Study On The Mechanism Of Sulfur-containing Amino Acid Metabolism Involved In Cadmium Tolerance In Saccharomyces Cerevisiae

The heavy metal cadmium（Cd）is one of the most common environmental pollutants,and long-term exposure can cause damage to multiple tissues and organs in the human body.Its half-life is more than ten years,and it is known to be the most easily accumulated toxic substance in the body.Due to the harmfulness of cadmium and the universality of its pollution,biological control is an important measure.The mechanism of cadmium toxicity has been extensively studied.However,the effect of nutrients such as amino acids on cell cadmium tolerance,especially the molecular mechanism,is not clear.In this study,Saccharomyces cerevisiae was used as a cell model to explore the molecular mechanism of regulating the metabolism of sulfur-containing amino acids to alleviate cadmium toxicity in cells.Firstly,we found that the addition of exogenous sulfur-containing amino acids could partially compensate for the cadmium resistance of wild-type Saccharomyces cerevisiae cells,while the addition of other amino acids such as leucine,glutamic acid,and tryptophan did not show similar phenotypes.The sulfate assimilation pathway（SAP）is a process in which extracellular sulfate enters cells to synthesize cysteine,methionine,and other organic sulfides,which play an important role in heavy metal tolerance.The results of the literature showed that cadmium stress could activate SAP in yeast cells,and related genes were up-regulated.However,our experimental results showed that sulfate-transporter-related gene defects sul1Δand sul2Δshowed growth advantages over wild-type WT under cadmium stress.It is speculated that there are two possibilities,first,sul1Δ,and sul2Δmay have the transport function of cadmium ion,cadmium cannot be transported into the cell after loss,reducing the toxic effect on the cell;Secondly,defects in sulfate-transporter-related genes may compensatively increase cysteine desulphurase activity in cells,promoting the formation of reduced S^2-and forming preferred detoxicating sulfides with cadmium.The cadmium absorption of wild-type WT and sul1Δ,and sul2Δmutants under stress without cadmium concentration was detected by the ICP-AES method,and the data showed that there was no significant difference in ion content among the three.Further experiments showed that the hydrogen sulfide synthesis of sul1Δ,and sul2Δmutant increased compared with WT,and cysteine could promote the cell to produce hydrogen sulfide,and cadmium was preferentially combined with hydrogen sulfide.On the contrary,the mutation met5Δstrain,which was related to the deletion of genes related to synthetic hydrogen sulfide,reduced the accumulation of hydrogen sulfide in the cell and was very sensitive to cadmium.In addition,the exogenous addition of hydrogen sulfide can enhance the resistance of WT to cadmium.These results indicate that the second hypothesis is correct,and the reduced state S^2-is more important than mercaptan（SH）in combating cadmium toxicity in the SAP pathway.Secondly,cysteine is an important product and reverse regulator of the SAP pathway,as well as a component of the synthesis of glutathione（GSH）.Both can be detoxicated by the chelation of sulfhydryl（SH）with cadmium,and GSH can also remove ROS resistance generated by cadmium.However,the mechanism of GSH participating in the specific molecular pathway of cadmium resistance and being affected by cellular oxidation levels（reduced GSH and oxidized GSSG levels）is not very clear.We first verified that GSH synthetase deficient strains gsh1Δ,gsh2Δ,and the vacuole glutathione S-binding transporter deficient strains ycf1Δwere sensitive to cadmium stress.The addition of exogenous GSH can partially complement the sensitive phenotype of the former gshΔmutation to cadmium,but has little effect on the inhibition of ycf1Δunder cadmium stress,indicating that the transport of GSH-cadmium chelate（GS-Cd-SG）to the vacuole is a key step in detoxification.Cadmium stress（40μM semi-inhibitory concentration）and an oxidizing agent（H₂O₂）treatment have similar growth inhibition effects on the WT,gsh1Δ,gsh2Δ,cys3Δ,ycf1Δ,and contrary to the results