Lack Of Sugar Under The Conditions Of Grp75 And P53 Binding Mediated Protective Effect On Pc12 Cells

Heat shock protein (Hsp) family maintaining the intracellular homeostasis and proteins interaction integrity of the cell exists in almost all of the prokaryotes and eukaryotes. In the presence of heat shock proteins, DNA and protein molecules in cells execute their boilogical functions strictly according to biological theorem. Glucose regulated protein 75(Grp75), a member of the heat shock protein family, has been shown to be involed in many important cellular processes, such as fuctioning as a molecular chaperon, aiding new translated polypeptide correctly folding, stabilizing cytoskeleton, involving in DNA replication and transcription as well as taking part in the regulation of intracellular signal transduction. More and more studies showed that Grp75 can protect cells that under different kinds of stresses including glucose deprivation from injuring thorough several different pathways. Previous study reported that Grp75 was able to bind to p53 through its 253-282 amino acid residues and subsequently prevented the nucleus translocation of p53.It has been shown that p53 is a key componet and plays pivotal role in the cell cycle and apoptosis, therefor we postulate that the execution of cellular protective function of Grp75 may thorough binding to p53.In order to investigate whether the binding of Grp75 and p53 involved in the process of cellular protection exexuted by Grp75,the eukaryotic expression vector of Grp75 deletion mutant was constructed. The Grp75 deletion mutant gene (Grp75(Δ253-282)) was obtained by SOE-PCR (gene splicing by overlap extension), and was subsequently constructed into a eukaryotic expression vector pcDNA3.1(+), obtaining the resultant recombinant vector pcDNA3.1(+)/Grp75(Δ253-282).In order to detect the interaction between Grp75 deletion mutant and p53 much more conveniently, eukaryotic recombinant expression vector of p53 with a C-terminal fused c-myc tag was also constructed.The recombinant plasmids pcDNA3.1(+)/Grp75(Δ253-282) were transfected into PC12 cells via liposome method, and the stable cell strain PC12/Grp75(Δ253-282)(+) was constructed via G418(1 mg/mL) selection. Semi-quantitative RT-PCR and Western blot showed that both of the transcription and translation of Grp75 in PC12/Grp75(Δ253-282)(+) cells were higher than that in PC12 cells, indicating that Grp75(Δ253-282) overexpressing stable cell strain PC12/Grp75(Δ253-282)(+) was successfully constructed. The viability and apoptosis of cells undergoing 0 h,3 h,9 h,18 h and 36 h of glucose deprivation respectively was measured by MTT assay, Hoechst33324 and Gimsa staining. It is shown that PC12/Grp75(+) cells display the highest cell activities among the three groups of cells investigated and the activities of PC12/Grp75(Δ253-282)(+) cells were higher than that in PC12 cells. Hoechst33324 and Gimsa staining indicated the same results as MTT assay. These results suggested, to some extent, the deletion mutants of Grp75(Δ253-282) lost its protective function. Western Blot indicated that the expression of p53 in PC12/Grp75(Δ253-282)(+) cells, which showed the same experession level as PC 12 cells, was higher than that in PC12/Grp75(+)cells.The same results was obtained via immunofluoresence comfirmation.Immunofluorescence was also employed to detected the expression and subcellular localization of p53 in different varieties of cells. The results showed that the expression and nuleus translocation of p53 in PC12 cells distinctly increased as the time of glucose diprivation prolonged(Oh,3h,18h,36h), while the expression of p53 in PC12/Grp75(+) cells was less than that of PC 12 cells under the same condition. These results implied that to some extent the expression of p53 was inhibited by overexpression of Grp75.In addition, the deletion of 253-282 amino acid residues of Grp75 did not completely repress its nucleus translocation, implicating that there is existing other interaction site with p53 in Grp75 protein. To further test whether Grp75 is still able to bind to p53 after deletion, co-immunoprecipitation was carried out to detect the interaction between Grp75(Δ253-282) and p53.The results showed that the Grp75 deletion mutant still kept the ablility to bind to p53.This may be a consequence of the existing of other interaction site with p53 in Grp75.Whether there is existing other interaction site with p53 in Grp75 protein remains to be elucidated.In summary, Grp75 protein protects PC 12 cells under glucose deprivation from injurying partially via directly binding to p53,and the binding of Grp75 and p53 partially can inhibit the nucleus translocation of p53 and subsequently hamper p53 trancriptional activity depended apoptosis process.Excepting for the known interaction site with p53 in Grp75 protein, there may be other interaction site with p53 existed in Grp75.In addition, overexpression of Grp75 can inhibit the expression of p53 in the cells and this inhibiting effect could reduce p53 caused apoptosis.