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 flavivirus NS3 protein plays an important role in the cleavage and processing of the viral polyprotein and in the synthesis of the viral RNA. NS3 recruits NS2B and NS5 proteins to form complexes possessing protease and replicase activities through protease and nucleoside triphosphatase/helicase domains. We have found that NS3 also induces apoptosis. Expression of the Langat (LGT) virus NS3 protein resulted in a cleavage of cellular DNA and reduced the viability of cells. Coexpression of NS3 with apoptotic inhibitors (CrmA and P35) and addition of caspase peptide substrates (Z-VAD-FMK and Z-IETD-FMK) to NS3-transfected cells blocked NS3-induced apoptosis. In cotransfection experiments, NS3 bound to caspase-8 and enhanced caspase-8-mediated apoptosis. NS3 and caspase-8 colocalized in the cytoplasm of transfected cells. Deletion analysis demonstrated that at least two regions of NS3 contribute to its apoptotic activities. The protease and helicase domains are each able to bind to caspase-8, while the protease domain alone induces apoptosis. The protease domain and tetrahelix region of the helicase domain are required for NS3 to augment caspase-8-mediated apoptosis. Thus, the LGT virus NS3 protein is a multifunctional protein that binds to caspase-8 and induces apoptosis.
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PMID:Langat flavivirus protease NS3 binds caspase-8 and induces apoptosis. 1199 98

The RNA nucleoside triphosphatase (NTPase)/helicases represent a large family of proteins that are detected in almost all biological systems where RNA plays a central role. The enzymes are capable of enzymatically unwinding duplex RNA structures by disrupting the hydrogen bonds that keep the two strands together. The strand separating activity is associated with hydrolysis of nucleoside triphosphate (NTP). Because of this, potential specific inhibitors of NTPase/helicases could act by one or more of the following mechanisms: (i) inhibition of NTPase activity by interference with NTP binding, (ii) inhibition of NTPase activity by an allosteric mechanism and (iii) inhibition of the coupling of NTP hydrolysis at the unwinding reaction. There are also other inhibitory mechanisms conceivable, which may involve a modulation of the interaction of the enzyme with its RNA substrate, for example, (iv) the competitive inhibition of RNA binding and (v) the inhibition of the unwinding by sterical blockade of the translocation of the NTPase/helicase along the polynucleotide chain. NTPase/helicase has also been identified in the viral genome of hepatitis C virus (HCV) which is a member of the Flaviviridae family. It is conceivable that the inhibition of the unwinding activity of the enzyme leads to the inhibition of virus replication and this may represent a novel antiviral strategy. This review updates the current spectrum of inhibitors targeting different mechanisms by which the NTPase and/or helicase activities of the HCV NTPase/helicase are inhibited. Consequently, some of the compounds might be important as antiviral agents against HCV.
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PMID:Nucleotide triphosphatase/helicase of hepatitis C virus as a target for antiviral therapy. 1220 78

Bacteriophage T7 gp4A' protein is a hexameric helicase-primase protein that separates the strands of a duplex DNA in a reaction coupled to dTTP hydrolysis. Here we reexamine in more detail the kinetic mechanism of dTTP hydrolysis by a preassembled T7 helicase hexamer in the absence of DNA. Pre-steady state dTTP hydrolysis kinetics showed a distinct burst whose amplitude indicated that a preformed hexamer of T7 helicase hydrolyzes on an average one dTTP per hexamer. The pre-steady state chase-time experiments provided evidence for sequential hydrolysis of two dTTPs. The medium [(18)O]P(i) exchange experiments failed to detect dTTP synthesis, indicating that the less than six-site hydrolysis observed is not due to reversible dTTP hydrolysis on the helicase active site. The P(i)-release rate was measured directly using a stopped-flow fluorescence assay, and it was found that the rate of dTTP hydrolysis on the helicase active site is eight times faster than the P(i)-release rate, which in turn is three times faster than the dTDP release rate. Thus, the rate-limiting step in the pathway of helicase-catalyzed deoxythymidine triphosphatase (dTTPase) reaction is the release of dTDP. Chase-time dTTPase kinetics in the steady state phase provided evidence for two to three slowly hydrolyzing dTTPase sites on the hexamer. The results of this study are therefore consistent with those reported earlier (Hingorani, M. M., Washington, M. T., Moore, K. C., and Patel, S. S. (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 5012-5017), and they support a model of dTTP hydrolysis by T7 helicase hexamer that is similar to the binding change mechanism of F(1)-ATPase with dTTP hydrolysis occurring sequentially at the catalytic sites.
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PMID:Kinetic pathway of dTTP hydrolysis by hexameric T7 helicase-primase in the absence of DNA. 1222 5

