脂筏对跨膜区介导的膜蛋白GP Ib-IX跨膜信号转导的调控机制研究

Lipid raft domains, also known as glycosphingolipid-enriched membranes, can act as a platform for the assembly of downstream signaling molecules and adhesion receptors. As the most important membrane receptor complex on the platelet surface, the glycoprotein (GP) Ib-IX complex locates in lipid raft and regulates the platelet activation and aggregation through transmembrane signaling transduction. However, how this complex carries out its multi-faceted functions in the lipid raft is not clear. In this project, we aim to (1) elucidate the structural property of lipid raft of platelets and the molecular mechanism of GP Ib-IX complex within lipid raft, (2) characterize the structural property of signal transduction domain and functional roles of inter-subunit interactions of GP Ib-IX complex, (3) discover the molecular mechanism of GP Ib-IX complex transmembrane signaling transduction regulated by lipid raft. Dissociation of the GP Ib-IX complex from the lipid raft by membrane cholesterol depletion abolishes transmembrane signaling transduction. These investigations integrate diverse physical, chemical and cell biological approaches to provide new insights into platelet lipid raft structure and dynamics and their roles in GP Ib-IX complex transmembrane signaling transduction. Overall, this proposed study will help to provide a mechanistic guide for developing novel therapeutic strategies to combat various complex related bleeding disorders and cardiovascular disease.

血小板活化和聚集过程中，脂筏是结合黏附分子和信号分子的信号转导平台，参与活化受体并募集信号分子、介导跨膜传递信号。作为血小板表面最重要的膜受体复合物的GP Ib-IX位于脂筏平台中，通过跨膜信号传导调控着血小板的活化和聚集，但其具体的分子机制尚不清楚。本项目将结合体外与体内分析深入研究血小板脂筏微区的结构特征以及膜蛋白GP Ib-IX进出脂筏的分子机制；进一步鉴定GP Ib-IX的信号转导区的结构特征，阐明跨膜区介导的膜蛋白GP Ib-IX跨膜信号转导的分子机制；通过干扰脂筏和GP Ib-IX跨膜区以及信号转导结构域之间的相互作用，破坏膜蛋白GP Ib-IX跨膜信号转导，从而揭示脂筏调控GP Ib-IX跨膜信号转导的分子机制，进一步阐明脂筏对血小板黏附以及激活生理功能的调控作用，为去除血栓以及治疗出血性疾病发病机理的研究和治疗方法的开发提供理论依据和实验支持。

作为血小板表面最重要的膜受体复合物的GP Ib-IX位于脂筏平台中，通过跨膜信号传导调控着血小板的活化和聚集，但其具体的分子机制尚不清楚。本项目采用TOXCAT实验系统和体外SDS-PAGE以及CD分析，通过跨膜长度的筛选以及大量的突变扫描从而确定了GP Ib-IX跨膜区的二聚活性及其二聚界面，实验结果表明极性残基序列Gln129以及His139构成GP Ibb跨膜区同源二聚体二聚界面。在此基础上我们采取粗粒化模拟方法以的手段对GP Ibb跨膜区二聚体进行了模拟研究，确认了相同的二聚界面，其中极性残基Gln129以及His139对于GP Ibb的二聚起着至关重要的作用，同时亮氨酸拉链区的形成维持了GP Ibb的二聚。本项目进一步通过构建好高表达GP Ib-IX的CHO细胞系，通过流式细胞术和蛋白质印迹的方法，结合基因定点突变鉴定GP Ib-IX跨膜区形成了一个四个a-helix组成的四螺旋轴，而在这个四螺旋轴中相互作用界面的关键残基为GP Iba中的Ser503，GP Ibb中Gln129以及His139，GP IX中的Asp135。在此基础上通过破坏细胞膜上脂筏和体外构建模拟脂筏的方式，从而实现干扰脂筏和GP Ib-IX跨膜区以及信号转导结构域之间的相互作用，破坏了膜蛋白GP Ib-IX跨膜信号转导，从而揭示了脂筏调控GP Ib-IX跨膜信号转导的分子机制。