, Viola; Klumperman, Judith; Hay, Jesse C

    2012-07-01

    The arginine-type soluble N-ethylmaleimide-sensitive factor attachment protein receptor (R-SNARE) ykt6 possesses several atypical properties including selective high expression in neurons, a lipidated C-terminus, localization to punctae that do not correspond with known endomembrane markers, a potent ability to protect the secretory pathway from alpha-synuclein over-expression and specific up-regulation in tumors. We have followed up on several of these features that together suggest nontraditional SNARE structures and functions. A significant portion of ykt6 in PC12 cells was found in a protease-resistant state suggestive of a large complex or aggregate. Other endoplasmic reticulum/Golgi SNAREs were not protease resistant, demonstrating that SNARE complexes per se did not cause protease resistance. Mutagenesis indicated that lipidation of the ykt6 C-terminus was also not involved, implicating its longin domain in particle formation. Immunogold electron microscopy revealed ykt6 labeling of ∼100 nm electron densities associated with diverse membranes. Density gradient analysis of the protease-resistant structures confirmed their tight association with membranes. Since excess ykt6 has been correlated with tumorigenesis, we tested whether ykt6 over-expression in normal rat kidney cells that normally express little ykt6 affected the cell cycle. Ykt6 over-expression was found to result in altered cell division cycles as evidenced by significantly smaller cells, a higher mitotic index and increased DNA synthesis. Mutagenesis studies dis-correlated SNARE function with the cell cycle effects; instead, the cell cycle effects correlated better with ykt6 properties related to the longin domain or particle formation. The ykt6 particles/aggregates may represent ykt6 engaged in a non-SNARE function(s) or else nonfunctional, stored and/or excess ykt6. Whether the particulate ykt6 structures represent a means of buffering the apparent proliferative activity or are in fact

  8. SNARE-Mediated Cholesterol Movement to Mitochondria Supports Steroidogenesis in Rodent Cells

    Science.gov (United States)

    Lin, Ye; Hou, Xiaoming; Shen, Wen-Jun; Hanssen, Ruth; Khor, Victor K.; Cortez, Yuan; Roseman, Ann N.; Azhar, Salman

    2016-01-01

    Vesicular transport involving soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) proteins is known to be responsible for many major cellular activities. In steroidogenic tissues, chronic hormone stimulation results in increased expression of proteins involved in the steroidogenic pathway, whereas acute hormone stimulation prompts the rapid transfer of cholesterol to the inner mitochondrial membrane to be utilized as substrate for steroid hormone production. Several different pathways are involved in supplying cholesterol to mitochondria, but mobilization of stored cholesteryl esters appears to initially constitute the preferred source; however, the mechanisms mediating this cholesterol transfer are not fully understood. To study the potential contribution of SNARE proteins in steroidogenesis, we examined the expression levels of various SNARE proteins in response to hormone stimulation in steroidogenic tissues and cells and established an in vitro mitochondria reconstitution assay system to assess the contribution of various SNARE proteins on cholesterol delivery for steroidogenesis. Our results from reconstitution experiments along with knockdown studies in rat primary granulosa cells and in a Leydig cell line show that soluble N-ethylmaleimide sensitive factor attachment protein-α, synaptosomal-associated protein of 25 kDa, syntaxin-5, and syntaxin-17 facilitate the transport of cholesterol to mitochondria. Thus, although StAR is required for efficient cholesterol movement into mitochondria for steroidogenesis, specific SNAREs participate and are necessary to mediate cholesterol movement to mitochondria. PMID:26771535

  9. Interactions between N-Ethylmaleimide-sensitive factor and GluA2 contribute to effects of glucocorticoid hormones on AMPA receptor function in the rodent hippocampus.

    NARCIS (Netherlands)

    Xiong, H.; Cassé, F.; Zhou, M.; Xiong, Z.Q.; Joels, M.; Martin, S.; Krugers, H.J.

    2016-01-01

    Glucocorticoid hormones, via activation of their receptors, promote memory consolidation, but the exact underlying mechanisms remain elusive. We examined how corticosterone regulates AMPA receptor (AMPAR) availability in the synapse, which is important for synaptic plasticity and memory formation.

  10. Interactions between N-Ethylmaleimide-Sensitive Factor and GluA2 contribute to effects of glucocorticoid hormones on AMPA receptor function in the rodent hippocampus

    NARCIS (Netherlands)

    Xiong, Hui; Cassé, Frédéric; Zhou, Ming; Xiong, Zhi-Qi; Joels, Marian; Martin, Stéphane; Krugers, Harm J

    Glucocorticoid hormones, via activation of their receptors, promote memory consolidation, but the exact underlying mechanisms remain elusive. We examined how corticosterone regulates AMPA receptor (AMPAR) availability in the synapse, which is important for synaptic plasticity and memory formation.

  11. Engineering of vesicle trafficking improves heterologous protein secretion in Saccharomyces cerevisiae.

    Science.gov (United States)

    Hou, Jin; Tyo, Keith; Liu, Zihe; Petranovic, Dina; Nielsen, Jens

    2012-03-01

    The yeast Saccharomyces cerevisiae is a widely used platform for the production of heterologous proteins of medical or industrial interest. However, heterologous protein productivity is often restricted due to the limitations of the host strain. In the protein secretory pathway, the protein trafficking between different organelles is catalyzed by the soluble NSF (N-ethylmaleimide-sensitive factor) receptor (SNARE) complex and regulated by the Sec1/Munc18 (SM) proteins. In this study, we report that over-expression of the SM protein encoding genes SEC1 and SLY1, improves the protein secretion in S. cerevisiae. Engineering Sec1p, the SM protein that is involved in vesicle trafficking from Golgi to cell membrane, improves the secretion of heterologous proteins human insulin precursor and α-amylase, and also the secretion of an endogenous protein invertase. Enhancing Sly1p, the SM protein regulating the vesicle fusion from endoplasmic reticulum (ER) to Golgi, increases α-amylase production only. Our study demonstrates that strengthening the protein trafficking in ER-to-Golgi and Golgi-to-plasma membrane process is a novel secretory engineering strategy for improving heterologous protein production in S. cerevisiae. Copyright © 2012 Elsevier Inc. All rights reserved.

  12. The proteins of exocytosis: lessons from the sperm model.

    Science.gov (United States)

    Tomes, Claudia Nora

    2015-02-01

    Exocytosis is a highly regulated process that consists of multiple functionally, kinetically and/or morphologically definable stages such as recruitment, targeting, tethering and docking of secretory vesicles with the plasma membrane, priming of the fusion machinery and calcium-triggered membrane fusion. After fusion, the membrane around the secretory vesicle is incorporated into the plasma membrane and the granule releases its contents. The proteins involved in these processes belong to several highly conserved families: Rab GTPases, SNAREs (soluble NSF-attachment protein receptors), α-SNAP (α-NSF attachment protein), NSF (N-ethylmaleimide-sensitive factor), Munc13 and -18, complexins and synaptotagmins. In the present article, the molecules of exocytosis are reviewed, using human sperm as a model system. Sperm exocytosis is driven by isoforms of the same proteinaceous fusion machinery mentioned above, with their functions orchestrated in a hierarchically organized and unidirectional signalling cascade. In addition to the universal exocytosis regulator calcium, this cascade includes other second messengers such as diacylglycerol, inositol 1,4,5-trisphosphate and cAMP, as well as the enzymes that synthesize them and their target proteins. Of special interest is the cAMP-binding protein Epac (exchange protein directly activated by cAMP) due in part to its enzymatic activity towards Rap. The activation of Epac and Rap leads to a highly localized calcium signal which, together with assembly of the SNARE complex, governs the final stages of exocytosis. The source of this releasable calcium is the secretory granule itself.

  13. Immunohistochemical localization of SNARE core proteins in intrapulpal and intradentinal nerve fibers of rat molar teeth.

    Science.gov (United States)

    Honma, Shiho; Kadono, Kohki; Kawano, Akiyo; Wakisaka, Satoshi

    2017-01-01

    The present study was designed to elucidate whether three soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE) core proteins, syntaxin-1, synaptosomal-associated protein of 25kDa (SNAP-25), and vesicle-associated membrane protein-2 (VAMP-2), are present in the dental pulp of the rat molar at both the light and electron microscopic levels. Immunohistochemistry for protein gene product 9.5 (PGP 9.5), a pan-neuronal marker, syntaxin-1, SNAP-25, and VAMP-2 was performed on decalcified rat molars for light and electron microscopic analyses. Double-immunolabeling of PGP 9.5 and the SNARE core proteins, as well as combinations of the SNARE core proteins, was also carried out. PGP 9.5-immunoreactive nerve fibers ran toward the coronal region, ramified at the subodontoblast layer, and formed the subodontoblastic nerve plexus. Most nerve fibers penetrated the predentin and dentin along the dentinal tubules. Most, if not all, nerve fibers displayed immunoreactivity for syntaxin-1, SNAP-25, and VAMP-2. Immunoelectron microscopic analyses confirmed the presence of immunoreactivity for the SNARE core proteins within the intradental axonal elements. The present findings suggest that, since SNARE core proteins participate in the docking and exocytosis of synaptic vesicles in the central nervous system, they may contribute to vesicle exocytosis from the dental nerve fibers even though there are no apparent synapses. Copyright © 2016 Elsevier Ltd. All rights reserved.

  14. γ-SNAP stimulates disassembly of endosomal SNARE complexes and regulates endocytic trafficking pathways.

    Science.gov (United States)

    Inoue, Hiroki; Matsuzaki, Yuka; Tanaka, Ayaka; Hosoi, Kaori; Ichimura, Kaoru; Arasaki, Kohei; Wakana, Yuichi; Asano, Kenichi; Tanaka, Masato; Okuzaki, Daisuke; Yamamoto, Akitsugu; Tani, Katsuko; Tagaya, Mitsuo

    2015-08-01

    Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) that reside in the target membranes and transport vesicles assemble into specific SNARE complexes to drive membrane fusion. N-ethylmaleimide-sensitive factor (NSF) and its attachment protein, α-SNAP (encoded by NAPA), catalyze disassembly of the SNARE complexes in the secretory and endocytic pathways to recycle them for the next round of fusion events. γ-SNAP (encoded by NAPG) is a SNAP isoform, but its function in SNARE-mediated membrane trafficking remains unknown. Here, we show that γ-SNAP regulates the endosomal trafficking of epidermal growth factor (EGF) receptor (EGFR) and transferrin. Immunoprecipitation and mass spectrometry analyses revealed that γ-SNAP interacts with a limited range of SNAREs, including endosomal ones. γ-SNAP, as well as α-SNAP, mediated the disassembly of endosomal syntaxin-7-containing SNARE complexes. Overexpression and small interfering (si)RNA-mediated depletion of γ-SNAP changed the morphologies and intracellular distributions of endosomes. Moreover, the depletion partially suppressed the exit of EGFR and transferrin from EEA1-positive early endosomes to delay their degradation and uptake. Taken together, our findings suggest that γ-SNAP is a unique SNAP that functions in a limited range of organelles - including endosomes - and their trafficking pathways. © 2015. Published by The Company of Biologists Ltd.

  15. Canine Salivary Glands: Analysis of Rab and SNARE Protein Expression and SNARE Complex Formation With Diverse Tissue Properties.

    Science.gov (United States)

    Gomi, Hiroshi; Osawa, Hiromi; Uno, Rie; Yasui, Tadashi; Hosaka, Masahiro; Torii, Seiji; Tsukise, Azuma

    2017-11-01

    The comparative structure and expression of salivary components and vesicular transport proteins in the canine major salivary glands were investigated. Histochemical analysis revealed that the morphology of the five major salivary glands-parotid, submandibular, polystomatic sublingual, monostomatic sublingual, and zygomatic glands-was greatly diverse. Immunoblot analysis revealed that expression levels of α-amylase and antimicrobial proteins, such as lysozyme, lactoperoxidase, and lactoferrin, differed among the different glands. Similarly, Rab proteins (Rab3d, Rab11a, Rab11b, Rab27a, and Rab27b) and soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE) proteins VAMP4, VAMP8, syntaxin-2, syntaxin-3, syntaxin-4, and syntaxin-6 were expressed at various levels in individual glands. mmunohistochemistry of Rab3d, Rab11b, Rab27b, VAMP4, VAMP8, syntaxin-4, and syntaxin-6 revealed their predominant expression in serous acinar cells, demilunes, and ductal cells. The VAMP4/syntaxin-6 SNARE complex, which is thought to be involved in the maturation of secretory granules in the Golgi field, was found more predominantly in the monostomatic sublingual gland than in the parotid gland. These results suggest that protein expression profiles in canine salivary glands differ among individual glands and reflect the properties of their specialized functions.

  16. Differential expression of synaptic proteins in unilateral 6-OHDA lesioned rat model-A comparative proteomics approach.

    Science.gov (United States)

    Xiong, Yan; Zhang, Yongqian; Iqbal, Javed; Ke, Ming; Wang, Yun; Li, Yujuan; Qing, Hong; Deng, Yulin

    2014-08-01

    Parkinson's disease (PD) is characterized as a movement disorder due to lesions in the basal ganglia. As the major input region of the basal ganglia, striatum plays a vital role in coordinating movements. It receives afferents from the cerebral cortex and projects afferents to the internal segment of the globus pallidus and substantia nigra pars reticulate. Additionally, accumulating evidences support a role for synaptic dysfunction in PD. Therefore, the present study explores the changes in protein abundance involved in synaptic disorders in unilateral lesioned 6-OHDA rat model. Based on (18) O/(16) O-labeling technique, striatal proteins were separated using online 2D-LC, and identified by nano-ESI-quadrupole-TOF. A total of 370 proteins were identified, including 76 significantly differentially expressed proteins. Twenty-two downregulated proteins were found in composition of vesicle, ten of which were involved in neuronal transmission and recycling across synapses. These include N-ethylmaleimide-sensitive fusion protein attachment receptor proteins (SNAP-25, syntaxin-1A, syntaxin-1B, VAMP2), synapsin-1, septin-5, clathrin heavy chain 1, AP-2 complex subunit beta, dynamin-1, and endophilin-A1. Moreover, MS result for syntaxin-1A was confirmed by Western blot analysis. Overall, these synaptic changes induced by neurotoxin may serve as a reference for understanding the functional mechanism of striatum in PD. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. The Structure of the Yeast Plasma Membrane SNARE Complex Reveals Destabilizing Water Filled Cavities

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    Strop, P.; Kaiser, S.E.; Vrljic, M.; Brunger, A.T.

    2009-05-26

    Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins form a complex that leads to membrane fusion between vesicles, organelles, and plasma membrane in all eukaryotic cells. We report the 1.7{angstrom} resolution structure of the SNARE complex that mediates exocytosis at the plasma membrane in the yeast Saccharomyces cerevisiae. Similar to its neuronal and endosomal homologues, the S. cerevisiae SNARE complex forms a parallel four-helix bundle in the center of which is an ionic layer. The S. cerevisiae SNARE complex exhibits increased helix bending near the ionic layer, contains water-filled cavities in the complex core, and exhibits reduced thermal stability relative to mammalian SNARE complexes. Mutagenesis experiments suggest that the water-filled cavities contribute to the lower stability of the S. cerevisiae complex.

  18. Rapid identification of human SNAP-25 transcript variants by a miniaturized capillary electrophoresis system.

    Science.gov (United States)

    Németh, Nóra; Kerékgyártó, Márta; Sasvári-Székely, Mária; Rónai, Zsolt; Guttman, András

    2014-02-01

    The 25 kDa synaptosomal-associated protein (SNAP-25) is a crucial component of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex and plays an important role in neurotransmission in the central nervous system. SNAP-25 has two different splice variants, SNAP-25a and SNAP-25b, differing in nine amino acids that results in a slight functional alteration of the generated soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex. Two independent techniques, a PCR-miniaturized CE method and a real-time PCR based approach were elaborated for the specific and quantitative detection of the two SNAP-25 transcription variants. DNA-constructs coding for the two isoforms were used for optimization. Excellent specificity was observed with the use of our previously described highly sensitive miniaturized CE system in combination with quantitative PCR. The ratio of the two isoforms were reliably detected in a range of at least four orders of magnitude with a linear regression of R(2) = 0.987. Expression of the two isoforms was determined in human samples, where SNAP-25 was detected even in non-neural tissues, although at approximately a 100-fold lower level compared to the central nervous system. The relative amount of the SNAP-25b isoform was higher in the brain, whereas expression of SNAP-25a variant proved to be slightly higher in extra-neural cell types. The genomics approach in conjunction with the miniaturized CE system introduced in this paper is readily applicable for rapid alternative splice variant analysis. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. The Vtc proteins in vacuole fusion: coupling NSF activity to V(0) trans-complex formation

    DEFF Research Database (Denmark)

    Müller, Oliver; Bayer, Martin J; Peters, Christopher

    2002-01-01

    The fusion of cellular membranes comprises several steps; membrane attachment requires priming of SNAREs and tethering factors by Sec18p/NSF (N-ethylmaleimide sensitive factor) and LMA1. This leads to trans-SNARE pairing, i.e. formation of SNARE complexes between apposed membranes. The yeast...

  20. Characterization of SNARE proteins in human pituitary adenomas: targeted secretion inhibitors as a new strategy for the treatment of acromegaly?

    Science.gov (United States)

    Garcia, Edwin A; Trivellin, Giampaolo; Aflorei, Elena D; Powell, Michael; Grieve, Joana; Alusi, Ghassan; Pobereskin, Luis; Shariati, Babak; Cudlip, Simon; Roncaroli, Federico; Mendoza, Nigel; Grossman, Ashley B; Harper, Elaine A; Korbonits, Márta

    2013-12-01

    Targeted secretion inhibitors (TSIs), a new class of recombinant biotherapeutic proteins engineered from botulinum toxin, represent a novel approach for treating diseases with excess secretion. They inhibit hormone secretion from targeted cell types through cleavage of SNARE (soluble N-ethylmaleimide-sensitive factor-activating protein receptor) proteins. qGHRH-LH(N)/D is a TSI targeting pituitary somatotroph through binding to the GHRH-receptor and cleavage of the vesicle-associated membrane protein (VAMP) family of SNARE proteins. Our objective was to study SNARE protein expression in pituitary adenomas and to inhibit GH secretion from somatotropinomas using qGHRH-LH(N)/D. We analyzed human pituitary adenoma analysis for SNARE expression and response to qGHRH-LH(N)/D treatment. The study was conducted in University Hospitals. We used pituitary adenoma samples from 25 acromegaly and 47 nonfunctioning pituitary adenoma patients. Vesicle-SNARE (VAMP1-3), target-SNARE (syntaxin1, SNAP-23, and SNAP-25), and GHRH-receptor detection with RT-qPCR, immunocytochemistry, and immunoblotting. Assessment of TSI catalytic activity on VAMPs and release of GH from adenoma cells. SNARE proteins were variably expressed in pituitary samples. In vitro evidence using recombinant GFP-VAMP2&3 or pituitary adenoma lysates suggested sufficient catalytic activity of qGHRH-LH(N)/D to degrade VAMPs, but was unable to inhibit GH secretion in somatotropinoma cell cultures. SNARE proteins are present in human pituitary somatotroph adenomas that can be targeted by TSIs to inhibit GH secretion. qGHRH-LH(N)/D was unable to inhibit GH secretion from human somatotroph adenoma cells. Further studies are required to understand how the SNARE proteins drive GH secretion in human somatotrophs to allow the development of novel TSIs with a potential therapeutic benefit.

  1. The interaction of mammalian Class C Vps with nSec-1/Munc18-a and syntaxin 1A regulates pre-synaptic release.

    Science.gov (United States)

    Kim, Bong Yoon; Sahara, Yoshinori; Yamamoto, Akitsugu; Kominami, Eiki; Kohsaka, Shinichi; Akazawa, Chihiro

    2006-11-24

    Membrane docking and fusion in neurons is a highly regulated process requiring the participation of a large number of SNAREs (soluble N-ethylmaleimide sensitive factor attachment protein receptors) and SNARE-interacting proteins. We found that mammalian Class C Vps protein complex associated specifically with nSec-1/Munc18-a, and syntaxin 1A both in vivo and in vitro. In contrast, VAMP2 and SNAP-25, other neuronal core complex proteins, did not interact. When co-transfected with the human growth hormone (hGH) reporter gene, mammalian Class C Vps proteins enhanced Ca2+-dependent exocytosis, which was abolished by the Ca2+-channel blocker nifedipine. In hippocampal primary cultures, the lentivirus-mediated overexpression of hVps18 increased asynchronous spontaneous synaptic release without changing mEPSCs. These results indicate that mammalian Class C Vps proteins are involved in the regulation of membrane docking and fusion through an interaction with neuronal specific SNARE molecules, nSec-1/Munc18-a and syntaxin 1A.

  2. Turning CALM into excitement: AP180 and CALM in endocytosis and disease.

    Science.gov (United States)

    Maritzen, Tanja; Koo, Seong Joo; Haucke, Volker

    2012-10-01

    Dynamic flux of membrane between intracellular compartments is a key feature of all eukaryotic cells. Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) play a crucial role in membrane dynamics by facilitating membrane fusion, for example at synapses where small synaptic vesicles (SVs) undergo activity-regulated neuroexocytosis, followed by the endocytic re-cycling of SV proteins and lipids. Recent work shows that the assembly protein 180 (AP180) N-terminal homology (ANTH) domain containing proteins AP180 and clathrin assembly lymphoid myeloid leukaemia (CALM) not only regulate the assembly of the endocytic machinery but also act as sorters for a subset of SNAREs, the vesicle-associated membrane proteins (VAMPs), most notably VAMP/synaptobrevin 2 at synapses. In this review, we summarise the current state of knowledge about the roles of AP180 and CALM family members in clathrin-dependent membrane traffic, the molecular mechanistic basis for their activities and their potential involvement in human disease. Copyright © 2012 Soçiété Francaise des Microscopies and Société de Biologie Cellulaire de France.

  3. The SNAP-25 Linker as an Adaptation Toward Fast Exocytosis

    Science.gov (United States)

    Nagy, Gábor; Milosevic, Ira; Mohrmann, Ralf; Wiederhold, Katrin; Walter, Alexander M.

    2008-01-01

    The assembly of four soluble N-ethylmaleimide-sensitive factor attachment protein receptor domains into a complex is essential for membrane fusion. In most cases, the four SNARE-domains are encoded by separate membrane-targeted proteins. However, in the exocytotic pathway, two SNARE-domains are present in one protein, connected by a flexible linker. The significance of this arrangement is unknown. We characterized the role of the linker in SNAP-25, a neuronal SNARE, by using overexpression techniques in synaptosomal-associated protein of 25 kDa (SNAP-25) null mouse chromaffin cells and fast electrophysiological techniques. We confirm that the palmitoylated linker-cysteines are important for membrane association. A SNAP-25 mutant without cysteines supported exocytosis, but the fusion rate was slowed down and the fusion pore duration prolonged. Using chimeric proteins between SNAP-25 and its ubiquitous homologue SNAP-23, we show that the cysteine-containing part of the linkers is interchangeable. However, a stretch of 10 hydrophobic and charged amino acids in the C-terminal half of the SNAP-25 linker is required for fast exocytosis and in its absence the calcium dependence of exocytosis is shifted toward higher concentrations. The SNAP-25 linker therefore might have evolved as an adaptation toward calcium triggering and a high rate of execution of the fusion process, those features that distinguish exocytosis from other membrane fusion pathways. PMID:18579690

  4. Kar5p is required for multiple functions in both inner and outer nuclear envelope fusion in Saccharomyces cerevisiae.

    Science.gov (United States)

    Rogers, Jason V; Rose, Mark D

    2014-12-02

    During mating in the budding yeast Saccharomyces cerevisiae, two haploid nuclei fuse via two sequential membrane fusion steps. SNAREs (i.e., soluble N-ethylmaleimide-sensitive factor attachment protein receptors) and Prm3p mediate outer nuclear membrane fusion, but the inner membrane fusogen remains unknown. Kar5p is a highly conserved transmembrane protein that localizes adjacent to the spindle pole body (SPB), mediates nuclear envelope fusion, and recruits Prm3p adjacent to the SPB. To separate Kar5p's functions, we tested localization, Prm3p recruitment, and nuclear fusion efficiency in various kar5 mutants. All domains and the conserved cysteine residues were essential for nuclear fusion. Several kar5 mutant proteins localized properly but did not mediate Prm3p recruitment; other kar5 mutant proteins localized and recruited Prm3p but were nevertheless defective for nuclear fusion, demonstrating additional functions beyond Prm3p recruitment. We identified one Kar5p domain required for SPB localization, which is dependent on the half-bridge protein Mps3p. Electron microscopy revealed a kar5 mutant that arrests with expanded nuclear envelope bridges, suggesting that Kar5p is required after outer nuclear envelope fusion. Finally, a split-GFP assay demonstrated that Kar5p localizes to both the inner and outer nuclear envelope. These insights suggest a mechanism by which Kar5p mediates inner nuclear membrane fusion. Copyright © 2015 Rogers and Rose.

  5. Effect of thyroxine on SNARE complex and synaptotagmin-1 expression in the prefrontal cortex of rats with adult-onset hypothyroidism.

    Science.gov (United States)

    Yang, H Y; Sun, C P; Jia, X M; Gui, L; Zhu, D F; Ma, W Q

    2012-03-01

    Thyroid hormone insufficiency in adulthood causes a wide range of brain impairments, including altered synaptic proteins in the prefrontal cortex (PFC). The present study investigated whether adult-onset hypothyroidism altered the expression of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes and synaptotagmin-1 (syt-1) in the PFC of rats. Sprague-Dawley rats were randomly divided into 4 groups: control, hypothyroid, and hypothyroid treated with T(4) [5 or 20 μg/100 g body weight (BW)]. Adult-onset hypothyroidism was induced in rats with the antithyroid drug 6-n-propyl-2-thiouracil (ip injection). PFC levels of synaptosomal-associated protein of 25 kDa (SNAP-25), syntaxin-1, vesicle-associated membrane protein 2 (VAMP-2) and syt-1 were determined by immunohistochemistry and western blot analyses. The results showed that syntaxin-1 and syt-1 were expressed at significantly lower levels in hypothyroid rats, VAMP-2 levels were not altered, and SNAP-25 levels were much higher compared to controls. A 2-week treatment with 5 μg T(4)/100 g BW partially normalized levels of SNARE complex and syt-1, and 20 μg T(4)/100 g BW restored these proteins closer to normal levels. Our findings indicate that dysregulation of SNARE complex and syt-1 in PFC of adult-onset hypothyroidism can be restored by T(4) treatment. © 2012, Editrice Kurtis.

  6. Polyphenols differentially inhibit degranulation of distinct subsets of vesicles in mast cells by specific interaction with granule-type-dependent SNARE complexes

    Science.gov (United States)

    Yang, Yoosoo; Oh, Jung-Mi; Heo, Paul; Shin, Jae Yoon; Kong, Byoungjae; Shin, Jonghyeok; Lee, Ji-Chun; Oh, Jeong Su; Park, Kye Won; Lee, Choong Hwan; Shin, Yeon-Kyun; Kweon, Dae-Hyuk

    2016-01-01

    Anti-allergic effects of dietary polyphenols were extensively studied in numerous allergic disease models, but the molecular mechanisms of anti-allergic effects by polyphenols remain poorly understood. In the present study, we show that the release of granular cargo molecules, contained in distinct subsets of granules of mast cells, is specifically mediated by two sets of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins, and that various polyphenols differentially inhibit the formation of those SNARE complexes. Expression analysis of RBL-2H3 cells for 11 SNARE genes and a lipid mixing assay of 24 possible combinations of reconstituted SNAREs indicated that the only two active SNARE complexes involved in mast cell degranulation are Syn (syntaxin) 4/SNAP (23 kDa synaptosome-associated protein)-23/VAMP (vesicle-associated membrane protein) 2 and Syn4/SNAP-23/VAMP8. Various polyphenols selectively or commonly interfered with ternary complex formation of these two SNARE complexes, thereby stopping membrane fusion between granules and plasma membrane. This led to the differential effect of polyphenols on degranulation of three distinct subsets of granules. These results suggest the possibility that formation of a variety of SNARE complexes in numerous cell types is controlled by polyphenols which, in turn, might regulate corresponding membrane trafficking. PMID:23252429

  7. Decreased Levels of VAMP2 and Monomeric Alpha-Synuclein Correlate with Duration of Dementia.

    Science.gov (United States)

    Vallortigara, Julie; Whitfield, David; Quelch, William; Alghamdi, Amani; Howlett, David; Hortobágyi, Tibor; Johnson, Mary; Attems, Johannes; O'Brien, John T; Thomas, Alan; Ballard, Clive G; Aarsland, Dag; Francis, Paul T

    2016-01-01

    Alpha-synuclein (α-syn) aggregations are the key pathological hallmark of dementia with Lewy bodies (DLB) and Parkinson's disease dementia (PDD), but are also frequently present in Alzheimer's disease (AD). Much remains unknown about the role of α-syn in the synapse and the wider role of synaptic dysfunction in these dementias. Changes in concentrations of key 'SNAP (Soluble N-ethylmaleimide Sensitive Factor Attachment Protein) Receptor' (SNARE) proteins as a consequence of alterations in the aggregation state of α-syn may contribute to synaptic dysfunction in patients with DLB, PDD, and AD and result in impaired cognition. We have studied a large cohort (n = 130) of autopsy confirmed DLB, PDD, AD, and control brains. Using semi-quantitative western blotting, we have demonstrated significant changes across the diagnostic groups of DLB, PDD, and AD in the SNARE and vesicle proteins syntaxin, Munc18, VAMP2, and monomeric α-syn in the prefrontal cortex, with a significant reduction of Munc18 in AD patients (p score before death (p = 0.016). We also identified a significant negative correlation between the duration of dementia and the levels of the binding partners VAMP2 (p = 0.0004) and monomeric α-syn (p = 0.0002). Our findings may indicate that an upregulation of SNARE complex related proteins occurs in the early stages of disease as an attempt at compensating for failing synapses, prior to widespread deposition of pathological α-syn.

  8. Phosphorylation of syntaxin-3 at Thr 14 negatively regulates exocytosis in RBL-2H3 mast cells.

    Science.gov (United States)

    Tadokoro, Satoshi; Shibata, Tetsuhiro; Inoh, Yoshikazu; Amano, Toshiro; Nakanishi, Mamoru; Hirashima, Naohide; Utsunomiya-Tate, Naoko

    2016-05-01

    Recent studies have revealed that soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins interact with each other, forming a SNARE complex that induces exocytosis in mast cells. Previously, we reported that syntaxin-3, a SNARE protein, regulates mast cell exocytosis and is constantly phosphorylated. In this study, we tried to identify the amino acid residue that is phosphorylated in mast cells, and to elucidate the regulatory mechanism of exocytosis by phosphorylation in syntaxin-3. We found that Thr 14 of syntaxin-3 was a phosphorylation site in mast cells. In addition, the overexpression of a constitutively dephosphorylated syntaxin-3 (T14A) mutant enhanced mast cell exocytosis. We also showed that the phosphomimetic mutation of syntaxin-3 at Thr 14 (T14E) induced structural changes in syntaxin-3, and this mutation inhibited binding of syntaxin-3 to Munc18-2. These results suggest that phosphorylated syntaxin-3 at Thr 14 negatively regulates mast cell exocytosis by impairing the interaction between syntaxin-3 and Munc18-2. © 2016 International Federation for Cell Biology.

  9. The golgin tether giantin regulates the secretory pathway by controlling stack organization within Golgi apparatus.

    Science.gov (United States)

    Koreishi, Mayuko; Gniadek, Thomas J; Yu, Sidney; Masuda, Junko; Honjo, Yasuko; Satoh, Ayano

    2013-01-01

    Golgins are coiled-coil proteins that play a key role in the regulation of Golgi architecture and function. Giantin, the largest golgin in mammals, forms a complex with p115, rab1, GM130, and soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), thereby facilitating vesicle tethering and fusion processes around the Golgi apparatus. Treatment with the microtubule destabilizing drug nocodazole transforms the Golgi ribbon into individual Golgi stacks. Here we show that siRNA-mediated depletion of giantin resulted in more dispersed Golgi stacks after nocodazole treatment than by control treatment, without changing the average cisternal length. Furthermore, depletion of giantin caused an increase in cargo transport that was associated with altered cell surface protein glycosylation. Drosophila S2 cells are known to have dispersed Golgi stacks and no giantin homolog. The exogenous expression of mammalian giantin cDNA in S2 cells resulted in clustered Golgi stacks, similar to the Golgi ribbon in mammalian cells. These results suggest that the spatial organization of the Golgi ribbon is mediated by giantin, which also plays a role in cargo transport and sugar modifications.

  10. Differential Interaction of Tomosyn with Syntaxin and SNAP25 Depends on Domains in the WD40 β-Propeller Core and Determines Its Inhibitory Activity*

    Science.gov (United States)

    Bielopolski, Noa; Lam, Alice D.; Bar-On, Dana; Sauer, Markus; Stuenkel, Edward L.; Ashery, Uri

    2014-01-01

    Neuronal exocytosis depends on efficient formation of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes and is regulated by tomosyn, a SNARE-binding protein. To gain new information about tomosyn's activity, we characterized its mobility and organization on the plasma membrane (PM) in relation to other SNARE proteins and inhibition of exocytosis. By using direct stochastic optical reconstruction microscopy (dSTORM), we found tomosyn to be organized in small clusters adjacent to syntaxin clusters. In addition, we show that tomosyn is present in both syntaxin-tomosyn complexes and syntaxin-SNAP25-tomosyn complexes. Tomosyn mutants that lack residues 537–578 or 897–917 from its β-propeller core diffused faster on the PM and exhibited reduced binding to SNAP25, suggesting that these mutants shift the equilibrium between tomosyn-syntaxin-SNAP25 complexes on the PM to tomosyn-syntaxin complexes. As these deletion mutants impose less inhibition on exocytosis, we suggest that tomosyn inhibition is mediated via tomosyn-syntaxin-SNAP25 complexes and not tomosyn-syntaxin complexes. These findings characterize, for the first time, tomosyn's dynamics at the PM and its relation to its inhibition of exocytosis. PMID:24782308

  11. Accumulation of SNAP25 in mouse gustatory and somatosensory cortices in response to food and chemical stimulation.

    Science.gov (United States)

    Kawakami, S; Ohmoto, M; Itoh, S; Yuasa, R; Inagaki, H; Nishimura, E; Ito, T; Misaka, T

    2012-08-30

    Food intake stimuli, including taste, somatosensory, and tactile stimuli, are received by receptors in the oral cavity, and this information is then transferred to the cerebral cortex. Signals from recently ingested food during the weaning period can affect synaptic transmission, resulting in biochemical changes in the cerebral cortex that modify gustatory and somatosensory nervous system plasticity. In this study, we investigated the expression patterns of molecular markers in mouse gustatory and somatosensory cortices during the weaning period. The expression of synaptosomal-associated protein 25 (SNAP25), a component of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex, was increased in the insular and somatosensory cortices at postnatal week 3 compared to postnatal week 2. Additionally, SNAP25 protein in the cerebral cortex accumulated in weaning mice fed solid food but not in mice fed only mother's milk at the weaning stage. Chemical stimulation by saccharin or capsaicin at the weaning stage also increased SNAP25 immunoreactivity in the insular or somatosensory cortical area, respectively. These results suggest that recently ingested chemical signals in the oral cavity during weaning increase the accumulation of SNAP25 in the gustatory and somatosensory cortices and promote neural plasticity during the development of the gustatory and somatosensory nervous systems. Copyright © 2012 IBRO. Published by Elsevier Ltd. All rights reserved.

  12. Low energy cost for optimal speed and control of membrane fusion.

    Science.gov (United States)

    François-Martin, Claire; Rothman, James E; Pincet, Frederic

    2017-02-07

    Membrane fusion is the cell's delivery process, enabling its many compartments to receive cargo and machinery for cell growth and intercellular communication. The overall activation energy of the process must be large enough to prevent frequent and nonspecific spontaneous fusion events, yet must be low enough to allow it to be overcome upon demand by specific fusion proteins [such as soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs)]. Remarkably, to the best of our knowledge, the activation energy for spontaneous bilayer fusion has never been measured. Multiple models have been developed and refined to estimate the overall activation energy and its component parts, and they span a very broad range from 20 kBT to 150 kBT, depending on the assumptions. In this study, using a bulk lipid-mixing assay at various temperatures, we report that the activation energy of complete membrane fusion is at the lowest range of these theoretical values. Typical lipid vesicles were found to slowly and spontaneously fully fuse with activation energies of ∼30 kBT Our data demonstrate that the merging of membranes is not nearly as energy consuming as anticipated by many models and is ideally positioned to minimize spontaneous fusion while enabling rapid, SNARE-dependent fusion upon demand.

  13. ER-associated SNAREs and Sey1p mediate nuclear fusion at two distinct steps during yeast mating.

    Science.gov (United States)

    Rogers, Jason V; Arlow, Tim; Inkellis, Elizabeth R; Koo, Timothy S; Rose, Mark D

    2013-12-01

    During yeast mating, two haploid nuclei fuse membranes to form a single diploid nucleus. However, the known proteins required for nuclear fusion are unlikely to function as direct fusogens (i.e., they are unlikely to directly catalyze lipid bilayer fusion) based on their predicted structure and localization. Therefore we screened known fusogens from vesicle trafficking (soluble N-ethylmaleimide-sensitive factor attachment protein receptors [SNAREs]) and homotypic endoplasmic reticulum (ER) fusion (Sey1p) for additional roles in nuclear fusion. Here we demonstrate that the ER-localized SNAREs Sec20p, Ufe1p, Use1p, and Bos1p are required for efficient nuclear fusion. In contrast, Sey1p is required indirectly for nuclear fusion; sey1Δ zygotes accumulate ER at the zone of cell fusion, causing a block in nuclear congression. However, double mutants of Sey1p and Sec20p, Ufe1p, or Use1p, but not Bos1p, display extreme ER morphology defects, worse than either single mutant, suggesting that retrograde SNAREs fuse ER in the absence of Sey1p. Together these data demonstrate that SNAREs mediate nuclear fusion, ER fusion after cell fusion is necessary to complete nuclear congression, and there exists a SNARE-mediated, Sey1p-independent ER fusion pathway.

  14. Replacing SNAP-25b with SNAP-25a expression results in metabolic disease.

    Science.gov (United States)

    Valladolid-Acebes, Ismael; Daraio, Teresa; Brismar, Kerstin; Harkany, Tibor; Ögren, Sven Ove; Hökfelt, Tomas G M; Bark, Christina

    2015-08-04

    Synaptosomal-associated protein of 25 kDa (SNAP-25) is a key molecule in the soluble N-ethylmaleimide-sensitive factor attachment protein (SNARE) complex mediating fast Ca(2+)-triggered release of hormones and neurotransmitters, and both splice variants, SNAP-25a and SNAP-25b, can participate in this process. Here we explore the hypothesis that minor alterations in the machinery mediating regulated membrane fusion can increase the susceptibility for metabolic disease and precede obesity and type 2 diabetes. Thus, we used a mouse mutant engineered to express normal levels of SNAP-25 but only SNAP-25a. These SNAP-25b-deficient mice were exposed to either a control or a high-fat/high-sucrose diet. Monitoring of food intake, body weight, hypothalamic function, and lipid and glucose homeostases showed that SNAP-25b-deficient mice fed with control diet developed hyperglycemia, liver steatosis, and adipocyte hypertrophy, conditions dramatically exacerbated when combined with the high-fat/high-sucrose diet. Thus, modified SNARE function regulating stimulus-dependent exocytosis can increase the vulnerability to and even provoke metabolic disease. When combined with a high-fat/high-sucrose diet, this vulnerability resulted in diabesity. Our SNAP-25b-deficient mouse may represent a diabesity model.

  15. Synaptotagmin 1 Negatively Controls the Two Distinct Immune Secretory Pathways to Powdery Mildew Fungi in Arabidopsis.

    Science.gov (United States)

    Kim, Hyeran; Kwon, Hyeokjin; Kim, Soohong; Kim, Mi Kyung; Botella, Miguel A; Yun, Hye Sup; Kwon, Chian

    2016-06-01

    PEN1, one of the plasma membrane (PM) syntaxins, comprises an immune exocytic pathway by forming the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex with SNAP33 and VAMP721/722 in plants. Although this secretory pathway is also involved in plant growth and development, how plants control their exocytic activity is as yet poorly understood. Since constitutive PEN1 cycling between the PM and endocytosed vesicles is critical for its immune activity, we studied here the relationship of PEN1 to synaptotagmin 1 (SYT1) that is known to regulate endocytosis at the PM. Interestingly, syt1 plants showed enhanced disease resistance to the Arabidopsis-adapted Golovinomyces orontii fungus, and elevated protein but not transcript levels of PEN1 Calcium-dependent promotion of PEN1-SYT1 interaction suggests that SYT1 controls defense activities of the PEN1-associated secretory pathway by post-translationally modulating PEN1. Increased PEN1-SYT1 interaction and inhibited PEN1 SNARE complex induction by G. orontii additionally suggest that the adaption of phytopathogens to host plants might partly result from effective suppression of the PEN1-related secretory pathway. Further genetic analyses revealed that SYT1 also regulates the atypical peroxisomal myrosinase PEN2-associated secretory pathway. © The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  16. Peptide inhibitors of botulinum neurotoxin serotype A: design, inhibition, cocrystal structures, structure-activity relationship and pharmacophore modeling

    Energy Technology Data Exchange (ETDEWEB)

    Kumar G.; Swaminathan S.; Kumaran, D.; Ahmed, S. A.

    2012-05-01

    Clostridium botulinum neurotoxins are classified as Category A bioterrorism agents by the Centers for Disease Control and Prevention (CDC). The seven serotypes (A-G) of the botulinum neurotoxin, the causative agent of the disease botulism, block neurotransmitter release by specifically cleaving one of the three SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins and induce flaccid paralysis. Using a structure-based drug-design approach, a number of peptide inhibitors were designed and their inhibitory activity against botulinum serotype A (BoNT/A) protease was determined. The most potent peptide, RRGF, inhibited BoNT/A protease with an IC{sub 50} of 0.9 {micro}M and a K{sub i} of 358 nM. High-resolution crystal structures of various peptide inhibitors in complex with the BoNT/A protease domain were also determined. Based on the inhibitory activities and the atomic interactions deduced from the cocrystal structures, the structure-activity relationship was analyzed and a pharmacophore model was developed. Unlike the currently available models, this pharmacophore model is based on a number of enzyme-inhibitor peptide cocrystal structures and improved the existing models significantly, incorporating new features.

  17. The primed SNARE–complexin–synaptotagmin complex for neuronal exocytosis

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, Qiangjun; Zhou, Peng; Wang, Austin L.; Wu, Dick; Zhao, Minglei; Südhof, Thomas C.; Brunger, Axel T.

    2017-08-16

    Synaptotagmin, complexin, and neuronal SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptor) proteins mediate evoked synchronous neurotransmitter release, but the molecular mechanisms mediating the cooperation between these molecules remain unclear. Here we determine crystal structures of the primed pre-fusion SNARE–complexin–synaptotagmin-1 complex. These structures reveal an unexpected tripartite interface between synaptotagmin-1 and both the SNARE complex and complexin. Simultaneously, a second synaptotagmin-1 molecule interacts with the other side of the SNARE complex via the previously identified primary interface. Mutations that disrupt either interface in solution also severely impair evoked synchronous release in neurons, suggesting that both interfaces are essential for the primed pre-fusion state. Ca2+ binding to the synaptotagmin-1 molecules unlocks the complex, allows full zippering of the SNARE complex, and triggers membrane fusion. The tripartite SNARE–complexin–synaptotagmin-1 complex at a synaptic vesicle docking site has to be unlocked for triggered fusion to start, explaining the cooperation between complexin and synaptotagmin-1 in synchronizing evoked release on the sub-millisecond timescale.

  18. The Atg17-Atg31-Atg29 Complex Coordinates with Atg11 to Recruit the Vam7 SNARE and Mediate Autophagosome-Vacuole Fusion.

    Science.gov (United States)

    Liu, Xu; Mao, Kai; Yu, Angela Y H; Omairi-Nasser, Amin; Austin, Jotham; Glick, Benjamin S; Yip, Calvin K; Klionsky, Daniel J

    2016-01-25

    Macroautophagy (hereafter autophagy) is an evolutionarily conserved process in which portions of the cytoplasm are engulfed, degraded, and subsequently recycled. The Atg17-Atg31-Atg29 complex translocates to the phagophore assembly site (PAS), where an autophagosome forms, at a very early stage of autophagy, playing a vital role in autophagy induction. Here, we identified a novel role of this complex in a late stage of autophagy where it coordinates with Atg11 to regulate autophagy-specific fusion with the vacuole. Atg17 and Atg11 interact with the vacuolar SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) Vam7 independently of each other. Several hydrophobic residues in helix 1 and helix 4 of Atg17 and the SNARE domain of Vam7 mediate the Atg17-Vam7 interaction. An F317D mutation of Atg17, which diminishes its interaction with Vam7 without affecting its interaction with Atg13 or Atg31, leads to a defect in the fusion of autophagosomes with the vacuole and decreased autophagy activity. These results provide the first demonstration that the Atg17-Atg31-Atg29 complex functions in both early and late stages of autophagy and also provide a mechanistic explanation for the coordination of autophagosome completion and fusion with the vacuole. Copyright © 2016 Elsevier Ltd. All rights reserved.

  19. Amyloid-β Oligomers May Impair SNARE-Mediated Exocytosis by Direct Binding to Syntaxin 1a

    Directory of Open Access Journals (Sweden)

    Yoosoo Yang

    2015-08-01

    Full Text Available Alzheimer’s disease (AD is closely associated with synaptic dysfunction, and thus current treatments often aim to stimulate neurotransmission to improve cognitive impairment. Whereas the formation of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE complex is essential for synaptic transmission, the correlation between SNAREs and AD neuropathology is unknown. Here, we report that intracellular amyloid-β (Aβ oligomers directly inhibit SNARE-mediated exocytosis by impairing SNARE complex formation. We observe abnormal reduction of SNARE complex levels in the brains of APP/PS1 transgenic (TG mice compared to age-matched wild-types. We demonstrate that Aβ oligomers block SNARE complex assembly through the direct interaction with a target membrane (t-SNARE syntaxin 1a in vitro. Furthermore, the results of the in vitro single-vesicle content-mixing assay reveal that Aβ oligomers inhibit SNARE-mediated fusion pores. Thus, our study identifies a potential molecular mechanism by which intracellular Aβ oligomers hamper SNARE-mediated exocytosis, likely leading to AD-associated synaptic dysfunctions.

  20. Spatial, temporal and functional molecular architecture of the munc18-syntaxin interaction

    OpenAIRE

    Smyth, Annya Mary

    2012-01-01

    Regulation of soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors (SNARE) mediated exocytosis is dependent upon four key proteins; the vesicular SNARE synaptobrevin, target SNAREs SNAP-25 and syntaxin and the Sec1/Munc18 (SM) protein munc18-1. Despite the munc18-1-syntaxin interaction being central to regulated vesicle exocytosis the spatial and temporal pattern of their molecular distribution and interaction in neuroendocrine and neuronal cells remai...

  1. Super-resolution Imaging Reveals the Internal Architecture of Nano-sized Syntaxin Clusters*

    Science.gov (United States)

    Bar-On, Dana; Wolter, Steve; van de Linde, Sebastian; Heilemann, Mike; Nudelman, German; Nachliel, Esther; Gutman, Menachem; Sauer, Markus; Ashery, Uri

    2012-01-01

    Key synaptic proteins from the soluble SNARE (N-ethylmaleimide-sensitive factor attachment protein receptor) family, among many others, are organized at the plasma membrane of cells as clusters containing dozens to hundreds of protein copies. However, the exact membranal distribution of proteins into clusters or as single molecules, the organization of molecules inside the clusters, and the clustering mechanisms are unclear due to limitations of the imaging and analytical tools. Focusing on syntaxin 1 and SNAP-25, we implemented direct stochastic optical reconstruction microscopy together with quantitative clustering algorithms to demonstrate a novel approach to explore the distribution of clustered and nonclustered molecules at the membrane of PC12 cells with single-molecule precision. Direct stochastic optical reconstruction microscopy images reveal, for the first time, solitary syntaxin/SNAP-25 molecules and small clusters as well as larger clusters. The nonclustered syntaxin or SNAP-25 molecules are mostly concentrated in areas adjacent to their own clusters. In the clusters, the density of the molecules gradually decreases from the dense cluster core to the periphery. We further detected large clusters that contain several density gradients. This suggests that some of the clusters are formed by unification of several clusters that preserve their original organization or reorganize into a single unit. Although syntaxin and SNAP-25 share some common distributional features, their clusters differ markedly from each other. SNAP-25 clusters are significantly larger, more elliptical, and less dense. Finally, this study establishes methodological tools for the analysis of single-molecule-based super-resolution imaging data and paves the way for revealing new levels of membranal protein organization. PMID:22700970

  2. Super-resolution imaging reveals the internal architecture of nano-sized syntaxin clusters.

    Science.gov (United States)

    Bar-On, Dana; Wolter, Steve; van de Linde, Sebastian; Heilemann, Mike; Nudelman, German; Nachliel, Esther; Gutman, Menachem; Sauer, Markus; Ashery, Uri

    2012-08-03

    Key synaptic proteins from the soluble SNARE (N-ethylmaleimide-sensitive factor attachment protein receptor) family, among many others, are organized at the plasma membrane of cells as clusters containing dozens to hundreds of protein copies. However, the exact membranal distribution of proteins into clusters or as single molecules, the organization of molecules inside the clusters, and the clustering mechanisms are unclear due to limitations of the imaging and analytical tools. Focusing on syntaxin 1 and SNAP-25, we implemented direct stochastic optical reconstruction microscopy together with quantitative clustering algorithms to demonstrate a novel approach to explore the distribution of clustered and nonclustered molecules at the membrane of PC12 cells with single-molecule precision. Direct stochastic optical reconstruction microscopy images reveal, for the first time, solitary syntaxin/SNAP-25 molecules and small clusters as well as larger clusters. The nonclustered syntaxin or SNAP-25 molecules are mostly concentrated in areas adjacent to their own clusters. In the clusters, the density of the molecules gradually decreases from the dense cluster core to the periphery. We further detected large clusters that contain several density gradients. This suggests that some of the clusters are formed by unification of several clusters that preserve their original organization or reorganize into a single unit. Although syntaxin and SNAP-25 share some common distributional features, their clusters differ markedly from each other. SNAP-25 clusters are significantly larger, more elliptical, and less dense. Finally, this study establishes methodological tools for the analysis of single-molecule-based super-resolution imaging data and paves the way for revealing new levels of membranal protein organization.

  3. Neurotoxins from Clostridium botulinum (serotype A) isolated from the soil of Mendoza (Argentina) differ from the A-Hall archetype and from that causing infant botulism.

    Science.gov (United States)

    Caballero, P; Troncoso, M; Patterson, S I; López Gómez, C; Fernandez, R; Sosa, M A

    2016-10-01

    The type A of neurotoxin produced by Clostridium botulinum is the prevalent serotype in strains of Mendoza. The soil is the main reservoir for C.botulinum and is possibly one of the infection sources in infant botulism. In this study, we characterized and compared autochthonous C. botulinum strains and their neurotoxins. Bacterial samples were obtained from the soil and from fecal samples collected from children with infant botulism. We first observed differences in the appearance of the colonies between strains from each source and with the A Hall control strain. In addition, purified neurotoxins of both strains were found to be enriched in a band of 300 kDa, whereas the A-Hall strain was mainly made up of a band of ∼600 kDa. This finding is in line with the lack of hemagglutinating activity of the neurotoxins under study. Moreover, the proteolytic activity of C. botulinum neurotoxins was evaluated against SNARE (soluble N-ethylmaleimide-sensitive factor-attachment protein receptor) proteins from rat brain. It was observed that both, SNAP 25 (synaptosomal-associated protein 25) and VAMP 2 (vesicle-associated membrane protein) were cleaved by the neurotoxins isolated from the soil strains, whereas the neurotoxins from infant botulism strains only induced a partial cleavage of VAMP 2. On the other hand, the neurotoxin from the A-Hall strain was able to cleave both proteins, though at a lesser extent. Our data indicate that the C.botulinum strain isolated from the soil, and its BoNT, exhibit different properties compared to the strain obtained from infant botulism patients, and from the A-Hall archetype. Copyright © 2016 Elsevier Ltd. All rights reserved.

  4. Signalling in ciliates: long- and short-range signals and molecular determinants for cellular dynamics.

    Science.gov (United States)

    Plattner, Helmut

    2017-02-01

    In ciliates, unicellular representatives of the bikont branch of evolution, inter- and intracellular signalling pathways have been analysed mainly in Paramecium tetraurelia, Paramecium multimicronucleatum and Tetrahymena thermophila and in part also in Euplotes raikovi. Electrophysiology of ciliary activity in Paramecium spp. is a most successful example. Established signalling mechanisms include plasmalemmal ion channels, recently established intracellular Ca(2+) -release channels, as well as signalling by cyclic nucleotides and Ca(2+) . Ca(2+) -binding proteins (calmodulin, centrin) and Ca(2+) -activated enzymes (kinases, phosphatases) are involved. Many organelles are endowed with specific molecules cooperating in signalling for intracellular transport and targeted delivery. Among them are recently specified soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), monomeric GTPases, H(+) -ATPase/pump, actin, etc. Little specification is available for some key signal transducers including mechanosensitive Ca(2+) -channels, exocyst complexes and Ca(2+) -sensor proteins for vesicle-vesicle/membrane interactions. The existence of heterotrimeric G-proteins and of G-protein-coupled receptors is still under considerable debate. Serine/threonine kinases dominate by far over tyrosine kinases (some predicted by phosphoproteomic analyses). Besides short-range signalling, long-range signalling also exists, e.g. as firmly installed microtubular transport rails within epigenetically determined patterns, thus facilitating targeted vesicle delivery. By envisaging widely different phenomena of signalling and subcellular dynamics, it will be shown (i) that important pathways of signalling and cellular dynamics are established already in ciliates, (ii) that some mechanisms diverge from higher eukaryotes and (iii) that considerable uncertainties still exist about some essential aspects of signalling. © 2015 Cambridge Philosophical Society.

