Molecular assembly and regulation of the cerebral cavernous malformation complex

标题：脑海绵状血管畸形复合体的分子组装和调控 摘要 这三种蛋白的功能丧失，KRIT1(Krev/Rap1相互作用陷阱1；CCM1，脑海绵状突起 畸形1)、CCM2(脑海绵状血管畸形2；OSM，MEKK3渗透传感支架)和 CCM3(脑海绵状血管畸形2；PDCD10，程序性细胞死亡10)，导致家族性 毁灭性的脑海绵状血管畸形(CCM)病。这些蛋白质的功能丧失是 因此与中风、局灶性神经缺陷、癫痫发作和血管异常直接相关。目标是 这一应用的目的是了解这些蛋白质正常功能的分子基础。去做 为此，我们将进行基于细胞的、生化和结构的研究，以解决我们的两个核心假设： CCM复合体的分子水平组织调节关键信号事件以及细胞内或细胞间 分子“头-尾”KRIT1相互作用调节KRIT1功能。在我们的初步研究中，我们有 测定了CCM蛋白KRIT1、CCM2和CCM3的第一晶体结构，并发现 这些蛋白质中的每一种都包含先前预测不到的蛋白质相互作用支架结构域。此外，我们的 对CCM蛋白的功能研究突出了其细胞功能的重要新方面， 尤其是关于整合素激活状态和信号的调节。因此，在目标1中，我们将 利用我们的优势地位，从结晶学的角度组装CCM络合物，并研究其 细胞中的功能角色。我们以前的研究也研究了CCM蛋白与 合作伙伴，包括ICAP1和RAP1。这些蛋白质结合KRIT1并可能影响其构象状态， 这反过来又被认为影响CCM复合体的形成。因此，在《目标2》中，我们将发现 KRIT1构象调控的分子机制及KRIT1构象状态对其的影响 通过CCM复合体发送信号。在这份多名调查员的提案中，博贡和考德伍德实验室 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 他们的正常功能，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 从这项研究中获得的对CCM蛋白的更好的理解也将突出进一步的作用 对于神经血管系统外的CCM蛋白。

TITLE: Molecular assembly and Regulation of the Cerebral Cavernous Malformation Complex ABSTRACT 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 vascular 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 state 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.