Effects And Mechanisms Study Of Redox Protein DJ-1 On Ferroptosis

Objectives Ferroptosis is a newly characterized form of regulated cell death mediated by irondependent oxidative damage with accumulation of Lipid ROS and shrunken mitochondria.Ferroptosis holds great potential for cancer therapy.However,the molecular mechanisms underlying ferroptosis remain largely elusive and there is still a lack of drug targets based on ferroptosis.DJ-1,as an important intracellular redox protein,is highly expressed in lung cancer,liver cancer and other tumors,and is related to the poor prognosis.Though it has been well documented that DJ-1 can protect cells from oxidative damage by directly or indirectly removing intracellular water-soluble ROS,it's not clear whether it also participate in defensing the ferroptosis caused by Lipid ROS.In this study,we define an integrative role of DJ-1 in ferroptosis and ferroptosis-based anti-tumor therapy,elucidate the role of transsulfuration pathway in DJ-1 regulated ferroptosis,and highlight a candidate therapeutic target to potentially improve the effect of ferroptosis-based antitumor therapy.Methods(1)Redox protein DJ-1 negatively regulates ferroptosis: Lentivirus knockdown system,CRISPR/Cas9-mediated knockout system,and lentivirus overexpression system were used to establish DJ-1 knockdown/knockout/overexpression cells,respectively;C11-BODIPY staining plus flow cytometry was used to investigate the effect of DJ-1 manipulation on Lipid ROS under ferroptosis stimuli;CCK8 assay was used to investigate the effect of DJ-1 manipulation on Erastin-induced cell death;Transmission electron microscopy was used to investigate the role of DJ-1 knockdown on mitochondria morphology changes under ferroptosis stimuli;Nude mouse xenograft model was performed to determine the effect DJ-1 in ferroptosis-based anti-tumor therapy;Immunohistochemistry and TUNEL assay were used to investigate the impact of DJ-1 on proliferation and apoptosis in tumor tissues;RT-PCR and ELISA assay were used to investigate the impact of DJ-1 on ferroptosis tumor tissues;RT-PCR was used to investigate the impact of DJ-1 on iron metabolism and lipid peroxidation associated proteins,such as DMT1,FTH1,FTL,ACSL4,LPCTA3,15-LOX;Western blot was conducted to investigate the impact of DJ-1 on the expression of SLC7A11 and NRF2;Glutamate release kit was used to investigate the impact of DJ-1 on the function of SLC7A11;CCK8 assay was used to investigate the effect of DJ-1 knockdown on NRF2 knockdown enhanced cell death induced by Erastin.(2)Mechanism of DJ-1-regulated ferroptosis: GSH detection kit was used to investigate the role of DJ-1 knockdown on intracellular GSH level under ferroptosis stimuli;Western blot was applied to investigate the impact of DJ-1 on GSH biosynthesis associated proteins expression(?-GCS and GSS);BODIPY staining plus flow cytometry and CCK8 assay were used to investigate the impact of GSH,NAC,Met,SAM,SAH,Hcy on DJ-1-regulated ferroptosis as a metabolite rescue assays;Metabolic flux analysis was performed to identify the key mediates involved in DJ-1-regulated ferroptosis;ELISA assay was used to investigate the effect of DJ-1 manipulation on intracellular Hcy level;Enzymatic activity assay was performed to investigate the impact of DJ-1 manipulation on SAHH activity;Proximity-dependent Biotin identification assay was performed to find the transit interacting protein involved in DJ-1-regulated SAHH;Immunoprecipitation was conducted to investigate the role of DJ-1 on SAHH/AHCYL1 heterodimer formation;Size exclusion chromatography and DSS mediated cross-linking assay were applied to investigate the impact of DJ-1 on the tetrameric SAHH complex formation;C11-BODIPY staining plus flow cytometry and CCK8 assay were used to investigate the role of SAHH overexpression or AHCYL1 knockdown on DJ-1-regulated ferroptosis.