¦¢sgt Hsp70/hsp90 Interaction Study

PartⅠ:Identification of the Association ofβSGT with Hsp70/Hsp90 in Yeast Cells and vitroTo identify the association ofβSGT and Hsp70/Hsp90 in the Yeast cells and vitro.βSGT was identified as an isoform ofαSGT in 2003.To futher elucidate the biological functions ofβSGT,we searched for Hsp90βthat specifically associate withβSGT with yeast two-hybrid screen experiments.Moreover,in order to determine whetherβSGT interacted with Hsp90βin vitro,we performed a GST pull-down experiment.βSGT is composed of 304 amino acids,the molecular mass is 34kDa.The TPR domain is in the middle of the protein.The TPR domain found in many proteins is believed to mediate protein-protein interaction. In the present studyβSGT containing TPR domains have shown to interact with HspT0 or with Hsp90.Here,we demonstrated that the C-terminal pentapeptide of Hsp70 and Hsp90 has an important role in TPR-mediated cofactor binding.Constructing the deletion of C-terminal, we shown that the interaction of TPR with MEEVD of Hsp90 is identified as the core contact for SGT binding to Hsp90;the formation of the Hsp70·SGT complex depends on recognition of the PTIEEVD by TPR domain in the yeast.Asp and Val of the EEVD motif are identified as general anchor residues,which are critical in Hsp90 binding by TPR domain. PartⅡ:Tissue distribution ofβSGT and the effection on the cell cyclewe describe here an isoform ofαSGT with 60%amino acid sequence identity that nameβSGT.βSGT is almost exclusively expressed in brain.The amino acid sequence of ratβSGT is compared with sequences of expressed sequence tags forβSGT from mouse,human and with sequences ofβSGT from gallus,mouse,and canis.In this present study,we contructed eukaryotic expression plasmid pCDNA3.1-myc-Hsp90βand was introduced into Hela cells line.after being subcloned,The recombined plasmid was purified and identified by limited enzyme digestion.The growth of Hsp90βhighly expressing cell line affect the cell cycle and the DNA content of S phase was higher.By flow cytometry,we demonstrate that the effect of high level Hsp90βon cell prolife ration was to accelerate cell growth by affecting cell cycle.