KRIT1 and Vascular Integrity

描述（由申请方提供）：内皮细胞（EC）连接调节血管通透性，并在心血管系统的发育和功能中发挥核心作用。Rap1 GTdR的激活稳定了这些连接，申请人发现KRIT 1（KRIT 1的Rap1结合蛋白产物，一种与脑海绵状畸形（CCM）相关的基因）可能介导EC-细胞连接的Rap1 GTdR稳定。CCM是一种常见的血管异常，影响超过100万美国人，使他们终生面临中风和其他神经系统后遗症的风险。目前尚无预防CCM病变发生或临床进展的治疗方法。申请人还发现，与KRIT 1遗传连接的受体玻璃心（HEG 1）与KRIT 1相互作用，整联蛋白胞质结构域也是如此。KRIT 1直接与CCM 2结合，CCM 2是与CCM相关的另一种基因的产物，他发现EC中KRIT 1或CCM 2的缺失导致Rho激酶（ROCK）的活化增加。因此，他假设Rap1结合靶向含有KRIT 1和CCM 2的大分子复合物至EC-细胞连接处，其中Rap1通过结合至HEG1胞质尾而被保留，并通过抑制RhoA及其效应物Rho激酶（ROCK）来稳定连接完整性。为了测试这些想法，申请人将绘制KRIT 1上的Rap1结合区域，以制备破坏Rap1结合的突变体，并检查它们对KRIT 1功能和定位的影响。同样，他将鉴定Rap1的开关1区域的突变体，这些突变体破坏与KRIT 1的结合，并评估它们对Rap1稳定EC细胞连接的影响。这些信息将用于破译Rap1在KRIT 1的定位和功能中的作用。其次，他将研究KRIT 1与HEG1的直接相互作用，并创建每个合作伙伴的突变体，这些突变体不能相互作用。然后，他将测试这些突变体在稳定EC细胞连接和RhoA-ROCK活性方面的功能。第三，申请人将分析RhoA和ROCK抑制的机制，以扩展KRIT1-CCM 2抑制RhoA效应物ROCK的活性，从而稳定EC-细胞连接的假设。特别是，他将评估KRIT 1的结构特征，使其能够抑制RhoA和ROCK。他发现，抑制RhoA/ROCK活性需要CCM 2与KRIT 1的相互作用，他将使用从细胞核中排除并且不能结合CCM 2的KRIT 1突变体来测试CCM 2通过控制KRIT 1定位于细胞-细胞连接来调节RhoA的想法。他还将利用对KRIT 1如何与整合素结合的见解来测试KRIT 1招募到EC连接限制整合素信号（如RhoA激活）的假设，这些信号会破坏细胞-细胞连接。这些研究将为新发现的调节血管发育和屏障功能的多蛋白复合物提供基本见解，并有可能在CCM中确定新的治疗靶点，这是一个重要的未满足的医疗需求。 公共卫生相关性：血管渗漏在多种疾病中起主要作用，并且目前没有治疗方法来减少脑海绵状血管畸形（CCM）的发展，也没有显著的相关发病率，尽管事实上超过一百万美国人患有这些病变。我们的研究已经暗示RhoA/ROCK通路在这种疾病中;存在相对良好耐受的药物，可以调节这一通路，为血管渗漏和CCM的治疗提出了新的方向。这些研究将为CCM多蛋白复合物提供基本的见解，该复合物包含三种基因与CCM相关的蛋白质;这些见解可能导致识别这些疾病的其他治疗靶点。

DESCRIPTION (provided by applicant): Endothelial cell (EC) junctions regulate vascular permeability and play a central role in the development and function of the cardiovascular system. Activation of Rap1 GTPase stabilizes these junctions and the applicant found that KRIT1, the Rap1-binding protein product of KRIT1, a gene linked with cerebral cavernous malformations (CCM), may mediate Rap1 GTPase stabilization of EC-cell junctions. CCM is a common vascular anomaly affecting more than a million Americans, predisposing them to a lifetime risk of stroke and other neurologic sequelae. There is currently no therapy to prevent the genesis or clinical progression of CCM lesions. The applicant has also found that heart of glass (HEG1), a receptor genetically-linked to KRIT1, interacts with KRIT1, as do integrin cytoplasmic domains. KRIT1 binds directly to CCM2, the product of another gene associated with CCM and he found that loss of either KRIT1 or CCM2 in ECs, leads to increased activation of Rho Kinase (ROCK). Thus, he hypothesizes that Rap1 binding targets a macromolecular complex containing KRIT1 and CCM2 to EC-cell junctions where it is retained by binding to the HEG1 cytoplasmic tail and stabilizes junctional integrity by inhibiting RhoA and its effector, Rho Kinase (ROCK). To test these ideas the applicant will map the Rap1 binding region on KRIT1 to make mutants that disrupt Rap1 binding and examine their effects on KRIT1 function and localization. Similarly, he will identify mutants of the Switch 1 region of Rap1 that disrupt binding to KRIT1 and assess their effects on Rap1 stabilization of EC-cell junctions. This information will be used to decipher the role of Rap1 in the localization and function of KRIT1. Secondly he will examine the direct interaction of KRIT1 with HEG1 and create mutants of each partner that fail to interact. He will then test the function of these mutants in stabilizing EC-cell junctions and RhoA-ROCK activity. Thirdly, the applicant will analyze the mechanism of RhoA and ROCK inhibition to extend the hypothesis that KRIT1-CCM2 suppresses the activity of the RhoA effector, ROCK, thereby stabilizing EC-cell junctions. In particular, he will assess the structural features of KRIT1 that enable it to inhibit RhoA and ROCK. He has found that the interaction of CCM2 with KRIT1 is required for suppression of RhoA/ROCK activity and he will use KRIT1 mutants that are excluded from the nucleus and fail to bind CCM2 to test the idea that CCM2 regulates RhoA by controlling the localization of KRIT1 to cell-cell junctions. He will also use insights into how KRIT1 binds to integrins to test the hypothesis that KRIT1 recruitment to EC junctions limits integrin signals, such as RhoA activation, that disrupt the cell-cell junctions. These studies will provide fundamental insight into a newly discovered multiprotein complex that regulates vascular development and barrier function and have the potential to identify new therapeutic targets in CCM, a significant unmet medical need. PUBLIC HEALTH RELEVANCE: Vascular leak plays a major role in a wide variety of diseases and there is presently no therapy to reduce the development of cerebral cavernous malformations (CCM), nor the significant associated morbidity, despite the fact that more than one million Americans bear these lesions. Our studies have already implicated the RhoA/ROCK pathway in this disease; the existence of relatively well-tolerated drugs that can modulate this pathway, has suggested new directions for therapy of vascular leak and CCM. These studies will provide fundamental insight into the CCM multi-protein complex that contains three proteins whose genes are linked to CCM; these insights may lead to identification of additional therapeutic targets for these diseases.