  5. SNAP-23 and VAMP-3 contribute to the release of IL-6 and TNFα from a human synovial sarcoma cell line.

    Science.gov (United States)

    Boddul, Sanjay V; Meng, Jianghui; Dolly, James Oliver; Wang, Jiafu

    2014-02-01

    Fibroblast-like synoviocytes are important mediators of inflammatory joint damage in arthritis through the release of cytokines, but it is unknown whether their exocytosis from these particular cells is SNARE-dependent. Here, the complement of soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) in human synovial sarcoma cells (SW982) was examined with respect to the secretion of interleukin-6 (IL-6) and tumour necrosis factor α (TNFα), before and after knockdown of a synaptosome-associated protein of molecular mass 23 kDa (SNAP-23) or the vesicle-associated membrane protein 3 (VAMP-3). Wild-type SW982 cells expressed SNAP-23, VAMP-3, syntaxin isoforms 2-4 and synaptic vesicle protein 2C (SV2C). These cells showed Ca²⁺-dependent secretion of IL-6 and TNFα when stimulated by interleukin-1β (IL-1β) or in combination with K⁺ depolarization. Specific knockdown of SNAP-23 or VAMP-3 decreased the exocytosis of IL-6 and TNFα; the reduced expression of SNAP-23 caused accumulation of SV2 in the peri-nuclear area. A monoclonal antibody specific for VAMP-3 precipitated SNAP-23 and syntaxin-2 (and syntaxin-3 to a lesser extent). The formation of SDS-resistant complexes by SNAP-23 and VAMP-3 was reduced upon knockdown of SNAP-23. Although the syntaxin isoforms 2, 3 and 4 are expressed in SW982 cells, knockdown of each did not affect the release of cytokines. Collectively, these results show that SNAP-23 and VAMP-3 participate in IL-1β-induced Ca²⁺-dependent release of IL-6 and TNFα from SW982 cells. © 2013 FEBS.

  6. Exosomes Derived from Mesenchymal Stromal Cells Promote Axonal Growth of Cortical Neurons.

    Science.gov (United States)

    Zhang, Yi; Chopp, Michael; Liu, Xian Shuang; Katakowski, Mark; Wang, Xinli; Tian, Xinchu; Wu, David; Zhang, Zheng Gang

    2017-05-01

    Treatment of brain injury with exosomes derived from mesenchymal stromal cells (MSCs) enhances neurite growth. However, the direct effect of exosomes on axonal growth and molecular mechanisms underlying exosome-enhanced neurite growth are not known. Using primary cortical neurons cultured in a microfluidic device, we found that MSC-exosomes promoted axonal growth, whereas attenuation of argonaut 2 protein, one of the primary microRNA (miRNA) machinery proteins, in MSC-exosomes abolished their effect on axonal growth. Both neuronal cell bodies and axons internalized MSC-exosomes, which was blocked by botulinum neurotoxins (BoNTs) that cleave proteins of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex. Moreover, tailored MSC-exosomes carrying elevated miR-17-92 cluster further enhanced axonal growth compared to native MSC-exosomes. Quantitative RT-PCR and Western blot analysis showed that the tailored MSC-exosomes increased levels of individual members of this cluster and activated the PTEN/mTOR signaling pathway in recipient neurons, respectively. Together, our data demonstrate that native MSC-exosomes promote axonal growth while the tailored MSC-exosomes can further boost this effect and that tailored exosomes can deliver their selective cargo miRNAs into and activate their target signals in recipient neurons. Neuronal internalization of MSC-exosomes is mediated by the SNARE complex. This study reveals molecular mechanisms that contribute to MSC-exosome-promoted axonal growth, which provides a potential therapeutic strategy to enhance axonal growth.

  7. [Four-week simulated weightlessness increases the expression of atrial natriuretic peptide in the myocardium].

    Science.gov (United States)

    Zhang, Wen-Cheng; Lu, Yuan-Ming; Yang, Huai-Zhang; Xu, Peng-Tao; Chang, Hui; Yu, Zhi-Bin

    2013-04-25

    One of the major circulatory changes that occur in human during space flight and simulated weightlessness is a cerebral redistribution of body fluids, which is accompanied by an increase of blood volume in the upper body. Therefore, atrial myocardium should increase the secretion of atrial natriuretic peptide (ANP), but the researches lack common conclusion until now. The present study was to investigate the expression level of ANP in simulated weightlessness rats, and to confirm the changes of ANP by observing the associated proteins of soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). The tail-suspended rat model was used to simulate weightlessness. Western blots were carried out to examine the expression levels of ANP and SNARE proteins in atrial and left ventricular myocardium. The results showed that ANP expression in atrial myocardium showed an increase in 4-week tail-suspended rats (SUS) compared with that in the synchronous control rats (CON). We only detected a trace amount of ANP in the left ventricular myocardium of the CON, but found an enhanced expression of ANP in left ventricular myocardium of the SUS. Expression of VAMP-1/2 (vesicle associated SNARE) increased significantly in both atrial and left ventricular myocardium in the SUS compared with that in the CON. There was no difference of the expression of syntaxin-4 (target compartment associated SNARE) between the CON and SUS, but the expression of SNAP-23 showed an increase in atrial myocardium of the SUS compared with that in the CON. Synip and Munc-18c as regulators of SNAREs did not show significant difference between the CON and SUS. These results suggest that the expression of ANP shows an increase in atrial and left ventricular myocardium of 4-week tail-suspended rats. Enhanced expression of VAMP-1/2 associated with ANP vesicles confirms the increased expression of ANP in atrial and left ventricular myocardium.

  8. Interchromosomal Transfer of Immune Regulation During Infection of Barley with the Powdery Mildew Pathogen

    Directory of Open Access Journals (Sweden)

    Priyanka Surana

    2017-10-01

    Full Text Available Powdery mildew pathogens colonize over 9500 plant species, causing critical yield loss. The Ascomycete fungus, Blumeria graminis f. sp. hordei (Bgh, causes powdery mildew disease in barley (Hordeum vulgare L.. Successful infection begins with penetration of host epidermal cells, culminating in haustorial feeding structures, facilitating delivery of fungal effectors to the plant and exchange of nutrients from host to pathogen. We used expression Quantitative Trait Locus (eQTL analysis to dissect the temporal control of immunity-associated gene expression in a doubled haploid barley population challenged with Bgh. Two highly significant regions possessing trans eQTL were identified near the telomeric ends of chromosomes (Chr 2HL and 1HS. Within these regions reside diverse resistance loci derived from barley landrace H. laevigatum (MlLa and H. vulgare cv. Algerian (Mla1, which associate with the altered expression of 961 and 3296 genes during fungal penetration of the host and haustorial development, respectively. Regulatory control of transcript levels for 299 of the 961 genes is reprioritized from MlLa on 2HL to Mla1 on 1HS as infection progresses, with 292 of the 299 alternating the allele responsible for higher expression, including Adaptin Protein-2 subunit μ AP2M and Vesicle Associated Membrane Protein VAMP72 subfamily members VAMP721/722. AP2M mediates effector-triggered immunity (ETI via endocytosis of plasma membrane receptor components. VAMP721/722 and SNAP33 form a Soluble N-ethylmaleimide-sensitive factor Attachment Protein REceptor (SNARE complex with SYP121 (PEN1, which is engaged in pathogen associated molecular pattern (PAMP-triggered immunity via exocytosis. We postulate that genes regulated by alternate chromosomal positions are repurposed as part of a conserved immune complex to respond to different pathogen attack scenarios.

  9. Therapeutic use of botulinum toxin in migraine: mechanisms of action

    Science.gov (United States)

    Ramachandran, Roshni; Yaksh, Tony L

    2014-01-01

    Migraine pain represents sensations arising from the activation of trigeminal afferents, which innervate the meningeal vasculature and project to the trigeminal nucleus caudalis (TNC). Pain secondary to meningeal input is referred to extracranial regions innervated by somatic afferents that project to homologous regions in the TNC. Such viscerosomatic convergence accounts for referral of migraine pain arising from meningeal afferents to particular extracranial dermatomes. Botulinum toxins (BoNTs) delivered into extracranial dermatomes are effective in and approved for treating chronic migraine pain. Aside from their well-described effect upon motor endplates, BoNTs are also taken up in local afferent nerve terminals where they cleave soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, and prevent local terminal release. However, a local extracranial effect of BoNT cannot account for allthe effects of BoNT upon migraine. We now know that peripherally delivered BoNTs are taken up in sensory afferents and transported to cleave SNARE proteins in the ganglion and TNC, prevent evoked afferent release and downstream activation. Such effects upon somatic input (as from the face) likewise would not alone account for block of input from converging meningeal afferents. This current work suggests that BoNTs may undergo transcytosis to cleave SNAREs in second-order neurons or in adjacent afferent terminals. Finally, while SNAREs mediate exocytotic release, they are also involved in transport of channels and receptors involved in facilitated pain states. The role of such post-synaptic effects of BoNT action in migraine remains to be determined. PMID:24819339

  10. Rapid product analysis and increased sensitivity for quantitative determinations of botulinum neurotoxin proteolytic activity.

    Science.gov (United States)

    Rowe, Benjamin; Schmidt, James J; Smith, Leonard A; Ahmed, S Ashraf

    2010-01-15

    The ultimate molecular action of botulinum neurotoxin (BoNT) is a Zn-dependent endoproteolytic activity on one of the three SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins. There are seven serotypes (A-G) of BoNT having distinct cleavage sites on the SNARE substrates. The proteolytic activity is located on the N-terminal light chain (Lc) domain and is used extensively as the primary target toward therapeutic development against botulism. Here we describe an improved method using ultra-performance liquid chromatography (UPLC) whereby quantitative data were obtained in 1/10th the time using 1/20th the sample and solvent volumes compared with a widely used high-performance liquid chromatography (HPLC) method. We also synthesized a VAMP (vesicle-associated membrane protein)-based peptide containing an intact V1 motif that was efficiently used as a substrate by BoNT/D Lc. Although serotype C1 cleaves the serotype A substrate at a bond separated by only one residue, we were able to distinguish the two reactions by UPLC. The new method can accurately quantify as low as 7 pmol of the peptide substrates for BoNT serotypes A, B, C1, and D. We also report here that the catalytic efficiency of serotype A can be stimulated 35-fold by the addition of Triton X-100 to the reaction mixture. Combining the use of Triton X-100 with the newly introduced UPLC method, we were able to accurately detect very low levels of proteolytic activity in a very short time. Sensitivity of the assay and accuracy and rapidity of product analysis should greatly augment efforts in therapeutic development.

  11. The conserved SNARE SEC-22 localizes to late endosomes and negatively regulates RNA interference in Caenorhabditis elegans.

    Science.gov (United States)

    Zhao, Yani; Holmgren, Benjamin T; Hinas, Andrea

    2017-03-01

    Small RNA pathways, including RNA interference (RNAi), play crucial roles in regulation of gene expression. Initially considered to be cytoplasmic, these processes have later been demonstrated to associate with membranes. For example, maturation of late endosomes/multivesicular bodies (MVBs) is required for efficient RNAi, whereas fusion of MVBs to lysosomes appears to reduce silencing efficiency. SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) mediate membrane fusion and are thus at the core of membrane trafficking. In spite of this, no SNARE has previously been reported to affect RNAi. Here, we demonstrate that in Caenorhabditis elegans, loss of the conserved SNARE SEC-22 results in enhanced RNAi upon ingestion of double-stranded RNA. Furthermore, SEC-22 overexpression inhibits RNAi in wild-type animals. We find that overexpression of SEC-22 in the target tissue (body wall muscle) strongly suppresses the sec-22(-) enhanced RNAi phenotype, supporting a primary role for SEC-22 in import of RNAi silencing signals or cell autonomous RNAi. A functional mCherry::SEC-22 protein localizes primarily to late endosomes/MVBs and these compartments are enlarged in animals lacking sec-22 SEC-22 interacts with late endosome-associated RNA transport protein SID-5 in a yeast two-hybrid assay and functions in a sid-5-dependent manner. Taken together, our data indicate that SEC-22 reduces RNAi efficiency by affecting late endosome/MVB function, for example, by promoting fusion between late endosomes/MVBs and lysosomes. To our knowledge, this is the first report of a SNARE with a function in small RNA-mediated gene silencing. © 2017 Zhao et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  12. Interchromosomal Transfer of Immune Regulation During Infection of Barley with the Powdery Mildew Pathogen.

    Science.gov (United States)

    Surana, Priyanka; Xu, Ruo; Fuerst, Gregory; Chapman, Antony V E; Nettleton, Dan; Wise, Roger P

    2017-10-05

    Powdery mildew pathogens colonize over 9500 plant species, causing critical yield loss. The Ascomycete fungus, Blumeria graminis f. sp. hordei (Bgh), causes powdery mildew disease in barley (Hordeum vulgare L.). Successful infection begins with penetration of host epidermal cells, culminating in haustorial feeding structures, facilitating delivery of fungal effectors to the plant and exchange of nutrients from host to pathogen. We used expression Quantitative Trait Locus (eQTL) analysis to dissect the temporal control of immunity-associated gene expression in a doubled haploid barley population challenged with Bgh Two highly significant regions possessing trans eQTL were identified near the telomeric ends of chromosomes (Chr) 2HL and 1HS. Within these regions reside diverse resistance loci derived from barley landrace H. laevigatum (MlLa) and H. vulgare cv. Algerian (Mla1), which associate with the altered expression of 961 and 3296 genes during fungal penetration of the host and haustorial development, respectively. Regulatory control of transcript levels for 299 of the 961 genes is reprioritized from MlLa on 2HL to Mla1 on 1HS as infection progresses, with 292 of the 299 alternating the allele responsible for higher expression, including Adaptin Protein-2 subunit μ AP2M and Vesicle Associated Membrane Protein VAMP72 subfamily members VAMP721/722. AP2M mediates effector-triggered immunity (ETI) via endocytosis of plasma membrane receptor components. VAMP721/722 and SNAP33 form a Soluble N-ethylmaleimide-sensitive factor Attachment Protein REceptor (SNARE) complex with SYP121 (PEN1), which is engaged in pathogen associated molecular pattern (PAMP)-triggered immunity via exocytosis. We postulate that genes regulated by alternate chromosomal positions are repurposed as part of a conserved immune complex to respond to different pathogen attack scenarios. Copyright © 2017 Surana et al.

  13. α-Synuclein Delays Endoplasmic Reticulum (ER)-to-Golgi Transport in Mammalian Cells by Antagonizing ER/Golgi SNAREs

    Science.gov (United States)

    Thayanidhi, Nandhakumar; Helm, Jared R.; Nycz, Deborah C.; Bentley, Marvin; Liang, Yingjian

    2010-01-01

    Toxicity of human α-synuclein when expressed in simple organisms can be suppressed by overexpression of endoplasmic reticulum (ER)-to-Golgi transport machinery, suggesting that inhibition of constitutive secretion represents a fundamental cause of the toxicity. Whether similar inhibition in mammals represents a cause of familial Parkinson's disease has not been established. We tested elements of this hypothesis by expressing human α-synuclein in mammalian kidney and neuroendocrine cells and assessing ER-to-Golgi transport. Overexpression of wild type or the familial disease-associated A53T mutant α-synuclein delayed transport by up to 50%; however, A53T inhibited more potently. The secretory delay occurred at low expression levels and was not accompanied by insoluble α-synuclein aggregates or mistargeting of transport machinery, suggesting a direct action of soluble α-synuclein on trafficking proteins. Co-overexpression of ER/Golgi arginine soluble N-ethylmaleimide-sensitive factor attachment protein receptors (R-SNAREs) specifically rescued transport, indicating that α-synuclein antagonizes SNARE function. Ykt6 reversed α-synuclein inhibition much more effectively than sec22b, suggesting a possible neuroprotective role for the enigmatic high expression of ykt6 in neurons. In in vitro reconstitutions, purified α-synuclein A53T protein specifically inhibited COPII vesicle docking and fusion at a pre-Golgi step. Finally, soluble α-synuclein A53T directly bound ER/Golgi SNAREs and inhibited SNARE complex assembly, providing a potential mechanism for toxic effects in the early secretory pathway. PMID:20392839

  14. Alpha-synuclein delays endoplasmic reticulum (ER)-to-Golgi transport in mammalian cells by antagonizing ER/Golgi SNAREs.

    Science.gov (United States)

    Thayanidhi, Nandhakumar; Helm, Jared R; Nycz, Deborah C; Bentley, Marvin; Liang, Yingjian; Hay, Jesse C

    2010-06-01

    Toxicity of human alpha-synuclein when expressed in simple organisms can be suppressed by overexpression of endoplasmic reticulum (ER)-to-Golgi transport machinery, suggesting that inhibition of constitutive secretion represents a fundamental cause of the toxicity. Whether similar inhibition in mammals represents a cause of familial Parkinson's disease has not been established. We tested elements of this hypothesis by expressing human alpha-synuclein in mammalian kidney and neuroendocrine cells and assessing ER-to-Golgi transport. Overexpression of wild type or the familial disease-associated A53T mutant alpha-synuclein delayed transport by up to 50%; however, A53T inhibited more potently. The secretory delay occurred at low expression levels and was not accompanied by insoluble alpha-synuclein aggregates or mistargeting of transport machinery, suggesting a direct action of soluble alpha-synuclein on trafficking proteins. Co-overexpression of ER/Golgi arginine soluble N-ethylmaleimide-sensitive factor attachment protein receptors (R-SNAREs) specifically rescued transport, indicating that alpha-synuclein antagonizes SNARE function. Ykt6 reversed alpha-synuclein inhibition much more effectively than sec22b, suggesting a possible neuroprotective role for the enigmatic high expression of ykt6 in neurons. In in vitro reconstitutions, purified alpha-synuclein A53T protein specifically inhibited COPII vesicle docking and fusion at a pre-Golgi step. Finally, soluble alpha-synuclein A53T directly bound ER/Golgi SNAREs and inhibited SNARE complex assembly, providing a potential mechanism for toxic effects in the early secretory pathway.

  15. Energetics, kinetics, and pathway of SNARE folding and assembly revealed by optical tweezers.

    Science.gov (United States)

    Zhang, Yongli

    2017-07-01

    Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are universal molecular engines that drive membrane fusion. Particularly, synaptic SNAREs mediate fast calcium-triggered fusion of neurotransmitter-containing vesicles with plasma membranes for synaptic transmission, the basis of all thought and action. During membrane fusion, complementary SNAREs located on two apposed membranes (often called t- and v-SNAREs) join together to assemble into a parallel four-helix bundle, releasing the energy to overcome the energy barrier for fusion. A long-standing hypothesis suggests that SNAREs act like a zipper to draw the two membranes into proximity and thereby force them to fuse. However, a quantitative test of this SNARE zippering hypothesis was hindered by difficulties to determine the energetics and kinetics of SNARE assembly and to identify the relevant folding intermediates. Here, we first review different approaches that have been applied to study SNARE assembly and then focus on high-resolution optical tweezers. We summarize the folding energies, kinetics, and pathways of both wild-type and mutant SNARE complexes derived from this new approach. These results show that synaptic SNAREs assemble in four distinct stages with different functions: slow N-terminal domain association initiates SNARE assembly; a middle domain suspends and controls SNARE assembly; and rapid sequential zippering of the C-terminal domain and the linker domain directly drive membrane fusion. In addition, the kinetics and pathway of the stagewise assembly are shared by other SNARE complexes. These measurements prove the SNARE zippering hypothesis and suggest new mechanisms for SNARE assembly regulated by other proteins. © 2017 The Protein Society.

  16. Molecular Structures and Functional Relationships in Clostridial Neurotoxins

    Energy Technology Data Exchange (ETDEWEB)

    Swaminathan S.

    2011-12-01

    The seven serotypes of Clostridium botulinum neurotoxins (A-G) are the deadliest poison known to humans. They share significant sequence homology and hence possess similar structure-function relationships. Botulinum neurotoxins (BoNT) act via a four-step mechanism, viz., binding and internalization to neuronal cells, translocation of the catalytic domain into the cytosol and finally cleavage of one of the three soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) causing blockage of neurotransmitter release leading to flaccid paralysis. Crystal structures of three holotoxins, BoNT/A, B and E, are available to date. Although the individual domains are remarkably similar, their domain organization is different. These structures have helped in correlating the structural and functional domains. This has led to the determination of structures of individual domains and combinations of them. Crystal structures of catalytic domains of all serotypes and several binding domains are now available. The catalytic domains are zinc endopeptidases and share significant sequence and structural homology. The active site architecture and the catalytic mechanism are similar although the binding mode of individual substrates may be different, dictating substrate specificity and peptide cleavage selectivity. Crystal structures of catalytic domains with substrate peptides provide clues to specificity and selectivity unique to BoNTs. Crystal structures of the receptor domain in complex with ganglioside or the protein receptor have provided information about the binding of botulinum neurotoxin to the neuronal cell. An overview of the structure-function relationship correlating the 3D structures with biochemical and biophysical data and how they can be used for structure-based drug discovery is presented here.

  17. Eosinophil cytokines: Emerging roles in immunity

    Directory of Open Access Journals (Sweden)

    Paige eLacy

    2014-11-01

    Full Text Available Eosinophils derive from the bone marrow and circulate at low levels in the blood in healthy individuals. These granulated cells preferentially leave the circulation and marginate to tissues, where they are implicated in the regulation of innate and adaptive immunity. In diseases such as allergic inflammation, eosinophil numbers escalate markedly in the blood and tissues where inflammatory foci are located. Eosinophils possess a range of immunomodulatory factors that are released upon cell activation, including over 35 cytokines, growth factors, and chemokines. Unlike T and B cells, eosinophils can rapidly release cytokines within minutes in response to stimulation. While some cytokines are stored as preformed mediators in crystalloid granules and secretory vesicles, eosinophils are also capable of undergoing de novo synthesis and secretion of these immunological factors. Some of the molecular mechanisms that coordinate the final steps of cytokine secretion are hypothesized to involve binding of membrane fusion complexes comprised of soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNAREs. These intracellular receptors regulate the release of granules and vesicles containing a range of secreted proteins, among which are cytokines and chemokines. Emerging evidence from both human and animal model-based research has suggested an active participation of eosinophils in several physiological/pathological processes such as immunomodulation and tissue remodeling. The observed eosinophil effector functions in health and disease implicate eosinophil cytokine secretion as a fundamental immunoregulatory process. The focus of this review is to describe the cytokines, growth factors, and chemokines that are elaborated by eosinophils, and to illustrate some of the intracellular events leading to the release of eosinophil-derived cytokines.

  18. The Qb-SNARE Memb11 interacts specifically with Arf1 in the Golgi apparatus of Arabidopsis thaliana.

    Science.gov (United States)

    Marais, Claireline; Wattelet-Boyer, Valérie; Bouyssou, Guillaume; Hocquellet, Agnès; Dupuy, Jean-William; Batailler, Brigitte; Brocard, Lysiane; Boutté, Yohann; Maneta-Peyret, Lilly; Moreau, Patrick

    2015-11-01

    The SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins are critical for the function of the secretory pathway. The SNARE Memb11 is involved in membrane trafficking at the ER-Golgi interface. The aim of the work was to decipher molecular mechanisms acting in Memb11-mediated ER-Golgi traffic. In mammalian cells, the orthologue of Memb11 (membrin) is potentially involved in the recruitment of the GTPase Arf1 at the Golgi membrane. However molecular mechanisms associated to Memb11 remain unknown in plants. Memb11 was detected mainly at the cis-Golgi and co-immunoprecipitated with Arf1, suggesting that Arf1 may interact with Memb11. This interaction of Memb11 with Arf1 at the Golgi was confirmed by in vivo BiFC (Bimolecular Fluorescence Complementation) experiments. This interaction was found to be specific to Memb11 as compared to either Memb12 or Sec22. Using a structural bioinformatic approach, several sequences in the N-ter part of Memb11 were hypothesized to be critical for this interaction and were tested by BiFC on corresponding mutants. Finally, by using both in vitro and in vivo approaches, we determined that only the GDP-bound form of Arf1 interacts with Memb11. Together, our results indicate that Memb11 interacts with the GDP-bound form of Arf1 in the Golgi apparatus. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

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

    Science.gov (United States)

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

    2018-02-16

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

  20. Mechanisms of Intestinal Serotonin Transporter (SERT Upregulation by TGF-β1 Induced Non-Smad Pathways.

    Directory of Open Access Journals (Sweden)

    Saad Nazir

    Full Text Available TGF-β1 is an important multifunctional cytokine with numerous protective effects on intestinal mucosa. The influence of TGF-β1 on serotonin transporter (SERT activity, the critical mechanism regulating the extracellular availability of serotonin (5-HT, is not known. Current studies were designed to examine acute effects of TGF-β1 on SERT. Model human intestinal Caco-2 cells grown as monolayer's or as cysts in 3D culture and ex vivo mouse model were utilized. Treatment of Caco-2 cells with TGF-β1 (10 ng/ml, 60 min stimulated SERT activity (~2 fold, P<0.005. This stimulation of SERT function was dependent upon activation of TGF-β1 receptor (TGFRI as SB-431542, a specific TGF-βRI inhibitor blocked the SERT stimulation. SERT activation in response to TGF-β1 was attenuated by inhibition of PI3K and occurred via enhanced recruitment of SERT-GFP to apical surface in a PI3K dependent manner. The exocytosis inhibitor brefeldin A (2.5 μM attenuated the TGF-β1-mediated increase in SERT function. TGF-β1 increased the association of SERT with the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE syntaxin 3 (STX3 and promoted exocytosis of SERT. Caco-2 cells grown as cysts in 3D culture recapitulated the effects of TGF-β1 showing increased luminal staining of SERT. Ussing chamber studies revealed increase in 3H-5-HT uptake in mouse ileum treated ex vivo with TGF-β1 (10 ng/ml, 1h. These data demonstrate a novel mechanism rapidly regulating intestinal SERT via PI3K and STX3. Since decreased SERT is implicated in various gastro-intestinal disorders e.g IBD, IBS and diarrhea, understanding mechanisms stimulating SERT function by TGF-β1 offers a novel therapeutic strategy to treat GI disorders.

  1. Structural And Biochemical Studies of Botulinum Neurotoxin Serotype C1 Light Chain Protease: Implications for Dual Substrate Specificity

    Energy Technology Data Exchange (ETDEWEB)

    Jin, R.; Sikorra, S.; Stegmann, C.M.; Pich, A.; Binz, T.; Brunger, A.T.

    2009-06-01

    Clostridial neurotoxins are the causative agents of the neuroparalytic disease botulism and tetanus. They block neurotransmitter release through specific proteolysis of one of the three soluble N-ethylmaleimide-sensitive-factor attachment protein receptors (SNAREs) SNAP-25, syntaxin, and synaptobrevin, which constitute part of the synaptic vesicle fusion machinery. The catalytic component of the clostridial neurotoxins is their light chain (LC), a Zn2+ endopeptidase. There are seven structurally and functionally related botulinum neurotoxins (BoNTs), termed serotype A to G, and tetanus neurotoxin (TeNT). Each of them exhibits unique specificity for their target SNAREs and peptide bond(s) they cleave. The mechanisms of action for substrate recognition and target cleavage are largely unknown. Here, we report structural and biochemical studies of BoNT/C1-LC, which is unique among BoNTs in that it exhibits dual specificity toward both syntaxin and SNAP-25. A distinct pocket (S1') near the active site likely achieves the correct register for the cleavage site by only allowing Ala as the P1' residue for both SNAP-25 and syntaxin. Mutations of this SNAP-25 residue dramatically reduce enzymatic activity. The remote a-exosite that was previously identified in the complex of BoNT/A-LC and SNAP-25 is structurally conserved in BoNT/C1. However, mutagenesis experiments show that the a-exosite of BoNT/C1 plays a less stringent role in substrate discrimination in comparison to that of BoNT/A, which could account for its dual substrate specificity.

  2. Synaptotagmin-7 Functions to Replenish Insulin Granules for Exocytosis in Human Islet β-Cells.

    Science.gov (United States)

    Dolai, Subhankar; Xie, Li; Zhu, Dan; Liang, Tao; Qin, Tairan; Xie, Huanli; Kang, Youhou; Chapman, Edwin R; Gaisano, Herbert Y

    2016-07-01

    Synaptotagmin (Syt)-7, a major component of the exocytotic machinery in neurons, is also the major Syt in rodent pancreatic β-cells shown to mediate glucose-stimulated insulin secretion (GSIS). However, Syt-7's precise exocytotic actions in β-cells remain unknown. We show that Syt-7 is abundant in human β-cells. Adenovirus-short hairpin RNA knockdown (KD) of Syt-7 in human islets reduced first- and second-phase GSIS attributed to the reduction of exocytosis of predocked and newcomer insulin secretory granules (SGs). Glucose stimulation expectedly induced Syt-7 association in a Ca(2+)-dependent manner with syntaxin-3 and syntaxin-1A soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes known to mediate exocytosis of newcomer and predocked SGs, respectively. However, Syt-7-KD did not disrupt SNARE complex assembly. Instead, electron microscopy analysis showed that Syt-7-KD reduced the recruitment of SGs to the plasma membrane after glucose-stimulated depletion, which could not be rescued by glucagon-like peptide 1 pretreatment. To assess the possibility that this new action of Syt-7 on SG recruitment may involve calmodulin (CaM), pretreatment of islets with CaM blocker calmidazolium showed effects very similar to those of Syt-7-KD. Syt-7 therefore plays a novel more dominant function in the replenishment of releasable SG pools in human β-cells than its previously purported role in exocytotic fusion per se. © 2016 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.

  3. TaSYP71, a Qc-SNARE, Contributes to Wheat Resistance against Puccinia striiformis f. sp. tritici

    Directory of Open Access Journals (Sweden)

    Minjie eLiu

    2016-04-01

    Full Text Available N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs are involved in plant resistance; however, the role of SYP71 in the regulation of plant–pathogen interactions is not well known. In this study, we characterized a plant-specific SNARE in wheat, TaSYP71, which contains a Qc-SNARE domain. Three homologues are localized on chromosome 1AL, 1BL and 1DL. Using Agrobacterium-mediated transient expression, TaSYP71 was localized to the plasma membrane in Nicotiana benthamiana. Quantitative real-time PCR assays revealed that TaSYP71 homologues was induced by NaCl, H2O2 stress and infection by virulent and avirulent Puccinia striiformis f. sp. tritici (Pst isolates. Heterologous expression of TaSYP71 in Schizosaccharomyces pombe elevated tolerance to H2O2. Meanwhile, H2O2 scavenging gene (TaCAT was downregulated in TaSYP71 silenced plants treated by H2O2 compared to that in control, which indicated that TaSYP71 enhanced tolerance to H2O2 stress possibly by influencing the expression of TaCAT to remove the excessive H2O2 accumulation. When TaSYP71 homologues were all silenced in wheat by the virus-induced gene silencing system, wheat plants were more susceptible to Pst, with larger infection area and more haustoria number, but the necrotic area of wheat mesophyll cells were larger, one possible explanation that minor contribution of resistance to Pst was insufficient to hinder pathogen extension when TaSYP71were silenced, and the necrotic area was enlarged accompanied with the pathogen growth. Of course, later cell death could not be excluded. In addition, the expression of pathogenesis-related genes were down-regulated in TaSYP71 silenced wheat plants. These results together suggest that TaSYP71 play a positive role in wheat defence against Pst.

  4. SNAP23-Dependent Surface Translocation of Leukotriene B4 (LTB4) Receptor 1 Is Essential for NOX2-Mediated Exocytotic Degranulation in Human Mast Cells Induced by Trichomonas vaginalis-Secreted LTB4.

    Science.gov (United States)

    Min, Arim; Lee, Young Ah; Kim, Kyeong Ah; El-Benna, Jamel; Shin, Myeong Heon

    2017-01-01

    Trichomonas vaginalis is a sexually transmitted parasite that causes vaginitis in women and itself secretes lipid mediator leukotriene B4 (LTB4). Mast cells are important effector cells of tissue inflammation during infection with parasites. Membrane-bridging SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complexes are critical for fusion during exocytosis. Although T. vaginalis-derived secretory products (TvSP) have been shown to induce exocytosis in mast cells, information regarding the signaling mechanisms between mast cell activation and TvSP is limited. In this study, we found that SNAP23-dependent surface trafficking of LTB4 receptor 1 (BLT1) is required for nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2)-mediated exocytotic degranulation of mast cells induced by TvSP. First, stimulation with TvSP induced exocytotic degranulation and reactive oxygen species (ROS) generation in HMC-1 cells. Next, TvSP-induced ROS generation and exocytosis were strongly inhibited by transfection of BLT1 small interfering RNA (siRNA). TvSP induced trafficking of BLT1 from the cytosol to the plasma membrane. We also found that knockdown of SNAP23 abrogated TvSP-induced ROS generation, exocytosis, and surface trafficking of BLT1 in HMC-1 cells. By coimmunoprecipitation, there was a physical interaction between BLT1 and SNAP23 in TvSP-stimulated HMC-1 cells. Taken together, our results suggest that SNAP23-dependent surface trafficking of BLT1 is essential for exocytosis in human mast cells induced by T. vaginalis-secreted LTB4 Our data collectively demonstrate a novel regulatory mechanism for SNAP23-dependent mast cell activation of T. vaginalis-secreted LTB4 involving surface trafficking of BLT1. These results can help to explain how the cross talk mechanism between parasite and host can govern deliberately tissue inflammatory responses. Copyright © 2016 American Society for Microbiology.

  5. GABAB receptor deficiency causes failure of neuronal homeostasis in hippocampal networks.

    Science.gov (United States)

    Vertkin, Irena; Styr, Boaz; Slomowitz, Edden; Ofir, Nir; Shapira, Ilana; Berner, David; Fedorova, Tatiana; Laviv, Tal; Barak-Broner, Noa; Greitzer-Antes, Dafna; Gassmann, Martin; Bettler, Bernhard; Lotan, Ilana; Slutsky, Inna

    2015-06-23

    Stabilization of neuronal activity by homeostatic control systems is fundamental for proper functioning of neural circuits. Failure in neuronal homeostasis has been hypothesized to underlie common pathophysiological mechanisms in a variety of brain disorders. However, the key molecules regulating homeostasis in central mammalian neural circuits remain obscure. Here, we show that selective inactivation of GABAB, but not GABA(A), receptors impairs firing rate homeostasis by disrupting synaptic homeostatic plasticity in hippocampal networks. Pharmacological GABA(B) receptor (GABA(B)R) blockade or genetic deletion of the GB(1a) receptor subunit disrupts homeostatic regulation of synaptic vesicle release. GABA(B)Rs mediate adaptive presynaptic enhancement to neuronal inactivity by two principle mechanisms: First, neuronal silencing promotes syntaxin-1 switch from a closed to an open conformation to accelerate soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex assembly, and second, it boosts spike-evoked presynaptic calcium flux. In both cases, neuronal inactivity removes tonic block imposed by the presynaptic, GB(1a)-containing receptors on syntaxin-1 opening and calcium entry to enhance probability of vesicle fusion. We identified the GB(1a) intracellular domain essential for the presynaptic homeostatic response by tuning intermolecular interactions among the receptor, syntaxin-1, and the Ca(V)2.2 channel. The presynaptic adaptations were accompanied by scaling of excitatory quantal amplitude via the postsynaptic, GB(1b)-containing receptors. Thus, GABA(B)Rs sense chronic perturbations in GABA levels and transduce it to homeostatic changes in synaptic strength. Our results reveal a novel role for GABA(B)R as a key regulator of population firing stability and propose that disruption of homeostatic synaptic plasticity may underlie seizure's persistence in the absence of functional GABA(B)Rs.

  6. Atomoxetine affects transcription/translation of the NMDA receptor and the norepinephrine transporter in the rat brain – an in vivo study

    Directory of Open Access Journals (Sweden)

    Udvardi PT

    2013-12-01

    Full Text Available Patrick T Udvardi,1,2 Karl J Föhr,3 Carolin Henes,1,2 Stefan Liebau,2 Jens Dreyhaupt,4 Tobias M Boeckers,2 Andrea G Ludolph11Department of Child and Adolescent Psychiatry and Psychotherapy, 2Institute of Anatomy and Cell Biology, 3Department of Anaesthesiology, 4Institute of Epidemiology and Medical Biometry, University of Ulm, Ulm, GermanyAbstract: Attention-deficit/hyperactivity disorder (ADHD is the most frequently diagnosed neurodevelopmental disorder. The norepinephrine transporter (NET inhibitor atomoxetine, the first nonstimulant drug licensed for ADHD treatment, also acts as an N-methyl-D-aspartate receptor (NMDAR antagonist. The compound's effects on gene expression and protein levels of NET and NMDAR subunits (1, 2A, and 2B are unknown. Therefore, adolescent Sprague Dawley rats were treated with atomoxetine (3 mg/kg, intraperitoneal injection [ip] or saline (0.9%, ip for 21 consecutive days on postnatal days (PND 21–41. In humans, atomoxetine's earliest clinical therapeutic effects emerge after 2–3 weeks. Material from prefrontal cortex, striatum (STR, mesencephalon (MES, and hippocampus (HC was analyzed either directly after treatment (PND 42 or 2 months after termination of treatment (PND 101 to assess the compound's long-term effects. In rat brains analyzed immediately after treatment, protein analysis exhibited decreased levels of the NET in HC, and NMDAR subunit 2B in both STR and HC; the transcript levels were unaltered. In rat brains probed 2 months after final atomoxetine exposure, messenger RNA analysis also revealed significantly reduced levels of genes coding for NMDAR subunits in MES and STR. NMDAR protein levels were reduced in STR and HC. Furthermore, the levels of two SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor proteins, synaptophysin and synaptosomal-associated protein 25, were also significantly altered in both treatment groups. This in vivo study detected atomoxetine's effects

  7. The Root Hair Specific SYP123 Regulates the Localization of Cell Wall Components and Contributes to Rizhobacterial Priming of Induced Systemic Resistance

    Directory of Open Access Journals (Sweden)

    Cecilia Rodriguez-Furlán

    2016-07-01

    Full Text Available Root hairs are important for nutrient and water uptake and are also critically involved the interaction with soil inhabiting microbiota. Root hairs are tubular-shaped outgrowths that emerge from trichoblasts. This polarized elongation is maintained and regulated by a robust mechanism involving the endomembrane secretory and endocytic system. Members of the syntaxin family of SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptor in plants (SYP, have been implicated in regulation of the fusion of vesicles with the target membranes in both exocytic and endocytic pathways. One member of this family, SYP123, is expressed specifically in the root hairs and accumulated in the growing tip region. This study shows evidence of the SYP123 role in polarized trafficking using knockout insertional mutant plants. We were able to observe defects in the deposition of cell wall proline rich protein PRP3 and cell wall polysaccharides. In a complementary strategy, similar results were obtained using a plant expressing a dominant negative soluble version of SYP123 (SP2 fragment lacking the transmembrane domain. The evidence presented indicates that SYP123 is also regulating PRP3 protein distribution by recycling by endocytosis. We also present evidence that indicates that SYP123 is necessary for the response of roots to plant growth promoting rhizobacterium (PGPR in order to trigger trigger induced systemic response (ISR. Plants with a defective SYP123 function were unable to mount a systemic acquired resistance (SAR in response to bacterial pathogen infection and induced systemic resistance (ISR upon interaction with rhizobacteria. These results indicated that SYP123 was involved in the polarized localization of protein and polysaccharides in growing root hairs and that this activity also contributed to the establishment of effective plant defense responses. Root hairs represent very plastic structures were many biotic and abiotic factors

  8. Syntaxin-4 is essential for IgE secretion by plasma cells

    Energy Technology Data Exchange (ETDEWEB)

    Rahman, Arman; DeCourcey, Joseph; Larbi, Nadia Ben [Immunomodulation Group, School of Biotechnology, Dublin City University (Ireland); Loughran, Sinéad T.; Walls, Dermot [School of Biotechnology and National Centre for Sensor Research, Dublin City University (Ireland); Loscher, Christine E., E-mail: christine.loscher@dcu.ie [Immunomodulation Group, School of Biotechnology, Dublin City University (Ireland)

    2013-10-11

    Highlights: •Knock-down of syntaxin-4 in U266 plasma cells resulted in reduction of IgE secretion. •Knock-down of syntaxin-4 also leads to the accumulation of IgE in the cell. •Immuno-fluorescence staining shows co-localisation of IgE and syntaxin-4 in U266 cells. •Findings suggest a critical requirement for syntaxin-4 in IgE secretion from plasma cells. -- Abstract: The humoral immune system provides a crucial first defense against the invasion of microbial pathogens via the secretion of antigen specific immunoglobulins (Ig). The secretion of Ig is carried out by terminally differentiated B-lymphocytes called plasma cells. Despite the key role of plasma cells in the immune response, the mechanisms by which they constitutively traffic large volumes of Ig out of the cell is poorly understood. The involvement of Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins in the regulation of protein trafficking from cells has been well documented. Syntaxin-4, a member of the Qa SNARE syntaxin family has been implicated in fusion events at the plasma membrane in a number of cells in the immune system. In this work we show that knock-down of syntaxin-4 in the multiple myeloma U266 human plasma cell line results in a loss of IgE secretion and accumulation of IgE within the cells. Furthermore, we show that IgE co-localises with syntaxin-4 in U266 plasma cells suggesting direct involvement in secretion at the plasma membrane. This study demonstrates that syntaxin-4 plays a critical role in the secretion of IgE from plasma cells and sheds some light on the mechanisms by which these cells constitutively traffic vesicles to the surface for secretion. An understanding of this machinery may be beneficial in identifying potential therapeutic targets in multiple myeloma and autoimmune disease where over-production of Ig leads to severe pathology in patients.

  9. SNAP25 is associated with schizophrenia and major depressive disorder in the Han Chinese population.

    Science.gov (United States)

    Wang, Qingzhong; Wang, Yanlin; Ji, Weidong; Zhou, Guoquan; He, Kuanjun; Li, Zhiqiang; Chen, Jianhua; Li, Wenjin; Wen, Zujia; Shen, Jiawei; Qiang, Yu; Ji, Jue; Wang, Yujiong; Shi, Yongyong; Yi, Qizhong; Wang, Yonggang

    2015-01-01

    Synaptosomal-associated protein of 25 kDa (SNAP25) is a member of the soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) protein complex, which plays essential roles in the modulation of different voltage-gated calcium channels and neurotransmitter release. Many previous studies have reported the SNAP25 gene to be significantly associated with attention-deficit/hyperactivity disorder (ADHD). Recently, shared genetic variants have been demonstrated in 5 major psychiatric disorders, including schizophrenia, major depressive disorder, bipolar disorder, autism spectrum disorders, and ADHD. However, no compelling, convincing evidence has suggested an association between SNAP25 and schizophrenia or major depressive disorder. Thus, we investigated the association between SNAP25 and both schizophrenia and major depressive disorder in the Han Chinese population. We performed a large-scale case-control study to test the association between SNAP25 and 2 major mental disorders, schizophrenia (DSM-IV criteria) and major depressive disorder (DSM-IV criteria), in the Han Chinese population. Seven single-nucleotide polymorphisms (SNPs) were genotyped in 1,330 schizophrenia patients, 1,045 major depressive disorder patients, and 1,520 healthy controls of Han Chinese origin. Two SNPs, rs3787283 and rs3746544, were found to be associated with both schizophrenia (rs3746544, adjusted P = .00257) and major depressive disorder (rs3746544, adjusted P = .0485; rs3787283, adjusted P = .00387) in this study. The AG haplotype consisting of rs3787283 and rs3746544 was also significantly associated with both schizophrenia and major depressive disorder (schizophrenia: adjusted P = .0126; major depressive disorder: adjusted P = .000580). Additionally, we carried out a meta-analysis of the current data and published association results and further confirmed the association between rs3746544 and schizophrenia (Pmeta = .002, ORmeta = 1.213 [95% CI, 1.077-1.367]). Our results

  10. The assembly of lipid droplets and its relation to cellular insulin sensitivity

    DEFF Research Database (Denmark)

    Boström, Pontus; Andersson, Linda; Li, Lu

    2009-01-01

    to be transported on microtubules. Lipid droplets grow in size by fusion, which is dependent on dynein and the transfer on microtubules, and is catalysed by the SNARE (soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptor) proteins SNAP-23 (23 kDa synaptosome-associated protein), syntaxin-5...... and VAMP-4 (vesicle-associated protein 4). SNAP-23 is also involved in the insulin-dependent translocation of the glucose transporter GLUT4 to the plasma membrane. Fatty acids induce a missorting of SNAP-23, from the plasma membrane to the interior of the cell, resulting in cellular insulin resistance...... that can be overcome by increasing the levels of SNAP-23. The same missorting of SNAP-23 occurs in vivo in skeletal-muscle biopsies from patients with T2D (Type 2 diabetes). Moreover, there was a linear relation between the amount of SNAP-23 in the plasma membrane from human skeletal-muscles biopsies...

  11. Multiple Ca2+ sensors in secretion

    DEFF Research Database (Denmark)

    Walter, Alexander M; Groffen, Alexander J; Sørensen, Jakob Balslev

    2011-01-01

    Regulated neurotransmitter secretion depends on Ca(2+) sensors, C2 domain proteins that associate with phospholipids and soluble N-ethylmaleimide-sensitive fusion attachment protein receptor (SNARE) complexes to trigger release upon Ca(2+) binding. Ca(2+) sensors are thought to prevent spontaneous...... fusion at rest (clamping) and to promote fusion upon Ca(2+) activation. At least eight, often coexpressed, Ca(2+) sensors have been identified in mammals. Accumulating evidence suggests that multiple Ca(2+) sensors interact, rather than work autonomously, to produce the complex secretory response...... observed in neurons and secretory cells. In this review, we present several working models to describe how different sensors might be arranged to mediate synchronous, asynchronous and spontaneous neurotransmitter release. We discuss the scenario that different Ca(2+) sensors typically act on one shared...

  12. Actin-Sorting Nexin 27 (SNX27)-Retromer Complex Mediates Rapid Parathyroid Hormone Receptor Recycling*

    Science.gov (United States)

    McGarvey, Jennifer C.; Xiao, Kunhong; Bowman, Shanna L.; Mamonova, Tatyana; Zhang, Qiangmin; Bisello, Alessandro; Sneddon, W. Bruce; Ardura, Juan A.; Jean-Alphonse, Frederic; Vilardaga, Jean-Pierre; Puthenveedu, Manojkumar A.; Friedman, Peter A.

    2016-01-01

    The G protein-coupled parathyroid hormone receptor (PTHR) regulates mineral-ion homeostasis and bone remodeling. Upon parathyroid hormone (PTH) stimulation, the PTHR internalizes into early endosomes and subsequently traffics to the retromer complex, a sorting platform on early endosomes that promotes recycling of surface receptors. The C terminus of the PTHR contains a type I PDZ ligand that binds PDZ domain-containing proteins. Mass spectrometry identified sorting nexin 27 (SNX27) in isolated endosomes as a PTHR binding partner. PTH treatment enriched endosomal PTHR. SNX27 contains a PDZ domain and serves as a cargo selector for the retromer complex. VPS26, VPS29, and VPS35 retromer subunits were isolated with PTHR in endosomes from cells stimulated with PTH. Molecular dynamics and protein binding studies establish that PTHR and SNX27 interactions depend on the PDZ recognition motif in PTHR and the PDZ domain of SNX27. Depletion of either SNX27 or VPS35 or actin depolymerization decreased the rate of PTHR recycling following agonist stimulation. Mutating the PDZ ligand of PTHR abolished the interaction with SNX27 but did not affect the overall rate of recycling, suggesting that PTHR may directly engage the retromer complex. Coimmunoprecipitation and overlay experiments show that both intact and mutated PTHR bind retromer through the VPS26 protomer and sequentially assemble a ternary complex with PTHR and SNX27. SNX27-independent recycling may involve N-ethylmaleimide-sensitive factor, which binds both PDZ intact and mutant PTHRs. We conclude that PTHR recycles rapidly through at least two pathways, one involving the ASRT complex of actin, SNX27, and retromer and another possibly involving N-ethylmaleimide-sensitive factor. PMID:27008860

  13. Protein Foods

    Science.gov (United States)

    ... Text Size: A A A Listen En Español Protein Foods Foods high in protein such as fish, ... for the vegetarian proteins, whether they have carbohydrate. Protein Choices Plant-Based Proteins Plant-based protein foods ...

  14. Protein-protein interactions

    DEFF Research Database (Denmark)

    Byron, Olwyn; Vestergaard, Bente

    2015-01-01

    Responsive formation of protein:protein interaction (PPI) upon diverse stimuli is a fundament of cellular function. As a consequence, PPIs are complex, adaptive entities, and exist in structurally heterogeneous interplays defined by the energetic states of the free and complexed protomers. The bi...

  15. Proteomic profiling of the hypothalamus in a mouse model of cancer-induced anorexia-cachexia.

    Science.gov (United States)

    Ihnatko, R; Post, C; Blomqvist, A

    2013-10-01

    Anorexia-cachexia is a common and severe cancer-related complication but the underlying mechanisms are largely unknown. Here, using a mouse model for tumour-induced anorexia-cachexia, we screened for proteins that are differentially expressed in the hypothalamus, the brain's metabolic control centre. The hypothalamus of tumour-bearing mice with implanted methylcholanthrene-induced sarcoma (MCG 101) displaying anorexia and their sham-implanted pair-fed or free-fed littermates was examined using two-dimensional electrophoresis (2-DE)-based comparative proteomics. Differentially expressed proteins were identified by liquid chromatography-tandem mass spectrometry. The 2-DE data showed an increased expression of dynamin 1, hexokinase, pyruvate carboxylase, oxoglutarate dehydrogenase, and N-ethylmaleimide-sensitive factor in tumour-bearing mice, whereas heat-shock 70 kDa cognate protein, selenium-binding protein 1, and guanine nucleotide-binding protein Gα0 were downregulated. The expression of several of the identified proteins was similarly altered also in the caloric-restricted pair-fed mice, suggesting an involvement of these proteins in brain metabolic adaptation to restricted nutrient availability. However, the expression of dynamin 1, which is required for receptor internalisation, and of hexokinase, and pyruvate carboxylase were specifically changed in tumour-bearing mice with anorexia. The identified differentially expressed proteins may be new candidate molecules involved in the pathophysiology of tumour-induced anorexia-cachexia.

  16. Differential roles for NSF and GRIP/ABP in AMPA receptor cycling.

    Science.gov (United States)

    Braithwaite, Steven P; Xia, Houhui; Malenka, Robert C

    2002-05-14

    alpha-Amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor (AMPAR) stability and movement at synapses are important factors controlling synaptic strength. Here, we study the roles of proteins [N-ethylmaleimide-sensitive fusion protein (NSF), glutamate receptor AMPAR binding protein (ABP)-interacting protein (GRIP)/(ABP), and protein interacting with C-kinase-1 (PICK1) that interact with the GluR2 subunit in the control of the surface expression and cycling of AMPARs. Epitope-tagged GluR2 formed functional receptors that exhibited targeting to synaptic sites. Constructs in which binding to NSF, PDZ proteins (GRIP/ABP and PICK1), or GRIP/ABP alone was eliminated each exhibited normal surface targeting and constitutive cycling. The lack of NSF binding, however, resulted in receptors that were endocytosed to a greater extent than wild-type receptors in response to application of AMPA or N-methyl-d-aspartate (NMDA). Conversely, the behavior of the GluR2 mutants incapable of binding to GRIP/ABP suggests that these PDZ proteins play a role in the stabilization of an intracellular pool of AMPARs that have been internalized on stimulation, thus inhibiting their recycling to the synaptic membrane. These results provide further evidence for distinct functional roles of GluR2-interacting proteins in AMPAR trafficking.

  17. Complexin regulates the closure of the fusion pore during regulated vesicle exocytosis.

    Science.gov (United States)

    Archer, Deborah A; Graham, Margaret E; Burgoyne, Robert D

    2002-05-24

    Membrane fusion during exocytosis and throughout the cell is believed to involve members of the SNARE (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors) family of proteins. The assembly of these proteins into a four-helix bundle may be part of the driving force for bilayer fusion. Regulated exocytosis in neurons and related cell types is specialized to be fast and Ca(2+)-dependent suggesting the involvement of other regulatory proteins specific for regulated exocytosis. Among these are the complexins, two closely related proteins that bind only to the assembled SNARE complex. We have investigated the function of complexin by analysis of single vesicle release events in adrenal chromaffin cells using carbon fiber amperometry. These cells express complexin II, and overexpression of this protein modified the kinetics of vesicle release events so that their time course was shortened. This effect depended on complexin interaction with the SNARE complex as introduction of a mutation of Arg-59, a residue that interacts with synaptobrevin in the SNARE complex, abolished its effects. The data are consistent with a function for complexin in stabilizing an intermediate of the SNARE complex to allow kiss-and-run recycling of the exocytosed vesicle.

  18. α-SNAP is expressed in mouse ovarian granulosa cells and plays a key role in folliculogenesis and female fertility.

