Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:O95477 (membrane-bound)
 29,236 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The protein C system provides important control of blood coagulation by regulating the activities of factor VIIIa (FVIIIa) and factor Va (FVa), cofactors in the activation of factor X and prothrombin, respectively. The system comprises membrane-bound and circulating proteins that assemble into multi-molecular complexes on cell surfaces. Vitamin K-dependent protein C, the key component of the system, circulates in blood as zymogen to an anticoagulant serine protease. It is efficiently activated on the surface of endothelial cells by thrombin bound to the membrane protein thrombomodulin. The endothelial protein C receptor (EPCR) further stimulates the protein C activation. Activated protein C (APC) together with its cofactor protein S inhibits coagulation by degrading FVIIIa and FVa on the surface of negatively charged phospholipid membranes. Efficient FVIIIa degradation by APC requires not only protein S but also intact FV, which like thrombin is a Janus-faced protein with both procoagulant and anticoagulant potential. In addition to its anticoagulant properties, APC has antiinflammatory and antiapoptotic functions, which are exerted when APC binds to EPCR and proteolytic cleaves protease-activated receptor 1 (PAR-1). The protein C system is physiologically important, and genetic defects affecting the system are the most common risk factors of venous thrombosis. The proteins of the protein C system are composed of multiple domains and the 3-dimensional structures of several of the proteins are known. The molecular recognition of the protein C system is progressively being unraveled, giving us new insights into this fascinating and intricate molecular scenario at the atomic level.
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PMID:Regulation of blood coagulation by the protein C anticoagulant pathway: novel insights into structure-function relationships and molecular recognition. 1586 Jul 36

Epithin is a type II transmembrane serine protease that exists in a soluble and membrane-bound form. Shedding is thought to be important in regulating its action, but little is known regarding the intracellular events that trigger such shedding. Here, we show that phorbol myristate acetate (PMA) causes the release of epithin. It also causes accumulation of the protein at the site of cell-cell contacts, and this accumulation is dependent on the formation of cortical actin. In addition, we have identified the actin-binding protein, filamin, as the linker between epithin and the actin cytoskeleton. The interaction of epithin and filamin was enhanced by PMA, and epithin was not released from filamin-deficient M2 cells. We also show that the release of epithin does not require its own activity and is blocked by a metalloprotease inhibitor, GM6001. These results show that filamin has an essential role in shedding by linking epithin to the as yet unidentified metalloprotease-shedding enzyme(s).
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PMID:Filamin is essential for shedding of the transmembrane serine protease, epithin. 1617 Mar 3

In the past 20 years, our understanding of the workings of complement regulatory protein, CD46 (membrane cofactor protein), has grown as has the impressive list of pathogens interacting with this membrane-bound complement inhibitor. Referred to as a "pathogen magnet," CD46 serves as a receptor for seven human pathogens. Initially discovered as a widely expressed C3b- and C4b-binding protein, it was subsequently shown to be a cofactor for the serine protease factor I to inactivate by limited proteolysis these two opsonins and components of the convertases. The involvement of CD46 in reproductive processes continues to be an emerging story. It is a protector of placental tissue, but it may also play a more direct role in reproduction through its expression on the inner acrosomal membrane of spermatozoa. Cross-linking CD46 with antibodies or natural or pathogenic ligands induces rapid turnover and signaling events. In this regard, much attention is currently focused on generating human T lymphocyte regulatory cells by cross-linking CD46. Finally, highlighting its importance in protecting cells against excessive complement activation is the discovery that even a heterozygous deficiency of CD46 predisposes to hemolytic uremic syndrome.
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PMID:Emerging roles and new functions of CD46. 1620 Apr 5

Tryptases are trypsin-like serine proteases whose expression is restricted to cells of hematopoietic origin, notably mast cells. gamma-Tryptase, a recently described member of the family also known as transmembrane tryptase (TMT), is a membrane-bound serine protease found in the secretory granules or on the surface of degranulated mast cells. The 321 amino acid protein contains an 18 amino acid propeptide linked to the catalytic domain (cd), followed by a single-span transmembrane domain. gamma-Tryptase is distinguished from other human mast cell tryptases by the presence of two unique cysteine residues, Cys(26) and Cys(145), that are predicted to form an intra-molecular disulfide bond linking the propeptide to the catalytic domain to form the mature, membrane-anchored two-chain enzyme. We expressed gamma-tryptase as either a soluble, single-chain enzyme with a C-terminal His tag (cd gamma-tryptase) or as a soluble pseudozymogen activated by enterokinase cleavage to form a two-chain protein with an N-terminal His tag (tc gamma-tryptase). Both recombinant proteins were expressed at high levels in Pichia pastoris and purified by affinity chromatography. The two forms of gamma-tryptase exhibit comparable kinetic parameters, indicating the propeptide does not contribute significantly to the substrate affinity or activity of the protease. Substrate and inhibitor library screening indicate that gamma-tryptase possesses a substrate preference and inhibitor profile distinct from that of beta-tryptase. Although the role of gamma-tryptase in mast cell function is unknown, our results suggest that it is likely to be distinct from that of beta-tryptase.
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PMID:Expression and characterization of recombinant gamma-tryptase. 1681 34

