MDM2介导RXRβ泛素化降解在动脉粥样硬化血管内皮线粒体稳态失衡中的作用及机制研究

RXRβ has been found as a potential new target for treatment of arteriosclerosis (AS) using proteomics based on iTRAQ mass spectrometry in our preliminary study. Furthermore, PGC-1α, a key regulator of mitochondrial homeostasis, was the target of RXRβ. The function of RXRβ was closely related to the mitochondrial homeostasis of vascular endothelial cells in AS environment, and such relationship was only dependent on protein expression of RXRβ. Next, we found that the stability of RXRβ protein was regulated by ubiquitin proteasome system. And MDM2, which has ubiquitin E3 ligase activity, could combine with RXRβ and promote ubiquitination degradation of RXRβ. Thus, this research will use pharmacological methods, molecular biology and gene technology to verify the following scientific hypothesis from the perspective of the interaction of protein and protein specific amino acid fragments: under the stimulation of atherogenic risk factors, MDM2 combines with RXRβ in the vascular endothelial cells and promotes RXRβ degradation by its E3 ubiquitination activity, which decreases the function of PGC-1α, leads to the imbalance of mitochondrial homeostasis and promotes the process of atherosclerosis. Targeted inhibition of MDM2 or interference with MDM2- RXRβ binding may have the effect to maintain mitochondrial homeostasis in vascular endothelial cells, which is a potential strategy for prevention and treatment of atherosclerosis.

我们前期利用基于iTRAQ质谱技术的蛋白质组学等方法发现动脉粥样硬化(AS)防治的潜在新靶点：RXRβ。进一步发现，线粒体稳态的关键调节因子PGC-1α是RXRβ作用靶点，RXRβ功能与AS环境下的血管内皮细胞线粒体稳态密切相关，但这相关性只与RXRβ蛋白表达有关，且发现RXRβ的蛋白稳定性受到泛素-蛋白酶体系统调控。初步研究表明具有泛素E3连接酶活性的MDM2能够与RXRβ结合促进RXRβ泛素化降解。因此，本课题拟利用药理学方法、分子生物和基因技术，从蛋白与蛋白特定氨基酸片段相互作用等角度验证以下科学假说：在致AS危险因子作用下，血管内皮细胞MDM2与RXRβ结合，利用其E3泛素连接酶活性促使RXRβ降解，进而导致PGC-1α信号功能减弱和细胞线粒体稳态失衡的发生，促进AS进程。靶向抑制MDM2或干扰MDM2-RXRβ结合具有潜在维持血管内皮细胞线粒体稳态作用，是调控AS发生的潜在策略。

探寻动脉粥样硬化(AS)调控新靶标是研发AS全新治疗药物的关键。我们前期发现AS防治的潜在新靶点RXRβ，及MDM2与RXRβ蛋白降解有关。MDM2在肿瘤疾病研究和应用中较多。我们首次将MDM2的功能研究扩展到AS。本研究旨在揭示MDM2-RXRβ参与AS发生、发展的机制及方式。主要研究内容包括以下三部分：1.分析MDM2在血管内皮细胞线粒体稳态中的作用；2.研究MDM2通过RXRβ调节血管内皮细胞线粒体稳态的分子基础及对RXRβ蛋白稳定性调控的具体机制；3.研究血管内皮依赖于MDM2的RXRβ泛素化降解在整体动物动脉粥样硬化病理生理过程中的作用。我们的研究发现，MDM2蛋白表达上调、RXRβ蛋白表达下调均参与致AS危险因子ox-LDL诱导的血管内皮细胞线粒体稳态失衡及炎症反应。进一步研究发现，在致AS危险因子作用下，血管内皮细胞MDM2与RXRβ结合，利用其E3泛素连接酶活性促使RXRβ多聚泛素化并蛋白降解，随之使PGC-1α信号功能减弱，进而导致血管内皮细胞线粒体稳态失衡的发生，由此介导TLR9/NF-κB信号通路和NLRP3/caspase-1炎症体的激活，进一步产生促炎细胞因子TNF-α、IL-6和IL-1β。功能缺失突变体MDM2-C464A、MDM2抑制剂、siRNA MDM2均减弱MDM2对RXRβ的多聚泛素化及蛋白降解，且靶向抑制MDM2或干扰MDM2-RXRβ系统能维持血管内皮细胞线粒体稳态，减少血管炎症反应，防治动脉粥样硬化发生。根据本项目的结果，我们提出以MDM2-RXRβ调节轴为靶点可能代表人类动脉粥样硬化治疗的新策略。