    Science.gov (United States)

    Arcos, Alexis; Paola, Matilde de; Gianetti, Diego; Acuña, Diego; Velásquez, Zahady D; Miró, María Paz; Toro, Gabriela; Hinrichsen, Bryan; Muñoz, Rosa Iris; Lin, Yimo; Mardones, Gonzalo A; Ehrenfeld, Pamela; Rivera, Francisco J; Michaut, Marcela A; Batiz, Luis Federico

    2017-09-18

    The balance between ovarian folliculogenesis and follicular atresia is critical for female fertility and is strictly regulated by a complex network of neuroendocrine and intra-ovarian signals. Despite the numerous functions executed by granulosa cells (GCs) in ovarian physiology, the role of multifunctional proteins able to simultaneously coordinate/modulate several cellular pathways is unclear. Soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein (α-SNAP) is a multifunctional protein that participates in SNARE-mediated membrane fusion events. In addition, it regulates cell-to-cell adhesion, AMPK signaling, autophagy and apoptosis in different cell types. In this study we examined the expression pattern of α-SNAP in ovarian tissue and the consequences of α-SNAP (M105I) mutation (hyh mutation) in folliculogenesis and female fertility. Our results showed that α-SNAP protein is highly expressed in GCs and its expression is modulated by gonadotropin stimuli. On the other hand, α-SNAP-mutant mice show a reduction in α-SNAP protein levels. Moreover, increased apoptosis of GCs and follicular atresia, reduced ovulation rate, and a dramatic decline in fertility is observed in α-SNAP-mutant females. In conclusion, α-SNAP plays a critical role in the balance between follicular development and atresia. Consequently, a reduction in its expression/function (M105I mutation) causes early depletion of ovarian follicles and female subfertility.

  19. Evaluation of the heterogeneous reactivity of the syntaxin molecules on the inner leaflet of the plasma membrane.

    Science.gov (United States)

    Bar-On, Dana; Gutman, Menachem; Mezer, Aviv; Ashery, Uri; Lang, Thorsten; Nachliel, Esther

    2009-09-30

    The soluble N-ethylmaleimide-sensitive fusion (NSF) attachment protein (SNAP) receptor (SNARE) protein syntaxin 1A forms nano-sized clusters (membrane rafts) on the plasma membrane (PM) that are in equilibrium with freely diffusing syntaxin molecules. SNARE-complex formation between syntaxin 1A and SNAP-25 (synaptosome-associated protein of 25 kDa) on the PM and synaptobrevin 2 on the vesicles (trans-SNAREs) is crucial for vesicle priming and fusion. This process might be impeded by the spontaneous accumulation of non-fusogenic cis-SNARE complexes formed when all three SNARE proteins reside on the PM. We investigated the kinetics of cis-SNARE complex assembly and disassembly and both exhibited biphasic behavior. The experimental measurements were analyzed through integration of differential rate equations pertinent to the reaction mechanism and through the application of a heuristic search for time constants and concentrations using a genetic algorithm. Reconstruction of the measurements necessitated the partitioning of syntaxin into two phases that might represent the syntaxin clusters and free syntaxin outside the clusters. The analysis suggests that most of the syntaxin in the clusters is concentrated in a nonreactive form. Consequently, cis-SNARE complex assembly in the clusters is substantially slower than outside the rafts. Interestingly, the clusters also mediate efficient disassembly of cis-SNARE complexes possibly attributable to the high local concentration of complexes in the clusters area that allows efficient disassembly by the enzymatic reaction of NSF.

  20. Focal defects in single-celled tubes mutant for Cerebral cavernous malformation 3, GCKIII, or NSF2.

    Science.gov (United States)

    Song, Yanjun; Eng, Melissa; Ghabrial, Amin S

    2013-06-10

    Tubes of differing cellular architecture connect into networks. In the Drosophila tracheal system, two tube types connect within single cells (terminal cells); however, the genes that mediate this interconnection are unknown. Here we characterize two genes that are essential for this process: lotus, required for maintaining a connection between the tubes, and wheezy, required to prevent local tube dilation. We find that lotus encodes N-ethylmaleimide sensitive factor 2 (NSF2), whereas wheezy encodes Germinal center kinase III (GCKIII). GCKIIIs are effectors of Cerebral cavernous malformation 3 (CCM3), a protein mutated in vascular disease. Depletion of Ccm3 by RNA interference phenocopies wheezy; thus, CCM3 and GCKIII, which prevent capillary dilation in humans, prevent tube dilation in Drosophila trachea. Ectopic junctional and apical proteins are present in wheezy terminal cells, and we show that tube dilation is suppressed by reduction of NSF2, of the apical determinant Crumbs, or of septate junction protein Varicose. Copyright © 2013 Elsevier Inc. All rights reserved.

  1. Trachynilysin mediates SNARE-dependent release of catecholamines from chromaffin cells via external and stored Ca2+.

    Science.gov (United States)

    Meunier, F A; Mattei, C; Chameau, P; Lawrence, G; Colasante, C; Kreger, A S; Dolly, J O; Molgó, J

    2000-04-01

    Trachynilysin, a 159 kDa dimeric protein purified from stonefish (Synanceia trachynis) venom, dramatically increases spontaneous quantal transmitter release at the frog neuromuscular junction, depleting small clear synaptic vesicles, whilst not affecting large dense core vesicles. The basis of this insensitivity of large dense core vesicles exocytosis was examined using a fluorimetric assay to determine whether the toxin could elicit catecholamine release from bovine chromaffin cells. Unlike the case of the motor nerve endings, nanomolar concentrations of trachynilysin evoked sustained Soluble N-ethylmaleimide-sensitive fusion protein Attachment Protein REceptor-dependent exocytosis of large dense core vesicles, but only in the presence of extracellular Ca2+. However, this response to trachynilysin does not rely on Ca2+ influx through voltage-activated Ca2+ channels because the secretion was only slightly affected by blockers of L, N and P/Q types. Instead, trachynilysin elicited a localized increase in intracellular fluorescence monitored with fluo-3/AM, that precisely co-localized with the increase of fluorescence resulting from caffeine-induced release of Ca2+ from intracellular stores. Moreover, depletion of the latter stores inhibited trachynilysin-induced exocytosis. Thus, the observed requirement of external Ca2+ for stimulation of large dense core vesicles exocytosis from chromaffin cells implicates plasma membrane channels that signal efflux of Ca2+ from intracellular stores. This study also suggests that the bases of exocytosis of large dense core vesicles from motor nerve terminals and neuroendocrine cells are distinct.

  2. Total protein

    Science.gov (United States)

    ... page: //medlineplus.gov/ency/article/003483.htm Total protein To use the sharing features on this page, please enable JavaScript. The total protein test measures the total amount of two classes ...

  3. Protein Structure

    Science.gov (United States)

    Asmus, Elaine Garbarino

    2007-01-01

    Individual students model specific amino acids and then, through dehydration synthesis, a class of students models a protein. The students clearly learn amino acid structure, primary, secondary, tertiary, and quaternary structure in proteins and the nature of the bonds maintaining a protein's shape. This activity is fun, concrete, inexpensive and…

  4. Network motif comparison rationalizes Sec1/Munc18-SNARE regulation mechanism in exocytosis

    Directory of Open Access Journals (Sweden)

    Xia Tian

    2012-03-01

    Full Text Available Abstract Background Network motifs, recurring subnetwork patterns, provide significant insight into the biological networks which are believed to govern cellular processes. Methods We present a comparative network motif experimental approach, which helps to explain complex biological phenomena and increases the understanding of biological functions at the molecular level by exploring evolutionary design principles of network motifs. Results Using this framework to analyze the SM (Sec1/Munc18-SNARE (N-ethylmaleimide-sensitive factor activating protein receptor system in exocytic membrane fusion in yeast and neurons, we find that the SM-SNARE network motifs of yeast and neurons show distinct dynamical behaviors. We identify the closed binding mode of neuronal SM (Munc18-1 and SNARE (syntaxin-1 as the key factor leading to mechanistic divergence of membrane fusion systems in yeast and neurons. We also predict that it underlies the conflicting observations in SM overexpression experiments. Furthermore, hypothesis-driven lipid mixing assays validated the prediction. Conclusion Therefore this study provides a new method to solve the discrepancies and to generalize the functional role of SM proteins.

  5. VAMP-1, VAMP-2, and syntaxin-4 regulate ANP release from cardiac myocytes.

    Science.gov (United States)

    Ferlito, Marcella; Fulton, William B; Zauher, Mohamed A; Marbán, Eduardo; Steenbergen, Charles; Lowenstein, Charles J

    2010-11-01

    ANP is a peptide released by cardiac myocytes that regulates blood pressure and natriuresis. However, the molecular mechanisms controlling ANP release from cardiac myocytes are not defined. We now identify three components of the exocytic machinery that regulate ANP release from atrial myocytes. We found that cardiac myocytes express N-ethylmaleimide sensitive factor (NSF), soluble NSF attachment protein (α-SNAP), and SNAP receptors (SNAREs). Additionally we found that specific SNARE molecules, VAMP-1 and VAMP-2, both co-sediment and co-localize with ANP. Also, one SNARE molecule, syntaxin-4, partially co-sediments and partially co-localizes with ANP. Furthermore, these three SNAREs, syntaxin-4 and VAMP-1 and VAMP-2, form a SNARE complex inside cardiac myocytes. Finally, knockdown of VAMP-1, VAMP-2, or syntaxin-4 blocks regulated release of ANP. In contrast, silencing of VAMP-3 did not have an effect on ANP release. Our data suggest that three specific SNAREs regulate cardiac myocyte exocytosis of ANP. Pathways that modify the exocytic machinery may influence natriuresis and blood pressure. Copyright © 2010 Elsevier Ltd. All rights reserved.

  6. Yeast Lipin 1 Orthologue Pah1p Regulates Vacuole Homeostasis and Membrane Fusion*

    Science.gov (United States)

    Sasser, Terry; Qiu, Quan-Sheng; Karunakaran, Surya; Padolina, Mark; Reyes, Anna; Flood, Blake; Smith, Sheena; Gonzales, Chad; Fratti, Rutilio A.

    2012-01-01

    Vacuole homotypic fusion requires a group of regulatory lipids that includes diacylglycerol, a fusogenic lipid that is produced through multiple metabolic pathways including the dephosphorylation of phosphatidic acid (PA). Here we examined the relationship between membrane fusion and PA phosphatase activity. Pah1p is the single yeast homologue of the Lipin family of PA phosphatases. Deletion of PAH1 was sufficient to cause marked vacuole fragmentation and abolish vacuole fusion. The function of Pah1p solely depended on its phosphatase activity as complementation studies showed that wild type Pah1p restored fusion, whereas the phosphatase dead mutant Pah1pD398E had no effect. We discovered that the lack of PA phosphatase activity blocked fusion by inhibiting the binding of SNAREs to Sec18p, an N-ethylmaleimide-sensitive factor homologue responsible for priming inactive cis-SNARE complexes. In addition, pah1Δ vacuoles were devoid of the late endosome/vacuolar Rab Ypt7p, the phosphatidylinositol 3-kinase Vps34p, and Vps39p, a subunit of the HOPS (homotypic fusion and vacuole protein sorting) tethering complex, all of which are required for vacuole fusion. The lack of Vps34p resulted in the absence of phosphatidylinositol 3-phosphate, a lipid required for SNARE activity and vacuole fusion. These findings demonstrate that Pah1p and PA phosphatase activity are critical for vacuole homeostasis and fusion. PMID:22121197

  7. Yeast lipin 1 orthologue pah1p regulates vacuole homeostasis and membrane fusion.

    Science.gov (United States)

    Sasser, Terry; Qiu, Quan-Sheng; Karunakaran, Surya; Padolina, Mark; Reyes, Anna; Flood, Blake; Smith, Sheena; Gonzales, Chad; Fratti, Rutilio A

    2012-01-13

    Vacuole homotypic fusion requires a group of regulatory lipids that includes diacylglycerol, a fusogenic lipid that is produced through multiple metabolic pathways including the dephosphorylation of phosphatidic acid (PA). Here we examined the relationship between membrane fusion and PA phosphatase activity. Pah1p is the single yeast homologue of the Lipin family of PA phosphatases. Deletion of PAH1 was sufficient to cause marked vacuole fragmentation and abolish vacuole fusion. The function of Pah1p solely depended on its phosphatase activity as complementation studies showed that wild type Pah1p restored fusion, whereas the phosphatase dead mutant Pah1p(D398E) had no effect. We discovered that the lack of PA phosphatase activity blocked fusion by inhibiting the binding of SNAREs to Sec18p, an N-ethylmaleimide-sensitive factor homologue responsible for priming inactive cis-SNARE complexes. In addition, pah1Δ vacuoles were devoid of the late endosome/vacuolar Rab Ypt7p, the phosphatidylinositol 3-kinase Vps34p, and Vps39p, a subunit of the HOPS (homotypic fusion and vacuole protein sorting) tethering complex, all of which are required for vacuole fusion. The lack of Vps34p resulted in the absence of phosphatidylinositol 3-phosphate, a lipid required for SNARE activity and vacuole fusion. These findings demonstrate that Pah1p and PA phosphatase activity are critical for vacuole homeostasis and fusion.

  8. Lower brain-derived neurotrophic factor levels associated with worsening fatigue in prostate cancer patients during repeated stress from radiation therapy.

    Science.gov (United States)

    Saligan, L N; Lukkahatai, N; Holder, G; Walitt, B; Machado-Vieira, R

    2016-12-01

    Fatigue during cancer treatment is associated with depression. Neurotrophic factors play a major role in depression and stress and might provide insight into mechanisms of fatigue. This study investigated the association between plasma concentrations of three neurotrophic factors (BDNF, brain-derived neurotrophic factor; GDNF, glial-derived neurotrophic factor; and SNAPIN, soluble N-ethylmaleimide sensitive fusion attachment receptor-associated protein) and initial fatigue intensification during external beam radiation therapy (EBRT) in euthymic non-metastatic prostate cancer men. Fatigue, as measured by the 13-item Functional Assessment of Cancer Therapy-Fatigue (FACT-F), and plasma neurotrophic factors were collected at baseline (prior to EBRT) and mid-EBRT. Subjects were categorized into fatigue and no fatigue groups using a > 3-point change in FACT-F scores between the two time points. Multiple linear regressions analysed the associations between fatigue and neurotrophic factors. FACT-F scores of 47 subjects decreased from baseline (43.95 ± 1.3) to mid-EBRT (38.36 ± 1.5, P < 0.001), indicating worsening fatigue. SNAPIN levels were associated with fatigue scores (rs = 0.43, P = 0.005) at baseline. A significant decrease of BDNF concentration (P = 0.008) was found in fatigued subjects during EBRT (n = 39). Baseline SNAPIN and decreasing BDNF levels may influence worsening fatigue during EBRT. Further investigations are warranted to confirm their role in the pathophysiology and therapeutics of fatigue.

  9. Synaptophysin 1 Clears Synaptobrevin 2 from the Presynaptic Active Zone to Prevent Short-Term Depression

    Directory of Open Access Journals (Sweden)

    Rajit Rajappa

    2016-02-01

    Full Text Available Release site clearance is an important process during synaptic vesicle (SV recycling. However, little is known about its molecular mechanism. Here we identify self-assembly of exocytosed Synaptobrevin 2 (Syb2 and Synaptophysin 1 (Syp1 by homo- and hetero-oligomerization into clusters as key mechanisms mediating release site clearance for preventing cis-SNARE complex formation at the active zone (AZ. In hippocampal neurons from Syp1 knockout mice, neurons expressing a monomeric Syb2 mutant, or after acute block of the ATPase N-ethylmaleimide-sensitive factor (NSF, responsible for cis-SNARE complex disassembly, we found strong frequency-dependent short-term depression (STD, whereas retrieval of Syb2 by compensatory endocytosis was only affected weakly. Defects in Syb2 endocytosis were stimulus- and frequency-dependent, indicating that Syp1 is not essential for Syb2 retrieval, but for its efficient clearance upstream of endocytosis. Our findings identify an SV protein as a release site clearance factor.

  10. α-SNAP Enhances SNARE Zippering by Stabilizing the SNARE Four-Helix Bundle.

    Science.gov (United States)

    Ma, Lu; Kang, Yuhao; Jiao, Junyi; Rebane, Aleksander A; Cha, Hyo Keun; Xi, Zhiqun; Qu, Hong; Zhang, Yongli

    2016-04-19

    Intracellular membrane fusion is mediated by dynamic assembly and disassembly of soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein (SNAP) receptors (SNAREs). α-SNAP guides NSF to disassemble SNARE complexes after membrane fusion. Recent experiments showed that α-SNAP also dramatically enhances SNARE assembly and membrane fusion. How α-SNAP is involved in these opposing activities is not known. Here, we examine the effect of α-SNAP on the stepwise assembly of the synaptic SNARE complex using optical tweezers. We found that α-SNAP destabilized the linker domain (LD) of the SNARE complex but stabilized its C-terminal domain (CTD) through a conformational selection mechanism. In contrast, α-SNAP minimally affected assembly of the SNARE N-terminal domain (NTD), indicating that α-SNAP barely bound the partially assembled trans-SNARE complex. Thus, α-SNAP recognizes the folded CTD for SNARE disassembly with NSF and subtly modulates membrane fusion by altering the stabilities of the SNARE CTD and LD. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  11. P120-Catenin Regulates Early Trafficking Stages of the N-Cadherin Precursor Complex.

    Directory of Open Access Journals (Sweden)

    Diana P Wehrendt

    Full Text Available It is well established that binding of p120 catenin to the cytoplasmic domain of surface cadherin prevents cadherin endocytosis and degradation, contributing to cell-cell adhesion. In the present work we show that p120 catenin bound to the N-cadherin precursor, contributes to its anterograde movement from the endoplasmic reticulum (ER to the Golgi complex. In HeLa cells, depletion of p120 expression, or blocking its binding to N-cadherin, increased the accumulation of the precursor in the ER, while it decreased the localization of mature N-cadherin at intercellular junctions. Reconstitution experiments in p120-deficient SW48 cells with all three major isoforms of p120 (1, 3 and 4 had similar capacity to promote the processing of the N-cadherin precursor to the mature form, and its localization at cell-cell junctions. P120 catenin and protein tyrosine phosphatase PTP1B facilitated the recruitment of the N-ethylmaleimide sensitive factor (NSF, an ATPase involved in vesicular trafficking, to the N-cadherin precursor complex. Dominant negative NSF E329Q impaired N-cadherin trafficking, maturation and localization at cell-cell junctions. Our results uncover a new role for p120 catenin bound to the N-cadherin precursor ensuring its trafficking through the biosynthetic pathway towards the cell surface.

  12. SNARE priming is essential for maturation of autophagosomes but not for their formation.

    Science.gov (United States)

    Abada, Adi; Levin-Zaidman, Smadar; Porat, Ziv; Dadosh, Tali; Elazar, Zvulun

    2017-11-28

    Autophagy, a unique intracellular membrane-trafficking pathway, is initiated by the formation of an isolation membrane (phagophore) that engulfs cytoplasmic constituents, leading to generation of the autophagosome, a double-membrane vesicle, which is targeted to the lysosome. The outer autophagosomal membrane consequently fuses with the lysosomal membrane. Multiple membrane-fusion events mediated by SNARE molecules have been postulated to promote autophagy. αSNAP, the adaptor molecule for the SNARE-priming enzyme N -ethylmaleimide-sensitive factor ( NSF ) is known to be crucial for intracellular membrane fusion processes, but its role in autophagy remains unclear. Here we demonstrated that knockdown of αSNAP leads to inhibition of autophagy, manifested by an accumulation of sealed autophagosomes located in close proximity to lysosomes but not fused with them. Under these conditions, moreover, association of both Atg9 and the autophagy-related SNARE protein syntaxin17 with the autophagosome remained unaffected. Finally, our results suggested that under starvation conditions, the levels of αSNAP, although low, are nevertheless sufficient to partially promote the SNARE priming required for autophagy. Taken together, these findings indicate that while autophagosomal-lysosomal membrane fusion is sensitive to inhibition of SNARE priming, the initial stages of autophagosome biogenesis and autophagosome expansion remain resistant to its loss. Copyright © 2017 the Author(s). Published by PNAS.

  13. Whey Protein

    Science.gov (United States)

    ... protein daily for 2 years does not improve bone density in postmenopausal women with osteoporosis. Weight loss. Most research suggests that taking whey protein alone, along with diet modifications, or while following an exercise plan does not seem to reduce weight for ...

  14. Protein Extractability

    African Journals Online (AJOL)

    limited to high oleic acid oil and water purification property (Katayon et al., 2006; Foid et al., 2001 and. Folkard et al., 1993), whereas it contains up to. 332.5 g of crude protein per kg of sample (Jose et al., 1999). Studies to characterize the interaction effects of pH and salts on the extraction of. PROTEIN EXTRACTABILITY ...

  15. Tau protein

    DEFF Research Database (Denmark)

    Frederiksen, Jette Lautrup Battistini; Kristensen, Kim; Bahl, Jmc

    2011-01-01

    Background: Tau protein has been proposed as biomarker of axonal damage leading to irreversible neurological impairment in MS. CSF concentrations may be useful when determining risk of progression from ON to MS. Objective: To investigate the association between tau protein concentration and 14......-3-3 protein in the cerebrospinal fluid (CSF) of patients with monosymptomatic optic neuritis (ON) versus patients with monosymptomatic onset who progressed to multiple sclerosis (MS). To evaluate results against data found in a complete literature review. Methods: A total of 66 patients with MS and/or ON from...... the Department of Neurology of Glostrup Hospital, University of Copenhagen, Denmark, were included. CSF samples were analysed for tau protein and 14-3-3 protein, and clinical and paraclinical information was obtained from medical records. Results: The study shows a significantly increased concentration of tau...

  16. Protein-Protein Interaction Databases

    DEFF Research Database (Denmark)

    Szklarczyk, Damian; Jensen, Lars Juhl

    2015-01-01

    of research are explored. Here we present an overview of the most widely used protein-protein interaction databases and the methods they employ to gather, combine, and predict interactions. We also point out the trade-off between comprehensiveness and accuracy and the main pitfall scientists have to be aware...

  17. Dietary Proteins

    Science.gov (United States)

    ... because your body doesn't store it the way it stores fats or carbohydrates. How much you need depends on your age, sex, health, and level of physical activity. Most Americans eat enough protein in their diet.

  18. Protein Crystallization

    Science.gov (United States)

    Chernov, Alexander A.

    2005-01-01

    Nucleation, growth and perfection of protein crystals will be overviewed along with crystal mechanical properties. The knowledge is based on experiments using optical and force crystals behave similar to inorganic crystals, though with a difference in orders of magnitude in growing parameters. For example, the low incorporation rate of large biomolecules requires up to 100 times larger supersaturation to grow protein, rather than inorganic crystals. Nucleation is often poorly reproducible, partly because of turbulence accompanying the mixing of precipitant with protein solution. Light scattering reveals fluctuations of molecular cluster size, its growth, surface energies and increased clustering as protein ages. Growth most often occurs layer-by-layer resulting in faceted crystals. New molecular layer on crystal face is terminated by a step where molecular incorporation occurs. Quantitative data on the incorporation rate will be discussed. Rounded crystals with molecularly disordered interfaces will be explained. Defects in crystals compromise the x-ray diffraction resolution crucially needed to find the 3D atomic structure of biomolecules. The defects are immobile so that birth defects stay forever. All lattice defects known for inorganics are revealed in protein crystals. Contribution of molecular conformations to lattice disorder is important, but not studied. This contribution may be enhanced by stress field from other defects. Homologous impurities (e.g., dimers, acetylated molecules) are trapped more willingly by a growing crystal than foreign protein impurities. The trapped impurities induce internal stress eliminated in crystals exceeding a critical size (part of mni for ferritin, lysozyme). Lesser impurities are trapped from stagnant, as compared to the flowing, solution. Freezing may induce much more defects unless quickly amorphysizing intracrystalline water.

  19. Septin dynamics are essential for exocytosis.

    Science.gov (United States)

    Tokhtaeva, Elmira; Capri, Joe; Marcus, Elizabeth A; Whitelegge, Julian P; Khuzakhmetova, Venera; Bukharaeva, Ellya; Deiss-Yehiely, Nimrod; Dada, Laura A; Sachs, George; Fernandez-Salas, Ester; Vagin, Olga

    2015-02-27

    Septins are a family of 14 cytoskeletal proteins that dynamically form hetero-oligomers and organize membrane microdomains for protein complexes. The previously reported interactions with SNARE proteins suggested the involvement of septins in exocytosis. However, the contradictory results of up- or down-regulation of septin-5 in various cells and mouse models or septin-4 in mice suggested either an inhibitory or a stimulatory role for these septins in exocytosis. The involvement of the ubiquitously expressed septin-2 or general septin polymerization in exocytosis has not been explored to date. Here, by nano-LC with tandem MS and immunoblot analyses of the septin-2 interactome in mouse brain, we identified not only SNARE proteins but also Munc-18-1 (stabilizes assembled SNARE complexes), N-ethylmaleimide-sensitive factor (NSF) (disassembles SNARE complexes after each membrane fusion event), and the chaperones Hsc70 and synucleins (maintain functional conformation of SNARE proteins after complex disassembly). Importantly, α-soluble NSF attachment protein (SNAP), the adaptor protein that mediates NSF binding to the SNARE complex, did not interact with septin-2, indicating that septins undergo reorganization during each exocytosis cycle. Partial depletion of septin-2 by siRNA or impairment of septin dynamics by forchlorfenuron inhibited constitutive and stimulated exocytosis of secreted and transmembrane proteins in various cell types. Forchlorfenuron impaired the interaction between SNAP-25 and its chaperone Hsc70, decreasing SNAP-25 levels in cultured neuroendocrine cells, and inhibited both spontaneous and stimulated acetylcholine secretion in mouse motor neurons. The results demonstrate a stimulatory role of septin-2 and the dynamic reorganization of septin oligomers in exocytosis. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. Septin Dynamics Are Essential for Exocytosis*

    Science.gov (United States)

    Tokhtaeva, Elmira; Capri, Joe; Marcus, Elizabeth A.; Whitelegge, Julian P.; Khuzakhmetova, Venera; Bukharaeva, Ellya; Deiss-Yehiely, Nimrod; Dada, Laura A.; Sachs, George; Fernandez-Salas, Ester; Vagin, Olga

    2015-01-01

    Septins are a family of 14 cytoskeletal proteins that dynamically form hetero-oligomers and organize membrane microdomains for protein complexes. The previously reported interactions with SNARE proteins suggested the involvement of septins in exocytosis. However, the contradictory results of up- or down-regulation of septin-5 in various cells and mouse models or septin-4 in mice suggested either an inhibitory or a stimulatory role for these septins in exocytosis. The involvement of the ubiquitously expressed septin-2 or general septin polymerization in exocytosis has not been explored to date. Here, by nano-LC with tandem MS and immunoblot analyses of the septin-2 interactome in mouse brain, we identified not only SNARE proteins but also Munc-18-1 (stabilizes assembled SNARE complexes), N-ethylmaleimide-sensitive factor (NSF) (disassembles SNARE complexes after each membrane fusion event), and the chaperones Hsc70 and synucleins (maintain functional conformation of SNARE proteins after complex disassembly). Importantly, α-soluble NSF attachment protein (SNAP), the adaptor protein that mediates NSF binding to the SNARE complex, did not interact with septin-2, indicating that septins undergo reorganization during each exocytosis cycle. Partial depletion of septin-2 by siRNA or impairment of septin dynamics by forchlorfenuron inhibited constitutive and stimulated exocytosis of secreted and transmembrane proteins in various cell types. Forchlorfenuron impaired the interaction between SNAP-25 and its chaperone Hsc70, decreasing SNAP-25 levels in cultured neuroendocrine cells, and inhibited both spontaneous and stimulated acetylcholine secretion in mouse motor neurons. The results demonstrate a stimulatory role of septin-2 and the dynamic reorganization of septin oligomers in exocytosis. PMID:25575596

  1. SNARE zippering is hindered by polyphenols in the neuron

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Yoosoo [Department of Genetic Engineering and Center for Human Interface Nanotechnology, Sungkyunkwan University, Suwon 440-746 (Korea, Republic of); Biomedical Research Institute, Korea Institute of Science and Technology, Seoul 136-791 (Korea, Republic of); Kim, Se-Hyun; Heo, Paul; Kong, Byoungjae; Shin, Jonghyeok; Jung, Young-Hun; Yoon, Keejung; Chung, Woo-Jae [Department of Genetic Engineering and Center for Human Interface Nanotechnology, Sungkyunkwan University, Suwon 440-746 (Korea, Republic of); Shin, Yeon-Kyun [Biomedical Research Institute, Korea Institute of Science and Technology, Seoul 136-791 (Korea, Republic of); Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, IA 50011 (United States); Kweon, Dae-Hyuk, E-mail: dhkweon@skku.edu [Department of Genetic Engineering and Center for Human Interface Nanotechnology, Sungkyunkwan University, Suwon 440-746 (Korea, Republic of)

    2014-07-18

    Highlights: • Membrane fusion driven by SNARE complex is hindered by several polyphenols. • Distinctive inhibitory effect of each polyphenol on SNARE zippering in neuron was examined. • FRET between fluorescence protein-tagged SNAREs probed well SNARE zippering in PC12 cells. • Delphinidin and cyanidin inhibit N-terminal SNARE nucleation in Ca{sup 2+}-independent manner. • Myricetin inhibits Ca{sup 2+}-dependent transmembrane association of SNARE complex. - Abstract: Fusion of synaptic vesicles with the presynaptic plasma membrane in the neuron is mediated by soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptor (SNARE) proteins. SNARE complex formation is a zippering-like process which initiates at the N-terminus and proceeds to the C-terminal membrane-proximal region. Previously, we showed that this zippering-like process is regulated by several polyphenols, leading to the arrest of membrane fusion and the inhibition of neuroexocytosis. In vitro studies using purified SNARE proteins reconstituted in liposomes revealed that each polyphenol uniquely regulates SNARE zippering. However, the unique regulatory effect of each polyphenol in cells has not yet been examined. In the present study, we observed SNARE zippering in neuronal PC12 cells by measuring the fluorescence resonance energy transfer (FRET) changes of a cyan fluorescence protein (CFP) and a yellow fluorescence protein (YFP) fused to the N-termini or C-termini of SNARE proteins. We show that delphinidin and cyanidin inhibit the initial N-terminal nucleation of SNARE complex formation in a Ca{sup 2+}-independent manner, while myricetin inhibits Ca{sup 2+}-dependent transmembrane domain association of the SNARE complex in the cell. This result explains how polyphenols exhibit botulinum neurotoxin-like activity in vivo.

  2. Aquaporin Protein-Protein Interactions

    Directory of Open Access Journals (Sweden)

    Jennifer Virginia Roche

    2017-10-01

    Full Text Available Aquaporins are tetrameric membrane-bound channels that facilitate transport of water and other small solutes across cell membranes. In eukaryotes, they are frequently regulated by gating or trafficking, allowing for the cell to control membrane permeability in a specific manner. Protein–protein interactions play crucial roles in both regulatory processes and also mediate alternative functions such as cell adhesion. In this review, we summarize recent knowledge about aquaporin protein–protein interactions; dividing the interactions into three types: (1 interactions between aquaporin tetramers; (2 interactions between aquaporin monomers within a tetramer (hetero-tetramerization; and (3 transient interactions with regulatory proteins. We particularly focus on the structural aspects of the interactions, discussing the small differences within a conserved overall fold that allow for aquaporins to be differentially regulated in an organism-, tissue- and trigger-specific manner. A deep knowledge about these differences is needed to fully understand aquaporin function and regulation in many physiological processes, and may enable design of compounds targeting specific aquaporins for treatment of human disease.

  3. Protein immobilization strategies for protein biochips

    NARCIS (Netherlands)

    Rusmini, F.; Rusmini, Federica; Zhong, Zhiyuan; Feijen, Jan

    2007-01-01

    In the past few years, protein biochips have emerged as promising proteomic and diagnostic tools for obtaining information about protein functions and interactions. Important technological innovations have been made. However, considerable development is still required, especially regarding protein

  4. Interaction entropy for protein-protein binding

    Science.gov (United States)

    Sun, Zhaoxi; Yan, Yu N.; Yang, Maoyou; Zhang, John Z. H.

    2017-03-01

    Protein-protein interactions are at the heart of signal transduction and are central to the function of protein machine in biology. The highly specific protein-protein binding is quantitatively characterized by the binding free energy whose accurate calculation from the first principle is a grand challenge in computational biology. In this paper, we show how the interaction entropy approach, which was recently proposed for protein-ligand binding free energy calculation, can be applied to computing the entropic contribution to the protein-protein binding free energy. Explicit theoretical derivation of the interaction entropy approach for protein-protein interaction system is given in detail from the basic definition. Extensive computational studies for a dozen realistic protein-protein interaction systems are carried out using the present approach and comparisons of the results for these protein-protein systems with those from the standard normal mode method are presented. Analysis of the present method for application in protein-protein binding as well as the limitation of the method in numerical computation is discussed. Our study and analysis of the results provided useful information for extracting correct entropic contribution in protein-protein binding from molecular dynamics simulations.

  5. Learning about Proteins

    Science.gov (United States)

    ... Videos for Educators Search English Español Learning About Proteins KidsHealth / For Kids / Learning About Proteins What's in ... from the foods you eat. Different Kinds of Protein Protein from animal sources, such as meat and ...

  6. Regulation of Exocytotic Fusion Pores by SNARE Protein Transmembrane Domains

    Directory of Open Access Journals (Sweden)

    Zhenyong Wu

    2017-10-01

    Full Text Available Calcium-triggered exocytotic release of neurotransmitters and hormones from neurons and neuroendocrine cells underlies neuronal communication, motor activity and endocrine functions. The core of the neuronal exocytotic machinery is composed of soluble N-ethyl maleimide sensitive factor attachment protein receptors (SNAREs. Formation of complexes between vesicle-attached v- and plasma-membrane anchored t-SNAREs in a highly regulated fashion brings the membranes into close apposition. Small, soluble proteins called Complexins (Cpx and calcium-sensing Synaptotagmins cooperate to block fusion at low resting calcium concentrations, but trigger release upon calcium increase. A growing body of evidence suggests that the transmembrane domains (TMDs of SNARE proteins play important roles in regulating the processes of fusion and release, but the mechanisms involved are only starting to be uncovered. Here we review recent evidence that SNARE TMDs exert influence by regulating the dynamics of the fusion pore, the initial aqueous connection between the vesicular lumen and the extracellular space. Even after the fusion pore is established, hormone release by neuroendocrine cells is tightly controlled, and the same may be true of neurotransmitter release by neurons. The dynamics of the fusion pore can regulate the kinetics of cargo release and the net amount released, and can determine the mode of vesicle recycling. Manipulations of SNARE TMDs were found to affect fusion pore properties profoundly, both during exocytosis and in biochemical reconstitutions. To explain these effects, TMD flexibility, and interactions among TMDs or between TMDs and lipids have been invoked. Exocytosis has provided the best setting in which to unravel the underlying mechanisms, being unique among membrane fusion reactions in that single fusion pores can be probed using high-resolution methods. An important role will likely be played by methods that can probe single fusion pores

  7. Efficient protein alignment algorithm for protein search.

    Science.gov (United States)

    Lu, Zaixin; Zhao, Zhiyu; Fu, Bin

    2010-01-18

    Proteins show a great variety of 3D conformations, which can be used to infer their evolutionary relationship and to classify them into more general groups; therefore protein structure alignment algorithms are very helpful for protein biologists. However, an accurate alignment algorithm itself may be insufficient for effective discovering of structural relationships among tens of thousands of proteins. Due to the exponentially increasing amount of protein structural data, a fast and accurate structure alignment tool is necessary to access protein classification and protein similarity search; however, the complexity of current alignment algorithms are usually too high to make a fully alignment-based classification and search practical. We have developed an efficient protein pairwise alignment algorithm and applied it to our protein search tool, which aligns a query protein structure in the pairwise manner with all protein structures in the Protein Data Bank (PDB) to output similar protein structures. The algorithm can align hundreds of pairs of protein structures in one second. Given a protein structure, the tool efficiently discovers similar structures from tens of thousands of structures stored in the PDB always in 2 minutes in a single machine and 20 seconds in our cluster of 6 machines. The algorithm has been fully implemented and is accessible online at our webserver, which is supported by a cluster of computers. Our algorithm can work out hundreds of pairs of protein alignments in one second. Therefore, it is very suitable for protein search. Our experimental results show that it is more accurate than other well known protein search systems in finding proteins which are structurally similar at SCOP family and superfamily levels, and its speed is also competitive with those systems. In terms of the pairwise alignment performance, it is as good as some well known alignment algorithms.

  8. Small heat shock proteins, protein degradation and protein aggregation diseases

    NARCIS (Netherlands)

    Vos, Michel J.; Zijlstra, Marianne P.; Carra, Serena; Sibon, Ody C. M.; Kampinga, Harm H.

    Small heat shock proteins have been characterized in vitro as ATP-independent molecular chaperones that can prevent aggregation of un- or misfolded proteins and assist in their refolding with the help of ATP-dependent chaperone machines (e. g., the Hsp70 proteins). Comparison of the functionality of

  9. EDITORIAL: Precision proteins Precision proteins

    Science.gov (United States)

    Demming, Anna

    2010-06-01

    Since the birth of modern day medicine, during the times of Hippocrates in ancient Greece, the profession has developed from the rudimentary classification of disease into a rigorous science with an inspiring capability to treat and cure. Scientific methodology has distilled clinical diagnostic tools from the early arts of prognosis, which used to rely as much on revelation and prophecy, as intuition and judgement [1]. Over the past decade, research into the interactions between proteins and nanosystems has provided some ingenious and apt techniques for delving into the intricacies of anatomical systems. In vivo biosensing has emerged as a vibrant field of research, as much of medical diagnosis relies on the detection of substances or an imbalance in the chemicals in the body. The inherent properties of nanoscale structures, such as cantilevers, make them well suited to biosensing applications that demand the detection of molecules at very low concentrations. Measurable deflections in cantilevers functionalised with antibodies provide quantitative indicators of the presence of specific antigens when the two react. Such developments have roused mounting interest in the interactions of proteins with nanostructures, such as carbon nanotubes [3], which have demonstrated great potential as generic biomarkers. Plasmonic properties are also being exploited in sensing applications, such as the molecular sentinel recently devised by researchers in the US. The device uses the plasmonic properties of a silver nanoparticle linked to a Raman labelled hairpin DNA probe to signal changes in the probe geometry resulting from interactions with substances in the environment. Success stories so far include the detection of two specific genes associated with breast cancer [4]. A greater understanding of how RNA interference regulates gene expression has highlighted the potential of using this natural process as another agent for combating disease in personalized medicine. However, the

  10. Protein docking prediction using predicted protein-protein interface

    Directory of Open Access Journals (Sweden)

    Li Bin

    2012-01-01

    Full Text Available Abstract Background Many important cellular processes are carried out by protein complexes. To provide physical pictures of interacting proteins, many computational protein-protein prediction methods have been developed in the past. However, it is still difficult to identify the correct docking complex structure within top ranks among alternative conformations. Results We present a novel protein docking algorithm that utilizes imperfect protein-protein binding interface prediction for guiding protein docking. Since the accuracy of protein binding site prediction varies depending on cases, the challenge is to develop a method which does not deteriorate but improves docking results by using a binding site prediction which may not be 100% accurate. The algorithm, named PI-LZerD (using Predicted Interface with Local 3D Zernike descriptor-based Docking algorithm, is based on a pair wise protein docking prediction algorithm, LZerD, which we have developed earlier. PI-LZerD starts from performing docking prediction using the provided protein-protein binding interface prediction as constraints, which is followed by the second round of docking with updated docking interface information to further improve docking conformation. Benchmark results on bound and unbound cases show that PI-LZerD consistently improves the docking prediction accuracy as compared with docking without using binding site prediction or using the binding site prediction as post-filtering. Conclusion We have developed PI-LZerD, a pairwise docking algorithm, which uses imperfect protein-protein binding interface prediction to improve docking accuracy. PI-LZerD consistently showed better prediction accuracy over alternative methods in the series of benchmark experiments including docking using actual docking interface site predictions as well as unbound docking cases.

  11. Our interests in protein-protein interactions

    Indian Academy of Sciences (India)

    protein interactions. Evolution of P-P partnerships. Evolution of P-P structures. Evolutionary dynamics of P-P interactions. Dynamics of P-P interaction network. Host-pathogen interactions. CryoEM mapping of gigantic protein assemblies.

  12. Evolution of protein-protein interactions

    Indian Academy of Sciences (India)

    Evolution of protein-protein interactions · Our interests in protein-protein interactions · Slide 3 · Slide 4 · Slide 5 · Slide 6 · Slide 7 · Slide 8 · Slide 9 · Slide 10 · Slide 11 · Slide 12 · Slide 13 · Slide 14 · Slide 15 · Slide 16 · Slide 17 · Slide 18 · Slide 19 · Slide 20.

  13. 24-hour urine protein

    Science.gov (United States)

    Urine protein - 24 hour; Chronic kidney disease - urine protein; Kidney failure - urine protein ... Bladder tumor Heart failure High blood pressure during pregnancy ( preeclampsia ) Kidney disease caused by diabetes, high blood pressure, autoimmune disorders, ...

  14. Protein in diet

    Science.gov (United States)

    Diet - protein ... Protein foods are broken down into parts called amino acids during digestion. The human body needs a ... to eat animal products to get all the protein you need in your diet. Amino acids are ...

  15. Protein-losing enteropathy

    Science.gov (United States)

    ... this page: //medlineplus.gov/ency/article/007338.htm Protein-losing enteropathy To use the sharing features on this page, please enable JavaScript. Protein-losing enteropathy is an abnormal loss of protein ...

  16. Nanotechnologies in protein microarrays.

    Science.gov (United States)

    Krizkova, Sona; Heger, Zbynek; Zalewska, Marta; Moulick, Amitava; Adam, Vojtech; Kizek, Rene

    2015-01-01

    Protein microarray technology became an important research tool for study and detection of proteins, protein-protein interactions and a number of other applications. The utilization of nanoparticle-based materials and nanotechnology-based techniques for immobilization allows us not only to extend the surface for biomolecule immobilization resulting in enhanced substrate binding properties, decreased background signals and enhanced reporter systems for more sensitive assays. Generally in contemporarily developed microarray systems, multiple nanotechnology-based techniques are combined. In this review, applications of nanoparticles and nanotechnologies in creating protein microarrays, proteins immobilization and detection are summarized. We anticipate that advanced nanotechnologies can be exploited to expand promising fields of proteins identification, monitoring of protein-protein or drug-protein interactions, or proteins structures.

  17. Protein sequence comparison and protein evolution

    Energy Technology Data Exchange (ETDEWEB)

    Pearson, W.R. [Univ. of Virginia, Charlottesville, VA (United States). Dept. of Biochemistry

    1995-12-31

    This tutorial was one of eight tutorials selected to be presented at the Third International Conference on Intelligent Systems for Molecular Biology which was held in the United Kingdom from July 16 to 19, 1995. This tutorial examines how the information conserved during the evolution of a protein molecule can be used to infer reliably homology, and thus a shared proteinfold and possibly a shared active site or function. The authors start by reviewing a geological/evolutionary time scale. Next they look at the evolution of several protein families. During the tutorial, these families will be used to demonstrate that homologous protein ancestry can be inferred with confidence. They also examine different modes of protein evolution and consider some hypotheses that have been presented to explain the very earliest events in protein evolution. The next part of the tutorial will examine the technical aspects of protein sequence comparison. Both optimal and heuristic algorithms and their associated parameters that are used to characterize protein sequence similarities are discussed. Perhaps more importantly, they survey the statistics of local similarity scores, and how these statistics can both be used to improve the selectivity of a search and to evaluate the significance of a match. They them examine distantly related members of three protein families, the serine proteases, the glutathione transferases, and the G-protein-coupled receptors (GCRs). Finally, the discuss how sequence similarity can be used to examine internal repeated or mosaic structures in proteins.

  18. Protein- protein interaction detection system using fluorescent protein microdomains

    Science.gov (United States)

    Waldo, Geoffrey S.; Cabantous, Stephanie

    2010-02-23

    The invention provides a protein labeling and interaction detection system based on engineered fragments of fluorescent and chromophoric proteins that require fused interacting polypeptides to drive the association of the fragments, and further are soluble and stable, and do not change the solubility of polypeptides to which they are fused. In one embodiment, a test protein X is fused to a sixteen amino acid fragment of GFP (.beta.-strand 10, amino acids 198-214), engineered to not perturb fusion protein solubility. A second test protein Y is fused to a sixteen amino acid fragment of GFP (.beta.-strand 11, amino acids 215-230), engineered to not perturb fusion protein solubility. When X and Y interact, they bring the GFP strands into proximity, and are detected by complementation with a third GFP fragment consisting of GFP amino acids 1-198 (strands 1-9). When GFP strands 10 and 11 are held together by interaction of protein X and Y, they spontaneous association with GFP strands 1-9, resulting in structural complementation, folding, and concomitant GFP fluorescence.

  19. Comparing side chain packing in soluble proteins, protein-protein interfaces, and transmembrane proteins.

    Science.gov (United States)

    Gaines, J C; Acebes, S; Virrueta, A; Butler, M; Regan, L; O'Hern, C S

    2018-02-10

    We compare side chain prediction and packing of core and non-core regions of soluble proteins, protein-protein interfaces, and transmembrane proteins. We first identified or created comparable databases of high-resolution crystal structures of these 3 protein classes. We show that the solvent-inaccessible cores of the 3 classes of proteins are equally densely packed. As a result, the side chains of core residues at protein-protein interfaces and in the membrane-exposed regions of transmembrane proteins can be predicted by the hard-sphere plus stereochemical constraint model with the same high prediction accuracies (>90%) as core residues in soluble proteins. We also find that for all 3 classes of proteins, as one moves away from the solvent-inaccessible core, the packing fraction decreases as the solvent accessibility increases. However, the side chain predictability remains high (80% within 30°) up to a relative solvent accessibility, rSASA≲0.3, for all 3 protein classes. Our results show that ≈40% of the interface regions in protein complexes are "core", that is, densely packed with side chain conformations that can be accurately predicted using the hard-sphere model. We propose packing fraction as a metric that can be used to distinguish real protein-protein interactions from designed, non-binding, decoys. Our results also show that cores of membrane proteins are the same as cores of soluble proteins. Thus, the computational methods we are developing for the analysis of the effect of hydrophobic core mutations in soluble proteins will be equally applicable to analyses of mutations in membrane proteins. © 2018 Wiley Periodicals, Inc.

  20. IGSF9 Family Proteins

    DEFF Research Database (Denmark)

    Hansen, Maria; Walmod, Peter Schledermann

    2013-01-01

    The Drosophila protein Turtle and the vertebrate proteins immunoglobulin superfamily (IgSF), member 9 (IGSF9/Dasm1) and IGSF9B are members of an evolutionarily ancient protein family. A bioinformatics analysis of the protein family revealed that invertebrates contain only a single IGSF9 family gene......, whereas vertebrates contain two to four genes. In cnidarians, the gene appears to encode a secreted protein, but transmembrane isoforms of the protein have also evolved, and in many species, alternative splicing facilitates the expression of both transmembrane and secreted isoforms. In most species......, the longest isoforms of the proteins have the same general organization as the neural cell adhesion molecule family of cell adhesion molecule proteins, and like this family of proteins, IGSF9 family members are expressed in the nervous system. A review of the literature revealed that Drosophila Turtle...

  1. Peptide segments in protein-protein interfaces

    Indian Academy of Sciences (India)

    Prakash

    2006-09-06

    Sep 6, 2006 ... contact surface from the rest of the protein surface have been used to identify the interaction sites (Jones and Thornton. 1997; Neuvirth et al 2004). Protein antigenic sites (epitopes that are recognized by antibodies) could be generally confined to continuous motifs of about 8–24 amino acid residues, or may ...

  2. Surface Mediated Protein Disaggregation

    Science.gov (United States)

    Radhakrishna, Mithun; Kumar, Sanat K.

    2014-03-01

    Preventing protein aggregation is of both biological and industrial importance. Biologically these aggregates are known to cause amyloid type diseases like Alzheimer's and Parkinson's disease. Protein aggregation leads to reduced activity of the enzymes in industrial applications. Inter-protein interactions between the hydrophobic residues of the protein are known to be the major driving force for protein aggregation. In the current paper we show how surface chemistry and curvature can be tuned to mitigate these inter-protein interactions. Our results calculated in the framework of the Hydrophobic-Polar (HP) lattice model show that, inter-protein interactions can be drastically reduced by increasing the surface hydrophobicity to a critical value corresponding to the adsorption transition of the protein. At this value of surface hydrophobicity, proteins lose inter-protein contacts to gain surface contacts and thus the surface helps in reducing the inter-protein interactions. Further, we show that the adsorption of the proteins inside hydrophobic pores of optimal sizes are most efficient both in reducing inter-protein contacts and simultaneously retaining most of the native-contacts due to strong protein-surface interactions coupled with stabilization due to the confinement. Department of Energy (Grant No DE-FG02-11ER46811).

  3. The Sec1/Munc18 protein Vps45 regulates cellular levels of its SNARE binding partners Tlg2 and Snc2 in Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Scott G Shanks

    Full Text Available Intracellular membrane trafficking pathways must be tightly regulated to ensure proper functioning of all eukaryotic cells. Central to membrane trafficking is the formation of specific SNARE (soluble N-ethylmeleimide-sensitive factor attachment protein receptor complexes between proteins on opposing lipid bilayers. The Sec1/Munc18 (SM family of proteins play an essential role in SNARE-mediated membrane fusion, and like the SNAREs are conserved through evolution from yeast to humans. The SM protein Vps45 is required for the formation of yeast endosomal SNARE complexes and is thus essential for traffic through the endosomal system. Here we report that, in addition to its role in regulating SNARE complex assembly, Vps45 regulates cellular levels of its SNARE binding partners: the syntaxin Tlg2 and the v-SNARE Snc2: Cells lacking Vps45 have reduced cellular levels of Tlg2 and Snc2; and elevation of Vps45 levels results in concomitant increases in the levels of both Tlg2 and Snc2. As well as regulating traffic through the endosomal system, the Snc v-SNAREs are also required for exocytosis. Unlike most vps mutants, cells lacking Vps45 display multiple growth phenotypes. Here we report that these can be reversed by selectively restoring Snc2 levels in vps45 mutant cells. Our data indicate that as well as functioning as part of the machinery that controls SNARE complex assembly, Vps45 also plays a key role in determining the levels of its cognate SNARE proteins; another key factor in regulation of membrane traffic.

  4. Physics of protein motility and motor proteins

    Science.gov (United States)

    Kolomeisky, Anatoly B.

    2013-09-01

    Motor proteins are enzymatic molecules that transform chemical energy into mechanical motion and work. They are critically important for supporting various cellular activities and functions. In the last 15 years significant progress in understanding the functioning of motor proteins has been achieved due to revolutionary breakthroughs in single-molecule experimental techniques and strong advances in theoretical modelling. However, microscopic mechanisms of protein motility are still not well explained, and the collective efforts of many scientists are needed in order to solve these complex problems. In this special section the reader will find the latest advances on the difficult road to mapping motor proteins dynamics in various systems. Recent experimental developments have allowed researchers to monitor and to influence the activity of single motor proteins with a high spatial and temporal resolution. It has stimulated significant theoretical efforts to understand the non-equilibrium nature of protein motility phenomena. The latest results from all these advances are presented and discussed in this special section. We would like to thank the scientists from all over the world who have reported their latest research results for this special section. We are also grateful to the staff and editors of Journal of Physics: Condensed Matter for their invaluable help in handling all the administrative and refereeing activities. The field of motor proteins and protein motility is fast moving, and we hope that this collection of articles will be a useful source of information in this highly interdisciplinary area. Physics of protein motility and motor proteins contents Physics of protein motility and motor proteinsAnatoly B Kolomeisky Identification of unique interactions between the flexible linker and the RecA-like domains of DEAD-box helicase Mss116 Yuan Zhang, Mirkó Palla, Andrew Sun and Jung-Chi Liao The load dependence of the physical properties of a molecular motor

  5. Polymer Directed Protein Assemblies

    Directory of Open Access Journals (Sweden)

    Patrick van Rijn

    2013-05-01

    Full Text Available Protein aggregation and protein self-assembly is an important occurrence in natural systems, and is in some form or other dictated by biopolymers. Very obvious influences of biopolymers on protein assemblies are, e.g., virus particles. Viruses are a multi-protein assembly of which the morphology is dictated by poly-nucleotides namely RNA or DNA. This “biopolymer” directs the proteins and imposes limitations on the structure like the length or diameter of the particle. Not only do these bionanoparticles use polymer-directed self-assembly, also processes like amyloid formation are in a way a result of directed protein assembly by partial unfolded/misfolded biopolymers namely, polypeptides. The combination of proteins and synthetic polymers, inspired by the natural processes, are therefore regarded as a highly promising area of research. Directed protein assembly is versatile with respect to the possible interactions which brings together the protein and polymer, e.g., electrostatic, v.d. Waals forces or covalent conjugation, and possible combinations are numerous due to the large amounts of different polymers and proteins available. The protein-polymer interacting behavior and overall morphology is envisioned to aid in clarifying protein-protein interactions and are thought to entail some interesting new functions and properties which will ultimately lead to novel bio-hybrid materials.

  6. Protein Data Bank (PDB)

    Data.gov (United States)

    U.S. Department of Health & Human Services — The Protein Data Bank (PDB) archive is the single worldwide repository of information about the 3D structures of large biological molecules, including proteins and...

  7. Urine protein electrophoresis test

    Science.gov (United States)

    Urine protein electrophoresis; UPEP; Multiple myeloma - UPEP; Waldenström macroglobulinemia - UPEP; Amyloidosis - UPEP ... special paper and apply an electric current. The proteins move and form visible bands. These reveal the ...

  8. Protein electrophoresis - serum

    Science.gov (United States)

    ... this page: //medlineplus.gov/ency/article/003540.htm Protein electrophoresis - serum To use the sharing features on ... JavaScript. This lab test measures the types of protein in the fluid (serum) part of a blood ...

