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)

Many secretory proteins are synthesized as proforms that become biologically active through a proteolytic cleavage in the trans-Golgi complex or at a later stage in the secretory pathway. Haptoglobin (Hp) is unusual in that it is cleaved in the endoplasmic reticulum before it enters the Golgi. Here, we present evidence that the recently discovered complement C1r-like protein (C1r-LP) mediates this cleavage. C1r-LP has not previously been shown to possess proteolytic activity, despite its homology to trypsin-like Ser proteinases. We demonstrate that coexpression of the proform of Hp (proHp) and C1r-LP in COS-1 cells effected cleavage of proHp in the endoplasmic reticulum. This cleavage depended on proteolytic activity of C1r-LP because mutation of the putative active-site Ser residue abolished the reaction. Furthermore, incubation of affinity-purified C1r-LP and proHp led to the cleavage of the latter protein. ProHp appeared to be cleaved at the expected site because substitution of Gly for Arg-161 blocked the reaction. C1r-LP showed specificity for proHp, in that it did not cleave the proform of complement C1s, a protein similar to Hp particularly around the cleavage site. C1r-LP accounts for at least part of the endogenous proHp-cleavage activity because suppression of the C1r-LP expression by RNA interference reduced the cleavage of proHp by up to 45% in the cells of a human hepatoma cell line (HepG2).
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PMID:Prohaptoglobin is proteolytically cleaved in the endoplasmic reticulum by the complement C1r-like protein. 1538 75

We have isolated a cDNA that encodes a novel serine protease, prosemin, from human brain. The cDNA of human prosemin is 1306 bp, encoding 317 amino acids. It showed significant homology with the sequence of a chromosome 16 cosmid clone (accession no. NT_037887.4). The prosemin gene contains six exons and five introns. The amino acid sequence of prosemin shows significant homology to prostasin, gamma-tryptase, and testisin (43%, 41%, and 38% identity, respectively), the genes of which are also located on chromosome 16. Northern hybridization showed that prosemin is expressed predominantly in the pancreas and weakly in the prostate and cerebellum. However, western blot and RT-PCR analyses showed that prosemin is expressed and secreted from various kinds of cancer cells, such as glioma, pancreas, prostate, and ovarian cell lines. Prosemin is secreted in the cystic fluid of clinical ovarian cancers. Furthermore, immunohistochemistry showed prosemin protein localized in the apical parts of ovarian carcinomas. Recombinant prosemin was expressed in COS cells and was purified by immunoaffinity chromatography. Recombinant prosemin preferentially cleaved benzyloxycarbonyl (Z)-His-Glu-Lys-methylcoumaryl amidide (MCA) and t-butyloxycarbonyl (Boc)-Gln-Ala-Arg-MCA. Our results suggest that prosemin is a novel serine protease of the chromosome 16 cluster that is highly expressed in the pancreas. The usefulness of this serine protease as a candidate tumor marker should be further examined.
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PMID:A novel serine protease highly expressed in the pancreas is expressed in various kinds of cancer cells. 1617 65

Prostate-specific membrane antigen (PSMA) is an integral cell-surface membrane glycoprotein that is overexpressed in prostate carcinomas rendering it an appropriate target for antibody-based therapeutic strategies. The biosynthesis of PSMA in transfected COS-1 cells reveals a slow conversion of mannose-rich to complex glycosylated PSMA compatible with slow transport kinetics from the endoplasmic reticulum to the Golgi. Importantly, mannose-rich PSMA persists as a trypsin-sensitive protein throughout its entire life cycle, and only Golgi-located PSMA glycoforms acquire trypsin resistance. This resistance, used here as a tool to examine correct folding, does not depend on the type of glycosylation, because different PSMA glycoforms generated in the presence of inhibitors of carbohydrate processing in the Golgi are also trypsin resistant. The conformational transition of PSMA to a correctly folded molecule is likely to occur in the Golgi and does not implicate ER molecular chaperones, such as BiP. We show here that PSMA is not only heavily N-but also O-glycosylated. The question arising is whether glycans, which do not play a role in folding of PSMA, are implicated in its transport to the cell surface. Neither the cell-surface expression of PSMA nor its efficient apical sorting in polarized Madin-Darby canine kidney cells are influenced by modulators of N- and O-glycosylation. The acquisition of folding determinants in the Golgi, therefore, is an essential prerequisite for protein trafficking and sorting of PSMA and suggests that altered or aberrant glycosylation often occurring during tumorigenesis has no regulatory effect on the cell-surface expression of PSMA.
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PMID:Apical transport and folding of prostate-specific membrane antigen occurs independent of glycan processing. 1622 66

