Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Assembly of the three neuronal membrane proteins synaptobrevin, syntaxin, and SNAP-25 is thought to be one of the key steps in mediating exocytosis of synaptic vesicles. In vivo and in vitro, these proteins form a tight complex. Assembly is associated with a large increase in alpha-helical content, suggesting that major structural and conformational changes are associated with the assembly reaction. Limited proteolysis by trypsin, chymotrypsin, and proteinase K of the ternary complex formed from recombinant proteins lacking their membrane anchors revealed a SDS-resistant minimal core. The components of this core complex were purified and characterized by N-terminal sequencing and mass spectrometry. They include a slightly shortened synaptobrevin fragment, C- and N-terminal fragments of SNAP-25, and a C-terminal fragment of syntaxin that is slightly larger than the previously characterized H3 domain. Recombinant proteins corresponding to these fragments are sufficient for assembly and disassembly. In addition, each of the two SNAP-25 fragments can individually form complexes with syntaxin and synaptobrevin, suggesting that they both contribute to the assembly of the SNARE complex. Upon complex assembly, a large increase in alpha-helical content is observed along with a significantly increased melting temperature (Tm). Like the full-length complex, the minimal complex tends to form an oligomeric species; global analysis of equilibrium ultracentrifugation data suggests a monomer-trimer equilibrium exists. These conserved biophysical properties may thus be of fundamental importance in the mechanism of membrane fusion.
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PMID:Identification of a minimal core of the synaptic SNARE complex sufficient for reversible assembly and disassembly. 967 3

Syncollin is a pancreatic zymogen granule protein that was isolated through its ability to bind to syntaxin. Here we show that syncollin has a cleavable signal sequence and can be removed from granule membranes by washing with sodium carbonate. When membranes were subjected to Triton X-114 partitioning, syncollin was found predominantly in the aqueous phase, indicating that it is not sufficiently hydrophobic to be embedded in the membrane. Syncollin has intramolecular disulfide bonds and was accessible to water-soluble cross-linking and biotinylating reagents only when granules were lysed by sonication. These results indicate that syncollin is tightly bound to the luminal surface of the granule membrane. In situ, syncollin was resistant to proteases such as trypsin. When granule membranes were solubilized in ionic detergents such as deoxycholate, this trypsin resistance was maintained, and syncollin migrated on sucrose density gradients as a large (150 kDa) protein. In contrast, in non-ionic detergents such as Triton X-100, syncollin became partially sensitive to trypsin and behaved as a monomer. Syncollin in alkaline extracts of granule membranes was also monomeric. However, reduction of the pH regenerated the oligomeric form, which was insoluble. We conclude that syncollin exists as a homo-oligomer and that its ability to self-associate can be reversibly modulated via changes in pH. In light of our findings, we reassess the likely role of syncollin in the pancreatic acinar cell.
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PMID:Analysis of the association of syncollin with the membrane of the pancreatic zymogen granule. 1075 42

The changes that SNAREs undergo during exocytosis were studied in permeabilised chromaffin cells treated with Ca(2+), MgATP or botulinum neurotoxin A. High-resolution 2D SDS-PAGE revealed multiple SDS-resistant SNARE complexes having a wide range of sizes and in which SNAP-25 and syntaxin predominate over synaptobrevin. Their formation increased upon Ca(2+)-stimulated exocytosis; notably, the 2D protocol proved much superior to 1D SDS-PAGE for the detection of large complexes and revealed that for forms with relative molecular mass greater than 100,000 stimulated induction was more significant than for smaller species. MgATP enhanced Ca(2+)-triggered catecholamine release but reduced the content of complexes. By contrast, botulinum neurotoxin type A inhibited exocytosis and altered the stoichiometry of the SNAP-25:syntaxin binary association, without lowering its abundance. The individual SNAREs were protected against trypsin proteolysis to varying extents in binary and ternary complexes of different sizes, suggestive of distinct folding intermediates. Our data suggest that Ca(2+) triggers an early stage of SNARE complex formation causing an accumulation of partially folded intermediates, especially of binary forms, as well as their maturation into smaller, more protease resistant states. In addition, botulinum neurotoxin A inhibits exocytosis by perturbing the syntaxin:SNAP-25 ratio in binary intermediates.
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PMID:Multiple forms of SNARE complexes in exocytosis from chromaffin cells: effects of Ca(2+), MgATP and botulinum toxin type A. 1186 72

