FAT10化修饰Lonp1诱导线粒体未折叠蛋白反应介导Apelin-13/APJ促血管平滑肌细胞增殖

Applicant reported that apelin-13, an endogenous ligand of G protein-coupled receptor APJ, promotes vascular smooth muscle cell (VSMC) proliferation. However，the molecular mechanism of apelin-13-induced VSMC proliferation remains unclear.Research studies revealed that mitochondrial unfolded protein response as the new stress mechanism increases cell proliferation. Preliminary experiments found that ubiquitin like protein FAT10 (human HLA-F adjacent transcript 10) interacts with ATP-dependent Lon protease 1 (Lonp1) in human aortic VSMC. Apelin-13 increases the expressions of FAT10 and Lonp1, and therefore induces mitochondrial unfolded protein response. According to that, the scientific hypothesis "FAT10ylation of Lonp1 inducing mitochondrial unfolded protein response mediates vascular smooth muscle cell proliferation induced by apelin-13"can be proposed. We adopt VSMC, ApoE knockout mice and other methods to explore whether mitochondrial unfolded protein response is involved in apelin-13/APJ-induced human aortic VSMC proliferation；To analyze whether FAT10ylation of Lonp1 promotes human aortic VSMC mitochondrial unfolded protein response induced by apelin-13/APJ. From the new perspective that FAT10ylation of Lonp1 mediates mitochondrial unfolded protein response, we can clarify a new molecular mechanism of apelin-13/APJ system to promote human aortic VSMC proliferation. The APJ receptor is a new drug target that provides the theoretical basis for vascular diseases via prevention and treatment of atherosclerosis and other vascular disorders.

申请者报道受体APJ的内源性配体Apelin-13促血管平滑肌细胞(VSMC)增殖，但机制不明。文献报道线粒体未折叠蛋白反应(mtUPR)作为新发现的细胞内应激机制，诱导细胞增殖。预实验发现类泛素蛋白FAT10与mtUPR标志物Lonp1有相互作用，Apelin-13增加FAT10及Lonp1的表达, 诱导mtUPR。据此提出“FAT10化修饰Lonp1诱导线粒体未折叠蛋白反应介导Apelin-13/APJ促血管平滑肌细胞增殖”的科学假说。课题拟采用VSMC及ApoE-/-小鼠模型等明确促进mtUPR介导Apelin-13/APJ促VSMC增殖；阐明FAT10化修饰Lonp1是Apelin-13/APJ诱导mtUPR关键环节。以FAT10化修饰Lonp1诱导mtUPR新视角，揭示Apelin-13/APJ促VSMC增殖新机理，并为APJ受体作为药物新靶标研发防治AS疾病新药物提供理论依据。

本项目以“FAT10化修饰Lonp1诱导线粒体未折叠蛋白反应”这一新视角，进一步揭示Apelin-13/APJ系统促血管平滑肌细胞增殖新机制，为以Apelin/APJ系统作为作用靶点的防治动脉粥样硬化等增生性心血管疾病提供理论依据。研究结果如下：1. Apelin-13诱导人主动脉血管平滑肌细胞(HA-VSMCs)中线粒体损伤和形态改变，包括线粒体内部出现空泡；APJ拮抗剂F13A可逆转Apelin-13引起HA-VSMCs线粒体形态不规则变化；2. Apelin-13引起HA-VSMCs中ROS水平升高，而F13A显著抑制Apelin-13诱导人主动脉血管平滑肌细胞ROS生成；3. Apelin-13引起HA-VSMCs中线粒体未折叠蛋白反应（mtUPR），包括mtUPR标志蛋白 Lonp1、HSP60、mtHSP70和 ClpP表达上调呈浓度性和时间性依赖性方式；F13A显著抑制Apelin-13引起HA-VSMCs中Lonp1、HSP60、mtHSP70和 ClpP的表达上调；免疫荧光实验亦证实，Apelin-13促进HA-VSMCs中Lonp1、HSP60、mtHSP70和 ClpP的表达上调；4. siRNA-Lonp1显著抑制Apelin-13诱导的HA-VSMCs中mtUPR标志蛋白HSP60、mtHSP70和 ClpP，表明Lonp1介导Apelin-13诱导mtUPR的发生；5.Apelin-13浓度和时间依赖性促进HA-VSMCs中FAT10的表达；6.激光共聚焦技术和免疫共沉淀技术均证实FAT10与Lonp1存在相互作用。进一步，Apelin-13显著促进HA-VSMCs中FAT10和Lonp1的共定位和相互作用；7. siRNA-FAT10显著减弱Apelin-13引起的HA-VSMCs中Lonp1、HSP60、 mtHSP70和 ClpP的表达，以上结果表明，FAT10对于Apelin-13 通过FAT10化修饰诱导血管平滑肌细胞mtUPR；8.Apelin-13/APJ 引起HA-VSMCs的PCNA和a-SMA的表达，F13A抑制Apelin-13 诱导HA-VSMCs中的PCNA和a-SMA的表达，结果表明Apelin-13促进HA-VSMCs增殖；