In this study, we aimed at establishing whether two previously identified thyroid disruptors, the insecticide 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT) and Aroclor 1254 (a complex mixture of polychlorinated water), may inhibit thyrotropin (TSH) receptor (TSHr) activity. DDT and Aroclor 1254 were shown to inhibit both the basal and bovine TSH (bTSH)-stimulated accumulation of cAMP in Chinese hamster ovary (CHO)-K1 cells stably transfected with the TSHr. Furthermore, both DDT and Aroclor 1254 did indeed prevent cAMP accumulation, as induced by the constitutive activity of a point mutant TSHr(I486M) transiently transfected in African green monkey kidney fibroblast (COS)-7 cells. Neither trypsin digestion of the extracellular domain (ECD) nor deletion of the ECD in a mutant TSHr trunk transiently transfected in COS-7 cells counteracted the inhibitory activity of DDT and Aroclor 1254. DDT exerted a weak inhibitory activity against forskolin in both CHO-K1 and COS-7 cells, whereas it was nil against the agonists dopamine and 5'-(N-ethyl-carboxamido)-adenosine (NECA) in CHO cells stably transfected with the dopamine D1 receptor and in COS-7 cells transiently transfected with the adenosine type 2a receptor (A2a) receptor. Furthermore, DDT was inactive against the stimulation by isoproterenol of the endogenously expressed beta2 adrenergic receptor in COS-7 cells. Conversely, Aroclor 1254 inhibited completely forskolin activity in CHO-K1 cells but not in COS-7 cells. Furthermore, it did not prevent accumulation of cAMP as induced by NECA in A2a transfected cells. The analog of DDT, diphenylethylene, was inactive against bTSH-induced increase in cAMP in CHO-K1 cells stably transfected with the TSHr. We interpreted these results as indicating that DDT and possibly Aroclor 1254 may have an uncompetitive inverse agonist activity for the TSHr.
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PMID:The thyroid disruptor 1,1,1-trichloro-2,2-bis(p-chlorophenyl)-ethane appears to be an uncompetitive inverse agonist for the thyrotropin receptor. 1706 16

Nicotine, a pharmacologically active constituent of tobacco smoke, decreases sex steroid production and impairs reproductive function. The rate-limiting step in steroid hormone biosynthesis is the transport of substrate cholesterol from the outer to inner mitochondrial membrane by the steroidogenic acute regulatory protein (StAR). StAR is a 37 kDa cytoplasmic phosphoprotein processed as a 32 kDa intermediate to a mature 30 kDa inactive mitochondrial protein. StAR's cholesterol transport capacity is proportional to its residency time at the outer mitochondrial membrane (OMM). Nonsteroidogenic COS-1 cells transfected with StAR/F2, steroidogenic MA-10 cells induced with cAMP or transfected with StAR or the isolated steroidogenic mitochondria preincubated with nicotine reduced StAR expression, import and activity. Mitochondria isolated from steroidogenic tissues or cells, pretreated with nicotine, also reduced the association of StAR with the OMM, but had no effect on the import of signal sequence substituted SCC/N-62StAR. The fluorescence emission maximum of StAR was unchanged with nicotine, but StAR's free energy of unfolding and the surface area (m) increased in the presence of nicotine. Nicotine also blocked StAR from proteolysis with trypsin, suggesting that nicotine partially stabilised protein conformation by insertion into the molten globule conformation of StAR.
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PMID:Folding, activity and import of steroidogenic acute regulatory protein into mitochondria changed by nicotine exposure. 1760 86

The microphthalmia-associated transcription factor (MITF) gene encodes a basic helix-loop-helix and leucin zipper protein. In this study, we identified a novel MITF isoform, MITF-CM, which possesses a unique amino terminus. Exon 1CM is located 84 kb upstream of the exon encoding the B1b domain. MITF-CM was expressed in the human mast cell line HMC-1, the human basophilic cell line KU812, and CB-derived mast cells cultured for 10 weeks as well as bone marrow mononuclear cells. Transient transfection of MITF-CM cDNA in COS-7 cells resulted in the expression of a 64-kDa protein, detected by Western blotting, and nuclear localization of the protein, detected by immunostaining. The transient cotransfection of a luciferase construct under the control of the tyrosinase promoter and MITF-CM cDNA increased luciferase activity threefold. In contrast, none of the MITF isoforms transactivated both the tryptase and chymase gene promoters, indicating differences in the gene transactivation system between humans and mice.
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PMID:MITF-CM, a newly identified isoform of microphthalmia-associated transcription factor, is expressed in cultured mast cells. 1828 17

Proteins that coat Plasmodium falciparum merozoite surface and those secreted from its apical secretory organelles are considered promising candidates for the vaccine against malaria. In the present study, we have identified an asparagine rich parasite protein (PfAARP; Gene ID PFD1105w), that harbors a predicted signal sequence, a C-terminal transmembrane region and whose transcription and translation patterns are similar to some well characterized merozoite surface/apical proteins. PfAARP was localized to the apical end of the merozoites by GFP-targeting approach using an inducible, schizont-stage expression system, by immunofluorescence assays using anti-PfAARP antibodies. Immuno-electron microsopic studies showed that PfAARP is localized in the apical ends of the rhoptries in the merozoites. RBC binding assays with PfAARP expressed on COS cells surface showed that it binds to RBCs through its N-terminal region with a receptor on the RBC surface that is sensitive to trypsin and neuraminidase treatments. Sequencing of PfAARP from different P. falciparum strains as well as field isolates showed that the N-terminal region is highly conserved. Recombinant protein corresponding to the N-terminal region of PfAARP (PfAARP-N) was produced in its functional form in E. coli. PfAARP-N showed reactivity with immune sera from individuals residing in P. falciparum endemic area. The anti-PfAARP-N rabbit antibodies significantly inhibited parasite invasion in vitro. Our data on localization, functional assays and invasion inhibition, suggest a role of PfAARP in erythrocyte binding and invasion by the merozoite.
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PMID:Identification and characterization of a novel Plasmodium falciparum merozoite apical protein involved in erythrocyte binding and invasion. 1832 51

