Research On The Mechanism Of DJ-1/CHAC1 Signaling Pathway In Glutathione Metabolism

The DJ-1 gene is the causative gene of autosomal recessive early-onset Parkinson’s disease(PD),and it is also an oncogene.It is a multifunctional protein with multiple functions such as transcription regulation,anti-oxidative stress response,chaperone protein,protease,and maintenance of mitochondrial homeostasis.A large number of studies have shown that DJ-1 is widely present in various organs of the human body,including brain,skeletal muscle,liver,heart,etc.Recent studies have found that DJ-1 dysfunction is widely involved in a series of diseases caused by oxidative stress,such as neurodegenerative diseases,type 2 diabetes,tumors,and male infertility.Research on the mechanism of this protein will be provide new ideas for the prevention and treatment of these diseases.PD,also known as tremor paralysis,is a common neurodegenerative disease.There are more than 6 million PD patients in my country.Its pathological manifestations are characterized by the massive loss of dopaminergic neurons in the substantia nigra compact part of the midbrain,accompanied by abnormal aggregation of α-synuclein(αs)to form Lewy bodies.In recent years,the role of genetic factors in the pathogenesis of PD has been paid more and more attention by scholars.Among them,the DJ-1 mutation was discovered in 2003 to cause autosomal recessive early-onset PD.In addition,the researchers found that the oxidation state DJ-1 is a component of Lewy bodies,suggesting that the oxidation state DJ-1 is deeply involved in the occurrence of PD.Overexpression of wild-type DJ-1 can protect dopaminergic neurons by increasing the level of heat shock protein 70 to inhibit protein aggregation and cytotoxicity caused by α-synuclein.The above research shows that DJ-1 plays an important role in the occurrence and development of PD.Glutathione(GSH)is a tripeptide widely found in mammals,and is a small molecule antioxidant containing sulfhydryl groups in cells.The γ-glutamyl bond gives glutathione special stability,making it insensitive to many cellular peptidases in the cell,and other special enzymes are needed to degrade it.In eukaryotes,glutathione is essential.Disrupting the biosynthesis of glutathione will cause embryonic death in mammals and plants.Astrocytes in the brain are the main donors of GSH.In addition to maintaining their own redox homeostasis,GSH derived from astrocytes can also help neurons resist external oxidative stress damage.It is worth noting that many diseases are related to changes in intracellular glutathione levels.Glutathione deficiency can induce heart disease,chronic infection,diabetes,cancer,and neurodegenerative diseases and other diseases.Therefore,the content of glutathione is very important for the health of cells and organisms.According to reports in the literature,knocking down DJ-1 can reduce GSH levels in cells,but the molecular mechanism of DJ-1 regulating GSH levels is still unclear.This article will start with astrocytes,the main source of GSH in the brain,observe the effect of DJ-1 on the level of GSH in primary mouse astrocytes,and verify the correlation between DJ-1 and GSH;then,the cells were pre-protected with gallic acid(GA),and the corresponding cytotoxicity was detected by MPP+ stimulation.It was found that gallic acid could reduce the cell damage induced by MPP+ stimulation by up-regulating DJ-1 in the cells,and then the Crispr-Cas9 technology was used to construct a DJ-1 knockout cell line to further clarify the role of DJ-1 in cell damage induced by MPP+ stimulation.Next,RNA sequencing was used to find the differentially expressed genes before and after DJ-1 knockout.Sequencing results showed that DJ-1 knockout could significantly up-regulate the expression of CHAC1(CHAC Glutathione Specific Gamma-Glutamylcyclotransferase 1);further molecular biology methods were used to verify whether DJ-1 regulates GSH levels through CHAC1;finally,transcription factors that may be involved in DJ-1 regulation of CHAC1 are sought through transcription factor enrichment,and how DJ-1 regulates the transcriptional activation activity of this transcription factor,and finally clarifies the molecular mechanism of DJ-1 regulating GSH levels.AIM:To clarify the molecular mechanism of astrocyte DJ-1 regulating GSH levels.METHODS:1.Use molecular cloning technology to construct plasmids that overexpress DJ-1(OE-DJ-1)and knock down DJ-1(sh-DJ-1),transfect in mouse primary astrocytes,and use GSH detection kit to detect the effect of DJ-1 on the level of GSH in mouse primary