点燃素-2介导的β3-型整合素激活的分子机理研究

Integrins are broadly expressed α/β heterodimeric transmembrane cell adhesion receptors and essentially involved in multiple pathological developments, such as in inflammation response, thrombosis formation and cancer metastasis. Integrin-mediate cell adhesion is critically regulated by integrin activation states, which are further determined by integrin cytoplasmic tail (CT) binding partners. Among multiple integrin CT binding proteins, kindlin family members (kindlin-1, -2 and -3) have been recently identified as essential integrin activators via direct interaction with integrin β CT. Even though the functional significance of kindlin family members in regulating integrin activation has been well demonstrated in cells, mouse models and kindlin-deficiency patients, the structural basis of kindlin members in supporting integrin activation still remains poorly understood. In this study, we will employ multiple structural and biochemical approaches to study the molecular details of kindlin-2 in interaction with integrin β3 CT during supporting integrin activation. First, we will determine the kindlin-2 structure by crystallization of kindlin-2 and its subdomain proteins. Second, we will determine the interactive specificity of kindlin-2/integrin β3 CT complex by co-crystallization. Alternatively, the essential kindlin-2 binding sites in integrin β3 CT will be mapped by mutagenesis and surface Plasmon resonance assays. Third, we will make efforts to identify the upstream signaling molecules of kindlin-2, thus establishing the kindlin-2 signaling pathway. Due to the high homology shared by kindin family members, the findings about kindlin-2 could be applicable to kindlin-1 and -3. We believe that these proposed studies will significantly enhance our understanding of the integrin activation mechanisms and may facilitate to generate the next generation of therapeutics for treating integrin-mediated cardiovascular disease and cancer.

整合素是细胞表面广泛表达的一类α/β异二聚体膜蛋白受体分子，主要介导细胞与细胞以及细胞与胞外基质的粘附，并在多种恶性病变中起重要作用。整合素的激活是由胞内激活信号通过调节整合素激活因子与整合素胞内序列的结合来实现的。点燃素是近年来发现的一类重要的整合素β亚基胞内结合蛋白，在整合素的激活中起重要作用。目前，我们对点燃素与整合素相互结合的结构基础及其调控还不甚了解。基于此，本申请课题将重点研究点燃素家族代表成员点燃素-2与β3-型整合素结合的结构基础和信号通路。首先通过蛋白结晶的手段研究点燃素-2蛋白的结构基础；其次通过共结晶并结合等离子表面共振技术来鉴定点燃素-2与β3-型整合素胞内序列肽段结合的特异性；然后通过酵母双杂交筛选和功能鉴定来完善点燃素-2激活整合素的信号通路。通过以上研究，我们将进一步阐明整合素激活的分子机理，为治疗整合素介导的心脑血管疾病和恶性肿瘤提供新的思路和特异药物靶位点。

本项目主要内容是通过研究血小板中整合素激活因子点燃素对β3型整合素的激活作用和两者相互结合的分子基础来鉴定整合素激活的信号通路，并以此为基础阐述整合素激活的分子机理，为开发特异性的抗血栓形成的小分子药物提供理论基础。首先，我们构建了阻断整合素与点燃素结合的点燃素基因敲入小鼠模型，在血小板聚集，凝血块收缩和动脉血栓形成实验模型中，我们发现点燃素与整合素的直接结合对表达在血小板上的β3型整合素正常功能的行使有重要的支持作用。其次，通过酵母双杂交筛选我们获得的多个参与点燃素结合的蛋白结合因子并进一步分析了他们在整合素激活中的作用，证明了一种点燃素结合蛋白因子paxillin可以通过结合点燃素而调控整合素的激活。最后，我们通过生长点燃素蛋白的晶体解析了点燃素蛋白的结构。这些发现进一步完善了整合素激活的信号通路和分子机制，为开发更安全高效的抗栓药物奠定了理论依据。