  9. Statistical Properties of Protein-Protein Interfaces

    Directory of Open Access Journals (Sweden)

    Mihaly Mezei

    2015-04-01

    Full Text Available The properties of 1172 protein complexes (downloaded from the Protein Data Bank (PDB have been studied based on the concept of circular variance as a buriedness indicator and the concept of mutual proximity as a parameter-free definition of contact. The propensities of residues to be in the protein, on the surface or form contact, as well as residue pairs to form contact were calculated. In addition, the concept of circular variance has been used to compare the ruggedness and shape of the contact surface with the overall surface.

  10. Destabilized bioluminescent proteins

    Energy Technology Data Exchange (ETDEWEB)

    Allen, Michael S. (Knoxville, TN); Rakesh, Gupta (New Delhi, IN); Gary, Sayler S. (Blaine, TN)

    2007-07-31

    Purified nucleic acids, vectors and cells containing a gene cassette encoding at least one modified bioluminescent protein, wherein the modification includes the addition of a peptide sequence. The duration of bioluminescence emitted by the modified bioluminescent protein is shorter than the duration of bioluminescence emitted by an unmodified form of the bioluminescent protein.

  11. CSF total protein

    Science.gov (United States)

    CSF total protein is a test to determine the amount of protein in your spinal fluid, also called cerebrospinal fluid (CSF). ... The normal protein range varies from lab to lab, but is typically about 15 to 60 milligrams per deciliter (mg/dL) ...

  12. Protein - Which is Best?

    Science.gov (United States)

    Hoffman, Jay R; Falvo, Michael J

    2004-09-01

    Protein intake that exceeds the recommended daily allowance is widely accepted for both endurance and power athletes. However, considering the variety of proteins that are available much less is known concerning the benefits of consuming one protein versus another. The purpose of this paper is to identify and analyze key factors in order to make responsible recommendations to both the general and athletic populations. Evaluation of a protein is fundamental in determining its appropriateness in the human diet. Proteins that are of inferior content and digestibility are important to recognize and restrict or limit in the diet. Similarly, such knowledge will provide an ability to identify proteins that provide the greatest benefit and should be consumed. The various techniques utilized to rate protein will be discussed. Traditionally, sources of dietary protein are seen as either being of animal or vegetable origin. Animal sources provide a complete source of protein (i.e. containing all essential amino acids), whereas vegetable sources generally lack one or more of the essential amino acids. Animal sources of dietary protein, despite providing a complete protein and numerous vitamins and minerals, have some health professionals concerned about the amount of saturated fat common in these foods compared to vegetable sources. The advent of processing techniques has shifted some of this attention and ignited the sports supplement marketplace with derivative products such as whey, casein and soy. Individually, these products vary in quality and applicability to certain populations. The benefits that these particular proteins possess are discussed. In addition, the impact that elevated protein consumption has on health and safety issues (i.e. bone health, renal function) are also reviewed. Key PointsHigher protein needs are seen in athletic populations.Animal proteins is an important source of protein, however potential health concerns do exist from a diet of protein

  13. Antimicrobial proteins : from old proteins, new tricks

    OpenAIRE

    Smith, Val; Dyrynda, Elisabeth

    2015-01-01

    This review describes the main types of antimicrobial peptides (AMPs) synthesised by crustaceans, primarily those identified in shrimp, crayfish, crab and lobster. It includes an overview of their range of microbicidal activities and the current landscape of our understanding of their gene expression patterns in different body tissues. It further summarises how their expression might change following various types of immune challenges. Included in the review are proteins or protein fragments ...

  14. Protein utilization in correlation to protein intake.

    Science.gov (United States)

    Krajcovicová, M; Dibák, O

    1980-01-01

    In a 14-day experiment, weaned and adult rats were given ad libitum isocaloric diets with a mounting casein content (5, 10, 15, 25 and 40% by weight) and growth parameters of protein biological value, PER and NPR, and the utilization parameters NPU (body protein) and LPU (liver protein) were determined together with phosphoenolpyruvate carboxykinase (gluconeogenetic enzyme) and pyruvate kinase (glycolytic enzyme) activity in the animals' liver. The decrease in all the biological value parameters in weaned rats on 25% and 40% casein diets and in adult rats on 15%, 25% and 40% casein diets shows that these concentrations are too high for the organism. The decrease in PER and diminished weight and body and liver nitrogen increments in both age groups in animals with a low protein intake is evidence that 5% casein is an inadequate concentration. The optimum diet for weaned rats is thus a 15% casein diet and for adult rats a 10% casein diet, as confirmed by the linear correlation between weight increments, body and liver nitrogen and protein intake and also by gluconeogenetic enzyme activity. Under the given experimental conditions the study is a contribution to the determination of optimum physiological doses of proteins.

  15. Highly thermostable fluorescent proteins

    Science.gov (United States)

    Bradbury, Andrew M [Santa Fe, NM; Waldo, Geoffrey S [Santa Fe, NM; Kiss, Csaba [Los Alamos, NM

    2012-05-01

    Thermostable fluorescent proteins (TSFPs), methods for generating these and other stability-enhanced proteins, polynucleotides encoding such proteins, and assays and method for using the TSFPs and TSFP-encoding nucleic acid molecules are provided. The TSFPs of the invention show extremely enhanced levels of stability and thermotolerance. In one case, for example, a TSFP of the invention is so stable it can be heated to 99.degree. C. for short periods of time without denaturing, and retains 85% of its fluorescence when heated to 80.degree. C. for several minutes. The invention also provides a method for generating stability-enhanced variants of a protein, including but not limited to fluorescent proteins.

  16. Protein Function Prediction.

    Science.gov (United States)

    Cruz, Leonardo Magalhães; Trefflich, Sheyla; Weiss, Vinícius Almir; Castro, Mauro Antônio Alves

    2017-01-01

    Protein function is a concept that can have different interpretations in different biological contexts, and the number and diversity of novel proteins identified by large-scale "omics" technologies poses increasingly new challenges. In this review we explore current strategies used to predict protein function focused on high-throughput sequence analysis, as for example, inference based on sequence similarity, sequence composition, structure, and protein-protein interaction. Various prediction strategies are discussed together with illustrative workflows highlighting the use of some benchmark tools and knowledge bases in the field.

  17. Highly thermostable fluorescent proteins

    Science.gov (United States)

    Bradbury, Andrew M [Santa Fe, NM; Waldo, Geoffrey S [Santa Fe, NM; Kiss, Csaba [Los Alamos, NM

    2011-03-22

    Thermostable fluorescent proteins (TSFPs), methods for generating these and other stability-enhanced proteins, polynucleotides encoding such proteins, and assays and method for using the TSFPs and TSFP-encoding nucleic acid molecules are provided. The TSFPs of the invention show extremely enhanced levels of stability and thermotolerance. In one case, for example, a TSFP of the invention is so stable it can be heated to 99.degree. C. for short periods of time without denaturing, and retains 85% of its fluorescence when heated to 80.degree. C. for several minutes. The invention also provides a method for generating stability-enhanced variants of a protein, including but not limited to fluorescent proteins.

  18. Protein oxidation and peroxidation

    DEFF Research Database (Denmark)

    Davies, Michael Jonathan

    2016-01-01

    and chain reactions with alcohols and carbonyls as major products; the latter are commonly used markers of protein damage. Direct oxidation of cysteine (and less commonly) methionine residues is a major reaction; this is typically faster than with H2O2, and results in altered protein activity and function....... Unlike H2O2, which is rapidly removed by protective enzymes, protein peroxides are only slowly removed, and catabolism is a major fate. Although turnover of modified proteins by proteasomal and lysosomal enzymes, and other proteases (e.g. mitochondrial Lon), can be efficient, protein hydroperoxides...

  19. Pigment-protein complexes

    Energy Technology Data Exchange (ETDEWEB)

    Siegelman, H W

    1980-01-01

    The photosynthetically-active pigment protein complexes of procaryotes and eucaryotes include chlorophyll proteins, carotenochlorophyll proteins, and biliproteins. They are either integral components or attached to photosynthetic membranes. Detergents are frequently required to solubilize the pigment-protein complexes. The membrane localization and detergent solubilization strongly suggest that the pigment-protein complexes are bound to the membranes by hydrophobic interactions. Hydrophobic interactions of proteins are characterized by an increase in entropy. Their bonding energy is directly related to temperature and ionic strength. Hydrophobic-interaction chromatography, a relatively new separation procedure, can furnish an important method for the purification of pigment-protein complexes. Phycobilisome purification and properties provide an example of the need to maintain hydrophobic interactions to preserve structure and function.

  20. Protein solubility modeling

    Science.gov (United States)

    Agena, S. M.; Pusey, M. L.; Bogle, I. D.

    1999-01-01

    A thermodynamic framework (UNIQUAC model with temperature dependent parameters) is applied to model the salt-induced protein crystallization equilibrium, i.e., protein solubility. The framework introduces a term for the solubility product describing protein transfer between the liquid and solid phase and a term for the solution behavior describing deviation from ideal solution. Protein solubility is modeled as a function of salt concentration and temperature for a four-component system consisting of a protein, pseudo solvent (water and buffer), cation, and anion (salt). Two different systems, lysozyme with sodium chloride and concanavalin A with ammonium sulfate, are investigated. Comparison of the modeled and experimental protein solubility data results in an average root mean square deviation of 5.8%, demonstrating that the model closely follows the experimental behavior. Model calculations and model parameters are reviewed to examine the model and protein crystallization process. Copyright 1999 John Wiley & Sons, Inc.

  1. Packing in protein cores

    Science.gov (United States)

    Gaines, J. C.; Clark, A. H.; Regan, L.; O'Hern, C. S.

    2017-07-01

    Proteins are biological polymers that underlie all cellular functions. The first high-resolution protein structures were determined by x-ray crystallography in the 1960s. Since then, there has been continued interest in understanding and predicting protein structure and stability. It is well-established that a large contribution to protein stability originates from the sequestration from solvent of hydrophobic residues in the protein core. How are such hydrophobic residues arranged in the core; how can one best model the packing of these residues, and are residues loosely packed with multiple allowed side chain conformations or densely packed with a single allowed side chain conformation? Here we show that to properly model the packing of residues in protein cores it is essential that amino acids are represented by appropriately calibrated atom sizes, and that hydrogen atoms are explicitly included. We show that protein cores possess a packing fraction of φ ≈ 0.56 , which is significantly less than the typically quoted value of 0.74 obtained using the extended atom representation. We also compare the results for the packing of amino acids in protein cores to results obtained for jammed packings from discrete element simulations of spheres, elongated particles, and composite particles with bumpy surfaces. We show that amino acids in protein cores pack as densely as disordered jammed packings of particles with similar values for the aspect ratio and bumpiness as found for amino acids. Knowing the structural properties of protein cores is of both fundamental and practical importance. Practically, it enables the assessment of changes in the structure and stability of proteins arising from amino acid mutations (such as those identified as a result of the massive human genome sequencing efforts) and the design of new folded, stable proteins and protein-protein interactions with tunable specificity and affinity.

  2. Expressed protein ligation for a large dimeric protein

    NARCIS (Netherlands)

    Karagöz, G.E.; Sinnige, T; Hsieh, O.; Rüdiger, S.G.D.

    2011-01-01

    Expressed protein ligation (EPL) is a protein engineering tool for post-translational ligation of protein or peptide fragments. This technique allows modification of specific parts of proteins, opening possibilities for incorporating probes for biophysical applications such as nuclear magnetic

  3. Toxic proteins in plants.

    Science.gov (United States)

    Dang, Liuyi; Van Damme, Els J M

    2015-09-01

    Plants have evolved to synthesize a variety of noxious compounds to cope with unfavorable circumstances, among which a large group of toxic proteins that play a critical role in plant defense against predators and microbes. Up to now, a wide range of harmful proteins have been discovered in different plants, including lectins, ribosome-inactivating proteins, protease inhibitors, ureases, arcelins, antimicrobial peptides and pore-forming toxins. To fulfill their role in plant defense, these proteins exhibit various degrees of toxicity towards animals, insects, bacteria or fungi. Numerous studies have been carried out to investigate the toxic effects and mode of action of these plant proteins in order to explore their possible applications. Indeed, because of their biological activities, toxic plant proteins are also considered as potentially useful tools in crop protection and in biomedical applications, such as cancer treatment. Genes encoding toxic plant proteins have been introduced into crop genomes using genetic engineering technology in order to increase the plant's resistance against pathogens and diseases. Despite the availability of ample information on toxic plant proteins, very few publications have attempted to summarize the research progress made during the last decades. This review focuses on the diversity of toxic plant proteins in view of their toxicity as well as their mode of action. Furthermore, an outlook towards the biological role(s) of these proteins and their potential applications is discussed. Copyright © 2015 Elsevier Ltd. All rights reserved.

  4. PROTEIN - WHICH IS BEST?

    Directory of Open Access Journals (Sweden)

    Michael J. Falvo

    2004-09-01

    Full Text Available Protein intake that exceeds the recommended daily allowance is widely accepted for both endurance and power athletes. However, considering the variety of proteins that are available much less is known concerning the benefits of consuming one protein versus another. The purpose of this paper is to identify and analyze key factors in order to make responsible recommendations to both the general and athletic populations. Evaluation of a protein is fundamental in determining its appropriateness in the human diet. Proteins that are of inferior content and digestibility are important to recognize and restrict or limit in the diet. Similarly, such knowledge will provide an ability to identify proteins that provide the greatest benefit and should be consumed. The various techniques utilized to rate protein will be discussed. Traditionally, sources of dietary protein are seen as either being of animal or vegetable origin. Animal sources provide a complete source of protein (i.e. containing all essential amino acids, whereas vegetable sources generally lack one or more of the essential amino acids. Animal sources of dietary protein, despite providing a complete protein and numerous vitamins and minerals, have some health professionals concerned about the amount of saturated fat common in these foods compared to vegetable sources. The advent of processing techniques has shifted some of this attention and ignited the sports supplement marketplace with derivative products such as whey, casein and soy. Individually, these products vary in quality and applicability to certain populations. The benefits that these particular proteins possess are discussed. In addition, the impact that elevated protein consumption has on health and safety issues (i.e. bone health, renal function are also reviewed

  5. Protein kinesis: The dynamics of protein trafficking and stability

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    1995-12-31

    The purpose of this conference is to provide a multidisciplinary forum for exchange of state-of-the-art information on protein kinesis. This volume contains abstracts of papers in the following areas: protein folding and modification in the endoplasmic reticulum; protein trafficking; protein translocation and folding; protein degradation; polarity; nuclear trafficking; membrane dynamics; and protein import into organelles.

  6. Protein flexibility as a biosignal.

    Science.gov (United States)

    Zhao, Qinyi

    2010-01-01

    Dynamic properties of a protein are crucial for all protein functions, and those of signaling proteins are closely related to the biological function of living beings. The protein flexibility signal concept can be used to analyze this relationship. Protein flexibility controls the rate of protein conformational change and influences protein function. The modification of protein flexibility results in a change of protein activity. The logical nature of protein flexibility cannot be explained by applying the principles of protein three-dimensional structure theory or conformation concept. Signaling proteins show high protein flexibility. Many properties of signaling can be traced back to the dynamic natures of signaling protein. The action mechanism of volatile anesthetics and universal cellular reactions are related to flexibility in the change of signaling proteins. We conclude that protein dynamics is an enzyme-enhanced process, called dynamicase.

  7. Supramolecular Chemistry Targeting Proteins.

    Science.gov (United States)

    van Dun, Sam; Ottmann, Christian; Milroy, Lech-Gustav; Brunsveld, Luc

    2017-10-11

    The specific recognition of protein surface elements is a fundamental challenge in the life sciences. New developments in this field will form the basis of advanced therapeutic approaches and lead to applications such as sensors, affinity tags, immobilization techniques, and protein-based materials. Synthetic supramolecular molecules and materials are creating new opportunities for protein recognition that are orthogonal to classical small molecule and protein-based approaches. As outlined here, their unique molecular features enable the recognition of amino acids, peptides, and even whole protein surfaces, which can be applied to the modulation and assembly of proteins. We believe that structural insights into these processes are of great value for the further development of this field and have therefore focused this Perspective on contributions that provide such structural data.

  8. Computational Protein Design

    DEFF Research Database (Denmark)

    Johansson, Kristoffer Enøe

    Proteins are the major functional group of molecules in biology. The impact of protein science on medicine and chemical productions is rapidly increasing. However, the greatest potential remains to be realized. The fi eld of protein design has advanced computational modeling from a tool of support...... to a central method that enables new developments. For example, novel enzymes with functions not found in natural proteins have been de novo designed to give enough activity for experimental optimization. This thesis presents the current state-of-the-art within computational design methods together...... with a novel method based on probability theory. With the aim of assembling a complete pipeline for protein design, this work touches upon several aspects of protein design. The presented work is the computational half of a design project where the other half is dedicated to the experimental part...

  9. [Erythrocyte membrane proteins].

    Science.gov (United States)

    Delaunay, J

    1977-01-01

    Proteins are important constituents of the red blood cell plasma membrane. Several important breakthroughs have occurred in their analysis over the past few years. SDS-polyacrylamide gel electrophoresis lead to the separation of the major proteins and glycoproteins. Location of most of these proteins -- either on the external, the internal or both surfaces of the membrane -- was determined. The strenght of the binding of the protein to the membrane was established. Hydrophobicity of membrane proteins has so far hindered their purification. However, the major glycoprotein (glycophorin A) was isolated and recently sequenced. The description of several membrane-associated enzyme activities has been followed by some understanding of their specific role in the red blood cell physiology. Abnormalities of glycoproteins, Ca2+-ATPase and of membrane protein phosphorylation have been reported under various conditions: sickle cell disease, hereditary spherocytoses, progressive muscular dystrophy.

  10. Algorithms for protein design.

    Science.gov (United States)

    Gainza, Pablo; Nisonoff, Hunter M; Donald, Bruce R

    2016-08-01

    Computational structure-based protein design programs are becoming an increasingly important tool in molecular biology. These programs compute protein sequences that are predicted to fold to a target structure and perform a desired function. The success of a program's predictions largely relies on two components: first, the input biophysical model, and second, the algorithm that computes the best sequence(s) and structure(s) according to the biophysical model. Improving both the model and the algorithm in tandem is essential to improving the success rate of current programs, and here we review recent developments in algorithms for protein design, emphasizing how novel algorithms enable the use of more accurate biophysical models. We conclude with a list of algorithmic challenges in computational protein design that we believe will be especially important for the design of therapeutic proteins and protein assemblies. Copyright © 2016 Elsevier Ltd. All rights reserved.

  11. Mayaro virus proteins

    Directory of Open Access Journals (Sweden)

    J. M. S. Mezencio

    1993-06-01

    Full Text Available Mayaro virus was grown in BHK-21 cells and purified by centrifugation in a potassium-tartrate gradient (5-50%. The electron microscopy analyses of the purified virus showed an homogeneous population of enveloped particles with 69 ñ 2.3 nm in diameter. Three structural virus proteins were identified and designated pl, p2 and p3. Their average molecular weight were p1, 54 KDa; p2, 50 KDa and p3, 34 KDa. In Mayaro virus infected. Aedes albopictus cells and in BHK-21 infected cells we detected six viral proteins, in wich three of them are the structural virus proteins and the other three were products from processing of precursors of viral proteins, whose molecular weights are 62 KDa, 64 KDa and 110 KDa. The 34 KDa protein was the first viral protein sinthesized at 5 hours post-infection in both cell lines studied.

  12. Pressure cryocooling protein crystals

    Science.gov (United States)

    Kim, Chae Un [Ithaca, NY; Gruner, Sol M [Ithaca, NY

    2011-10-04

    Preparation of cryocooled protein crystal is provided by use of helium pressurizing and cryocooling to obtain cryocooled protein crystal allowing collection of high resolution data and by heavier noble gas (krypton or xenon) binding followed by helium pressurizing and cryocooling to obtain cryocooled protein crystal for collection of high resolution data and SAD phasing simultaneously. The helium pressurizing is carried out on crystal coated to prevent dehydration or on crystal grown in aqueous solution in a capillary.

  13. Protein carbonylation in plants

    DEFF Research Database (Denmark)

    Møller, Ian Max; Havelund, Jesper; Rogowska-Wrzesinska, Adelina

    2017-01-01

    This chapter provides an overview of the current knowledge on protein carbonylation in plants and its role in plant physiology. It starts with a brief outline of the turnover and production sites of reactive oxygen species (ROS) in plants and the causes of protein carbonylation. This is followed...... by a description of the methods used to study protein carbonylation in plants, which is also very brief as the methods are similar to those used in studies on animals. The chapter also focuses on protein carbonylation in plants in general and in mitochondria and in seeds in particular, as case stories where...

  14. Engineering therapeutic protein disaggregases.

    Science.gov (United States)

    Shorter, James

    2016-05-15

    Therapeutic agents are urgently required to cure several common and fatal neurodegenerative disorders caused by protein misfolding and aggregation, including amyotrophic lateral sclerosis (ALS), Parkinson's disease (PD), and Alzheimer's disease (AD). Protein disaggregases that reverse protein misfolding and restore proteins to native structure, function, and localization could mitigate neurodegeneration by simultaneously reversing 1) any toxic gain of function of the misfolded form and 2) any loss of function due to misfolding. Potentiated variants of Hsp104, a hexameric AAA+ ATPase and protein disaggregase from yeast, have been engineered to robustly disaggregate misfolded proteins connected with ALS (e.g., TDP-43 and FUS) and PD (e.g., α-synuclein). However, Hsp104 has no metazoan homologue. Metazoa possess protein disaggregase systems distinct from Hsp104, including Hsp110, Hsp70, and Hsp40, as well as HtrA1, which might be harnessed to reverse deleterious protein misfolding. Nevertheless, vicissitudes of aging, environment, or genetics conspire to negate these disaggregase systems in neurodegenerative disease. Thus, engineering potentiated human protein disaggregases or isolating small-molecule enhancers of their activity could yield transformative therapeutics for ALS, PD, and AD. © 2016 Shorter. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  15. Modular protein domains

    National Research Council Canada - National Science Library

    Cesareni, Giovanni

    2005-01-01

    ... encodes not only sequence, but somehow explicitly specifies folding, structure, and biological function as well. How, then, can one learn to read this 'language of proteins'? One of the most powerful approaches to 'cracking the protein code' has involved sequence comparisons between and within species, a task now greatly simplified by the ever...

  16. Advances in Protein Precipitation

    NARCIS (Netherlands)

    Golubovic, M.

    2009-01-01

    Proteins are biological macromolecules, which are among the key components of all living organisms. Proteins are nowadays present in all fields of biotech industry, such as food and feed, synthetic and pharmaceutical industry. They are isolated from their natural sources or produced in different

  17. Amino acids and proteins

    NARCIS (Netherlands)

    van Goudoever, Johannes B.; Vlaardingerbroek, Hester; van den Akker, Chris H.; de Groof, Femke; van der Schoor, Sophie R. D.

    2014-01-01

    Amino acids and protein are key factors for growth. The neonatal period requires the highest intake in life to meet the demands. Those demands include amino acids for growth, but proteins and amino acids also function as signalling molecules and function as neurotransmitters. Often the nutritional

  18. Poxviral Ankyrin Proteins

    Directory of Open Access Journals (Sweden)

    Michael H. Herbert

    2015-02-01

    Full Text Available Multiple repeats of the ankyrin motif (ANK are ubiquitous throughout the kingdoms of life but are absent from most viruses. The main exception to this is the poxvirus family, and specifically the chordopoxviruses, with ANK repeat proteins present in all but three species from separate genera. The poxviral ANK repeat proteins belong to distinct orthologue groups spread over different species, and align well with the phylogeny of their genera. This distribution throughout the chordopoxviruses indicates these proteins were present in an ancestral vertebrate poxvirus, and have since undergone numerous duplication events. Most poxviral ANK repeat proteins contain an unusual topology of multiple ANK motifs starting at the N-terminus with a C-terminal poxviral homologue of the cellular F-box enabling interaction with the cellular SCF ubiquitin ligase complex. The subtle variations between ANK repeat proteins of individual poxviruses suggest an array of different substrates may be bound by these protein-protein interaction domains and, via the F-box, potentially directed to cellular ubiquitination pathways and possible degradation. Known interaction partners of several of these proteins indicate that the NF-κB coordinated anti-viral response is a key target, whilst some poxviral ANK repeat domains also have an F-box independent affect on viral host-range.

  19. Multidomain proteins under force.

    Science.gov (United States)

    Valle-Orero, Jessica; Rivas-Pardo, Jaime Andrés; Popa, Ionel

    2017-04-28

    Advancements in single-molecule force spectroscopy techniques such as atomic force microscopy and magnetic tweezers allow investigation of how domain folding under force can play a physiological role. Combining these techniques with protein engineering and HaloTag covalent attachment, we investigate similarities and differences between four model proteins: I10 and I91-two immunoglobulin-like domains from the muscle protein titin, and two α + β fold proteins-ubiquitin and protein L. These proteins show a different mechanical response and have unique extensions under force. Remarkably, when normalized to their contour length, the size of the unfolding and refolding steps as a function of force reduces to a single master curve. This curve can be described using standard models of polymer elasticity, explaining the entropic nature of the measured steps. We further validate our measurements with a simple energy landscape model, which combines protein folding with polymer physics and accounts for the complex nature of tandem domains under force. This model can become a useful tool to help in deciphering the complexity of multidomain proteins operating under force.

  20. NMR of unfolded proteins

    Indian Academy of Sciences (India)

    In the post-genomic era, as more and more genome sequences are becoming known and hectic efforts are underway to decode the information content in them, it is becoming increasingly evident that flexibility in proteins plays a crucial role in many of the biological functions. Many proteins have intrinsic disorder either ...

  1. Stability of Hyperthermophilic Proteins

    DEFF Research Database (Denmark)

    Stiefler-Jensen, Daniel

    in the high stability of hyperthermophilic enzymes. The thesis starts with an introduction to the field of protein and enzyme stability with special focus on the thermophilic and hyperthermophilic enzymes and proteins. After the introduction three original research manuscripts present the experimental data...

  2. Protein expression-yeast.

    Science.gov (United States)

    Nielsen, Klaus H

    2014-01-01

    Yeast is an excellent system for the expression of recombinant eukaryotic proteins. Both endogenous and heterologous proteins can be overexpressed in yeast (Phan et al., 2001; Ton and Rao, 2004). Because yeast is easy to manipulate genetically, a strain can be optimized for the expression of a specific protein. Many eukaryotic proteins contain posttranslational modifications that can be performed in yeast but not in bacterial expression systems. In comparison with mammalian cell culture expression systems, growing yeast is both faster and less expensive, and large-scale cultures can be performed using fermentation. While several different yeast expression systems exist, this chapter focuses on the budding yeast Saccharomyces cerevisiae and will briefly describe some options to consider when selecting vectors and tags to be used for protein expression. Throughout this chapter, the expression and purification of yeast eIF3 is shown as an example alongside a general scheme outline. © 2014 Elsevier Inc. All rights reserved.

  3. MicroProteins

    DEFF Research Database (Denmark)

    Eguen, Teinai Ebimienere; Straub, Daniel; Graeff, Moritz

    2015-01-01

    MicroProteins (miPs) are short, usually single-domain proteins that, in analogy to miRNAs, heterodimerize with their targets and exert a dominant-negative effect. Recent bioinformatic attempts to identify miPs have resulted in a list of potential miPs, many of which lack the defining characterist......MicroProteins (miPs) are short, usually single-domain proteins that, in analogy to miRNAs, heterodimerize with their targets and exert a dominant-negative effect. Recent bioinformatic attempts to identify miPs have resulted in a list of potential miPs, many of which lack the defining...... can extend beyond transcription factors (TFs) to encompass different non-TF proteins that require dimerization for full function....

  4. Protein disulfide engineering.

    Science.gov (United States)

    Dombkowski, Alan A; Sultana, Kazi Zakia; Craig, Douglas B

    2014-01-21

    Improving the stability of proteins is an important goal in many biomedical and industrial applications. A logical approach is to emulate stabilizing molecular interactions found in nature. Disulfide bonds are covalent interactions that provide substantial stability to many proteins and conform to well-defined geometric conformations, thus making them appealing candidates in protein engineering efforts. Disulfide engineering is the directed design of novel disulfide bonds into target proteins. This important biotechnological tool has achieved considerable success in a wide range of applications, yet the rules that govern the stabilizing effects of disulfide bonds are not fully characterized. Contrary to expectations, many designed disulfide bonds have resulted in decreased stability of the modified protein. We review progress in disulfide engineering, with an emphasis on the issue of stability and computational methods that facilitate engineering efforts. Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  5. Artificially Engineered Protein Polymers.

    Science.gov (United States)

    Yang, Yun Jung; Holmberg, Angela L; Olsen, Bradley D

    2017-06-07

    Modern polymer science increasingly requires precise control over macromolecular structure and properties for engineering advanced materials and biomedical systems. The application of biological processes to design and synthesize artificial protein polymers offers a means for furthering macromolecular tunability, enabling polymers with dispersities of ∼1.0 and monomer-level sequence control. Taking inspiration from materials evolved in nature, scientists have created modular building blocks with simplified monomer sequences that replicate the function of natural systems. The corresponding protein engineering toolbox has enabled the systematic development of complex functional polymeric materials across areas as diverse as adhesives, responsive polymers, and medical materials. This review discusses the natural proteins that have inspired the development of key building blocks for protein polymer engineering and the function of these elements in material design. The prospects and progress for scalable commercialization of protein polymers are reviewed, discussing both technology needs and opportunities.

  6. Sensitizing properties of proteins

    DEFF Research Database (Denmark)

    Poulsen, Lars K.; Ladics, Gregory S; McClain, Scott

    2014-01-01

    scientists from academia, government, and industry participated in the symposium. Experts provided overviews on known mechanisms by which proteins in food may cause sensitization, discussed experimental models to predict protein sensitizing potential, and explored whether such experimental techniques may......The scope of allergy risk is diverse considering the myriad ways in which protein allergenicity is affected by physiochemical characteristics of proteins. The complexity created by the matrices of foods and the variability of the human immune system add additional challenges to understanding...... Allergenicity Technical Committee of the International Life Sciences Institute's Health and Environmental Sciences Institute, featured presentations on current methods, test systems, research trends, and unanswered questions in the field of protein sensitization. A diverse group of over 70 interdisciplinary...

  7. Sensitizing properties of proteins

    DEFF Research Database (Denmark)

    Poulsen, Lars K.; Ladics, Gregory S; McClain, Scott

    2014-01-01

    The scope of allergy risk is diverse considering the myriad ways in which protein allergenicity is affected by physiochemical characteristics of proteins. The complexity created by the matrices of foods and the variability of the human immune system add additional challenges to understanding...... the relationship between sensitization potential and allergy disease. To address these and other issues, an April 2012 international symposium was held in Prague, Czech Republic, to review and discuss the state-of-the-science of sensitizing properties of protein allergens. The symposium, organized by the Protein...... Allergenicity Technical Committee of the International Life Sciences Institute's Health and Environmental Sciences Institute, featured presentations on current methods, test systems, research trends, and unanswered questions in the field of protein sensitization. A diverse group of over 70 interdisciplinary...

  8. [Controversies around diet proteins].

    Science.gov (United States)

    Cichosz, Grazyna; Czeczot, Hanna

    2013-12-01

    Critical theories regarding proteins of anima origin are still and still popularized, though they are ungrounded from scientific point of view. Predominance of soya proteins over the animal ones in relation to their influence on calcium metabolism, bone break risk or risk of osteoporosis morbidity has not been confirmed in any honest, reliable research experiment. Statement, that sulphur amino acids influence disadvantageously on calcium metabolism of human organism and bone status, is completely groundless, the more so as presence of sulphur amino acids in diet (animal proteins are their best source) is the condition of endogenic synthesis of glutathione, the key antioxidant of the organism, and taurine stimulating brain functioning. Deficiency of proteins in the diet produce weakness of intellectual effectiveness and immune response. There is no doubt that limitation of consumption of animal proteins of standard value is not good for health.

  9. Coarse-grain modelling of protein-protein interactions

    NARCIS (Netherlands)

    Baaden, Marc; Marrink, Siewert J.

    2013-01-01

    Here, we review recent advances towards the modelling of protein-protein interactions (PPI) at the coarse-grained (CG) level, a technique that is now widely used to understand protein affinity, aggregation and self-assembly behaviour. PPI models of soluble proteins and membrane proteins are

  10. Swaps in protein sequences.

    Science.gov (United States)

    Fliess, Amit; Motro, Benny; Unger, Ron

    2002-08-01

    An important question in protein evolution is to what extent proteins may have undergone swaps (switches of domain or fragment order) during evolution. Such events might have occurred in several forms: Swaps of short fragments, swaps of structural and functional motifs, or recombination of domains in multidomain proteins. This question is important for the theoretical understanding of the evolution of proteins, and has practical implications for using swaps as a design tool in protein engineering. In order to analyze the question systematically, we conducted a large scale survey of possible swaps and permutations among all pairs of protein from the Swissport database. A swap is defined as a specific kind of sequence mutation between two proteins in which two fragments that appear in both sequences have different relative order in the two sequences. For example, aXbYc and dYeXf are defined as a swap, where X and Y represent sequence fragments that switched their order. Identifying such swaps is difficult using standard sequence comparison packages. One of the main problems in the analysis stems from the fact that many sequences contain repeats, which may be identified as false-positive swaps. We have used two different approaches to detect pairs of proteins with swaps. The first approach is based on the predefined list of domains in Pfam. We identified all the proteins that share at least two domains and analyzed their relative order, looking for pairs in which the order of these domains was switched. We designed an algorithm to distinguish between real swaps and duplications. In the second approach, we used Blast to detect pairs of proteins that share several fragments. Then, we used an automatic procedure to select pairs that are likely to contain swaps. Those pairs were analyzed visually, using a graphical tool, to eliminate duplications. Combining these approaches, about 140 different cases of swaps in the Swissprot database were found (after eliminating

  11. Anchored design of protein-protein interfaces.

    Directory of Open Access Journals (Sweden)

    Steven M Lewis

    Full Text Available Few existing protein-protein interface design methods allow for extensive backbone rearrangements during the design process. There is also a dichotomy between redesign methods, which take advantage of the native interface, and de novo methods, which produce novel binders.Here, we propose a new method for designing novel protein reagents that combines advantages of redesign and de novo methods and allows for extensive backbone motion. This method requires a bound structure of a target and one of its natural binding partners. A key interaction in this interface, the anchor, is computationally grafted out of the partner and into a surface loop on the design scaffold. The design scaffold's surface is then redesigned with backbone flexibility to create a new binding partner for the target. Careful choice of a scaffold will bring experimentally desirable characteristics into the new complex. The use of an anchor both expedites the design process and ensures that binding proceeds against a known location on the target. The use of surface loops on the scaffold allows for flexible-backbone redesign to properly search conformational space.This protocol was implemented within the Rosetta3 software suite. To demonstrate and evaluate this protocol, we have developed a benchmarking set of structures from the PDB with loop-mediated interfaces. This protocol can recover the correct loop-mediated interface in 15 out of 16 tested structures, using only a single residue as an anchor.

  12. Antimicrobial proteins: From old proteins, new tricks.

    Science.gov (United States)

    Smith, Valerie J; Dyrynda, Elisabeth A

    2015-12-01

    This review describes the main types of antimicrobial peptides (AMPs) synthesised by crustaceans, primarily those identified in shrimp, crayfish, crab and lobster. It includes an overview of their range of microbicidal activities and the current landscape of our understanding of their gene expression patterns in different body tissues. It further summarises how their expression might change following various types of immune challenges. The review further considers proteins or protein fragments from crustaceans that have antimicrobial properties but are more usually associated with other biological functions, or are derived from such proteins. It discusses how these unconventional AMPs might be generated at, or delivered to, sites of infection and how they might contribute to crustacean host defence in vivo. It also highlights recent work that is starting to reveal the extent of multi-functionality displayed by some decapod AMPs, particularly their participation in other aspects of host protection. Examples of such activities include proteinase inhibition, phagocytosis, antiviral activity and haematopoiesis. Copyright © 2015 Elsevier Ltd. All rights reserved.

  13. Multidomain proteins under force

    Science.gov (United States)

    Valle-Orero, Jessica; Andrés Rivas-Pardo, Jaime; Popa, Ionel

    2017-04-01

    Advancements in single-molecule force spectroscopy techniques such as atomic force microscopy and magnetic tweezers allow investigation of how domain folding under force can play a physiological role. Combining these techniques with protein engineering and HaloTag covalent attachment, we investigate similarities and differences between four model proteins: I10 and I91—two immunoglobulin-like domains from the muscle protein titin, and two α + β fold proteins—ubiquitin and protein L. These proteins show a different mechanical response and have unique extensions under force. Remarkably, when normalized to their contour length, the size of the unfolding and refolding steps as a function of force reduces to a single master curve. This curve can be described using standard models of polymer elasticity, explaining the entropic nature of the measured steps. We further validate our measurements with a simple energy landscape model, which combines protein folding with polymer physics and accounts for the complex nature of tandem domains under force. This model can become a useful tool to help in deciphering the complexity of multidomain proteins operating under force.

  14. Protein oxidation in aquatic foods

    DEFF Research Database (Denmark)

    Baron, Caroline P.

    2014-01-01

    The chapter discusses general considerations about protein oxidation and reviews the mechanisms involved in protein oxidation and consequences of protein oxidation on fish proteins. It presents two case studies, the first deals with protein and lipid oxidation in frozen rainbow trout......, and the second with oxidation in salted herring. The mechanisms responsible for initiation of protein oxidation are unclear, but it is generally accepted that free radical species initiating lipid oxidation can also initiate protein oxidation. The chapter focuses on interaction between protein and lipid...... oxidation. The protein carbonyl group measurement is the widely used method for estimating protein oxidation in foods and has been used in fish muscle. The chapter also talks about the impact of protein oxidation on protein functionality, fish muscle texture, and food nutritional value. Protein oxidation...

  15. Sound of proteins

    DEFF Research Database (Denmark)

    2007-01-01

    In my group we work with Molecular Dynamics to model several different proteins and protein systems. We submit our modelled molecules to changes in temperature, changes in solvent composition and even external pulling forces. To analyze our simulation results we have so far used visual inspection...... and statistical analysis of the resulting molecular trajectories (as everybody else!). However, recently I started assigning a particular sound frequency to each amino acid in the protein, and by setting the amplitude of each frequency according to the movement amplitude we can "hear" whenever two aminoacids...

  16. PDP: protein domain parser.

    Science.gov (United States)

    Alexandrov, Nickolai; Shindyalov, Ilya

    2003-02-12

    We have developed a program for automatic identification of domains in protein three-dimensional structures. Performance of the program was assessed by three different benchmarks: (i) by comparison with the expert-curated SCOP database of structural domains; (ii) by comparison with a collection of manual domain assignments; and (iii) by comparison with a set of 55 proteins, frequently used as a benchmark for automatic domain assignment. In all these benchmarks PDP identified domains correctly in more than 80% of proteins. http://123d.ncifcrf.gov/.

  17. Alpha Shapes and Proteins

    DEFF Research Database (Denmark)

    Winter, Pawel; Sterner, Henrik; Sterner, Peter

    2009-01-01

    We provide a unified description of (weighted) alpha shapes, beta shapes and the corresponding simplicialcomplexes. We discuss their applicability to various protein-related problems. We also discuss filtrations of alpha shapes and touch upon related persistence issues.We claim that the full...... potential of alpha-shapes and related geometrical constructs in protein-related problems yet remains to be realized and verified. We suggest parallel algorithms for (weighted) alpha shapes, and we argue that future use of filtrations and kinetic variants for larger proteins will need such implementation....

  18. Designing microcapsules based on protein fibrils and protein - polysaccharide complexes

    NARCIS (Netherlands)

    Hua, K.N.P.

    2012-01-01

    Keywords: encapsulation, microcapsule, protein, fibril, protein-polysaccharide complex, controlled release, interfacial rheology, lysozyme, ovalbumin This thesis describes the design of encapsulation systems using mesostructures from proteins and polysaccharides. The approach was to first

  19. Polymers for Protein Conjugation

    Directory of Open Access Journals (Sweden)

    Gianfranco Pasut

    2014-01-01

    Full Text Available Polyethylene glycol (PEG at the moment is considered the leading polymer for protein conjugation in view of its unique properties, as well as to its low toxicity in humans, qualities which have been confirmed by its extensive use in clinical practice. Other polymers that are safe, biodegradable and custom-designed have, nevertheless, also been investigated as potential candidates for protein conjugation. This review will focus on natural polymers and synthetic linear polymers that have been used for protein delivery and the results associated with their use. Genetic fusion approaches for the preparation of protein-polypeptide conjugates will be also reviewed and compared with the best known chemical conjugation ones.

  20. Electron transfer in proteins

    DEFF Research Database (Denmark)

    Farver, O; Pecht, I

    1991-01-01

    Electron migration between and within proteins is one of the most prevalent forms of biological energy conversion processes. Electron transfer reactions take place between active centers such as transition metal ions or organic cofactors over considerable distances at fast rates and with remarkable...... specificity. The electron transfer is attained through weak electronic interaction between the active sites, so that considerable research efforts are centered on resolving the factors that control the rates of long-distance electron transfer reactions in proteins. These factors include (in addition......-containing proteins. These proteins serve almost exclusively in electron transfer reactions, and as it turns out, their metal coordination sites are endowed with properties uniquely optimized for their function....

  1. Protein Colloidal Aggregation Project

    Science.gov (United States)

    Oliva-Buisson, Yvette J. (Compiler)

    2014-01-01

    To investigate the pathways and kinetics of protein aggregation to allow accurate predictive modeling of the process and evaluation of potential inhibitors to prevalent diseases including cataract formation, chronic traumatic encephalopathy, Alzheimer's Disease, Parkinson's Disease and others.

  2. Interactive protein manipulation

    Energy Technology Data Exchange (ETDEWEB)

    SNCrivelli@lbl.gov

    2003-07-01

    We describe an interactive visualization and modeling program for the creation of protein structures ''from scratch''. The input to our program is an amino acid sequence -decoded from a gene- and a sequence of predicted secondary structure types for each amino acid-provided by external structure prediction programs. Our program can be used in the set-up phase of a protein structure prediction process; the structures created with it serve as input for a subsequent global internal energy minimization, or another method of protein structure prediction. Our program supports basic visualization methods for protein structures, interactive manipulation based on inverse kinematics, and visualization guides to aid a user in creating ''good'' initial structures.

  3. Parallel Computational Protein Design.

    Science.gov (United States)

    Zhou, Yichao; Donald, Bruce R; Zeng, Jianyang

    2017-01-01

    Computational structure-based protein design (CSPD) is an important problem in computational biology, which aims to design or improve a prescribed protein function based on a protein structure template. It provides a practical tool for real-world protein engineering applications. A popular CSPD method that guarantees to find the global minimum energy solution (GMEC) is to combine both dead-end elimination (DEE) and A* tree search algorithms. However, in this framework, the A* search algorithm can run in exponential time in the worst case, which may become the computation bottleneck of large-scale computational protein design process. To address this issue, we extend and add a new module to the OSPREY program that was previously developed in the Donald lab (Gainza et al., Methods Enzymol 523:87, 2013) to implement a GPU-based massively parallel A* algorithm for improving protein design pipeline. By exploiting the modern GPU computational framework and optimizing the computation of the heuristic function for A* search, our new program, called gOSPREY, can provide up to four orders of magnitude speedups in large protein design cases with a small memory overhead comparing to the traditional A* search algorithm implementation, while still guaranteeing the optimality. In addition, gOSPREY can be configured to run in a bounded-memory mode to tackle the problems in which the conformation space is too large and the global optimal solution cannot be computed previously. Furthermore, the GPU-based A* algorithm implemented in the gOSPREY program can be combined with the state-of-the-art rotamer pruning algorithms such as iMinDEE (Gainza et al., PLoS Comput Biol 8:e1002335, 2012) and DEEPer (Hallen et al., Proteins 81:18-39, 2013) to also consider continuous backbone and side-chain flexibility.

  4. Protein Nitrogen Determination

    Science.gov (United States)

    Nielsen, S. Suzanne

    The protein content of foods can be determined by numerous methods. The Kjeldahl method and the nitrogen combustion (Dumas) method for protein analysis are based on nitrogen determination. Both methods are official for the purposes of nutrition labeling of foods. While the Kjeldahl method has been used widely for over a hundred years, the recent availability of automated instrumentation for the Dumas method in many cases is replacing use of the Kjeldahl method.

  5. Disease specific protein corona

    Science.gov (United States)

    Rahman, M.; Mahmoudi, M.

    2015-03-01

    It is now well accepted that upon their entrance into the biological environments, the surface of nanomaterials would be covered by various biomacromolecules (e.g., proteins and lipids). The absorption of these biomolecules, so called `protein corona', onto the surface of (nano)biomaterials confers them a new `biological identity'. Although the formation of protein coronas on the surface of nanoparticles has been widely investigated, there are few reports on the effect of various diseases on the biological identity of nanoparticles. As the type of diseases may tremendously changes the composition of the protein source (e.g., human plasma/serum), one can expect that amount and composition of associated proteins in the corona composition may be varied, in disease type manner. Here, we show that corona coated silica and polystyrene nanoparticles (after interaction with in the plasma of the healthy individuals) could induce unfolding of fibrinogen, which promotes release of the inflammatory cytokines. However, no considerable releases of inflammatory cytokines were observed for corona coated graphene sheets. In contrast, the obtained corona coated silica and polystyrene nanoparticles from the hypofibrinogenemia patients could not induce inflammatory cytokine release where graphene sheets do. Therefore, one can expect that disease-specific protein coronas can provide a novel approach for applying nanomedicine to personalized medicine, improving diagnosis and treatment of different diseases tailored to the specific conditions and circumstances.

  6. Fast protein folding kinetics

    Science.gov (United States)

    Gelman, Hannah; Gruebele, Martin

    2014-01-01

    Fast folding proteins have been a major focus of computational and experimental study because they are accessible to both techniques: they are small and fast enough to be reasonably simulated with current computational power, but have dynamics slow enough to be observed with specially developed experimental techniques. This coupled study of fast folding proteins has provided insight into the mechanisms which allow some proteins to find their native conformation well less than 1 ms and has uncovered examples of theoretically predicted phenomena such as downhill folding. The study of fast folders also informs our understanding of even “slow” folding processes: fast folders are small, relatively simple protein domains and the principles that govern their folding also govern the folding of more complex systems. This review summarizes the major theoretical and experimental techniques used to study fast folding proteins and provides an overview of the major findings of fast folding research. Finally, we examine the themes that have emerged from studying fast folders and briefly summarize their application to protein folding in general as well as some work that is left to do. PMID:24641816

  7. The effect of protein-protein and protein-membrane interactions on membrane fouling in ultrafiltration

    NARCIS (Netherlands)

    Huisman, I.H.; Prádanos, P.; Hernández, A.

    2000-01-01

    It was studied how protein-protein and protein-membrane interactions influence the filtration performance during the ultrafiltration of protein solutions over polymeric membranes. This was done by measuring flux, streaming potential, and protein transmission during filtration of bovine serum albumin

  8. Protein: MPA1 [TP Atlas

    Lifescience Database Archive (English)

    Full Text Available MPA1 TLR signaling molecules RSAD2 CIG5 Radical S-adenosyl methionine domain-containing protein 2 Cytomegalo...virus-induced gene 5 protein, Viperin, Virus inhibitory protein, endoplasmic reticu

  9. Protein hydrolysates in sports nutrition

    Directory of Open Access Journals (Sweden)

    Manninen Anssi H

    2009-09-01

    Full Text Available Abstract It has been suggested that protein hydrolysates providing mainly di- and tripeptides are superior to intact (whole proteins and free amino acids in terms of skeletal muscle protein anabolism. This review provides a critical examination of protein hydrolysate studies conducted in healthy humans with special reference to sports nutrition. The effects of protein hydrolysate ingestion on blood amino acid levels, muscle protein anabolism, body composition, exercise performance and muscle glycogen resynthesis are discussed.

  10. PROTEIN SYNTHESIS GAME

    Directory of Open Access Journals (Sweden)

    J.C.Q. Carvalho

    2004-05-01

    Full Text Available The theoretical explanation of biological concepts, associated with the use of teaching games andmodels, intensify the comprehension and increase students interest, stimulating them to participateactively on the teaching-learning process. The sta of dissemination from Centro de BiotecnologiaMolecular Estrutural (CBME, in partnership with the Centro de Divulgac~ao Cientca e Cultural(CDCC, presents, in this work, a new educational resource denoted: Protein Synthesis Game. Theapproach of the game involves the cytological aspects of protein synthesis, directed to high schoolstudents. Students are presented to day-by-day facts related to the function of a given protein in thehuman body. Such task leads players to the goal of solving out a problem through synthesizing aspecied protein. The game comprises: (1 a board illustrated with the transversal section of animalcell, with its main structures and organelles and sequences of hypothetical genes; (2 cards with thedescription of steps and other structures required for protein synthesis in eukaryotic cells; (3 piecesrepresenting nucleotides, polynucleotides, ribosome, amino acids, and polypeptide chains. In order toplay the game, students take cards that sequentially permit them to acquire the necessary pieces forproduction of the protein described in each objective. Players must move the pieces on the board andsimulate the steps of protein synthesis. The dynamic of the game allows students to easily comprehendprocesses of transcription and translation. This game was presented to dierent groups of high schoolteachers and students. Their judgments have been heard and indicated points to be improved, whichhelped us with the game development. Furthermore, the opinions colleted were always favorable forthe application of this game as a teaching resource in classrooms.

  11. Bioinformatics and moonlighting proteins

    Directory of Open Access Journals (Sweden)

    Sergio eHernández

    2015-06-01

    Full Text Available Multitasking or moonlighting is the capability of some proteins to execute two or more biochemical functions. Usually, moonlighting proteins are experimentally revealed by serendipity. For this reason, it would be helpful that Bioinformatics could predict this multifunctionality, especially because of the large amounts of sequences from genome projects. In the present work, we analyse and describe several approaches that use sequences, structures, interactomics and current bioinformatics algorithms and programs to try to overcome this problem. Among these approaches are: a remote homology searches using Psi-Blast, b detection of functional motifs and domains, c analysis of data from protein-protein interaction databases (PPIs, d match the query protein sequence to 3D databases (i.e., algorithms as PISITE, e mutation correlation analysis between amino acids by algorithms as MISTIC. Programs designed to identify functional motif/domains detect mainly the canonical function but usually fail in the detection of the moonlighting one, Pfam and ProDom being the best methods. Remote homology search by Psi-Blast combined with data from interactomics databases (PPIs have the best performance. Structural information and mutation correlation analysis can help us to map the functional sites. Mutation correlation analysis can only be used in very specific situations –it requires the existence of multialigned family protein sequences - but can suggest how the evolutionary process of second function acquisition took place. The multitasking protein database MultitaskProtDB (http://wallace.uab.es/multitask/, previously published by our group, has been used as a benchmark for the all of the analyses.

  12. Direct protein-protein conjugation by genetically introducing bioorthogonal functional groups into proteins.

    Science.gov (United States)

    Kim, Sanggil; Ko, Wooseok; Sung, Bong Hyun; Kim, Sun Chang; Lee, Hyun Soo

    2016-11-15

    Proteins often function as complex structures in conjunction with other proteins. Because these complex structures are essential for sophisticated functions, developing protein-protein conjugates has gained research interest. In this study, site-specific protein-protein conjugation was performed by genetically incorporating an azide-containing amino acid into one protein and a bicyclononyne (BCN)-containing amino acid into the other. Three to four sites in each of the proteins were tested for conjugation efficiency, and three combinations showed excellent conjugation efficiency. The genetic incorporation of unnatural amino acids (UAAs) is technically simple and produces the mutant protein in high yield. In addition, the conjugation reaction can be conducted by simple mixing, and does not require additional reagents or linker molecules. Therefore, this method may prove very useful for generating protein-protein conjugates and protein complexes of biochemical significance. Copyright © 2016. Published by Elsevier Ltd.

  13. Benchtop Detection of Proteins

    Science.gov (United States)

    Scardelletti, Maximilian C.; Varaljay, Vanessa

    2007-01-01

    A process, and a benchtop-scale apparatus for implementing the process, have been developed to detect proteins associated with specific microbes in water. The process and apparatus may also be useful for detection of proteins in other, more complex liquids. There may be numerous potential applications, including monitoring lakes and streams for contamination, testing of blood and other bodily fluids in medical laboratories, and testing for microbial contamination of liquids in restaurants and industrial food-processing facilities. A sample can be prepared and analyzed by use of this process and apparatus within minutes, whereas an equivalent analysis performed by use of other processes and equipment can often take hours to days. The process begins with the conjugation of near-infrared-fluorescent dyes to antibodies that are specific to a particular protein. Initially, the research has focused on using near-infrared dyes to detect antigens or associated proteins in solution, which has proven successful vs. microbial cells, and streamlining the technique in use for surface protein detection on microbes would theoretically render similar results. However, it is noted that additional work is needed to transition protein-based techniques to microbial cell detection. Consequently, multiple such dye/antibody pairs could be prepared to enable detection of multiple selected microbial species, using a different dye for each species. When excited by near-infrared light of a suitable wavelength, each dye fluoresces at a unique longer wavelength that differs from those of the other dyes, enabling discrimination among the various species. In initial tests, the dye/antibody pairs are mixed into a solution suspected of containing the selected proteins, causing the binding of the dye/antibody pairs to such suspect proteins that may be present. The solution is then run through a microcentrifuge that includes a membrane that acts as a filter in that it retains the dye/antibody/protein

  14. Self-Assembling Protein Microarrays

    Science.gov (United States)

    Ramachandran, Niroshan; Hainsworth, Eugenie; Bhullar, Bhupinder; Eisenstein, Samuel; Rosen, Benjamin; Lau, Albert Y.; C. Walter, Johannes; LaBaer, Joshua

    2004-07-01

    Protein microarrays provide a powerful tool for the study of protein function. However, they are not widely used, in part because of the challenges in producing proteins to spot on the arrays. We generated protein microarrays by printing complementary DNAs onto glass slides and then translating target proteins with mammalian reticulocyte lysate. Epitope tags fused to the proteins allowed them to be immobilized in situ. This obviated the need to purify proteins, avoided protein stability problems during storage, and captured sufficient protein for functional studies. We used the technology to map pairwise interactions among 29 human DNA replication initiation proteins, recapitulate the regulation of Cdt1 binding to select replication proteins, and map its geminin-binding domain.

