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）： 我们的目标是了解高度保守的八蛋白外囊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“组成部分”，囊肿形成的缺陷与常染色体显性多囊肾病 (ADPKD) 等人类疾病有关。 ADPKD 是最常见的潜在致命遗传病，影响约 1/500 人，也是终末期肾病的第四大原因。目前尚无批准的治疗方法可用于治疗 500,000 名 ADPKD 美国人，其中包括 100,000 名退伍军人及其家属。 在肾小管细胞中，我们最近表明，外囊复合体定位于初级纤毛的形成，并且是初级纤毛形成所必需的，初级纤毛是一种与 ADPKD 发病机制密切相关的细胞器。外囊与中央纤毛蛋白 Par3 共定位并共免疫沉淀。敲低外囊成分 Sec10 后，初级纤毛发生不再发生。我们的初步研究还显示了 Sec10 和 Par3 之间的相互作用，这是目标 1 的基础。分泌途径对于正常的细胞功能至关重要，而外囊因介导携带膜蛋白的分泌囊泡的靶向和对接而闻名。多种 Rho 和 Rab 家族 GTPases 调节外囊。在酵母中，我们发现 Cdc42（一种 Rho GTP 酶）将外囊定位于特定的质膜结构域，我们的初步研究表明：Sec10 和 Cdc42 是结合伴侣，Cdc42 和 Sec10 共同定位于初级纤毛，并且 Cdc42 扰动抑制纤毛发生。我们还表明，外囊与 Rab8 发生共免疫沉淀，Rab8 是纤毛发生所必需的一种小型 GTP 酶，存在于携带纤毛蛋白的囊泡表面。这些是目标 2 的关键数据。我们的初步研究表明，Sec10 敲低细胞在几个关键方面与 ADPKD 细胞相似：它们的细胞内钙水平较低，不会随着液体流动而增加，有丝分裂原激活蛋白激酶 (MAPK) 途径被打开，它们就会过度增殖。重要的是，我们证明了外囊 Sec10 和多囊蛋白-2 之间的生化相互作用。 PKD2 编码钙通道多囊蛋白-2，是两个基因之一，突变后会导致 ADPKD。我们在斑马鱼中使用 sec10 反义吗啉代显示了体内相关性，结果与注射 pkd2 吗啉代后的表型非常相似，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 pkd2 和 sec10 之间的遗传相互作用，即每种吗啉代的少量单独没有作用，共同导致上述表型。我们的合作者证明了外囊在人类 ADPKD 细胞中的细胞内错误定位，从而在外囊定位与 ADPKD 之间建立了联系。这些数据是目标 3 的基础。因此，我们提出的研究针对这样的假设：外囊在纤毛发生和囊肿发生中发挥的核心作用是通过 Cdc42 在初级纤毛上的定位来介导的，通过与 Par3 结合使其稳定，以及随后携带纤毛蛋白的 Rab8 阳性囊泡的靶向和对接。因此，我们将基于我们的发现，即外囊对于纤毛发生是必要的，通过执行三个目标。在目标 1 中，我们将识别并绘制外囊组件 Sec10 和 Par3 之间的相互作用域。然后我们将破坏相互作用的域并确定纤毛发生和囊肿发生的后果。在目标 2 中，我们将研究 Rho 和 Rab GTPases，特别是 Cdc42 和 Rab8，在将外囊定位到初级纤毛以运输携带纤毛蛋白的分泌囊泡中的作用。最后，在目标 3 中，我们将使用 sec10 和 Rho/Rab GTPases 的反义吗啉代确定外囊在斑马鱼体内纤毛发生和囊肿发生中的作用。成功完成这些目标将提高我们对细胞和分子水平上的蛋白质递送、纤毛发生和囊肿发生的理解，并确定 ADPKD 治疗干预的新靶点。 公共卫生相关性： 常染色体显性多囊肾病 (ADPKD) 影响大约 1/500 人，即 500,000 名美国人，是最常见的潜在致命遗传病，也是肾衰竭的第四大原因。鉴于 2500 万退伍军人和另外 4500 万家庭成员，至少有 100,000 名退伍军人或家属患有 ADPKD。作为费城 VAMC 的肾脏内科主任和宾夕法尼亚陆军国民警卫队的医生，我经常接诊 ADPKD 患者，但很难告诉他们没有经过批准的治疗方法。 ADPKD 的特点是囊性过度生长，从而破坏肾脏。一种称为初级纤毛的细胞器与 ADPKD 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的形成是必需的。在这里，我们提出实验来测试外囊如何构建初级纤毛，成功完成后可以确定干预 ADPKD 的新候选靶点。