Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.1 (chymotrypsin)
 10,938 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Three alternatively spliced forms of the amyloid precursor protein (APP), APP-695, APP-751, and APP-770, were expressed in the baculovirus expression vector system. The recombinant proteins were secreted into the culture medium by infected insect cells, and APP molecules were detected in insect cells and medium 2 days after infection with the recombinant APP-baculoviruses. A partial sequence of the NH2 terminus of the secreted protein revealed identity with the native secreted protein and showed that the signal peptide was recognized and properly cleaved in insect cells. Purified secreted recombinant APP-751 comigrated with protease nexin 2 purified from platelets and fibroblasts. A 15-kDa COOH-terminal fragment of APP was also detected in cells infected with recombinant baculoviruses, suggesting that recombinant APP proteins were cleaved at the COOH-terminal end like native APP protein. Recombinant APP-751 and APP-770 formed complexes with epidermal growth factor-binding protein, whereas APP-695 did not. In addition, recombinant APP-751 and APP-770 inhibited trypsin and chymotrypsin activity, whereas APP-695 did not. Growth of a human fibroblast cell line, A-1, that required APP for complete growth, was restored upon addition of secreted recombinant APP-695 or APP-751. Thus, the appropriately sized, secreted recombinant APP proteins produced in this expression system are biologically active.
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PMID:Expression of active secreted forms of human amyloid beta-protein precursor by recombinant baculovirus-infected insect cells. 194 49

Although multiple forms of the HIV-1 Tat protein are synthesized during infection from alternatively spliced mRNAs, only 72 amino acid residues encoded in the first Tat exon are necessary for full transactivation activity. We used limited proteolytic digestions of proteins expressed in vitro to study the structures of three Tat proteins from isolate HXB2: 72R Tat (first Tat exon); 86R Tat (first and second Tat exons), Tev or TNV (first Tat exon plus Env and Rev exons). For the 86R and Tev proteins, either trypsin or chymotrypsin cleaved the majority of carboxyl residues from the first exon. Moreover, when released from carboxyl residues, the first exon of 86R and Tev was relatively resistant to subsequent proteolysis. The entire 72R Tat protein was relatively resistant to proteolysis. The protease-resistant first exon in all Tat proteins was abolished by EDTA treatment, suggesting that divalent cations are required for its conformation. Our results suggest that the first exon in the 86R, 72R, and Tev proteins is folded into a similar structure which, as defined by partial proteolysis, acts as a single biochemical domain.
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PMID:Limited proteolytic digestions identify common structural features of HIV-1 Tat proteins expressed during infection from alternatively spliced mRNAs. 845 40

The alternatively spliced type III connecting segment (IIICS) of fibronectin (Fn) contains an amino acid sequence, CS-1, which is recognized by the integrin receptor, alpha 4 beta 1. Plasma Fn inhibits alpha 4 beta 1-dependent binding of lymphocytes and monocytes to CS-1 containing Fn derivatives poorly, suggesting limited exposure of the CS-1 sequence in Fn. To test the availability of CS-1 in plasma Fn, an antibody was raised to the synthetic peptide CS-1. The CS-1 sequence was found to be minimally exposed in plasma Fn; and immobilization of Fn, a model of matrix deposition, caused only a modest increase in its exposure. Digestion of Fn with selected proteases, however, induced substantial expression of the CS-1 sequence. The acid protease cathepsin D generated fragments of 31-33.5 kDa from the COOH-terminal heparin-binding domain of Fn which possessed high immunoreactivity with anti-CS-1. Digestion of Fn with cathepsin B also resulted in the exposure of CS-1 sequence in a 140 kDa fragment. Although the digestion of Fn with neutral proteases (neutrophil elastase, cathepsin G, chymotrypsin, trypsin) generated fragments from the COOH-terminal heparin-binding domain of similar molecular weight as with cathepsin D, the exposure of CS-1 did not occur. Exposure of the CS-1 region by the cathepsins was supported by cell adhesion experiments; digestion of Fn with cathepsins D and B transformed inert plasma Fn to an effective inhibitor of adhesion of lymphoblastoid B and T cells (Ramos, Jurkat, Molt-4) to an immobilized CS-1 conjugate. These results suggest that exposure of the CS-1 sequence in plasma Fn by proteolysis with cathepsins D and B, enzymes implicated in several pathological processes, may serve a regulatory function in cell adhesion. The adhesive function of the CS-1 region in intact Fn appears to be suppressed by the native conformation of the molecule.
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PMID:Proteolysis regulates exposure of the IIICS-1 adhesive sequence in plasma fibronectin. 871 84