  15. Changes in protein composition and protein phosphorylation during ...

    African Journals Online (AJOL)

    Changes in protein profiles and protein phosphorylation were studied in various stages of germinating somatic and zygotic embryos. Many proteins, which were expressed in cotyledonary stage somatic embryos, were also present in the zygotic embryos obtained from mature dry seed. The intensity of 22 kDa protein was ...

  16. Electrochemical nanomoulding through proteins

    Science.gov (United States)

    Allred, Daniel B.

    The continued improvements in performance of modern electronic devices are directly related to the manufacturing of smaller, denser features on surfaces. Electrochemical fabrication has played a large role in continuing this trend due to its low cost and ease of scaleability toward ever smaller dimensions. This work introduces the concept of using proteins, essentially monodisperse complex polymers whose three-dimensional structures are fixed by their encoded amino acid sequences, as "moulds" around which nanostructures can be built by electrochemical fabrication. Bacterial cell-surface layer proteins, or "S-layer" proteins, from two organisms---Deinococcus radiodurans and Sporosarcina ureae---were used as the "moulds" for electrochemical fabrication. The proteins are easily purified as micron-sized sheets of periodic molecular complexes with 18-nm hexagonal and 13-nm square unit cell lattices, respectively. Direct imaging by transmission electron microscopy on ultrathin noble metal films without sample preparation eliminates potential artifacts to the high surface energy substrates necessary for high nucleation densities. Characterization involved imaging, electron diffraction, spectroscopy, and three-dimensional reconstruction. The S-layer protein of D. radiodurans was further subjected to an atomic force microscope based assay to determine the integrity of its structure and long-range order and was found to be useful for fabrication from around pH 3 to 12.

  17. Protein Denaturation in Foam.

    Science.gov (United States)

    Clarkson; Cui; Darton

    1999-07-15

    The aim of this study was to elucidate the mechanism by which protein molecules become denatured in foam. It was found that damage to the protein is mainly due to surface denaturation at the gas-liquid interface. A fraction of the molecules adsorbed do not refold to their native state when they desorb. The degree of denaturation was found to correlate directly with the interfacial exposure, which, for mobile or partially mobile interfaces, is increased by drainage. Experiments with two different proteins showed that, under the conditions of the tests, around 10% of BSA molecules which had adsorbed at the surface remained denatured when they desorbed. For pepsin the figure was around 75%. Oxidation, which was previously thought to be a major cause of protein damage in foam, was found to be minimal. Neither do the high shear stresses in the liquid bulk encountered during bubble bursting cause denaturation, because energy is dissipated at a much greater length scale than that of the protein molecule. Copyright 1999 Academic Press.

  18. Protein (Cyanobacteria): 654346314 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available protein Mastigocoleus testarum MLEQIELKPNWERNQVAFLDFIVNGTSLHDQFDHPQVRDLCTVFTSDQYEFDGKSSAAIHASWFLGYGETPFPDDRIPVYICSSGDFDCGTVTAYLTVNDGTIKWSEFRIERLTEELQDQPIELTSVKQCVFERNAYEKLFQPFLRKVID

  19. Protein (Cyanobacteria): 654344406 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available protein Mastigocoleus testarum MNKTWRVYLSGEIHTDWREQIEAGTKAAGLPVSFAAPVTDHASSDACGAEILGPEENEFWFDNKGAKVNAIRTSTLIKDADIVVVRFGDKYKQWNAAFDAGYAAALGKPIITLHDAELRHPLKEVDGAALAWAQEPSQVVRLLKYVIEGTL

  20. Polarizable protein packing

    KAUST Repository

    Ng, Albert H.

    2011-01-24

    To incorporate protein polarization effects within a protein combinatorial optimization framework, we decompose the polarizable force field AMOEBA into low order terms. Including terms up to the third-order provides a fair approximation to the full energy while maintaining tractability. We represent the polarizable packing problem for protein G as a hypergraph and solve for optimal rotamers with the FASTER combinatorial optimization algorithm. These approximate energy models can be improved to high accuracy [root mean square deviation (rmsd) < 1 kJ mol -1] via ridge regression. The resulting trained approximations are used to efficiently identify new, low-energy solutions. The approach is general and should allow combinatorial optimization of other many-body problems. © 2011 Wiley Periodicals, Inc. J Comput Chem, 2011 Copyright © 2011 Wiley Periodicals, Inc.

  1. Thermodynamics of Protein Aggregation

    Science.gov (United States)

    Osborne, Kenneth L.; Barz, Bogdan; Bachmann, Michael; Strodel, Birgit

    Amyloid protein aggregation characterizes many neurodegenerative disorders, including Alzheimer's, Parkinson's, and Creutz- feldt-Jakob disease. Evidence suggests that amyloid aggregates may share similar aggregation pathways, implying simulation of full-length amyloid proteins is not necessary for understanding amyloid formation. In this study we simulate GNNQQNY, the N-terminal prion-determining domain of the yeast protein Sup35 to investigate the thermodynamics of structural transitions during aggregation. We use a coarse-grained model with replica-exchange molecular dynamics to investigate the association of 3-, 6-, and 12-chain GNNQQNY systems and we determine the aggregation pathway by studying aggregation states of GN- NQQNY. We find that the aggregation of the hydrophilic GNNQQNY sequence is mainly driven by H-bond formation, leading to the formation of /3-sheets from the very beginning of the assembly process. Condensation (aggregation) and ordering take place simultaneously, which is underpinned by the occurrence of a single heat capacity peak only.

  2. Thermal hysteresis proteins.

    Science.gov (United States)

    Barrett, J

    2001-02-01

    Extreme environments present a wealth of biochemical adaptations. Thermal hysteresis proteins (THPs) have been found in vertebrates, invertebrates, plants, bacteria and fungi and are able to depress the freezing point of water (in the presence of ice crystals) in a non-colligative manner by binding to the surface of nascent ice crystals. The THPs comprise a disparate group of proteins with a variety of tertiary structures and often no common sequence similarities or structural motifs. Different THPs bind to different faces of the ice crystal, and no single mechanism has been proposed to account for THP ice binding affinity and specificity. Experimentally THPs have been used in the cryopreservation of tissues and cells and to induce cold tolerance in freeze susceptible organisms. THPs represent a remarkable example of parallel and convergent evolution with different proteins being adapted for an anti-freeze role.

  3. Accessory Proteins at ERES

    DEFF Research Database (Denmark)

    Klinkenberg, Rafael David

    proteins. Together these components co‐operate in cargo‐selection as well as forming, loading and releasing budding vesicles from specific regions on the membrane surface of the ER. Coat components furthermore convey vesicle targeting towards the Golgi. However, not much is known about the mechanisms...... that regulate the COPII assembly at the vesicle bud site. This thesis provides the first regulatory mechanism of COPII assembly in relation to ER‐membrane lipid‐signal recognition by the accessory protein p125A (Sec23IP). The aim of the project was to characterize p125A function by dissecting two main domains...... in the protein; a putative lipid‐associating domain termed the DDHD domain that is defined by the four amino acid motif that gives the domain its name; and a ubiquitously found domain termed Sterile α‐motif (SAM), which is mostly associated with oligomerization and polymerization. We first show, that the DDHD...

  4. Matricellular proteins and biomaterials.

    Science.gov (United States)

    Morris, Aaron H; Kyriakides, Themis R

    2014-07-01

    Biomaterials are essential to modern medicine as components of reconstructive implants, implantable sensors, and vehicles for localized drug delivery. Advances in biomaterials have led to progression from simply making implants that are nontoxic to making implants that are specifically designed to elicit particular functions within the host. The interaction of implants and the extracellular matrix during the foreign body response is a growing area of concern for the field of biomaterials, because it can lead to implant failure. Expression of matricellular proteins is modulated during the foreign body response and these proteins interact with biomaterials. The design of biomaterials to specifically alter the levels of matricellular proteins surrounding implants provides a new avenue for the design and fabrication of biomimetic biomaterials. Copyright © 2014 International Society of Matrix Biology. Published by Elsevier B.V. All rights reserved.

  5. Trisulfides in Proteins

    DEFF Research Database (Denmark)

    Nielsen, Rasmus W.; Tachibana, Christine; Hansen, Niels Erik

    2011-01-01

    Trisulfides and other oligosulfides are widely distributed in the biological world. In plants, e.g., garlic, trisulfides are associated with potentially beneficial properties. However, an extra neutral sulfur atom covalently bound between the two sulfur atoms of a pair of cysteines is not a commo...... post-translational modification, and the number of proteins in which a trisulfide has been unambiguously identified is small. Nevertheless, we believe that its prevalence may be underestimated, particularly with the increasing evidence for significant pools of sulfides in living tissues...... and their possible roles in cellular metabolism. This review focuses on examples of proteins that are known to contain a trisulfide bridge, and gives an overview of the chemistry of trisulfide formation, and the methods by which it is detected in proteins....

  6. Epistasis in protein evolution

    Science.gov (United States)

    Starr, Tyler N.

    2016-01-01

    Abstract The structure, function, and evolution of proteins depend on physical and genetic interactions among amino acids. Recent studies have used new strategies to explore the prevalence, biochemical mechanisms, and evolutionary implications of these interactions—called epistasis—within proteins. Here we describe an emerging picture of pervasive epistasis in which the physical and biological effects of mutations change over the course of evolution in a lineage‐specific fashion. Epistasis can restrict the trajectories available to an evolving protein or open new paths to sequences and functions that would otherwise have been inaccessible. We describe two broad classes of epistatic interactions, which arise from different physical mechanisms and have different effects on evolutionary processes. Specific epistasis—in which one mutation influences the phenotypic effect of few other mutations—is caused by direct and indirect physical interactions between mutations, which nonadditively change the protein's physical properties, such as conformation, stability, or affinity for ligands. In contrast, nonspecific epistasis describes mutations that modify the effect of many others; these typically behave additively with respect to the physical properties of a protein but exhibit epistasis because of a nonlinear relationship between the physical properties and their biological effects, such as function or fitness. Both types of interaction are rampant, but specific epistasis has stronger effects on the rate and outcomes of evolution, because it imposes stricter constraints and modulates evolutionary potential more dramatically; it therefore makes evolution more contingent on low‐probability historical events and leaves stronger marks on the sequences, structures, and functions of protein families. PMID:26833806

  7. Protein biosynthesis in mitochondria.

    Science.gov (United States)

    Kuzmenko, A V; Levitskii, S A; Vinogradova, E N; Atkinson, G C; Hauryliuk, V; Zenkin, N; Kamenski, P A

    2013-08-01

    Translation, that is biosynthesis of polypeptides in accordance with information encoded in the genome, is one of the most important processes in the living cell, and it has been in the spotlight of international research for many years. The mechanisms of protein biosynthesis in bacteria and in the eukaryotic cytoplasm are now understood in great detail. However, significantly less is known about translation in eukaryotic mitochondria, which is characterized by a number of unusual features. In this review, we summarize current knowledge about mitochondrial translation in different organisms while paying special attention to the aspects of this process that differ from cytoplasmic protein biosynthesis.

  8. Water-transporting proteins

    DEFF Research Database (Denmark)

    Zeuthen, Thomas

    2010-01-01

    Transport through lipids and aquaporins is osmotic and entirely driven by the difference in osmotic pressure. Water transport in cotransporters and uniporters is different: Water can be cotransported, energized by coupling to the substrate flux by a mechanism closely associated with protein...... is not clear. It is associated with the substrate movements in aqueous pathways within the protein; a conventional unstirred layer mechanism can be ruled out, due to high rates of diffusion in the cytoplasm. The physiological roles of the various modes of water transport are reviewed in relation to epithelial...

  9. Cold gelation of globular proteins

    NARCIS (Netherlands)

    Alting, A.C.

    2003-01-01

    Keywords : globular proteins, whey protein, ovalbumin, cold gelation, disulfide bonds, texture, gel hardnessProtein gelation in food products is important to obtain desirable sensory and textural properties. Cold gelation is a novel method to produce protein-based gels. It is a two step process in

  10. The Formation of Protein Structure

    DEFF Research Database (Denmark)

    Bohr, Jakob; Bohr, Henrik; Brunak, Søren

    1996-01-01

    Dynamically induced curvature owing to long-range excitations along the backbones of protein molecules with non-linear elastic properties may control the folding of proteins.......Dynamically induced curvature owing to long-range excitations along the backbones of protein molecules with non-linear elastic properties may control the folding of proteins....

  11. A simple dependence between protein evolution rate and the number of protein-protein interactions

    Directory of Open Access Journals (Sweden)

    Hirsh Aaron E

    2003-05-01

    Full Text Available Abstract Background It has been shown for an evolutionarily distant genomic comparison that the number of protein-protein interactions a protein has correlates negatively with their rates of evolution. However, the generality of this observation has recently been challenged. Here we examine the problem using protein-protein interaction data from the yeast Saccharomyces cerevisiae and genome sequences from two other yeast species. Results In contrast to a previous study that used an incomplete set of protein-protein interactions, we observed a highly significant correlation between number of interactions and evolutionary distance to either Candida albicans or Schizosaccharomyces pombe. This study differs from the previous one in that it includes all known protein interactions from S. cerevisiae, and a larger set of protein evolutionary rates. In both evolutionary comparisons, a simple monotonic relationship was found across the entire range of the number of protein-protein interactions. In agreement with our earlier findings, this relationship cannot be explained by the fact that proteins with many interactions tend to be important to yeast. The generality of these correlations in other kingdoms of life unfortunately cannot be addressed at this time, due to the incompleteness of protein-protein interaction data from organisms other than S. cerevisiae. Conclusions Protein-protein interactions tend to slow the rate at which proteins evolve. This may be due to structural constraints that must be met to maintain interactions, but more work is needed to definitively establish the mechanism(s behind the correlations we have observed.

  12. Modelling of proteins in membranes

    DEFF Research Database (Denmark)

    Sperotto, Maria Maddalena; May, S.; Baumgaertner, A.

    2006-01-01

    This review describes some recent theories and simulations of mesoscopic and microscopic models of lipid membranes with embedded or attached proteins. We summarize results supporting our understanding of phenomena for which the activities of proteins in membranes are expected to be significantly...... affected by the lipid environment. Theoretical predictions are pointed out, and compared to experimental findings, if available. Among others, the following phenomena are discussed: interactions of interfacially adsorbed peptides, pore-forming amphipathic peptides, adsorption of charged proteins onto...... oppositely charged lipid membranes, lipid-induced tilting of proteins embedded in lipid bilayers, protein-induced bilayer deformations, protein insertion and assembly, and lipid-controlled functioning of membrane proteins....

  13. Protein degradation systems in platelets.

    Science.gov (United States)

    Kraemer, B F; Weyrich, A S; Lindemann, S

    2013-11-01

    Protein synthesis and degradation are essential processes that allow cells to survive and adapt to their surrounding milieu. In nucleated cells, the degradation and/or cleavage of proteins is required to eliminate aberrant proteins. Cells also degrade proteins as a mechanism for cell signalling and complex cellular functions. Although the last decade has convincingly shown that platelets synthesise proteins, the roles of protein degradation in these anucleate cytoplasts are less clear. Here we review what is known about protein degradation in platelets placing particular emphasis on the proteasome and the cysteine protease calpain.

  14. Truly Absorbed Microbial Protein Synthesis, Rumen Bypass Protein, Endogenous Protein, and Total Metabolizable Protein from Starchy and Protein-Rich Raw Materials

    NARCIS (Netherlands)

    Parand, Ehsan; Vakili, Alireza; Mesgaran, Mohsen Danesh; Duinkerken, Van Gert; Yu, Peiqiang

    2015-01-01

    This study was carried out to measure truly absorbed microbial protein synthesis, rumen bypass protein, and endogenous protein loss, as well as total metabolizable protein, from starchy and protein-rich raw feed materials with model comparisons. Predictions by the DVE2010 system as a more

  15. Protein requirements of Penaeid shrimp.

    OpenAIRE

    Kanazawa, A

    1989-01-01

    Proteins are indispensable nutrients for growth and maintenance of live of all animals. The optimum protein levels in diets for shrimps are different among the various species. Squid meal is an effective protein source for many penaeids. The effects of dietary protein, lipid, and carbohydrate levels on the growth and survival of larvae of Penaeus japonicus were examined by feeding trials using purified diet with carrageenan as a binder. As a result, the effects of protein levels on growth and...

  16. Protein oxidation and ageing

    DEFF Research Database (Denmark)

    Linton, S; Davies, Michael Jonathan; Dean, R T

    2001-01-01

    of redox-active metal ions that could catalyse oxidant formation. As a result of this decrease in antioxidant defences, and increased rate of ROS formation, it is possible that the impact of ROS increases with age. ROS are known to oxidise biological macromolecules, with proteins an important target...

  17. Thermodynamics of meat proteins

    NARCIS (Netherlands)

    Sman, van der R.G.M.

    2012-01-01

    We describe the water activity of meat, being a mixture of proteins, salts and water, by the Free-Volume-Flory–Huggins (FVFH) theory augmented with the equation. Earlier, the FVFH theory is successfully applied to describe the thermodynamics to glucose homopolymers like starch, dextrans and

  18. Protein digestion in ruminants

    African Journals Online (AJOL)

    Animal Nutrition, Animal and Dairy Science Research Institute, Irene, 1675Republic of South Africa. Although the protein requirement of domestic ruminants may be calculated from a simple one-compartment model, this approach ignores factors such as microbial fermentation in the rumen and the non-equality of feed.

  19. Protein Sorting Prediction

    DEFF Research Database (Denmark)

    Nielsen, Henrik

    2017-01-01

    Many computational methods are available for predicting protein sorting in bacteria. When comparing them, it is important to know that they can be grouped into three fundamentally different approaches: signal-based, global-property-based and homology-based prediction. In this chapter, the strengt...

  20. Allosteric Regulation of Proteins

    Indian Academy of Sciences (India)

    ... Lecture Workshops · Refresher Courses · Symposia · Live Streaming. Home; Journals; Resonance – Journal of Science Education; Volume 22; Issue 1. Allosteric Regulation of Proteins: A Historical Perspective on the Development of Concepts and Techniques. General Article Volume 22 Issue 1 January 2017 pp 37-50 ...

  1. Markers of protein oxidation

    DEFF Research Database (Denmark)

    Headlam, Henrietta A; Davies, Michael Jonathan

    2004-01-01

    Exposure of proteins to radicals in the presence of O2 gives both side-chain oxidation and backbone fragmentation. These processes can be interrelated, with initial side-chain oxidation giving rise to backbone damage via transfer reactions. We have shown previously that alkoxyl radicals formed on...

  2. Protein digestion in ruminants

    African Journals Online (AJOL)

    acids absorbed into the circulation of the animal. Ideally, therefore, the biological value of a feed protein should be determined from the amount and type of amino acid appearing in the portal circulation of the animal, and not simplythe dissappearance of amino acids from the tract. Ruminant digestion may be more easily ...

  3. Antifreeze Proteins of Bacteria

    Indian Academy of Sciences (India)

    Home; Journals; Resonance – Journal of Science Education; Volume 12; Issue 12. Antifreeze Proteins of Bacteria. M K Chattopadhyay. General Article Volume 12 Issue 12 December 2007 pp 25-30. Fulltext. Click here to view fulltext PDF. Permanent link: http://www.ias.ac.in/article/fulltext/reso/012/12/0025-0030. Keywords.

  4. NMR of unfolded proteins

    Indian Academy of Sciences (India)

    Unknown

    2005-01-03

    Jan 3, 2005 ... deposition of data and advanced search on the pattern of PDB.12. Detailed characterization of the unfolded state and consequent identification of the folding initiation sites in a given protein provide valuable insight into its folding mechanism.18 Well-formed or transient residual structures in the unfolded ...

  5. Protein Requirements during Aging

    Directory of Open Access Journals (Sweden)

    Glenda Courtney-Martin

    2016-08-01

    Full Text Available Protein recommendations for elderly, both men and women, are based on nitrogen balance studies. They are set at 0.66 and 0.8 g/kg/day as the estimated average requirement (EAR and recommended dietary allowance (RDA, respectively, similar to young adults. This recommendation is based on single linear regression of available nitrogen balance data obtained at test protein intakes close to or below zero balance. Using the indicator amino acid oxidation (IAAO method, we estimated the protein requirement in young adults and in both elderly men and women to be 0.9 and 1.2 g/kg/day as the EAR and RDA, respectively. This suggests that there is no difference in requirement on a gender basis or on a per kg body weight basis between younger and older adults. The requirement estimates however are ~40% higher than the current protein recommendations on a body weight basis. They are also 40% higher than our estimates in young men when calculated on the basis of fat free mass. Thus, current recommendations may need to be re-assessed. Potential rationale for this difference includes a decreased sensitivity to dietary amino acids and increased insulin resistance in the elderly compared with younger individuals.

  6. Protein: CAD [Trypanosomes Database

    Lifescience Database Archive (English)

    Full Text Available CAD carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotaseCAD... trifunctional proteincarbamoylphosphate synthetase 2/aspartate transcarbamylase/dihydroorotasemultifunctional protein CAD... H.sapiens 47458828 18105007 790 P27708 CAD_(gene) 2.1.3.2|3.5.2.3|6.3.5.5 114010 2p22-p21 hsa00250|hsa00240 ...

  7. Measuring protein breakdown in individual proteins in vivo

    DEFF Research Database (Denmark)

    Holm, Lars; Kjær, Michael

    2010-01-01

    be used to determine the breakdown rate of specific proteins and, therefore, do not keep up to the preceding methodological demands in physiological research. A newly developed approach to determine the fractional breakdown rate of single proteins seems promising. Its conceptual advantage......PURPOSE OF REVIEW: To outline different approaches of how protein breakdown can be quantified and to present a new approach to determine the fractional breakdown rate of individual slow turnover proteins in vivo. RECENT FINDINGS: None of the available methods for determining protein breakdown can...... is that the proteins of interest are the site of measurement. Hence, the application initially demands the proteins to be labeled with stable isotopically labeled amino acids. Subsequently, the loss of label from the proteins will be dependent on the protein breakdown rate when no labeled amino acids...

  8. Interaction between plate make and protein in protein crystallisation screening.

    Directory of Open Access Journals (Sweden)

    Gordon J King

    Full Text Available BACKGROUND: Protein crystallisation screening involves the parallel testing of large numbers of candidate conditions with the aim of identifying conditions suitable as a starting point for the production of diffraction quality crystals. Generally, condition screening is performed in 96-well plates. While previous studies have examined the effects of protein construct, protein purity, or crystallisation condition ingredients on protein crystallisation, few have examined the effect of the crystallisation plate. METHODOLOGY/PRINCIPAL FINDINGS: We performed a statistically rigorous examination of protein crystallisation, and evaluated interactions between crystallisation success and plate row/column, different plates of same make, different plate makes and different proteins. From our analysis of protein crystallisation, we found a significant interaction between plate make and the specific protein being crystallised. CONCLUSIONS/SIGNIFICANCE: Protein crystal structure determination is the principal method for determining protein structure but is limited by the need to produce crystals of the protein under study. Many important proteins are difficult to crystallize, so that identification of factors that assist crystallisation could open up the structure determination of these more challenging targets. Our findings suggest that protein crystallisation success may be improved by matching a protein with its optimal plate make.

  9. Minireview: protein arginine methylation of nonhistone proteins in transcriptional regulation.

    Science.gov (United States)

    Lee, Young-Ho; Stallcup, Michael R

    2009-04-01

    Endocrine regulation frequently culminates in altered transcription of specific genes. The signal transduction pathways, which transmit the endocrine signal from cell surface to the transcription machinery, often involve posttranslational modifications of proteins. Although phosphorylation has been by far the most widely studied protein modification, recent studies have indicated important roles for other types of modification, including protein arginine methylation. Ten different protein arginine methyltransferase (PRMT) family members have been identified in mammalian cells, and numerous substrates are being identified for these PRMTs. Whereas major attention has been focused on the methylation of histones and its role in chromatin remodeling and transcriptional regulation, there are many nonhistone substrates methylated by PRMTs. This review primarily focuses on recent progress on the roles of the nonhistone protein methylation in transcription. Protein methylation of coactivators, transcription factors, and signal transducers, among other proteins, plays important roles in transcriptional regulation. Protein methylation may affect protein-protein interaction, protein-DNA or protein-RNA interaction, protein stability, subcellular localization, or enzymatic activity. Thus, protein arginine methylation is critical for regulation of transcription and potentially for various physiological/pathological processes.

  10. HIV protein sequence hotspots for crosstalk with host hub proteins.

    Directory of Open Access Journals (Sweden)

    Mahdi Sarmady

    Full Text Available HIV proteins target host hub proteins for transient binding interactions. The presence of viral proteins in the infected cell results in out-competition of host proteins in their interaction with hub proteins, drastically affecting cell physiology. Functional genomics and interactome datasets can be used to quantify the sequence hotspots on the HIV proteome mediating interactions with host hub proteins. In this study, we used the HIV and human interactome databases to identify HIV targeted host hub proteins and their host binding partners (H2. We developed a high throughput computational procedure utilizing motif discovery algorithms on sets of protein sequences, including sequences of HIV and H2 proteins. We identified as HIV sequence hotspots those linear motifs that are highly conserved on HIV sequences and at the same time have a statistically enriched presence on the sequences of H2 proteins. The HIV protein motifs discovered in this study are expressed by subsets of H2 host proteins potentially outcompeted by HIV proteins. A large subset of these motifs is involved in cleavage, nuclear localization, phosphorylation, and transcription factor binding events. Many such motifs are clustered on an HIV sequence in the form of hotspots. The sequential positions of these hotspots are consistent with the curated literature on phenotype altering residue mutations, as well as with existing binding site data. The hotspot map produced in this study is the first global portrayal of HIV motifs involved in altering the host protein network at highly connected hub nodes.

  11. Fragments of protein A eluted during protein A affinity chromatography.

    Science.gov (United States)

    Carter-Franklin, Jayme N; Victa, Corazon; McDonald, Paul; Fahrner, Robert

    2007-09-07

    Protein A affinity chromatography is a common method for process scale purification of monoclonal antibodies. During protein A affinity chromatography, protein A ligand co-elutes with the antibody (commonly called leaching), which is a potential disadvantage since the leached protein A may need to be cleared for pharmaceutical antibodies. To determine the mechanism of protein A leaching and characterize the leached protein A, we fluorescently labeled the protein A ligand in situ on protein A affinity chromatography media. We found that intact protein A leaches when loading either purified antibody or unpurified antibody in harvested cell culture fluid (HCCF), and that additionally fragments of protein A leach when loading HCCF. The leaching of protein A fragments can be reduced by EDTA, suggesting that proteinases contribute to the generation of protein A fragments. We found that protein A fragments larger than about 6000 Da can be measured by enzyme linked immunosorbent assay, and that they can be more difficult to clear than whole protein A by cation-exchange chromatography.

  12. Exploring NMR ensembles of calcium binding proteins: Perspectives to design inhibitors of protein-protein interactions

    Directory of Open Access Journals (Sweden)

    Craescu Constantin T

    2011-05-01

    Full Text Available Abstract Background Disrupting protein-protein interactions by small organic molecules is nowadays a promising strategy employed to block protein targets involved in different pathologies. However, structural changes occurring at the binding interfaces make difficult drug discovery processes using structure-based drug design/virtual screening approaches. Here we focused on two homologous calcium binding proteins, calmodulin and human centrin 2, involved in different cellular functions via protein-protein interactions, and known to undergo important conformational changes upon ligand binding. Results In order to find suitable protein conformations of calmodulin and centrin for further structure-based drug design/virtual screening, we performed in silico structural/energetic analysis and molecular docking of terphenyl (a mimicking alpha-helical molecule known to inhibit protein-protein interactions of calmodulin into X-ray and NMR ensembles of calmodulin and centrin. We employed several scoring methods in order to find the best protein conformations. Our results show that docking on NMR structures of calmodulin and centrin can be very helpful to take into account conformational changes occurring at protein-protein interfaces. Conclusions NMR structures of protein-protein complexes nowadays available could efficiently be exploited for further structure-based drug design/virtual screening processes employed to design small molecule inhibitors of protein-protein interactions.

  13. Inferring protein function by domain context similarities in protein-protein interaction networks

    Directory of Open Access Journals (Sweden)

    Sun Zhirong

    2009-12-01

    Full Text Available Abstract Background Genome sequencing projects generate massive amounts of sequence data but there are still many proteins whose functions remain unknown. The availability of large scale protein-protein interaction data sets makes it possible to develop new function prediction methods based on protein-protein interaction (PPI networks. Although several existing methods combine multiple information resources, there is no study that integrates protein domain information and PPI networks to predict protein functions. Results The domain context similarity can be a useful index to predict protein function similarity. The prediction accuracy of our method in yeast is between 63%-67%, which outperforms the other methods in terms of ROC curves. Conclusion This paper presents a novel protein function prediction method that combines protein domain composition information and PPI networks. Performance evaluations show that this method outperforms existing methods.

  14. High quality protein microarray using in situ protein purification

    Directory of Open Access Journals (Sweden)

    Fleischmann Robert D

    2009-08-01

    Full Text Available Abstract Background In the postgenomic era, high throughput protein expression and protein microarray technologies have progressed markedly permitting screening of therapeutic reagents and discovery of novel protein functions. Hexa-histidine is one of the most commonly used fusion tags for protein expression due to its small size and convenient purification via immobilized metal ion affinity chromatography (IMAC. This purification process has been adapted to the protein microarray format, but the quality of in situ His-tagged protein purification on slides has not been systematically evaluated. We established methods to determine the level of purification of such proteins on metal chelate-modified slide surfaces. Optimized in situ purification of His-tagged recombinant proteins has the potential to become the new gold standard for cost-effective generation of high-quality and high-density protein microarrays. Results Two slide surfaces were examined, chelated Cu2+ slides suspended on a polyethylene glycol (PEG coating and chelated Ni2+ slides immobilized on a support without PEG coating. Using PEG-coated chelated Cu2+ slides, consistently higher purities of recombinant proteins were measured. An optimized wash buffer (PBST composed of 10 mM phosphate buffer, 2.7 mM KCl, 140 mM NaCl and 0.05% Tween 20, pH 7.4, further improved protein purity levels. Using Escherichia coli cell lysates expressing 90 recombinant Streptococcus pneumoniae proteins, 73 proteins were successfully immobilized, and 66 proteins were in situ purified with greater than 90% purity. We identified several antigens among the in situ-purified proteins via assays with anti-S. pneumoniae rabbit antibodies and a human patient antiserum, as a demonstration project of large scale microarray-based immunoproteomics profiling. The methodology is compatible with higher throughput formats of in vivo protein expression, eliminates the need for resin-based purification and circumvents

  15. Metabolism of minor isoforms of prion proteins: Cytosolic prion protein and transmembrane prion protein

    OpenAIRE

    Song, Zhiqi; Zhao, Deming; Yang, Lifeng

    2013-01-01

    Transmissible spongiform encephalopathy or prion disease is triggered by the conversion from cellular prion protein to pathogenic prion protein. Growing evidence has concentrated on prion protein configuration changes and their correlation with prion disease transmissibility and pathogenicity. In vivo and in vitro studies have shown that several cytosolic forms of prion protein with specific topological structure can destroy intracellular stability and contribute to prion protein pathogenicit...

  16. Shape complementarity and hydrogen bond preferences in protein-protein interfaces: implications for antibody modeling and protein-protein docking.

    Science.gov (United States)

    Kuroda, Daisuke; Gray, Jeffrey J

    2016-08-15

    Characterizing protein-protein interfaces and the hydrogen bonds is a first step to better understand proteins' structures and functions toward high-resolution protein design. However, there are few large-scale surveys of hydrogen bonds of interfaces. In addition, previous work of shape complementarity of protein complexes suggested that lower shape complementarity in antibody-antigen interfaces is related to their evolutionary origin. Using 6637 non-redundant protein-protein interfaces, we revealed peculiar features of various protein complex types. In contrast to previous findings, the shape complementarity of antibody-antigen interfaces resembles that of the other interface types. These results highlight the importance of hydrogen bonds during evolution of protein interfaces and rectify the prevailing belief that antibodies have lower shape complementarity. jgray@jhu.edu Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  17. Dairy Proteins and Energy Balance

    DEFF Research Database (Denmark)

    Bendtsen, Line Quist

    High protein diets affect energy balance beneficially through decreased hunger, enhanced satiety and increased energy expenditure. Dairy products are a major source of protein. Dairy proteins are comprised of two classes, casein (80%) and whey proteins (20%), which are both of high quality......, but casein is absorbed slowly and whey is absorbed rapidly. The present PhD study investigated the effects of total dairy proteins, whey, and casein, on energy balance and the mechanisms behind any differences in the effects of the specific proteins. The results do not support the hypothesis that dairy...... proteins, whey or casein are more beneficial than other protein sources in the regulation of energy balance, and suggest that dairy proteins, whey or casein seem to play only a minor role, if any, in the prevention and treatment of obesity....

  18. Discovering Protein-Protein Interactions Using Nucleic Acid Programmable Protein Arrays.

    Science.gov (United States)

    Tang, Yanyang; Qiu, Ji; Machner, Matthias; LaBaer, Joshua

    2017-03-03

    We have developed a protocol enabling the study of protein-protein interactions (PPIs) at the proteome level using in vitro-synthesized proteins. Assay preparation requires molecular cloning of the query gene into a vector that supports in vitro transcription/translation (IVTT) and appends a HaloTag to the query protein of interest. In parallel, protein microarrays are prepared by printing plasmids encoding glutathione S-transferase (GST)-tagged target proteins onto a carrier matrix/glass slide coated with antibody directed against GST. At the time of the experiment, the query protein and the target protein are produced separately through IVTT. The query protein is then applied to nucleic acid programmable protein arrays (NAPPA) that display thousands of freshly produced target proteins captured by anti-GST antibody. Interactions between the query and immobilized target proteins are detected through addition of a fluorophore-labeled HaloTag ligand. Our protocol allows the elucidation of PPIs in a high-throughput fashion using proteins produced in vitro, obviating the scientific challenges, high cost, and laborious work, as well as concerns about protein stability, which are usually present in protocols using conventional protein arrays. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

  19. Circular dichroism spectroscopy of fluorescent proteins

    NARCIS (Netherlands)

    Visser, N.V.; Hink, M.A.; Borst, J.W.; Krogt, van der G.N.M.; Visser, A.J.W.G.

    2002-01-01

    Circular dichroism (CD) spectra have been obtained from several variants of green fluorescent protein: blue fluorescent protein (BFP), enhanced cyan fluorescent protein (CFP), enhanced green fluorescent protein (GFP), enhanced yellow fluorescent protein (YFP), all from Aequorea victoria, and the red

  20. Competitive Protein Adsorption - Multilayer Adsorption and Surface Induced Protein Aggregation

    DEFF Research Database (Denmark)

    Holmberg, Maria; Hou, Xiaolin

    2009-01-01

    In this study, competitive adsorption of albumin and IgG (immunoglobulin G) from human serum solutions and protein mixtures onto polymer surfaces is studied by means of radioactive labeling. By using two different radiolabels (125I and 131I), albumin and IgG adsorption to polymer surfaces...... is monitored simultaneously and the influence from the presence of other human serum proteins on albumin and IgG adsorption, as well as their mutual influence during adsorption processes, is investigated. Exploring protein adsorption by combining analysis of competitive adsorption from complex solutions...... of high concentration with investigation of single protein adsorption and interdependent adsorption between two specific proteins enables us to map protein adsorption sequences during competitive protein adsorption. Our study shows that proteins can adsorb in a multilayer fashion onto the polymer surfaces...

  1. Protein Functionalized Nanodiamond Arrays

    Directory of Open Access Journals (Sweden)

    Liu YL

    2010-01-01

    Full Text Available Abstract Various nanoscale elements are currently being explored for bio-applications, such as in bio-images, bio-detection, and bio-sensors. Among them, nanodiamonds possess remarkable features such as low bio-cytotoxicity, good optical property in fluorescent and Raman spectra, and good photostability for bio-applications. In this work, we devise techniques to position functionalized nanodiamonds on self-assembled monolayer (SAMs arrays adsorbed on silicon and ITO substrates surface using electron beam lithography techniques. The nanodiamond arrays were functionalized with lysozyme to target a certain biomolecule or protein specifically. The optical properties of the nanodiamond-protein complex arrays were characterized by a high throughput confocal microscope. The synthesized nanodiamond-lysozyme complex arrays were found to still retain their functionality in interacting with E. coli.

  2. Problems in Protein Biosynthesis

    Science.gov (United States)

    Lengyel, Peter

    1966-01-01

    Outline of the steps in protein synthesis. Nature of the genetic code. The use of synthetic oligo- and polynucleotides in deciphering the code. Structure of the code: relatedness of synonym codons. The wobble hypothesis. Chain initiation and N-formyl-methionine. Chain termination and nonsense codons. Mistakes in translation: ambiguity in vitro. Suppressor mutations resulting in ambiguity. Limitations in the universality of the code. Attempts to determine the particular codons used by a species. Mechanisms of suppression, caused by (a) abnormal aminoacyl-tRNA, (b) ribosomal malfunction. Effect of streptomycin. The problem of "reading" a nucleic acid template. Different ribosomal mutants and DNA polymerase mutants might cause different mistakes. The possibility of involvement of allosteric proteins in template reading. PMID:5338560

  3. Accessory Proteins at ERES

    DEFF Research Database (Denmark)

    Klinkenberg, Rafael David

    distribution of mSec16B. We further dissect both mSec16A and mSec16B, and show that the region in human mSec16B encompassing residues 35‐194 and the region in human mSec16A comprising residues 1096‐1190 maintain membrane binding irrespective of the removal of membrane associating proteins by salt wash...... or proteolytic digestion. However, neither mSec16B (35‐194) nor mSec16A (1096‐1190) maintain ERES targeting. These findings support previous observations of the need for the membrane binding regions to be expressed in cis with a Central Conserved Domain (CCD) in both proteins to convey ERES targeting....

  4. Porcine prion protein amyloid

    OpenAIRE

    Hammarstr?m, Per; Nystr?m, Sofie

    2015-01-01

    ABSTRACT Mammalian prions are composed of misfolded aggregated prion protein (PrP) with amyloid-like features. Prions are zoonotic disease agents that infect a wide variety of mammalian species including humans. Mammals and by-products thereof which are frequently encountered in daily life are most important for human health. It is established that bovine prions (BSE) can infect humans while there is no such evidence for any other prion susceptible species in the human food chain (sheep, goat...

  5. Engineering ancestral protein hyperstability.

    Science.gov (United States)

    Romero-Romero, M Luisa; Risso, Valeria A; Martinez-Rodriguez, Sergio; Ibarra-Molero, Beatriz; Sanchez-Ruiz, Jose M

    2016-10-15

    Many experimental analyses and proposed scenarios support that ancient life was thermophilic. In congruence with this hypothesis, proteins encoded by reconstructed sequences corresponding to ancient phylogenetic nodes often display very high stability. Here, we show that such 'reconstructed ancestral hyperstability' can be further engineered on the basis of a straightforward approach that uses exclusively information afforded by the ancestral reconstruction process itself. Since evolution does not imply continuous progression, screening of the mutations between two evolutionarily related resurrected ancestral proteins may identify mutations that further stabilize the most stable one. To explore this approach, we have used a resurrected thioredoxin corresponding to the last common ancestor of the cyanobacterial, Deinococcus and Thermus groups (LPBCA thioredoxin), which has a denaturation temperature of ∼123°C. This high value is within the top 0.1% of the denaturation temperatures in the ProTherm database and, therefore, achieving further stabilization appears a priori as a challenging task. Nevertheless, experimental comparison with a resurrected thioredoxin corresponding to the last common ancestor of bacteria (denaturation temperature of ∼115°C) immediately identifies three mutations that increase the denaturation temperature of LPBCA thioredoxin to ∼128°C. Comparison between evolutionarily related resurrected ancestral proteins thus emerges as a simple approach to expand the capability of ancestral reconstruction to search sequence space for extreme protein properties of biotechnological interest. The fact that ancestral sequences for many phylogenetic nodes can be derived from a single alignment of modern sequences should contribute to the general applicability of this approach. © 2016 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

  6. Immunoprecipitation-based analysis of protein-protein interactions.

    Science.gov (United States)

    Speth, Corinna; Toledo-Filho, Luis A A; Laubinger, Sascha

    2014-01-01

    Several techniques allow the detection of protein-protein interactions. In vivo co-immunoprecipitation (Co-IP) studies are an important complement to other commonly used techniques such as yeast two-hybrid or fluorescence complementation, as they reveal interactions between functional proteins at physiological relevant concentrations. Here, we describe an in vivo Co-IP approach using either GFP affinity matrix or specific antibodies to purify proteins of interests and their interacting partners.

  7. Neutron protein crystallography

    Energy Technology Data Exchange (ETDEWEB)

    Niimura, Nobuo [Japan Atomic Energy Research Inst., Tokai, Ibaraki (Japan). Tokai Research Establishment

    1998-10-01

    X-ray diffraction of single crystal has enriched the knowledge of various biological molecules such as proteins, DNA, t-RNA, viruses, etc. It is difficult to make structural analysis of hydrogen atoms in a protein using X-ray crystallography, whereas neutron diffraction seems usable to directly determine the location of those hydrogen atoms. Here, neutron diffraction method was applied to structural analysis of hen egg-white lysozyme. Since the crystal size of a protein to analyze is generally small (5 mm{sup 3} at most), the neutron beam at the sample position in monochromator system was set to less than 5 x 5 mm{sup 2} and beam divergence to 0.4 degree or less. Neutron imaging plate with {sup 6}Li or Gd mixed with photostimulated luminescence material was used and about 2500 Bragg reflections were recorded in one crystal setting. A total of 38278 reflections for 2.0 A resolution were collected in less than 10 days. Thus, stereo views of Trp-111 omit map around the indol ring of Trp-111 was presented and the three-dimensional arrangement of 696H and 264D atoms in the lysozyme molecules was determined using the omit map. (M.N.)

  8. Metabolism of minor isoforms of prion proteins: Cytosolic prion protein and transmembrane prion protein

    Science.gov (United States)

    Song, Zhiqi; Zhao, Deming; Yang, Lifeng

    2013-01-01

    Transmissible spongiform encephalopathy or prion disease is triggered by the conversion from cellular prion protein to pathogenic prion protein. Growing evidence has concentrated on prion protein configuration changes and their correlation with prion disease transmissibility and pathogenicity. In vivo and in vitro studies have shown that several cytosolic forms of prion protein with specific topological structure can destroy intracellular stability and contribute to prion protein pathogenicity. In this study, the latest molecular chaperone system associated with endoplasmic reticulum-associated protein degradation, the endoplasmic reticulum resident protein quality-control system and the ubiquitination proteasome system, is outlined. The molecular chaperone system directly correlates with the prion protein degradation pathway. Understanding the molecular mechanisms will help provide a fascinating avenue for further investigations on prion disease treatment and prion protein-induced neurodegenerative diseases. PMID:25206608

  9. Understanding Protein Non-Folding

    Science.gov (United States)

    Uversky, Vladimir N.; Dunker, A. Keith

    2010-01-01

    This review describes the family of intrinsically disordered proteins, members of which fail to form rigid 3-D structures under physiological conditions, either along their entire lengths or only in localized regions. Instead, these intriguing proteins/regions exist as dynamic ensembles within which atom positions and backbone Ramachandran angles exhibit extreme temporal fluctuations without specific equilibrium values. Many of these intrinsically disordered proteins are known to carry out important biological functions which, in fact, depend on the absence of specific 3-D structure. The existence of such proteins does not fit the prevailing structure-function paradigm, which states that unique 3-D structure is a prerequisite to function. Thus, the protein structure-function paradigm has to be expanded to include intrinsically disordered proteins and alternative relationships among protein sequence, structure, and function. This shift in the paradigm represents a major breakthrough for biochemistry, biophysics and molecular biology, as it opens new levels of understanding with regard to the complex life of proteins. This review will try to answer the following questions: How were intrinsically disordered proteins discovered? Why don't these proteins fold? What is so special about intrinsic disorder? What are the functional advantages of disordered proteins/regions? What is the functional repertoire of these proteins? What are the relationships between intrinsically disordered proteins and human diseases? PMID:20117254

  10. Regulation of protein function by ‘microProteins'

    OpenAIRE

    Staudt, Annica-Carolin; Wenkel, Stephan

    2010-01-01

    Elegant post-translational regulation is achieved by ‘microProteins', which form homotypic dimers with their targets and act through the dominant–negative suppression of protein complex function. The recent identification of new microProteins suggests their role is general and has evolved in both the plant and animal kingdoms.

  11. Digestion of protein and protein gels in simulated gastric environment

    NARCIS (Netherlands)

    Luo, Q.; Boom, R.M.; Janssen, A.E.M.

    2015-01-01

    Despite the increasing attention to food digestion research, food scientists still need to better understand the underlying mechanisms of digestion. Most in vitro studies on protein digestion are based on experiments with protein solutions. In this study, the digestion of egg white protein and whey

  12. Molecular simulations of lipid-mediated protein-protein interactions

    NARCIS (Netherlands)

    de Meyer, F.J.M.; Venturoli, M.; Smit, B.

    2008-01-01

    Recent experimental results revealed that lipid-mediated interactions due to hydrophobic forces may be important in determining the protein topology after insertion in the membrane, in regulating the protein activity, in protein aggregation and in signal transduction. To gain insight into the

  13. The interface of protein structure, protein biophysics, and molecular evolution

    Science.gov (United States)

    Liberles, David A; Teichmann, Sarah A; Bahar, Ivet; Bastolla, Ugo; Bloom, Jesse; Bornberg-Bauer, Erich; Colwell, Lucy J; de Koning, A P Jason; Dokholyan, Nikolay V; Echave, Julian; Elofsson, Arne; Gerloff, Dietlind L; Goldstein, Richard A; Grahnen, Johan A; Holder, Mark T; Lakner, Clemens; Lartillot, Nicholas; Lovell, Simon C; Naylor, Gavin; Perica, Tina; Pollock, David D; Pupko, Tal; Regan, Lynne; Roger, Andrew; Rubinstein, Nimrod; Shakhnovich, Eugene; Sjölander, Kimmen; Sunyaev, Shamil; Teufel, Ashley I; Thorne, Jeffrey L; Thornton, Joseph W; Weinreich, Daniel M; Whelan, Simon

    2012-01-01

    Abstract The interface of protein structural biology, protein biophysics, molecular evolution, and molecular population genetics forms the foundations for a mechanistic understanding of many aspects of protein biochemistry. Current efforts in interdisciplinary protein modeling are in their infancy and the state-of-the art of such models is described. Beyond the relationship between amino acid substitution and static protein structure, protein function, and corresponding organismal fitness, other considerations are also discussed. More complex mutational processes such as insertion and deletion and domain rearrangements and even circular permutations should be evaluated. The role of intrinsically disordered proteins is still controversial, but may be increasingly important to consider. Protein geometry and protein dynamics as a deviation from static considerations of protein structure are also important. Protein expression level is known to be a major determinant of evolutionary rate and several considerations including selection at the mRNA level and the role of interaction specificity are discussed. Lastly, the relationship between modeling and needed high-throughput experimental data as well as experimental examination of protein evolution using ancestral sequence resurrection and in vitro biochemistry are presented, towards an aim of ultimately generating better models for biological inference and prediction. PMID:22528593

  14. Utilization of soya protein as an alternative protein source in ...

    African Journals Online (AJOL)

    In contrast, no significant differences were found in feed and protein utilization parameters. For carcass trait, ash, crude fat, and energy varied significantly with soya protein incorporation in fish diet. Concerning organoleptic characteristics, odour and texture in mouth were not affected by incorporation of soya protein in diet.

  15. Protein engineering techniques gateways to synthetic protein universe

    CERN Document Server

    Poluri, Krishna Mohan

    2017-01-01

    This brief provides a broad overview of protein-engineering research, offering a glimpse of the most common experimental methods. It also presents various computational programs with applications that are widely used in directed evolution, computational and de novo protein design. Further, it sheds light on the advantages and pitfalls of existing methodologies and future perspectives of protein engineering techniques.

  16. Recent excitements in protein NMR: Large proteins and biologically ...

    Indian Academy of Sciences (India)

    The advent of Transverse Relaxation Optimized SpectroscopY (TROSY) and perdeuteration allowed biomolecularNMR spectroscopists to overcome the size limitation barrier (~20 kDa) in de novo structure determination of proteins.The utility of these techniques was immediately demonstrated on large proteins and protein ...

  17. Protein stress and stress proteins: implications in aging and disease

    Indian Academy of Sciences (India)

    2007-04-02

    Apr 2, 2007 ... Environmantal stress induces damage that activates an adaptive response in any organism. The cellular stress response is based on the induction of cytoprotective proteins, the so called stress or heat shock proteins. The stress response as well as stress proteins are ubiquitous, highly conserved ...

  18. Protein: MPA1 [TP Atlas

    Lifescience Database Archive (English)

    Full Text Available feron stimulator, Mediator of IRF3 activation, Stimulator of interferon genes protein 9606 Homo sapiens Q86WV6 340061 ... ...MPA1 TLR signaling molecules TMEM173 ERIS, MITA, STING Transmembrane protein 173 Endoplasmic reticulum inter

  19. Epitope tagging of recombinant proteins.

    Science.gov (United States)

    Brizzard, B; Chubet, R

    2001-05-01

    Epitope tagging is a method of expressing proteins whereby an epitope for a specific monoclonal antibody is fused to a target protein using recombinant DNA techniques. The fusion gene is cloned into an appropriate expression vector for the experimental cell type and host cells are transfected. The fusion protein can then be detected and/or purified using a monoclonal antibody specific for the epitope tag. This unit presents protocols for detection and purification of proteins tagged with a particular epitope, the FLAG tag, although the same general approach can be applied to other epitope tags. The protocols in this unit employ the anti-FLAG M2 antibody to detect and purify FLAG-tagged proteins. The methods presented are immunoprecipitation of FLAG fusion proteins from cells using an anti-FLAG M2 affinity gel, detection of FLAG fusion proteins by western blotting, and purification of FLAG fusion proteins by anti-FLAG M2 affinity chromatography.

  20. Protein: FBA3 [TP Atlas

    Lifescience Database Archive (English)

    Full Text Available FBA3 Atg1 kinase complex TOR1 DRR1 Serine/threonine-protein kinase TOR1 Dominant rapamycin... resistance protein 1, Phosphatidylinositol kinase homolog TOR1, Target of rapamycin kinase 1 559292

  1. Functional aspects of protein flexibility

    DEFF Research Database (Denmark)

    Teilum, Kaare; Olsen, Johan G; Kragelund, Birthe B

    2009-01-01

    Proteins are dynamic entities, and they possess an inherent flexibility that allows them to function through molecular interactions within the cell, among cells and even between organisms. Appreciation of the non-static nature of proteins is emerging, but to describe and incorporate...... this into an intuitive perception of protein function is challenging. Flexibility is of overwhelming importance for protein function, and the changes in protein structure during interactions with binding partners can be dramatic. The present review addresses protein flexibility, focusing on protein-ligand interactions....... The thermodynamics involved are reviewed, and examples of structure-function studies involving experimentally determined flexibility descriptions are presented. While much remains to be understood about protein flexibility, it is clear that it is encoded within their amino acid sequence and should be viewed...

  2. Protein Linked to Atopic Dermatitis

    Science.gov (United States)

    ... Research Matters January 14, 2013 Protein Linked to Atopic Dermatitis Normal skin from a mouse (left) shows no ... that lack of a certain protein may trigger atopic dermatitis, the most common type of eczema. The finding ...

  3. Protein-ECE MEtallopincer Hybrids

    NARCIS (Netherlands)

    Kruithof, C.A.

    2007-01-01

    Modification of proteins with metal complexes is a promising and a relatively new field which conceals many challenges and potential applications. The field is a balance of contributions from the biological (protein engineering, bioconjugation) and chemical sciences (organic, inorganic and

  4. Leptospira Protein Expression During Infection

    Science.gov (United States)

    We are characterizing protein expression in vivo during experimental leptospirosis using immunofluorescence microscopy. Coding regions for several proteins were identified through analysis of Leptospira interrogans serovar Copenhageni and L. borgpetersenii serovar Hardjo genomes. In addition, codi...

  5. Yeast Interacting Proteins Database: YJL199C, YJL199C [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available d in closely related Saccharomyces species; protein detected in large-scale protein-protein interaction studies...cies; protein detected in large-scale protein-protein interaction studies Rows with this prey as prey (4) Ro...n; not conserved in closely related Saccharomyces species; protein detected in large-scale protein-protein interaction studies... species; protein detected in large-scale protein-protein interaction studies Rows with this prey as prey Ro

  6. Dependence of Golgi apparatus integrity on nitric oxide in vascular cells: implications in pulmonary arterial hypertension

    Science.gov (United States)

    Lee, Jason E.; Patel, Kirit; Almodóvar, Sharilyn; Tuder, Rubin M.; Flores, Sonia C.

