线粒体膜心磷脂与GPX4蛋白互作介导铁死亡依赖性肝毒性的调控机制研究

Membrane lipid homeostasis serves as a new cell transduction signal, which maintains the regulation of the integrity of the plasma membrane and organelles and mediates diverse modes of regulatory cell death (RCD). We have confirmed that the necrosomes of necroptosis complex were prone to mitochondria translocalization, and this is associated with the regulation of targeting dephosphorylation as well as the mitochondrial membrane externalization of the cardiolipin (CL) in our additional lastest analysis. However, the lipid-protein interaction between the CL externalizated in the mitochondrial membrane and the ferroptosis marker glutathione peroxidase 4 (GPX4) is still unknown, so is the regulatory mechanism. This project for the first time proposed the hypothesis that the modulation of the CL-GPX4 interaction signaling axis via the mitochondrial-associated protein phosphatase (mtPPs) cascade is a new pathway for the regulation of induced ferroptosis mode. We plan to geneticaly establish the engineered human hepatocytes, used in 3D spheroids system, co-culture, and huamn liver chimeric (HLC) mice for in vitro and in vivo experiments administered with representative hepatotoxic xenobiotics. The metabolic pathways associated with CL composition and externalization in mitochondrial membrane induced by xenobiotics were screened and identifed. The GPX4 translocation to the mitochondrial membrane and its formation of protein complex mediated by the dephosphorylation of GPX4 functional sites were explored. The regulation for different signaling targets in the process of CL-GPX4 interactions via mitochondrial-associated protein phosphatases (mtPPs) cascade were investigated. The modulation for the functional homeostasis in multi-organelles associated with mitochondrial membrane contact sites (MCS) and their relationship with the hepatotoxicity and damage effects induced by xenobiotics were evaluated. The aim of the project is to elucidate the new regulatory mechanism involved in the mtPPs cascade mediates multi-target protein quality control for the homeostasis of mitochondrial membrane lipid-protein complex in CL-GPX4 interaction signaling axis, and to explore a new steady GPX4 complex body-based damage identified molecular patterns related to the xenobiotics-induced hepatotoxicity mediated by membrane-lipid-associated cell death signaling pathways. The results could provide novel targetings for molecular characterizations, toxicity assessments, and effective interventions to the environmental exposure via the application of targets and their dephosphorylation in the membrane lipid-protein interaction signaling axis.

膜脂质稳态作为新的细胞信号平台，调控细胞器和膜的完整性，介导调节性细胞死亡。我们已证实调节性细胞死亡蛋白复合物可转位线粒体并与去磷酸化调控有关；新近发现与线粒体膜心磷脂(CL)外化相关的端倪；但CL与谷胱甘肽过氧化物酶4(GPX4)互作和调控机制尚未知。本项目首次提出“线粒体关联性蛋白磷酸酶(mtPPs)级联调节CL-GPX4互作信号轴作为诱导铁死亡调控新通路”假设。外源物暴露的人肝细胞模型3D培养和共培养及嵌合小鼠体内外试验；鉴定暴露相关的CL变化和外化；探明GPX4线粒体膜转位与CL互作并招募蛋白复合物和诱导铁死亡表型，探究CL-GPX4互作和mtPPs对信号轴蛋白质量控制的调控；探讨线粒体膜关联的细胞器稳态调节细胞毒性和肝损伤。阐明CL-GPX4互作介导铁死亡依赖性肝毒性的信号级联调控新机制；为环境暴露中膜脂质-蛋白互作的脂质死亡信号表征、线粒体膜毒性评价和细胞铁死亡干预提供新靶向。

背景：膜脂质-蛋白质互作和稳态调控作为新型信号平台，可介导调节性细胞死亡(RCD)。我们研究已发现不同RCD生物标志可经由翻译后修饰(PTM)调控和调节线粒体(mito)相关多细胞器通讯。本项目聚焦“线粒体依赖性铁死亡调控外源物诱导肝毒性”科学问题，探明“心磷脂(CL)-谷胱甘肽过氧化物酶4(GPX4)信号轴级联调控”的新机制。.完成的内容：(1)表型上探索不同外源物诱导细胞铁死亡；(2)机制上探明线粒体CL依赖性结构和功能对铁死亡调节、及线粒体p-GPX4Ser2和COX-2等靶分子及其线粒体关联性蛋白磷酸酶(PPs)介导PTM调控；(3)潜在应用上探讨铁死亡在外源物诱导不同细胞转归中的毒理学评价和靶向干预作用。.主要结果：(1)探讨线粒体p-GPX4Ser2依赖性铁死亡介导外源物诱导肝毒性的调控机制。发现线粒体p-GPX4经由调节p53逆行信号介导铁死亡；探明PP2A-B55调控线粒体p-GPX4Ser2及其与p53互作介导铁死亡转归。(2)探究线粒体CL参与线粒体质量控制(MQC)调节介导外源物诱导铁死亡的调控机制。发现CL参与Drp1依赖性MQC对铁死亡的调节；探明镉诱导铁死亡依赖性肝毒性的CL重构及其PGAM5调控机制。(3)探索外源物诱导GPX4依赖性铁死亡敏感性的线粒体调控机制。发现线粒体铁硫簇代谢调节嵴重塑介导CL-GPX4相关铁死亡的分子机制及其抗肝癌敏感性；线粒体PD-L1-GPX4信号轴介导IFN-γ诱导肝癌干细胞代谢重编程和铁死亡耐受。(4)探讨细胞器间通讯调节GPX4依赖性铁死亡介导外源物诱导毒性作用的机制及靶向干预。发现线粒体-内质网通讯调控BDE47诱导铁死亡；BDE47抑制UPRER诱导铁死亡的线粒体自噬调节机制；提示PPs靶向STAU1调控参与BDE47诱发细胞铁死亡。.科学意义：理论上阐明“线粒体CL-GPX4-PPs级联信号轴、经由线粒体关联性多细胞器通讯和MQC调节、介导外源物诱导铁死亡依赖性肝毒性和转归”的新分子机制。在“线粒体CL-GPX4/COX-2互作-蛋白PTM调控级联新通路”方面具有创新性。提供筛选线粒体CL-GPX4-PPs调控轴作为新生物标志和潜在干预新靶点的证据。