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)

Protease-activated receptor-2 (PAR-2), a G protein-coupled receptor for trypsin and tryptase, exerts important physiological and pathological functions in multiple systems. However, unlike PAR-1, the PAR-2-mediated intracellular signal transductions are hardly known. Here, using yeast two-hybrid screening with a human brain cDNA library, we identified an interacting partner of human PAR-2, the Jun activation domain-binding protein 1 (Jab1). The interaction was confirmed by glutathione S-transferase pull-down assays in vitro, and by co-immunoprecipitation assays in vivo. Jab1 was also shown to be colocalized with PAR-2 in both transfected HEK293 cells and in normal primary human astrocytes by double immunofluorescence staining. Further experiments demonstrated that multiple intracellular domains of PAR-2 are required for the interaction with Jab1. We then showed that agonist stimulation of PAR-2 disrupted the interaction, which could be prevented by the inhibitor of receptor endocytosis phenylarsine oxide, but not by the lysosomal protease inhibitor ZPAD. Importantly, we found that activation of PAR-2 induced the redistribution of Jab1 from the plasma membrane to the cytosol, but did not influence expression of Jab1. Furthermore, Jab1 mediated PAR-2-induced c-Jun activation, which was followed by increased activation of activator protein-1. Loss-of-function studies, using Jab1 small interfering RNA, demonstrated that Jab1 knockdown blocked PAR-2-induced activator protein-1 activation. Taken together, our data demonstrate that Jab1 is an important effector that mediates a novel signal transduction pathway for PAR-2-dependent gene expression.
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PMID:Jab1, a novel protease-activated receptor-2 (PAR-2)-interacting protein, is involved in PAR-2-induced activation of activator protein-1. 1641 Feb 50

Rat liver microsomal glutathione S-transferase (MGST1) is known to be activated by trypsin, however, it has not been clarified whether MGST1 is activated by a protease present in liver. In the present study we purified the MGST1 activating protease from liver microsomes and finally identified that the protease is hepsin, a type II transmembrane serine protease. When the protease was incubated with the purified MGST1 or liposomal MGST1 at 4 degrees C, MGST1 activity was increased 3-4.5 fold after 3-6 d. In electrophoretic and immunoblot analyses after the incubation of MGST1 with the protease MGST1 dimer and its degraded fragment were detected. These results suggest that the rat liver microsomal hepsin functions as MGST1 activating/degrading enzyme.
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PMID:Purification of liver serine protease which activates microsomal glutathione S-transferase: possible involvement of hepsin. 1665 11

Degenerate primers were designed based on all possible sequences of the N-terminal and C-terminal regions of Delonix regia trypsin inhibitor (DrTI). Five hundred sixty-one bp of polymerase chain reaction (PCR) product was amplified using the above degenerate primers and genomic DNA and cDNA of Delonix regia as a template. The amplified PCR products were cloned and sequenced. DNA sequence analysis of cDNA and genomic clones of DrTI have the same nucleotide sequence in the coding region, and manifested a genomic clone without intervening sequences in the coding region. The amino acid sequence deduced from the DrTI genomic and cDNA clones agreed with that identified via amino acid sequencing analysis, except that two amino acid residues, Ser and Lys, existed between residues Lys141 and Ser142. DrTI open reading frame was then amplified and cloned in-frame with GST in pGEX4T-1 and overexpressed in Escherichia coli to yield a glutathione S-transferase (GST)-fusion protein with a calculated molecular mass of about 45 kDa. The recombinant DrTI (reDrTI) was derived by treating the GST-DrTI fusion protein with thrombin. Both the reDrTI and GST-DrTI fusion protein exhibited a strong identical inhibitory effect on trypsin activity.
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PMID:Genomic and cDNA cloning, characterization of Delonix regia trypsin inhibitor (DrTI) gene, and expression of DrTI in Escherichia coli. 1721 72