    2011-01-01

    Although reduced bioavailability of nitric oxide (NO) has been implicated in the pathogenesis of pulmonary arterial hypertension (PAH), its consequences on organellar structure and function within vascular cells is largely unexplored. We investigated the effect of reduced NO on the structure of the Golgi apparatus as assayed by giantin or GM130 immunofluorescence in human pulmonary arterial endothelial (HPAECs) and smooth muscle (HPASMCs) cells, bovine PAECs, and human EA.hy926 endothelial cells. Golgi structure was also investigated in cells in tissue sections of pulmonary vascular lesions in idiopathic PAH (IPAH) and in macaques infected with a chimeric simian immunodeficiency virus containing the human immunodeficiency virus (HIV)-nef gene (SHIV-nef) with subcellular three-dimensional (3D) immunoimaging. Compounds with NO scavenging activity including 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO), methylene blue, N-acetylcysteine, and hemoglobin markedly fragmented the Golgi in all cell types evaluated as did monocrotaline pyrrole, while LY-83583, sildenafil, fasudil, Y-27632, Tiron, Tempol, or H2O2 did not. Golgi fragmentation by NO scavengers was inhibited by diethylamine NONOate, was evident in HPAECs after selective knockdown of endothelial nitric oxide synthase using small interfering RNA (siRNA), was independent of microtubule organization, required the GTPase dynamin 2, and was accompanied by depletion of α-soluble N-ethylmaleimide-sensitive factor (NSF) acceptor protein (α-SNAP) from Golgi membranes and codispersal of the SNAP receptor (SNARE) Vti1a with giantin. Golgi fragmentation was confirmed in endothelial and smooth muscle cells in pulmonary arterial lesions in IPAH and the SHIV-nef-infected macaque with subcellular 3D immunoimaging. In SHIV-nef-infected macaques Golgi fragmentation was observed in cells containing HIV-nef-bearing endosomes. The observed Golgi fragmentation suggests that NO plays a significant role in

  7. Protein: MPA6 [TP Atlas

    Lifescience Database Archive (English)

    Full Text Available MPA6 Adionectin and its receptors Adipoq Acdc, Acrp30, Apm1 Adiponectin 30 kDa adipocyte complement-relate...d protein, Adipocyte complement-related 30 kDa protein, Adipocyte, C1q and collagen domain-containing prote...in, Adipocyte-specific protein AdipoQ 10090 Mus musculus 11450 Q60994 1C28, 1C3H Q60994 18446001, 19788607 ...

  8. Dipolar response of hydrated proteins

    OpenAIRE

    Matyushov, Dmitry V.

    2011-01-01

    The paper presents an analytical theory and numerical simulations of the dipolar response of hydrated proteins. The effective dielectric constant of the solvated protein, representing the average dipole moment induced at the protein by a uniform external field, shows a remarkable variation among the proteins studied by numerical simulations. It changes from 0.5 for ubiquitin to 640 for cytochrome c. The former value implies a negative dipolar susceptibility of ubiquitin, that is a dia-electri...

  9. Protein corona: Opportunities and challenges

    Science.gov (United States)

    Zanganeh, Saeid; Spitler, Ryan; Erfanzadeh, Mohsen; Alkilany, Alaaldin M.; Mahmoudi, Morteza

    2017-01-01

    In contact with biological fluids diverse type of biomolecules (e.g., proteins) adsorb onto nanoparticles forming protein corona. Surface properties of the coated nanoparticles, in terms of type and amount of associated proteins, dictate their interactions with biological systems and thus biological fate, therapeutic efficiency and toxicity. In this perspective, we will focus on the recent advances and pitfalls in the protein corona field. PMID:26783938

  10. The papillomavirus E2 proteins.

    Science.gov (United States)

    McBride, Alison A

    2013-10-01

    The papillomavirus E2 proteins are pivotal to the viral life cycle and have well characterized functions in transcriptional regulation, initiation of DNA replication and partitioning the viral genome. The E2 proteins also function in vegetative DNA replication, post-transcriptional processes and possibly packaging. This review describes structural and functional aspects of the E2 proteins and their binding sites on the viral genome. It is intended to be a reference guide to this viral protein. Published by Elsevier Inc.

  11. Protein corona: Opportunities and challenges.

    Science.gov (United States)

    Zanganeh, Saeid; Spitler, Ryan; Erfanzadeh, Mohsen; Alkilany, Alaaldin M; Mahmoudi, Morteza

    2016-06-01

    In contact with biological fluids diverse type of biomolecules (e.g., proteins) adsorb onto nanoparticles forming protein corona. Surface properties of the coated nanoparticles, in terms of type and amount of associated proteins, dictate their interactions with biological systems and thus biological fate, therapeutic efficiency and toxicity. In this perspective, we will focus on the recent advances and pitfalls in the protein corona field. Copyright © 2016 Elsevier Ltd. All rights reserved.

  12. A Novel Approach for Protein-Named Entity Recognition and Protein-Protein Interaction Extraction

    Directory of Open Access Journals (Sweden)

    Meijing Li

    2015-01-01

    Full Text Available Many researchers focus on developing protein-named entity recognition (Protein-NER or PPI extraction systems. However, the studies about these two topics cannot be merged well; then existing PPI extraction systems’ Protein-NER still needs to improve. In this paper, we developed the protein-protein interaction extraction system named PPIMiner based on Support Vector Machine (SVM and parsing tree. PPIMiner consists of three main models: natural language processing (NLP model, Protein-NER model, and PPI discovery model. The Protein-NER model, which is named ProNER, identifies the protein names based on two methods: dictionary-based method and machine learning-based method. ProNER is capable of identifying more proteins than dictionary-based Protein-NER model in other existing systems. The final discovered PPIs extracted via PPI discovery model are represented in detail because we showed the protein interaction types and the occurrence frequency through two different methods. In the experiments, the result shows that the performances achieved by our ProNER and PPI discovery model are better than other existing tools. PPIMiner applied this protein-named entity recognition approach and parsing tree based PPI extraction method to improve the performance of PPI extraction. We also provide an easy-to-use interface to access PPIs database and an online system for PPIs extraction and Protein-NER.

  13. Proteins: Chemistry, Characterization, and Quality

    NARCIS (Netherlands)

    Sforza, S.; Tedeschi, T.; Wierenga, P.A.

    2016-01-01

    Proteins are one of the major macronutrients in food, and several traditional food commodities are good sources of proteins (meat, egg, milk and dairy products, fish, and soya). Proteins are polymers made by 20 different amino acids. They might undergo desired or undesired chemical or enzymatic

  14. Protein: MPA3 [TP Atlas

    Lifescience Database Archive (English)

    Full Text Available MPA3 NADPH oxidase regulators NOXO1 P41NOX, SH3PXD5 NOXO1 NADPH oxidase organizer 1... NADPH oxidase regulatory protein, Nox organizer 1, Nox-organizing protein 1, SH3 and PX domain-containing protein 5 9606 Homo sapiens Q8NFA2 124056 2L73 ...

  15. Protein: MPA3 [TP Atlas

    Lifescience Database Archive (English)

    Full Text Available 1 47 kDa autosomal chronic granulomatous disease protein, 47 kDa neutrophil oxidase factor, NCF-47K, Neutro...phil NADPH oxidase factor 1, Nox organizer 2, Nox-organizing protein 2, SH3 and PX domain-containing protein

  16. Protein: MPB1 [TP Atlas

    Lifescience Database Archive (English)

    Full Text Available MPB1 Related chemokines IL8 CXCL8 Interleukin_8 Interleukin-8 C-X-C motif chemokine... 8, Emoctakin, Granulocyte chemotactic protein 1, Monocyte-derived neutrophil chemotactic factor, Monocyte-d...erived neutrophil-activating peptide, Neutrophil-activating protein 1, Protein 3-10C, T-cell chemotactic fac

  17. Protein: FBA4 [TP Atlas

    Lifescience Database Archive (English)

    Full Text Available ng kinase assembly factor MAT1 CDK7/cyclin-H assembly factor, Cyclin-G1-interacting protein, Menage a trois, RING finger prote...in 66, RING finger protein MAT1, p35, p36 9606 Homo sapiens P51948 4331 1G25 4331 P51948 ...

  18. Photoreceptor proteins from purple bacteria

    NARCIS (Netherlands)

    Hendriks, J.; van der Horst, M.A.; Chua, T.K.; Ávila Pérez, M.; van Wilderen, L.J.; Alexandre, M.T.A.; Groot, M.-L.; Kennis, J.T.M.; Hellingwerf, K.J.; Hunter, C.N.; Daldal, F.; Thurnauer, M.C.; Beatty, J.T.

    2009-01-01

    Purple bacteria contain representatives of four of the six main families of photoreceptor proteins: phytochromes, BLUF domain containing proteins, xanthopsins (i.e., photoactive yellow proteins), and phototropins (containing one or more light, oxygen, or voltage (LOV) domains). Most of them have a

  19. Protein quality of pig diets

    NARCIS (Netherlands)

    Hulshof, Tetske

    2016-01-01

    The increasing world population and per capita income imposes a risk for protein scarcity. It is, therefore, necessary to use current ingredients more efficiently which includes the accurate assessment of protein quality before inclusion in animal diets. Protein quality is defined in this thesis as

  20. Modeling complexes of modeled proteins.

    Science.gov (United States)

    Anishchenko, Ivan; Kundrotas, Petras J; Vakser, Ilya A

    2017-03-01

    Structural characterization of proteins is essential for understanding life processes at the molecular level. However, only a fraction of known proteins have experimentally determined structures. This fraction is even smaller for protein-protein complexes. Thus, structural modeling of protein-protein interactions (docking) primarily has to rely on modeled structures of the individual proteins, which typically are less accurate than the experimentally determined ones. Such "double" modeling is the Grand Challenge of structural reconstruction of the interactome. Yet it remains so far largely untested in a systematic way. We present a comprehensive validation of template-based and free docking on a set of 165 complexes, where each protein model has six levels of structural accuracy, from 1 to 6 Å C α RMSD. Many template-based docking predictions fall into acceptable quality category, according to the CAPRI criteria, even for highly inaccurate proteins (5-6 Å RMSD), although the number of such models (and, consequently, the docking success rate) drops significantly for models with RMSD > 4 Å. The results show that the existing docking methodologies can be successfully applied to protein models with a broad range of structural accuracy, and the template-based docking is much less sensitive to inaccuracies of protein models than the free docking. Proteins 2017; 85:470-478. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  1. Structuring high-protein foods

    NARCIS (Netherlands)

    Purwanti, N.

    2012-01-01

    Increased protein consumption gives rise to various health benefits. High-protein intake can lead to muscle development, body weight control and suppression of sarcopenia progression. However, increasing the protein content in food products leads to textural changes over time. These changes result

  2. Functional Foods Containing Whey Proteins

    Science.gov (United States)

    Whey proteins, modified whey proteins, and whey components are useful as nutrients or supplements for health maintenance. Extrusion modified whey proteins can easily fit into new products such as beverages, confectionery items (e.g., candies), convenience foods, desserts, baked goods, sauces, and in...

  3. Protein Quantitation Using Mass Spectrometry

    Science.gov (United States)

    Zhang, Guoan; Ueberheide, Beatrix M.; Waldemarson, Sofia; Myung, Sunnie; Molloy, Kelly; Eriksson, Jan; Chait, Brian T.; Neubert, Thomas A.; Fenyö, David

    2013-01-01

    Mass spectrometry is a method of choice for quantifying low-abundance proteins and peptides in many biological studies. Here, we describe a range of computational aspects of protein and peptide quantitation, including methods for finding and integrating mass spectrometric peptide peaks, and detecting interference to obtain a robust measure of the amount of proteins present in samples. PMID:20835801

  4. Biophysics of protein evolution and evolutionary protein biophysics

    Science.gov (United States)

    Sikosek, Tobias; Chan, Hue Sun

    2014-01-01

    The study of molecular evolution at the level of protein-coding genes often entails comparing large datasets of sequences to infer their evolutionary relationships. Despite the importance of a protein's structure and conformational dynamics to its function and thus its fitness, common phylogenetic methods embody minimal biophysical knowledge of proteins. To underscore the biophysical constraints on natural selection, we survey effects of protein mutations, highlighting the physical basis for marginal stability of natural globular proteins and how requirement for kinetic stability and avoidance of misfolding and misinteractions might have affected protein evolution. The biophysical underpinnings of these effects have been addressed by models with an explicit coarse-grained spatial representation of the polypeptide chain. Sequence–structure mappings based on such models are powerful conceptual tools that rationalize mutational robustness, evolvability, epistasis, promiscuous function performed by ‘hidden’ conformational states, resolution of adaptive conflicts and conformational switches in the evolution from one protein fold to another. Recently, protein biophysics has been applied to derive more accurate evolutionary accounts of sequence data. Methods have also been developed to exploit sequence-based evolutionary information to predict biophysical behaviours of proteins. The success of these approaches demonstrates a deep synergy between the fields of protein biophysics and protein evolution. PMID:25165599

  5. Protein-protein interactions and cancer: targeting the central dogma.

    Science.gov (United States)

    Garner, Amanda L; Janda, Kim D

    2011-01-01

    Between 40,000 and 200,000 protein-protein interactions have been predicted to exist within the human interactome. As these interactions are of a critical nature in many important cellular functions and their dysregulation is causal of disease, the modulation of these binding events has emerged as a leading, yet difficult therapeutic arena. In particular, the targeting of protein-protein interactions relevant to cancer is of fundamental importance as the tumor-promoting function of several aberrantly expressed proteins in the cancerous state is directly resultant of its ability to interact with a protein-binding partner. Of significance, these protein complexes play a crucial role in each of the steps of the central dogma of molecular biology, the fundamental processes of genetic transmission. With the many important discoveries being made regarding the mechanisms of these genetic process, the identification of new chemical probes are needed to better understand and validate the druggability of protein-protein interactions related to the central dogma. In this review, we provide an overview of current small molecule-based protein-protein interaction inhibitors for each stage of the central dogma: transcription, mRNA splicing and translation. Importantly, through our analysis we have uncovered a lack of necessary probes targeting mRNA splicing and translation, thus, opening up the possibility for expansion of these fields.

  6. The Proteins API: accessing key integrated protein and genome information.

    Science.gov (United States)

    Nightingale, Andrew; Antunes, Ricardo; Alpi, Emanuele; Bursteinas, Borisas; Gonzales, Leonardo; Liu, Wudong; Luo, Jie; Qi, Guoying; Turner, Edd; Martin, Maria

    2017-07-03

    The Proteins API provides searching and programmatic access to protein and associated genomics data such as curated protein sequence positional annotations from UniProtKB, as well as mapped variation and proteomics data from large scale data sources (LSS). Using the coordinates service, researchers are able to retrieve the genomic sequence coordinates for proteins in UniProtKB. This, the LSS genomics and proteomics data for UniProt proteins is programmatically only available through this service. A Swagger UI has been implemented to provide documentation, an interface for users, with little or no programming experience, to 'talk' to the services to quickly and easily formulate queries with the services and obtain dynamically generated source code for popular programming languages, such as Java, Perl, Python and Ruby. Search results are returned as standard JSON, XML or GFF data objects. The Proteins API is a scalable, reliable, fast, easy to use RESTful services that provides a broad protein information resource for users to ask questions based upon their field of expertise and allowing them to gain an integrated overview of protein annotations available to aid their knowledge gain on proteins in biological processes. The Proteins API is available at (http://www.ebi.ac.uk/proteins/api/doc). © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  7. The Proteins API: accessing key integrated protein and genome information

    Science.gov (United States)

    Antunes, Ricardo; Alpi, Emanuele; Gonzales, Leonardo; Liu, Wudong; Luo, Jie; Qi, Guoying; Turner, Edd

    2017-01-01

    Abstract The Proteins API provides searching and programmatic access to protein and associated genomics data such as curated protein sequence positional annotations from UniProtKB, as well as mapped variation and proteomics data from large scale data sources (LSS). Using the coordinates service, researchers are able to retrieve the genomic sequence coordinates for proteins in UniProtKB. This, the LSS genomics and proteomics data for UniProt proteins is programmatically only available through this service. A Swagger UI has been implemented to provide documentation, an interface for users, with little or no programming experience, to ‘talk’ to the services to quickly and easily formulate queries with the services and obtain dynamically generated source code for popular programming languages, such as Java, Perl, Python and Ruby. Search results are returned as standard JSON, XML or GFF data objects. The Proteins API is a scalable, reliable, fast, easy to use RESTful services that provides a broad protein information resource for users to ask questions based upon their field of expertise and allowing them to gain an integrated overview of protein annotations available to aid their knowledge gain on proteins in biological processes. The Proteins API is available at (http://www.ebi.ac.uk/proteins/api/doc). PMID:28383659

  8. Characterization of protein-protein interactions by isothermal titration calorimetry.

    Science.gov (United States)

    Velazquez-Campoy, Adrian; Leavitt, Stephanie A; Freire, Ernesto

    2004-01-01

    Isothermal titration calorimetry (ITC) is a powerful technique to study both protein-ligand and protein-protein interactions. This methods chapter is devoted to describing protein-protein interactions, in particular, the association between two different proteins and the self-association of a protein into homodimers. ITC is the only technique that determines directly the thermodynamic parameters of a given reaction: DeltaG, DeltaH, DeltaS, and DeltaCP. Isothermal titration calorimeters have evolved over the years and one of the latest models is the VP-ITC produced by Microcal, Inc. In this chapter we will be describing the general procedure for performing an ITC experiment as well as for the specific cases of porcine pancreatic trypsin binding to soybean trypsin inhibitor and the dissociation of bovine pancreatic alpha-chymotrypsin.

  9. Understanding Protein-Protein Interactions Using Local Structural Features

    DEFF Research Database (Denmark)

    Planas-Iglesias, Joan; Bonet, Jaume; García-García, Javier

    2013-01-01

    Protein-protein interactions (PPIs) play a relevant role among the different functions of a cell. Identifying the PPI network of a given organism (interactome) is useful to shed light on the key molecular mechanisms within a biological system. In this work, we show the role of structural features...... (loops and domains) to comprehend the molecular mechanisms of PPIs. A paradox in protein-protein binding is to explain how the unbound proteins of a binary complex recognize each other among a large population within a cell and how they find their best docking interface in a short timescale. We use...... interacting and non-interacting protein pairs to classify the structural features that sustain the binding (or non-binding) behavior. Our study indicates that not only the interacting region but also the rest of the protein surface are important for the interaction fate. The interpretation...

  10. Protein subcellular localization assays using split fluorescent proteins

    Science.gov (United States)

    Waldo, Geoffrey S [Santa Fe, NM; Cabantous, Stephanie [Los Alamos, NM

    2009-09-08

    The invention provides protein subcellular localization assays using split fluorescent protein systems. The assays are conducted in living cells, do not require fixation and washing steps inherent in existing immunostaining and related techniques, and permit rapid, non-invasive, direct visualization of protein localization in living cells. The split fluorescent protein systems used in the practice of the invention generally comprise two or more self-complementing fragments of a fluorescent protein, such as GFP, wherein one or more of the fragments correspond to one or more beta-strand microdomains and are used to "tag" proteins of interest, and a complementary "assay" fragment of the fluorescent protein. Either or both of the fragments may be functionalized with a subcellular targeting sequence enabling it to be expressed in or directed to a particular subcellular compartment (i.e., the nucleus).

  11. Diffusion of Integral Membrane Proteins in Protein-Rich Membranes

    DEFF Research Database (Denmark)

    Javanainen, Matti; Martinez-Seara, Hector; Metzler, Ralf

    2017-01-01

    of being protein-poor, native cell membranes are extremely crowded with proteins. On the basis of extensive molecular simulations, we here demonstrate that protein crowding of the membrane at physiological levels leads to deviations from the SD relation and to the emergence of a stronger Stokes......-like dependence D ∝ 1/R. We propose that this 1/R law mainly arises due to geometrical factors: smaller proteins are able to avoid confinement effects much better than their larger counterparts. The results highlight that the lateral dynamics in the crowded setting found in native membranes is radically different......The lateral diffusion of embedded proteins along lipid membranes in protein-poor conditions has been successfully described in terms of the Saffman-Delbrück (SD) model, which predicts that the protein diffusion coefficient D is weakly dependent on its radius R as D ∝ ln(1/R). However, instead...

  12. Protein from methanol

    Energy Technology Data Exchange (ETDEWEB)

    Rosenzweig, M.; Ushio, S.

    1974-01-07

    The biosynthesis of proteins from methanol produced from natural gas can provide an attractive alternative to the already commercially proven technique of protein synthesis from gas oil and n-paraffin feedstocks if current pilot-plant tests in England and Japan prove successful. The methanol route also provides other advantages as a protein feedstock: it is water soluble, contains no polycyclic aromatic compounds, and requires less oxygen than methane. Its lower boiling point helps ease the separation of feedstock from the product stream. Finally, it will require lower investment costs. Both ICI and Mitsubishi Gas Chemical Co. are large methanol producers. ICI already has a 1000 ton/yr plant operating at Teeside, England, and expects to decide on a 100,000 m ton/yr plant later this year. Mitsubishi is constructing a large-scale pilot plant scheduled to come onstream this year. ICI will use a Pseudomona bacterium at 98.6/sup 0/F (37/sup 0/C) in the fermenter. Mitsubishi has not yet decided on a yeast or a bacteria, and is searching for a strain capable of withstanding up to 115/sup 0/F (46/sup 0/C). In the more advanced ICI process, methanol will be mixed with phosphoric acid, potassium sulfate, sodium chloride, and traces of iron, copper, zinc, and molybdenum; diluted with water; passed through a sterilization tank; and fermented at pH 7 in a pressure cycle fermenter. The product stream, containing a 3 percent suspension of cellular dry matter, is taken near the top of the fermenter riser, then passed through a flotation vessel and a centrifuge to pack the cell concentration to 20 percent. Water is recycled. Whatever methanol remains in the fermenter product stream is either used up by the microorganisms in subsequent processing or vaporized in the dryer. (auth)

  13. NMR Studies of Protein Hydration and Protein-Ligand Interactions

    Science.gov (United States)

    Chong, Yuan

    Water on the surface of a protein is called hydration water. Hydration water is known to play a crucial role in a variety of biological processes including protein folding, enzymatic activation, and drug binding. Although the significance of hydration water has been recognized, the underlying mechanism remains far from being understood. This dissertation employs a unique in-situ nuclear magnetic resonance (NMR) technique to study the mechanism of protein hydration and the role of hydration in alcohol-protein interactions. Water isotherms in proteins are measured at different temperatures via the in-situ NMR technique. Water is found to interact differently with hydrophilic and hydrophobic groups on the protein. Water adsorption on hydrophilic groups is hardly affected by the temperature, while water adsorption on hydrophobic groups strongly depends on the temperature around 10 C, below which the adsorption is substantially reduced. This effect is induced by the dramatic decrease in the protein flexibility below 10 C. Furthermore, nanosecond to microsecond protein dynamics and the free energy, enthalpy, and entropy of protein hydration are studied as a function of hydration level and temperature. A crossover at 10 C in protein dynamics and thermodynamics is revealed. The effect of water at hydrophilic groups on protein dynamics and thermodynamics shows little temperature dependence, whereas water at hydrophobic groups has stronger effect above 10 C. In addition, I investigate the role of water in alcohol binding to the protein using the in-situ NMR detection. The isotherms of alcohols are first measured on dry proteins, then on proteins with a series of controlled hydration levels. The free energy, enthalpy, and entropy of alcohol binding are also determined. Two distinct types of alcohol binding are identified. On the one hand, alcohols can directly bind to a few specific sites on the protein. This type of binding is independent of temperature and can be

  14. Protein-Protein Interactions: Structurally Conserved Residues Distinguish between Binding Sites and Exposed Protein Surfaces

    National Research Council Canada - National Science Library

    Buyong Ma; Tal Elkayam; Haim Wolfson; Ruth Nussinov

    2003-01-01

    Polar residue hot spots have been observed at protein-protein binding sites. Here we show that hot spots occur predominantly at the interfaces of macromolecular complexes, distinguishing binding sites from the remainder of the surface...

  15. Information contained in protein shapes

    Science.gov (United States)

    Sundaram, K.; Viswanadhan, V. N.; Macelroy, R. D.

    1983-01-01

    The sequence of local conformations at C-alpha atoms of a protein has been considered as an informational message string. The total self-information contents and self-information per letter have been evaluated for 83 globular proteins whose structures are known from X-ray crystallography. The derived information contents provide a method of quantitating structural specificity of proteins. This method of analysis enables repeating, intricate structural features to be recognized. Among the globular proteins whose structures have been solved, high potential iron protein stands out with the largest three-letter dependence.

  16. Protein-protein interaction network-based detection of functionally similar proteins within species.

    Science.gov (United States)

    Song, Baoxing; Wang, Fen; Guo, Yang; Sang, Qing; Liu, Min; Li, Dengyun; Fang, Wei; Zhang, Deli

    2012-07-01

    Although functionally similar proteins across species have been widely studied, functionally similar proteins within species showing low sequence similarity have not been examined in detail. Identification of these proteins is of significant importance for understanding biological functions, evolution of protein families, progression of co-evolution, and convergent evolution and others which cannot be obtained by detection of functionally similar proteins across species. Here, we explored a method of detecting functionally similar proteins within species based on graph theory. After denoting protein-protein interaction networks using graphs, we split the graphs into subgraphs using the 1-hop method. Proteins with functional similarities in a species were detected using a method of modified shortest path to compare these subgraphs and to find the eligible optimal results. Using seven protein-protein interaction networks and this method, some functionally similar proteins with low sequence similarity that cannot detected by sequence alignment were identified. By analyzing the results, we found that, sometimes, it is difficult to separate homologous from convergent evolution. Evaluation of the performance of our method by gene ontology term overlap showed that the precision of our method was excellent. Copyright © 2012 Wiley Periodicals, Inc.

  17. Tetramer formation in Arabidopsis MADS domain proteins: analysis of a protein-protein interaction network

    NARCIS (Netherlands)

    Espinosa-Soto, C.; Immink, R.G.H.; Angenent, G.C.; Alvarez-Buylla, E.R.; Folter, de S.

    2014-01-01

    Background: MADS domain proteins are transcription factors that coordinate several important developmental processes in plants. These proteins interact with other MADS domain proteins to form dimers, and it has been proposed that they are able to associate as tetrameric complexes that regulate

  18. Discover Protein Complexes in Protein-Protein Interaction Networks Using Parametric Local Modularity

    Directory of Open Access Journals (Sweden)

    Tan Kai

    2010-10-01

    Full Text Available Abstract Background Recent advances in proteomic technologies have enabled us to create detailed protein-protein interaction maps in multiple species and in both normal and diseased cells. As the size of the interaction dataset increases, powerful computational methods are required in order to effectively distil network models from large-scale interactome data. Results We present an algorithm, miPALM (Module Inference by Parametric Local Modularity, to infer protein complexes in a protein-protein interaction network. The algorithm uses a novel graph theoretic measure, parametric local modularity, to identify highly connected sub-networks as candidate protein complexes. Using gold standard sets of protein complexes and protein function and localization annotations, we show our algorithm achieved an overall improvement over previous algorithms in terms of precision, recall, and biological relevance of the predicted complexes. We applied our algorithm to predict and characterize a set of 138 novel protein complexes in S. cerevisiae. Conclusions miPALM is a novel algorithm for detecting protein complexes from large protein-protein interaction networks with improved accuracy than previous methods. The software is implemented in Matlab and is freely available at http://www.medicine.uiowa.edu/Labs/tan/software.html.

  19. Detection of protein complex from protein-protein interaction network using Markov clustering

    Science.gov (United States)

    Ochieng, P. J.; Kusuma, W. A.; Haryanto, T.

    2017-05-01

    Detection of complexes, or groups of functionally related proteins, is an important challenge while analysing biological networks. However, existing algorithms to identify protein complexes are insufficient when applied to dense networks of experimentally derived interaction data. Therefore, we introduced a graph clustering method based on Markov clustering algorithm to identify protein complex within highly interconnected protein-protein interaction networks. Protein-protein interaction network was first constructed to develop geometrical network, the network was then partitioned using Markov clustering to detect protein complexes. The interest of the proposed method was illustrated by its application to Human Proteins associated to type II diabetes mellitus. Flow simulation of MCL algorithm was initially performed and topological properties of the resultant network were analysed for detection of the protein complex. The results indicated the proposed method successfully detect an overall of 34 complexes with 11 complexes consisting of overlapping modules and 20 non-overlapping modules. The major complex consisted of 102 proteins and 521 interactions with cluster modularity and density of 0.745 and 0.101 respectively. The comparison analysis revealed MCL out perform AP, MCODE and SCPS algorithms with high clustering coefficient (0.751) network density and modularity index (0.630). This demonstrated MCL was the most reliable and efficient graph clustering algorithm for detection of protein complexes from PPI networks.

  20. Human cancer protein-protein interaction network: a structural perspective.

    Directory of Open Access Journals (Sweden)

    Gozde Kar

    2009-12-01

    Full Text Available Protein-protein interaction networks provide a global picture of cellular function and biological processes. Some proteins act as hub proteins, highly connected to others, whereas some others have few interactions. The dysfunction of some interactions causes many diseases, including cancer. Proteins interact through their interfaces. Therefore, studying the interface properties of cancer-related proteins will help explain their role in the interaction networks. Similar or overlapping binding sites should be used repeatedly in single interface hub proteins, making them promiscuous. Alternatively, multi-interface hub proteins make use of several distinct binding sites to bind to different partners. We propose a methodology to integrate protein interfaces into cancer interaction networks (ciSPIN, cancer structural protein interface network. The interactions in the human protein interaction network are replaced by interfaces, coming from either known or predicted complexes. We provide a detailed analysis of cancer related human protein-protein interfaces and the topological properties of the cancer network. The results reveal that cancer-related proteins have smaller, more planar, more charged and less hydrophobic binding sites than non-cancer proteins, which may indicate low affinity and high specificity of the cancer-related interactions. We also classified the genes in ciSPIN according to phenotypes. Within phenotypes, for breast cancer, colorectal cancer and leukemia, interface properties were found to be discriminating from non-cancer interfaces with an accuracy of 71%, 67%, 61%, respectively. In addition, cancer-related proteins tend to interact with their partners through distinct interfaces, corresponding mostly to multi-interface hubs, which comprise 56% of cancer-related proteins, and constituting the nodes with higher essentiality in the network (76%. We illustrate the interface related affinity properties of two cancer-related hub

  1. Molecular principles of human virus protein-protein interactions.

    Science.gov (United States)

    Halehalli, Rachita Ramachandra; Nagarajaram, Hampapathalu Adimurthy

    2015-04-01

    Viruses, from the human protein-protein interaction network perspective, target hubs, bottlenecks and interconnected nodes enriched in certain biological pathways. However, not much is known about the general characteristic features of the human proteins interacting with viral proteins (referred to as hVIPs) as well as the motifs and domains utilized by human-virus protein-protein interactions (referred to as Hu-Vir PPIs). Our study has revealed that hVIPs are mostly disordered proteins, whereas viral proteins are mostly ordered proteins. Protein disorder in viral proteins and hVIPs varies from one subcellular location to another. In any given viral-human PPI pair, at least one of the two proteins is structurally disordered suggesting that disorder associated conformational flexibility as one of the characteristic features of virus-host interaction. Further analyses reveal that hVIPs are (i) slowly evolving proteins, (ii) associated with high centrality scores in human-PPI network, (iii) involved in multiple pathways, (iv) enriched in eukaryotic linear motifs (ELMs) associated with protein modification, degradation and regulatory processes, (v) associated with high number of splice variants and (vi) expressed abundantly across multiple tissues. These aforementioned findings suggest that conformational flexibility, spatial diversity, abundance and slow evolution are the characteristic features of the human proteins targeted by viral proteins. Hu-Vir PPIs are mostly mediated via domain-motif interactions (DMIs) where viral proteins employ motifs that mimic host ELMs to bind to domains in human proteins. DMIs are shared among viruses belonging to different families indicating a possible convergent evolution of these motifs to help viruses to adopt common strategies to subvert host cellular pathways. Hu-Vir PPI data, DDI and DMI data for human-virus PPI can be downloaded from http://cdfd.org.in/labpages/computational_biology_datasets.html. Supplementary data are

  2. Introduction to protein crystallization.

    Science.gov (United States)

    McPherson, Alexander; Gavira, Jose A

    2014-01-01

    Protein crystallization was discovered by chance about 150 years ago and was developed in the late 19th century as a powerful purification tool and as a demonstration of chemical purity. The crystallization of proteins, nucleic acids and large biological complexes, such as viruses, depends on the creation of a solution that is supersaturated in the macromolecule but exhibits conditions that do not significantly perturb its natural state. Supersaturation is produced through the addition of mild precipitating agents such as neutral salts or polymers, and by the manipulation of various parameters that include temperature, ionic strength and pH. Also important in the crystallization process are factors that can affect the structural state of the macromolecule, such as metal ions, inhibitors, cofactors or other conventional small molecules. A variety of approaches have been developed that combine the spectrum of factors that effect and promote crystallization, and among the most widely used are vapor diffusion, dialysis, batch and liquid-liquid diffusion. Successes in macromolecular crystallization have multiplied rapidly in recent years owing to the advent of practical, easy-to-use screening kits and the application of laboratory robotics. A brief review will be given here of the most popular methods, some guiding principles and an overview of current technologies.

  3. Bioactive proteins from pipefishes

    Directory of Open Access Journals (Sweden)

    E. Rethna Priya

    2013-08-01

    Full Text Available Objective: To screen antimicrobial potence of some pipefish species collected from Tuticorin coastal environment. Methods: Antimicrobial activity of pipefishes in methanol extract was investigated against 10 bacterial and 10 fungal human pathogenic strains. Results: Among the tested strains, in Centriscus scutatus, pipefish showed maximum zone of inhibition against Vibrio cholerae (8 mm and minimum in the sample of Hippichthys cyanospilos against Klebseilla pneumoniae (2 mm. In positive control, maximum zone of inhibition was recorded in Vibrio cholerae (9 mm and minimum in Klebseilla pneumoniae, and Salmonella paratyphi (5 mm. Chemical investigation indicated the presence of peptides as evidenced by ninhydrin positive spots on thin layer chromatography and presence of peptide. In SDS PAGE, in Centriscus scutatus, four bands were detected in the gel that represented the presence of proteins in the range nearly 25.8-75 kDa. In Hippichthys cyanospilos, five bands were detected in the gel that represented the presence of proteins in the range nearly 20.5-78 kDa. The result of FT-IR spectrum revealed that the pipe fishes extracts compriseed to have peptide derivatives as their predominant chemical groups. Conclusions: It can be conclude that this present investigation suggests the tested pipe fishes will be a potential source of natural bioactive compounds.

  4. Bioactive proteins from pipefishes

    Directory of Open Access Journals (Sweden)

    E. Rethna Priya

    2013-01-01

    Full Text Available Objective: To screen antimicrobial potence of some pipefish species collected from Tuticorin coastal environment. Methods: Antimicrobial activity of pipefishes in methanol extract was investigated against 10 bacterial and 10 fungal human pathogenic strains. Results: Among the tested strains, in Centriscus scutatus, pipefish showed maximum zone of inhibition against Vibrio cholerae (8 mm and minimum in the sample of Hippichthys cyanospilos against Klebseilla pneumoniae (2 mm. In positive control, maximum zone of inhibition was recorded in Vibrio cholerae (9 mm and minimum in Klebseilla pneumoniae, and Salmonella paratyphi (5 mm. Chemical investigation indicated the presence of peptides as evidenced by ninhydrin positive spots on thin layer chromatography and presence of peptide. In SDS PAGE, in Centriscus scutatus, four bands were detected in the gel that represented the presence of proteins in the range nearly 25.8-75 kDa. In Hippichthys cyanospilos, five bands were detected in the gel that represented the presence of proteins in the range nearly 20.5-78 kDa. The result of FT-IR spectrum revealed that the pipe fishes extracts compriseed to have peptide derivatives as their predominant chemical groups. Conclusions: It can be conclude that this present investigation suggests the tested pipe fishes will be a potential source of natural bioactive compounds.

  5. Protein Chemical Shift Prediction

    CERN Document Server

    Larsen, Anders S

    2014-01-01

    The protein chemical shifts holds a large amount of information about the 3-dimensional structure of the protein. A number of chemical shift predictors based on the relationship between structures resolved with X-ray crystallography and the corresponding experimental chemical shifts have been developed. These empirical predictors are very accurate on X-ray structures but tends to be insensitive to small structural changes. To overcome this limitation it has been suggested to make chemical shift predictors based on quantum mechanical(QM) calculations. In this thesis the development of the QM derived chemical shift predictor Procs14 is presented. Procs14 is based on 2.35 million density functional theory(DFT) calculations on tripeptides and contains corrections for hydrogen bonding, ring current and the effect of the previous and following residue. Procs14 is capable at performing predictions for the 13CA, 13CB, 13CO, 15NH, 1HN and 1HA backbone atoms. In order to benchmark Procs14, a number of QM NMR calculatio...

  6. Water-transporting proteins.

    Science.gov (United States)

    Zeuthen, Thomas

    2010-04-01

    Transport through lipids and aquaporins is osmotic and entirely driven by the difference in osmotic pressure. Water transport in cotransporters and uniporters is different: Water can be cotransported, energized by coupling to the substrate flux by a mechanism closely associated with protein. In the K(+)/Cl(-) and the Na(+)/K(+)/2Cl(-) cotransporters, water is entirely cotransported, while water transport in glucose uniporters and Na(+)-coupled transporters of nutrients and neurotransmitters takes place by both osmosis and cotransport. The molecular mechanism behind cotransport of water is not clear. It is associated with the substrate movements in aqueous pathways within the protein; a conventional unstirred layer mechanism can be ruled out, due to high rates of diffusion in the cytoplasm. The physiological roles of the various modes of water transport are reviewed in relation to epithelial transport. Epithelial water transport is energized by the movements of ions, but how the coupling takes place is uncertain. All epithelia can transport water uphill against an osmotic gradient, which is hard to explain by simple osmosis. Furthermore, genetic removal of aquaporins has not given support to osmosis as the exclusive mode of transport. Water cotransport can explain the coupling between ion and water transport, a major fraction of transepithelial water transport and uphill water transport. Aquaporins enhance water transport by utilizing osmotic gradients and cause the osmolarity of the transportate to approach isotonicity.

  7. Mathematical methods for protein science

    Energy Technology Data Exchange (ETDEWEB)

    Hart, W.; Istrail, S.; Atkins, J. [Sandia National Labs., Albuquerque, NM (United States)

    1997-12-31

    Understanding the structure and function of proteins is a fundamental endeavor in molecular biology. Currently, over 100,000 protein sequences have been determined by experimental methods. The three dimensional structure of the protein determines its function, but there are currently less than 4,000 structures known to atomic resolution. Accordingly, techniques to predict protein structure from sequence have an important role in aiding the understanding of the Genome and the effects of mutations in genetic disease. The authors describe current efforts at Sandia to better understand the structure of proteins through rigorous mathematical analyses of simple lattice models. The efforts have focused on two aspects of protein science: mathematical structure prediction, and inverse protein folding.

  8. Metagenomics and the protein universe

    Science.gov (United States)

    Godzik, Adam

    2011-01-01

    Metagenomics sequencing projects have dramatically increased our knowledge of the protein universe and provided over one-half of currently known protein sequences; they have also introduced a much broader phylogenetic diversity into the protein databases. The full analysis of metagenomic datasets is only beginning, but it has already led to the discovery of thousands of new protein families, likely representing novel functions specific to given environments. At the same time, a deeper analysis of such novel families, including experimental structure determination of some representatives, suggests that most of them represent distant homologs of already characterized protein families, and thus most of the protein diversity present in the new environments are due to functional divergence of the known protein families rather than the emergence of new ones. PMID:21497084

  9. The Papillomavirus E2 proteins

    Energy Technology Data Exchange (ETDEWEB)

    McBride, Alison A., E-mail: amcbride@nih.gov

    2013-10-15

    The papillomavirus E2 proteins are pivotal to the viral life cycle and have well characterized functions in transcriptional regulation, initiation of DNA replication and partitioning the viral genome. The E2 proteins also function in vegetative DNA replication, post-transcriptional processes and possibly packaging. This review describes structural and functional aspects of the E2 proteins and their binding sites on the viral genome. It is intended to be a reference guide to this viral protein. - Highlights: • Overview of E2 protein functions. • Structural domains of the papillomavirus E2 proteins. • Analysis of E2 binding sites in different genera of papillomaviruses. • Compilation of E2 associated proteins. • Comparison of key mutations in distinct E2 functions.

  10. Protein folding and wring resonances

    DEFF Research Database (Denmark)

    Bohr, Jakob; Bohr, Henrik; Brunak, Søren

    1997-01-01

    The polypeptide chain of a protein is shown to obey topological contraints which enable long range excitations in the form of wring modes of the protein backbone. Wring modes of proteins of specific lengths can therefore resonate with molecular modes present in the cell. It is suggested...... that protein folding takes place when the amplitude of a wring excitation becomes so large that it is energetically favorable to bend the protein backbone. The condition under which such structural transformations can occur is found, and it is shown that both cold and hot denaturation (the unfolding...... of proteins) are natural consequences of the suggested wring mode model. Native (folded) proteins are found to possess an intrinsic standing wring mode....

  11. Advantages of proteins being disordered.

    Science.gov (United States)

    Liu, Zhirong; Huang, Yongqi

    2014-05-01

    The past decade has witnessed great advances in our understanding of protein structure-function relationships in terms of the ubiquitous existence of intrinsically disordered proteins (IDPs) and intrinsically disordered regions (IDRs). The structural disorder of IDPs/IDRs enables them to play essential functions that are complementary to those of ordered proteins. In addition, IDPs/IDRs are persistent in evolution. Therefore, they are expected to possess some advantages over ordered proteins. In this review, we summarize and survey nine possible advantages of IDPs/IDRs: economizing genome/protein resources, overcoming steric restrictions in binding, achieving high specificity with low affinity, increasing binding rate, facilitating posttranslational modifications, enabling flexible linkers, preventing aggregation, providing resistance to non-native conditions, and allowing compatibility with more available sequences. Some potential advantages of IDPs/IDRs are not well understood and require both experimental and theoretical approaches to decipher. The connection with protein design is also briefly discussed. © 2014 The Protein Society.

  12. Protein Adsorption in Three Dimensions

    Science.gov (United States)

    Vogler, Erwin A.

    2011-01-01

    Recent experimental and theoretical work clarifying the physical chemistry of blood-protein adsorption from aqueous-buffer solution to various kinds of surfaces is reviewed and interpreted within the context of biomaterial applications, especially toward development of cardiovascular biomaterials. The importance of this subject in biomaterials surface science is emphasized by reducing the “protein-adsorption problem” to three core questions that require quantitative answer. An overview of the protein-adsorption literature identifies some of the sources of inconsistency among many investigators participating in more than five decades of focused research. A tutorial on the fundamental biophysical chemistry of protein adsorption sets the stage for a detailed discussion of the kinetics and thermodynamics of protein adsorption, including adsorption competition between two proteins for the same adsorbent immersed in a binary-protein mixture. Both kinetics and steady-state adsorption can be rationalized using a single interpretive paradigm asserting that protein molecules partition from solution into a three-dimensional (3D) interphase separating bulk solution from the physical-adsorbent surface. Adsorbed protein collects in one-or-more adsorbed layers, depending on protein size, solution concentration, and adsorbent surface energy (water wettability). The adsorption process begins with the hydration of an adsorbent surface brought into contact with an aqueous-protein solution. Surface hydration reactions instantaneously form a thin, pseudo-2D interface between the adsorbent and protein solution. Protein molecules rapidly diffuse into this newly-formed interface, creating a truly 3D interphase that inflates with arriving proteins and fills to capacity within milliseconds at mg/mL bulk-solution concentrations CB. This inflated interphase subsequently undergoes time-dependent (minutes-to-hours) decrease in volume VI by expulsion of either-or-both interphase water and

  13. Protein function prediction via graph kernels

    National Research Council Canada - National Science Library

    Borgwardt, Karsten M; Ong, Cheng Soon; Schönauer, Stefan; Vishwanathan, S V N; Smola, Alex J; Kriegel, Hans-Peter

    2005-01-01

    Computational approaches to protein function prediction infer protein function by finding proteins with similar sequence, structure, surface clefts, chemical properties, amino acid motifs, interaction...

  14. Protein oxidation in aging and the removal of oxidized proteins.

    Science.gov (United States)

    Höhn, Annika; König, Jeannette; Grune, Tilman

    2013-10-30

    Reactive oxygen species (ROS) are generated constantly within cells at low concentrations even under physiological conditions. During aging the levels of ROS can increase due to a limited capacity of antioxidant systems and repair mechanisms. Proteins are among the main targets for oxidants due to their high rate constants for several reactions with ROS and their abundance in biological systems. Protein damage has an important influence on cellular viability since most protein damage is non-repairable, and has deleterious consequences on protein structure and function. In addition, damaged and modified proteins can form cross-links and provide a basis for many senescence-associated alterations and may contribute to a range of human pathologies. Two proteolytic systems are responsible to ensure the maintenance of cellular functions: the proteasomal (UPS) and the lysosomal system. Those degrading systems provide a last line of antioxidative protection, removing irreversible damaged proteins and recycling amino acids for the continuous protein synthesis. But during aging, both systems are affected and their proteolytic activity declines significantly. Here we highlight the recent advantages in the understanding of protein oxidation and the fate of these damaged proteins during aging. This article is part of a Special Issue entitled: Posttranslational Protein modifications in biology and Medicine. Copyright © 2013 Elsevier B.V. All rights reserved.

  15. Protein-protein interaction based on pairwise similarity

    Directory of Open Access Journals (Sweden)

    Zaki Nazar

    2009-05-01

    Full Text Available Abstract Background Protein-protein interaction (PPI is essential to most biological processes. Abnormal interactions may have implications in a number of neurological syndromes. Given that the association and dissociation of protein molecules is crucial, computational tools capable of effectively identifying PPI are desirable. In this paper, we propose a simple yet effective method to detect PPI based on pairwise similarity and using only the primary structure of the protein. The PPI based on Pairwise Similarity (PPI-PS method consists of a representation of each protein sequence by a vector of pairwise similarities against large subsequences of amino acids created by a shifting window which passes over concatenated protein training sequences. Each coordinate of this vector is typically the E-value of the Smith-Waterman score. These vectors are then used to compute the kernel matrix which will be exploited in conjunction with support vector machines. Results To assess the ability of the proposed method to recognize the difference between "interacted" and "non-interacted" proteins pairs, we applied it on different datasets from the available yeast saccharomyces cerevisiae protein interaction. The proposed method achieved reasonable improvement over the existing state-of-the-art methods for PPI prediction. Conclusion Pairwise similarity score provides a relevant measure of similarity between protein sequences. This similarity incorporates biological knowledge about proteins and it is extremely powerful when combined with support vector machine to predict PPI.

  16. Bioinformatic Prediction of WSSV-Host Protein-Protein Interaction

    Directory of Open Access Journals (Sweden)

    Zheng Sun

    2014-01-01

    Full Text Available WSSV is one of the most dangerous pathogens in shrimp aquaculture. However, the molecular mechanism of how WSSV interacts with shrimp is still not very clear. In the present study, bioinformatic approaches were used to predict interactions between proteins from WSSV and shrimp. The genome data of WSSV (NC_003225.1 and the constructed transcriptome data of F. chinensis were used to screen potentially interacting proteins by searching in protein interaction databases, including STRING, Reactome, and DIP. Forty-four pairs of proteins were suggested to have interactions between WSSV and the shrimp. Gene ontology analysis revealed that 6 pairs of these interacting proteins were classified into “extracellular region” or “receptor complex” GO-terms. KEGG pathway analysis showed that they were involved in the “ECM-receptor interaction pathway.” In the 6 pairs of interacting proteins, an envelope protein called “collagen-like protein” (WSSV-CLP encoded by an early virus gene “wsv001” in WSSV interacted with 6 deduced proteins from the shrimp, including three integrin alpha (ITGA, two integrin beta (ITGB, and one syndecan (SDC. Sequence analysis on WSSV-CLP, ITGA, ITGB, and SDC revealed that they possessed the sequence features for protein-protein interactions. This study might provide new insights into the interaction mechanisms between WSSV and shrimp.

  17. A new protein structure representation for efficient protein function prediction.

    Science.gov (United States)

    Maghawry, Huda A; Mostafa, Mostafa G M; Gharib, Tarek F

    2014-12-01

    One of the challenging problems in bioinformatics is the prediction of protein function. Protein function is the main key that can be used to classify different proteins. Protein function can be inferred experimentally with very small throughput or computationally with very high throughput. Computational methods are sequence based or structure based. Structure-based methods produce more accurate protein function prediction. In this article, we propose a new protein structure representation for efficient protein function prediction. The representation is based on three-dimensional patterns of protein residues. In the analysis, we used protein function based on enzyme activity through six mechanistically diverse enzyme superfamilies: amidohydrolase, crotonase, haloacid dehalogenase, isoprenoid synthase type I, and vicinal oxygen chelate. We applied three different classification methods, naïve Bayes, k-nearest neighbors, and random forest, to predict the enzyme superfamily of a given protein. The prediction accuracy using the proposed representation outperforms a recently introduced representation method that is based only on the distance patterns. The results show that the proposed representation achieved prediction accuracy up to 98%, with improvement of about 10% on average.

  18. Role for protein-protein interaction databases in human genetics.

    Science.gov (United States)

    Pattin, Kristine A; Moore, Jason H

    2009-12-01

    Proteomics and the study of protein-protein interactions are becoming increasingly important in our effort to understand human diseases on a system-wide level. Thanks to the development and curation of protein-interaction databases, up-to-date information on these interaction networks is accessible and publicly available to the scientific community. As our knowledge of protein-protein interactions increases, it is important to give thought to the different ways that these resources can impact biomedical research. In this article, we highlight the importance of protein-protein interactions in human genetics and genetic epidemiology. Since protein-protein interactions demonstrate one of the strongest functional relationships between genes, combining genomic data with available proteomic data may provide us with a more in-depth understanding of common human diseases. In this review, we will discuss some of the fundamentals of protein interactions, the databases that are publicly available and how information from these databases can be used to facilitate genome-wide genetic studies.

  19. Mapping Protein-Protein Interactions by Quantitative Proteomics

    DEFF Research Database (Denmark)

    Dengjel, Joern; Kratchmarova, Irina; Blagoev, Blagoy

    2010-01-01

    Proteins exert their function inside a cell generally in multiprotein complexes. These complexes are highly dynamic structures changing their composition over time and cell state. The same protein may thereby fulfill different functions depending on its binding partners. Quantitative mass...... spectrometry (MS)-based proteomics in combination with affinity purification protocols has become the method of choice to map and track the dynamic changes in protein-protein interactions, including the ones occurring during cellular signaling events. Different quantitative MS strategies have been used...... to characterize protein interaction networks. In this chapter we describe in detail the use of stable isotope labeling by amino acids in cell culture (SILAC) for the quantitative analysis of stimulus-dependent dynamic protein interactions....

  20. Revisiting the Voronoi description of protein-protein interfaces.

    Science.gov (United States)

    Cazals, Frédéric; Proust, Flavien; Bahadur, Ranjit P; Janin, Joël

    2006-09-01

    We developed a model of macromolecular interfaces based on the Voronoi diagram and the related alpha-complex, and we tested its properties on a set of 96 protein-protein complexes taken from the Protein Data Bank. The Voronoi model provides a natural definition of the interfaces, and it yields values of the number of interface atoms and of the interface area that have excellent correlation coefficients with those of the classical model based on solvent accessibility. Nevertheless, some atoms that do not lose solvent accessibility are part of the interface defined by the Voronoi model. The Voronoi model provides robust definitions of the curvature and of the connectivity of the interfaces, and leads to estimates of these features that generally agree with other approaches. Our implementation of the model allows an analysis of protein-water contacts that highlights the role of structural water molecules at protein-protein interfaces.

  1. Duchenne Muscular Dystrophy (DMD) Protein-Protein Interaction Mapping.

    Science.gov (United States)

    Rezaei Tavirani, Mostafa; OkHOVATIAN, Farshad; Zamanian Azodi, Mona; Rezaei Tavirani, Majid

    2017-01-01

    Duchenne muscular dystrophy (DMD) is one of the mortal diseases, subjected to study in terms of molecular investigation. In this study, the protein interaction map of this muscle-wasting condition was generated to gain a better knowledge of interactome profile of DMD. Applying Cytoscape and String Database, the protein-protein interaction network was constructed and the gene ontology of the constructed network was analyzed for biological process, molecular function, and cellular component annotations. Among 100 proteins related to DMD, dystrophin, utrophin, caveolin 3, and myogenic differentiation 1 play key roles in DMD network. In addition, the gene ontology analysis showed that regulation processes, kinase activity, and sarcoplasmic reticulum were the highlighted biological processes, molecular function, and cell component enrichments respectively for the proteins related to DMD. The central proteins and the enriched ontologies can be suggested as possible prominent agents in DMD; however, the validation studies may be required.

  2. On the role of electrostatics on protein-protein interactions

    Science.gov (United States)

    Zhang, Zhe; Witham, Shawn; Alexov, Emil

    2011-01-01

    The role of electrostatics on protein-protein interactions and binding is reviewed in this article. A brief outline of the computational modeling, in the framework of continuum electrostatics, is presented and basic electrostatic effects occurring upon the formation of the complex are discussed. The role of the salt concentration and pH of the water phase on protein-protein binding free energy is demonstrated and indicates that the increase of the salt concentration tends to weaken the binding, an observation that is attributed to the optimization of the charge-charge interactions across the interface. It is pointed out that the pH-optimum (pH of optimal binding affinity) varies among the protein-protein complexes, and perhaps is a result of their adaptation to particular subcellular compartment. At the end, the similarities and differences between hetero- and homo-complexes are outlined and discussed with respect to the binding mode and charge complementarity. PMID:21572182

  3. Methods for detection of protein-protein and protein-DNA interactions using HaloTag.

    Science.gov (United States)

    Urh, Marjeta; Hartzell, Danette; Mendez, Jacqui; Klaubert, Dieter H; Wood, Keith

    2008-01-01

    HaloTag is a protein fusion tag which was genetically engineered to covalently bind a series of specific synthetic ligands. All ligands carry two groups, the reactive group and the functional/reporter group. The reactive group, the choloroalkane, is the same in all the ligands and is involved in binding to the HaloTag. The functional reporter group is variable and can carry many different moieties including fluorescent dyes, affinity handles like biotin or solid surfaces such as agarose beads. Thus, HaloTag can serve either as a labeling tag or as a protein immobilization tag depending on which ligand is bound to it. Here, we describe a procedure for immobilization of HaloTag fusion proteins and how immobilized proteins can be used to study protein-protein and protein-DNA interactions in vivo and in vitro.