(3)Anti-tumor intervention strategy based on the dimeric DJ-1 structure: Immunoprecipitation was conducted to investigate the formation of DJ-1 homodimer;Point mutation combined with DSS mediated cross-linking assay were applied to investigate the impact of C terminal amino acids of DJ-1 on its dimerization;Nacetylcysteine deglycation assay was performed to investigate the impact of dimer deficient DJ-1 on its deglycase activity;C11-BODIPY staining plus flow cytometry and SRB assay were used to investigate the impact of dimer deficient DJ-1 on ferroptosis;Design and synthesis of dimeric DJ-1 inhibitors and N-acetylcysteine deglycation assay was performed to investigate the inhibitory potency on DJ-1 deglycase activity at the purified protein level in vitro;C11-BODIPY staining plus flow cytometry and SRB assay were used to investigate the role of DJ-1 inhibitor on ferroptosis.Results(1)Redox protein DJ-1 negatively regulates ferroptosis: Both DJ-1 knockdown and DJ-1 knockout enhanced the accumulation of Lipid ROS and cell death under ferroptosis stimuli;Wildtype DJ-1 overexpression reduced the accumulation of Lipid ROS and cell death under ferroptosis stimuli in DJ-1 knockout MEFs;DJ-1 knockdown showed more shrunken mitochondria under ferroptosis stimuli;Nude mouse xenograft model showed that suppression of DJ-1 enhanced ferroptosis in vivo;DJ-1 depletion did not impact tumor cell proliferation and apoptosis during piperazine Erastin-based anti-tumor therapy in vivo;RT-PCR and ELISA assay showed that DJ-1 depletion enhanced ferroptosis with increased PTGS2 level and 4-HNE level during piperazine Erastin-based anti-tumor therapy in vivo;DJ-1 could not affect iron metabolism and lipid peroxidation;The expression and the function of SLC7A11 were not affected by manipulating DJ-1 expression;The expression of NRF2 protein and well-characterized NRF2 target genes were significantly upregulated after Erastin treatment in DJ-1 knockdown H1299 cells;DJ-1 knockdown enhanced ferroptosis even in NRF2 knockdown H1299 cells.(2)Mechanism of DJ-1-regulated ferroptosis: DJ-1 suppresses ferroptosis through the transsulfuration pathway by increasing the formation of the SAHH tetramer and preserving its activity via reducing the formation of SAHH/AHCYL1 heterodimer.Both DJ-1 knockdown and DJ-1 knockout reduced the GSH levels under ferroptotic stimuli;DJ-1 did not affect glutathione biosynthesis associated proteins expression(?-GCS and GSS);GSH,NAC,Hcy totally blocked the Lipid ROS and cell death enhanced by DJ-1 knockdown under ferroptosis stimuli;Metabolic analysis showed DJ-1 knockdown reduced the Hcy levels;DJ-1 promoted the enzymatic activity of SAHH;Proximitydependent Biotin Identification assay showed DJ-1 had an interaction with AHCYL1;DJ-1 impaired the formation of SAHH/AHCYL1 heterodimer;DJ-1 enhanced the formation of SAHH tetramer;Both SAHH overexpression and AHCYL1 knockdown reduced the Lipid ROS and cell death enhanced by DJ-1 knockdown under ferroptosis stimuli.(3)Anti-tumor intervention strategy based on the dimeric DJ-1 structure: DJ-1 represented as homodimer in cells;The last three amino acids of C terminus determined dimeric DJ-1 formation;Dimer deficient DJ-1 lost its deglycase activity;Dimer deficient DJ-1 could not suppress Erastin-induced Lipid ROS and cell death in MEFs;Design and synthesis of DJ-1 inhibitors based on DJ-1 homodimer structure,named as DMs;DMs inhibited DJ-1 deglycase activity and DM10 showed the best inhibitory property in vitro, which could bind to the homodimer of DJ-1;DM10 enhanced Erastin-induced ferroptosis.Conclusion In this study,we define a negative role of DJ-1 in ferroptosis.Of note,DJ-1 knockdown or knockout potently enhances the accumulation of Lipid ROS and cell death induced by several ferroptosis inducers,while DJ-1 overexpression inhibits ferroptosis.Mechanistically,metabolic analysis and metabolite rescue assays reveal that DJ-1 depletion inhibits the transsulfuration pathway by disrupting the formation of the SAHH tetramer and impairing its activity via enhancing the formation of SAHH/AHCYL1 heterodimer.Consequently,increased ferroptosis is induced when homocysteine generation is decreased,which might be the only source of glutathione biosynthesis when cysteine uptake is blocked.DJ-1 depletion potently enhances the sensitivity of tumor cells to ferroptosis inducers both in vitro and in vivo.The homodimer sturuture is required ofr the biological function of DJ-1 and the last 3 amino acids of C terminus determines the DJ-1 homodimer.Based on these,a series of dimeric DJ-1 inhibitors were designed and synthesized,and the dimeric DJ-1 inhibitor DM10 can effiently enhance the effect of ferroptosis based anti-tumor therapy.