Molecular assembly and regulation of the cerebral cavernous malformation complex

描述(由申请人提供)：KRIT1(Krev/Rap1相互作用陷阱1；CCM1，脑海绵状畸形1)、CCM2(脑海绵状畸形2；OSM，MEKK3渗透传感支架)和CCM3(脑海绵状畸形2；PDCD10，程序性细胞死亡10)三种蛋白质功能丧失，导致毁灭性脑海绵状畸形(CCM)疾病的家族形式。因此，这些蛋白功能的丧失与中风、局灶性神经缺陷、癫痫发作和血管异常直接相关。这项应用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、生化的和结构的研究，以解决我们的两个核心假设：CCM复合体的分子水平组织调控关键信号事件，分子内或分子间的“头-尾”KRIT1相互作用调节KRIT1功能。在我们的初步研究中，我们确定了每个CCM蛋白质KRIT1、CCM2和CCM3的第一晶体结构，并发现每个蛋白质都含有先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，特别是在整合素激活状态和信号调节方面。因此，在目标1中，我们将利用我们的优势地位，以结晶学的方式组装CCM络合物，并研究其在细胞中的功能作用。我们之前的研究 还研究了CCM蛋白与包括ICAP1和Rap1在内的伙伴的直接相互作用。这些蛋白与KRIT1结合，可能影响其构象状态，进而影响CCM复合体的形成。因此，在目标2中，我们将发现调节KRIT1构象的分子机制，以及KRIT1构象状态对通过CCM复合体传递信号的影响。在这项多研究者计划中，Boggon和Calderwood实验室将对这些蛋白质进行高度协作的结构导向功能研究，以更好地了解它们的正常功能，特别是CCM疾病相关的细胞功能。此外，由于CCM蛋白都广泛表达，并在进化过程中具有高度的序列保守性，我们预计通过本研究获得的对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.