astrocytes;use Cell Counting Kit-8(CCK-8)method and use lactate dehydrogenase(LDH)assay kit to detect cell viability and LDH release;2.Using Crispr-Cas9 technology to construct DJ-1 knockout cell line,use Western blot(WB)to detect the protein expression of DJ-1 in wild-type cells and DJ-1 knockout cells level to evaluate whether DJ-1 was successfully knocked out;3.Extract RNA from wild-type and DJ-1 knockout cells for RNA-Seq,GO enrichment analysis of the biological processes involved in differentially expressed genes,volcano map and heat map analysis of differentially expressed genes before and after DJ-1 knockout;4.Apply real-time fluorescent quantitative PCR(qRT-PCR)method and Western blot method to detect the effect of DJ-1 on CHAC1 mRNA and protein expression,and use GSH detection kit to detect the effect of DJ-1 on GSH levels;5.Transfect in HEK 293T cells and mouse primary astrocytes with siRNA technology to observe the effect of down-regulation of CHAC1 or activating transcription factor 3(ATF3)on DJ-1’s regulation of GSH levels;6.Use ThiolTrackerTM Violet fluorescent probe to detect GSH in cells;7.Enrichment of transcription factors to search for transcription factors involved in DJ-1 regulation of CHAC 1;8.Use molecular cloning technology to construct VC155-DJ-1,VN173-JUN and VN173-ATF3 plasmids,and use bimolecular fluorescence complementation(bifc)technology to determine the transcription factor that DJ-1 regulates CHAC1;9.Use molecular cloning technology to construct a truncated plasmid of DJ-1 nuclear localization signal sequence(VC155-DJ-1 Δ NLS),co-transfect HEK 293T cells with VN173-ATF3 plasmid,and use bimolecular fluorescence complementation technology to observe the cell location where DJ-1 interacts with ATF3;10.Use molecular cloning technology to construct a plasmid that overexpresses DJ-1(OE-DJ-1-flag)and overexpresses DJ-1 with deletion of nuclear localization signal sequence(OE-DJ-1-flagΔNLS)with flag tags,transfect HEK 293T cells,use real-time fluorescent quantitative PCR and Western blot to detect the effect of DJ-1 on CHAC1 mRNA and protein expression,and use GSH detection kit to detect the effect of DJ-1 on GSH levels;11.Use molecular cloning technology to construct VN173-ATF3 truncated bodies(VN173-ATF3ΔN,VN173-ATF3ΔBasic domain,VN 173-ATF3ΔLeucine Zipper,VN173-ATF3ΔbZIP and VN173-ATF3 ΔC),co-transfect HEK 293T cells with VC155-DJ-1 plasmid,and the specific area of DJ-1 and ATF3 binding was observed by using bimolecular fluorescence complementation technology;12.Use molecular cloning technology to construct overexpressing ATF3(OE-ATF3)and overexpressing bZIP domain deleted ATF3(OE-ATF3ΔbZIP)plasmids,transfect HEK 293T cells,and use Western blot to detect the effect of bZIP domain of ATF3 on the protein expression of CHAC1,and the application of GSH detection kit to detect the influence of bZIP domain of ATF3 on GSH levels.RESULTS:1.DJ-1 up-regulates intracellular glutathione levels to reduce cell damage induced byMPP+stimulationCompared with the control group,the level of GSH in mouse primary astrocytes was significantly increased after DJ-1 was overexpressed,and the level of GSH was significantly decreased after DJ-1 was knocked down.The results show that DJ-1 has a positive regulatory effect on the level of glutathione in mouse primary astrocytes.Western blot results show that GA or OE-DJ-1 can up-regulate DJ-1 to reverse the down-regulation of DJ-1 induced by MPP+stimulation.CCK8 and LDH test results show that GA or OE-DJ-1 can significantly increase cell viability and significantly reduce LDH release under MPP+stimulation.In addition,GSH test results show that GA or OE-DJ-1 can significantly increase the level of GSH in cells stimulated by MPP+.Furthermore,the Crispr-Cas9 technology was used to knock out DJ-1 in HEK 293T cells to clarify the role of DJ-1 in cell damage induced by MPP+ stimulation.The results showed that compared with wild-type cells,the cell viability after DJ-1 knockout was significantly reduced and the amount of LDH released was significantly increased under MPP+ stimulation.At the same time,the GSH test results showed that compared with the wild-type cells,the intracellular GSH level was significantly reduced regardless of the basic state or MPP+stimulation after DJ-1 knockout.The above results indicate that DJ-1 can reduce cell damage induced by MPP+ stimulation by up-regulating the level of glutathione in cells.2.The expression of glutathione degrading enzyme CHAC1 is significantly up-regulated after DJ-1 is knocked out by Crispr-Cas9Crispr-Cas9 technology was used