The Exocyst in Synthesis, Cystogenesis and Tubulogenesis

合成、胞囊发生和管管发生中的胞外囊

基本信息

批准号：
7921099
负责人：
JOSHUA H LIPSCHUTZ
金额：
$ 5.19万
依托单位：
UNIVERSITY OF PENNSYLVANIA
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-09-30 至 2011-09-29
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7921099
关键词：
Affect Affinity Apical Autosomal Dominant Polycystic Kidney Binding Biological Assay Biology Cell model Cell physiology Cells Chimera organism Chimeric Proteins Cilia Co-Immunoprecipitations Collagen Complex Consensus Sequence Cyst Defect Development Disease Docking Endoplasmic Reticulum Engineering Epithelial Epithelial Cells Gel Goals In Vitro Kidney Location Mammalian Cell Maps Mediating Membrane Proteins Messenger RNA Molecular Mutagenesis Mutate Mutation Organ Organelles Pathogenesis Pathway interactions Phosphorylation Play Protein Biosynthesis Protein translocation Proteins Regulation Research Research Personnel Ribosomes Role Rough endoplasmic reticulum Staging System Testing Translating Translations Tubular formation Vesicle apical membrane basolateral membrane design human disease novel novel therapeutics overexpression programs protein complex protein transport yeast two hybrid system

项目摘要

DESCRIPTION (provided by applicant): Cysts and tubules are basic "building blocks" for epithelial organs such as the kidney, and defects in cyst and tubule formation are implicated in disorders such as autosomal dominant polycystic kidney disease. Our goal is to understand the biology of cystogenesis and tubulogenesis as it relates to development and disease. Using a well-described in vitro collagen gel system, we have shown that a central factor in cystogenesis and tubulogenesis is the exocyst, an evolutionarily conserved eight-protein complex involved in the secretory pathway. The secretory pathway is essential for proper cellular function, and the exocyst is known for mediating the targeting and docking of vesicles carrying secretory and basolateral proteins during the final stage of this pathway. We recently showed that the exocyst, particularly the Sec10 component, also has the novel and unexpected function of specifically regulating protein synthesis, the first stage of the secretory pathway, by interacting with the Sec61 a component of the endoplasmic reticulum (ER) translocon. In mammalian cells, proteins are simultaneously translated and translocated across the rough ER via the translocon. Our proposal is directed toward the hypothesis that the central role played by the exocyst in cyst and tubule formation is a result of its specific effects on protein synthesis. Accordingly, we will build on our findings by asking the following questions: How does the exocyst/Sec61 a interaction regulate protein synthesis? What are the interacting domains between Secl0 and Sec61a? Finally, how does the exocyst/translocon, recognize and then specifically regulate basolateral, but not apical, protein synthesis? To answer these questions we will use in vitro systems, including cell-free assays, to test whether the exocyst regulates protein translation and/or translocation (Aim 1). We will then identify and map the SeclO/Sec61 a interacting domain, and determine the functional consequences of mutating this domain, with respect to protein synthesis and cyst and tubule formation (Aim 2). Lastly, we will identify sequences that direct exocyst/translocon regulation of basolateral protein synthesis. This will be done using existing basolateral proteins that traffic to the apical membrane, due to mutations in the basolateral targeting sequence, and chimeras composed of portions of apical and basolateral proteins (Aim 3). Completion of these studies will enhance the understanding of the mechanisms of protein synthesis in cyst and tubule formation at the cellular and molecular levels and lay the groundwork for the development of novel therapeutics.

描述（由申请人提供）：囊肿和肾小管是上皮器官（例如肾脏）的基本“构件”，囊肿和肾小管形成的缺陷与常染色体显性多囊肾病等疾病有关。我们的目标是了解与发育和疾病相关的囊肿发生和管状发生的生物学。使用充分描述的体外胶原凝胶系统，我们已经证明囊肿发生和肾小管发生的核心因素是外囊，这是一种参与分泌途径的进化上保守的八蛋白复合物。分泌途径对于正常的细胞功能至关重要，并且众所周知，外囊在该途径的最后阶段介导携带分泌蛋白和基底外侧蛋白的囊泡的靶向和对接。我们最近表明，外囊，特别是 Sec10 成分，还具有特异性调节蛋白质合成（分泌途径的第一阶段）的新颖且意想不到的功能，通过与内质网（ER）易位子的 Sec61 成分相互作用。在哺乳动物细胞中，蛋白质通过易位子同时翻译并易位穿过粗糙的内质网。我们的建议针对这样的假设：外囊在囊肿和小管形成中发挥的核心作用是其对蛋白质合成的特定作用的结果。因此，我们将通过提出以下问题来建立我们的发现：exocyst/Sec61 a 相互作用如何调节蛋白质合成？ Secl0 和 Sec61a 之间的交互域是什么？最后，外囊/易位子如何识别并特异性调节基底外侧而非顶端的蛋白质合成？为了回答这些问题，我们将使用体外系统（包括无细胞测定）来测试外囊是否调节蛋白质翻译和/或易位（目标 1）。然后，我们将识别并绘制 SeclO/Sec61 相互作用结构域，并确定该结构域突变对蛋白质合成以及囊肿和小管形成的功能影响（目标 2）。最后，我们将鉴定指导基底外侧蛋白质合成的外囊/易位子调节的序列。这将使用由于基底外侧靶向序列的突变而运输至顶膜的现有基底外侧蛋白以及由顶端和基底外侧蛋白部分组成的嵌合体来完成（目标3）。这些研究的完成将增强对细胞和分子水平上囊肿和肾小管形成中蛋白质合成机制的理解，并为新型疗法的开发奠定基础。