E3 Ubiquitin Ligase Siah-1 Molecular Mechanisms Of Regulation Of The Hepatitis B Virus X Protein Stability

Hepatocellular carcinoma (HCC) is a common malignancy. In China, chronic hepatitis B virus (HBV) infection is a major risk of developing HCC. HBV encoded X protein (HBx) is a multifunctional protein, and has a wide range of trans-activation, which can regulate HBV replication and infection, affect the intracellular signaling transduction pathway, cause the abnormal expression of host genes resulting in cell malignant proliferation, transformation and leading to the occurrence of HCC.The transgenic mice overexpressing HBx protein are susceptible to HCC, and knockdown the expression of HBx by RNAi can inhibit the tumorigenesis. Therefore, the expression level of HBx plays an important role in the progression of HCC. HBx can be actively ubiquitinated and subjected to degradation through ubiquitin-proteasome pathway. However, E3ligase in charge of HBx ubiquitination is still unknown. Our present study identified Siah-1as a novel E3ligase to promote HBx ubiquitination and subsequent proteasomal degradation.Tumor suppressor p53is shown to increase HBx ubiqitination, however, p53itselt is not the E3ligase. Therefore, we speculate that the specific E3ligases for facilitating HBx ubiquitiantion exsist in these p53-induciable E3ligases. Many studies have demonstrated that p53can induce the expression of Siah-1function as E3ligase. Siah-1expression was down-regulated in HCC samples, which correlates with larger, poorly differentiated and/or advanced staged HCC. Furthermore, Siah-1is reported to target herpesvirus-encoded ORF45protein for degradation via ubiquitin-proteasome pathway. So we investigated whether Siah-1was the candidate E3ligase for mediating HBx ubiquitination and degradation.Our results indicate that HBx protein level was significantly reduced in a dose-dependent manner when coexpressed with Siah-1but not with the ligase-deficient mutant. However, treatment with proteasome inhibitor MG132rescued HBx reduction mediated by Siah-1.Additionally, knockdown of endogenous Siah-1by siRNA resulted in up-regulation of HBx protein level. Moreover, Siah-1accelerated HBx protein degradation. Thus, these results suggested that E3ligase Siah-1stimulated the proteasomal degradation of HBx protein. Luciferase reporter assay showed that co-expressed Siah-1attenuated the GRE, HSE, CRE signal pathways transactivated by ectopically expressed HBx, and these signalings may account for the cell migration and proliferation. We found that Siah-1could interact with HBx both in vitro and in vivo. The immunofluorescence staining showed that Siah-1and HBx could co-localize in cytoplasm around the envelope of nuclear. Further studies showed that the middle and C-terminus of HBx, and the cysteine-rich region of Siah-1are essential for their association. Ubiquitination assay in vivo indicated that Siah-1could facilitate HBx poly-ubiquitination depending on the E3ligase activity of Siah-1.In addition, Siah-1could help to the association between HBx and the C8subunit of20S proteasome, which contribute to the degradation of HBx. Since the C-terminal deletion of HBx is a highly frequent event in HBV-infected HCC samples, and these HBx C-terminal truncates have more potential to cause carcinogenesis. Accordingly, we constructed three C-terminal truncates of HBx detected in HCC. Our result found that Siah-1could not degrade these truncates, which may account for improving the stability of these truncates probably. We also confirmed that wild type p53induced Siah-1expression. Importantly, when Siah-1expression was reduced, p53-mediated HBx reduction was reversed, the result demonstratd that Siah-1participated in p53-mediated HBx reduction. In addition, we determined the expression difference of Siah-1between tumors and adjacent non-tumorous counterparts in39pairs of HCC samples. Our results confirmed that Siah-1level was down-regulated in HCCs. Meanwhile, we performed the mutation detection within the ORF of Siah-1in270HCC cases and9hepatoma cell lines. Our results further confirmed that Siah-1gene is highly conservative and the mutation frequency is quite low in HCCs. Thereby, these results indicated that expression level of Siah-1may be more important to impact HCC but not Siah-1mutation.In summary, our study firstly identified Siah-1as a novel E3ligase for HBx ubiquitination and degradation. Therefore, we proposed that Siah-1may be significant in ubiquitin-dependent degradation of HBx. Meanwhile, our study provides some new clues for a comprehensive understanding the molecular mechanism of HCC.