Role of Programmed Cell Death10 (PDCD10) in p38 MAP kinase activation and PDK1 si

描述(由申请人提供)：了解和操纵血管生成和血管生成过程中生理信号所需的复杂蛋白质-蛋白质相互作用是开发出血性中风预防和治疗的重要步骤。这些相互作用的破坏或信号蛋白的失调可能导致血管形成缺陷和随后的疾病。脑海绵畸形(CCM)是大脑血管异常的一个例子，它是由信号分子的破坏引起的，自然发生在0.1%到0.5%的人口中。CCM的遗传性发生与三个基因座的基因突变有关。它们编码三种CCM蛋白：Krit1(Krev相互作用陷阱-1)、OSM(MEKK3或Malcalvernin的渗透传感支架)和PDCD10(程序性细胞死亡10)。我们发现这三种CCM蛋白相互作用，在细胞内形成信号复合体。PDCD10在这些蛋白质中脱颖而出，因为它的氨基酸序列从无脊椎动物到哺乳动物都非常保守。PDCD10突变约占所有CCM的10%；然而，后续病变是最严重的形式。PDCD10也是唯一一个没有已知结构域或基序的CCM蛋白，可以为功能提供可能的洞察。我们的长期目标是了解PDCD10如何在生理性血管发育和病理性CCM发育中发挥作用，并将这一基本知识从CCM系统转化为出血性中风和其他血管畸形的新预防/治疗干预措施。我们假设(1)PDCD10通过与OSM的相互作用调节p38的激活，以及(2)PDCD10通过与磷脂和PDK1的相互作用参与PDK1信号转导。我们进一步认为，这些相互作用是内皮细胞迁移和血管形成所必需的。本研究提出了两个目标：明确PDCD10在内皮细胞p38MAPK激活中的作用(目标1)和明确PDCD10的膜磷脂结合与通过PDK1的信号转导之间的功能关系(目标2)。利用内皮细胞系，我们将使用RNAi和含有影响其与OSM、磷脂和PDK1相互作用能力的定点突变的突变体来检测PDCD10被敲除所导致的功能变化。还将研究复合体的局部相互作用、下游激酶的激活以及内皮迁移和血管形成。公共卫生相关性：了解和操纵血管生成和血管生成过程中生理信号所需的复杂蛋白质-蛋白质相互作用是开发出血性中风预防和治疗的重要步骤。这些拟议的研究将是第一批确定PDCD10蛋白未知功能的研究之一。PDCD10基因突变导致一种称为脑海绵状畸形(CCM)的遗传性出血性中风。这项工作将CCM状况与两个重要的细胞信号转导通路联系起来，包括p38 MAP激酶和PI3K/PDK1，并为出血性中风的新治疗靶点提供了新的靶点。

DESCRIPTION (provided by applicant): Understanding and manipulating complex protein-protein interactions required for physiological signaling during angiogenesis and vasculogenesis is an important step in developing prevention and treatment for hemorrhagic stroke. Disruption of these interactions or dysregulation of signaling proteins can result in defective vascular formation and subsequent disease. Cerebral cavernous malformation (CCM) is an example of a vascular anomaly in the brain caused by disruptions of signaling molecules that occurs naturally in 0.1 to 0.5% of the population. Genetic mutations at three loci are responsible for the inherited development of CCM. They encode three CCM proteins: Krit1 (Krev interaction trapped-1), OSM (Osmosensing scaffold for MEKK3 or malcalvernin), and PDCD10 (Programmed cell death 10). We have found that all three CCM proteins interact with each other and form a signaling complex in the cell. PDCD10 stands out among these proteins because of its extremely conserved amino acid sequence from invertebrates to mammals. Mutations of PDCD10 represent about 10% of all CCM; however, the subsequent lesions are the most severe form. PDCD10 is also the only CCM protein that has no known domain or motif that would provide possible insight into function. Our long term goals are to understand how PDCD10 functions in physiologic vascular development and pathologic CCM development and to translate this basic knowledge from this CCM system to new preventive/therapeutic interventions for hemorrhagic stroke and other vascular malformations. We hypothesize that (1) PDCD10 regulates p38 activation through its interaction with OSM, and (2) PDCD10 participates in PDK1 signaling via its interactions with phospholipids, and PDK1. We further propose that these interactions are essential for endothelial migration and vessel formation. Two aims are proposed in this study including define the role of PDCD10 in endothelial p38 MAPK activation (Aim 1) and define the functional relationship between membrane phospholipid binding of PDCD10 and signaling through PDK1 (Aim 2). Using endothelial cell line, we will examine the functional changes resulting from PDCD10 knockdown using RNAi and mutants containing site-specific mutations that impact its ability to interact with OSM, phospholipids, and PDK1. The localized interaction of complex, activation of downstream kinases, as well as endothelial migration and vessel formation will also be examined. PUBLIC HEALTH RELEVANCE: Understanding and manipulating complex protein-protein interactions required for physiological signaling during angiogenesis and vasculogenesis is an important step in developing prevention and treatment for hemorrhagic stroke. These proposed studies will be one of the first to define the unknown function of PDCD10 protein. Mutations of PDCD10 gene contribute to a genetically-predisposed hemorrhagic stroke condition called Cerebral Cavernous Malformation (CCM). This work links the CCM condition to two important cell signaling cascades including p38 MAP kinase and PI3K/PDK1, and providing us to new therapeutic targets for hemorrhagic stroke.