  4. Manipulating protein adsorption using a patchy protein-resistant brush.

    Science.gov (United States)

    Gon, Saugata; Bendersky, Marina; Ross, Jennifer L; Santore, Maria M

    2010-07-20

    Toward the development of surfaces for the precise manipulation of proteins, this study explores the fabrication and protein-interactive behavior of a new type of surface containing extremely small (on the order of 10 nm or less) flat adhesive "patches" or islands embedded in and partially concealed by a protein-repellant PEG (poly(ethylene glycol)) brush. The adsorption of fibrinogen, the model protein chosen to probe the biomaterial interactions of these surfaces, is very sensitive to the surface density of the adhesive patches, occurring only above a threshold. This suggests that two or more adhesive patches are needed to capture each protein. When the average spacing of the adhesive patches exceeds the fibrinogen length, no adsorption occurs because individual patches are too weakly binding for protein capture, as a result of being at least partially obstructed by the brush. The small size of the adhesive patches relative to the 47 nm fibrinogen length thus defines a limiting regime of surface design, distinct from surfaces where larger features can adhere single isolated proteins or multiple proteins together. The restricted protein-surface contact may comprise a means of preserving protein structure and function in the adsorbed state. This article demonstrates several additional interesting features of PEG brushes relevant to biomaterial design. First a moderate amount of adhesive material can be buried at the base of a brush without a measurable impact on the corona density. Second, a different amount of material at the base of a brush can be rendered ineffective to capturing adhesive proteins, despite a modest compromise of the brush corona. From this will follow insight into the design of patterned biomaterial surfaces, the bioactivity of the edges of patterned features, and an understanding of how flaws in brushes compromise protein resistance or allow access to small adhesive sites.

  5. Concentration dependent model of protein-protein interaction networks

    CERN Document Server

    Zhang, Jingshan

    2007-01-01

    The scale free structure p(k)~k^{-gamma} of protein-protein interaction networks can be produced by a static physical model. We find the earlier study of deterministic threshold models with exponential fitness distributions can be generalized to explain the apparent scale free degree distribution of the physical model, and this explanation provides a generic mechanism of "scale free" networks. We predict the dependence of gamma on experimental protein concentrations. The clustering coefficient distribution of the model is also studied.

  6. Proteins aggregation and human diseases

    Science.gov (United States)

    Hu, Chin-Kun

    2015-04-01

    Many human diseases and the death of most supercentenarians are related to protein aggregation. Neurodegenerative diseases include Alzheimer's disease (AD), Huntington's disease (HD), Parkinson's disease (PD), frontotemporallobar degeneration, etc. Such diseases are due to progressive loss of structure or function of neurons caused by protein aggregation. For example, AD is considered to be related to aggregation of Aβ40 (peptide with 40 amino acids) and Aβ42 (peptide with 42 amino acids) and HD is considered to be related to aggregation of polyQ (polyglutamine) peptides. In this paper, we briefly review our recent discovery of key factors for protein aggregation. We used a lattice model to study the aggregation rates of proteins and found that the probability for a protein sequence to appear in the conformation of the aggregated state can be used to determine the temperature at which proteins can aggregate most quickly. We used molecular dynamics and simple models of polymer chains to study relaxation and aggregation of proteins under various conditions and found that when the bending-angle dependent and torsion-angle dependent interactions are zero or very small, then protein chains tend to aggregate at lower temperatures. All atom models were used to identify a key peptide chain for the aggregation of insulin chains and to find that two polyQ chains prefer anti-parallel conformation. It is pointed out that in many cases, protein aggregation does not result from protein mis-folding. A potential drug from Chinese medicine was found for Alzheimer's disease.

  7. Viral organization of human proteins.

    Directory of Open Access Journals (Sweden)

    Stefan Wuchty

    2010-08-01

    Full Text Available Although maps of intracellular interactions are increasingly well characterized, little is known about large-scale maps of host-pathogen protein interactions. The investigation of host-pathogen interactions can reveal features of pathogenesis and provide a foundation for the development of drugs and disease prevention strategies. A compilation of experimentally verified interactions between HIV-1 and human proteins and a set of HIV-dependency factors (HDF allowed insights into the topology and intricate interplay between viral and host proteins on a large scale. We found that targeted and HDF proteins appear predominantly in rich-clubs, groups of human proteins that are strongly intertwined among each other. These assemblies of proteins may serve as an infection gateway, allowing the virus to take control of the human host by reaching protein pathways and diversified cellular functions in a pronounced and focused way. Particular transcription factors and protein kinases facilitate indirect interactions between HDFs and viral proteins. Discerning the entanglement of directly targeted and indirectly interacting proteins may uncover molecular and functional sites that can provide novel perspectives on the progression of HIV infection and highlight new avenues to fight this virus.

  8. Protein Adaptations in Archaeal Extremophiles

    Directory of Open Access Journals (Sweden)

    Christopher J. Reed

    2013-01-01

    Full Text Available Extremophiles, especially those in Archaea, have a myriad of adaptations that keep their cellular proteins stable and active under the extreme conditions in which they live. Rather than having one basic set of adaptations that works for all environments, Archaea have evolved separate protein features that are customized for each environment. We categorized the Archaea into three general groups to describe what is known about their protein adaptations: thermophilic, psychrophilic, and halophilic. Thermophilic proteins tend to have a prominent hydrophobic core and increased electrostatic interactions to maintain activity at high temperatures. Psychrophilic proteins have a reduced hydrophobic core and a less charged protein surface to maintain flexibility and activity under cold temperatures. Halophilic proteins are characterized by increased negative surface charge due to increased acidic amino acid content and peptide insertions, which compensates for the extreme ionic conditions. While acidophiles, alkaliphiles, and piezophiles are their own class of Archaea, their protein adaptations toward pH and pressure are less discernible. By understanding the protein adaptations used by archaeal extremophiles, we hope to be able to engineer and utilize proteins for industrial, environmental, and biotechnological applications where function in extreme conditions is required for activity.

  9. Protein detection system

    Science.gov (United States)

    Fruetel, Julie A [Livermore, CA; Fiechtner, Gregory J [Bethesda, MD; Kliner, Dahv A. V. [San Ramon, CA; McIlroy, Andrew [Livermore, CA

    2009-05-05

    The present embodiment describes a miniature, microfluidic, absorption-based sensor to detect proteins at sensitivities comparable to LIF but without the need for tagging. This instrument utilizes fiber-based evanescent-field cavity-ringdown spectroscopy, in combination with faceted prism microchannels. The combination of these techniques will increase the effective absorption path length by a factor of 10.sup.3 to 10.sup.4 (to .about.1-m), thereby providing unprecedented sensitivity using direct absorption. The coupling of high-sensitivity absorption with high-performance microfluidic separation will enable real-time sensing of biological agents in aqueous samples (including aerosol collector fluids) and will provide a general method with spectral fingerprint capability for detecting specific bio-agents.

  10. JAK protein kinase inhibitors.

    Science.gov (United States)

    Thompson, James E

    2005-06-01

    In humans, the Janus protein tyrosine kinase family (JAKs) contains four members: JAK1, JAK2, JAK3 and TYK2. JAKs phosphorylate signal transducers and activators of transcription (STATs) simultaneously with other phosphorylations required for activation, and there are several cellular mechanisms in place to inhibit JAK/STAT signaling. That one might be able to modulate selected JAK/STAT-mediated cellular signals by inhibiting JAK kinase activity to effect a positive therapeutic outcome is a tantalizing prospect, as yet incompletely realized. While current data suggest no therapeutic use for JAK1 and TYK2 inhibition, JAK2 inhibition seems a promising but not definitively tested mechanism for treatment of leukemia. More promising, however, are data indicating a possible therapeutic use of JAK3 inhibition. The restriction of the JAK3-deficient phenotype to the hematopoietic system and the resulting profound immune suppression suggest that JAK3 could be a target for immunosuppressive therapies used to prevent organ transplant rejection.

  11. Protein Polymers and Amyloids

    DEFF Research Database (Denmark)

    Risør, Michael Wulff

    2014-01-01

    that inhibits its target protease through a large conformational change but mutations compromise this function and cause premature structural collapse into hyperstable polymers. Understanding the conformational disorders at a molecular level is not only important for our general knowledge on protein folding...... of this mechanism were investigated through a series of interaction experiments. Despite a very buried location in the native structure, evidence here suggest that the C-terminal tail is labile under slightly destabilizing conditions, providing new detail to this matter. A small infectious polymer unit was also...... constructed and used to show how polymerogenic seeding and polymer propagation might happen inside the body. The locking of central structural elements during α1AT folding or in the native state represents a therapeutic strategy to prevent polymerization. Using Molecular Dynamics simulations, we identified...

  12. Protein Hormones and Immunity‡

    Science.gov (United States)

    Kelley, Keith W.; Weigent, Douglas A.; Kooijman, Ron

    2007-01-01

    A number of observations and discoveries over the past 20 years support the concept of important physiological interactions between the endocrine and immune systems. The best known pathway for transmission of information from the immune system to the neuroendocrine system is humoral in the form of cytokines, although neural transmission via the afferent vagus is well documented also. In the other direction, efferent signals from the nervous system to the immune system are conveyed by both the neuroendocrine and autonomic nervous systems. Communication is possible because the nervous and immune systems share a common biochemical language involving shared ligands and receptors, including neurotransmitters, neuropeptides, growth factors, neuroendocrine hormones and cytokines. This means that the brain functions as an immune-regulating organ participating in immune responses. A great deal of evidence has accumulated and confirmed that hormones secreted by the neuroendocrine system play an important role in communication and regulation of the cells of the immune system. Among protein hormones, this has been most clearly documented for prolactin (PRL), growth hormone (GH), and insulin-like growth factor-1 (IGF-I), but significant influences on immunity by thyroid stimulating hormone (TSH) have also been demonstrated. Here we review evidence obtained during the past 20 years to clearly demonstrate that neuroendocrine protein hormones influence immunity and that immune processes affect the neuroendocrine system. New findings highlight a previously undiscovered route of communication between the immune and endocrine systems that is now known to occur at the cellular level. This communication system is activated when inflammatory processes induced by proinflammatory cytokines antagonize the function of a variety of hormones, which then causes endocrine resistance in both the periphery and brain. Homeostasis during inflammation is achieved by a balance between cytokines and

  13. Novel protein-protein interactions inferred from literature context.

    Directory of Open Access Journals (Sweden)

    Herman H H B M van Haagen

    Full Text Available We have developed a method that predicts Protein-Protein Interactions (PPIs based on the similarity of the context in which proteins appear in literature. This method outperforms previously developed PPI prediction algorithms that rely on the conjunction of two protein names in MEDLINE abstracts. We show significant increases in coverage (76% versus 32% and sensitivity (66% versus 41% at a specificity of 95% for the prediction of PPIs currently archived in 6 PPI databases. A retrospective analysis shows that PPIs can efficiently be predicted before they enter PPI databases and before their interaction is explicitly described in the literature. The practical value of the method for discovery of novel PPIs is illustrated by the experimental confirmation of the inferred physical interaction between CAPN3 and PARVB, which was based on frequent co-occurrence of both proteins with concepts like Z-disc, dysferlin, and alpha-actinin. The relationships between proteins predicted by our method are broader than PPIs, and include proteins in the same complex or pathway. Dependent on the type of relationships deemed useful, the precision of our method can be as high as 90%. The full set of predicted interactions is available in a downloadable matrix and through the webtool Nermal, which lists the most likely interaction partners for a given protein. Our framework can be used for prioritizing potential interaction partners, hitherto undiscovered, for follow-up studies and to aid the generation of accurate protein interaction maps.

  14. Protein complexes predictions within protein interaction networks using genetic algorithms.

    Science.gov (United States)

    Ramadan, Emad; Naef, Ahmed; Ahmed, Moataz

    2016-07-25

    Protein-protein interaction networks are receiving increased attention due to their importance in understanding life at the cellular level. A major challenge in systems biology is to understand the modular structure of such biological networks. Although clustering techniques have been proposed for clustering protein-protein interaction networks, those techniques suffer from some drawbacks. The application of earlier clustering techniques to protein-protein interaction networks in order to predict protein complexes within the networks does not yield good results due to the small-world and power-law properties of these networks. In this paper, we construct a new clustering algorithm for predicting protein complexes through the use of genetic algorithms. We design an objective function for exclusive clustering and overlapping clustering. We assess the quality of our proposed clustering algorithm using two gold-standard data sets. Our algorithm can identify protein complexes that are significantly enriched in the gold-standard data sets. Furthermore, our method surpasses three competing methods: MCL, ClusterOne, and MCODE in terms of the quality of the predicted complexes. The source code and accompanying examples are freely available at http://faculty.kfupm.edu.sa/ics/eramadan/GACluster.zip .

  15. Water-Protein Interactions: The Secret of Protein Dynamics

    Directory of Open Access Journals (Sweden)

    Silvia Martini

    2013-01-01

    Full Text Available Water-protein interactions help to maintain flexible conformation conditions which are required for multifunctional protein recognition processes. The intimate relationship between the protein surface and hydration water can be analyzed by studying experimental water properties measured in protein systems in solution. In particular, proteins in solution modify the structure and the dynamics of the bulk water at the solute-solvent interface. The ordering effects of proteins on hydration water are extended for several angstroms. In this paper we propose a method for analyzing the dynamical properties of the water molecules present in the hydration shells of proteins. The approach is based on the analysis of the effects of protein-solvent interactions on water protons NMR relaxation parameters. NMR relaxation parameters, especially the nonselective (R1NS and selective (R1SE spin-lattice relaxation rates of water protons, are useful for investigating the solvent dynamics at the macromolecule-solvent interfaces as well as the perturbation effects caused by the water-macromolecule interactions on the solvent dynamical properties. In this paper we demonstrate that Nuclear Magnetic Resonance Spectroscopy can be used to determine the dynamical contributions of proteins to the water molecules belonging to their hydration shells.

  16. Protein intake, body composition, and protein status following bariatric surgery.

    Science.gov (United States)

    Andreu, Alba; Moizé, Violeta; Rodríguez, Lucía; Flores, Lilliam; Vidal, Josep

    2010-11-01

    Daily protein intake recommendations have recently been proposed for the bariatric patient. We aimed to evaluate the accomplishment of these recommendations, and the influence of protein intake (PI) on fat free mass (FFM) and protein status changes following bariatric surgery. We examined 101 consecutive patients undergoing laparoscopic Roux-in-Y gastric gypass (LGBP) or laparoscopic sleeve gastrectomy (LSG). Based on 3-day food records, PI from food and supplements were quantified at 4, 8, and 12 months after surgery. The association between PI and body composition (bioelectrical impedance), plasma albumin and pre-albumin was evaluated at all study time points. A PI protein supplementation, supplements were taken only by 63.4, 50.5, and 33.7% of the participants at 4, 8, and 12 months. However, protein supplementation was effective in helping patients to achieve the daily protein intake goal. In linear regression analysis, male gender and weight loss, but not PI, were significantly associated with loss of FFM (p protein supplementation for the achievement of the recommended daily protein intake in the bariatric patient. However, our data does not help to define a PI goal as critical in determining the FFM and protein status changes following LGBP or LSG.

  17. Protein-Protein Interaction Detection: Methods and Analysis

    Directory of Open Access Journals (Sweden)

    V. Srinivasa Rao

    2014-01-01

    Full Text Available Protein-protein interaction plays key role in predicting the protein function of target protein and drug ability of molecules. The majority of genes and proteins realize resulting phenotype functions as a set of interactions. The in vitro and in vivo methods like affinity purification, Y2H (yeast 2 hybrid, TAP (tandem affinity purification, and so forth have their own limitations like cost, time, and so forth, and the resultant data sets are noisy and have more false positives to annotate the function of drug molecules. Thus, in silico methods which include sequence-based approaches, structure-based approaches, chromosome proximity, gene fusion, in silico 2 hybrid, phylogenetic tree, phylogenetic profile, and gene expression-based approaches were developed. Elucidation of protein interaction networks also contributes greatly to the analysis of signal transduction pathways. Recent developments have also led to the construction of networks having all the protein-protein interactions using computational methods for signaling pathways and protein complex identification in specific diseases.

  18. Modular protein switches derived from antibody mimetic proteins.

    Science.gov (United States)

    Nicholes, N; Date, A; Beaujean, P; Hauk, P; Kanwar, M; Ostermeier, M

    2016-02-01

    Protein switches have potential applications as biosensors and selective protein therapeutics. Protein switches built by fusion of proteins with the prerequisite input and output functions are currently developed using an ad hoc process. A modular switch platform in which existing switches could be readily adapted to respond to any ligand would be advantageous. We investigated the feasibility of a modular protein switch platform based on fusions of the enzyme TEM-1 β-lactamase (BLA) with two different antibody mimetic proteins: designed ankyrin repeat proteins (DARPins) and monobodies. We created libraries of random insertions of the gene encoding BLA into genes encoding a DARPin or a monobody designed to bind maltose-binding protein (MBP). From these libraries, we used a genetic selection system for β-lactamase activity to identify genes that conferred MBP-dependent ampicillin resistance to Escherichia coli. Some of these selected genes encoded switch proteins whose enzymatic activity increased up to 14-fold in the presence of MBP. We next introduced mutations into the antibody mimetic domain of these switches that were known to cause binding to different ligands. To different degrees, introduction of the mutations resulted in switches with the desired specificity, illustrating the potential modularity of these platforms. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  19. Noninvasive imaging of protein-protein interactions in living animals

    Science.gov (United States)

    Luker, Gary D.; Sharma, Vijay; Pica, Christina M.; Dahlheimer, Julie L.; Li, Wei; Ochesky, Joseph; Ryan, Christine E.; Piwnica-Worms, Helen; Piwnica-Worms, David

    2002-05-01

    Protein-protein interactions control transcription, cell division, and cell proliferation as well as mediate signal transduction, oncogenic transformation, and regulation of cell death. Although a variety of methods have been used to investigate protein interactions in vitro and in cultured cells, none can analyze these interactions in intact, living animals. To enable noninvasive molecular imaging of protein-protein interactions in vivo by positron-emission tomography and fluorescence imaging, we engineered a fusion reporter gene comprising a mutant herpes simplex virus 1 thymidine kinase and green fluorescent protein for readout of a tetracycline-inducible, two-hybrid system in vivo. By using micro-positron-emission tomography, interactions between p53 tumor suppressor and the large T antigen of simian virus 40 were visualized in tumor xenografts of HeLa cells stably transfected with the imaging constructs. Imaging protein-binding partners in vivo will enable functional proteomics in whole animals and provide a tool for screening compounds targeted to specific protein-protein interactions in living animals.

  20. Protein (Cyanobacteria): 553733356 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available ransporter family protein Lyngbya aestuarii MTQYSRYYSQTPNYPNSNHPSLENINLTIDPGKTVALVGKNGAGKTTLTKLLCRLYDPDCGKILWKGEDLRALELEDLRQKIAVVLQNYARFPLTVRENIALGNLEKLNCDRTLFKAIEKAGITRKIHSLPNPLDTPL

  1. Protein (Cyanobacteria): 515863728 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available hypothetical protein Geminocystis herdmanii MNRINKLVSITSAIICSGITTITSQLPAVAGDVSPLCENLNMGTQILISTKEFNAAICDKYYIEPQSGCPMPLEYFYVGQSRKTGESIVLPASDVSTSNPFMRIYKAQNGNYTYQIASSGAYGGNSWTSLSVFNKGY

  2. Protein (Cyanobacteria): 515864564 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available ypothetical protein Geminocystis herdmanii MASIVGNSRNNLLEGTLGNDTILGLGGNDTLDGEGGNDLLNGGDGNDLLDGGSGNDTLIGGNGNDT...LDGEGGNDLLNGGSGNDLLDGGSGNDTLIGGNGNDTLDGESGNDLLNGGSGNDLLDGEGGNDTLIGGLGRDRLDGGAGADFYLYNSPNEGRDLIDDYSVTNDTFLFRRNGFNGGLSLGTLNANQFTYGSSASDGNDRFIYNRSTGELFFDIDGTGSSSQQLIAKLIDPIGVLNRNDIVII

  3. Protein (Cyanobacteria): 504951340 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available tical protein Nostoc sp. PCC 7524 MTTVVQEYELKTLQRLKEEENQGAIQFEAAIEQGLLIVVDFESEQEEESYINYAAILGDDGESATCAIAVHRQWAIATDDKRAISFIQKEASNIQILSTPEIIKNWSEVASLDNSELRNILNSIRLKGRYLPAKTHPLRNWWLGILK

  4. Protein (Cyanobacteria): 516359091 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available tical protein Scytonema hofmanni MTHFFEFEADFVESLRCIPMQVRLKLDTCGIKLKLNQWNQFSEKERLALVERPCNTEETIQEYREFLRQLVQQHTGESATDLPVEEAPLWLDEQNIPNSVTSKAQEFGIEMTPNQWSNLLPVQRFALIKLSRSSHENKNFLPALKEFHVV

  5. Protein (Cyanobacteria): 515866305 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available hypothetical protein Nodosilinea nodulosa MISFSPIPWIAVLGRLGIAVALGASIGVDREYSQKAAGLRTNMLVALGAALFILVTIQSGMAQADSTALARSLQGVITGVGFVGAGSILRTGRVRGLTSATAIWVSAGVGLAAGLGQWQLGLLGTGLALMILRLLKFAED

  6. Protein (Cyanobacteria): 515875839 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available DLTATLAQNQAEDCFLVDALGTWVANGLEWSEEEWQGKVRDLLELLPTLPGVVILVGEETGWGLVPVYPLGRQFRDRLGSLLRYVGTLATSVYLVTGGYALDLTQLGIPLLGNRGGEGEGERGRGGAGE ...pothetical protein Spirulina subsalsa MIHSPTIILVTGPARSGKSEWAESLAMQSGKRVSYLATARLNPEDSEWQARIEKHQARRPPDWKTLWVPE

  7. Protein (Cyanobacteria): 441045 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available hetical protein Microcystis aeruginosa PCC 9432 MKKRKIANTLRKALLEDGKMERALYEYELEEHLDYWYEGLKSDREQFVFAVTENSGDVAMVLITPDKTIYVNEEAREKLAEFWIKAYRNNINRLIPMMAENLANNIISVTGVKMVSPNQHRHWVSLRP ...

  8. Protein (Cyanobacteria): 648456548 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available thetical protein Leptolyngbya boryana MSLALLEQYAMKLIDLETAVDPVLEEQLFDLLLVRDRIECLRKDYDAANLQKLLHLDQRLQQQGTRIAQFLNLPNCRTTVKPTEDAWWWWFEPAGDWRDRYDWLWSALCVPMMATSGALLLDLSGRFLSGGIDTFGALMWSVKVY

  9. Protein (Cyanobacteria): 441039 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available hetical protein Microcystis aeruginosa PCC 9717 MKKRKIANTLRKALLEDGKMERALYEYELEEHIDYWYEGLKSDRDQFVFAVTENSGDVAMVLITPGKTIYVNEEAREKLSQFWIKAYENNINQLIPMMAENLANDIISVTGVKMVSPNQKRRWVSLRP ...

  10. Protein (Viridiplantae): 308798659 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available named protein product Ostreococcus tauri MAMRGMKMAAKAPTTGRRARRTRADARTPARFVAARVNADDLTDAARDKFDEVTTTLSEYWEDSDEKPALVTLGVYGIVGLVAANGTLRAVDGLPLIPDFLELVGILFSGFFVYQNLLYKPDRAALRETISKIYNKIL ...

  11. Protein (Cyanobacteria): 499441265 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available thetical protein Synechococcus sp. WH 8102 MLRLVWLLPLALLQACAGSPVAEELQRSFESPELMATEAEAPIPEQPQVVDPTPIDRSQEVEVEQEAATKSDTDTNPDTDGDGIDVQQPISKSLQPPAPYRITIRLAGADPAAPAEAVTRALRQSEVVFSVERIERITP

  12. Protein (Cyanobacteria): 516316998 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available cal protein Prochlorothrix hollandica MGCLLEFWGMSATETVVITFGLDETEFEDEERLRFAKKLLPLMRKECDAVERVERAEDLNPEAGSKPGIATLIGLLTAEVGLDSIKEFIGFLGDRMGDQPMTVTVGEVTITARSRHELEQLEPMALRLLDAQRQPQGEAKNV

  13. Protein (Cyanobacteria): 495464035 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available hypothetical protein Moorea producens MTVRQPRYSKEEFARRGDEIYETQVRPKVEAGNHGKIVAIDIETGDFEVDPREIAACDRLEARNPDAQRISEKFFDTEFCPPSPPILGGTRINLLVEVPQNWGTNGGLDVANETFQTTSQIWIVRIGSRYVRRFGGRGKRTG

  14. Protein (Cyanobacteria): 516354103 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available cal protein Scytonema hofmanni MTRVYLDTSIYNRPFDDQTQPKIFLETQAVILILQMIEGKSIELVSSSVLEYENSRNPFPLKQQAMQQYLQMATVRQQADETIKQRAKQLEQQGLKAIDALHVACAEASGTNYLITSDKRLINRCQKLTFRVINPTNFILEVEDDYQGT

  15. Protein (Viridiplantae): 159463846 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available 2036 predicted protein Chlamydomonas reinhardtii MRRLQVCACCAGAWRLVRHGGGWRLGVCQRAMKACASLFLHASTRTVSRCMPACVRPATQCDQSTGMHVNRKRDCAFIMYSKGSAGKSTARWGAARSRQAAHVYAALCLCRSELGPRPLTCCRGYRQTP ...

  16. Protein (Viridiplantae): 159468077 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available hypothetical protein CHLREDRAFT_171030 Chlamydomonas reinhardtii MWATKLEAQLQLMFMPTRLHRRPLHQGTCRNYSTAPGITGVIELTSAFYRMYPNATFVFNKETAAKGTYRGEEETAASWWLKHVGSKLEIYLSPLLGLWAMSPPGPSGAGTR ...

  17. Protein (Viridiplantae): 159470305 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available predicted protein Chlamydomonas reinhardtii MSSRPKRAASANMANVIAAEKANKAAALHAWPKMWATKLEAQLQLMFMPTRLHRRPLHQGTCRNYSTAPGITGVIELTSAFYRMYPNATFVFNKETAAKGTYRGEEETAASWWLKHVGSKLEIYLSPLRCRPEVSR ...

  18. Protein (Cyanobacteria): 493210752 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available protein CheY Nodularia spumigena MNNTTAIMPIEVLLVEDNPGDAELTRIALEDSKISVNLNVVEDGVEAMAFLQKQGNYANAPHPDIVLLDLNLPKKDGREVLAEIKADKKLRRIPVVVLTTSQSEEDILKAYNLSANCFITKPVDFDQFVKIVQSIENFWFAIVKLPPE

  19. Protein (Cyanobacteria): 295749 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available ceiver protein Nostoc sp. PCC 7107 MPIEVLLVEDNPGDAELTRIALQDSKISINLNIVEDGVEAMAFLRKQDSYTRKPHPDIVLLDLNLPRKDGREVLAEMKSDDHLKRIPVVVLTTSQSEEDILKAYNLAANCYITKPVDFDQFVKIVQSIENFWFAIVKLPPE ...

  20. Protein (Cyanobacteria): 497073171 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available pothetical protein Fischerella sp. JSC-11 MHYYVHPFQLELHKLENMIVHVQHVNNQEVKQIADSRLFTSQAIGEEGGDTVTTKAIGEEGGDTVTTQAIGEEGGDTVTTKAIGEEGGDTVTTQAIGEEGGDTVTTQAIGEEGGDTVTTKAIGEEGGDTVTTLAFGEEGGF

  1. Protein (Cyanobacteria): 518320325 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available ... hypothetical protein Calothrix sp. PCC 7103 MDYVHPFQMELHKLESMIVHVQYADIKEVDKTLASNDAVSTQAVGEEGGTKVSTRALGEEGGNILTTYAVGEEGGNILTTYAVGEEGGDKVTTQAVGEEGGTRVTTYAVGEEGGGRVTTKAVGEEGGSIIRR

  2. Protein (Cyanobacteria): 424444 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available hetical protein Microcystis aeruginosa PCC 9809 MSLEENPYPNDESAEFTQAIEEVEAALASLKDRYRQISEAEQQKKDLEAQFSQIEPQWRENPLPELEKELVQIREQIQELEVILESNLLKEGELKRLFWEGIRRGLLGEVFWQIVRFGGIGVLLGWILRSCSG ...

  3. Protein (Cyanobacteria): 515881707 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available thetical protein Fischerella sp. PCC 9339 MNTLIAFVKKLRLRQVLTVFLAGLLFLTGSIYTSGYAQAAQLKSQVFLADAGQQSELLYPGAETPVGRAYKEGELPIKSEKDFRPNAGNLIQNEPSVTQRAKDRIETVKEAVEEASGFLKDKGNEATKRPELQPNPAVNK

  4. Protein (Cyanobacteria): 424446 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available hetical protein Microcystis aeruginosa PCC 9806 MSLEENPYPNDESAEFTQAIAEVEAAITALKDRYRQINEAERQKKDLEAQFSQIEPQWRENPLPELEKELVQIREQIQELEVILESNLLKEGELKRLFWEGIRRGLLGEVFWQIVRFGGIGVLLGWILRSCTG ...

  5. Protein (Cyanobacteria): 76081 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available protein Nodularia spumigena CCY9414 MNDQPVHVAIAILYQEDKFLMQLRDNIPGILYPGYWGLFGGHIELGETPDVAVKREVIEEIGYTLPSFAEFGCYADDAVVRHVFHAPLLVELDQLVLNEGWDMGLLTPEDIRQGKCYSPIADEVRLLGAIHQRIMLDFISH ...

  6. Protein (Cyanobacteria): 497312480 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available domain-containing protein Pseudanabaena biceps MKKRVNLTFPKRAISIPITYRLAKDFNIAANIIRAQVAPNKVGKMVLELSGDIDQLEEALDWMRSQDIEVSLHGREIVIDDTTCVDCGLCTGVCPTEALTLDSKTFQLNFLRSRCVVCEQCITACPVNAISINL

  7. Protein (Cyanobacteria): 499683197 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available othetical protein Synechococcus sp. CC9605 MSNHKINRYDAMPPHIIKALTLCANGSTWVDAAAAVGIKAPCLRKWYRDRRAEEFIESLVRENLNVANNLLTSAAPRLADELIQIALDPNVKAYARTQAISESFKILRENVLEAEQRRQLQEIRQTLQSLEDSKTVTV

  8. Protein (Cyanobacteria): 499682832 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available othetical protein Synechococcus sp. CC9605 MSENQLVNRFDAIPPHIIKALTLCANGSTWADAAAAVGIKAPCLRKWYRDRRAEEFIETLVRENLNVANNLLTSAAPRLADELIQIALDPNVKAYARTQAISESFKILRENVLEAEQRRQLQEIRQTLQSLEDSKTVTV

  9. Protein (Cyanobacteria): 499440544 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available othetical protein Synechococcus sp. WH 8102 MSYTEINRYDSIPPHIIKGLTLCANGSTWADAAAAVGVKAPCLRKWYRDSRAEEFIESLVRENINVANNLLTSAAPRLADELIKIALDPKVKAYARTQAISESFKILRENVLEAEQRKQLQEIRRTLQAIEDGKAVDV

  10. Protein (Cyanobacteria): 24305 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available ing protein Anabaena sp. 90 MNKLIFLDTNILGMVTNPKSSNSNCQECKEWLDELPLKSYQIILPEIADYEVRRELLRAGKTKGIKRLDQLKQAITYLPITTATMLLAAQFWAEIRNTGKPTADPKSLDGDVILAAQAKIEELNGDQVIVATTNVKHLSLFVDAREWQMIN ...

  11. Protein (Cyanobacteria): 653152304 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available protein Aphanizomenon flos-aquae MSKVIILDSAPVGLITNPKGNPLSVQCQEWFYSLFERGYEVILPEIIDYEIRRELLRANKLSGIRKLNQLKSEIIYLPITTEVMLKAAELWAEVRNKGKSTADNKALDGDVILAAQSILVANYGNEVIIATSNKKHLSLFIDAREWQEI

  12. Protein (Cyanobacteria): 500464022 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available thetical protein Synechococcus sp. WH 7803 MSRQRFRGLYLQNTGHPLCFSFVTYTPQTREQMVACGDLRADEEYFSPVLFDFLLFVSEGILGASPGVAFPFGYDDLAIVASRIRGTGVQHEYLIAINASAWNESKQAVLQQLRDILSRDLWDGARLRRGNDHPSPSE

  13. Protein (Cyanobacteria): 499305066 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available ical protein Nostoc sp. PCC 7120 MFKILFDSDLILDAVMNRTELAEDVRTLLENLHPSIRLYLTDVGLQKVSTYTYCLKNSQIPEIIVDWLQEQIQICPIDQGLLQKARYSPLRDFESAVELACINHYQLNAIVTNKPEDFIVTAHPLCVWSFADLWLRVNLESQLQATIHS

  14. Protein (Cyanobacteria): 515856463 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available othetical protein Leptolyngbya boryana MLQDALTWIQNLGATGAIVFILLYMCAAVLWIPGTLLTLGAGLVYGLFLGSLYVAIGATLGAIAAFLVG...RYVARDWVSQRIEANAKWKAIDQAVAKEGLKIVILTRLSPVFPFTLLNYAFGVTQVSLKDYALGCFGMIPGIIMYVYIGSLAGNLATLGKAPLSSEAQLAQWGLRIVGLIATVVVTVYVTRIARKALQDSGVEDS

  15. Protein (Cyanobacteria): 504938346 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available pothetical protein Synechococcus sp. PCC 6312 MSRKQHGLDWIVFYSDAVFAIAITLISVEIKLPFESGQLNSTELSHDLLNLFPEHQSYIFTFLIIGFFWINQYQYFTYIKHCDYKLFWLNTILLMCIDFLPFPASVLNDYRRQPVAVIFYACSMIATGLIKMVVRI

  16. Protein (Cyanobacteria): 515858423 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available hetical protein Leptolyngbya boryana MFRRILVVLEDAPSQHCVFNTALRFARANQAQLCLVDLRTNPAAIAHSLAEMAIGLGIQVDISELSEKTEQALIRTARNWYADLIVIGHALHPTLSPILPCTVLIVQQEQEHTISMTMQLRPQVPDGAVRNRLERLLDLTPSS

  17. Protein (Viridiplantae): 224125616 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available TPYLAHRSSFALPTAEAIKKVVAQTHAADRIRKAVEDAAATRIQAVFRSYLARKALCALRGLVKLQALVRGHQVRKQTTAT...0 predicted protein Populus trichocarpa MGKASRWMINFLLGKKEEKTKKNDISFHAEKETTPTATPAYKRRWSFGKSAKKERVYRGRRSLDSIIT

  18. Protein (Cyanobacteria): 516355738 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available tical protein Scytonema hofmanni MARIRLLHAKLHQVRVTDANVNYVGSVTIDSELIDKVGILPLQEVEIWNVSNGNRLSTYVLSGEPGSGVICLNGAAAHLCEPGDFVIIAAYEERDRAEVFRTGHEARVVIADEHNRCKKFFSQTLDPCQGKLLFHAEVTEITATTNF

  19. Protein (Cyanobacteria): 12321 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available protein Synechococcus sp. RCC307 MQGRSPAIGATTGLDEAYRLCRQQGLRLSRQRRLVLEILWRSGEHLSARDIFDRLNADGRRIGHTSVYQNLESLHSNGVIECLEKAQGRLYGHRADPHSHLTCLESGRISDLDIELPADLVEAIEQRTGFSIESYSLNLQGRPLP ...

  20. Protein (Cyanobacteria): 546232768 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available hypothetical protein Crocosphaera watsonii MKLNHSLVFISLTTIGLSLITPAKADAQLRNIGPNISIPSRECIPGAINCGGEIHRENMRHNRQLYFQTPEKILQHFHRERTERACLERTMTTPPPPIKANCNQYLEQIENFNQQDAVIDQRLLQQQEIDRLYPNGSNF

  1. Protein (Cyanobacteria): 494522819 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available hypothetical protein Crocosphaera watsonii MKLNHSLVFISLTTIGLSLITPAKADAQLRNIGPNISIPSRECIPGAINCGGEIHRENMRHNRQLYFQTPEKILQHFHRERTERACLERTMTTPPPPINANCNQYLEQIENFNQQDAVIDQRLLQQQEIDRLYPNGSNF

  2. Protein (Cyanobacteria): 654346332 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available protein Mastigocoleus testarum MAGINLFDMLTQTNNGAAVQQVGQQTGLQPDMAQTAIKVLLPAIAGGLQRNVQQPGGLQSLLGALQNGHHEQYLDQP...ETLGKPESIADGNAILGHLLGSKDTSRAVAAQAAQKTGLSEQVLKSVLPMVASMAMASLSKQTRKPDMAGALAGMLSGQQPQPAQAGLGGLIGGLLGGGSKSQPQSGAMGMLGGLLDADGDGNAMDEIFQMVMNRR

  3. Protein (Cyanobacteria): 504939852 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available ical protein Calothrix sp. PCC 7507 MGLSDGLANPDKKAIVVEDCCSMIDAQLASKSGISGMAIKAAFGALKGVKPGYIAYVVEQILPQCFTALDPIWSEGLQTGDPVGYLNANRDRTADALLSVTDARAQNLKRQIVKGTYDKLRGSAKQNVEEAVPELAKIIDKYTKT

  4. Protein (Cyanobacteria): 499683514 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available pothetical protein Synechococcus sp. CC9605 MQLEQIEQALQAPVMDAVIALSERVQTLETNPEGRIYTAYRAIDQTLSLGYSDNIDSITEQLHERDFVLLASRRGTRREQRLLLLTLKEIGIASSYSENCFTASQNTVNHLRHLGWPLGNFKQGANSTKTHKRFNLER

  5. Protein (Cyanobacteria): 493680837 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available FVATGLVNKEIEKRTVYLLVAKPISRAELIVGKHLGLSAVLAVLVAAMTVIYLAILSLSRIPFPLGSILIASLFIWFELCLMAGVGILFGVFSSSLLATLLTFGVYLM...thetical protein Microcoleus vaginatus MNLRRILTVATNVFWEVIRDRILYLIIIFALLMGASVRLIPELAATTEKKIILDVGLAAMSILGLIATV

  6. Protein (Cyanobacteria): 115179 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available domain protein Calothrix sp. PCC 6303 MGTICEDKLYITNTKSTMSTMSTPTIDQLKQSDVPVIVADHEGIVVDINSNFEIIFGWTAEEIIGQPLTVILPAFFRDSHNLGFARFSATGQATVLNHPLNLKAVTKDNREIESEHFIIAEKQDGQWLFAAKLRPLEMA ...

  7. Protein (Cyanobacteria): 648292043 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available hypothetical protein Nodosilinea nodulosa MLDTNLWISALLFGGLPAQLIKLAQDGHVEIYTSQDLLAELADVLGYPKFQSRLKRLSSTSEALLINVTRLATICESPPPLAVPELRDQDDMIVLQAAVAAQAIAIVSGDDDLLALEQIGEISILTVRAFLFRYFPDSS

  8. Protein (Cyanobacteria): 497312160 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available ILYLIFSIKLPNLDWYQALFTIASTSGLIGLVIGGAIGTLYGWFFKSSLSISCRGCLERGQYLLMLEGSETLTRKGREILDNYTVKPH ...thetical protein Pseudanabaena biceps MIAVLPDESSAFEAYRLLQCHGISPEHLALVGKGYSSPDSVGLFNPTYTTWRYAKRGMFWLGVISTVTGV

  9. Protein (Cyanobacteria): 553733132 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available pothetical protein Lyngbya aestuarii MKRKTRTSPARSTFNYTALAVIGGVLILGIGIGIAISSTTTFSPENVASSQFIDRSAPSTETCIKFGASAM...VTDMRVFVTLNPFNVYISQPRMQPGCVLRTSNWTILKKNNLISSEQERDCKQRMNTFGYTGELESSPEISCIYQNNSAENLFLSQPGGGGMTPARPRAESDRF

  10. Protein (Cyanobacteria): 553729546 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available ypothetical protein Lyngbya aestuarii MTSLTQNSIILEKAIKYVLKKSPDKPKTEAVVEALIEQEKTASKLKEPSNFSQFLGTWRLCFITGTQKTR...RKIGTALGPGRYLPNWVKIYLSYSDSSASPQVNLEQAFEAGNVENSVKLGGLKLTLSGPVKFQEKKNILAFDFTRMKVILFGVKLYDGYIRGGAESEEKFYSDRINKQAFFAYFYIQEKAIAARGRGGGLALWGRES

  11. Protein (Cyanobacteria): 495458053 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available ... hypothetical protein, partial Moorea producens DASNLSRTDGVDISWSTAEGVLINATTYSIENSQLLSRFDLTQSEEQWQVQGEMQGKPVSSVLEHKGELLSDYGSYLVSLELLGSEQDVVTQNMWVAEADPISATAVKMSKIADNEHANVKMDIGTICSRIFS

  12. Protein (Cyanobacteria): 648401911 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available ... hypothetical protein Calothrix sp. PCC 7103 MAPTICSFGGLILMASPMLLQVPADYQKFETKRRTSDSEDINRARIKERKETANLLQKTGLLREGKTLTIRDYEDDSKEKPGISNRTLLSYLEDEEVYVYDFKRMCIGKIKSRRFYWKHHYKGICDNAPTVTDN

  13. Protein (Viridiplantae): 159472102 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available 4474 predicted protein, partial Chlamydomonas reinhardtii PPSPAPPSPEPGSPPPSPAPPSPQPPSPAPPSPEPGSPPPSPAPPSPKPPSPAPPSPEQPGSPPPSPPPPRPQPPSPAPPSPEPGSPPPSPAPPSPQPPSPAPPSPEPGSPPPSPAPTQP ...

  14. Protein (Cyanobacteria): 515860616 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available pothetical protein Leptolyngbya boryana MHKIKNRVVGTSTASRQKENPMNTRNLLSGLALFALPMALGLAMPSLAVPNNGGSGTKMDELKKGGYTCERVSVNFIECTKDGSPTYWCTDNGECQQQARRHVTFPGQLPGAADPGRPTVVIEAQPILSPSNLGVRNGAQF

  15. Protein (Cyanobacteria): 550281717 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available hypothetical protein Rubidibacter lacunae MDTDMSVELINLSLKPREYLKGLTTIVIAYLAVNLLSLERIGALLRKFKRSSCQELNTCEAEIIWAAIHKSSLYFPGRVACLELSLAFTIYALISKRSSIWCVGVAVDPIRAHAWVEVEQKPFHEKNDLYLYFKKILVV

  16. Protein (Cyanobacteria): 553732548 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available ypothetical protein Lyngbya aestuarii MKGLRVDLSRSDVKVALDLCYYDFETILQALILWALYCEEEGKPLQFPNKTLTQAIAQQWKPREYSPWSDKILSNPRFQSPGTKWWIAAAEGLGRDVRNQLIADVDEKGSQQYVLFRNGLTLRLNTALNWDWEKIRAYGERQTR

  17. Protein (Cyanobacteria): 500469187 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available pothetical protein Synechococcus sp. RCC307 MKILLSLLLLLAPTAALAQEQKKPQSMRDAADSFRICRTIPEERRDESAGRRVAQAWIDSAPSGAEERLPRRELMEAMVKAYAAYMGERKAYGAIGCSEGILDRVENQNWSSFHQGIREVLMKQGMGDLMTPGTPPGQ

  18. Protein (Viridiplantae): 159488149 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available 8105 predicted protein Chlamydomonas reinhardtii MSTSGLLFQRRSVTAATYKRSSNRQTRLNVVAFGGQQGAAPEHAARARTTPQASMAASTMPGPQGAELGNWLRQLDLFFSKSRDTRSLSEISDFNMSDEDHDDDHASHMYVSHLAARMAMEPLPGRE ...

  19. Protein (Cyanobacteria): 546232644 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available ypothetical protein Crocosphaera watsonii MEKQTFSGKGKAGIMGLKLPSVPRISEGNRNSSYHWYLSVICNKSDRAYDVVVEGLLQPVALEANVQQQLATANLEERIKLYQTYDLWHENLDTLATMRRSQPQNSRASQQLGQLLQSVKLDPSIGQQPLLGIQTLTSRR

  20. Protein (Cyanobacteria): 504941098 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available al protein Calothrix sp. PCC 7507 MSATYQADFNLWIDKTAKLLREHRWQEIDLEHLIAEVEDLGKSERRAIISQLIRLLLHLLKWQYQPQRRSDSWLDSITDARTQIELAIQDSPSLKSYPIEQLKESYQKARRQAAKQTGMIISVFPEGCPYSLELVLDEDWLPEASE

  1. Protein (Cyanobacteria): 515871072 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available GGTAYIGPHIQLPENFINVLENVFHELLENKSSAKENHIHLHTNKDINNNKSSSCLASRRILLLSANPQKTESLHRRKEIEEIENALNRATVARLKEGKGDPVFEPLL...ypothetical protein Nodosilinea nodulosa MPDPVDKLCQIIAQELRSNKNITTHELIDHVNKKISQDSELKEALISDQRIQQINRDNSVNFQTLLE

  2. Dipolar response of hydrated proteins.

    Science.gov (United States)

    Matyushov, Dmitry V

    2012-02-28

    The paper presents an analytical theory and numerical simulations of the dipolar response of hydrated proteins in solution. We calculate the effective dielectric constant representing the average dipole moment induced at the protein by a uniform external field. The dielectric constant shows a remarkable variation among the proteins, changing from 0.5 for ubiquitin to 640 for cytochrome c. The former value implies a negative dipolar susceptibility, that is a dia-electric dipolar response and negative dielectrophoresis. It means that ubiquitin, carrying an average dipole of ≃240 D, is expected to repel from the region of a stronger electric field. This outcome is the result of a negative cross-correlation between the protein and water dipoles, compensating for the positive variance of the intrinsic protein dipole in the overall dipolar susceptibility. In contrast to the neutral ubiquitin, charged proteins studied here show para-electric dipolar response and positive dielectrophoresis. The study suggests that the dipolar response of proteins in solution is strongly affected by the coupling of the protein surface charge to the hydration water. The protein-water dipolar cross-correlations are long-ranged, extending ~2 nm from the protein surface into the bulk. A similar correlation length of about 1 nm is seen for the electrostatic potential produced by the hydration water inside the protein. The analysis of numerical simulations suggests that the polarization of the protein-water interface is highly heterogeneous and does not follow the standard dielectric results for cavities carved in dielectrics. The polarization of the water shell gains in importance, relative to the intrinsic protein dipole, at high frequencies, above the protein Debye peak. The induced interfacial dipole can be either parallel or antiparallel to the protein dipole, depending on the distribution of the protein surface charge. As a result, the high-frequency absorption of the protein solution can

  3. Hydrogels Constructed from Engineered Proteins.

    Science.gov (United States)

    Li, Hongbin; Kong, Na; Laver, Bryce; Liu, Junqiu

    2016-02-24

    Due to their various potential biomedical applications, hydrogels based on engineered proteins have attracted considerable interest. Benefitting from significant progress in recombinant DNA technology and protein engineering/design techniques, the field of protein hydrogels has made amazing progress. The latest progress of hydrogels constructed from engineered recombinant proteins are presented, mainly focused on biorecognition-driven physical hydrogels as well as chemically crosslinked hydrogels. The various bio-recognition based physical crosslinking strategies are discussed, as well as chemical crosslinking chemistries used to engineer protein hydrogels, and protein hydrogels' various biomedical applications. The future perspectives of this fast evolving field of biomaterials are also discussed. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Maintaining protein composition in cilia.

    Science.gov (United States)

    Stephen, Louise A; Elmaghloob, Yasmin; Ismail, Shehab

    2017-12-20

    The primary cilium is a sensory organelle that is vital in regulating several signalling pathways. Unlike most organelles cilia are open to the rest of the cell, not enclosed by membranes. The distinct protein composition is crucial to the function of cilia and many signalling proteins and receptors are specifically concentrated within distinct compartments. To maintain this composition, a mechanism is required to deliver proteins to the cilium whilst another must counter the entropic tendency of proteins to distribute throughout the cell. The combination of the two mechanisms should result in the concentration of ciliary proteins to the cilium. In this review we will look at different cellular mechanisms that play a role in maintaining the distinct composition of cilia, including regulation of ciliary access and trafficking of ciliary proteins to, from and within the cilium.

  5. Protein stability, flexibility and function

    DEFF Research Database (Denmark)

    Teilum, Kaare; Olsen, Johan G; Kragelund, Birthe B

    2011-01-01

    for a delineation of the molecular details of their function. Several of these mutations interfered with the binding of a specific ligand with a concomitant effect on the stability of the protein scaffold. It has been ambiguous and not straightforward to recognize if any relationships exist between the stability...... presented is it clear that there are specific sites (flexibility hotspots) in proteins that are important for both binding and stability. This article is part of a Special Issue entitled: Protein Dynamics: Experimental and Computational Approaches.......Proteins rely on flexibility to respond to environmental changes, ligand binding and chemical modifications. Potentially, a perturbation that changes the flexibility of a protein may interfere with its function. Millions of mutations have been performed on thousands of proteins in quests...

  6. Seed Storage Proteins In Coffee

    OpenAIRE

    Bau S.M.T.; Mazzafera P.; Santoro L.G.

    2001-01-01

    It has been reported that Coffea arabica seeds contain as the main reserve protein, a legumin-like protein, constituted of two subunits, alpha and beta, of approximately 35 and 20 kDa. In this work the seed proteins of several coffee species and varieties were investigated by SDS-PAGE and gel filtration. No differences were observed in the electrophoretic profiles among varieties of C. arabica, however, marked differences were observed among species, or even among individuals of some species....

  7. Protein: FBB5 [TP Atlas

    Lifescience Database Archive (English)

    Full Text Available FBB5 RNA silencing EIF2C2 AGO2 EIF2C2 Protein argonaute-2 Eukaryotic translation in...itiation factor 2C 2, PAZ Piwi domain protein, Protein slicer 9606 Homo sapiens Q9UKV8 27161 3LUK, 3LUH, 3LUG, 3QX8, 3QX9, 3LUD, 3LUJ, 3LUC 27161 Q9UKV8 18524951 ...

  8. Epicutaneous sensitization with protein antigen

    Directory of Open Access Journals (Sweden)

    I-Lin Liu

    2012-12-01

    Full Text Available In the past few decades there has been a progressive understanding that epicutaneous sensitization with protein antigen is an important sensitization route in patients with atopic dermatitis. A murine protein-patch model has been established, and an abundance of data has been obtained from experiments using this model. This review discusses the characteristics of epicutaneous sensitization with protein antigen, the induced immune responses, the underlying mechanisms, and the therapeutic potential.

  9. Dynamic identifying protein functional modules based on adaptive density modularity in protein-protein interaction networks.

    Science.gov (United States)

    Shen, Xianjun; Yi, Li; Yi, Yang; Yang, Jincai; He, Tingting; Hu, Xiaohua

    2015-01-01

    The identification of protein functional modules would be a great aid in furthering our knowledge of the principles of cellular organization. Most existing algorithms for identifying protein functional modules have a common defect -- once a protein node is assigned to a functional module, there is no chance to move the protein to the other functional modules during the follow-up processes, which lead the erroneous partitioning occurred at previous step to accumulate till to the end. In this paper, we design a new algorithm ADM (Adaptive Density Modularity) to detect protein functional modules based on adaptive density modularity. In ADM algorithm, according to the comparison between external closely associated degree and internal closely associated degree, the partitioning of a protein-protein interaction network into functional modules always evolves quickly to increase the density modularity of the network. The integration of density modularity into the new algorithm not only overcomes the drawback mentioned above, but also contributes to identifying protein functional modules more effectively. The experimental result reveals that the performance of ADM algorithm is superior to many state-of-the-art protein functional modules detection techniques in aspect of the accuracy of prediction. Moreover, the identified protein functional modules are statistically significant in terms of "Biological Process" annotated in Gene Ontology, which provides substantial support for revealing the principles of cellular organization.

  10. Assessment and significance of protein-protein interactions during development of protein biopharmaceuticals.

    Science.gov (United States)

    Yadav, Sandeep; Liu, Jun; Scherer, Thomas M; Gokarn, Yatin; Demeule, Barthélemy; Kanai, Sonoko; Andya, James D; Shire, Steven J

    2013-06-01

    Early development of protein biotherapeutics using recombinant DNA technology involved progress in the areas of cloning, screening, expression and recovery/purification. As the biotechnology industry matured, resulting in marketed products, a greater emphasis was placed on development of formulations and delivery systems requiring a better understanding of the chemical and physical properties of newly developed protein drugs. Biophysical techniques such as analytical ultracentrifugation, dynamic and static light scattering, and circular dichroism were used to study protein-protein interactions during various stages of development of protein therapeutics. These studies included investigation of protein self-association in many of the early development projects including analysis of highly glycosylated proteins expressed in mammalian CHO cell cultures. Assessment of protein-protein interactions during development of an IgG1 monoclonal antibody that binds to IgE were important in understanding the pharmacokinetics and dosing for this important biotherapeutic used to treat severe allergic IgE-mediated asthma. These studies were extended to the investigation of monoclonal antibody-antigen interactions in human serum using the fluorescent detection system of the analytical ultracentrifuge. Analysis by sedimentation velocity analytical ultracentrifugation was also used to investigate competitive binding to monoclonal antibody targets. Recent development of high concentration protein formulations for subcutaneous administration of therapeutics posed challenges, which resulted in the use of dynamic and static light scattering, and preparative analytical ultracentrifugation to understand the self-association and rheological properties of concentrated monoclonal antibody solutions.

