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

---

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

C1-inhibitor is a member of the serpin family of proteinase inhibitors and is an important inhibitor of complement and contact system proteinases. The native protein has the characteristic serpin feature of being in a kinetically trapped metastable state rather than in the most stable state it could adopt. A consequence of this is that it readily forms loop-sheet dimers and polymers, by a mechanism believed to be the same as observed with other serpins. An unusual feature of C1-inhibitor is that it has a unique amino-terminal domain, of unknown function, held to the serpin domain by two disulfide bonds not found in other serpins. We report here that reduction of these bonds by DTT, causes a conformational change such that the reactive center loop inserts into beta-sheet A. This form of C1-inhibitor is less stable to heat and urea than the native protein, and is more susceptible to extensive degradation by trypsin. These data show that the disulfide bonds in C1-inhibitor are required for the protein to be stabilized in the metastable state with the reactive center loop expelled from beta-sheet A.
...
PMID:The native metastable fold of C1-inhibitor is stabilized by disulfide bonds. 1100 79

Determination of the structures of fibroblast growth factors and interleukin-1s has previously revealed that they both adopt a beta-trefoil fold, similar to those found in Kunitz soybean trypsin inhibitors, ricin-like toxins, plant agglutinins and hisactophilin. These families possess distinct functions and occur in different subcellular localisations, and they appear to lack significant similarities in their sequences, ligands and modes of ligand binding. We have analysed the significance of sequence identities observed after structure alignment and provide statistical evidence that these beta-trefoil proteins are all homologues, having arisen from a common ancestor. In addition, we have explored the sequence space of all beta-trefoil proteins and have determined that the actin-binding proteins fascins, and other proteins of unknown function, are beta-trefoil family homologues. Unlike other beta-trefoil proteins, the triplicated repeats in each of the four beta-trefoil domains of fascins are significantly similar in sequence. This hints at how the beta-trefoil fold arose from the duplication of an ancestral gene encoding a homotrimeric single-repeat protein. The combined analysis of structure and sequence databases for detecting significant similarities is suggested as a highly sensitive approach to determining the common ancestry of extremely divergent homologues.
...
PMID:Identification of distant homologues of fibroblast growth factors suggests a common ancestor for all beta-trefoil proteins. 1118 73

Time-course analysis of root protein profiles was studied by two-dimensional gel electrophoresis and silver staining in the model plant Medicago truncatula, inoculated either with the arbuscular mycorrhizal fungus Glomus mosseae or with the nitrogen fixing bacterium Sinorhizobium meliloti. Protein modifications in relation to the development of both symbioses included down- and upregulations, as well as newly induced polypeptides. Matrix assisted laser desorption/ionization-time of flight-mass spectrometry after trypsin digestion clearly identified one polypeptide induced in nodulated roots as a M. truncatula leghemoglobin. Internal sequencing with a quadrupole time-of-flight mass spectrometer and database searches confirmed the induction of proteins previously described in root symbioses, and revealed the implication of other proteins. In nodulated roots, one polypeptide was identified as an elongation factor Tu from S. meliloti, while another one could not be assigned a function. In mycorrhizal roots, analyzed proteins also included a protein of unknown function, as well as a glutathione-S-transferase, a fucosidase, a myosin-like protein, a serine hydroxymethyltransferase and a cytochrome-c-oxidase. These results emphasize the usefulness of proteome analysis in identifying molecular events occurring in plant root symbioses.
...
PMID:Proteome analysis and identification of symbiosis-related proteins from Medicago truncatula Gaertn. by two-dimensional electrophoresis and mass spectrometry. 1182 12

Syncollin is a small protein that is abundantly expressed in pancreatic acinar cells and that is tightly associated with the lumenal side of the zymogen granule membrane. To shed light on the hitherto unknown function of syncollin, we have generated syncollin-deficient mice. The mice are viable and show a normal pancreatic morphology as well as normal release kinetics in response to secretagogue stimulation. Although syncollin is highly enriched in zymogen granules, no change was found in the overall protein content and in the levels of chymotrypsin, trypsin, and amylase. However, syncollin-deficient mice reacted to caerulein hyperstimulation with a more severe pancreatitis. Furthermore, the rates of both protein synthesis and intracellular transport of secretory proteins were reduced. We conclude that syncollin plays a role in maturation and/or concentration of zymogens in zymogen granules.
...
PMID:Loss of the zymogen granule protein syncollin affects pancreatic protein synthesis and transport but not secretion. 1183 20

