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Wenjin Su - One of the best experts on this subject based on the ideXlab platform.
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purification characterization and cdna cloning of a myofibril bound Serine Proteinase from the skeletal muscle of crucian carp carassius auratus
Journal of Agricultural and Food Chemistry, 2007Co-Authors: Kenji Hara, Wenjin SuAbstract:A myofibril-bound Serine Proteinase (MBSP) was highly purified from the skeletal muscle of crucian carp (Carasius auratus) by acidic treatment of myofibril solution and chromatographies on Q-Sepharose and benzamidine-Sepharose 6B. MBSP revealed a main protein band of approximately 28 kDa on SDS-polyacrylamide gel electrophoresis (PAGE) and was particularly inhibited by Serine Proteinase inhibitors. Substrate-specificity analysis revealed that the enzyme specifically cleaved at the carboxyl side of arginine and lysine residues, suggesting the characteristics of a trypsin-type Serine Proteinase. MBSP gene was cloned on the basis of the N-terminal sequence and the conserved active site peptide of Serine Proteinases together with 5‘-rapid amplification of cDNA ends (5‘-RACE) and 3‘-RACE. The coding region gave an amino acid sequence of 242 residues including the initiation methionine and a signal peptide of 20 residues. Amino acid residues of His60, Asp106, and Ser196 consisting of the catalytic triad of seri...
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the effect of soybean trypsin inhibitor on the degradation of myofibrillar proteins by an endogenous Serine Proteinase of crucian carp
Food Chemistry, 2006Co-Authors: Xinjing Jiang, Kenji Hara, Zhijun Zhang, Wenjin SuAbstract:Abstract The effect of soybean trypsin inhibitor (STI) on the degradation of crucian carp myofibrillar proteins caused by an endogenous myofibril-bound Serine Proteinase (MBSP) was studied. Soybean trypsin inhibitor was purified to high homogeneity and mixed with myofibrils and its inhibitory effect on myofibrillar protein degradation was investigated. In the absence of STI, proteolysis of myofibrillar proteins including myosin heavy chain, α-actinin, actin and tropomyosin could be identified after incubation at 55 °C for 5–20 min depending on the kind of the protein. In contrast, in the presence of STI, with final concentration of 0.75 μg/ml, proteolysis of these proteins was greatly suppressed even after incubation for 1 h, suggesting STI is an effective inhibitor in preventing myofibrillar protein degradation caused by a Serine Proteinase that is quite possibly MBSP. Though STI has disadvantages for food digestion, as a native food grade inhibitor, it is safe as a food additive, especially at low concentration. Because in surimi production, the decrease of elasticity is always accompanied with the degradation of myofibrillar proteins, thus, the present result suggested the possibility that STI could be applicable in surimi production in order to enhance the elasticity that is the quality of the final products.
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degradation of myofibrillar proteins by a myofibril bound Serine Proteinase in the skeletal muscle of crucian carp carasius auratus
Food Chemistry, 2006Co-Authors: Xinjing Jiang, Zhijun Zhang, Huichang Zhong, Wenjin SuAbstract:A myofibril-bound Serine Proteinase (MBSP) in the skeletal muscle of crucian carp (Carasius auratus) was identified. Hydrolysis of myofibrillar proteins by the endogenous MBSP was studied. Myosin heavy chain (MHC) was degraded markedly when crucian carp myofibril was incubated at 55 °C, as shown by SDS-PAGE. Prolonged incubation of myofibril at 55 °C also caused the obvious degradation of tropomyosin, while the decomposition of other myofibrillar proteins, such as α-actinin and actin, was slight, as detected by Western blotting. The results suggest the existence of an endogenous myofibril-associated Proteinase in crucian carp myofibril, which efficiently cleaves MHC and tropomyosin. Serine Proteinase inhibitors (Lima bean trypsin inhibitor, PMSF and benzamidine) greatly suppressed the degradation of MHC, caused by the enzyme, while inhibitors for cysteine, metallo-, and aspartic Proteinases showed only partial or incomplete inhibitory effects, indicating that the endogenous Proteinase is a Serine Proteinase. Substrate specificity analysis, using partially purified crucian carp MBSP, suggested that the enzyme is a trypsin-like Serine Proteinase.