  11. Developing algorithms for predicting protein-protein interactions of homology modeled proteins.

    Energy Technology Data Exchange (ETDEWEB)

    Martin, Shawn Bryan; Sale, Kenneth L.; Faulon, Jean-Loup Michel; Roe, Diana C.

    2006-01-01

    The goal of this project was to examine the protein-protein docking problem, especially as it relates to homology-based structures, identify the key bottlenecks in current software tools, and evaluate and prototype new algorithms that may be developed to improve these bottlenecks. This report describes the current challenges in the protein-protein docking problem: correctly predicting the binding site for the protein-protein interaction and correctly placing the sidechains. Two different and complementary approaches are taken that can help with the protein-protein docking problem. The first approach is to predict interaction sites prior to docking, and uses bioinformatics studies of protein-protein interactions to predict theses interaction site. The second approach is to improve validation of predicted complexes after docking, and uses an improved scoring function for evaluating proposed docked poses, incorporating a solvation term. This scoring function demonstrates significant improvement over current state-of-the art functions. Initial studies on both these approaches are promising, and argue for full development of these algorithms.

  12. Protein function prediction using neighbor relativity in protein-protein interaction network.

    Science.gov (United States)

    Moosavi, Sobhan; Rahgozar, Masoud; Rahimi, Amir

    2013-04-01

    There is a large gap between the number of discovered proteins and the number of functionally annotated ones. Due to the high cost of determining protein function by wet-lab research, function prediction has become a major task for computational biology and bioinformatics. Some researches utilize the proteins interaction information to predict function for un-annotated proteins. In this paper, we propose a novel approach called "Neighbor Relativity Coefficient" (NRC) based on interaction network topology which estimates the functional similarity between two proteins. NRC is calculated for each pair of proteins based on their graph-based features including distance, common neighbors and the number of paths between them. In order to ascribe function to an un-annotated protein, NRC estimates a weight for each neighbor to transfer its annotation to the unknown protein. Finally, the unknown protein will be annotated by the top score transferred functions. We also investigate the effect of using different coefficients for various types of functions. The proposed method has been evaluated on Saccharomyces cerevisiae and Homo sapiens interaction networks. The performance analysis demonstrates that NRC yields better results in comparison with previous protein function prediction approaches that utilize interaction network. Copyright © 2012 Elsevier Ltd. All rights reserved.

  13. Protein aggregation kinetics during Protein A chromatography. Case study for an Fc fusion protein.

    Science.gov (United States)

    Shukla, Abhinav A; Gupta, Priyanka; Han, Xuejun

    2007-11-09

    Protein A chromatography has come to be widely adopted for large-scale purification of monoclonal antibodies and Fc fusion proteins. The low pH conditions required for Protein A elution can often lead to aggregation issues for these products. A concerted study of the kinetics of aggregate formation and their relation to chromatography on Protein A media has been lacking. This paper provides a framework to describe aggregation kinetics for an Fc fusion protein that was highly susceptible to aggregate formation under low pH conditions. In contrast to what is usually expected to be a higher order reaction, first order aggregation kinetics were observed for this protein over a wide range of conditions. A comparison of the rate constants of aggregation forms an effective means of comparing various stabilizing additives to the elution buffer with one another. Inclusion of urea in the elution buffer at moderate concentrations (Protein A column were both found to be effective solutions to the aggregation issue. Elution from the Protein A resin was found to increase the aggregation rate constants over and above what would be expected from exposure to low pH conditions in solution alone. This demonstrates that Protein A-Fc interactions can destabilize product structure and increase the tendency to aggregate. The results presented here are anticipated to assist the development of Protein A process conditions for products that are prone to form high molecular weight aggregates during column elution.

  14. The clinical expression of hereditary protein C and protein S deficiency: : a relation to clinical thrombotic risk-factors and to levels of protein C and protein S

    NARCIS (Netherlands)

    Henkens, C. M. A.; van der Meer, J.; Hillege, J. L.; Bom, V. J. J.; Halie, M. R.; van der Schaaf, W.

    We investigated 103 first-degree relatives of 13 unrelated protein C or protein S deficient patients to assess the role of additional thrombotic risk factors and of protein C and protein S levels in the clinical expression of hereditary protein C and protein S deficiency. Fifty-seven relatives were

  15. Recovery of protein from green leaves

    NARCIS (Netherlands)

    Tamayo Tenorio, Angelica; Gieteling, Jarno; Jong, De Govardus A.H.; Boom, Remko M.; Goot, Van Der Atze J.

    2016-01-01

    Plant leaves are a major potential source of novel food proteins. Till now, leaf protein extraction methods mainly focus on the extraction of soluble proteins, like rubisco protein, leaving more than half of all protein unextracted. Here, we report on the total protein extraction from sugar beet

  16. Update on protein structure prediction

    DEFF Research Database (Denmark)

    Hubbard, T; Tramontano, A; Barton, G

    1996-01-01

    Computational tools for protein structure prediction are of great interest to molecular, structural and theoretical biologists due to a rapidly increasing number of protein sequences with no known structure. In October 1995, a workshop was held at IRBM to predict as much as possible about a number...... of proteins of biological interest using ab initio pre!diction of fold recognition methods. 112 protein sequences were collected via an open invitation for target submissions. 17 were selected for prediction during the workshop and for 11 of these a prediction of some reliability could be made. We believe...

  17. Dewetting Transitions in Protein Cavities *

    Science.gov (United States)

    Young, Tom; Hua, Lan; Huang, Xuhui; Abel, Robert; Friesner, Richard; Berne, B. J.

    2010-01-01

    In a previous analysis of the solvation of protein active sites, a drying transition was observed in the narrow hydrophobic binding cavity of Cox-2. With the use of a crude metric that often seems able to discriminate those protein cavities that dry from those that do not, we made an extensive search of the pdb, and identified five other proteins that, in molecular dynamics simulations, undergo drying transitions in their active sites. Because such cavities need not desolvate before binding hydrophobic ligands they often exhibit very large binding affinities. This paper gives evidence that drying in protein cavities is not unique to Cox-2. PMID:20225258

  18. Structure Prediction of Membrane Proteins

    Science.gov (United States)

    Hu, Xiche

    Membrane proteins play a central role in many cellular and physiological processes. It is estimated that integral membrane proteins make up about 20-30% of the proteome (Krogh et al., 2001b; Stevens and Arkin, 2000; von Heijne, 1999). They are essential mediators of material and information transfer across cell membranes. Their functions include active and passive transport of molecules into and out of cells and organelles; transduction of energy among various forms (light, electrical, and chemical energy); as well as reception and transduction of chemical and electrical signals across membranes (Avdonin, 2005; Bockaert et al., 2002; Pahl, 1999; Rehling et al., 2004; Stack et al., 1995). Identifying these transmembrane (TM) proteins and deciphering their molecular mechanisms, then, is of great importance, particularly as applied to biomedicine. Membrane proteins are the targets of a large number of pharmacologically and toxicologically active substances, and are directly involved in their uptake, metabolism, and clearance (Bettler et al., 1998; Cohen, 2002; Heusser and Jardieu, 1997; Tibes et al., 2005; Xu et al., 2005). Despite the importance of membrane proteins, the knowledge of their high-resolution structures and mechanisms of action has lagged far behind in comparison to that of water-soluble proteins: less than 1% of all three-dimensional structures deposited in the Protein Data Bank are of membrane proteins. This unfortunate disparity stems from difficulties in overexpression and the crystallization of membrane proteins (Grisshammer and Tate, 1995; Michel, 1991).

  19. Borrowed proteins in bacterial bioluminescence.

    Science.gov (United States)

    O'Kane, D J; Woodward, B; Lee, J; Prasher, D C

    1991-01-01

    A library of Photobacterium phosphoreum DNA was screened in lambda 2001 for the lumazine protein gene, using two degenerate 17-mer oligonucleotide probes that were deduced from a partial protein primary sequence. The lumazine protein gene was localized to a 3.4-kilobase BamHI/EcoRI fragment in one clone. The fragment contained an open reading frame, encoding a 189-residue protein, that had a predicted amino acid sequence that concurred with the partial sequence determined for lumazine protein. Considerable sequence similarity was detected between lumazine protein, the yellow fluorescence protein from Vibrio fischeri, and the alpha subunit of riboflavin synthetase (EC 2.5.1.9). A highly conserved sequence in lumazine protein corresponds to the proposed lumazine binding sites in the alpha subunit of riboflavin synthetase. Several secondary structure programs predict the conformation of this site in lumazine protein to be a beta-sheet. A minimal model with three interactions between the ligand and this beta-sheet structure is proposed, which is consistent with the results of NMR and ligand binding studies. Images PMID:1996310

  20. Reduced protein adsorption by osmolytes.

    Science.gov (United States)

    Evers, Florian; Steitz, Roland; Tolan, Metin; Czeslik, Claus

    2011-06-07

    Osmolytes are substances that affect osmosis and are used by cells to adapt to environmental stress. Here, we report a neutron reflectivity study on the influence of some osmolytes on protein adsorption at solid-liquid interfaces. Bovine ribonuclease A (RNase) and bovine insulin were used as model proteins adsorbing at a hydrophilic silica and at a hydrophobic polystyrene surface. From the neutron reflectivity data, the adsorbed protein layers were characterized in terms of layer thickness, protein packing density, and adsorbed protein mass in the absence and presence of urea, trehalose, sucrose, and glycerol. All data point to the clear effect of these nonionic cosolvents on the degree of protein adsorption. For example, 1 M sucrose leads to a reduction of the adsorbed amount of RNase by 39% on a silica surface and by 71% on a polystyrene surface. Trehalose was found to exhibit activity similar to that of sucrose. The changes in adsorbed protein mass can be attributed to a decreased packing density of the proteins in the adsorbed layers. Moreover, we investigated insulin adsorption at a hydrophobic surface in the absence and presence of glycerol. The degree of insulin adsorption is decreased by even 80% in the presence of 4 M of glycerol. The results of this study demonstrate that nonionic cosolvents can be used to tune and control nonspecific protein adsorption at aqueous-solid interfaces, which might be relevant for biomedical applications.

  1. High throughput protein production screening

    Science.gov (United States)

    Beernink, Peter T [Walnut Creek, CA; Coleman, Matthew A [Oakland, CA; Segelke, Brent W [San Ramon, CA

    2009-09-08

    Methods, compositions, and kits for the cell-free production and analysis of proteins are provided. The invention allows for the production of proteins from prokaryotic sequences or eukaryotic sequences, including human cDNAs using PCR and IVT methods and detecting the proteins through fluorescence or immunoblot techniques. This invention can be used to identify optimized PCR and WT conditions, codon usages and mutations. The methods are readily automated and can be used for high throughput analysis of protein expression levels, interactions, and functional states.

  2. Protein intrinsic disorder in plants

    Directory of Open Access Journals (Sweden)

    Florencio ePazos

    2013-09-01

    Full Text Available To some extent contradicting the classical paradigm of the relationship between protein 3D structure and function, now it is clear that large portions of the proteomes, especially in higher organisms, lack a fixed structure and still perform very important functions. Proteins completely or partially unstructured in their native (functional form are involved in key cellular processes underlain by complex networks of protein interactions. The intrinsic conformational flexibility of these disordered proteins allows them to bind multiple partners in transient interactions of high specificity and low affinity. In concordance, in plants this type of proteins has been found in processes requiring these complex and versatile interaction networks. These include transcription factor networks, where disordered proteins act as integrators of different signals or link different transcription factor subnetworks due to their ability to interact (in many cases simultaneously with different partners. Similarly, they also serve as signal integrators in signalling cascades, such as those related to response to external stimuli. Disordered proteins have also been found in plants in many stress-response processes, acting as protein chaperones or protecting other cellular components and structures. In plants, it is especially important to have complex and versatile networks able to quickly and efficiently respond to changing environmental conditions since these organisms can not escape and have no other choice than adapting to them. Consequently, protein disorder can play an especially important role in plants, providing them with a fast mechanism to obtain complex, interconnected and versatile molecular networks.

  3. Computational protein design: a review

    Science.gov (United States)

    Coluzza, Ivan

    2017-04-01

    Proteins are one of the most versatile modular assembling systems in nature. Experimentally, more than 110 000 protein structures have been identified and more are deposited every day in the Protein Data Bank. Such an enormous structural variety is to a first approximation controlled by the sequence of amino acids along the peptide chain of each protein. Understanding how the structural and functional properties of the target can be encoded in this sequence is the main objective of protein design. Unfortunately, rational protein design remains one of the major challenges across the disciplines of biology, physics and chemistry. The implications of solving this problem are enormous and branch into materials science, drug design, evolution and even cryptography. For instance, in the field of drug design an effective computational method to design protein-based ligands for biological targets such as viruses, bacteria or tumour cells, could give a significant boost to the development of new therapies with reduced side effects. In materials science, self-assembly is a highly desired property and soon artificial proteins could represent a new class of designable self-assembling materials. The scope of this review is to describe the state of the art in computational protein design methods and give the reader an outline of what developments could be expected in the near future.

  4. Protein-stabilized magnetic fluids

    Energy Technology Data Exchange (ETDEWEB)

    Soenen, S.J.H. [Interdisciplinary Research Center, Katholieke Universiteit Leuven-Campus Kortrijk, University Campus, B-8500 Kortrijk (Belgium); Hodenius, M.; Schmitz-Rode, T. [Helmholtz Institute, Applied Medical Engineering, RWTH Aachen University, Aachen (Germany); De Cuyper, M. [Interdisciplinary Research Center, Katholieke Universiteit Leuven-Campus Kortrijk, University Campus, B-8500 Kortrijk (Belgium)], E-mail: Marcel.DeCuyper@KULeuven-Kortrijk.be

    2008-03-15

    The adsorption of bovine serum albumin (BSA) and egg yolk phosvitin on magnetic fluid particles was investigated. Incubation mixtures were prepared by mixing an alkaline suspension of tetramethylammonium-coated magnetite cores with protein solutions at various protein/Fe{sub 3}O{sub 4} ratios, followed by dialysis against a 5 mM TES buffer (pH 7.0), after which separation of bound and non-bound protein by high-gradient magnetophoresis was executed. Both the kinetic profiles as well as the isotherms of adsorption strongly differed for both proteins. In case of the spherical BSA, initially, abundant adsorption occurred, then it decreased and-at high protein concentrations-it slowly raised again. In contrast, with the highly phosphorylated phosvitin, binding slowly started and the extent of protein adsorption remained unchanged both as a function of time and phosvitin concentration. Competition binding studies, using binary protein mixtures composed of equal weight amounts of BSA and phosvitin, showed that binding of the latter protein is 'unrealistically' high. Based on the geometry of the two proteins, putative pictures on their orientation on the particle's surface in the various experimental conditions were deduced.

  5. Protein Misfolding and Human Disease

    DEFF Research Database (Denmark)

    Gregersen, Niels; Bross, Peter Gerd; Vang, Søren

    2006-01-01

    phenylketonuria, Parkinson's disease, α-1-antitrypsin deficiency, familial neurohypophyseal diabetes insipidus, and short-chain acyl-CoA dehydrogenase deficiency. Despite the differences, an emerging paradigm suggests that the cellular effects of protein misfolding provide a common framework that may contribute...... to the elucidation of the cell pathology and guide intervention and treatment strategies of many genetic and age-dependent diseases.......Protein misfolding is a common event in living cells. In young and healthy cells, the misfolded protein load is disposed of by protein quality control (PQC) systems. In aging cells and in cells from certain individuals with genetic diseases, the load may overwhelm the PQC capacity, resulting...

  6. [Protein toxins of Staphylococcus aureus].

    Science.gov (United States)

    Shamsutdinov, A F; Tiurin, Iu A

    2014-01-01

    Main scientific-research studies regarding protein bacterial toxins of the most widespread bacteria that belong to Staphylococcus spp. genus and in particular the most pathogenic species for humans--Staphylococcus aureus, are analyzed. Structural and biological properties of protein toxins that have received the name of staphylococcus pyrogenic toxins (PTSAg) are presented. Data regarding genetic regulation of secretion and synthesis of these toxins and 3 main regulatory genetic systems (agr--accessory gene regulator, xpr--extracellular protein regulator, sar--staphylococcal accessory regulator) that coordinate synthesis of the most important protein toxins and enzymes for virulence of S. aureus, are presented.

  7. Prion protein dynamics before aggregation

    National Research Council Canada - National Science Library

    Srivastava, Kinshuk Raj; Lapidusa, Lisa J

    2017-01-01

      Prion diseases, like Alzheimer's disease and Parkinson disease, are rapidly progressive neurodegenerative disorders caused by misfolding followed by aggregation and accumulation of protein deposits in neuronal cells...

  8. Protein linguistics - a grammar for modular protein assembly?

    Science.gov (United States)

    Gimona, Mario

    2006-01-01

    The correspondence between biology and linguistics at the level of sequence and lexical inventories, and of structure and syntax, has fuelled attempts to describe genome structure by the rules of formal linguistics. But how can we define protein linguistic rules? And how could compositional semantics improve our understanding of protein organization and functional plasticity?

  9. Inactivation of Tor proteins affects the dynamics of endocytic proteins ...

    Indian Academy of Sciences (India)

    Tor2 is an activator of the Rom2/Rho1 pathway that regulates -factor internalization. Since the recruitment of endocytic proteins such as actin-binding proteins and the amphiphysins precedes the internalization of -factor, we hypothesized that loss of Tor function leads to an alteration in the dynamics of the endocytic ...

  10. Spot Accession Protein Protein Unique Secuence Number number ...

    Indian Academy of Sciences (India)

    Classification of the proteins identified as altered in the cardiac left ventricles from TPCN1 KO vs. WT mice by 2-DE-MADI-MS. The spot number, SwissProt accession number, protein name, relative fold-change and P-value. (given by the software SameSpots), experimental and theoretical pI and Mw values, Mascot score, ...

  11. Human Serum Protein-Bound iodine and Protein Fractions at ...

    African Journals Online (AJOL)

    Iodine profile of Nigerians at different ages in both sexes and in pregnant women, and under narcotic influence, such as alcoholism, cigarette smoking and marijuana addiction were studied. Their serum total protein, albumin and globulin concentrations were also determined. Results of the study showed that serum protein ...

  12. Protein stress and stress proteins: implications in aging and disease

    Indian Academy of Sciences (India)

    Madhu Sudhan

    2007-04-02

    Apr 2, 2007 ... cells reaching 1–5% of total cellular protein, which shows that a continuous intense demand is present to .... stem (and tumor) cell proliferation and cell survival. Hsp90 ensures, amongst several hundred ... interventions focusing to preserve the protein turnover is an attractive therapy in anti-aging research.

  13. Website on Protein Interaction and Protein Structure Related Work

    Science.gov (United States)

    Samanta, Manoj; Liang, Shoudan; Biegel, Bryan (Technical Monitor)

    2003-01-01

    In today's world, three seemingly diverse fields - computer information technology, nanotechnology and biotechnology are joining forces to enlarge our scientific knowledge and solve complex technological problems. Our group is dedicated to conduct theoretical research exploring the challenges in this area. The major areas of research include: 1) Yeast Protein Interactions; 2) Protein Structures; and 3) Current Transport through Small Molecules.

  14. Detecting protein-protein interactions in living cells

    DEFF Research Database (Denmark)

    Gottschalk, Marie; Bach, Anders; Hansen, Jakob Lerche

    2009-01-01

    to the endogenous C-terminal peptide of the NMDA receptor, as evaluated by a cell-free protein-protein interaction assay. However, it is important to address both membrane permeability and effect in living cells. Therefore a bioluminescence resonance energy transfer (BRET) assay was established, where the C...

  15. Eukaryotic LYR Proteins Interact with Mitochondrial Protein Complexes

    Directory of Open Access Journals (Sweden)

    Heike Angerer

    2015-02-01

    Full Text Available In eukaryotic cells, mitochondria host ancient essential bioenergetic and biosynthetic pathways. LYR (leucine/tyrosine/arginine motif proteins (LYRMs of the Complex1_LYR-like superfamily interact with protein complexes of bacterial origin. Many LYR proteins function as extra subunits (LYRM3 and LYRM6 or novel assembly factors (LYRM7, LYRM8, ACN9 and FMC1 of the oxidative phosphorylation (OXPHOS core complexes. Structural insights into complex I accessory subunits LYRM6 and LYRM3 have been provided by analyses of EM and X-ray structures of complex I from bovine and the yeast Yarrowia lipolytica, respectively. Combined structural and biochemical studies revealed that LYRM6 resides at the matrix arm close to the ubiquinone reduction site. For LYRM3, a position at the distal proton-pumping membrane arm facing the matrix space is suggested. Both LYRMs are supposed to anchor an acyl-carrier protein (ACPM independently to complex I. The function of this duplicated protein interaction of ACPM with respiratory complex I is still unknown. Analysis of protein-protein interaction screens, genetic analyses and predicted multi-domain LYRMs offer further clues on an interaction network and adaptor-like function of LYR proteins in mitochondria.

  16. Analysis of protein folds using protein contact networks

    Indian Academy of Sciences (India)

    Proteins are important biomolecules, which perform diverse structural and functional roles in living systems. Starting from a linear chain of amino acids, proteins fold to different secondary structures, which then fold through short- and long-range interactions to give rise to the final three-dimensional shapes useful to carry out ...

  17. Protein scissors: Photocleavage of proteins at specific locations

    Indian Academy of Sciences (India)

    Unknown

    suggested mechanism of protein cleavage. The origin of the specificity of photocleavage is discussed and specificity is valuable in targeting desired sites of proteins with small molecules. Keywords. Photocleavage; serum albumin; lysozyme; fluorescence; gelelectrophoresis. 1. Introduction. The binding of small molecules ...

  18. Protein-Protein Interactions (PPI) reagents: | Office of Cancer Genomics

    Science.gov (United States)

    The CTD2 Center at Emory University has a library of genes used to study protein-protein interactions in mammalian cells. These genes are cloned in different mammalian expression vectors. A list of available cancer-associated genes can be accessed below.

  19. Protein-Protein Interaction Reagents | Office of Cancer Genomics

    Science.gov (United States)

    The CTD2 Center at Emory University has a library of genes used to study protein-protein interactions in mammalian cells. These genes are cloned in different mammalian expression vectors. A list of available cancer-associated genes can be accessed below. Emory_CTD^2_PPI_Reagents.xlsx Contact: Haian Fu

  20. Protein stability: a crystallographer’s perspective

    Energy Technology Data Exchange (ETDEWEB)

    Deller, Marc C., E-mail: mdeller@stanford.edu [Stanford University, Shriram Center, 443 Via Ortega, Room 097, MC5082, Stanford, CA 94305-4125 (United States); Kong, Leopold [National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), National Institutes of Health (NIH), Building 8, Room 1A03, 8 Center Drive, Bethesda, MD 20814 (United States); Rupp, Bernhard [k.-k. Hofkristallamt, 91 Audrey Place, Vista, CA 92084 (United States); Medical University of Innsbruck, Schöpfstrasse 41, A-6020 Innsbruck (Austria)

    2016-01-26

    An understanding of protein stability is essential for optimizing the expression, purification and crystallization of proteins. In this review, discussion will focus on factors affecting protein stability on a somewhat practical level, particularly from the view of a protein crystallographer. Protein stability is a topic of major interest for the biotechnology, pharmaceutical and food industries, in addition to being a daily consideration for academic researchers studying proteins. An understanding of protein stability is essential for optimizing the expression, purification, formulation, storage and structural studies of proteins. In this review, discussion will focus on factors affecting protein stability, on a somewhat practical level, particularly from the view of a protein crystallographer. The differences between protein conformational stability and protein compositional stability will be discussed, along with a brief introduction to key methods useful for analyzing protein stability. Finally, tactics for addressing protein-stability issues during protein expression, purification and crystallization will be discussed.

  1. Protein-protein interaction predictions using text mining methods.

    Science.gov (United States)

    Papanikolaou, Nikolas; Pavlopoulos, Georgios A; Theodosiou, Theodosios; Iliopoulos, Ioannis

    2015-03-01

    It is beyond any doubt that proteins and their interactions play an essential role in most complex biological processes. The understanding of their function individually, but also in the form of protein complexes is of a great importance. Nowadays, despite the plethora of various high-throughput experimental approaches for detecting protein-protein interactions, many computational methods aiming to predict new interactions have appeared and gained interest. In this review, we focus on text-mining based computational methodologies, aiming to extract information for proteins and their interactions from public repositories such as literature and various biological databases. We discuss their strengths, their weaknesses and how they complement existing experimental techniques by simultaneously commenting on the biological databases which hold such information and the benchmark datasets that can be used for evaluating new tools. Copyright © 2014 Elsevier Inc. All rights reserved.

  2. Understanding Protein Evolution: From Protein Physics to Darwinian Selection

    Science.gov (United States)

    Zeldovich, Konstantin B.; Shakhnovich, Eugene I.

    2008-05-01

    Efforts in whole-genome sequencing and structural proteomics start to provide a global view of the protein universe, the set of existing protein structures and sequences. However, approaches based on the selection of individual sequences have not been entirely successful at the quantitative description of the distribution of structures and sequences in the protein universe because evolutionary pressure acts on the entire organism, rather than on a particular molecule. In parallel to this line of study, studies in population genetics and phenomenological molecular evolution established a mathematical framework to describe the changes in genome sequences in populations of organisms over time. Here, we review both microscopic (physics-based) and macroscopic (organism-level) models of protein-sequence evolution and demonstrate that bridging the two scales provides the most complete description of the protein universe starting from clearly defined, testable, and physiologically relevant assumptions.

  3. Composition of Overlapping Protein-Protein and Protein-Ligand Interfaces.

    Directory of Open Access Journals (Sweden)

    Ruzianisra Mohamed

    Full Text Available Protein-protein interactions (PPIs play a major role in many biological processes and they represent an important class of targets for therapeutic intervention. However, targeting PPIs is challenging because often no convenient natural substrates are available as starting point for small-molecule design. Here, we explored the characteristics of protein interfaces in five non-redundant datasets of 174 protein-protein (PP complexes, and 161 protein-ligand (PL complexes from the ABC database, 436 PP complexes, and 196 PL complexes from the PIBASE database and a dataset of 89 PL complexes from the Timbal database. In all cases, the small molecule ligands must bind at the respective PP interface. We observed similar amino acid frequencies in all three datasets. Remarkably, also the characteristics of PP contacts and overlapping PL contacts are highly similar.

  4. Text Mining for Protein Docking.

    Science.gov (United States)

    Badal, Varsha D; Kundrotas, Petras J; Vakser, Ilya A

    2015-12-01

    The rapidly growing amount of publicly available information from biomedical research is readily accessible on the Internet, providing a powerful resource for predictive biomolecular modeling. The accumulated data on experimentally determined structures transformed structure prediction of proteins and protein complexes. Instead of exploring the enormous search space, predictive tools can simply proceed to the solution based on similarity to the existing, previously determined structures. A similar major paradigm shift is emerging due to the rapidly expanding amount of information, other than experimentally determined structures, which still can be used as constraints in biomolecular structure prediction. Automated text mining has been widely used in recreating protein interaction networks, as well as in detecting small ligand binding sites on protein structures. Combining and expanding these two well-developed areas of research, we applied the text mining to structural modeling of protein-protein complexes (protein docking). Protein docking can be significantly improved when constraints on the docking mode are available. We developed a procedure that retrieves published abstracts on a specific protein-protein interaction and extracts information relevant to docking. The procedure was assessed on protein complexes from Dockground (http://dockground.compbio.ku.edu). The results show that correct information on binding residues can be extracted for about half of the complexes. The amount of irrelevant information was reduced by conceptual analysis of a subset of the retrieved abstracts, based on the bag-of-words (features) approach. Support Vector Machine models were trained and validated on the subset. The remaining abstracts were filtered by the best-performing models, which decreased the irrelevant information for ~ 25% complexes in the dataset. The extracted constraints were incorporated in the docking protocol and tested on the Dockground unbound benchmark set

  5. Text Mining for Protein Docking.

    Directory of Open Access Journals (Sweden)

    Varsha D Badal

    2015-12-01

    Full Text Available The rapidly growing amount of publicly available information from biomedical research is readily accessible on the Internet, providing a powerful resource for predictive biomolecular modeling. The accumulated data on experimentally determined structures transformed structure prediction of proteins and protein complexes. Instead of exploring the enormous search space, predictive tools can simply proceed to the solution based on similarity to the existing, previously determined structures. A similar major paradigm shift is emerging due to the rapidly expanding amount of information, other than experimentally determined structures, which still can be used as constraints in biomolecular structure prediction. Automated text mining has been widely used in recreating protein interaction networks, as well as in detecting small ligand binding sites on protein structures. Combining and expanding these two well-developed areas of research, we applied the text mining to structural modeling of protein-protein complexes (protein docking. Protein docking can be significantly improved when constraints on the docking mode are available. We developed a procedure that retrieves published abstracts on a specific protein-protein interaction and extracts information relevant to docking. The procedure was assessed on protein complexes from Dockground (http://dockground.compbio.ku.edu. The results show that correct information on binding residues can be extracted for about half of the complexes. The amount of irrelevant information was reduced by conceptual analysis of a subset of the retrieved abstracts, based on the bag-of-words (features approach. Support Vector Machine models were trained and validated on the subset. The remaining abstracts were filtered by the best-performing models, which decreased the irrelevant information for ~ 25% complexes in the dataset. The extracted constraints were incorporated in the docking protocol and tested on the Dockground unbound

  6. Protein-protein interactions within late pre-40S ribosomes.

    Directory of Open Access Journals (Sweden)

    Melody G Campbell

    2011-01-01

    Full Text Available Ribosome assembly in eukaryotic organisms requires more than 200 assembly factors to facilitate and coordinate rRNA transcription, processing, and folding with the binding of the ribosomal proteins. Many of these assembly factors bind and dissociate at defined times giving rise to discrete assembly intermediates, some of which have been partially characterized with regards to their protein and RNA composition. Here, we have analyzed the protein-protein interactions between the seven assembly factors bound to late cytoplasmic pre-40S ribosomes using recombinant proteins in binding assays. Our data show that these factors form two modules: one comprising Enp1 and the export adaptor Ltv1 near the beak structure, and the second comprising the kinase Rio2, the nuclease Nob1, and a regulatory RNA binding protein Dim2/Pno1 on the front of the head. The GTPase-like Tsr1 and the universally conserved methylase Dim1 are also peripherally connected to this second module. Additionally, in an effort to further define the locations for these essential proteins, we have analyzed the interactions between these assembly factors and six ribosomal proteins: Rps0, Rps3, Rps5, Rps14, Rps15 and Rps29. Together, these results and previous RNA-protein crosslinking data allow us to propose a model for the binding sites of these seven assembly factors. Furthermore, our data show that the essential kinase Rio2 is located at the center of the pre-ribosomal particle and interacts, directly or indirectly, with every other assembly factor, as well as three ribosomal proteins required for cytoplasmic 40S maturation. These data suggest that Rio2 could play a central role in regulating cytoplasmic maturation steps.

  7. Termination of protein synthesis.

    Science.gov (United States)

    Tuite, M F; Stansfield, I

    1994-05-01

    One of three mRNA codons--UAA, UAG and UGA--is used to signal to the elongating ribosome that translation should be terminated at this point. Upon the arrival of the stop codon at the ribosomal acceptor(A)-site, a protein release factor (RF) binds to the ribosome resulting in the peptidyl transferase centre of the ribosome switching to a hydrolytic function to remove the completed polypeptide chain from the peptidyl-tRNA bound at the adjacent ribosomal peptidyl(P)-site. In this review recent advances in our understanding of the mechanism of termination in the bacterium Escherichia coli will be summarised, paying particular attention to the roles of 16S ribosomal RNA and the release factors RF-1, RF-2 and RF-3 in stop codon recognition. Our understanding of the translation termination process in eukaryotes is much more rudimentary with the identity of the single eukaryotic release factor (eRF) still remaining elusive. Finally, several examples of how the termination mechanism can be subverted either to expand the genetic code (e.g. selenocysteine insertion at UGA codons) or to regulate the expression of mammalian retroviral or plant viral genomes will be discussed.

  8. Porcine prion protein amyloid.

    Science.gov (United States)

    Hammarström, Per; Nyström, Sofie

    2015-01-01

    Mammalian prions are composed of misfolded aggregated prion protein (PrP) with amyloid-like features. Prions are zoonotic disease agents that infect a wide variety of mammalian species including humans. Mammals and by-products thereof which are frequently encountered in daily life are most important for human health. It is established that bovine prions (BSE) can infect humans while there is no such evidence for any other prion susceptible species in the human food chain (sheep, goat, elk, deer) and largely prion resistant species (pig) or susceptible and resistant pets (cat and dogs, respectively). PrPs from these species have been characterized using biochemistry, biophysics and neurobiology. Recently we studied PrPs from several mammals in vitro and found evidence for generic amyloidogenicity as well as cross-seeding fibril formation activity of all PrPs on the human PrP sequence regardless if the original species was resistant or susceptible to prion disease. Porcine PrP amyloidogenicity was among the studied. Experimentally inoculated pigs as well as transgenic mouse lines overexpressing porcine PrP have, in the past, been used to investigate the possibility of prion transmission in pigs. The pig is a species with extraordinarily wide use within human daily life with over a billion pigs harvested for human consumption each year. Here we discuss the possibility that the largely prion disease resistant pig can be a clinically silent carrier of replicating prions.

  9. Identifying protein complexes based on density and modularity in protein-protein interaction network.

    Science.gov (United States)

    Ren, Jun; Wang, Jianxin; Li, Min; Wang, Lusheng

    2013-01-01

    Identifying protein complexes is crucial to understanding principles of cellular organization and functional mechanisms. As many evidences have indicated that the subgraphs with high density or with high modularity in PPI network usually correspond to protein complexes, protein complexes detection methods based on PPI network focused on subgraph's density or its modularity in PPI network. However, dense subgraphs may have low modularity and subgraph with high modularity may have low density, which results that protein complexes may be subgraphs with low modularity or with low density in the PPI network. As the density-based methods are difficult to mine protein complexes with low density, and the modularity-based methods are difficult to mine protein complexes with low modularity, both two methods have limitation for identifying protein complexes with various density and modularity. To identify protein complexes with various density and modularity, including those have low density but high modularity and those have low modularity but high density, we define a novel subgraph's fitness, fρ, as fρ= (density)(ρ*)(modularity)(1-ρ), and propose a novel algorithm, named LF_PIN, to identify protein complexes by expanding seed edges to subgraphs with the local maximum fitness value. Experimental results of LF-PIN in S.cerevisiae show that compared with the results of fitness equal to density (ρ = 1) or equal to modularity (ρ = 0), the LF-PIN identifies known protein complexes more effectively when the fitness value is decided by both density and modularity (0modularity. By considering both the density and the modularity, LF-PIN outperforms other protein complexes detection methods that only consider density or modularity, especially in identifying known protein complexes with low density or low modularity.

  10. Analysis of leukocyte membrane protein interactions using protein microarrays

    Directory of Open Access Journals (Sweden)

    Foster-Cuevas Mildred

    2005-03-01

    Full Text Available Abstract Background Protein microarrays represent an emerging class of proteomic tools to investigate multiple protein-protein interactions in parallel. A sufficient proportion of immobilized proteins must maintain an active conformation and an orientation that allows for the sensitive and specific detection of antibody and ligand binding. In order to establish protein array technology for the characterization of the weak interactions between leukocyte membrane proteins, we selected the human leukocyte membrane protein CD200 (OX2 and its cell surface receptor (hCD200R as a model system. As antibody-antigen reactions are generally of higher affinity than receptor-ligand binding, we first analyzed the reactivity of monoclonal antibodies (mAb to normal and mutant forms of immobilized CD200R. Results Fluorescently labelled mAb DX147, DX136 and OX108 were specifically reactive with immobilized recombinant hCD200R extracellular region, over a range of 0.1–40 μg ml-1 corresponding to a limit of sensitivity of 0.01–0.05 femtomol per spot. Orientating hCD200R using capture antibodies, showed that DX147 reacts with an epitope spatially distinct from the more closely related DX136 and OX108 epitopes. A panel of soluble recombinant proteins with mutations in hCD200R domain 1 produced by transiently transfected cells, was arrayed directly without purification and screened for binding to the three mAb. Several showed decreased binding to the blocking mAb DX136 and OX108, suggesting close proximity of these epitopes to the CD200 binding site. Binding of hCD200 to directly immobilized rat, mouse, and hCD200R was achieved with multimeric ligands, in the form of biotinylated-hCD200 coupled to FITC-labelled avidin coated beads. Conclusion We have achieved sensitive, specific and reproducible detection of immobilized CD200R with different antibodies and mapped antigenic epitopes for two mAb in the vicinity of the ligand binding site using protein microarrays

  11. Increasing Alfalfa Rumen Bypass Protein

    Science.gov (United States)

    Alfalfa has one of the highest crude protein contents among forage crops, but is is rapidly and extensively degraded by rumen microorganisms. To examine differential protein digestion, three distinct varieties of alfalfa, grown from single plants, were subjected to fermentation in the rumen of a ca...

  12. Protein: MPA3 [TP Atlas

    Lifescience Database Archive (English)

    Full Text Available MPA3 Neutrophil oxidase factors BEM1 SRO1 Bud emergence protein 1 Suppressor of RHO3 pro...tein 1 559292 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) 852499 P29366 2V6V, 1IPG, 2CZO, 1IP9 2RQW, 2KFK, 2RQV 20410294, 19451149 ...

  13. Teaching computers to fold proteins

    DEFF Research Database (Denmark)

    Winther, Ole; Krogh, Anders Stærmose

    2004-01-01

    A new general algorithm for optimization of potential functions for protein folding is introduced. It is based upon gradient optimization of the thermodynamic stability of native folds of a training set of proteins with known structure. The iterative update rule contains two thermodynamic average...

  14. Extraction of Proteins with ABS

    NARCIS (Netherlands)

    Desai, R.K.; Streefland, M.; Wijffels, R.H.; Eppink, M.H.M.

    2016-01-01

    Over the past years, there has been an increasing trend in research on the extraction and purification of proteins using aqueous biphasic systems (ABS) formed by polymers, e.g., polyethylene glycol (PEG). In general, when dealing with protein purification processes, it is essential to maintain their

  15. Protein Electrophoresis/Immunofixation Electrophoresis

    Science.gov (United States)

    ... High-sensitivity C-reactive Protein (hs-CRP) Histamine Histone Antibody HIV Antibody and HIV Antigen (p24) HIV ... online.com . Accessed May 2010. (© 1995–2010). Unit Code 80085: Electrophoresis, Protein, Serum. Mayo Clinic Mayo Medical ...

  16. Protein species as diagnostic markers

    NARCIS (Netherlands)

    Steffen, Pascal; Kwiatkowski, Marcel; Robertson, Wesley D.; Zarrine-Afsar, Mash; Deterra, Diana; Richter, Verena; Schlueter, Hartmut

    2016-01-01

    Many diseases are associated with protein species perturbations. A prominent example of an established diagnostic marker is the glycated protein species of hemoglobin, termed HbA1c. HbA1c concentration is increased in the blood of diabetes mellitus patients due to their poor control of blood glucose

  17. Protein: MPB4 [TP Atlas

    Lifescience Database Archive (English)

    Full Text Available MPB4 Sema3A signaling molecules DPYSL2 CRMP2, ULIP2 DPYSL2 Dihydropyrimidinase-related prote...in 2 Collapsin response mediator protein 2, N2A3, Unc-33-like phosphoprotein 2 9606 Homo sapiens Q16555 1808 2VM8, 2GSE 1808 Q16555 ...

  18. Protein: MPA3 [TP Atlas

    Lifescience Database Archive (English)

    Full Text Available MPA3 RACs RAC1 TC25 Rac1 Ras-related C3 botulinum toxin substrate 1 Cell migration-inducing gene 5 prote...in, Ras-like protein TC25, p21-Rac1 9606 Homo sapiens P63000 5879 3BJI, 1FOE, 3SU8, 1RY

  19. Protein: FBA4 [TP Atlas

    Lifescience Database Archive (English)

    Full Text Available FBA4 general transcription factor TFIIE SND1 TDRD11 SND1 Staphylococcal nuclease domain-containing prote...in 1 100 kDa coactivator, EBNA2 coactivator p100, Tudor domain-containing protein 11, p

  20. Protein: FEA3 [TP Atlas

    Lifescience Database Archive (English)

    Full Text Available FEA3 AREB pathway: Signaling proteins SRK2I 41K, OSKL2, SNRK2.3 Serine/threonine-prote...in kinase SRK2I OST1-kinase-like 2, Protein ATHPROKIN B, SNF1-related kinase 2.3 3702 Arabidopsis thaliana 836822 Q39193 3UC3 19880399 ...

  1. Characterization of carrot arabinogalactan proteins

    NARCIS (Netherlands)

    Immerzeel, P.

    2005-01-01

    Arabinogalactan proteins (AGPs) are highly glycosylated proteins. Besides galactose and arabinose the carbohydrate part of AGPs contains other neutral sugars and uronic acids. AGPs are widely distributed in the plant kingdom, probably occurring in all tissues of every plant. Yariv phenylglycoside is

  2. Use of Protein Folding Reagents

    Science.gov (United States)

    Wingfield, Paul T.

    2016-01-01

    The reagents and methods for purification of the most commonly used denaturants guanidine hydrochloride (guanidine-HCl) and urea are described. Other protein denaturants and reagents used to fold proteins are briefly mentioned. Sulfhydryl reagents (reducing agents) and “oxido-shuffling” (or oxidative regeneration) systems are also described. PMID:18429069

  3. PROTEIN CRYSTALS AND THEIR STABILITY

    NARCIS (Netherlands)

    DRENTH, J; HAAS, C

    Assuming a simple model, it can be derived that the free energy difference between protein molecules in the crystalline state and in a saturated solution is determined by C(sol)/C(cr), in which C(sol) is the concentration of the protein in the solution and C(cr) that in the crystal. It is estimated

  4. Fluorescent Proteins for Flow Cytometry.

    Science.gov (United States)

    Hawley, Teresa S; Hawley, Robert G; Telford, William G

    2017-04-03

    Fluorescent proteins have become standard tools for cell and molecular biologists. The color palette of fluorescent proteins spans the ultraviolet, visible, and near-infrared spectrum. Utility of fluorescent proteins has been greatly facilitated by the availability of compact and affordable solid state lasers capable of providing various excitation wavelengths. In theory, the plethora of fluorescent proteins and lasers make it easy to detect multiple fluorescent proteins simultaneously. However, in practice, heavy spectral overlap due to broad excitation and emission spectra presents a challenge. In conventional flow cytometry, careful selection of excitation wavelengths and detection filters is necessary. Spectral flow cytometry, an emerging methodology that is not confined by the "one color, one detector" paradigm, shows promise in the facile detection of multiple fluorescent proteins. This chapter provides a synopsis of fluorescent protein development, a list of commonly used fluorescent proteins, some practical considerations and strategies for detection, and examples of applications. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

  5. Protein: MPA1 [TP Atlas

    Lifescience Database Archive (English)

    Full Text Available ning protein 2 Viperin, Virus inhibitory protein, endoplasmic reticulum-associated, interferon-inducible 10090 Mus musculus 58185 Q8CBB9 21435586 ... ...MPA1 TLR signaling molecules Rsad2 Vig1 Radical S-adenosyl methionine domain-contai

  6. Adjustable chain trees for proteins

    DEFF Research Database (Denmark)

    Winter, Pawel; Fonseca, Rasmus

    2012-01-01

    A chain tree is a data structure for changing protein conformations. It enables very fast detection of clashes and free energy potential calculations. A modified version of chain trees that adjust themselves to the changing conformations of folding proteins is introduced. This results in much...

  7. Cohesion and Adhesion with Proteins

    Science.gov (United States)

    Charles R. Frihart

    2016-01-01

    With increasing interest in bio-based adhesives, research on proteins has expanded because historically they have been used by both nature and humans as adhesives. A wide variety of proteins have been used as wood adhesives. Ancient Egyptians most likely used collagens tobond veneer to wood furniture, then came casein (milk), blood, fish scales, and soy adhesives, with...

  8. Protein folding on a chip

    CERN Multimedia

    2004-01-01

    "Scientists at the U.S. Department of Energy's Brookhaven National Laboratory are proposing to use a super- computer originally developed to simulate elementary particles in high- energy physics to help determine the structures and functions of proteins, including, for example, the 30,000 or so proteins encoded by the human genome" (1 page)

  9. Protein: FBA5 [TP Atlas

    Lifescience Database Archive (English)

    Full Text Available FBA5 VSOP(voltage sensor-only protein1) Hvcn1 Bts, Vsop Voltage-gated hydrogen chan...nel 1 Hydrogen voltage-gated channel 1, Voltage sensor domain-only protein 10090 Mus musculus 74096 Q3U2S8 Q3U2S8 20018719 ...

  10. Protein: FBA5 [TP Atlas

    Lifescience Database Archive (English)

    Full Text Available FBA5 VSOP(voltage sensor-only protein1) HVCN1 VSOP, VSX1 Voltage-gated hydrogen cha...nnel 1 Hydrogen voltage-gated channel 1, Voltage sensor domain-only protein 7719 Ciona intestinalis 778897 Q1JV40 ...

  11. Protein: FBA5 [TP Atlas

    Lifescience Database Archive (English)

    Full Text Available FBA5 VSOP(voltage sensor-only protein1) HVCN1 VSOP Voltage-gated hydrogen channel 1... Hydrogen voltage-gated channel 1, Voltage sensor domain-only protein 9606 Homo sapiens Q96D96 84329 3A2A 18583477, 19285483 ...

  12. Direct electrochemistry of redox proteins

    NARCIS (Netherlands)

    Heering, H.A.

    1995-01-01

    The goal of the project was to obtain more detailed insight in interactions between redox proteins and solid electrodes and the mechanisms of electron transfer. In addition to this, the influence of the protein environment on the redox properties of the active site and the possible

  13. Protein Electrochemistry: Questions and Answers.

    Science.gov (United States)

    Fourmond, V; Léger, C

    This chapter presents the fundamentals of electrochemistry in the context of protein electrochemistry. We discuss redox proteins and enzymes that are not photoactive. Of course, the principles described herein also apply to photobioelectrochemistry, as discussed in later chapters of this book. Depending on which experiment is considered, electron transfer between proteins and electrodes can be either direct or mediated, and achieved in a variety of configurations: with the protein and/or the mediator free to diffuse in solution, immobilized in a thick, hydrated film, or adsorbed as a sub-monolayer on the electrode. The experiments can be performed with the goal to study the protein or to use it. Here emphasis is on mechanistic studies, which are easier in the configuration where the protein is adsorbed and electron transfer is direct, but we also explain the interpretation of signals obtained when diffusion processes affect the response.This chapter is organized as a series of responses to questions. Questions 1-5 are related to the basics of electrochemistry: what does "potential" or "current" mean, what does an electrochemical set-up look like? Questions 6-9 are related to the distinction between adsorbed and diffusive redox species. The answers to questions 10-13 explain the interpretation of slow and fast scan voltammetry with redox proteins. Questions 14-19 deal with catalytic electrochemistry, when the protein studied is actually an enzyme. Questions 20, 21 and 22 are general.

  14. Prions: Beyond a Single Protein.

    Science.gov (United States)

    Das, Alvin S; Zou, Wen-Quan

    2016-07-01

    Since the term protein was first coined in 1838 and protein was discovered to be the essential component of fibrin and albumin, all cellular proteins were presumed to play beneficial roles in plants and mammals. However, in 1967, Griffith proposed that proteins could be infectious pathogens and postulated their involvement in scrapie, a universally fatal transmissible spongiform encephalopathy in goats and sheep. Nevertheless, this novel hypothesis had not been evidenced until 1982, when Prusiner and coworkers purified infectious particles from scrapie-infected hamster brains and demonstrated that they consisted of a specific protein that he called a "prion." Unprecedentedly, the infectious prion pathogen is actually derived from its endogenous cellular form in the central nervous system. Unlike other infectious agents, such as bacteria, viruses, and fungi, prions do not contain genetic materials such as DNA or RNA. The unique traits and genetic information of prions are believed to be encoded within the conformational structure and posttranslational modifications of the proteins. Remarkably, prion-like behavior has been recently observed in other cellular proteins-not only in pathogenic roles but also serving physiological functions. The significance of these fascinating developments in prion biology is far beyond the scope of a single cellular protein and its related disease. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  15. Protein release from collagen matrices.

    Science.gov (United States)

    Sano; Hojo; Maeda; Fujioka

    1998-05-04

    The effective delivery of protein drugs is an important research subject in the field of pharmacology, and to prolong the effect of protein drugs, many studies are being conducted to control the release of proteins from various carrier materials. Collagen is one of the most useful candidates for this purpose, and many studies have been reported; pharmaceutical formulations containing collagen in gel, film and sponge form are used to incorporate low-molecular-weight compounds such as antibiotics and carcinostatics, and the release of these compounds is controlled by the concentration of the gel as well as the shape and degree of crosslinking of the matrix. However, it is still difficult to retain protein drugs in the collagen. In this article, we report on the controlled release of protein drugs using collagen which exhibits good biocompatibility as a carrier, focusing on a new drug delivery system, the Minipellet, which we have developed.

  16. Soy protein modification: A review

    Directory of Open Access Journals (Sweden)

    Barać Miroljub B.

    2004-01-01

    Full Text Available Soy protein products such as flour, concentrates and isolates are used in food formulation because of their functionality, nutritional value and low cost. To obtain their optimal nutritive and functional properties as well as desirable flavor different treatments are used. Soybean proteins can be modified by physical, chemical and enzymatic treatments. Different thermal treatments are most commonly used, while the most appropriate way of modifying soy proteins from the standpoint of safety is their limited proteolysis. These treatments cause physical and chemical changes that affect their functional properties. This review discusses three principal methods used for modification of soy protein products, their effects on dominant soy protein properties and some biologically active compounds.

  17. Validation of protein carbonyl measurement

    DEFF Research Database (Denmark)

    Augustyniak, Edyta; Adam, Aisha; Wojdyla, Katarzyna

    2015-01-01

    Protein carbonyls are widely analysed as a measure of protein oxidation. Several different methods exist for their determination. A previous study had described orders of magnitude variance that existed when protein carbonyls were analysed in a single laboratory by ELISA using different commercial...... kits. We have further explored the potential causes of variance in carbonyl analysis in a ring study. A soluble protein fraction was prepared from rat liver and exposed to 0, 5 and 15min of UV irradiation. Lyophilised preparations were distributed to six different laboratories that routinely undertook...... and control liver proteins, only seven were common in all three liver preparations. Lysine and arginine residues modified by carbonyls are likely to be resistant to tryptic proteolysis. Use of a cocktail of proteases may increase the recovery of oxidised peptides. In conclusion, standardisation is critical...

  18. Structural Genomics of Protein Phosphatases

    Energy Technology Data Exchange (ETDEWEB)

    Almo,S.; Bonanno, J.; Sauder, J.; Emtage, S.; Dilorenzo, T.; Malashkevich, V.; Wasserman, S.; Swaminathan, S.; Eswaramoorthy, S.; et al

    2007-01-01

    The New York SGX Research Center for Structural Genomics (NYSGXRC) of the NIGMS Protein Structure Initiative (PSI) has applied its high-throughput X-ray crystallographic structure determination platform to systematic studies of all human protein phosphatases and protein phosphatases from biomedically-relevant pathogens. To date, the NYSGXRC has determined structures of 21 distinct protein phosphatases: 14 from human, 2 from mouse, 2 from the pathogen Toxoplasma gondii, 1 from Trypanosoma brucei, the parasite responsible for African sleeping sickness, and 2 from the principal mosquito vector of malaria in Africa, Anopheles gambiae. These structures provide insights into both normal and pathophysiologic processes, including transcriptional regulation, regulation of major signaling pathways, neural development, and type 1 diabetes. In conjunction with the contributions of other international structural genomics consortia, these efforts promise to provide an unprecedented database and materials repository for structure-guided experimental and computational discovery of inhibitors for all classes of protein phosphatases.

  19. Bacterial Protein-Tyrosine Kinases

    DEFF Research Database (Denmark)

    Shi, Lei; Kobir, Ahasanul; Jers, Carsten

    2010-01-01

    enzymes that are unique in exploiting the ATP/GTP-binding Walker motif to catalyze phosphorylation of protein tyrosine residues. Characterized for the first time only a decade ago, BY-kinases have now come to the fore. Important regulatory roles have been linked with these enzymes, via their involvement......Bacteria and Eukarya share essentially the same family of protein-serine/threonine kinases, also known as the Hanks-type kinases. However, when it comes to protein-tyrosine phosphorylation, bacteria seem to have gone their own way. Bacterial protein-tyrosine kinases (BY-kinases) are bacterial...... in exopolysaccharide production, virulence, DNA metabolism, stress response and other key functions of the bacterial cell. BY-kinases act through autophosphorylation (mainly in exopolysaccharide production) and phosphorylation of other proteins, which have in most cases been shown to be activated by tyrosine...

  20. Metabolism of biologics: biotherapeutic proteins.

    Science.gov (United States)

    Hamuro, Lora L; Kishnani, Narendra S

    2012-01-01

    Recombinant therapeutic protein drugs have now been in clinical use for nearly three decades and have advanced considerably in complexity over this time period. Regulatory approvals of some early pioneering protein drugs did not require characterization of metabolism, but more recently regulatory expectations and guidance have appropriately evolved. Sponsors may now be expected to investigate metabolism of newer biologics as the structural complexity of proteins has increased markedly, particularly with the introduction of conjugated and modified proteins. This review discusses the value and need for metabolite characterization of some therapeutic proteins by presenting select examples. Regulatory expectations will undoubtedly evolve further with the development of other novel macromolecular biologic therapeutics based on modified nucleic acids, novel conjugated lipids and polysaccharides.