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

描述（由申请人提供）：了解和控制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卒中预防和治疗的重要一步。这些相互作用的破坏或信号蛋白的失调可导致血管形成缺陷和随后的疾病。脑海绵状血管瘤（CCM）是由信号分子破坏引起的大脑血管异常的一个例子，在0.1%至0.5%的人群中自然发生。三个位点的基因突变是导致CCM遗传发展的原因。它们编码三种CCM蛋白：Krit1 (Krev相互作用捕获-1),OSM （MEKK3或malcalvernin的渗透感应支架）和PDCD10（程序性细胞死亡10）。我们发现这三种CCM蛋白相互作用并在细胞中形成信号复合物。PDCD10因其从无脊椎动物到哺乳动物的极其保守的氨基酸序列而在这些蛋白质中脱颖而出。PDCD10突变约占所有CCM的10%；然而，随后的病变是最严重的形式。PDCD10也是唯一没有已知结构域或基序的CCM蛋白，这将提供可能的功能见解。我们的长期目标是了解PDCD10在生理血管发育和病理CCM发展中的作用，并将CCM系统的基础知识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，(2)PDCD10通过与磷脂和PDK1的相互作用参与PDK1信号传导。我们进一步提出，这些相互作用对内皮迁移和血管形成至关重要。本研究提出了两个目标，包括确定PDCD10在内皮p38 MAPK激活中的作用（Aim 1）和确定PDCD10膜磷脂结合与PDK1信号传导之间的功能关系（Aim 2）。使用内皮细胞系，我们将使用RNAi和含有影响其与OSM、磷脂和PDK1相互作用能力的位点特异性突变的突变体来检查PDCD10敲低导致的功能变化。复合物的局部相互作用，下游激酶的激活，以及内皮迁移和血管形成也将被检查。公共卫生相关性：了解和控制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卒中预防和治疗的重要一步。这些拟议的研究将是首次确定PDCD10蛋白未知功能的研究之一。PDCD10基因的突变导致了一种遗传易感性的出血性中风，称为脑海绵状血管瘤（CCM）。这项研究将CCM与p38 MAP激酶和PI3K/PDK1这两个重要的细胞信号级联联系起来，为出血性中风的治疗提供了新的靶点。

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.