Study On The Molecular Mechanism Of Elp3 Involved In S.cerevisiae Oxidative Stress Reaction Via Yap1

The Elongator complex is a six-subunit complex with histone acetyltransferase activity and functional integrity.Its catalytic subunit ELP3 has two conserved domains,the HAT domain and the SAM domain.The deletion of this subunit or the occurrence of a point mutation that attenuates HAT activity can affect Elongator function,and both can make the hydrogen peroxide hypersensitive phenotype of Saccharomyces cerevisiae.In Saccharomyces cerevisiae,the mechanism of oxidative stress response is mainly regulated and amplified by the transcription factor Yap1.Under normal conditions,Yap1 is diffusely distributed in the cytoplasm,but under the treatment with hydrogen peroxide,Yap1 translocates and accumulates in the nuclear,and then activates a series of Antioxidant gene expression.To investigate whether the ELP3 subunit affects the yeast oxidative stress through the Yapl pathway.Wild-type(WT)and elp3? strains was used as experimental materials and treated with 2 mM hydrogen peroxide to detect Yap1 nuclear localization,activity,and expression of Yap1 target gene.The results showed that ELP3 deletion affected the nuclear localization level of Yap1.The nuclear localization level in elp3? strain was significantly decreased,but the activity of Yap1 in elp3? strain was significantly increased by the reporter gene assay.qRT-PCR experiments shown thatthe Elp3 deletion severely affected the expression of the Yap1 target gene too.This indicates that the Elp3 subunits influence the mRNA levels of a series of redox-related genes by affecting the nuclear localization level of transcription factor Yap1 and its activity,thus affecting the oxidative stress response of Saccharomyces cerevisiae.Saccharomyces cerevisiae,like other eukaryotic organisms,contains two superoxide dismutases(SOD1,SOD2)and plays an important role in the clearance of intracellular ROS,and heat shock proteins have multiple functions such as molecular chaperones in response to oxidative stress.tThe expression of SOD1,SOD2 and two types of heat shock proteins(HSP75,HSP105)were measured by qRT-PCR and Western blot.The results showed that the deletion of Elp3 subunit affects the expression of SODBoth at mRNA and protein level,and the expression of HSP105 protein was significantly decreased.This suggests that Elp3 may be involved in the regulation of the expression of oxidative stress related genes.ROS content and cellular MDA levels can be used to visualize the intracellular redox state.Wild type(WT)and elp3? strains were used as materials and treated with 2 mM hydrogen peroxide,intracellular ROS contents and MDA level were deected by NBT and TBA methods,respectively.The results showed that the ROS content and MDA levels in the elp3? strains increased significantly.It shows that under the oxidative stress condition,the deletion of ELP3 makes the yeast cells highly oxidized.Then the intracellular glutathione peroxidase(GPX)activity and protein content were measured with Athar and Iqbel's method.The results showed that both the GPX enzyme activity and protein content of elp3? strain were lower than WT significantly This shows that the reduced resistance of oxidants to ELP3-deficient strains is associated with changes in their antioxidant enzyme activity and intracellular ROS levels.Diamide is a very common oxidant,and its mechanism of induction of yeast oxidative stress is different from that of hydrogen peroxide.In order to further study whether ELP3 deletion is also affected by diamide resistance,wild type(WT),elp3? and intact and partially deleted ELP3 transformants were used as materials,sensitivity experiments and growth curves were performed and drawn for different concentrations of diamide-treated strains.The results showed that the elp3? strain was highly sensitive to the diamide.Under the oxidant treatment,the growth status ofhElp3,HAT and SAM domains deleted yElp3 transformants were similar to that of elp3? strain,while yeast full length ELP3 transformant was similar to the WT phenotype.And high temperature stress can aggravate the susceptibility of each strain to oxidants.The results show that the deletion of Elp3 and its two conserved domains can reduce the diamide resistance of Saccharomyces cerevisiae,and human Elp3 has no compensation for the diamide-sensitive phenotype of elp3? strain.These results confirm that the two conserved domains of Elp3 are critical for the regulation of yeast oxidative stress by Elp3.