Ca2+-triggered catecholamine exocytosis from chromaffin cells involves SNAP-25, synaptobrevin and syntaxin (known as SNAREs). Synaptotagmin I has been implicated as the Ca2+-sensor because it binds Ca2+, and this enhances its binding to syntaxin, SNAP-25 and phospholipids in vitro. However, most of these interactions are only mediated by [Ca2+]i two orders of magnitude higher than that needed to elicit secretion. Thus, the Ca2+ sensitivities of synaptotagmin I and the other SNAREs were quantified in situ. Secretion elicited from permeabilised cells by microM Ca2+ was accompanied, with almost identical Ca2+ dependencies, by changes in synaptotagmin I, SNAP-25, syntaxin and synaptobrevin that rendered them less susceptible to trypsin. The majority of the trypsin-resistant SNAREs were not associated with SDS-resistant complexes. None of these proteins acquired trypsin resistance in cells rendered incompetent for exocytosis by run-down. Removal of nine C-terminal residues from SNAP-25 by botulinum toxin A reduced both exocytosis and the SNAREs' acquisition of trypsin resistance but did not alter the Ca2+ sensitivity, except for synaptotagmin I. Even after synaptobrevin had been cleaved by botulinum toxin B, all the other proteins still responded to Ca2+. These data support a model whereby Ca2+ is sensed, probably by synaptotagmin I, and the signal passed to syntaxin and SNAP-25 before they interact with synaptobrevin.
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PMID:Ca2+-induced changes in SNAREs and synaptotagmin I correlate with triggered exocytosis from chromaffin cells: insights gleaned into the signal transduction using trypsin and botulinum toxins. 1207 69

Protein phosphorylation is a crucial post-translational modification mechanism in the regulation of synaptic organization and function. Here, we analyzed synaptosome fractions from human cerebral cortex obtained during therapeutic surgery. To minimize changes in the phosphorylation state of proteins, the tissue was homogenized within two minutes of excision. Synaptosomal proteins were digested with trypsin and phosphopeptides were isolated by immobilized metal affinity chromatography and analyzed by liquid chromatography and tandem mass spectrometry. The method allowed the detection of residues on synaptic proteins that were presumably phosphorylated in the intact cell, including synapsin 1, syntaxin 1, and SNIP, PSD-93, NCAM, GABA-B receptor, chaperone molecules, and protein kinases. Some of the residues identified are the same or homologous to sites that had been previously described to be phosphorylated in mammals whereas others appear to be novel sites which, to our knowledge, have not been reported previously. The study shows that new phosphoproteomic strategies can be used to analyze subcellular fractions from small amounts of tissue for the identification of phosphorylated residues for research and potentially for diagnostic purposes.
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PMID:Phosphoproteomic analysis of synaptosomes from human cerebral cortex. 1582 5

Vasopressin acts on renal collecting duct cells to stimulate translocation of aquaporin-2 (AQP2)-containing membrane vesicles from throughout the cytoplasm to the apical region. The vesicles fuse with the plasma membrane to increase water permeability. To identify the intracellular membrane compartments that contain AQP2, we carried out LC-MS/MS-based proteomic analysis of immunoisolated AQP2-containing intracellular vesicles from rat inner medullary collecting duct. Immunogold electron microscopy and immunoblotting confirmed heavy AQP2 labeling of immunoisolated vesicles. Vesicle proteins were separated by SDS-PAGE followed by in-gel trypsin digestion in consecutive gel slices and identification by LC-MS/MS. Identification of Rab GTPases 4, 5, 18, and 21 (associated with early endosomes); Rab7 (late endosomes); and Rab11 and Rab25 (recycling endosomes) indicate that a substantial fraction of intracellular AQP2 is present in endosomal compartments. In addition, several endosome-associated SNARE proteins were identified including syntaxin-7, syntaxin-12, syntaxin-13, Vti1a, vesicle-associated membrane protein 2, and vesicle-associated membrane protein 3. Rab3 was not found, however, either by mass spectrometry or immunoblotting, suggesting a relative lack of AQP2 in secretory vesicles. Additionally, we identified markers of the trans-Golgi network, components of the exocyst complex, and several motor proteins including myosin 1C, non-muscle myosins IIA and IIB, myosin VI, and myosin IXB. Beyond this, identification of multiple endoplasmic reticulum-resident proteins and ribosomal proteins indicated that a substantial fraction of intracellular AQP2 is present in rough endoplasmic reticulum. These results show that AQP2-containing vesicles are heterogeneous and that intracellular AQP2 resides chiefly in endosomes, trans-Golgi network, and rough endoplasmic reticulum.
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PMID:Large scale protein identification in intracellular aquaporin-2 vesicles from renal inner medullary collecting duct. 1590 45

