Identification of Androgen Receptor Associated Proteins Related with HBV Enhanced AR Activity

• Main Authors: Ya-Wen Cheng, 鄭雅紋
• Other Authors: Shiou-Hwei Yeh
• Format: Others
• Language: zh-TW
• Published: 2008
• Online Access: http://ndltd.ncl.edu.tw/handle/95183627114047932709

碩士 === 國立臺灣大學 === 微生物學研究所 === 96 === Taiwan is a hyperendemic area for hepatitis B virus (HBV) infection, which significantly increase the risk of HCC. One characteristic for HBV-related HCC is the male gender preference. Our previous studies identified a possible mechanism underlying the male gender preference, showing that HBx viral protein can up-regulate the transcriptional activity of androgen receptor (AR) in an androgen concentration dependent manner. However, how does HBx enhances the AR activity still remained un-clarified. According to the findings in prostate cancers that the transcriptional activity of AR can be regulated by its associated co-regulators. We propose to test the possibility that HBx can also through affecting the AR associated co-regulators to enhance its transcriptional activity as one possible mechanism. Therefore, the specific aim of this study is to investigate the AR associated proteins involved in HBV enhanced AR activation. The strategy is to identify the specific AR coregulators changing the affinity with AR in the presence of HBx, and then further investigate their functional roles in the HBx-enhanced AR transactivation. The Tandem Affinity Purification (TAP) system was used to purify the AR associated proteins in the current study, due to its great advantages for isolation of proteins with high specificity under physiological conditions. We have constructed pNTAP-AR and pNCTAP-AR plasmids, and found that HBx can enhance the transcriptional activity of pNTAP-AR (but not pNCTAP-AR) close to the level of wild type AR without tag. We thus co-transfected pNTAP-AR and 1.5-mer HBV constructs into Huh-7 cells for the subsequent TAP purification studies. Aided by the MALDI-TOF MS analysis, we have identified one protein, Acetyl-coA carboxylase alpha (ACCα), which was enriched in the fraction of HBV+AR compared with AR only. Further study pointed out that the increased interaction is caused by HBx viral protein. Aided by co-immunoprecipitation analysis, we found that ACCαcan indeed interact with AR. ACCα is an enzyme involved in the synthesis of fatty acid through catalyzing the condensation of malonyl-CoA. To further investigate the functional effect of the interaction between ACCαand AR on AR activity, we approached by knockdown the expression of ACCαby lentivirus based si-RNA in Huh-7 cells. Intriguingly, we found that both the protein level and transcriptional activity of AR were significantly deceased. Aided by quantitative PCR, we found that the effect of si-ACCαon AR protein did not occur at the RNA level. Instead, the results from MG132 treatment suggested that the effect might be attributed by the effect ACCαon regulating the protein degradation of AR. Moreover, we have identified that the interaction of AR and ACCα might mainly occur at the cytosol rather than in the nucleus. In summary, we have identified a novel AR associated protein, ACCα, with its association with AR enhanced by HBx. It might provide a possible regulatory mechanism for HBx enhanced AR activation. Although how does the cytosolic ACCα regulate the protein amount of AR through affecting its degradation process awaits further investigation, the current study points out ACCα as one potential target for blocking the AR activation in male hepatocarcinogenesis.