of reducing agent（DTT）treatment,indicating that cellular oxidative stress is an important mechanism of cadmium toxicity.The endoplasmic REDOX reductase ERO1p promotes the formation of disulfide bonds through protein disulfide isomerase（PDI）and is involved in maintaining the REDOX balance of endoplasmic reticulum（ER）.Our experimental results showed that the WT strain overexpressing ERO1 became sensitive to cadmium stress,while the overexpression of ERO1 in gsh1Δand ycf1Δdid not change the strain’s sensitivity to cadmium stress.The exogenous GSH can improve the cadmium tolerance of WT and gsh1Δgenotypes except for ycf1Δ.The exogenous DTT can significantly improve the cadmium resistance in the WT strain overexpressing ERO1,indicating that the overexpression of ERO1 improves the cell oxidation level,promotes the transformation from reduced GSH to oxidized GSSG,and reduces the cadmium resistance of cells.Further tests showed that the reduced GSH level of WT strain significantly decreased with the increase of cadmium concentration.The degree of lipid peroxidation（MDA）increased,and exogenous cysteine and GSH had an obvious replenishment effect.Overexpression of ERO1 promoted the cellular effect of cadmium toxicity,which again demonstrated that thiol-containing（SH）cysteine and GSH content and intracellular oxidation level affected the cadmium resistance of yeast cells.Finally,the TOR pathway in cells is an important regulatory platform that connects growth processes such as sugar,lipid,amino acid metabolism,and protein synthesis.We found that the tor1Δ（TOR pathway PIK-related protein kinase rapamycin target gene mutant）and sfp1Δ（its downstream effect factor ribosome protein transcription and biogenetic gene deletion mutant）exhibited growth advantages over the wild-type under cadmium stress.Compared with the inhibitory effects of other metal ions such as copper and iron,tor1Δand sfp1Δshowed specific resistance to cadmium stress.It was speculated that the TOR pathway is involved in the cadmium resistance of cells through its mediated metabolism of sulfur-containing amino acids.The results showed that the growth advantage of sfp1Δand tor1Δon cadmium disappeared in a sulfur-amino acid-restricted medium.Exogenous rapamycin can also partially eliminate the inhibition of cadmium stress on WT growth.We demonstrated that treatment with rapamycin inhibited TORC1 kinase in cells leading to dephosphorylation of Sfp1p from the nucleus to the cytoplasm.We also demonstrated that cadmium treatment mimics the nuclear localization of Sfp1-GFP induced by rapamycin into the cytoplasm,and the addition of exogenous cysteine causes Sfp1-GFP to reposition in the nucleus.Subsequently,we detected the expression of downstream response genes of Sfp1p,and the results showed that the expression level of ribosomal protein gene RPS11B decreased under cadmium treatment,while the expression level of hydrogen sulfide synthesis gene MET5 increased under cadmium treatment.Further biochemical analysis showed that compared with the wild type,the content of reduced sulfur ions in sfp1Δ,and tor1Δcells increased.The above data are consistent with the results of the first part,suggesting that cadmium stress can activate the SAP pathway,especially induce the reduced state S^2-accumulation,and that the Tor C1-mediated sulfur-containing amino acid metabolic pathway plays an important role in the resistance of cells to cadmium.In conclusion,Saccharomyces cerevisiae was used as a cell model to explore the regulatory relationship between heavy metal cadmium stress,the involvement of sulfur-containing amino acids such as cysteine in the SAP pathway,and the TOR signaling pathway.The results showed that cadmium stress can activate the SAP pathway and change cell oxidation levels to induce alterations in the metabolism of sulfur-containing amino acids such as cysteine.The TOR signaling pathway connects and coordinates the relationship between sulfur-containing amino acid nutrition metabolism and cell growth inhibition caused by cadmium stress.These findings provide new ideas for the breeding of cadmium-resistant strains or plants and provide a theoretical basis for the biological control of heavy metals.