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)

Yeast Mas70p and NADH cytochrome b5 reductase are bitopic integral proteins of the mitochondrial outer membrane and are inserted into the lipid-bilayer in an Nin-Ccyto orientation via an NH2-terminal signal-anchor sequence. The signal anchor of both proteins is comprised of a short, positively charged domain followed by the predicted transmembrane segment. The positively charged domain is capable of functioning independently as a matrix-targeting signal in yeast mitochondria in vitro but does not support import into mammalian mitochondria (rat or human). Rather, this domain represents a cryptic signal that can direct import into mammalian mitochondria only if proximal components of the outer membrane import machinery are removed. This can be accomplished either by treating the surface of the intact mitochondria with trypsin or by generating mitoplasts. The import receptor Tom20p (Mas20p/MOM19) is responsible for excluding the cryptic matrix-targeting signal from mammalian mitochondria since replacement of yeast Tom20p with the human receptor confers this property to the yeast organelle while at the same time maintaining import of other proteins. In addition to contributing to positive recognition of precursor proteins, therefore, the results suggest that hTom20p may also have the ability to screen potential matrix-targeting sequences and exclude certain proteins that would otherwise be recognized and imported by distal components of the outer and inner membrane protein-translocation machinery. These findings also indicate, however, that cryptic signals, if they exist within otherwise native precursor proteins, may remain topogenically silent until the precursor successfully clears hTom20p, at which time the activity of the cryptic signal is manifested and can contribute to subsequent translocation and sorting of the polypeptide.
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PMID:The human mitochondrial import receptor, hTom20p, prevents a cryptic matrix targeting sequence from gaining access to the protein translocation machinery. 870 17

Three monoclonal antibodies (mAbs), M344, M300 and M75, were shown to define a unique tumour-associated antigen (TAA) of superficial bladder tumours. The antigenic determinants are expressed on a very-high-molecular-mass component and, in about 50% of the positive samples, one determinant is also detected on a 62 kDa molecular species, observed only under reducing conditions. The objectives of the present study were to characterize further this TAA by analysing (1) the biochemical nature of the epitopes recognized by the three mAbs, and (2) the biochemical and structural features of the molecule bearing them. The antigenicity was resistant to heat denaturation, trypsin and alpha-chymotrypsin treatments but highly sensitive to papain and Pronase digestion. NaIO4 oxidation decreased reactivity to mAbs M344 and M300 but enhanced reactivity to mAb M75. The three determinants were insensitive to beta-galactosidase and alpha-L-fucosidase but were sensitive to Vibrio cholerae neuraminidase. None of the three mAbs reacted with ovine, bovine or porcine submaxillary mucins. Deglycosylation with O-glycosidase or trifluoromethanesulphonic acid completely abolished the reactivity of the mAbs whereas N-glycosidase F deglycosylation had no appreciable effect. The presence on the molecule of cryptic Gal(beta(1-3))GalNAc as a major core disaccharide was demonstrated by a heterologous sandwich assay using mAb M75 and peanut agglutinin. Thiol reduction using beta-mercaptoethanol increased mobility of the high-molecular-mass component in polyacrylamide gels. We thus conclude that mAbs M344 and M300 react with sialylated carbohydrate epitopes, and mAb M75 reacts with a partially cryptic and periodate-resistant sialylated epitope expressed on a typical secreted high-molecular-mass oligomeric mucin which we named MAUB for mucin antigen of the urinary bladder.
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PMID:Biochemical analysis of a bladder-cancer-associated mucin: structural features and epitope characterization. 903 80