Tissue factor, the physiologic trigger of blood clotting, is the membrane-anchored protein cofactor for the plasma serine protease, factor VIIa. Tissue factor is hypothesized to position and align the active site of factor VIIa relative to the membrane surface for optimum proteolytic attack on the scissile bonds of membrane-bound protein substrates such as factor X. We tested this hypothesis by raising the factor VIIa binding site above the membrane surface by creating chimeras containing the tissue factor ectodomain linked to varying portions of the membrane-anchored protein, P-selectin. The tissue factor/P-selectin chimeras bound factor VIIa with high affinity and supported full allosteric activation of factor VIIa toward tripeptidyl-amide substrates. That the active site of factor VIIa was raised above the membrane surface when bound to tissue factor/P-selectin chimeras was confirmed using resonance energy transfer techniques in which appropriate fluorescent dyes were placed in the active site of factor VIIa and at the membrane surface. The chimeras were deficient in supporting factor X activation by factor VIIa due to decreased k(cat). The chimeras were also markedly deficient in clotting plasma, although incubating factor VII or VIIa with the chimeras prior to the addition of plasma restored much of their procoagulant activity. Interestingly, all chimeras fully supported tissue factor-dependent factor VII autoactivation. These studies indicate that proper positioning of the factor VII/VIIa binding site on tissue factor above the membrane surface is important for efficient rates of activation of factor X by this membrane-bound enzyme/cofactor complex.
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PMID:Raising the active site of factor VIIa above the membrane surface reduces its procoagulant activity but not factor VII autoactivation. 1683 45

Examples abound of membrane-bound enzymes for which the local membrane environment plays an important role, including the ectoenzyme that triggers blood clotting, the plasma serine protease, factor VIIa, bound to the integral membrane protein, tissue factor. The activity of this enzyme complex is markedly influenced by lipid bilayer composition and further by tissue factor partitioning into membrane microdomains on some cell surfaces. Unfortunately, little is known about how membrane microdomain composition controls factor VIIa-tissue factor activity, as reactions catalyzed by membrane-tethered enzymes are typically studied under conditions in which the experimenter cannot control the composition of the membrane in the immediate vicinity of the enzyme. To overcome this problem, we used a nanoscale approach that afforded complete control over the membrane environment surrounding tissue factor by assembling the factor VIIa.tissue factor complex on stable bilayers containing 67 +/- 1 phospholipid molecules/leaflet (Nanodiscs). We investigated how local changes in phospholipid bilayer composition modulate the activity of the factor VIIa.tissue factor complex. We also addressed whether this enzyme requires a pool of membrane-bound protein substrate (factor X) for efficient catalysis, or alternatively if it could efficiently activate factor X, which binds directly to the membrane nanodomain adjacent to tissue factor. We have shown that full proteolytic activity of the factor VIIa.tissue factor complex requires extremely high local concentrations of anionic phospholipids and further that a large pool of membrane-bound factor X is not required to support sustained catalysis.
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PMID:The local phospholipid environment modulates the activation of blood clotting. 1720 Jan 19

Dengue viruses (DENV) have 5'-capped RNA genomes of (+) polarity and encode a single polyprotein precursor that is processed into mature viral proteins. NS2B, NS3 and NS5 proteins catalyse/activate enzyme activities that are required for key processes in the virus life cycle. The heterodimeric NS2B/NS3 is a serine protease required for processing. Using a high-throughput protease assay, we screened a small molecule chemical library and identified -200 compounds having > or = 50% inhibition. Moreover, NS3 exhibits RNA-stimulated NTPase, RNA helicase and the 5'-RNA triphosphatase activities. The NTPase and the 5'-RTPase activities of NS3 are stimulated by interaction with NS5. Moreover, the conserved, positively charged motif in DENV-2 NS3, 184RKRK, is required for RNA binding and modulates the RNA-dependent enzyme activities of NS3. To study viral replication, a variety of methods are used such as the in vitro RNA-dependent RNA polymerase assays that utilize lysates from DENV-2-infected mosquito or mammalian cells or the purified NS5 along with exogenous short subgenomic viral RNAs or the replicative intracellular membrane-bound viral RNAs as templates. In addition, a cell-based DENV-2 replicon RNA encoding a luciferase reporter is also used to examine the role of cis-acting elements within the 3' UTR and the RKRK motif in viral replication.
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PMID:Multiple enzyme activities of flavivirus proteins. 1731 55