Semicarbazide (SEM) was considered to be a characteristic protein-bound side-chain metabolite of the banned veterinary drug nitrofurazone and used as a marker of nitrofurazone abuse. It was recently discovered that SEM can arise in food from sources other than nitrofurazone. This uncertainty over the source of SEM may be overcome if alternative markers specific to tissue-bound nitrofurazone residues can be determined. The structure of nitrofurazone metabolites in vivo and particular proteins to which they are bound are not known. These proteins with altered structure due to the presence of the drug metabolites can be considered as potential alternative biomarkers of nitrofurazone abuse. The proteins implicated in the in vivo binding of nitrofurazone were separated and identified. A crude mixture of proteins extracted from the liver of a rat treated with the drug was separated using a series of different techniques such as preparative isoelectric focusing and size exclusion HPLC. Multiple fractions were assayed by LC-MS/MS to detect the presence of SEM. The proteins containing SEM residues were identified by peptide mass mapping using trypsin digestion and MALDI-TOF. The first protein identified as containing high concentration of SEM was albumin. It was also shown that low molecular weight species within a protein mixture whose main constituent was glutathione S-transferase contained a high concentration of SEM. The chemical composition of these components is under investigation. Preliminary data suggest the SEM forms part of a nitrofurazone metabolite conjugated to glutathione.
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PMID:The identification of potential alternative biomarkers of nitrofurazone abuse in animal derived food products. 1824 64

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

X-linked inhibitor of apoptosis protein (XIAP) inhibits apoptosis mainly through inhibition of caspase-9 and executioner caspases of -3 and -7. The inhibition of the former protease is implemented through the bacculoviral inhibitory repeat-3 (Bir3) domain, while the inhibition of the latter is accomplished by the interaction of the linker region located between the Bir1 and the Bir2 domains with their active sites. Both modes of inhibition are antagonized by SMAC, which is released from mitochondria during the initiation of the intrinsic apoptosis pathway. Although the mechanism of SMAC interference in Bir3 inhibition of caspase-9 is clearly established, the mechanism by which SMAC interferes with the inhibition of the executioner caspases by XIAP remains largely unknown. To address this issue, we performed a limited proteolysis of glutathione S-transferase (GST)-tagged XIAP-Bir2 by trypsin in the presence and in the absence of SMAC peptide. Under these conditions, the proteolysis of the linker region was diminished considerably. Furthermore, the rate of association of caspase-3 and -7 with XIAP in the presence of the SMAC peptide was reduced drastically, suggesting that SMAC peptide restricts the exposure of the linker region. A limited proteolysis of caspase-7 in the presence of GST-Bir2 and GST-NBir3 (the Bir3 domain of human NAIP) as negative controls was also performed. Matrix-assisted laser desorption/ionization time-of-flight analysis of the fragments revealed the identity of protected sites, suggesting that the Bir2 domain makes numerous contacts with the large subunit of caspase-7. These, combined with the results from Far-Western experiments, strongly suggest that the groove for the inhibitor(s)-of-apoptosis-protein-binding motif on the Bir2 favors binding to the N-terminus of the large subunit rather than to the small subunit of caspase-7. Our results further show that the active-site pocket of caspase-7 is first occupied by the linker region, followed by the interaction of the N-terminus of the enzyme with the SMAC-binding site of the Bir2 domain.
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PMID:A mechanistic insight into SMAC peptide interference with XIAP-Bir2 inhibition of executioner caspases. 1861 10

Tri a Bd 27K is the predominant allergen in wheat. In the present study, this allergen was purified to homogeneity from wheat flour. The N-terminal amino acid sequences of the purified allergen and the peptides obtained by its digestion, with trypsin were determined, and the allergen was shown to be a glycoprotein with an Asn-linked sugar moiety containing fucose residues. A cDNA encoding the allergen was obtained by polymerase chain reaction (PCR). The cDNA codes for a protein of 203 amino acid residues, with a molecular mass of 22,803 Da, that has two tentative sites glycosylated at Asn residues. Homology analysis suggested that the allergen might belong to a family of gamma-interferon-inducible thiol reductases. The cDNA was expressed as a fusion protein with glutathione S-transferase in Escherichia coli. However, unlike the allergen purified from wheat, recombinant Tri a Bd 27K was not immunoblotted with IgE antibodies in the serum of a wheat-sensitive patient.
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PMID:Isolation and molecular cloning of a major wheat allergen, Tri a Bd 27K. 1912 51

