Study On The Effect And Related Mechanism Of MPND On The Protein Level Of Hepatitis B Virus X Protein (HBx)

Hepatitis B virus(HBV)infection resulted in chronic hepatitis,and closely associated with the development of cirrhosis and hepatocellular carcinoma(HCC).The genome of HBV is a partially double-stranded circular DNA of about 3.2 kb pairs with four open reading frames(ORF)of P,C,S and X.The X ORF encodes the HBx protein,which plays an essential role in virus replication and pathogenesis.However,HBx is an extremely unstable protein,which is rapidly degraded within a short time after translation.Degradation of HBx is mainly through the ubiquitin-dependent and ubiquitin-independent proteasome pathway,while the specific mechanisms are not fully elucidated.MPND belongs to the JAMMs family of depubiquitinases(DUBs),which is the only one which possesses metalloproteinase activities in the DUBs.According to different domains,JAMMs can be divided into two groups including JAMM /MPN+proteins and MPN-proteins.MPND belongs to the former.JAMM / MPN+ proteins are zinc dependent homopeptidases,in which the catalytic center composed of JAMM can selectively hydrolyze the peptide chain between ubiquitin / ubiquitin like protein and target protein or between ubiquitin monomers in polymer chains.At present,the function of MPND is not clear.Our previous study confirmed the interaction between MPND and HBx in yeast by Matchmaker two-hybrid screening,however,whether the interaction still exists in mammalian cells and the effect of MPND on HBx are still unknown.The aim of the present study is to investigate the effect of MPND on HBx and elucidate the underlying mechanisms.The first part of this study aims to further confirm the interaction between MPND and HBx in vivo.Firstly,co-immunoprecipitation(Co-IP)was used to confirm the interaction between MPND and HBx in vivo.The co-localization of MPND and HBx in mammalian cells was verified by confocal microscopy.In addition,interaction domains between MPND and HBx were determined by Matchmaker two-hybrid system.The second part of this study aims to investigate the effects of MPND on HBx stability.We detected the effects of MPND on HBx protein level,and the results demonstrated that over expression of MPND could significantly increase the HBx protein level.We also confirmed that the 272-349 aa of MPND also be able to increase increase HBx protein level.The results also showed that over expression of MPND could increase the HBx protein level of encoded by HBV.The third part of this study aims to further explore the inhibition mechanisms of MPND on the degradation of HBx and the effects of MPND on trans-activation activities of HBx.To address these issues,firstly,MG132,a proteasome inhibitor,was subjected and the results revealed that it could reverse the degradation of HBx by MPND.However,the level of polyubiquitination of HBx did not change,suggesting that MPND inhibited the degradation of HBx by ubiquitin-independent proteasome pathway.Finally,the luciferase reporter assay was adapted to evaluate the effects of MPND on the trans-activation activities of HBx.The results showed that MPND could significantly enhance the effects of HBx on transcription activities of NF –?B.