Anchalee Tassanakajon - One of the best experts on this subject based on the ideXlab platform.
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a snake like Serine Proteinase pmsnake activates prophenoloxidase activating system in black tiger shrimp penaeus monodon
Developmental and Comparative Immunology, 2017Co-Authors: Warunthorn Monwan, Piti Amparyup, Anchalee TassanakajonAbstract:Clip domain Serine Proteinases (ClipSPs) play critical roles in the activation of proteolytic cascade in invertebrate immune systems including the prophenoloxidase (proPO) activating system. In this study, we characterized a snake-like Serine protease, namely PmSnake, from the shrimp Penaeus monodon which has previously been identified based on the subtractive cDNA library of proPO double-stranded RNA (dsRNA)-treated hemocytes. An open reading frame of PmSnake contains 1068 bp encoding a predicted protein of 355 amino acid residues with a putative signal peptide of 22 amino acids and two conserved domains (N-terminal clip domain and C-terminal trypsin-like Serine Proteinase domain). Sequence analysis revealed that PmSnake was closest to the AeSnake from ant Acromyrmex echinatior (53% similarity), but was quite relatively distant from other shrimp PmclipSPs. PmSnake transcript was mainly expressed in shrimp hemocytes and up-regulated after systemic Vibrio harveyi infection indicating that it is an immune-responsive gene. Suppression of PmSnake expression by dsRNA interference reduced both transcript and protein levels leading to a reduction of the hemolymph phenoloxidase (PO) activity (36%), compared to the control, suggesting that the PmSnake functions as a clip-SP in shrimp proPO system. Western blot analysis using anti-PmSnake showed that the PmSnake was detected in hemocytes but not in cell-free plasma. In vitro PO activity and Serine Proteinase activity assays showed that adding rPmSnake into the shrimp hemolymph could increase PO activity as well as Serine Proteinase activity suggesting that the rPmSnake activates the proPO system via Serine Proteinase cascade.
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molecular cloning characterization and expression of a masquerade like Serine Proteinase homologue from black tiger shrimp penaeus monodon
Fish & Shellfish Immunology, 2007Co-Authors: Piti Amparyup, Rungrat Jitvaropas, Naritsara Pulsook, Anchalee TassanakajonAbstract:Abstract A full-length cDNA of a masquerade-like Serine Proteinase homologue ( Pm MasSPH) of Penaeus monodon was cloned and characterized by rapid amplification cDNA end (RACE) method. The complete cDNA sequence of 1958 bp contains an open reading frame (ORF) of 1572 bp, encoding a 523 amino acid protein including a 19 amino acid signal peptide. The calculated molecular mass of the mature protein (504 amino acids) is 51.58 kDa with an estimated p I of 4.86. Pm MasSPH has most of the structural characteristics of insect prophenoloxidase activating factors (PPAFs) (the N -terminal clip domain and the C -terminal Serine Proteinase-like domain) but in the N -terminal region there are extensive glycine-rich repeats (LGGQGGG). Sequence comparison showed that the deduced amino acid of Pm MasSPH has an overall similarity of 69%, 68% and 61% to those of Apis mellifera PPAF, Callinectes sapidus PPAF and Tenebrio molitor PPAF, respectively. A neighbour-joining tree revealed a clear differentiation of each species and also indicated that Pm MasSPH and C. sapidus PPAF are closely related phylogenetically. In situ hybridisation and real-time RT–PCR analyses showed that Pm MasSPH transcript in haemocytes of P. monodon increased within 24 h after Vibrio harveyi injection.
Kenji Hara - One of the best experts on this subject based on the ideXlab platform.