to knock out DJ-1 in HEK 293T cells.The RNA of wild-type and DJ-1 knockout cells was collected,and differentially expressed genes were detected by second-generation sequencing.GO enrichment analysis found that the biological processes involved in these differentially expressed genes included glutathione metabolism,indicating that DJ-1 and GSH have strong correlation.Among them,the expression of CHAC1,a glutathione degrading enzyme,was significantly up-regulated after DJ-1 was knocked out,suggesting that DJ-1 has a negative regulatory effect on the expression of CHAC1.3.DJ-1 reduces GSH degradation by inhibiting the expression of glutathione degrading enzyme CHAC 1Compared with the control group,the protein expression level of CHAC1 in HEK 293T cells and mouse primary astrocytes was significantly reduced after DJ-1 was overexpressed,while the protein expression level of CHAC1 was significantly increased after DJ-1 was knocked down.The GSH test results showed that compared with the control group,the level of GSH in HEK 293T cells and mouse primary astrocytes was significantly increased after DJ-1 was overexpressed,while the level of GSH was significantly reduced after DJ-1 was knocked down.In addition,the mRNA level of CHAC1 in HEK 293T cells was significantly reduced after DJ-1 was overexpressed,and the mRNA level of CHAC1 was significantly increased after DJ-1 was knocked down.The results showed that DJ-1 can regulate the transcription of CHAC1.Furthermore,while knocking down DJ-1,siRNA was used to interfere with down-regulating the expression of CHAC1.The results showed that knocking down CHAC1 can cancel the reduction of GSH level induced by knocking down DJ-1,suggesting that DJ-1 reduces the degradation of GSH by inhibiting the expression of CHAC1.4.DJ-1 inhibits its transcriptional activation activity by binding to the bZIP domain of ATF3,thereby down-regulating the expression of the glutathione degrading enzyme CHAC1In order to explore how DJ-1 inhibits the expression of CHAC1,JUN and ATF3,two transcription factors that may regulate CHAC1,were screened through transcription factor enrichment.Further using bimolecular fluorescence complementation technology,it is found that ATF3 binds to DJ-1 in the nucleus,suggesting that DJ-1 may regulate the expression of CHAC1 by affecting the transcription factor activity of ATF3.Furthermore,siRNA interference was used to down-regulate the expression of ATF3.Compared with the control group,the protein expression level of CHAC1 in HEK 293T cells and mouse primary astrocytes was significantly reduced after ATF3 was knocked down.When knocking down DJ-1 while knocking down ATF3,the protein expression level of CHAC1 did not change significantly compared with the ATF3 group alone,suggesting that DJ-1 down-regulated the expression of CHAC1 by inhibiting the transcriptional activation activity of ATF3.Next,in order to study which domain of DJ-1 directly binds to ATF3,we constructed ATF3 plasmids with different domain deletions.Using bimolecular fluorescence complementation technology,we found that bZIP domain deletion abolished the interaction between ATF3 and DJ-1.Western blot results showed that,compared with the control group,the protein expression level of CHAC1 in HEK 293T cells was significantly increased after overexpression of ATF3,while the protein expression of CHAC1 did not change significantly after overexpression of ATF3 without the bZIP domain.At the same time,the GSH test results showed that,compared with the control group,the level of GSH in HEK 293T cells was significantly reduced after overexpression of ATF3,while the level of GSH did not change significantly after overexpression of ATF3 lacking the bZIP domain,suggesting that DJ-1 through binding The bZIP domain of ATF3 inhibits its transcriptional activation activity and down-regulates the expression of CHAC1 to reduce GSH degradation.CONCLUSIONS:It is proved that DJ-1 enters the nucleus and binds to the bZIP domain of the transcription factor ATF3,inhibits its binding to the CHAC1 promoter,thereby down-regulates the expression of CHAC1 to reduce GSH degradation,and clarifies the molecular mechanism of DJ-1 regulating GSH levels.The major contributions of the present study lie in:1.The new function of DJ-1 combined with ATF3 to inhibit its transcriptional activation activity is reported;2.Prove that astrocyte DJ-1 down-regulates the expression of CHAC1 by inhibiting the transcriptional activation activity of ATF3,thereby reducing GSH degradation,and elucidating the molecular mechanism of DJ-1 regulating GSH levels.