Abstract
Hepatitis B virus X protein (HBx) plays important roles in viral replication and the development of hepatocellular carcinoma. HBx is a rapid turnover protein and ubiquitin-proteasome pathway has been suggested to influence HBx stability as treatment with proteasome inhibitors increases the levels of HBx protein and causes accumulation of the polyubiquitinated forms of HBx. Deubiquitinases (DUBs) are known to act by removing ubiquitin moieties from proteins and thereby reverse their stability and/or activity. However, no information is available regarding the involvement of DUBs in regulation of ubiquitylation-dependent proteasomal degradation of HBx protein. This study identified the deubiquitylating enzyme USP15 as a critical regulator of HBx protein level. USP15 was found to directly interact with HBx via binding to the HBx region between amino acid residues 51 and 80. USP15 increased HBx protein levels in a dose-dependent manner and siRNA-mediated knockdown of endogenous USP15 reduced HBx protein levels. Increased HBx stability and steady-state level by USP15 were attributable to reduced HBx ubiquitination and proteasomal degradation. Importantly, the transcriptional transactivation function of HBx is enhanced by overexpression of USP15. These results suggest that USP15 plays an essential role in stabilizing HBx and subsequently affects the biological function of HBx.
Highlights
Hepatocellular carcinoma (HCC) is a common malignancy and chronic hepatitis B virus (HBV) infection is one of the major risks of developing HCC, which accounts for more than 80% of HCC cases in high-incidence region[1]
To confirm the interaction of Hepatitis B virus X protein (HBx) with ubiquitin-specific peptidase 15 (USP15) and map the domains of HBx involved in this interaction, HBx and a series of deletion mutants (Fig. 1A) were examined by CytoTrap two-hybrid assay for their ability to interact with USP15
Yeast transformant colonies harboring pMyr-USP15 with deletion mutant of HBxΔ5 1–80 or HBxΔ8 1–120 could not grow in galactose at 37 °C (Fig. 1B, rows 8 and 9), which indicates that the region between HBx amino acid residues 51 and 120 is required for the interaction with USP15
Summary
Hepatocellular carcinoma (HCC) is a common malignancy and chronic hepatitis B virus (HBV) infection is one of the major risks of developing HCC, which accounts for more than 80% of HCC cases in high-incidence region[1]. No deubiquitylating enzymes or deubiquitinases (DUBs) have been identified so far that might account for regulation of ubiquitylation-dependent proteasomal degradation of HBx protein. Many of the HBx binding partners have been identified using immunoprecipitation or using classical yeast two-hybrid (Y2H) screenings While some of these interactions with HBx have been validated, the physiologic relevance of such interactions remains largely uncertain. We found that USP15-mediated deubiquitylation protects HBx from proteasomal degradation increasing the stability and level of HBx protein as well as its transactivation activity. These results suggest that interventions directed at suppressing the level or functional activity of USP15 may be of therapeutic value in HBx-related hepatocarcinogenesis
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