Purified rice (Oryza sativa) mitochondrial proteins have been arrayed by isoelectric focusing/polyacrylamide gel electrophoresis (PAGE), by blue-native (BN) PAGE, and by reverse-phase high-performance liquid chromatography (LC) separation (LC-mass spectrometry [MS]). From these protein arrays, we have identified a range of rice mitochondrial proteins, including hydrophilic/hydrophobic proteins (grand average of hydropathicity = -1.27 to +0.84), highly basic and acid proteins (isoelectric point = 4.0-12.5), and proteins over a large molecular mass range (6.7-252 kD), using proteomic approaches. BN PAGE provided a detailed picture of electron transport chain protein complexes. A total of 232 protein spots from isoelectric focusing/PAGE and BN PAGE separations were excised, trypsin digested, and analyzed by tandem MS (MS/MS). Using this dataset, 149 of the protein spots (the products of 91 nonredundant genes) were identified by searching translated rice open reading frames from genomic sequence and six-frame translated rice expressed sequence tags. Sequence comparison allowed us to assign functions to a subset of 85 proteins, including many of the major function categories expected for this organelle. A further six spots were matched to rice sequences for which no specific function has yet been determined. Complete digestion of mitochondrial proteins with trypsin yielded a peptide mixture that was analyzed directly by reverse-phase LC via organic solvent elution from a C-18 column (LC-MS). These data yielded 170 MS/MS spectra that matched 72 sequence entries from open reading frame and expressed sequence tag databases. Forty-five of these were obtained using LC-MS alone, whereas 28 proteins were identified by both LC-MS and gel-based separations. In total, 136 nonredundant rice proteins were identified, including a new set of 23 proteins of unknown function located in plant mitochondria. We also report the first direct identification, to our knowledge, of PPR (pentatricopeptide repeat) proteins in the plant mitochondrial proteome. This dataset provides the first extensive picture, to our knowledge, of mitochondrial functions in a model monocot plant.
...
PMID:Towards an analysis of the rice mitochondrial proteome. 1274 28

Xanthomonas sp. secretes an extracellular protein (Mr approximately 70+/-5 kDa) during growth on purified natural rubber [poly(1,4-cis-isoprene)] but not during growth on water-soluble carbon sources such as glucose or gluconate. A 1.3 kbp DNA fragment coding for an internal part of the structural gene of the 70 kDa protein was amplified by nested polymerase chain reaction (PCR) using amino acid sequence information obtained after Edman degradation of selected trypsin-generated peptides of the purified 70 kDa protein. The PCR product was used as a DNA probe to clone the complete structural gene from genomic DNA of Xanthomonas sp. The sequenced DNA contained a 2037 bp open reading frame which coded for a polypeptide of 678 amino acids (Mr 74.6 kDa) and which included the features of the N-terminal signal peptidase cleavage site (Mr approximately 72.9 kDa for the mature protein). Analysis of the amino acid sequence revealed the presence of two heme binding motifs (CXXCH) and a approximately 20 amino acids long sequence that is conserved in the Paracoccus denitrificans and Pseudomonas aeruginosa diheme cytochrome c peroxidases (CCPs). This region includes a histidine residue (H519 in Xanthomonas sp. and H265 and H271 in the Pseudomonas strains, respectively) that is essential for activity in CCPs and that is also conserved in other bacterial oxidases. Blast analysis confirmed the relatedness of the 70 kDa protein to heme-containing oxidases and suggested that it is a member of a new family of relatively large (approximately 500 to approximately 1000 amino acids) extracellular proteins with so far unknown function being only far related in amino acid sequence to P. denitrificans and P. aeruginosa CCPs.
...
PMID:Sequence analysis of a gene product synthesized by Xanthomonas sp. during growth on natural rubber latex. 1285 68

The social amoeba Dictyostelium discoideum exhibits high activities of phospholipase and lysophospholipase [Ferber, Munder, Fischer and Gerisch (1970) Eur. J. Biochem. 14, 253-257]. We assayed Dictyostelium lysates to demonstrate the presence of a highly active phospholipase B (PLB) enzyme that removed both fatty-acid chains from phosphatidylcholine and produced the water-soluble glycerophosphorylcholine. We purified the PLB activity from Dictyostelium cytosol using standard agarose media (size exclusion and ion exchange), and combined this with an affinity purification step using myristoylated ARF1 (ADP-ribosylation factor 1), a protein which has a single fatty acid at its N-terminus. Two proteins co-purified (48 kDa and 65 kDa), and the 48 kDa protein was digested with trypsin, peptide fragments were separated by reverse-phase chromatography, and the resultant peptides were sequenced by Edman degradation. From the peptide sequences obtained, database searches revealed a gene which encodes a protein of 65 kDa with unknown function. The 48 kDa protein therefore appears to be a fragment of the full-length 65 kDa product. Expression of the gene in Escherichia coli confirmed that it encodes a PLB. Characterization of its substrate specificity indicated that, in addition to phosphatidylcholine deacylation, the enzyme also hydrolysed phosphatidylinositol and phosphatidylethanolamine. The PLB identified in the present study is not related to existing PLBs found in bacteria, fungi or mammals. There are, however, genes similar to Dictyostelium PLB in mammals, flies, worms and Giardia, but not in yeast. We therefore have identified a novel family of intracellular PLBs.
...
PMID:Identification of phospholipase B from Dictyostelium discoideum reveals a new lipase family present in mammals, flies and nematodes, but not yeast. 1519 48