Nonstructural protein 3 (NS3) of Hepatitis C virus (HCV) is a multifunctional protein and possesses protease, nucleotide triphosphatase and helicase activities. The N-terminal domain of NS3 (amino acids 1027-1218; delta NS3) has a trypsin-like protease activity and is essential for processing of viral polyprotein. Accordingly it is a potential target for anti-HCV drugs and we isolated RNA aptamers (Kd = 10 nM, Ki = 100 nM) using in vitro selection strategy. To study the interaction between delta NS3 and its aptamer, we applied alanine scanning mutagenesis and constructed seven mutant proteins at positive amino acid residues on the surface of delta NS3. Binding and inhibitory activities of the NS3 aptamer against mutant proteins were kinetically analyzed. These results clarified that especially Arg161 and Arg130 are important for interaction with the NS3 aptamer.
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PMID:Analysis of aptamer binding site for HCV-NS3 protease by alanine scanning mutagenesis. 1290 64

Hepatitis C virus (HCV) causes a persistent infection, chronic hepatitis, and leads to hepatocellular carcinoma. Nonstructural protein 3 (NS3) of HCV possesses protease, nucleoside triphosphatase, and helicase activities. Using the yeast two hybrid assay, we identified Sm-D1, a host protein that binds to NS3. Sm-D1 is a component of small nuclear ribonucleoprotein (snRNP) complexes which are associated with autoimmune disease. Sm-D1 has Gly-Arg (GR) repeats at the C terminus, which contains dimethylarginine modified by post-translational modification and may constitute an immunoreactive determinant. Deletion mutants revealed that the C-terminal region of Sm-D1 containing the GR repeats was the binding region for NS3, and the expression feature of Sm-D1 was affected by co-expression of NS3. Immunostaining assay demonstrated that NS3 was also present in the nucleus of cells overexpressing Sm-D1, although it was usually found in cytoplasm. The localization of NS3 could change following interaction with Sm-D1 and affect the function of Sm-D1.
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PMID:Hepatitis C virus nonstructural protein NS3 binds to Sm-D1, a small nuclear ribonucleoprotein associated with autoimmune disease. 1452 21

The VP6 protein of bluetongue virus possesses a number of activities, including nucleoside triphosphatase, RNA binding, and helicase activity (N. Stauber, J. Martinez-Costas, G. Sutton, K. Monastyrskaya, and P. Roy, J. Virol. 71:7220-7226, 1997). Although the enzymatic functions of the protein have been documented, a detailed structure and function study has not been completed and the oligomeric form of the protein in solution has not been described. In this study, we have characterized VP6 activity by creating site-directed mutations in the putative functional helicase domains. Mutant proteins were expressed at high levels in an insect cell by using recombinant baculoviruses purified and analyzed for ATP binding, ATP hydrolysis, and RNA unwinding activities. UV cross-linking experiments indicated that the lysine residue in the conserved motif AXXGXGK(110)V is directly involved in ATP binding, whereas mutant R(205)Q in the arginine-rich motif ER(205)XGRXXR bound ATP at a level comparable to that of the wild-type protein. The RNA binding activity was drastically altered in the R(205)Q mutant and was also affected in the K(110)N mutant. Helicase activity was altered in both mutants. The mutation E(157)N in the DEXX sequence, presumed to act as a Walker B motif, showed an intermediate activity, implying that this motif does not play a crucial role in VP6 function. Purified protein demonstrated stable oligomers with a ring-like morphology in the presence of nucleic acids similar to those shown by other helicases. Gel filtration chromatography, native gel electrophoresis, and glycerol gradient analysis clearly indicated multiple oligomeric forms of VP6.
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PMID:Defining the structure-function relationships of bluetongue virus helicase protein VP6. 1455 20