The VP60 capsid protein of rabbit haemorrhagic disease virus (60 kDa) has been fused to the C-terminus of beta-galactosidase and produced in E. coli from two related expression vectors. One of these vectors, carries a 429 bp DNA segment encoding the N-terminus peptide of VP60, and directs the synthesis of a larger fusion that contains the entire viral protein. Both fusion proteins are efficiently cleaved at a presumed trypsin-like target site within the carboxy moiety of beta-galactosidase (Arg 611-Thr 612), which is activated by the presence of the viral partner. In the larger fusion, VP60 is released by a cleavage within the linker region that affects about 10% of the chimeric proteins. In this situation, the resulting beta-galactosidase-like fragment recovers its natural proteolytic stability. These results prove that cryptic cleavage sites in beta-galactosidase can be efficiently activated in a fusion protein and suggest that this activation is based on reversible steric constraints generated by the fusion partner.
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PMID:Reversible activation of a cryptic cleavage site within E. coli beta-galactosidase in beta-galactosidase fusion proteins. 943 12

Treatment of low density lipoprotein (LDL) with degrading enzymes transforms the molecule to a moiety that is micromorphologically indistinguishable from lipoproteinaceous particles that are present in atherosclerotic plaques, and enzymatically modified LDL (E-LDL), but not oxidized LDL (ox-LDL), spontaneously activates the alternative complement pathway, as do lesion lipoprotein derivatives. Furthermore, because E-LDL is a potent inducer of macrophage foam cell formation, we propose that enzymatic degradation may be the key process that renders LDL atherogenic. In this article, we report the production of two murine monoclonal antibodies recognizing cryptic epitopes in human apolipoprotein B that become exposed after enzymatic attack on LDL. One antibody reacted with LDL after single treatment with trypsin, whereas recognition by the second antibody required combined treatment of LDL with trypsin and cholesterol esterase. In ELISAs, both antibodies reacted with E-LDL produced in vitro and with lesion complement activator derived from human atherosclerotic plaques, but they were unreactive with native LDL or ox-LDL. The antibodies stained E-LDL, but not native LDL or ox-LDL, that had been artificially injected into arterial vessel walls. With the use of these antibodies, we have demonstrated that early human atherosclerotic coronary lesions obtained at autopsy as well as lesions examined in freshly explanted hearts always contain extensive extracellular deposits of E-LDL. Terminal complement complexes, detected with a monoclonal antibody specific for a C5b-9 neoepitope, colocalized with E-LDL within the intima, which is compatible with the proposal that subendothelially deposited LDL is enzymatically transformed to a complement activator at the earliest stages in lesion development.
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PMID:Immunohistochemical demonstration of enzymatically modified human LDL and its colocalization with the terminal complement complex in the early atherosclerotic lesion. 951 5

Previously, we produced the whole extracellular region of metabotropic glutamate receptor subtype 1 (mGluR1) in a soluble form. The soluble receptor retained a ligand affinity comparable with that of the full-length membrane-bound receptor and formed a disulfide-linked dimer. Here, we have identified a cysteine residue responsible for the intermolecular disulfide bond and determined domain organization of the extracellular region of mGluR1. A mutant, C140A, was a monomer under nonreduced conditions by SDS-polyacrylamide gel electrophoresis; however, C140A was eluted at the position similar to that of mGluR113, the wild type soluble receptor, by size exclusion column chromatography. Furthermore, C140A bound a ligand, [(3)H]quisqualate, with an affinity similar to that obtained by mGluR113. Oocytes injected with RNA for full-length mGluR1 containing C140A mutation showed responses to ligands at magnitudes similar to those with wild type full-length RNA. Thus, elimination of the disulfide linkage did not perturb the dimer formation and ligand signaling, suggesting that cryptic dimer interface(s) possibly exist in mGluR1. Limited proteolysis of the whole extracellular fragment (residue 33-592) revealed two trypsin-sensitive sites, after the residues Arg(139) and Arg(521). A 15-kDa NH(2)-terminal proteolytic fragment (residue 33-139) was associated with the downstream part after the digestion. Arg(521) was located before a cysteine-rich stretch preceding the transmembrane region. A new shorter soluble receptor (residue 33-522) lacking the cysteine-rich region was designed based on the protease-sensitive boundary. The purified receptor protein gave a K(d) value of 58.1 +/- 0.84 nm, which is compatible to a reported value of the full-length receptor. The B(max) value was 7.06 +/- 0. 82 nmol/mg of protein. These results indicated that the ligand-binding specificity of mGluR1 is confined to the NH(2)-terminal 490-amino acid region of the mature protein.
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PMID:Cryptic dimer interface and domain organization of the extracellular region of metabotropic glutamate receptor subtype 1. 1087 32

