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:3.6.1.25 (triphosphatase)
1,529 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The nonstructural protein NS3 of hepatitis C virus (HCV) possesses two enzymatic domains which are thought to be essential for the virus life cycle: an N-terminal serine-type proteinase, responsible for the processing of nonstructural polypeptides, and a C-terminal nucleoside triphosphatase/helicase, presumably involved in the unwinding of the viral genome. The human antibody response to NS3 usually appears early in the course of HCV infection and is predominantly directed against the carboxyl-terminal portion; however, its fine specificity and clinical significance are largely unknown. We have generated a human monoclonal antibody (hMAb), designated CM3.B6, from a cloned B-cell line obtained from the peripheral blood of a patient with chronic HCV infection, which selectively recognized the purified NS3 protein expressed in bacteria or in eukaryotic cells transfected with full-length or NS3 cDNA. Fine-specificity studies revealed that CM3.B6 recognized a 92-amino-acid sequence (clone 8, amino acids 1363 to 1454) selected from an NS3 DNase fragment library but failed to bind to 12-mer peptides synthesized from the same region, suggesting recognition of a conformational B-cell epitope. Experiments using deletion mutants of clone 8 and competitive inhibition studies using a panel of NS3 peptide-specific murine MAbs indicated that limited N-terminal and C-terminal deletions resulted in a significant reduction of hMAb binding to clone 8, thus identifying a minimal antibody binding domain within clone 8. Competition experiments showed that binding of CM3.B6 to the NS3 protein was efficiently inhibited by 39 of 44 (89%) sera from HCV-infected patients, suggesting that the hMAb recognized an immunodominant epitope within the NS3 region. More importantly, recognition of the sequence defined by CM3.B6 appeared to accurately discriminate between viremic and nonviremic anti-HCV positive sera, suggesting potentially relevant clinical applications in the diagnosis and treatment of HCV infection.
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PMID:Significance of the immune response to a major, conformational B-cell epitope on the hepatitis C virus NS3 region defined by a human monoclonal antibody. 751 28

Neurofibromatosis type 1 (NF1) is a common human genetic disease involving various neural crest (NC)-derived cell types, in particular, Schwann cells and melanocytes. The gene responsible for NF1 encodes the protein neurofibromin, which contains a domain with amino acid sequence homology to the ras-guanosine triphosphatase activating protein, suggesting that neurofibromin may play a role in intracellular signaling pathways regulating cellular proliferation or differentiation, or both. To determine whether neurofibromin plays a role in NC cell development, we used antibodies raised against human neurofibromin fusion proteins in western blot and immunocytochemical studies of early avian embryos. These antibodies specifically recognized the 235 kD chicken neurofibromin protein, which was expressed in migrating trunk and cranial NC cells of early embryos (E1.5 to E2), as well as in endothelial and smooth muscle cells of blood vessels and in a subpopulation of non-NC-derived cells in the dermamyotome. At slightly later stages (E3 to E5), neurofibromin immunostaining was observed in various NC derivatives, including dorsal root ganglia and peripheral nerves, as well as non-NC-derived cell types, including heart, skeletal muscle, and kidney. At still later stages (E7 to E9), neurofibromin immunoreactivity was found in almost all tissues in vivo. To determine whether the levels of neurofibromin changed during melanocyte and Schwann cell development, tissue culture experiments were performed. Cultured NC cells were found to express neurofibromin at early time points in culture, but the levels of immunoreactivity decreased as the cells underwent pigmentation. Schwann cells, on the other hand, continued to express neurofibromin in culture. These data suggest, therefore, that neurofibromin may play a role in the development of both NC cells and a variety of non-NC-derived tissues.
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PMID:Regulated expression of neurofibromin in migrating neural crest cells of avian embryos. 756 32

The Hepatitis C Virus (HCV) NS3 protein contains amino acid motifs of a serine proteinase, a nucleotide triphosphatase (NTPase), and an RNA helicase based on amino acid sequence analysis. Proteinase and NTPase activities of the HCV NS3 protein were reported by several investigators. Here, we show that the recombinant HCV NS3 protein purified from a T7 promoter and His-tag expression system possesses an RNA helicase activity. The recombinant HCV NS3 protein consists of 466 amino acids from the carboxy terminal of a HCV NS3 open reading frame and 25 additional residues from the vector. The recombinant HCV NS3 protein was purified by metal-binding chromatography. The helicase activity requires ATP and divalent cations such as Mg2+ and Mn2+. The helicase activity was abolished by monoclonal antibody specific to the HCV NS3 protein.
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PMID:C-terminal domain of the hepatitis C virus NS3 protein contains an RNA helicase activity. 757 85

