The Molecular Mechanisms Of SIRT2 Regulating HSP90 Activity

Objective: To discover proteins interacting with SIRT2 and study the mechanism of SIRT2 regulating heat shock protein HSP90 activity.To provide a theoretical basis for the further study of the signal pathway mediated by SIRT2 and lay a foundation for the further research of the mechanism that SIRT2 regulates cellular stress response.Methods: SIRT2-pGEX-6P-1 eukaryotic expression plasmid was constructed by a series of methods including PCR.GST-SIRT2 fusion protein was induced and purified. Pull-down assay was used to screen proteins interacting with SIRT2 in rat glioma cells.Mass spectrometry and Western Blotting were employed to verify the proteins.The cells were collected and lysed by RIPA buffer.Then the supernatant,anti-SIRT2 antibody and proteinA/G beads were incubated in low temperature.The coimmunoprecipitation assay was employed to further verify the interactions between proteins.The SIRT2 siRNA,whose effect was obvious, was screened. SIRT2 lentivirus expression vector and SIRT2 lentivirus interference vector were constructed and stable SIRT2 overexpression and interference cell lines were selected by puromycin. The cells integrated with lentivirus were cultured,collected and lysed by RIPA.Then the supernatant,anti-HSP90 antibody and proteinA/G beads were incubated in low temperature.The immunoprecipitation assay was used to detect the deacetylation of HSP90 by SIRT2. The SIRT2 gene fulllength fragment was obtained from SIRT2-pIRES,and then it was subcloned into the eukaryotic expression vector pEGFP-C2 by a series of methods,such as PCR, plastic recycling, T-vector cloning and so on, making the target gene and GFP gene express simultaneously. The recombinant plasmid was transfected into 293 T cell lines by Lipofectamine 2000. The subcellular localization of the fusion protein was monitored by autofluorescence microscopy.The reagent making cytoplasm and nucleus separate and Western Blotting were used to detect the subcellular localization of the fusion protein.The nuclear export inhibitor leptomycin B was added and then the same processing was performed.Results:(1) SIRT2-pGEX-6P-1 prokaryotic expression plasmid was constructed successfully.(2)After being induced by IPTG,GST-SIRT2 fusion protein was highly expressed and purified.(3) Through the experiment of pull-down and mass spectrometry,the proteins interacted with SIRT2 were discovered.(4) The coimmunoprecipitation experiment proved that SIRT2 interacted with HSP90.(5) The SIRT2 siRNA repressed SIRT2 expression obviously.(6) The construction of SIRT2 lentivirus expression vector and SIRT2 lentivirus interference vector were successfully completed.(7) Stable SIRT2 overexpression and interference cell lines were chosen.(8) The immunoprecipitation experiment suggested that the acetylation level of HSP90 increased or decreased when SIRT2 was overexpressed or inhibited.(9) The eukaryotic expression plasmid SIRT2-pEGFP-C2 was constructed successfully.(10)After transfection of eukaryotic expression plasmid of SIRT2-pEGFP-C2,SIRT2 was observed by a fluorescence microscope that it was mainly distributed in the cytoplasm, almost no distribution in the nucleus.After adding the nuclear inhibitor leptomycinB,there was obvious aggregation of SIRT2 in the nucleus and there was still a lot of SIRT2 in the cytoplasm.About 12 h later,there was no significant difference between the cytoplasm and nucleus for the distribution.Remove leptomycin B, SIRT2 was still mainly distributed in the cytoplasm,which is the same with the results before adding leptomycin B.After use the reagent making cytoplasm and nucleus separate and Western Blotting, SIRT2 was thought that it was mainly expressed in the cytoplasm and almost no expression in the nucleus. After adding leptomycinB, SIRT2 was still expressed highly in the cytoplasm and the expression in the nucleus significantly increased. Removing leptomycin B, SIRT2 was still mainly expressed in the cytoplasm,which was the same with the results before adding leptomycinB.Conclusion:(1) SIRT2 interacted with HSP90.(2) SIRT2 deacetylated HSP90.(3) SIRT2 mainly was distributed in the cytoplasm.SIRT2 may be a nucIeocytopIasmic shuttIe protein,and its subcellular localization is a dynamic process.