Hypophosphatasia, a congenital metabolic disease related to the tissue-nonspecific alkaline phosphatase gene (TNSALP), is characterized by reduced serum alkaline phosphatase levels and defective mineralization of hard tissues. A replacement of valine with alanine at position 406, located in the crown domain of TNSALP, was reported in a perinatal form of hypophosphatasia. To understand the molecular defect of the TNSALP (V406A) molecule, we examined this missense mutant protein in transiently transfected COS-1 cells and in stable CHO-K1 Tet-On cells. Compared with the wild-type enzyme, the mutant protein showed a markedly reduced alkaline phosphatase activity. This was not the result of defective transport and resultant degradation of TNSALP (V406A) in the endoplasmic reticulum, as the majority of newly synthesized TNSALP (V406A) was conveyed to the Golgi apparatus and incorporated into a cold detergent insoluble fraction (raft) at a rate similar to that of the wild-type TNSALP. TNSALP (V406A) consisted of a dimer, as judged by sucrose gradient centrifugation, suggestive of its proper folding and correct assembly, although this mutant showed increased susceptibility to digestion by trypsin or proteinase K. When purified as a glycosylphosphatidylinositol-anchorless soluble form, the mutant protein exhibited a remarkably lower Kcat/Km value compared with that of the wild-type TNSALP. Interestingly, leucine and isoleucine, but not phenylalanine, were able to substitute for valine, pointing to the indispensable role of residues with a longer aliphatic side chain at position 406 of TNSALP. Taken together, this particular mutation highlights the structural importance of the crown domain with respect to the catalytic function of TNSALP.
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PMID:Molecular basis of perinatal hypophosphatasia with tissue-nonspecific alkaline phosphatase bearing a conservative replacement of valine by alanine at position 406. Structural importance of the crown domain. 1842 67

The swine influenza virus (SIV) strain A/Swine/TianJin/01/2004(H1N1) (A/S/TJ/04) was rescued successfully by an eight-plasmid rescue system. The cDNAs of SIV 8 gene segments were synthesized by RT-PCR and cloned into the RNA polymerase I/II bidirection expression vector PHW2000 independently, resulting in 8 recombinant plasmids. The 8 recombinant plasmids were cotransfected into COS-1 cell, 30 h later TPCK-trypsin was added to 0.5 microg/mL. The COS-1 cell and supernatant were harvested 48 h after cotransfection and were inoculated into the allantoic cavity of 9-day-old specific-pathogen free (SPF) chicken eggs. The allantoic fluid of dead eggs was harvested and passaged 3 generations in SPF chicken eggs to get infective virus. The successful rescue of A/S/TJ/04 SIV was identified by hemagglutination assay, hemagglutination inhibition assay, sequence analysis and electron microscope observation. The successful rescue of SIV built a platform for the research of the relationship between genome structure and function of SIV, the mechanisms of trans-species transmission of influenza virus and for the generation of new SIV as vaccine.
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PMID:[The rescue of H1N1 subtype swine influenza virus]. 1872 8

The use of peptides as drugs in pharmaceutical applications is hindered by their susceptibility to proteolysis and therefore low bioavailability. beta-Peptides that contain an additional methylene group in the backbone, are gaining recognition from a pharmaceutical stand point as they are considerably more resilient to proteolysis and metabolism. Recently, we reported two new classes of beta-peptides, beta(3)- and beta(2)-peptides derived from L-aspartic acid and L-diaminopropionic acid, respectively. Here, we report the proteolytic stability of these beta-peptidic compounds and a mixed alpha /beta-peptide against three enzymes (pronase, trypsin and elastase), as well as, human serum. The stability of these peptides was compared to an alpha-peptide. Peptides containing beta-linkages were resistant to all conditions. The mixed alpha /beta-peptide, however, exhibited proteolysis in the presence of trypsin and pronase but not elastase. The rate of degradation of the mixed alpha /beta-peptide was slower than that would be expected for an alpha-peptide. In addition, these beta-peptides were not toxic to HeLa and COS-1 cell lines as observed by MTT cytotoxicity assay. These results expand the scope of mixed alpha /beta-peptides containing beta-amino acids or small beta-peptide fragments as therapeutic peptides.
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PMID:The proteolytic stability and cytotoxicity studies of L-aspartic acid and L-diaminopropionic acid derived beta-peptides and a mixed alpha/beta-peptide. 1931 48


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