Activity-based proteome profiling of hepatoma cells during hepatitis C virus replication using protease substrate probes.
Activity-based protein profiling (ABPP) offers direct insight into changes in catalytic activity of enzyme classes in complex proteomes, rather than protein or transcript abundance. Here, ABPP was performed in Huh7 hepatoma cell lines with a group of ABPP probes composed of an N-acetylated amino acid, that mimic the P(1) position in protease peptide substrates. Five different probes bearing distinct amino acids (Ser, Thr, Phe, Glu and His) labeled 54 differentially active proteins, including proteases, other hydrolases, oxidoreductases and isomerases. Four of the six protease families were targeted based on their P(1) substrate preferences. The broader specificity of the labeling observed could be explained by the substrate-based targeting nature and the electrophilic properties of the ABPP probes. When applied to Huh7 cells stably replicating hepatitis C virus (HCV) subgenomic replicon RNA, four proteins showed reduced activity, while three proteins had increased activity during HCV replication. These differentially active hits included carboxylesterase 1, cathepsin D, HSP105, protein disulfide isomerase 1 and A6, chaperonin containing TCP1 and isochorismatase domain containing 1, which demonstrated substrate preferences by being labeled by specific substrate probes. This illustrates the broader activity-based profiling capabilities of these substrate-based probes to reveal novel enzyme candidates and their potential roles during HCV replication.
Blais
David R
DR
Steacie Institute for Molecular Sciences, National Research Council Canada, 100 Sussex Drive, Ottawa, ON, K1A 0R6, Canada.
Brûlotte
Marc
M
Qian
Yiming
Y
Bélanger
Sylvie
S
Yao
Shao Q
SQ
Pezacki
John Paul
JP
D001483:Base Sequence; D015153:Blotting, Western; D045744:Cell Line, Tumor; D017931:DNA Primers; D016174:Hepacivirus / Q000235:genetics / Q000502:physiology*; D006801:Humans; D020543:Proteome; D012367:RNA, Viral / Q000235:genetics; D014779:Virus Replication
D017931:DNA Primers; D020543:Proteome; D012367:RNA, Viral