Erastin Modifies GPX4 Ubiquitination Via Trim46 And Induces Ferroptosis

Ferroptosis is a novel mode of programmed cell death that is iron-dependent.It is characterized by the inactivation of intracellular glutathione-dependent antioxidant systems and the massive accumulation of lipid peroxides.Current studies have shown that ferroptosis is closely associated with the development of numerous serious life-threatening human health diseases.Therefore,it is essential to elucidate the molecular mechanisms involved in the development of ferroptosis and to search for its potential targets,which can help provide new ideas for the treatment of ferroptosis-related diseases.GPX4 is an antioxidant enzyme that catalyzes the reduction of multiple peroxides,thereby maintaining the balance of ROS in the body and thereby protecting cells from peroxide damage.It has been shown that GPX4 is a key negative regulator of ferroptosis and can scavenge intracellular accumulation of peroxides and oxygen radicals by hydrolyzing lipid peroxides.Therefore,how to enhance ferroptosis and thus contribute to cancer cell death by inhibiting GPX4 expression or enzyme activity in tumor cells is a promising new strategy for cancer therapy.Ubiquitination is a widespread post-translational modification that plays an important biological regulatory role in the cell.It is catalyzed by a variety of enzymes and can specifically ubiquitinate substrate proteins,which in turn are recognized and degraded in the proteasome.In recent years,the role of protein post-translational modifications in the process of ferroptosis has received much attention from scientists.It has been found that deubiquitinase inhibitors can trigger ferroptosis and that this process is accompanied by the degradation of GPX4 protein.Furthermore,in retinal endothelial cells,high glucose treatment can modulate the level of ubiquitination modifications of GPX4 via Trim46.It is intriguing whether Erastin,as an ferroptosis inducer,can also mediate the ubiquitination modification of GPX4 through Trim46 and thus regulate the ferroptosis process.This is the scientific question that this thesis aims to explore and answer.In this study,we used HEK293 T as a cell model,and determined the occurrenceV of ferroptosis by measuring ferroptosis-related indicators after treatment with Erastin and other ferroptosis regulators.Then,the effect of Trim46 on the protein stability of GPX4,the ubiquitination level of GPX4,and the potential ubiquitination sites of GPX4 was examined to clarify the ubiquitination effect of Trim46 on GPX4 protein and the molecular mechanism;to elucidate whether Erastin mediates the ubiquitination modification of GPX4 through Trim46 to regulate ferroptosis in cells.The results of the present study will provide a new theoretical basis for the targeting of many tumor interventions that target key factors related to ferroptosis regulation.The related work is summarized as follows.First,to clarify the effects of Erastin on ferroptosis and Trim46 expression,we used different concentrations of Erastin alone or in combination with other regulators of ferroptosis.By using ROS,MDA,GSH,CCK-8 and Western Blot,we determined that Erastin induced ferroptosis in 293 T cells,reduced cellular value-added capacity,and was accompanied by an upregulation of Trim46 protein expression.Second,to clarify the potential relationship between Trim46 and GPX4,we purchased the PC3.1-Flag-Trim46 plasmid and successfully constructed the PC3.1-Trim46 plasmid to establish the overexpression of Trim46 system.Then,we added the proteasome inhibitor MG132 in groups to detect the protein expression level of GPX4,and the results showed that overexpression of Trim46 down-regulated the protein stability of GPX4.Third,to clarify whether Trim46 can directly mediate the ubiquitination modification of GPX4,we successfully constructed the PC3.1-Flag-GPX4 plasmid and established the overexpression GPX4 system;transfected Flag-GPX4 and PC3.1-Trim46 vectors alone or in combination in 293 T cells,and detected the interaction between Trim46 and GPX4 by Flag IP.The results showed that Trim46 could directly interact with GPX4 and promote the upregulation of GPX4 ubiquitination level.Fourth,to determine the molecular mechanism of Trim46-mediated GPX4 ubiquitination regulation,we constructed GPX4 deubiquitination point mutation plasmids(K107R,K126 R,K167R,K191R)based on the Phosphositeplus website to predict the potential ubiquitination sites of GPX4,and verified the successful construction of the point mutation vector by Western Blot.The results of ubiquitination IP suggested that the K191 site is the regulatory site for ubiquitination modification of GPX4 catalyzed by Trim46.Fifth,to examine the possible mechanism of Erastin inhibition of GPX4 expression,we applied Erastin alone or in combination with MG132,and the Western Blot results showed that Erastin treatment could reduce the stability of GPX4 protein.To determine the effect of Erastin on the ubiquitination degradation of GPX4,we transiently transfected Flag-GPX4 and HA-Ubi vectors in 293 T cells and treated the cells with MG132,and the Flag IP results showed that Erastin induced the ubiquitination degradation of GPX4.We showed that Erastin could catalyze the ubiquitination degradation of GPX4 through Trim46 in 293 T cells transfected with Trim46 and GPX4 vector alone or in combination with Erastin treatment,and Western Bloting and Flag IP results showed that Erastin could catalyze the ubiquitination degradation of GPX4 through Trim46.Sixth,to clarify the role of Trim46-GPX4 pathway in the induction of ferroptosis by Erastin,we transfected the overexpressed Trim46 vector in 293 T cells and performed a reversion assay by overexpressing GPX4 after treatment with Erastin to detect ROS,MDA,GSH,and MTT,and the results demonstrated the role of Trim46-GPX4 pathway in the induction of ferroptosis by Erastin.The results demonstrated that the Trim46-GPX4 pathway plays an important role in the induction of ferroptosis by Erastin.In summary,it is hypothesized that Erastin can regulate the ubiquitinated degradation of GPX4 and induce ferroptosis in cells through the ubiquitination modification of GPX4 K191 site catalyzed by Trim46.The experimental results obtained in this paper will provide new perspectives for the subsequent study of potential targets in the treatment of ferroptosis-related diseases.