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purification characterization and cdna cloning of a myofibril bound Serine Proteinase from the skeletal muscle of crucian carp carassius auratus
Journal of Agricultural and Food Chemistry, 2007Co-Authors: Kenji Hara, Wenjin SuAbstract:A myofibril-bound Serine Proteinase (MBSP) was highly purified from the skeletal muscle of crucian carp (Carasius auratus) by acidic treatment of myofibril solution and chromatographies on Q-Sepharose and benzamidine-Sepharose 6B. MBSP revealed a main protein band of approximately 28 kDa on SDS-polyacrylamide gel electrophoresis (PAGE) and was particularly inhibited by Serine Proteinase inhibitors. Substrate-specificity analysis revealed that the enzyme specifically cleaved at the carboxyl side of arginine and lysine residues, suggesting the characteristics of a trypsin-type Serine Proteinase. MBSP gene was cloned on the basis of the N-terminal sequence and the conserved active site peptide of Serine Proteinases together with 5‘-rapid amplification of cDNA ends (5‘-RACE) and 3‘-RACE. The coding region gave an amino acid sequence of 242 residues including the initiation methionine and a signal peptide of 20 residues. Amino acid residues of His60, Asp106, and Ser196 consisting of the catalytic triad of seri...
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purification characterization and cdna cloning of a myofibril bound Serine Proteinase from the skeletal muscle of crucian carp carassius auratus
Journal of Agricultural and Food Chemistry, 2007Co-Authors: Chuan Guo, Minjie Cao, Guangming Liu, Xiongshui Lin, Kenji HaraAbstract:A myofibril-bound Serine Proteinase (MBSP) was highly purified from the skeletal muscle of crucian carp (Carasius auratus) by acidic treatment of myofibril solution and chromatographies on Q-Sepharose and benzamidine-Sepharose 6B. MBSP revealed a main protein band of approximately 28 kDa on SDS-polyacrylamide gel electrophoresis (PAGE) and was particularly inhibited by Serine Proteinase inhibitors. Substrate-specificity analysis revealed that the enzyme specifically cleaved at the carboxyl side of arginine and lysine residues, suggesting the characteristics of a trypsin-type Serine Proteinase. MBSP gene was cloned on the basis of the N-terminal sequence and the conserved active site peptide of Serine Proteinases together with 5'-rapid amplification of cDNA ends (5'-RACE) and 3'-RACE. The coding region gave an amino acid sequence of 242 residues including the initiation methionine and a signal peptide of 20 residues. Amino acid residues of His60, Asp106, and Ser196 consisting of the catalytic triad of Serine Proteinases were conserved in the sequence. Crucian carp MBSP shared relatively high identities with other Serine Proteinases, especially in well-conserved regions.
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the effect of soybean trypsin inhibitor on the degradation of myofibrillar proteins by an endogenous Serine Proteinase of crucian carp
Food Chemistry, 2006Co-Authors: Xinjing Jiang, Kenji Hara, Zhijun Zhang, Wenjin SuAbstract:Abstract The effect of soybean trypsin inhibitor (STI) on the degradation of crucian carp myofibrillar proteins caused by an endogenous myofibril-bound Serine Proteinase (MBSP) was studied. Soybean trypsin inhibitor was purified to high homogeneity and mixed with myofibrils and its inhibitory effect on myofibrillar protein degradation was investigated. In the absence of STI, proteolysis of myofibrillar proteins including myosin heavy chain, α-actinin, actin and tropomyosin could be identified after incubation at 55 °C for 5–20 min depending on the kind of the protein. In contrast, in the presence of STI, with final concentration of 0.75 μg/ml, proteolysis of these proteins was greatly suppressed even after incubation for 1 h, suggesting STI is an effective inhibitor in preventing myofibrillar protein degradation caused by a Serine Proteinase that is quite possibly MBSP. Though STI has disadvantages for food digestion, as a native food grade inhibitor, it is safe as a food additive, especially at low concentration. Because in surimi production, the decrease of elasticity is always accompanied with the degradation of myofibrillar proteins, thus, the present result suggested the possibility that STI could be applicable in surimi production in order to enhance the elasticity that is the quality of the final products.