Enzymatic hydrolysates of Laminaria japonica were evaluated for antioxidative activities using hydroxyl radical scavenging activity and protective effects against H(2)O(2)-induced DNA and cell damage. In addition, activities of antioxidative enzymes, including catalase, glutathione peroxidase, and glutathione S-transferase, of the enzymatic hydrolysates from L. japonica were also estimated. L. japonica was first enzymatically hydrolyzed by seven carbohydrases (Dextrozyme, AMG, Promozyme, Maltogenase, Termamyl, Viscozyme, and Celluclast [all from Novo Co., Novozyme Nordisk, Bagsvaerd, Denmark]) and five proteinases (Flavourzyme, Neutrase, Protamex, Alcalase [all from Novo Co.], and pancreatic trypsin). The hydroxyl radical scavenging activities of Promozyme and pancreatic trypsin hydrolysates from L. japonica were the highest as compared to those of the other carbohydrases and proteinases, and their 50% inhibitory concentration values were 1.67 and 317.49 mug/mL, respectively. The pancreatic trypsin hydrolysates of L. japonica exerted a protective effect on H(2)O(2)-induced DNA damage. We also evaluated the protective effect on hydroxyl radical-induced oxidative damage in PC12 cells via propidium iodide staining using a flow cytometer. The AMG and pancreatic trypsin hydrolysates of L. japonica dose-dependently protected PC12 cells against cell death caused by hydroxyl radical-induced oxidative damage. Additionally, we analyzed the activity of antioxidative enzymes such as catalase, glutathione peroxidase, and the phase II biotransformation enzyme glutathione S-transferase in L. japonica-treated cells. The activity of all antioxidative enzymes was higher in L. japonica-treated cells compared with the nontreated cells. These results indicate that enzymatic hydrolysates of L. japonica possess antioxidative activity.
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PMID:Protective effects against H2O2-induced damage by enzymatic hydrolysates of an edible brown seaweed, sea tangle (Laminaria japonica). 1929 10

Protein abundance profiling from tissue using liquid chromatography-tandem mass spectrometry-based "shotgun" proteomics and label-free relative quantitation was evaluated for the investigation of estrogen-regulated protein expression in the mouse brain and uterus. Sample preparation involved a 30-min protein extraction in 8 M aqueous urea solution, followed by disulfide reduction, thiol alkylation, and trypsin digestion of the extracted proteins, and was performed on 3-4 mg of tissue to evaluate the suitability of this methodology to expedite the survey of cellular pathways that are affected in vivo by an experimental therapeutic intervention in an animal model. The label-free proteomic approach (spectral counting) was suitable to identify even subtle changes in cortical protein levels and revealed significant estrogen-induced upregulation of ATP synthase (both alpha- and beta-isoforms), aspartate aminotransferase 2, and mitochondrial malate dehydrogenase without any prior subcellular fractionation of the tissue or the use of multidimensional chromatographic separation. The methodology was also suitable to observe various up- and downregulated proteins in the uterine tissue of ovariectomized mice upon treatment with 17beta-estradiol. In addition to confirming a very significant decrease in the abundance of glutathione S-transferase recognized as a marker of estrogen's impact, our studies have also revealed potential new protein markers such as desmin and lumican that are critical components of cytoskeletal arrangement and, hence, regulation of their abundance could contribute to major morphological changes in the uterus occurring upon estrogenic stimulation.
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PMID:Rapid label-free identification of estrogen-induced differential protein expression in vivo from mouse brain and uterine tissue. 1954 49

It has previously been shown that a approximately 27 kDa serine protease of Schistosoma mansoni larvae, the cercarial elastase (CE), was a poor immunogen in as much as it failed to induce an antibody response. The CE has a critical role in enabling schistosome larvae to penetrate the skin of their definitive hosts, so the apparently poor immunogenicity of this enzyme is clearly of interest. To understand its lack of immunogenicity better and in particular to determine whether it is related to its proteolytic activity, we have measured antibody responses of mice to three different serine proteases. Groups of mice were immunized with porcine pancreatic trypsin (TRY), chymotrypsin (CHY) or elastase (ELA) and the resulting antibody response compared with antibody responses to two non-protease antigens, chicken egg albumin (OVA) and Schistosoma japonicum glutathione S-transferase (GST), all being administered with alum as an adjuvant. Of 12 mice that were injected five times at 14 day intervals with TRY, only one produced antibody reactive with this enzyme in ELISA. Immunizations with CHY or ELA induced somewhat better antibody responses than TRY, but the responses to the first and second injections of these two proteases nevertheless seemed comparatively lower than the responses to GST. Induction of antibody responses by OVA and GST was not affected when TRY was injected concomitantly. Thus, the antibody response to one of the serine proteases used in this study, mammalian trypsin, was anomalous.
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PMID:Anomalous immunogenic properties of serine proteases. 1975 Dec 73


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