Promote Survival Signaling Pathway In Grp75 Inhibition Of Bax Conformational Change In The Role

Apoptosis is essential for normal development and tissue homeostasis of all multicellular organisms.Two distinct pathways of apoptosis have been described,the death receptor pathway(the extrinsic apoptotic pathway) and the mitochondrial pathway(the intrinsic apoptotic pathway).Through various stresses,cells have the option of actively engaging the intrinsic apoptotic pathway,which subsequently leads to their death.Previous studies have shown that release of cytochrome c(Cyt C) and other pro-apoptogenic factors from mitochondria to cytosol is an important step in the intrinsic pathway of apoptosis.Stress to the cell causes DNA damage and protein denaturation.The stressors can also induce cellular defense mechanism,in which a lot of proteins include heat shock protein(HSP) involved.Glucose-regulated protein 75(Grp75) is a member belonging to the heat shock protein 70(HSP70) family.Grp75 achieves multiple functions including mitochondrial import,intracellular trafficking,control of cellular proliferation and differentiation.It can also respond to many forms of stress.Our previous observation showed Grp75 overexpression provides protection against injuries induces by glucose deprivation.However,the mechanisms underpinning this protection are not fully understood.Several studies have indicated that an upregulation of Grp75 suppresses the engagement of apoptosis from various stressors.Annexin V/PI double staining and cell morphology analysis showed that Grp75 overexpression inhibited the apoptosis induced by glucose withdrawal.Immunofluorescence of Cyt C indicated that Grp75 suppressed the intrinsic apoptotic pathway.The release of Cyt C is regulated by a complex balance of pro-apoptotic and anti-apoptotic proteins of the Bcl-2 family.Bcl-2,one of the most important anti-apoptotic proteins of the Bcl-2 family,can prevent cytochrome c release,whereas pro-apoptotic family members such as Bax can promote cytochrome c release.Studies suggested that the relative ratios of Bcl-2 to Bax,rather than the single expression of either protein,can influence survival or death to diverse apoptotic stimuli.The conformational change of Bax is also important to regulate cytochrome c release and induce apoptosis in stressed cells.Although Grp75 overexpression could slightly inhibit the increase of the Bax/Bcl-2 gene expression ratio,it was not the major reason for Grp75 protecting PC12 cells from GD injury.Grp75 could also inhibit Bax conformational change induced by GD.At the same time,the time-course of Bax conformational change inhibition was consistent with apoptosis,so we concluded that Grp75 blocks GD-induced apoptosis,at least partially,due to the inhibition of Bax conformational change,thereby preventing the release of Cyt C from the mitochondrial intermembrane space.Members of the Bcl-2 family have been shown in some situations to be targets of the kinases that are activated in stressed cells.Western Blotting, immunofluorescence and apoptotic analysis were performed to investigate the function of two pro-survival signal pathways(Raf/Mek/Erk and PI3K/AKT) in the inhibition of Bax conformational change by Grp75.Results showed that Grp75 suppressed Bax conformational change and apoptosis through maintaining the activity of Raf/Mek/Erk pathway and PI3K protein.Recent studies have shown the presence of cross-talk between the two signal pathways.Our results showed that Grp75 could activate AKT through Raf/Mek/Erk signal pathway under glucose deprivation. Molecular chaperone Grp75 can assist in the de novo folding of proteins,in the refolding of unfolded proteins;it can prevent aggregation of misfolded proteins.So the in vivo interaction of Grp75 and Akt,Erkl/2 protein was investigated with co-immunoprecipitation.The results showed there was no physical interaction between Grp75 and Akt.But the assay confirmed the direct interaction of Grp75 and Erk1/2 both under normal condition and glucose deprivation,which needs further study.In conclusion,Grp75 activated Akt through Raf/Mek/Erk signal pathway,and enhanced pro-survival signal through maintaining the activity of PI3K under glucose deprivation.And the activated Akt and Erk1/2 by Grp75 inhibited the Bax conformational change and apoptosis.