The exocyst in ciliogenesis and cystogenesis

纤毛发生和囊肿发生中的外囊

• 批准号: 8045088
• 负责人: JOSHUA H LIPSCHUTZ
• 金额: --
• 依托单位: PHILADELPHIA VA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-01-01 至 2014-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8045088
• 关键词: Affect; American; Animals; Attention; Autosomal Dominant Polycystic Kidney; Binding; Biochemical; Biological Assay; Calcium; Calcium Channel; Cell Culture Techniques; Cell membrane; Cell physiology; Cells; Cilia; Co-Immunoprecipitations; Complex; Cyst; Cytosol; Data; Databases; Defect; Disease; Docking; Dominant-Negative Mutation; End stage renal failure; Epithelial; Epithelial Cells; Family; Family member; Foundations; GTPase-Activating Proteins; Genes; Genetic; Goals; Guanine Nucleotide Exchange Factors; Guanosine Triphosphate; Guanosine Triphosphate Phosphohydrolases; Hereditary Disease; Human; In Vitro; Injection of therapeutic agent; Intervention; Kidney; Kidney Failure; Life; Link; Maps; Mediating; Medicine; Membrane Proteins; Mitogen-Activated Protein Kinases; Molecular; Monomeric GTP-Binding Proteins; Mutate; Organ; Organelles; PKD2 protein; Pathogenesis; Pathway interactions; Patients; Pennsylvania; Phenotype; Philadelphia; Physicians; Play; Proteins; RNA Splicing; Renal tubule structure; Research; Role; Secretory Vesicles; Series; Site; Site-Directed Mutagenesis; Supportive care; Surface; Tail; Testing; Therapeutic Intervention; Vesicle; Veterans; Yeasts; Zebrafish; cilium biogenesis; fluid flow; improved; in vivo; nephrogenesis; novel; overexpression; rab GTP-Binding Proteins; receptor; research study; rho; rho GTP-Binding Proteins; small hairpin RNA; trafficking; yeast two hybrid system

DESCRIPTION (provided by applicant): Our goal here is to understand the role of the highly conserved eight-protein exocyst complex in ciliogenesis and cystogenesis. Cysts are "building blocks" for epithelial organs such as the kidney, and defects in cyst formation are implicated in human disorders such as autosomal dominant polycystic kidney disease (ADPKD). ADPKD is the most common potentially lethal genetic disease, affecting approximately 1/500 people, and the fourth leading cause of end-stage kidney disease. There are currently no approved therapies for the 500,000 Americans, including 100,000 veterans and dependents, with ADPKD. In renal tubule cells, we recently showed that the exocyst complex localized to, and was necessary for, formation of the primary cilium, an organelle strongly implicated in ADPKD pathogenesis. The exocyst co- localized and co-immunoprecipitated with Par3, a central ciliary protein. Following knockdown of exocyst component Sec10, primary ciliogenesis no longer occurred. Our Preliminary Studies also show an interaction between Sec10 and Par3, which is the foundation for Aim 1. The secretory pathway is crucial for proper cell function and the exocyst is known for mediating the targeting and docking of secretory vesicles carrying membrane proteins. Multiple Rho and Rab family GTPases regulate the exocyst. In yeast, we showed that Cdc42, a Rho GTPase, localized the exocyst to specific plasma membrane domains and our Preliminary Studies demonstrate that: Sec10 and Cdc42 are binding partners, Cdc42 and Sec10 co-localize at primary cilia, and Cdc42 perturbation inhibits ciliogenesis. We also show that the exocyst co-immunoprecipitates with Rab8, a small GTPase necessary for ciliogenesis and found on the surface of vesicles carrying ciliary proteins. These are