Study On The Mechanism Of Endoplasmic Reticulum Stress Response Gene ERO1 Maintaining Endoplasmic Reticulum Homeostasis In Yeast Cells

Endoplasmic reticulum(ER)is the main place for the correct folding and maturation of eukaryotic proteins.It has a strict steady-state control mechanism.The protein and redox homeostasis could be disturbed by internal and external factors,resulting in ER stress(ERS).A large number of resident ER proteins are responsible for maintaining ER homeostasis.In the past decade,secondary cell damage caused by chronic ERS has been recognized as the core factor in the pathophysiology of widespread human diseases.Therefore,the study on the regulation and maintenance mechanism of ER homeostasis is of great significance for disease treatment,drug research and development.ERO1,a resistant gene to DTT(strong reductant),was screened from an over expression library of essential genes in S.cerevisiae.ERO1p plays an important role in maintaining the redox homeostasis of ER and promoting protein disulfide bonding.The DTT resistance was not dependent on the yeast IRE1/HAC1 pathway by preliminary drug sensitivity assay and RT-PCR.Further studies showed that glutathione synthase(GSH1p)plays a key role in the DTT resistance mediated by ERO1.Under different oxidants(H₂O₂,potassium dichromate,cadmium chloride),unfolded protein response(UPR)inducer(Tunicamycin,TM)and temperature stress,the overexpression of ERO1was more sensitive than that of wild type,suggesting that ERO1p overexpression promoted cell oxidation.Overexpression of ERO1 in the glutaredoxin system gene(GRX1,GRX2)and transketolase gene(TKL1)mutants showed resistance to DTT,however the H₂O₂ sensitive phenotype was lost.The results indicated that the mechanism(ERO1p overexpression promotes cell oxidation)was dependent on the formation of disulfide bonds in cells.The glutathione(GSH)is the main hydrogen donor within the cells.The strain overexpressed ERO1 after mutation of GSH1,a key gene for GSH synthesis,lost DTT resistance phenotype.When amino acids(Cys,Glu,Gly)related GSH synthesis were removed from the culture media,GSH1 mutants showed no sensitivity to DTT.The addition of above amino acids or GSH could weaken the resistance of WT overexpressed ERO1 to DTT,further demonstrating that the mechanism was dependent on the disulfide bond formation process in GSH.The measurement of cellular GSH and reactive oxygen species(ROS)showed that GSSG levels were increased,however the reduced GSH levels were decreased in the strain overexpressed ERO1 compared with the wild-type strain.Overexpression ERO1 in gsh1?decreased the level of ROS.It futher showed that reduced GSH could play a role in providing reduction potential in ERO1-mediated DTT resistance.ERO1 plays a key role in maintaining cell redox homeostasis and disulfide bond synthesis.Since protein disulfide isomerase 1(PDI1)plays a role of thiol electron transfer in the process of ERO1-mediated protein disulfide bond synthesis,we constructed overexpression strains of PDI1and its paralog EUG1.However,different from the strain overexpressed ERO1,the two strains are sensitive to DTT.It was speculated that the expression level of ERO1 plays a restrictive role in the maintenance of cellular redox homeostasis and the process of disulfide bond synthesis involved in PDI1/EUG1.Then we constructed strains(Pgal10-ERO1)and ERO1-Damp(reduced ERO1 RNA expression)that controlled the expression level of ERO1 by galactose regulatory elements,and overexpressed PDI1 and EUG1in these constructed strains.With the increase of ERO1 expression,the growth defects of the above strains at the low expression level of ERO1were supplemented,the sensitivities to H₂O₂ were weakened,and the resistances to DTT were increased.It was proved that the involvement of PDI1/EUG1 in the maintenance of cellular redox homeostasis and the process of disulfide bond synthesis was affected by the expression level of ERO1.Subsequently,biochemical detection of oxidative stress in strains with different ERO1 expression levels showed that oxidative stress indicators(ROS,OH-,MDA)of strains with high ERO1expression levels were increased,and antioxidant enzymes(SOD,POD,CAT)activities and GSH were also increased.The increase in H₂O₂ treatment aggravated oxidative stress,while DTT treatment reduced oxidative stress,indicating that the expression level of ERO1 plays an important role in the regulation of ER redox homeostasis.Finally,the functional conservation analysis of ERO1 showed that the amino acids in the key domain of ERO1p were highly conserved in different species.We cloned the Ca.ERO1 gene of Candida albicans and successfully expressed in Saccharomyces cerevisiae.Overexpression of Ca.ERO1 showed resistance to DTT and sensitivity to H₂O₂,indicating that its function was conservative in fungi.In conclusion,ERO1,a DTT resistant gene,was screened from yeast overexpression library and its function in maintaining the redox homeostasis of endoplasmic reticulum was investigated.The results demonstrate that GSH1 plays a key role in the DTT resistance mediated by ERO1,and the expression level of ERO1 plays a key role in the maintenance of redox homeostasis and disulfide bond formation,and has fungal conservatism.