The first evidence of autoproteolytic activity of the approximately 50-kDa light chain of the clostridial neurotoxins (NT) is traceable to the observations that the light chains of botulinum NT serotypes A and E, separated from their approximately 100-kDa heavy chain conjugate, were found cleaved at the amino side of Tyr250 and Arg244, respectively [DasGupta and Foley (1989). Biochimie 71: 1183-1200]. Specific cleavages of the recombinant light chain of NT type A, including at Tyr249-Tyr250, firmly established that the cleavages reported earlier were due to autoproteolysis [Ahmed et al. (2001). J. Protein Chem. 20: 221-231; Ahmed et al. (2003). Biochemistry 42:12539-12549] and not by contaminating proteases or non-enzymatic. We now report many cleavages in the NT types A, B and E and also in their separated light and heavy chains, and identification of several of the peptide bonds cleaved. None of the identified cleaved bonds (-P1-P1' -) in one serotype (except Asp-Pro) was found common in other serotypes or cleaved within itself at a second site. After separation from the heavy chain self-cleavages of the light chains of type A, B and E at Tyr249-Tyr250, Gln258-Ser259 and Ile243-Arg244, respectively indicate an intriguing feature (in the aligned sequences these bonds of type A and B are 2 and type A and E are 4 peptide bonds apart) that may have some role in the NT's structure-function relationship yet to be understood. We point out that autoproteolysis of a single peptide bond (Phe418-Thr419 or Phe422-Glu423) in NT type A reported by Ahmed et al. (2001) can potentially generate proteolytically active light chain freed of the heavy chain; this is an efficient pathway, that by-passes nicking by a trypsin-like protease(s) inside the intrachain disulfide bridge and its reductive cleavage. We offer probable explanations for the observed cleavages such as acid- and metal-mediated (non-catalytic and non-stoichiometric) reactions in addition to autoproteolysis but cannot predict which mechanism(s) of cleavage occur or prevail following NT's entry in the body as poison or therapeutic agent. The metal chelator O-phenanthroline (above critical miceller concentration) in the presence of dithiothreitol cleaved type E NT at limited sites generating discrete 114-, 87-, 49-, 42-, and 31-kDa fragments but degraded NTs type A and B extensively. The limited cleavage of type E NT was dependent on the presence of metal ion(s) bound to the protein and its native (urea sensitive) conformation. The self-cleavage of the NTs at specific sites prompted us to search for specific binding sites on the NTs analogous to SNARE-motifs-the 9-residuelong motifs present on the NT's natural substrates (SNAP-25, syntaxin, VAMP/synaptobrevin); such putative binding motifs (sites) noted on all clostridial NTs are reported here. Their relationship to the observed autoproteolysis remains to be determined experimentally. The dinucleotide NAD(+)/NADH associated with the NTs type A, B and E (2-3 NADH per protein molecule) via their H-chains, and a portion of the H-chain (toward the C-terminus) appears to exhibit limited amino acid sequence homology with lactate dehydrogenase-a representative NAD(+)/NADH binding protein.
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PMID:Botulinum neurotoxin types A, B, and E: fragmentations by autoproteolysis and other mechanisms including by O-phenanthroline-dithiothreitol, and association of the dinucleotides NAD(+)/NADH with the heavy chain of the three neurotoxins. 1632 41

The inner medullary collecting duct (IMCD) is an important site of vasopressin-regulated water and urea transport. Here we have used protein mass spectrometry to investigate the proteome of the IMCD cell and how it is altered in response to long-term vasopressin administration in rats. IMCDs were isolated from inner medullas of rats, and IMCD proteins were identified by liquid chromatography/tandem mass spectrometry (LC-MS/MS). We present a WWW-based "IMCD Proteome Database" containing all IMCD proteins identified in this study (n = 704) and prior MS-based identification studies (n = 301). We used the isotope-coded affinity tag (ICAT) technique to identify IMCD proteins that change in abundance in response to vasopressin. Vasopressin analog (dDAVP) or vehicle was infused subcutaneously in Brattleboro rats for 3 days, and IMCDs were isolated for proteomic analysis. dDAVP and control samples were labeled with different cleavable ICAT reagents (mass difference 9 amu) and mixed. This was followed by one-dimensional SDS-PAGE separation, in-gel trypsin digestion, biotin-avidin affinity purification, and LC-MS/MS identification and quantification. Responses to vasopressin for a total of 165 proteins were quantified. Quantification, based on semiquantitative immunoblotting of 16 proteins for which antibodies were available, showed a high degree of correlation with ICAT results. In addition to aquaporin-2 and gamma-epithelial Na channel (gamma-ENaC), five of the immunoblotted proteins were substantially altered in abundance in response to dDAVP, viz., syntaxin-7, Rap1, GAPDH, heat shock protein (HSP)70, and cathepsin D. A 28-protein vasopressin signaling network was constructed using literature-based network analysis software focusing on the newly identified proteins, providing several new hypotheses for future studies.
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PMID:High-throughput identification of IMCD proteins using LC-MS/MS. 1644 82

