The Nucleocytoplasmic Shuttling And Post-translational Modifications Study Of Silkworm Autophagy Receptor Protein P62 And Ref(2)p

Silkworm p62?also known as sequestosome-1,SilkDB:BGIBMGA002620-PA?is a protein with several functional domains,and plays important roles in many cellular processes such as autophagy,apoptosis.Compared to p62,the p62 paralog,Ref?2?P lacks the Phox and Bem1p?PB1?domain.In this study,the nucleocytoplasmic shuttling characteristics and post-translational modifications of silkworm p62 and Ref?2?P were mainly investigated.The results were showed as follows.1.Characterization of nucleocytoplasmic shuttling of p62 and Ref?2?P?Fusion expression vectors of eGFP with p62 and Ref?2?P was constructed,and then transfected into BmN cells.The confocal laser-scanning microscope analysis showed that p62 and Ref?2?P were distributed in the cytoplasm in a spot-like manner.By adding the nuclear export inhibitor lepramycin B?LMB?,p62 and Ref?2?P were displayed in the nucleus.These results suggested that p62 and Ref?2?P were CRM1-dependent nucleocytoplasmic shuttling proteins.In addition,the transport period of p62?K8A/D59A?into the nucleus is much shorter than that of wild type p62,indicating that PB1 domain played an important role in retention of p62 in the cytoplasm.?To determine the nuclear export-signals?NESs?and nuclear localization signals?NLSs?,Ref?2?P was taken as an example.Truncated and site-directed mutants of Ref?2?P were constructed,and then imaged under a Leica TCS SP8 confocal laser-scanning microscope.The NES sequences were determined to be aa?NES1?and aa?NES2?.There are at least NLSs aa 1-350.Additionally,mutation of aa?NES2?led to dispersed distribution of Ref?2?P,suggesting that aa?NES2?of Ref?2?P was not only an authentic NES but also played a role in formation of inclusion body.?Co-transfection of Ref?2?P and polyhedrin was performed.It was observed that Ref?2?P and polyhedrin were co-located at the inclusion body in the cytoplasm,indicating that Ref?2?P could transport the protein polyhedrin which originally located in the nucleus to the cytoplasm to form inclusion bodies.When aa?NES2?was mutated,the formation of inclusion body was disrupted.The ability of silkworm Ref?2?P to transport viral proteins from the nucleus to the cytoplasm to form inclusion body indicated that Ref?2?P displayed antiviral activity,which could be considered as a strategy of host against viral infection.?Additionally,upon BmNPV T3 infection,the subcellular localization of silkworm Ref?2?P was changed from main location in the cytoplasm to main distribution in the nucleus.Western blotinging analysis showed that there was an additional band in the protein expression profile of Ref?2?P in infected cells,which was predicted to be cleaved product of Ref?2?P.The cleavage site was determined to be located in aa 390-451,leading to separation of NLSs from NESs of Ref?2?P.The cleaved products may lose the ability to transport viral proteins from the nucleus to the cytoplasm to form inclusion body,which should be verified further.2.the post-translational modifications of silkworm p62 and Ref?2?POur previous studies suggested that silkworm p62 and Ref?2?P proteins may be modified by ubiquitin or ubiquitin-like proteins.By site-directed mutagenesis of p62and Ref?2?P,it was found that,upon mutation of lysine XXX(K_xxx)of p62 to arginine,the p62 inclusion body became smaller,and the number of inclusion bodies in individual cell increased,and the molecular weight obtained by Western blotinging analysis decreased significantly.Moreover,mutation of the K_xxxxx of Ref?2?P to arginine,Ref?2?P was dispersed in the whole cell,and the molecular weight of the protein decreased obviously.We speculate that K_xxxxx is the key post-translational modification site of silkworm p62 and Ref?2?P protein,and the modification of K_xxxxx may exert a great effect on the stability of Ref?2?P protein.