Molecular assembly and regulation of the cerebral cavernous malformation complex

描述（申请人提供）：三种蛋白KRIT 1功能丧失（Krev/Rap 1相互作用，1例被困; CCM 1，脑海绵状血管畸形1例），CCM 2（脑海绵状血管畸形2; OSM，MEKK 3的血管敏感支架）和CCM 3（脑海绵状血管畸形2例; PDCD 10，程序性细胞死亡10），导致毁灭性的脑海绵状血管畸形（CCM）疾病的家族形式。因此，这些蛋白质的功能丧失与中风、局灶性神经缺陷、癫痫发作和血管异常直接相关。该应用程序的目标是了解这些蛋白质正常功能的分子基础。为此，我们将进行基于细胞的生物化学和结构研究，以解决我们的两个中心假设：CCM复合物的分子水平组织调节关键信号传导事件，分子内或分子间的“头-尾”KRIT 1相互作用调节KRIT 1功能。在我们的初步研究中，我们已经确定了每个CCM蛋白KRIT 1，CCM 2和CCM 3的第一个晶体结构，并发现这些蛋白质中的每一个都含有以前未预测的蛋白质相互作用支架结构域。此外，我们对CCM蛋白的功能研究突出了其细胞功能的重要新方面，特别是关于整合素活化状态和信号传导的调节。因此，在目标1中，我们将使用我们的晶体位置来组装CCM复合物，并研究其在细胞中的功能作用。我们以前的研究 还研究了CCM蛋白与合作伙伴，包括ICAP 1和Rap 1的直接相互作用。这些蛋白质结合KRIT 1并可能影响其构象状态，这反过来又影响CCM复合物的形成。因此，在目标2中，我们将发现调节KRIT 1构象的分子机制以及KRIT 1构象状态对通过CCM复合物的信号传导的影响。在这项多研究者提案中，Boggon和Calderwood实验室将对这些蛋白质进行高度合作的结构导向功能研究，以更好地了解它们的正常功能，特别关注CCM疾病相关的细胞功能。此外，由于CCM蛋白各自广泛表达并且通过进化具有高序列保守性，我们期望从本研究中获得的对CCM蛋白的更好理解也将突出CCM蛋白在神经血管系统之外的进一步作用。

DESCRIPTION (provided by applicant): Loss of function of the three proteins, KRIT1 (Krev/Rap1 Interacting Trapped 1; CCM1, cerebral cavernous malformation 1), CCM2 (cerebral cavernous malformation 2; OSM, osmosensing scaffold for MEKK3) and CCM3 (cerebral cavernous malformation 2; PDCD10, programmed cell death 10), cause the familial form of the devastating Cerebral Cavernous Malformations (CCM) disease. Loss of function of these proteins is therefore directly linked with stroke, focal neurological defects, seizures and vascula abnormalities. The goal of this application is to understand the molecular underpinnings for normal function of these proteins. To do this we will conduct cell-based, biochemical and structural studies that will address our two central hypotheses: Molecular-level organization of the CCM complex regulates key signaling events and Intra- or inter- molecular "head-tail" KRIT1 interactions regulate KRIT1 function. In our preliminary studies we have determined the first crystal structures of each of the CCM proteins, KRIT1, CCM2 and CCM3, and have found each of these proteins to contain previously unpredicted protein interaction scaffold domains. Furthermore, our functional studies of the CCM proteins have highlighted important new aspects of their cellular function, particularly with regards to the regulation of integrin activation stat and signaling. Therefore, in Aim 1 we will use our advantaged position to assemble the CCM complex crystallographically and to investigate its functional roles in cells. Our previous studies also investigated the direct interactions of CCM proteins with partners, including ICAP1 and Rap1. These proteins bind KRIT1 and may impact its conformational status, which in turn is suggested to impact formation of the CCM complex. Therefore, in Aim 2 we will discover the molecular mechanisms that regulate KRIT1 conformation and the impact of KRIT1 conformational state on signaling via the CCM complex. In this Multi-Investigator proposal, the Boggon and Calderwood laboratories will conduct a highly collaborative structure-directed functional study of these proteins to better understand their normal functions, with particular attention to CCM disease-related cellular functions. Furthermore, as the CCM proteins are each widely expressed and have high sequence conservation through evolution, we expect that the improved understanding of the CCM proteins obtained from this study will also highlight further roles for the CCM proteins outside of the neurovasculature.