Sharpin-Kindlin-1复合物调控β1-型整合素激活的分子机制研究

The Kindlin family represents a new class of integrin binding proteins, which comprises three evolutionarily conserved members, Kindlin-1, Kindlin-2 and Kindlin-3. Kindlins can directly bind to the integrin β cytoplasmic tails (CTs) and are involved in regulating integrin activation and integrin-mediated cell adhesion and migration. Loss-of-function mutations in Kindlins can cause several kinds of human diseases. For example, deficiency of Kindlin-1 in patients leads to Kindler syndrome (KS), featured with skin abnormality due to the dysfunction of β1-integrin in epithelial cells. It is suggested that Kindlins function as an adaptor to bridge integrin and intracellular regulatory signaling in cells. However, how Kindlins interact with their upstream signaling molecules and regulate integrin activation is mechanistically unknown. To identify key Kindlin binding partners, we employed a high-throughput yeast two-hybrid system to screen human universal cDNA library and successfully identified one Kindlin-1 binding protein called Shaprin, which was previously recognized as an integrin activation inhibitor via binding to the integrin α CTs in the β1-integrin family. In this study, we will investigate the functional role and explore the molecular mechanism of the Sharpin-Kindlin-1 interaction in regulating integrin activation. Firstly, we will utilize multiple approaches, including yeast two-hybrid, co-immunoprecipitation, pull-down, and mutagenesis, to map the binding sites in Sharpin for Kindlin-1 and the integrin α CTs. Secondly, we will employ a well-established in vitro integrin activation assay to dissect the regulatory role of the Sharpin-Kindlin-1 complex in integrin activation. This study will reveal the molecular regulation of the Sharpin-Kindlin-1-integrin signaling pathway and provide new insights into the molecular mechanism of integrin activation. The expected results may also help to develop a novel therapeutic strategy for preventing and treating the related diseases.

Kindlin是近年来发现的一类重要的整合素胞内激活蛋白, 它们通过与整合素β亚基胞内结构域结合来调控整合素激活、并参与整合素介导的细胞粘附和迁移等多种生物学过程。人源Kindlin-1表达缺失会造成上皮细胞β1型整合素功能异常，导致Kindler综合征。目前，Kindlin诱导整合素激活的分子机制尚不清楚。我们通过酵母双杂交筛选，鉴定到Kindlin-1的重要结合蛋白Sharpin。Sharpin是已知的β1型整合素α亚基胞内结构域结合因子、并能抑制整合素激活。本项目将进一步运用酵母双杂交、pull-down、免疫共沉淀等生化和分子生物学研究方法来揭示Sharpin与Kindlin-1及整合素α亚基之间相互作用的分子基础，并运用整合素激活的细胞模型阐述Sharpin与Kindlin-1的相互作用对β1型整合素激活的调控功能及其分子机制。预期研究成果将有助于进一步阐明整合素激活的分子机理

生物体细胞与细胞以及细胞和胞外基质的选择性互动是依赖一系列细胞表面粘附分子实现的，整合素是其中重要一员。整合素功能行使受激活信号通路的精确调控，其失调就会产生相应的病变，如炎症反应、血栓形成与癌症发生等。Sharpin蛋白是一种已知的整合素α亚基胞内结合蛋白并能够抑制β1-型整合素的激活，但其对不同整合素激活的调控作用以及与其他整合素激活因子（talin和kindlin）的功能协同关系目前尚不明确。本项目运用多种生物化学、分子生物学和细胞生物学研究手段，对sharpin参与由talin与kindlin-1介导的β1-型整合素激活的调控作用和分子机理进行了系统深入的研究。利用整合素激活的细胞模型我们发现细胞中过表达Sharpin抑制整合素α5β1的激活，但对整合素αIIbβ3无明显抑制作用；利用靶向Sharpin的siRNA降低细胞中内源Sharpin的表达可以促进整合素α5β1的激活而对整合素αIIbβ3的激活无显著的影响，表明Sharpin对整合素激活的抑制功能并不具有普遍性。接下来，我们利用Pulldown和NMR实验证明了Sharpin可识别结合整合素α5β1的胞内序列，但与αIIbβ3胞内序列的相互作用不明显,从机制上解释了Sharpin对不同类型整合素调控的功能特异性。进一步我们通过Pull down实验发现Sharpin与TalinH都能够识别并结合到整合素β1亚基胞内段近膜端的NxxY基序并且Sharpin与β1亚基胞内段的结合能够抑制Talin与β1CT的相互作用。同时，Pull down与酵母双杂交实验证实了Sharpin与Kindlin之间存在直接的相互作用，并且Sharpin-Kindlin-1复合物结合到整合素β亚基的胞内序列对Talin-H结合到整合素β1亚基同样具有抑制作用。在Kindlin-1存在时，Sharpin结合到整合素β亚基的量比Sharpin单独时显著增多，推测细胞内Kindlin-1的存在可以起到募集Sharpin到β1亚基的作用，从而促进整合素从激活态到静息态的转变。综上，本论文的研究结果揭示了一种新的整合素激活的分子调控机制，进一步阐明了整合素激活信号通路调控的复杂性和精确性, 为相关疾病的治疗奠定了坚实的理论基础。