Guanosine triphosphatase activating protein (GAP) is an essential component of Ras signaling pathways. GAP functions in different cell types as a deactivator and a transmitter of cellular Ras signals. A domain (amino acids 275 to 351) encompassing the Src homology region 3 (SH3) of GAP was found to be essential for GAP signaling. A monoclonal antibody was used to block germinal vesicle breakdown (GVBD) induced by the oncogenic protein Ha-ras Lys12 in Xenopus oocytes. The monoclonal antibody, which was found to recognize the peptide containing amino acids 275 to 351 within the amino-terminal domain of GAP, did not modify the stimulation of the Ha-Ras-GTPase by GAP. Injection of peptides corresponding to amino acids 275 to 351 and 317 to 326 blocked GVBD induced by insulin or by Ha-Ras Lys12 but not that induced by progesterone. These findings confirm that GAP is an effector for Ras in Xenopus oocytes and that the SH3 domain is essential for signal transduction.
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PMID:Identification of the SH3 domain of GAP as an essential sequence for Ras-GAP-mediated signaling. 767 7

The pestivirus bovine viral diarrhea virus (BVDV) p80 protein (referred to here as the NS3 protein) contains amino acid sequence motifs predictive of three enzymatic activities: serine proteinase, nucleoside triphosphatase, and RNA helicase. We have previously demonstrated that the former two enzymatic activities are associated with this protein. Here, we show that a purified recombinant BVDV NS3 protein derived from baculovirus-infected insect cells possesses RNA helicase activity. BVDV NS3 RNA helicase activity was specifically inhibited by monoclonal antibodies to the p80 protein. The activity was dependent on the presence of nucleoside triphosphate and divalent cation, with a preference for ATP and Mn2+. Hydrolysis of the nucleoside triphosphate was necessary for strand displacement. The helicase activity required substrates with an un-base-paired region on the template strand 3' of the duplex region. As few as three un-base-paired nucleotides were sufficient for efficient oligonucleotide displacement. However, the enzyme did not act on substrates having a single-stranded region only to the 5' end of the duplex or on substrates lacking single-stranded regions altogether (blunt-ended duplex substrates), suggesting that the directionality of the BVDV RNA helicase was 3' to 5' with respect to the template strand. The BVDV helicase activity was able to displace both RNA and DNA oligonucleotides from RNA template strands but was unable to release oligonucleotides from DNA templates. The possible role of this activity in pestivirus replication is discussed.
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PMID:Pestivirus NS3 (p80) protein possesses RNA helicase activity. 785 9

The formation of microbicidal oxidants by stimulated phagocytes is a major mechanism of host defence against infection and may also cause unwanted damage to host tissues in the setting of inappropriate inflammation. Recently, the molecular basis for oxidant production has been defined by elucidating the structure, biochemistry and regulation of the phagocyte NADPH oxidase, a multicomponent enzyme that uses NADPH to reduce molecular oxygen to superoxide anion which is then converted to hydrogen peroxide. Many of the advances resulted from the study of phagocytes obtained from patients with inherited abnormalities of the NADPH oxidase system, known as the chronic granulomatous diseases of childhood (CGD). These patients are susceptible to life-threatening infections. The NADPH oxidase is a complex enzyme system that has been shown to contain cytosolic and membrane components that assemble at the plasma membrane with cell activation. These components include a membrane NADPH-binding flavoprotein, cytochrome b558, the cytosolic proteins p47phox, p67phox and a small ras-related guanosine triphosphatase or rac protein that confers guanosine triphosphate sensitivity to the NADPH oxidase. Clinically, the NADPH oxidase system can be stimulated with interferon-gamma, resulting in reduced infections in patients with CGD. In addition, the recent incorporation of genes for the components of the NADPH oxidase into retrovirus vectors has resulted in successful transduction of these genes into blood stem cells from CGD patients with correction of the functional defect. This suggests that gene therapy for correction of CGD will be possible in the near future.
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PMID:Delineation of the phagocyte NADPH oxidase through studies of chronic granulomatous diseases of childhood. 818 51

