The NTPase/helicase domain of hepatitis C virus nonstructural protein 3 inhibits protein kinase C independently of its NTPase activity

TY - JOUR

T1 - The NTPase/helicase domain of hepatitis C virus nonstructural protein 3 inhibits protein kinase C independently of its NTPase activity

AU - Hartjen, Philip

AU - Höchst, Bastian

AU - Heim, Denise

AU - von der Kammer, Henning

AU - Lucke, Judith

AU - Reinholz, Michael

AU - Baier, Andrea

AU - Smeets, Ralf

AU - Wege, Henning

AU - Borowski, Peter

AU - Schulze Zur Wiesch, Julian

PY - 2013/9/1

Y1 - 2013/9/1

N2 - Helicase motif VI is a short arginine-rich motif within the NTPase/helicase domain of the non-structural protein 3 (NS3) of the hepatitis C virus (HCV). We previously demonstrated that it reduces the catalytic activity and intracellular shuttling of protein kinase C (PKC). Thus, NS3-mediated PKC inhibition may be involved in HCV-associated hepatocellular carcinoma (HCC). In this study, we expand on our earlier results, which were obtained in experiments with short fragments of NS3, to show for the first time that the catalytically active, longer C-terminal NTPase/helicase of NS3 acts as a potent PKC inhibitor in vitro. PKC inhibition assays with the NTPase-inactive mutant NS3h-D1316A revealed a mixed type kinetic inhibition pattern. A broad range of 11 PKC isotypes was tested and all of the PKC isotypes were inhibited with IC₅₀-values in the low micromolar range. These findings were confirmed for the wild-type NTPase/helicase domain in a non-radiometric PKC inhibition assay with ATP regeneration to rule out any effect of ATP hydrolysis caused by its NTPase activity. PKCα was inhibited with a micromolar IC₅₀ in this assay, which compares well with our result for NS3h-D1316A (IC₅₀ = 0.7 μM). In summary, these results confirm that catalytically active NS3 NTPase/helicase can act in an analogous manner to shorter NS3 fragments as a pseudosubstrate inhibitor of PKC.

AB - Helicase motif VI is a short arginine-rich motif within the NTPase/helicase domain of the non-structural protein 3 (NS3) of the hepatitis C virus (HCV). We previously demonstrated that it reduces the catalytic activity and intracellular shuttling of protein kinase C (PKC). Thus, NS3-mediated PKC inhibition may be involved in HCV-associated hepatocellular carcinoma (HCC). In this study, we expand on our earlier results, which were obtained in experiments with short fragments of NS3, to show for the first time that the catalytically active, longer C-terminal NTPase/helicase of NS3 acts as a potent PKC inhibitor in vitro. PKC inhibition assays with the NTPase-inactive mutant NS3h-D1316A revealed a mixed type kinetic inhibition pattern. A broad range of 11 PKC isotypes was tested and all of the PKC isotypes were inhibited with IC₅₀-values in the low micromolar range. These findings were confirmed for the wild-type NTPase/helicase domain in a non-radiometric PKC inhibition assay with ATP regeneration to rule out any effect of ATP hydrolysis caused by its NTPase activity. PKCα was inhibited with a micromolar IC₅₀ in this assay, which compares well with our result for NS3h-D1316A (IC₅₀ = 0.7 μM). In summary, these results confirm that catalytically active NS3 NTPase/helicase can act in an analogous manner to shorter NS3 fragments as a pseudosubstrate inhibitor of PKC.

KW - Adenosine Triphosphate

KW - Amino Acid Sequence

KW - Binding Sites

KW - Biocatalysis

KW - Electrophoresis, Polyacrylamide Gel

KW - Hepacivirus

KW - Hydrolysis

KW - Kinetics

KW - Models, Molecular

KW - Mutation

KW - Nucleoside-Triphosphatase

KW - Protein Kinase C

KW - Protein Structure, Tertiary

KW - RNA Helicases

KW - Viral Nonstructural Proteins

U2 - 10.2478/s11658-013-0099-7

DO - 10.2478/s11658-013-0099-7

M3 - SCORING: Journal article

C2 - 23893289

VL - 18

SP - 447

EP - 458

JO - CELL MOL BIOL LETT

JF - CELL MOL BIOL LETT

SN - 1425-8153

IS - 3

ER -