载脂蛋白AI硝基化调控胆固醇外流的机制研究

Cholesterol efflux is a crucial step of the reverse cholesterol transport. It is reported that nitrated apolipoprotein AI (NT-apoAI) might be dysfunctional, related to reduced cholesterol efflux and atherosclerosis in small case-control studies. Our previous studies showed that serum NT-apoAI/apoAI ratio was higher in patients with coronary heart disease than controls and it was negatively associated with cholesterol efflux, but the mechanism remains unknown. We hypothesis that NT-apoAI might be related to atherosclerosis and cholesterol efflux and different nitrotyrosine sites of apoAI might have different impacts on cholesterol efflux. In the present study, we are going to measure the serum levels of NT-apoAI and cholesterol efflux between patients with coronary heart disease and controls in a relatively large Chinese cohort by using a novel ELISA method in order to establish the relationships between NT-apoAI, cholesterol efflux and the severity of coronary atherosclerosic lesions. Further, we are going to study the impact of the specific nitrotyrosine sites of apoAI on the cholesterol efflux by point mutation of the 4 known nitrityrosine sites and following nitriation in vitro and biogenesis of HDL from nitrated apoAI in vivo by injecting these nitrated apoAI into apoAI KO mice and measuring mice serum apoAI and HDL levels. Our study might preliminarily provide the mechanisms of the NT-apoAI on cholesterol efflux and the results might give novel therapeutic target on atherosclerosis.

胆固醇外流是胆固醇逆向转运的关键步骤。HDL的结构蛋白-apoAI是胆固醇外流的接受体，其发生硝基化后蛋白构象和性质发生改变，直接影响胆固醇外流。我们的前期研究结果显示冠心病患者血清NT-apoAI/apoAI比例升高，并与胆固醇外流负相关。为此我们提出假说：NT-apoAI通过减少胆固醇外流，参与动脉粥样硬化的发生发展，apoAI上不同酪氨酸残基位点的硝基化可能是影响胆固醇外流的关键所在。为验证这一假说，我们在人群研究中高通量测定冠心病患者及其对照组血清NT-apoAI，分析其与冠脉病变严重程度、胆固醇外流的关系；对apoAI的四个酪氨酸残基位点进行点突变及体外硝基化，研究一个或几个位点的突变对硝基化的影响及其对胆固醇外流的作用；在apoAI敲除小鼠体内研究硝基化对apoAI转化成HDL的影响，从人群资料、体外细胞、在体动物水平三个层次阐明apoAI硝基化调控胆固醇外流的作用及机制。

胆固醇外流是胆固醇逆向转运的关键步骤。HDL的结构蛋白-apoAI是胆固醇外流的接受体，其发生硝基化后蛋白构象和性质发生改变，直接影响胆固醇外流。我们的前期研究结果显示冠心病患者血清NT-apoAI/apoAI比例升高，并与胆固醇外流负相关。为此我们提出假说：NT-apoAI通过减少胆固醇外流，参与动脉粥样硬化的发生发展，apoAI上不同酪氨酸残基位点的硝基化可能是影响胆固醇外流的关键所在。为验证这一假说，我们在人群研究中高通量测定冠心病患者及其对照组血清NT-apoAI，分析其与冠脉病变严重程度、胆固醇外流的关系；对apoAI的Tyrosine 192残基位点进行点突变及体外硝基化，研究该位点的突变对硝基化的影响及其对胆固醇外流的作用；在apoAI敲除小鼠体内研究硝基化对apoAI转化成HDL的影响。研究结果发现，胆固醇外流与NT-apoAI浓度呈负相关，apoAI的Tyr-192酪氨酸残基硝基化不能减少ABCA1依赖的胆固醇外流。