In live cells, autoactivation of matriptase, a membrane-bound serine protease, can be induced by lysophospholipids, androgens, and the polyanionic compound suramin. These structurally distinct chemicals induce different signaling pathways and cellular events that somehow, in a cell type-specific manner, lead to activation of matriptase immediately followed by inhibition of matriptase by hepatocyte growth factor activator inhibitor 1 (HAI-1). In the current study, we established an analogous matriptase autoactivation system in an in vitro cell-free setting and showed that a burst of matriptase activation and HAI-1-mediated inhibition spontaneously occurred in the insoluble fractions of cell homogenates and that this in vitro activation could be attenuated by a soluble suppressive factor(s) in cytosolic fractions. Immunofluorescence staining and subcellular fractionation studies revealed that matriptase activation occurred in the perinuclear regions. Solubilization of matriptase from cell homogenates by Triton X-100 or sonication of cell homogenates completely inhibited the effect, suggesting that matriptase activation requires proper lipid bilayer microenvironments, potentially allowing appropriate interactions of matriptase zymogens with HAI-1 and other components. Matriptase activation occurred in a narrow pH range (from pH 5.2 to 7.2), with a sharp increase in activation at the transition from pH 5.2 to 5.4, and could be completely suppressed by moderately increased ionic strength. Protease inhibitors only modestly affected activation, whereas 30 nM (5 microg/ml) of anti-matriptase LDL receptor domain 3 monoclonal antibodies completely blocked activation. These atypical biochemical features are consistent with a mechanism for autoactivation of matriptase that requires protein-protein interactions but not active proteases.
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PMID:Autoactivation of matriptase in vitro: requirement for biomembrane and LDL receptor domain. 1734 10

The complement system is important for protection from invading pathogens, removal of waste products and guidance of the immune response. Furthermore, complement can be also targeted to cancer cells. However, membrane-bound inhibitors over-expressed by certain types of tumor cells restrict the cytotoxic activity of complement. Herein we report that non-small cell lung cancer (NSCLC) cells produce soluble complement inhibitors factor I (FI) and C4b-binding protein (C4BP). FI is a serine protease capable of degrading the activated complement components C3b and C4b, whilst C4BP acts as its cofactor. Furthermore, NSCLC cells express membrane-bound regulators and shed membrane cofactor protein (MCP), which shares cofactor function with C4BP. Secretion of FI from NSCLC cells was higher than previously reported for any non-hepatic source and FI produced by these cells could efficiently support cleavage of C3b and C4b. In vitro functional assays revealed that additional FI significantly decreased C3 deposition and complement-dependent lysis, particularly when cofactors were added. Our results demonstrate that soluble inhibitors produced by NSCLC cells may provide further protection from complement beyond the level ensured by membrane-bound inhibitors and, as such, contribute to the aggressive phenotype of these lung cancer cells.
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PMID:Non-small cell lung cancer cells produce a functional set of complement factor I and its soluble cofactors. 1754 10

It is well known that the Escherichia coli inner membrane-bound protease DegS is a periplasmic stress sensor for unfolded outer membrane proteins (OMPs). Previous studies have also shown that the outer membrane protease OmpT activates plasminogen in vitro and this may be exploited by bacteria in the course of pathogenesis. However, there has been no research on the plasminogen activation ability of the important periplasmic protein DegS. Accordingly, in this study, the whole-length and truncated degS genes were separately overexpressed in Escherichia coli, the recombinant proteins purified by affinity chromatography, and their plasminogen activator role tested in vitro. The results suggested that the whole-length DegS was able to activate plasminogen on a plasma plate. The truncated form of DegS (residues 80-345), designated delta DegS, also acted as a plasminogen activator, as confirmed by different assays. The serine protease property of delta DegS was verified based on the complete inhibition of its enzyme activity by PMSF (phenylmethanesulfonyl fluoride). Therefore, the present results indicate that DegS is a plasminogen activator in vitro.
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PMID:Abridged region from Escherichia coli periplasmic stress sensor DegS acts as plasminogen activator in vitro. 1805 Dec 69


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