SNARE proteins direct membrane fusion events required for platelet granule secretion. These proteins are oriented in cell membranes such that most of the protein resides in a cytosolic compartment. Evaluation of SNARE protein localization in activated platelets using immunonanogold staining and electron microscopy, however, demonstrated expression of SNAP-23 and syntaxin-2 on the extracellular surface of the platelet plasma membrane. Flow cytometry of intact platelets confirmed trypsin-sensitive SNAP-23 and syntaxin-2 localization to the extracellular surface of the plasma membrane. Acyl-protein thioesterase 1 and botulinum toxin C light chain released SNAP-23 and syntaxin-2, respectively, from the surface of intact platelets. When resting platelets were incubated with both acyl-protein thioesterase 1 and botulinum toxin C light chain, a complex that included both SNAP-23 and syntaxin-2 was detected in supernatants, indicating that extracellular SNARE proteins retain their ability to bind one another. These observations represent the first description of SNARE proteins on the extracellular surface of a cell.
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PMID:SNAP-23 and syntaxin-2 localize to the extracellular surface of the platelet plasma membrane. 1791 51

The reinforcing effects and long-term consequences of cocaine self-administration have been associated with brain regions of the mesolimbic dopamine pathway, namely the nucleus accumbens (NAc). Studies of cocaine-induced biochemical adaptations in rodent models have advanced our knowledge; however, unbiased detailed assessments of intracellular alterations in the primate brain are scarce, yet essential, to develop a comprehensive understanding of cocaine addiction. To this end, two-dimensional difference in gel electrophoresis (2D-DIGE) was used to compare changes in cytosolic protein abundance in the NAc between rhesus monkeys self-administering cocaine and controls. Following image normalization, spots with significantly differential image intensities (P<0.05) were identified, excised, trypsin digested and analyzed by matrix-assisted laser-desorption ionization time-of-flight time-of-flight (MALDI-TOF-TOF). In total, 1098 spots were subjected to statistical analysis with 22 spots found to be differentially abundant of which 18 proteins were positively identified by mass spectrometry. In addition, approximately 1000 protein spots were constitutively expressed of which 21 proteins were positively identified by mass spectrometry. Increased levels of proteins in the cocaine-exposed monkeys include glial fibrillary acidic protein, syntaxin-binding protein 3, protein kinase C isoform, adenylate kinase isoenzyme 5 and mitochondrial-related proteins, whereas decreased levels of proteins included beta-soluble N-ethylmaleimide-sensitive factor attachment protein and neural and non-neural enolase. Using a complimentary proteomics approach, the differential expression of phosphorylated proteins in the cytosolic fraction of these subjects was examined. Two-dimensional gel electrophoresis (2DGE) was followed by gel staining with Pro-Q Diamond phosphoprotein gel stain, enabling differentiation of approximately 150 phosphoprotein spots between the groups. Following excision and trypsin digestions, MALDI-TOF-TOF was used to confirm the identity of 15 cocaine-altered phosphoproteins. Significant increased levels were detected for gamma-aminobutyric acid type A receptor-associated protein 1, 14-3-3 gamma-protein, glutathione S-transferase and brain-type aldolase, whereas significant decreases were observed for beta-actin, Rab GDP-dissociation inhibitor, guanine deaminase, peroxiredoxin 2 isoform b and several mitochondrial proteins. Results from these studies indicate coordinated dysregulation of proteins related to cell structure, signaling, metabolism and mitochondrial function. These data extend and compliment previous studies of cocaine-induced biochemical alterations in human postmortem brain tissue, using an animal model that closely recapitulates the human condition and provide new insight into the molecular basis of the disease and potential targets for pharmacotherapeutic intervention.
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PMID:Integrative proteomic analysis of the nucleus accumbens in rhesus monkeys following cocaine self-administration. 1850 25


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