Differential Proteomics Reveals Discrete Functions of Proteins Interacting with Hypo- versus Hyper-phosphorylated NS5A of the Hepatitis C Virus
Journal
Journal of Proteome Research
Journal Volume
18
Journal Issue
7
Pages
2813-2825
Date Issued
2019
Author(s)
Abstract
Protein phosphorylation is a reversible post-translational modification that regulates many biological processes in almost all living forms. In the case of the hepatitis C virus (HCV), the nonstructural protein 5A (NS5A) is believed to transit between hypo- and hyper-phosphorylated forms that interact with host proteins to execute different functions; however, little was known about the proteins that bind either form of NS5A. Here, we generated two high-quality antibodies specific to serine 235 nonphosphorylated hypo- vs serine 235 phosphorylated (pS235) hyper-phosphorylated form of NS5A and for the first time segregated these two forms of NS5A plus their interacting proteins for dimethyl-labeling based proteomics. We identified 629 proteins, of which 238 were quantified in three replicates. Bioinformatics showed 46 proteins that preferentially bind hypo-phosphorylated NS5A are involved in antiviral response and another 46 proteins that bind pS235 hyper-phosphorylated NS5A are involved in liver cancer progression. We further identified a DNA-dependent kinase (DNA-PK) that binds hypo-phosphorylated NS5A. Inhibition of DNA-PK with an inhibitor or via gene-specific knockdown significantly reduced S232 phosphorylation and NS5A hyper-phosphorylation. Because S232 phosphorylation initiates sequential S232/S235/S238 phosphorylation leading to NS5A hyper-phosphorylation, we identified a new protein kinase that regulates a delicate balance of NS5A between hypo- and hyper-phosphorylation states, respectively, involved in host antiviral responses and liver cancer progression. ? 2019 American Chemical Society.
SDGs
Other Subjects
antivirus agent; nonstructural protein 5A; protein kinase; serine; sodium thiosulfate; DNA dependent protein kinase; NS-5 protein, hepatitis C virus; protein binding; viral protein; Article; bioinformatics; cancer growth; controlled study; Hepatitis C virus; Huh-7 cell line; human; human cell; liver cancer; priority journal; protein analysis; protein binding; protein function; protein interaction; protein localization; protein phosphorylation; proteomics; chemistry; complication; Hepacivirus; hepatitis C; immunology; liver tumor; metabolism; pathology; phosphorylation; procedures; proteomics; DNA-Activated Protein Kinase; Hepacivirus; Hepatitis C; Humans; Liver Neoplasms; Phosphorylation; Protein Binding; Proteomics; Viral Nonstructural Proteins
Publisher
American Chemical Society
Type
journal article