critical data for Aim 2. Our Preliminary Studies show that Sec10 knockdown cells are similar to ADPKD cells in several crucial ways: they have low levels of intracellular calcium that do not increase with fluid flow, the mitogen activated protein kinase (MAPK) pathway is turned on, and they are hyperproliferative. Importantly, we demonstrate a biochemical interaction between exocyst Sec10 and polycystin-2. PKD2, encoding the calcium channel polycystin-2, is one of two genes, which when mutated, causes ADPKD. We show in vivo correlation using sec10 antisense morpholinos in zebrafish, which results in a remarkably similar phenotype to that following injection of pkd2 morpholinos, with a "curly up" tail, dilated glomeruli, disorganized cilia, and MAPK pathway activation. Finally, we demonstrate a genetic interaction between pkd2 and sec10, in that small amounts of each morpholino that individually have no effect, together result in the phenotype noted above. Our collaborators showed the exocyst mislocalized intracellularly in human ADPKD cells, thereby establishing a link between exocyst localization and ADPKD. These data are the basis for Aim 3. Our proposed research, therefore, is directed toward the hypothesis that the central role the exocyst plays in ciliogenesis and cystogenesis is mediated via its localization at the primary cilium by Cdc42, its stabilization by binding to Par3, and the subsequent targeting and docking of Rab8 positive vesicles carrying ciliary proteins. Accordingly, we will build on our findings that the exocyst is necessary for ciliogenesis by performing three Aims. In Aim 1, we will identify and map the interacting domain between exocyst component Sec10 and Par3. We will then disrupt the interacting domain and determine the consequences with respect to ciliogenesis and cystogenesis. In Aim 2, we will investigate the role of Rho and Rab GTPases, especially Cdc42 and Rab8, in localizing the exocyst to primary cilia for trafficking of secretory vesicles carrying ciliary proteins. Finally, in Aim 3, we will determine the role of the exocyst in ciliogenesis and cystogenesis in vivo using antisense morpholinos of sec10 and Rho/Rab GTPases in zebrafish. Successful completion of these Aims will improve our understanding of protein delivery, ciliogenesis, and cystogenesis at the cell and molecular levels, and identify novel targets for therapeutic intervention in ADPKD. PUBLIC HEALTH RELEVANCE: Autosomal dominant polycystic kidney disease (ADPKD), affecting approximately 1/500 people, or 500,000 Americans, is the most common potentially lethal genetic disease and 4th leading cause of kidney failure. Given 25 million veterans and another 45 million family members, there are at least 100,000 veterans or dependents with ADPKD. As Chief of Kidney Medicine at the Philadelphia VAMC and physician in the Pennsylvania Army National Guard, I frequently see patients with ADPKD, and it is difficult to tell them that there are no approved therapies. ADPKD is characterized by cystic overgrowth that destroys the kidney. A cellular organelle called the primary cilium has been strongly implicated in ADPKD pathogenesis and our Preliminary Studies show that the eight-protein exocyst complex is necessary for cilia and cyst formation, both in cell culture and living animals. Here, we propose experiments to test how the exocyst builds primary cilia, with successful completion leading to identification of novel candidate targets for intervention in ADPKD.