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identification of a myofibril bound Serine Proteinase mbsp in the skeletal muscle of lizard fish saurida wanieso which specifically cleaves the arginine site
Comparative Biochemistry and Physiology B, 2000Co-Authors: Kiyoshi Osatomi, Kenji Hara, Tadashi IshiharaAbstract:Abstract A myofibril-bound Serine Proteinase (MBSP) from the skeletal muscle of lizard fish (Saurida wanieso) was purified to homogeneity by a heating treatment followed by a series of column chromatographies on DEAE-Sephacel, Sephacryl S-200, Q-Sepharose, Hydroxyapatite and Benzamidine-Sepharose 6B, and characterized enzymatically. On SDS-polyacrylamide gel electrophoresis (SDS-PAGE), the purified enzyme showed a band with molecular mass of ≈29 kDa under reducing conditions, while 60 kDa under non-reducing conditions. The optimum temperature of the enzyme was 50°C using t-butyloxycarbonyl-Phe-Ser-Arg-4-methylcoumaryl-7-amide (Boc-Phe-Ser-Arg-MCA) as a substrate. Substrate specificity analysis both using MCA-substrates and peptides showed that MBSP specifically cleaved at the carboxyl side of the arginine residue. Inhibitor susceptibility analysis revealed that MBSP was inhibited effectively by Pefabloc SC, soybean trypsin inhibitor (STI) and aprotinin, indicating the characteristic of a Serine Proteinase. When myofibril was incubated with the enzyme, it optically degraded myosin heavy chain at 55–60°C, while α-actinin and actin were not at all hydrolyzed as detected by immunoblotting. The N-terminal amino acid sequence of MBSP was partially determined as IVGGAEXVPY- and was very homologous to other Serine proteases.
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myofibril bound Serine Proteinase mbp and its degradation of myofibrillar proteins
Journal of Food Science, 1999Co-Authors: Kenji Hara, Kiyoshi Osatomi, Katsuyasu Tachibana, T Izumi, Tadashi IshiharaAbstract:Proteolysis of a myofibril-bound Serine Proteinase (MBP) from carp Cyprinus carpio on myofibrillar proteins and their gel formation ability were investigated. MBP readily decomposed myosin heavy chain as indicated by SDS-PAGE. In the preparation of kamaboko, the gel formation ability was diminished by addition of MBP. The optimum degradation temperatures of MBP to myosin heavy chain in myofibril and kamaboko gel were 55°C and 60°C, respectively. The degradation effects of MBP on actin, α-actinin and tropomyosin were studied by the immunoblotting method. Because of its myofibril-bound and myofibrillar protein degradation characteristics, MBP was regarded as the Proteinase most probably involved in the modori effect.
Michael R. Kanost - One of the best experts on this subject based on the ideXlab platform.
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prophenoloxidase activating Proteinase 3 pap 3 from manduca sexta hemolymph a clip domain Serine Proteinase regulated by serpin 1j and Serine Proteinase homologs
Insect Biochemistry and Molecular Biology, 2003Co-Authors: Haobo Jiang, Yang Wang, Yifei Zhu, Michael R. KanostAbstract:Phenoloxidase (PO) is a key enzyme implicated in several defense mechanisms in insects and crustaceans. It is converted from prophenoloxidase (proPO) through limited proteolysis by prophenoloxidase-activating Proteinase (PAP). We previously isolated PAP-1 from integument and PAP-2 from hemolymph of the tobacco hornworm, Manduca sexta. Here, we report the purification, characterization, and regulation of PAP-3 from the hemolymph. Similar to M. sexta PAP-2, PAP-3 consists of two amino-terminal clip domains followed by a carboxyl-terminal catalytic domain, whereas PAP-1 contains only one clip domain at its amino-terminus. Purified PAP-3 cleaved proPO at Arg51 and generated a low level of PO activity. However, the enzyme efficiently activated proPO when M. sexta Serine Proteinase homolog-1 and -2 were present. These Proteinase-like proteins associate with immulectin-2, a pattern-recognition receptor for lipopolysaccharide. M. sexta PAP-3 was inhibited by recombinant serpin-1J, which formed an SDS-stable complex with the enzyme. PAP-3 mRNA was detected at a low level in the fat body or hemocytes of naive larvae, but was elevated in insects that had been challenged with bacteria. These data, along with our previous results on PAP-1 and PAP-2, indicate that proPO activation by PAPs is a tightly regulated process. Individual PAPs could play different roles during immune responses and developmental processes.
