线粒体Trx2在血管内皮细胞内调控ASK1的分子机制及其作为动脉粥样硬化防治靶点的研究

Mitochondrial dysfunction in endothelial cells plays an important role in the vascular disease. We have reported that the Trx2, a key mitochondrial protein, is critical to maintain mitochondria in reduced state and prevent endothelial cell dysfunction and atherosclerotic development. Trx2 is able to suppress mitochondrial reactive oxygen species (ROS) generation and enhance nitric oxide (NO) bioavailability. Further, Trx2 binds to ASK1 to regulate mitochondria-dependent apoptotic pathway. Interestingly, a single Cys mutant within the catalytic site of Trx2 (Trx2-C93S which lack the redox-catalyzing activity) retains the ability in ASK1 binding, inhibiting ASK1-induced EC apoptosis, suggesting a direct association of Trx2 with ASK1 is critical for the inhibitory effect of Trx2 on ASK1. However, the molecular mechanism of Trx2 regulates the activity of mitochondrial ASK1 (mtASK1) and further modulates the function of endothelial cells is not known. Our latest preliminary data shows that cellular protein within Trx2 siRNA treated endothelial progenitor cells exhibits a lower amount of S-nitrosylation, an important post-translational modification mediated by NO. S-nitrosylation can regulate a wide variety of signaling events and cellular function, including ASK1. Further, the expression of mtASK1 by adenoviral infection induces an opening of mitochondrial permeability pore, ROS generation and cyto c release. Therefore, we hypothesize that Trx2 modulates mtASK1 activity via Trx2-augmented NO bioavailability mediated S-nitrosylation, and direct binding of Trx2 and mtASK1 is critical to maintain the integrity and redox state of mitochondria. We will determine whether Trx2 upregulates S-nitrosylation of ASK1 and other related key mitochondrial proteins, and whether such modification would contribute to protecting endothelial cells. Further, we will investigate Trx2-ASK1 binding could maintain the integrity of mitochondria function and cellular redox hemostasis. Besides, Trx2-expressing lentiviral vector will be delivered to endothelial cells in ApoE-/- mice before and post the onset of atherosclerosis. This will test the potential utility of mitochondrial anti-oxidative gene therapy for prevention and treatment of atherosclerosis. This project will demonstrate a novel mechanism of mitochondria regulates endothelial cells, and this will provide supporting evidence for development of Trx2 as cardiovascular treatment target.

我们已报道作为一个抗氧化剂，线粒体Trx2在内皮细胞表达能减少活性氧簇(ROS)，增强NO生物活性，抑制线粒体促凋亡应激激酶(ASK1)介导的细胞凋亡，缓解动脉粥样硬化疾病进程。但Trx2如何调控ASK1并保护内皮细胞功能的分子机制未明。新近数据表明内皮细胞敲低Trx2后，NO介导的总巯基亚硝基化修饰水平下降, 同时ASK1能调节线粒体通透孔，并促进ROS生成和细胞凋亡，提示Trx2可能通过巯基亚硝基化修饰来影响ASK1及线粒体关键蛋白活性，并通过结合ASK1调控线粒体氧化还原状态及细胞存活。在前期基础上，拟用生物素交换、免疫共沉淀探讨由Trx2调控发生巯基亚硝基化的线粒体蛋白，构建Trx2突变体解析Trx2-ASK1结合对内皮细胞功能的影响，利用Trx2基因治疗验证其对动脉粥样硬化小鼠疾病模型的防治意义。本项目将揭示线粒体调控内皮细胞新机制，为Trx2作为血管疾病防治新靶点提供重要依据。

动脉粥样硬化是一种慢性炎症疾病，活性氧簇(ROS)及炎性分子等外部因素将进一步促进动脉壁炎症的发展，而内皮细胞在控制炎症和动脉粥样硬化过程中起到了重要作用。前期我们已报道，作为一个有效的抗氧化物，线粒体硫氧还蛋白亚型2（Trx2）在内皮细胞的表达能减少ROS，抑制促凋亡应激激酶(ASK1)介导的细胞凋亡，在保护心血管系统上起到了至关重要的作用。在此基础上，本课题继续深入研究在过量ROS刺激下Trx2保护内皮细胞功能的机制。RNA-seq分析发现，在内皮细胞中沉默Trx2，NF-κB信号通路被激活。尽管Trx2敲低后不能诱导细胞衰老（senescence）；过量ROS处理后，Trx2敲低的内皮细胞中衰老相关标志物如p-p53、p21和p16水平均明显升高，并分泌大量IL-6和IL-1α，提示Trx2可以保护细胞免于ROS诱导的细胞衰老。另一方面，我们还发现ROS能抑制参与Trx2成熟的蛋白α-MPP（PMPCA）的类泛素化修饰（SUMOylation），从而损伤Trx2的加工和功能，导致血管内皮细胞衰老。在这过程中，Trx2独特的SUMO相互作用基序（SUMO-interacting motif）对Trx2在线粒体的加工和随后抗氧化和抗衰老活性至关重要。另外，由于Trx2在内皮细胞中能抑制ASK1介导的细胞凋亡，我们也拓展关注了ASK1互作蛋白AIP1(ASK1-interacting protein-1)在内皮细胞内的作用。AIP1在内皮细胞中也能调控ROS生成，这主要通过抑制p47phox-NOX2/p22phox复合物的形成，进而阻断NOX2(NADPH oxidase-2)活性来实现抗ROS的功能。该项目为Trx2及AIP1作为血管疾病的防治新靶点提供重要依据，具有崭新的理论价值和潜在的应用价值。