Using thiol-specific fluorescence labelling, over 30 putative target proteins of thioredoxin h with diverse structures and functions have been identified in seeds of barley and other plants. To gain insight at the structural level into the specificity of target protein reduction by thioredoxin h, thioredoxin h-reducible disulphide bonds in individual target proteins are identified using a novel strategy based on differential alkylation of cysteine thiol groups by iodoacetamide and 4-vinylpyridine. This method enables the accessible cysteine side chains in the thiol form (carbamidomethylated) to be distinguished from those inaccessible or disulphide bound form (pyridylethylated) according to the mass difference in the peptide mass maps obtained by matrix-assistend laser desorption/ionisation-time of flight mass spectrometry. Using this approach, in vitro reduction of disulphides in recombinant barley alpha-amylase/subtilisin inhibitor (BASI) by barley thioredoxin h isoform 1 was analysed. Furthermore, the method was coupled with two-dimensional electrophoresis for convenient thioredoxin h-reducible disulphide identification in barley seed extracts without the need for protein purification or production of recombinant proteins. Mass shifts of 15 peptides, induced by treatment with thioredoxin h and differential alkylation, identified specific reduction of nine disulphides in BASI, four alpha-amylase/trypsin inhibitors and a protein of unknown function. Two specific disulphides, located structurally close to the alpha-amylase binding surfaces of BASI and alpha-amylase inhibitor BMAI-1 were demonstrated to be reduced to a particularly high extent. For the first time, specificity of thioredoxin h for particular disulphide bonds is demonstrated, providing a basis to study structural aspects of the recognition mechanism and regulation of target proteins.
...
PMID:Identification of thioredoxin h-reducible disulphides in proteomes by differential labelling of cysteines: insight into recognition and regulation of proteins in barley seeds by thioredoxin h. 1576 94

The field of proteomics aims to assign functions to the numerous protein products encoded by eukaryotic and prokaryotic genomes. Toward this end, chemical strategies have emerged as a powerful means to enrich specific classes of proteins based on shared functional properties, such as catalytic activity [activity-based protein profiling (ABPP)], and post-translational modification state. The theoretical information content in chemical proteomic experiments greatly exceeds the actual data procured, due in large part to limitations in existing analytical technologies. Here, we present a tandem orthogonal proteolysis (TOP) strategy for high-content chemical proteomics that enables the parallel characterization of probe-labeled proteins and sites of probe modification. The TOP approach exploits "click chemistry" to introduce a multifunctional tag onto probe-labeled proteins that contains both a biotin group for protein enrichment and a tobacco etch virus (TEV) protease cleavage site for selective release of probe-modified peptides. Following capture on streptavidin beads, protein targets of probes and their sites of labeling are sequentially identified by a two-step proteolysis strategy (trypsin and TEV, respectively). We apply the TOP method to characterize targets of sulfonate ester ABPP probes in tissue proteomes, resulting in the discovery of numerous active site-labeled enzymes. Enzymes modified on regulatory sites and proteins of unknown function were also identified. These findings indicate that a wide range of functional residues are targeted by sulfonate ester probes and highlight the value of TOP-based chemical proteomics for the characterization of proteins and the residues that regulate their activity.
...
PMID:A tandem orthogonal proteolysis strategy for high-content chemical proteomics. 1601 63

Synechococcus sp. PCC 7002 and all other cyanobacteria that synthesize phycocyanin have a gene, cpcT, that is paralogous to cpeT, a gene of unknown function affecting phycoerythrin synthesis in Fremyella diplosiphon. A cpcT null mutant contains 40% less phycocyanin than wild type and produces smaller phycobilisomes with red-shifted absorbance and fluorescence emission maxima. Phycocyanin from the cpcT mutant has an absorbance maximum at 634 nm compared with 626 nm for the wild type. The phycocyanin beta-subunit from the cpcT mutant has slightly smaller apparent molecular weight on SDS-PAGE. Purified phycocyanins from the cpcT mutant and wild type were cleaved with formic acid, and the products were analyzed by SDS-PAGE. No phycocyanobilin chromophore was bound to the peptide containing Cys-153 derived from the phycocyanin beta-subunit of the cpcT mutant. Recombinant CpcT was used to perform in vitro bilin addition assays with apophycocyanin (CpcA/CpcB) and phycocyanobilin. Depending on the source of phycocyanobilin, reaction products with CpcT had absorbance maxima between 597 and 603 nm as compared with 638 nm for the control reactions, in which mesobiliverdin becomes covalently bound. After trypsin digestion and reverse phase high performance liquid chromatography, the CpcT reaction product produced one major phycocyanobilin-containing peptide. This peptide had a retention time identical to that of the tryptic peptide that includes phycocyanobilin-bound, cysteine 153 of wild-type phycocyanin. The results from characterization of the cpcT mutant as well as the in vitro biochemical assays demonstrate that CpcT is a new phycocyanobilin lyase that specifically attaches phycocyanobilin to Cys-153 of the phycocyanin beta-subunit.
...
PMID:Identification and characterization of a new class of bilin lyase: the cpcT gene encodes a bilin lyase responsible for attachment of phycocyanobilin to Cys-153 on the beta-subunit of phycocyanin in Synechococcus sp. PCC 7002. 1664 22


<< Previous 1 2 3 4 5 Next >>

---