Sequence motifs within the nonstructural protein NS3 of members of the Flaviviridae family suggest that this protein possesses nucleoside triphosphatase (NTPase) and RNA helicase activity. The RNA-stimulated NTPase activity of this protein from prototypic members of the Pestivirus and Flavivirus genera has recently been established and enzymologically characterized. Here, we experimentally demonstrate that the NS3 protein from a member of the third genus of Flaviviridae, human hepatitis C virus (HCV), also possesses a polynucleotide-stimulated NTPase activity. Characterization of the purified HCV NTPase activity showed that it exhibited reaction condition optima with respect to pH, MgCl2, and salt identical to those of the representative pestivirus and flavivirus enzymes. However, each NTPase also possessed several unique properties when compared with one another. Notably, the profile of polynucleotide stimulation of the NTPase activity was distinct for the three enzymes. The HCV NTPase was the only one whose activity was significantly enhanced by a deoxyribopolynucleotide. Additional distinguishing features among the three enzymes relating to the kinetic properties of their NTPase activities are discussed. These studies provide a foundation for investigation of the putative RNA helicase activity of these proteins and for further study of the role of the NS3 proteins of members of the Flaviviridae in the replication cycle of these viruses.
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PMID:Hepatitis C virus NS3 protein polynucleotide-stimulated nucleoside triphosphatase and comparison with the related pestivirus and flavivirus enzymes. 839 75

Unlike the alpha subunits of heterotrimeric guanosine triphosphate (GTP)-binding proteins, Ras-related GTP-binding proteins have hitherto been considered not to bind or become activated by tetrafluoroaluminate (AIF4-). However, the product of the proto-oncogene ras in its guanosine diphosphate (GDP)-bound form interacted with AIF4 - in the presence of stoichiometric amounts of either of the guanosine triphosphatase (GTPase)-activating proteins (GAPs) p120GAP and neurofibromin. Neither oncogenic Ras nor a GAP mutant without catalytic activity produced such a complex. Together with the finding that the Ras-binding domain of the protein kinase c-Raf, whose binding site on Ras overlaps that of the GAPs, did not induce formation of such a complex, this result suggests that GAP and neurofibromin stabilize the transition state of the GTPase reaction of Ras.
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PMID:Formation of a transition-state analog of the Ras GTPase reaction by Ras-GDP, tetrafluoroaluminate, and GTPase-activating proteins. 865 79

The NS3 protein of flaviviruses is a multifunctional polypeptide required for virus replication. Enzymic activities that have been demonstrated or predicted from the presence of sequence motifs include protease, NTPase, helicase and RNA triphosphatase. Both full-length and truncated forms of NS3 have been identified in infected cells. To examine internal cleavage of the NS3 protein of dengue virus 2 (DEN-2), infected cells or COS cells transfected with cDNA encoding NS2B/3 were radiolabelled and immunoprecipitated with antiserum against NS3 or hyperimmune mouse ascitic fluid. The polypeptides detected were NS2B/3 (Mr 83000), NS3 (Mr 69000), NS3' (Mr 50000) and NS3" (Mr 19000). The latter polypeptide has not been previously identified. For DEN-2, it has been proposed that NS3' results from cleavage at the site ...R457R / GR460... within an RNA helicase sequence motif of NS3. Our results demonstrated that cleavage occurred at this site, and that prior cleavage between NS2B/NS3 was not necessary.
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PMID:Internal proteolysis of the NS3 protein specified by dengue virus 2. 901 55

Transport of macromolecules into and out of nuclei, essential steps in gene expression, are potential points of control. The matrix protein (M protein) of vesicular stomatitis virus (VSV) was shown to block transport of RNAs and proteins between the nucleus and cytoplasm of Xenopus laevis oocytes. The pattern of inhibition indicated that M protein interfered with transport that is dependent on the ras-like nuclear guanosine triphosphatase (GTPase) Ran-TC4 and its associated factors. This inhibition of nuclear transport by M protein explains several observations about the effects of VSV infection on host cell gene expression and suggests that RNA export is closely coupled to protein import.
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PMID:Inhibition of Ran guanosine triphosphatase-dependent nuclear transport by the matrix protein of vesicular stomatitis virus. 920 40


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