The protease-activated receptor (PAR) belongs to the large superfamily of G-protein-coupled seven trans-membrane domain receptors. The activation of PARs is achieved by proteolytic unmasking of the cryptic N-terminal receptor-activating sequence that binds to the body of the same receptor molecule. PARs-1, -3 and -4 are activated by thrombin, while PAR-2 is activated by trypsin or mast cell tryptase, but not by thrombin. PARs are widely distributed to a variety of tissues and participate in a number of physiological or pathophysiological phenomena such as platelet aggregation, inflammation and cardiovascular, digestive or respiratory functions. Thus, PARs are of physiological importance and also of pharmacological interest as the novel target for drug development.
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PMID:Protease-activated receptor (PAR), a novel family of G protein-coupled seven trans-membrane domain receptors: activation mechanisms and physiological roles. 1088 46

Mutations in the fibrillin-1 gene (FBN1) cause Marfan syndrome (MFS), an autosomal dominant disorder of connective tissue with highly variable clinical manifestations. FBN1 contains 47 epidermal growth factor (EGF)-like modules, 43 of which display a consensus sequence for calcium binding (cbEGF). Calcium binding by cbEGF modules is thought to be essential for the conformation and stability of fibrillin-1. Missense mutations in cbEGF modules are the most common mutations found in MFS and generally affect one of the six highly conserved cysteines or residues of the calcium-binding consensus sequence. We have generated a series of recombinant fibrillin-1 fragments containing six cbEGF modules (cbEGF nos. 15-20) with various mutations at different positions of cbEGF module no. 17, which is known to contain a cryptic cleavage site for trypsin. A mutation affecting a residue of the calcium-binding consensus sequence (K1300E) found in a patient with relatively mild clinical manifestations of classic MFS caused a modest increase in susceptibility to in vitro proteolysis by trypsin, whereas a mutation affecting the sixth cysteine residue of the same cbEGF module (C1320S) reported in a severely affected patient caused a dramatic increase in susceptibility to in vitro proteolysis by trypsin. A mutation at the cryptic cleavage site for trypsin abolished sensitivity of wild-type fragments and fragments containing K1300E to trypsin proteolysis. Whereas the relevance of in vitro proteolysis to the in vivo pathogenesis of MFS remains unclear, our findings demonstrate that individual mutations in cbEGF modules can affect these modules differentially and may suggest an explanation for some genotype-phenotype relationships in MFS.
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PMID:Differential effect of FBN1 mutations on in vitro proteolysis of recombinant fibrillin-1 fragments. 1107 82

Western blot experiments showed that sera from mice infected with the mouse hepatitis virus strain A59 (MHV-A59) contained autoantibodies (autoAb) that bound to a 40-kDa protein present in liver and kidney extracts. No reaction was observed with extracts of the heart, muscles, spleen, brain and lung. The Ab cross-reacted with a 40-kDa protein from human, rat and sheep liver, but not with liver extracts from the silver side fish (Odontesthes bonariensis). No correlation was found between the development of the hypergammaglobulinemia that followed the viral infection and the occurrence of the autoAb. Reactive immunoglobulins pertained to the IgG1, IgG2a and IgG2b subclasses, recognized cryptic epitopes and were detected from 10 days up to 8 weeks after MHV-infection. The 40-kDa protein was purified from mouse liver extracts by ion-exchange chromatography, gel filtration and SDS-PAGE. Because the N-terminal was blocked, we digested the protein in-gel with trypsin and sequenced various peptides. Results indicated a 100% homology of sequence between the protein recognized by the autoAb and liver fumarylacetoacetate hydrolase (FAH), the enzyme that mediates the last step of tyrosine catabolism. Additionally, a second protein recognized by the autoAb was detected during FAH purification steps and was identified as liver alcohol dehydrogenase.
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PMID:Identification of two liver proteins recognized by autoantibodies elicited in mice infected with mouse hepatitis virus A59. 1146 1

