Determining the Functional Significance of the CCM complex

确定 CCM 复合体的功能意义

• 批准号: 9056341
• 负责人: Kyle Margaret Draheim
• 金额: $ 6.2万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-04-01 至 2017-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9056341
• 关键词: Affect; Ankyrin Repeat; Apoptosis; Binding; Binding Sites; Biological Assay; Biological Process; Blood Vessels; Blood capillaries; Brain hemorrhage; CCM1 gene; Cell Nucleus; Cell physiology; Cells; Complex; Data; Dimerization; Dysplasia; Endothelial Cells; Engineering; Focal Adhesions; Gene Proteins; Genes; Human; Individual; Inherited; Integrins; Intercellular Junctions; Lead; Lesion; Mediating; Modeling; Molecular; Mutation; Neuraxis; Nuclear; Nuclear Export; PTB Domain; Pathway interactions; Permeability; Physiological; Population; Prevalence; Process; Proteins; Role; Seizures; Signal Transduction; Stimulus; Stroke; Tertiary Protein Structure; Testing; Tube; Work; base; capillary; cell motility; cerebral cavernous malformations; knock-down; loss of function mutation; mRNA Expression; member; migration; monolayer; multicatalytic endopeptidase complex; mutant; protein complex; public health relevance; research study; response; rho; shear stress

 DESCRIPTION (provided by applicant): Cerebral cavernous malformations (CCMs) are sporadically acquired or inherited vascular lesions of the central nervous system consisting of clusters of dilated, thin-walled blood vessels that predispose individuals to seizures and stroke. CCMs have a prevalence of 0.1-0.5% in the human population and are associated with inherited or sporadic mutations in three genes and their resulting proteins: KRIT1 (Krev/Rap1 Interacting Trapped 1, CCM1), CCM2 (malcavernin) and CCM3 (programmed cell death 10, PDCD10). Early work suggests the direct interaction of the CCM proteins with one another forms a signaling platform around a ternary KRIT1:CCM2:CCM3: complex termed the CCM complex. In this model, CCM2 acts as the central hub using independent binding sites to simultaneously interact with KRIT1 and CCM3. It is believed the KRIT1 binding partner ICAP1 (integrin cytoplasmic associated protein 1) is also part of this complex. However, the CCM complex independent and dependent roles of the CCM proteins is yet to be determined. Using knockdown studies, I will by evaluate how the loss of one CCM protein affects the mRNA expression, protein levels and cellular localization of the other CCM-complex members. Mutants which interrupt CCM protein interactions or change CCM protein localization will be used in knockdown/rescue experiments to determine the importance CCM complex formation and proper localization of CCM proteins. Tube formation, cell migration, response to shear stress, integrin activation, Rho/ROCK activation and monolayer permeability assays will determine the functional consequences of these mutations for endothelial processes. Additionally, endogenous CCM protein levels and localization in endothelial cells will be assessed after activating pathways previously connected to CCM proteins. The functional focus of the studies proposed will provide a significant enhancement to the molecular-level understanding of the proteins involved in CCM, and a consequent improvement in understanding the molecular basis for CCM acquisition and CCM-associated hemorrhagic stroke.

 描述（由申请方提供）：脑海绵状血管畸形（CCM）是中枢神经系统的偶发性获得性或遗传性血管病变，由扩张的薄壁血管簇组成，使个体易于癫痫发作和卒中。CCM在人群中的患病率为0.1 - 0.5%，并且与三种基因及其产生的蛋白质中的遗传性或散发性突变相关：KRIT 1（Krev/Rap1相互作用捕获1，CCM 1），CCM 2（malcavernin）和CCM 3（程序性细胞死亡10，PDCD 10）。早期的工作表明CCM蛋白彼此之间的直接相互作用形成了一个围绕三元KRIT 1：CCM 2：CCM 3：复合物的信号平台，称为CCM复合物。在该模型中，CCM 2作为中心枢纽，使用独立的结合位点同时与KRIT 1和CCM 3相互作用。据信KRIT 1结合伴侣ICAP1（整合素细胞质相关蛋白1）也是该复合物的一部分。然而，CCM复合物的CCM蛋白的独立和依赖的作用还有待确定。使用敲除研究，我将评估一个CCM蛋白的丢失如何影响其他CCM复合体成员的mRNA表达、蛋白水平和细胞定位。中断CCM蛋白相互作用或改变CCM蛋白定位的突变体将用于敲减/拯救实验，以确定CCM复合物形成的重要性和CCM蛋白的适当定位。管形成，细胞迁移，响应剪切应力，整合素活化，Rho/ROCK活化和单层渗透性测定将确定这些突变的内皮过程的功能后果。此外，将在激活先前与CCM蛋白相关的途径后评估内皮细胞中的内源性CCM蛋白水平和定位。拟议的研究的功能重点将提供一个显着的增强，以分子水平的了解CCM涉及的蛋白质，并因此改善了解CCM收购和CCM相关的出血性中风的分子基础。