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prophenoloxidase activating Proteinase 2 from hemolymph of manduca sexta a bacteria inducible Serine Proteinase containing two clip domains
Journal of Biological Chemistry, 2003Co-Authors: Haobo Jiang, Yang Wang, Michael R. KanostAbstract:Proteolytic activation of prophenoloxidase in insects is a component of the host defense system against invading pathogens and parasites. We have purified from hemolymph of the tobacco hornworm, Manduca sexta, a new Serine Proteinase that cleaves prophenoloxidase. This enzyme, designated prophenoloxidase-activating Proteinase-2 (PAP-2), differs from another PAP, previously isolated from integuments of the same insect (PAP-1). PAP-2 contains two clip domains at its amino terminus and a catalytic domain at its carboxyl terminus, whereas PAP-1 has only one clip domain. Purified PAP-2 cleaved prophenoloxidase at Arg51but yielded a product that has little phenoloxidase activity. However, in the presence of two Serine Proteinase homologs, active phenoloxidase was generated at a much higher level, and it formed covalently linked, high molecular weight oligomers. The Serine Proteinase homologs associate with a bacteria-binding lectin in M. sextahemolymph, indicating that they may be important for ensuring that the activation of prophenoloxidase occurs only in the vicinity of invading microorganisms. PAP-2 mRNA was not detected in naive larval fat body or hemocytes, but it became abundant in these tissues after the insects were injected with bacteria.
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Nonproteolytic Serine Proteinase homologs are involved in prophenoloxidase activation in the tobacco hornworm, Manduca sexta.
Insect biochemistry and molecular biology, 2003Co-Authors: Haobo Jiang, Yang Wang, Michael R. KanostAbstract:In insects, the prophenoloxidase activation system is a defense mechanism against parasites and pathogens. Recognition of parasites or pathogens by pattern recognition receptors triggers activation of a Serine Proteinase cascade, leading to activation of prophenoloxidase-activating Proteinase (PAP). PAP converts inactive prophenoloxidase (proPO) to active phenoloxidase (PO), which then catalyzes oxidation of phenolic compounds that can polymerize to form melanin. Because quinone intermediates and melanin are toxic to both hosts and pathogens, activation of proPO must be tightly regulated and localized. We report here purification and cDNA cloning of Serine Proteinase homologs (SPHs) from the tobacco hornworm, Manduca sexta, which interact with PAP-1 in proPO activation. Two SPHs were co-purified from plasma of M. sexta larvae with immulectin-2, a C-type lectin that binds to bacterial lipopolysaccharide. They contain an amino-terminal clip domain connected to a carboxyl-terminal Serine Proteinase-like domain. PAP-1 alone cannot efficiently activate proPO, but a mixture of SPHs and PAP-1 was much more effective for proPO activation. Immulectin-2, proPO and PAP-1 in hemolymph bound to the immobilized recombinant Proteinase-like domain of SPH-1, indicating that a complex containing these proteins may exist in hemolymph. Since immulectin-2 is a pattern recognition receptor that binds to surface carbohydrates on pathogens, such a protein complex may localize activation of proPO on the surface of pathogens. SPH, which binds to immulectin-2, may function as a mediator to recruit proPO and PAP to the site of infection.