Benign mesenchymal neoplasms associated with rearrangements of the DNA architectural factor gene HMGIC on chromosome 12 include lipomas, uterine leiomyomata, pulmonary chondroid hamartomas, endometrial polyps, salivary gland pleomorphic adenomas, and breast fibroadenomas. Although HMGIC also has been implicated in the pathobiology of aggressive angiomyxoma of the vulva, the molecular mechanisms pertaining to this neoplasm are unclear. Tissue from a recurrent aggressive angiomyxoma was investigated by cytogenetic and expression analysis for HMGIC and HMGIY. The trypsin-Giemsa-banded karyotype showed a clonal translocation between chromosomes 8 and 12 [46,XX,t(8;12)(p12;q15)]. Fluorescence in situ hybridization (FISH) analysis with whole chromosome paint probes for chromosomes 8 and 12 excluded cryptic involvement of other chromosomes. The chromosome 12 breakpoint was mapped with two-color FISH analysis using cosmid probes at the 5' and 3' termini of HMGIC. Both cosmid probes showed hybridization to the normal chromosome 12 and the der(12) chromosome, indicating that the breakpoint was 3' (telomeric) to the gene. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed HMGIC expression in the tumor, and immunohistochemistry localized HMGIC expression to the tumor's spindle cells. Like numerous benign mesenchymal tumors, this locally aggressive tumor is associated with rearrangements near or within HMGIC, but chimeric gene formation was not required for tumorigenesis. Inappropriate expression of this DNA binding protein, however, may be important in the pathobiology of this tumor. Understanding the pathogenetic mechanism may also be helpful in developing new diagnostic tools for identifying residual disease.
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PMID:Chromosomal translocation t(8;12) induces aberrant HMGIC expression in aggressive angiomyxoma of the vulva. 1155 Feb 85

The talin rod contains approximately 11 vinculin binding sites (VBSs), each defined by hydrophobic residues in a series of amphipathic helices that are normally buried within the helical bundles that make up the rod. Consistent with this, talin failed to compete for binding of the vinculin Vd1 domain to an immobilized talin polypeptide containing a constitutively active VBS. However, talin did bind to GST-Vd1 in pull-down assays, and isothermal titration calorimetry measurements indicate a K(d) of approximately 9 mum. Interestingly, Vd1 binding exposed a trypsin cleavage site in the talin rod between residues 898 and 899, indicating that there are one or more active VBSs in the N-terminal part of the talin rod. This region comprises a five helix bundle (residues 482-655) followed by a seven-helix bundle (656-889) and contains five VBSs (helices 4, 6, 9, 11, and 12). The single VBS within 482-655 is cryptic at room temperature. In contrast, talin 482-889 binds Vd1 with high affinity (K(d) approximately 0.14 mum), indicating that one or more of the four VBSs within 656-889 are active, and this likely represents the vinculin binding region in intact talin. In support of this, hemagglutinin-tagged talin 482-889 localized efficiently to focal adhesions, whereas 482-655 did not. Differential scanning calorimetry showed a strong negative correlation between Vd1 binding and helical bundle stability, and a 755-889 mutant with a more stable fold bound Vd1 much less well than wild type. We conclude that the stability of the helical bundles that make up the talin rod is an important factor determining the activity of the individual VBSs.
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PMID:The activity of the vinculin binding sites in talin is influenced by the stability of the helical bundles that make up the talin rod. 1640 2


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