描述（由申请人提供）： 我们的目的是了解高度保守的八蛋白外囊蛋白复合物在纤毛发生和囊肿发生中的作用。囊肿是肾脏等上皮器官的“基础”，囊肿形成的缺陷与常染色体显性多囊肾脏病（ADPKD）等人类疾病有关。 ADPKD是最常见的致命遗传疾病，影响了约1/500人，也是终末期肾脏疾病的第四个主要原因。目前尚无针对500,000名美国人的批准疗法，包括100,000名退伍军人和受抚养人，以及ADPKD。 在肾小管细胞中，我们最近表明，外旋转复合物位于原发性纤毛的形成所必需的，这是一种与ADPKD发病机理相关的细胞器。与中央睫状蛋白Par3共同局部并共免疫沉淀。在敲除外囊肿成分SEC10之后，不再发生原发性纤毛发生。我们的初步研究还表明，SEC10和PAR3之间的相互作用是目标1的基础。分泌途径对于适当的细胞功能至关重要，并且胞外囊肿以介导携带膜蛋白的分泌囊泡的靶向和对接而闻名。多个RHO和RAB家族GTPases调节外囊肿。在酵母中，我们表明，Rho GTPase Cdc42将外旋植物定位于特定的质膜结构域，而我们的初步研究表明：SEC10和CDC42是结合伙伴，Cdc42和Sec10在原代纤毛中的Cdc42和Sec10共定位在原发性纤毛，以及CDC42扰动抑制cili cilienagin cilienseption。我们还表明，外囊肿共免疫沉淀与Rab8是纤毛发生所需的小GTPase，并且在携带纤毛蛋白的囊泡表面发现。这些是AIM 2的关键数据。我们的初步研究表明，SEC10敲低细胞以几种关键方式与ADPKD细胞相似：它们的细胞内钙水平较低，不会随液体流动而增加，有丝分裂激活的蛋白激活蛋白激酶（MAPK）途径被打开，并且是高增生性的。重要的是，我们证明了外囊肿Sec10和Polycystin-2之间的生化相互作用。编码钙通道Polycystin-2的PKD2是两个基因之一，当突变时会导致ADPKD。我们在斑马鱼中使用SEC10反义形态的形态学显示了体内相关性，这与注射PKD2形态学后具有非常相似的表型，并带有“卷发”尾巴，扩张的肾小球，混乱的纤毛纤毛和MAPK途径激活。最后，我们证明了PKD2和SEC10之间的遗传相互作用，其中少量的每个形态诺里诺人都没有影响，从而导致上述表型。我们的合作者在人ADPKD细胞中表现出胞外细胞内的胞外囊性错误，从而在外囊肿定位和ADPKD之间建立了联系。这些数据是目标3的基础。因此，我们提出的研究是针对以下假设，即外循环症在纤毛生成和囊肿发生中所扮演的核心作用是通过cdc42在原发性纤毛中的定位来介导的，它通过与PAR3结合而稳定在cillium cilium cdilium，并通过与PAR3的稳定性以及随后的靶标和cockicles car car ciily car car ciliary cortee car ciliyring cilliary protee蛋白。因此，我们将基于我们的发现，即通过执行三个目标，纤毛发生对于纤毛生成是必要的。在AIM 1中，我们将识别并绘制exocyst组件SEC10和PAR3之间的相互作用域。然后，我们将破坏相互作用的结构域，并确定有关纤毛发生和囊肿发生的后果。在AIM 2中，我们将研究Rho和Rab GTPases，尤其是Cdc42和Rab8在将外囊囊定位到原发性纤毛中的作用，用于运输携带纤毛蛋白的分泌囊泡。最后，在AIM 3中，我们将使用Sec10和Rho/Rab GTPases在斑马鱼中的反义吗啡在体内的纤毛生成和囊肿发生中的作用。这些目标的成功完成将提高我们对细胞和分子水平上蛋白质递送，纤毛生成和囊肿发生的理解，并确定在ADPKD中治疗干预的新靶标。 公共卫生相关性： 常染色体显性多囊性肾脏疾病（ADPKD）影响约1/500人或500,000美国人，是最常见的潜在致命遗传疾病，也是肾衰竭的第四个主要原因。给予2500万退伍军人和另外4500万家庭成员，至少有100,000名退伍军人或ADPKD的家属。作为费城VAMC的肾脏医学主管和宾夕法尼亚州陆军国民警卫队的医师，我经常看到患有ADPKD的患者，很难告诉他们没有批准的疗法。 ADPKD的特征是囊性过度生长会破坏肾脏。一个称为原发性纤毛的细胞细胞器在ADPKD发病机理中强烈涉及，我们的初步研究表明，八蛋白蛋白胞外囊肿复合物对于纤毛和囊肿的形成是在细胞培养和活动物中所必需的。在这里，我们提出了实验，以测试外囊肿如何构建原发性纤毛，并成功完成，从而确定了新型候选目标的干预ADPKD。