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Serine Proteinase inhibitors in arthropod immunity
Developmental and Comparative Immunology, 1999Co-Authors: Michael R. KanostAbstract:Abstract Arthropod hemolymph contains proteins with Serine Proteinase inhibitory activity. These inhibitors may exist in plasma or in hemocyte granules. Serine Proteinase inhibitors from the Kazal, Kunitz, α-macroglobulin, and serpin families have been identified in arthropod hemolymph and have been characterized biochemically. Two new families of low molecular weight Serine Proteinase inhibitors have recently been discovered: one in silkworms (the Bombyx family) and another in locusts and a crayfish. The Serine Proteinase inhibitors in arthropod hemolymph are likely to function in protecting their hosts from infection by pathogens or parasites. Some may inhibit fungal or bacterial Proteinases. Others probably have roles in regulating endogenous Proteinases involved in coagulation, prophenol oxidase activation, or cytokine activation.
Charles M Rice - One of the best experts on this subject based on the ideXlab platform.
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bovine viral diarrhea virus ns3 Serine Proteinase polyprotein cleavage sites cofactor requirements and molecular model of an enzyme essential for pestivirus replication
Journal of Virology, 1997Co-Authors: Ernesto Mendez, Paul R Caron, Chao Lin, Mark A Murcko, Marc S Collett, Charles M RiceAbstract:Members of the Flaviviridae encode a Serine Proteinase termed NS3 that is responsible for processing at several sites in the viral polyproteins. In this report, we show that the NS3 Proteinase of the pestivirus bovine viral diarrhea virus (BVDV) (NADL strain) is required for processing at nonstructural (NS) protein sites 3/4A, 4A/4B, 4B/5A, and 5A/5B but not for cleavage at the junction between NS2 and NS3. Cleavage sites of the Proteinase were determined by amino-terminal sequence analysis of the NS4A, NS4B, NS5A, and NS5B proteins. A conserved leucine residue is found at the P1 position of all four cleavage sites, followed by either Serine (3/4A, 4B/5A, and 5A/5B sites) or alanine (4A/4B site) at the P1' position. Consistent with this cleavage site preference, a structural model of the pestivirus NS3 Proteinase predicts a highly hydrophobic P1 specificity pocket. trans-Processing experiments implicate the 64-residue NS4A protein as an NS3 Proteinase cofactor required for cleavage at the 4B/5A and 5A/5B sites. Finally, using a full-length functional BVDV cDNA clone, we demonstrate that a catalytically active NS3 Serine Proteinase is essential for pestivirus replication.
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the hepatitis c virus ns3 Serine Proteinase and ns4a cofactor establishment of a cell free trans processing assay
Proceedings of the National Academy of Sciences of the United States of America, 1995Co-Authors: Chao Lin, Charles M RiceAbstract:Abstract The hepatitis C virus RNA genome encodes a long polyprotein that is proteolytically processed into at least 10 products. The order of these cleavage products in the polyprotein is NH2-C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B -COOH. A Serine Proteinase domain located in the N-terminal one-third of nonstructural protein NS3 mediates cleavage at four downstream sites (the 3/4A, 4A/4B, 4B/5A, and 5A/5B sites). In addition to the Proteinase catalytic domain, the NS4A protein is required for processing at the 4B/5A site but not at the 5A/5B site. These cleavage events are likely to be essential for virus replication, making the Serine Proteinase an attractive antiviral target. Here we describe an in vitro assay where the NS3-4A polyprotein, NS3, the Serine Proteinase domain (the N-terminal 181 residues of NS3), and the NS4A cofactor were produced by cell-free translation and tested for trans-processing of radiolabeled substrates. Polyprotein substrates, NS4A-4B or truncated NS5A-5B, were cleaved in trans by all forms of the Proteinase, whereas NS4A was also required for NS4B-5A processing. Proteolysis was abolished by substitution mutations previously shown to inactivate the Proteinase or block cleavage at specific sites in vivo. Furthermore, N-terminal sequence analysis established that cleavage in vitro occurred at the authentic 4A/4B site. Translation in the presence of microsomal membranes enhanced processing for some, but not all, Proteinase-substrate combinations. Trans-processing was both time and temperature dependent and was eliminated by treatment with a variety of detergents above their critical micelle concentrations. Among many common Proteinase inhibitors tested, only high (millimolar) concentrations of Serine Proteinase inhibitors tosyllysyl chloromethyl ketone and 4-(2-aminoethyl)benzenesulfonyl fluoride inactivated the NS3 Proteinase. This in vitro assay should facilitate purification and further characterization of the viral Serine Proteinase and identification of molecules which selectively inhibit its activity.
