The exocyst in ciliogenesis and cystogenesis

纤毛发生和囊肿发生中的外囊

• 批准号: 8242625
• 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/8242625
• 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; public health relevance; 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）： 我们的目标是了解高度保守的八蛋白外囊复合物在纤毛发生和囊发生中的作用。囊肿是上皮器官（如肾脏）的“构建块”，囊肿形成缺陷与人类疾病（如常染色体显性遗传性多囊肾病（ADPKD））有关。ADPKD是最常见的潜在致命遗传疾病，影响约1/500的人，也是终末期肾病的第四大原因。目前，50万美国人，包括10万退伍军人和家属，没有批准的治疗方法。 在肾小管细胞中，我们最近发现，外囊复合物定位于初级纤毛的形成，并且是初级纤毛形成所必需的，初级纤毛是与ADPKD发病机制密切相关的细胞器。外囊与中央纤毛蛋白Par 3共定位和共免疫沉淀。敲除外囊组分Sec 10后，不再发生初级纤毛发生。我们的初步研究还显示Sec 10和Par 3之间的相互作用，这是目标1的基础。分泌途径对于适当的细胞功能是至关重要的，并且已知外囊介导携带膜蛋白的分泌囊泡的靶向和对接。多个Rho和Rab家族GTP酶调节外囊。在酵母中，我们发现，Cdc 42，Rho GTdR，本地化的exocyst特定的质膜结构域和我们的初步研究表明：Sec 10和Cdc 42是结合伙伴，Cdc 42和Sec 10共定位在初级纤毛，Cdc 42扰动抑制纤毛发生。我们还表明，外囊共免疫沉淀与Rab 8，纤毛发生所需的小GTdR和携带纤毛蛋白的囊泡表面上发现。这些是目标2的关键数据。我们的初步研究表明，Sec 10敲低细胞在几个关键方面与ADPKD细胞相似：它们具有低水平的细胞内钙，不随流体流动而增加，丝裂原活化蛋白激酶（MAPK）通路被打开，并且它们是过度增殖的。重要的是，我们证明了外囊Sec 10和多囊蛋白-2之间的生物化学相互作用。PKD 2编码钙通道多囊蛋白-2，是两个基因之一，当突变时，导致ADPKD。我们显示在斑马鱼中使用sec 10反义morpholinos的体内相关性，这导致了与注射pkd 2 morpholinos后的表型非常相似的表型，具有“卷曲”的尾巴、扩张的肾小球、紊乱的纤毛和MAPK通路激活。最后，我们证明了pkd 2和sec 10之间的遗传相互作用，即少量的每个吗啉代单独没有影响，共同导致上述表型。我们的合作者显示，外囊在人ADPKD细胞中错误定位于细胞内，从而建立了外囊定位和ADPKD之间的联系。这些数据是目标3的基础。 因此，我们提出的研究是针对这样的假设，即外囊在纤毛发生和囊形成中发挥的中心作用是通过Cdc 42在初级纤毛处的定位、通过与Par 3结合的稳定以及随后的Rab 8阳性囊泡携带纤毛蛋白的靶向和对接来介导的。因此，我们将建立在我们的研究结果，外囊是必要的纤毛发生进行三个目标。在目标1中，我们将识别和映射外囊组分Sec 10和Par 3之间的相互作用域。然后，我们将破坏相互作用的结构域，并确定与纤毛和囊肿发生的后果。在目的2中，我们将研究Rho和Rab GTP酶，特别是Cdc 42和Rab 8，在定位外囊到初级纤毛运输携带纤毛蛋白的分泌囊泡中的作用。最后，在目标3中，我们将确定的作用外囊在纤毛和cystogenesis在体内使用反义morpholinos的sec 10和Rho/Rab GTP酶在斑马鱼。这些目标的成功完成将提高我们在细胞和分子水平上对蛋白质递送、纤毛发生和囊形成的理解，并确定ADPKD治疗干预的新靶点。 公共卫生关系： 常染色体显性遗传性多囊肾病（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和肾衰竭的第四大原因。考虑到2500万退伍军人和另外4500万家庭成员，至少有10万退伍军人或家属患有ADPKD。作为费城VAMC肾脏医学主任和宾夕法尼亚州陆军国民警卫队的医生，我经常见到ADPKD患者，很难告诉他们没有批准的治疗方法。ADPKD的特征是囊性过度生长，破坏肾脏。一种称为初级纤毛的细胞器与ADPKD的发病机制密切相关，我们的初步研究表明，在细胞培养物和活体动物中，八蛋白外囊复合物对于纤毛和囊肿形成是必需的。在这里，我们提出了实验来测试外囊如何建立初级纤毛，成功完成导致ADPKD干预新的候选目标的识别。