Activity-based protein profiling of the hepatitis C virus replication in Huh-7 hepatoma cells using a non-directed active site probe

Proteome Science

Research

David R Blais¹  Ragunath Singaravelu²  John Paul Pezacki³  Craig S McKay⁴ 

[1] Steacie Institute for Molecular Sciences, National Research Council Canada, K1A 0R6, Ottawa, Ontario, Canada;Steacie Institute for Molecular Sciences, National Research Council Canada, K1A 0R6, Ottawa, Ontario, Canada;Department of Biochemistry, Microbiology and Immunology, University of Ottawa, K1N 6N5, Ottawa, Ontario, Canada;Steacie Institute for Molecular Sciences, National Research Council Canada, K1A 0R6, Ottawa, Ontario, Canada;Department of Biochemistry, Microbiology and Immunology, University of Ottawa, K1N 6N5, Ottawa, Ontario, Canada;Department of Chemistry, University of Ottawa, K1N 6N5, Ottawa, Ontario, Canada;Steacie Institute for Molecular Sciences, National Research Council Canada, K1A 0R6, Ottawa, Ontario, Canada;Department of Chemistry, University of Ottawa, K1N 6N5, Ottawa, Ontario, Canada;

关键词: Protein Disulfide Isomerase;    Purine Nucleoside Phosphorylase;    ABPP;    Phenyl Sulfonate;    Subgenomic Replicon;   

DOI  :  10.1186/1477-5956-8-5

 received in 2009-10-08, accepted in 2010-02-04,  发布年份 2010

来源: Springer

【 摘 要 】

BackgroundHepatitis C virus (HCV) poses a growing threat to global health as it often leads to serious liver diseases and is one of the primary causes for liver transplantation. Currently, no vaccines are available to prevent HCV infection and clinical treatments have limited success. Since HCV has a small proteome, it relies on many host cell proteins to complete its life cycle. In this study, we used a non-directed phenyl sulfonate ester probe (PS4≡) to selectively target a broad range of enzyme families that show differential activity during HCV replication in Huh-7 cells.ResultsThe PS4≡ probe successfully targeted 19 active proteins in nine distinct protein families, some that were predominantly labeled in situ compared to the in vitro labeled cell homogenate. Nine proteins revealed altered activity levels during HCV replication. Some candidates identified, such as heat shock 70 kDa protein 8 (or HSP70 cognate), have been shown to influence viral release and abundance of cellular lipid droplets. Other differentially active PS4≡ targets, such as electron transfer flavoprotein alpha, protein disulfide isomerase A5, and nuclear distribution gene C homolog, constitute novel proteins that potentially mediate HCV propagation.ConclusionsThese findings demonstrate the practicality and versatility of non-directed activity-based protein profiling (ABPP) to complement directed methods and accelerate the discovery of altered protein activities associated with pathological states such as HCV replication. Collectively, these results highlight the ability of in situ ABPP approaches to facilitate the identification of enzymes that are either predominantly or exclusively labeled in living cells. Several of these differentially active enzymes represent possible HCV-host interactions that could be targeted for diagnostic or therapeutic purposes.

【 授权许可】

Unknown   
© Singaravelu et al; licensee BioMed Central Ltd. 2010. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.