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a central region in the hepatitis c virus ns4a protein allows formation of an active ns3 ns4a Serine Proteinase complex in vivo and in vitro
Journal of Virology, 1995Co-Authors: Chao Lin, J A Thomson, Charles M RiceAbstract:A virus-encoded Serine Proteinase mediates four site-specific cleavages in the hepatitis C virus polyprotein. In addition to the catalytic domain, which is located in the N-terminal one-third of nonstructural protein NS3, the 54-residue NS4A protein is required for cleavage at some but not all sites. Here, we provide evidence for a non-ionic detergent-stable interaction between NS4A and the NS3 Serine Proteinase domain and demonstrate that the central region of NS4A plays a key role in NS4A-dependent processing. Hydrophobic residues, in particular Ile-29, were shown to be important for NS4A activity, and a synthetic peptide, spanning NS4A residues 22 to 34, could substitute for intact NS4A in a cell-free trans cleavage assay. Furthermore, NS4A mutations, which abolished or inhibited processing, correlated with destabilization of the NS3-NS4A complex. These results suggest that a stable interaction exists between the central region of NS4A and the NS3 catalytic domain which is required for NS4A-dependent processing. Since NS4A is required for processing at certain Serine Proteinase-dependent cleavage sites, this interaction may represent a new target for development of antiviral compounds.
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hepatitis c virus ns3 Serine Proteinase trans cleavage requirements and processing kinetics
Journal of Virology, 1994Co-Authors: Chao Lin, Bela Pragai, Arash Grakoui, Charles M RiceAbstract:The hepatitis C virus H strain (HCV-H) polyprotein is cleaved to produce at least 10 distinct products, in the order of NH2-C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B -COOH. An HCV-encoded Serine Proteinase activity in NS3 is required for cleavage at four sites in the nonstructural region (3/4A, 4A/4B, 4B/5A, and 5A/5B). In this report, the HCV-H Serine Proteinase domain (the N-terminal 181 residues of NS3) was tested for its ability to mediate trans-processing at these four sites. By using an NS3-5B substrate with an inactivated Serine Proteinase domain, trans-cleavage was observed at all sites except for the 3/4A site. Deletion of the inactive Proteinase domain led to efficient trans-processing at the 3/4A site. Smaller NS4A-4B and NS5A-5B substrates were processed efficiently in trans; however, cleavage of an NS4B-5A substrate occurred only when the Serine Proteinase domain was coexpressed with NS4A. Only the N-terminal 35 amino acids of NS4A were required for this activity. Thus, while NS4A appears to be absolutely required for trans-cleavage at the 4B/5A site, it is not an essential cofactor for Serine Proteinase activity. To begin to examine the conservation (or divergence) of Serine Proteinase-substrate interactions during HCV evolution, we demonstrated that similar trans-processing occurred when the Proteinase domains and substrates were derived from two different HCV subtypes. These results are encouraging for the development of broadly effective HCV Serine Proteinase inhibitors as antiviral agents. Finally, the kinetics of processing in the nonstructural region was examined by pulse-chase analysis. NS3-containing precursors were absent, indicating that the 2/3 and 3/4A cleavages occur rapidly. In contrast, processing of the NS4A-5B region appeared to involve multiple pathways, and significant quantities of various polyprotein intermediates were observed. NS5B, the putative RNA polymerase, was found to be significantly less stable than the other mature cleavage products. This instability appeared to be an inherent property of NS5B and did not depend on expression of other viral polypeptides, including the HCV-encoded Proteinases.