Ion transport and regulation of Na(+)-Ca(2+) exchange were examined for two alternatively spliced isoforms of the canine cardiac Na(+)-Ca(2+) exchanger, NCX1.1, to assess the role(s) of the mutually exclusive A and B exons. The exchangers examined, NCX1.3 and NCX1.4, are commonly referred to as the kidney and brain splice variants and differ only in the expression of the BD or AD exons, respectively. Outward Na(+)-Ca(2+) exchange activity was assessed in giant, excised membrane patches from Xenopus laevis oocytes expressing the cloned exchangers, and the characteristics of Na(+)(i)- (i.e., I(1)) and Ca(2+)(i)- (i.e., I(2)) dependent regulation of exchange currents were examined using a variety of experimental protocols. No remarkable differences were observed in the current-voltage relationships of NCX1.3 and NCX1.4, whereas these isoforms differed appreciably in terms of their I(1) and I(2) regulatory properties. Sodium-dependent inactivation of NCX1.3 was considerably more pronounced than that of NCX1.4 and resulted in nearly complete inhibition of steady state currents. This novel feature could be abolished by proteolysis with alpha-chymotrypsin. It appears that expression of the B exon in NCX1.3 imparts a substantially more stable I(1) inactive state of the exchanger than does the A exon of NCX1.4. With respect to I(2) regulation, significant differences were also found between NCX1.3 and NCX1.4. While both exchangers were stimulated by low concentrations of regulatory Ca(2+)(i), NCX1.3 showed a prominent decrease at higher concentrations (>1 microM). This does not appear to be due solely to competition between Ca(2+)(i) and Na(+)(i) at the transport site, as the Ca(2+)(i) affinities of inward currents were nearly identical between the two exchangers. Furthermore, regulatory Ca(2+)(i) had only modest effects on Na(+)(i)-dependent inactivation of NCX1.3, whereas I(1) inactivation of NCX1.4 could be completely eliminated by Ca(2+)(i). Our results establish an important role for the mutually exclusive A and B exons of NCX1 in modulating the characteristics of ionic regulation and provide insight into how alternative splicing tailors the regulatory properties of Na(+)-Ca(2+) exchange to fulfill tissue-specific requirements of Ca(2+) homeostasis.
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PMID:Ionic regulatory properties of brain and kidney splice variants of the NCX1 Na(+)-Ca(2+) exchanger. 1053 74

The human ov-serpin monocyte neutrophil elastase inhibitor (MNEI) is encoded by a single gene SERPINB1. It is a highly efficient inhibitor of neutrophil granule proteases. Four murine genes with high sequence identity with MNEI were identified and fully sequenced, and these were named EIA, EIB, EIC, and EID. EIA, EIB and EIC showed the same seven-exon gene structure as SERPINB1. However, EIC included an additional, alternatively spliced, exon due to the insertion of an endogenous retrovirus-like sequence. EID lacked several exons and is a pseudogene. Reverse transcriptase-PCR showed that EIA, like MNEI, is expressed at high levels in many tissues. EIB is mainly expressed in brain, and EIC was only expressed as splicing variants unlikely to encode a functional serpin. Upon incubation with serine proteases, EIA formed inhibitory covalent complexes with pancreatic and neutrophil elastases, cathepsin G, proteinase-3, and chymotrypsin, as previously shown for MNEI, whereas EIB was only able to do so with cathepsin G. According to the new serpin nomenclature, the genes encoding EIA, EIB, EIC, and EID will be called Serpinb1, Serpinb1b, Serpinb1c, and Serpinb1-ps1. These data demonstrate that the four murine homologs of MNEI have met different evolutionary fates, and that EIA is the mouse ortholog of MNEI.
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PMID:Characterization of four murine homologs of the human ov-serpin monocyte neutrophil elastase inhibitor MNEI (SERPINB1). 1218 54