5'-O-(4-fluorosulphonylbenzoyl)-esters of ribavirin (FSBR), adenosine (FSBA), guanosine (FSBG) and inosine (FSBI) were obtained by acylation of the 5'-OH of adenosine, guanosine, inosine, and ribavirin with 4-fluorosulphonylbenzoyl chloride (FSBCI) in HMPA. The above derivatives were tested as inhibitors of nucleoside triphosphatase (NTPase)/helicase activities of Flaviviridae: hepatitis C virus (HCV), West Nile virus (WNV), Japanese encephalitis virus (JEV) and dengue virus (DENV) and polymerase activity of HCV and WNV. When the unwinding activity of viral NTPase/helicases was tested under standard conditions, only weak inhibition was obtained with FSBI (IC50 > or = 120 microM) and in the case of FSBG even an activation was seen. The preincubation of the NTPase/helicases with the 5'-O-FSB derivatives increased the inhibitory effect. Screening of the 5'-O-FSB derivatives on inhibition of the WNV and HCV RNA polymerases employing GTP or UTP substrates revealed rather modest inhibitory effect. FSBI exhibited the highest inhibitory activity against WNV (IC50 = 70 microM with UTP substrate) and HCV polymerase (IC50 = 80 microM with GTP substrate). Other 5'-O-FSB derivatives were very weak inhibitors or completely failed to show any activity against HCV and WNV enzymes. In contrast to the NTPase/helicases the preincubation of the polymerases did not influence the inhibition.
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PMID:Synthesis and evaluation of ATP-binding site directed potential inhibitors of nucleoside triphosphatases/helicases and polymerases of hepatitis C and other selected Flaviviridae viruses. 1507 13

Severe acute respiratory syndrome coronavirus (SARS-CoV), a newly identified group 2 coronavirus, is the causative agent of severe acute respiratory syndrome, a life-threatening form of pneumonia in humans. Coronavirus replication and transcription are highly specialized processes of cytoplasmic RNA synthesis that localize to virus-induced membrane structures and were recently proposed to involve a complex enzymatic machinery that, besides RNA-dependent RNA polymerase, helicase, and protease activities, also involves a series of RNA-processing enzymes that are not found in most other RNA virus families. Here, we characterized the enzymatic activities of a recombinant form of the SARS-CoV helicase (nonstructural protein [nsp] 13), a superfamily 1 helicase with an N-terminal zinc-binding domain. We report that nsp13 has both RNA and DNA duplex-unwinding activities. SARS-CoV nsp13 unwinds its substrates in a 5'-to-3' direction and features a remarkable processivity, allowing efficient strand separation of extended regions of double-stranded RNA and DNA. Characterization of the nsp13-associated (deoxy)nucleoside triphosphatase ([dNTPase) activities revealed that all natural nucleotides and deoxynucleotides are substrates of nsp13, with ATP, dATP, and GTP being hydrolyzed slightly more efficiently than other nucleotides. Furthermore, we established an RNA 5'-triphosphatase activity for the SARS-CoV nsp13 helicase which may be involved in the formation of the 5' cap structure of viral RNAs. The data suggest that the (d)NTPase and RNA 5'-triphosphatase activities of nsp13 have a common active site. Finally, we established that, in SARS-CoV-infected Vero E6 cells, nsp13 localizes to membranes that appear to be derived from the endoplasmic reticulum and are the likely site of SARS-CoV RNA synthesis.
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PMID:Multiple enzymatic activities associated with severe acute respiratory syndrome coronavirus helicase. 1514 Sep 59

The non-structural protein 3 (NS3) of hepatitis C virus (HCV) is a highly promising target for anti-HCV therapy because of its multiple enzymatic activities, such as RNA-stimulated nucleoside triphosphatase, RNA helicase and serine protease. The helicase domain of NS3 as well as domain 2 of the helicase were expressed in a baculovirus system to obtain in high yield active proteins for prospective studies of complexes of the helicase with its inhibitors. A novel direct fluorometric test of helicase activity with a quenched DNA substrate, 3' labeled with a Cy3 dye and 5' labeled with a Black Hole Quencher, was developed and optimal reaction conditions established. This test based on fluorescence resonance energy transfer is simple and fast. It allows for direct measurements of enzyme activity, circumventing laborious and complicated radioactive techniques that are poorly reproducible. The results obtained encourage us to propose this new fluorescent assay as a method enabling high throughput screening of anti-helicase compounds.
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PMID:Direct fluorometric measurement of hepatitis C virus helicase activity. 1517 32

The human coronavirus 229E (HCoV-229E) replicase gene-encoded nonstructural protein 13 (nsp13) contains an N-terminal zinc-binding domain and a C-terminal superfamily 1 helicase domain. A histidine-tagged form of nsp13, which was expressed in insect cells and purified, is reported to unwind efficiently both partial-duplex RNA and DNA of up to several hundred base pairs. Characterization of the nsp13-associated nucleoside triphosphatase (NTPase) activities revealed that all natural ribonucleotides and nucleotides are substrates of nsp13, with ATP, dATP, and GTP being hydrolyzed most efficiently. Using the NTPase active site, HCoV-229E nsp13 also mediates RNA 5'-triphosphatase activity, which may be involved in the capping of viral RNAs.
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PMID:Human coronavirus 229E nonstructural protein 13: characterization of duplex-unwinding, nucleoside triphosphatase, and RNA 5'-triphosphatase activities. 1522 Apr 59


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