Conformation-dependent regulation of KRIT1 and the CCM complex

摘要/摘要 血管屏障功能的调节是血管网络最重要的功能之一， 也是最容易受到疾病状态影响的功能之一。尽管大多数心血管疾病 疾病影响血管壁的通透性，我们对血管通透性的了解 在正常情况下受到监管的情况仍然不完整。CCM蛋白复合体，包含支架 蛋白质KRIT1、CCM2和PDCD10/CCM3对于内皮细胞-细胞接触的稳定性和 体内血管通透性的调节。然而，我们仍然对这些蛋白质是如何形成的知之甚少。 或者这种复合体实际上如何稳定细胞与细胞之间的接触。初步 数据表明，KRIT1在内皮细胞黏附连接(AJ)上的定位对于正常的 整个CCM复合体的功能，这种定位受KRIT1和KRIT1的构象控制 通过KRIT1与其已知结合伙伴的相互作用。我们假设KRIT1被贩卖到 细胞内部位控制CCM复合体调节内皮细胞通透性的能力。 因此，我们建议使用几种先进的成像和生化方法来确定 KRIT1在三个亚细胞位置的构象和结合作用：血浆 膜、细胞质和细胞核。这些信息将与功能分析结合使用。 构建完整内皮单分子层内KRIT1/CCM信号传导模型。这一信息将是 对未来针对CCM复合体的药物开发至关重要，并将显著增加我们的 对血管动态平衡的理解。

Summary/Abstract The regulation of blood vessel barrier function is one of the most important functions of the vascular network, and one of the first functions to be affected by the disease state. Despite the fact that most cardiovascular diseases affect the permeability of the blood vessel wall, our knowledge of how vascular permeability is regulated under normal conditions remains incomplete. The CCM protein complex, containing the scaffolding proteins KRIT1, CCM2, and PDCD10/CCM3, is critical for the stability of endothelial cell-cell contacts and regulation of vascular permeability in vivo. However, we still know relatively little about how these proteins are regulated in a normal functioning cell, or how this complex actually stabilizes cell-cell contacts. Preliminary data indicate that the localization of KRIT1 at endothelial adherens junctions (AJ) is essential to the proper functioning of the entire CCM complex, and that this localization is controlled by the conformation of KRIT1 and by the interaction of KRIT1 with its known binding partners. We hypothesize that the trafficking of KRIT1 to intracellular sites controls the ability of the CCM complex to regulate permeability in endothelial cells. Therefore, we propose to use several advanced imaging and biochemical approaches to determine the respective conformation and binding interactions of KRIT1 in three subcellular locations: the plasma membrane, the cytoplasm, and the nucleus. This information will be used in conjunction with functional assays to construct a model of KRIT1/CCM signaling within an intact endothelial monolayer. This information will be critical for future drug development targeting the CCM complex and will significantly increase our understanding of vascular homeostasis.