Intracellular Route of Secreted Apical Proteins In Renal Epithelial Cells

肾上皮细胞分泌顶端蛋白的细胞内途径

• 批准号: 8005034
• 负责人: Polly Elizabeth Mattila
• 金额: $ 5.47万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-12-01 至 2011-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8005034
• 关键词: Apical; Biochemical; Cell Line; Cell membrane; Cell physiology; Cell surface; Data; Detergents; Development; Diabetes Mellitus; Disease; Endosomes; Epithelial; Epithelial Cells; Flow Cytometry; Future; Glycosylphosphatidylinositols; Goals; Golgi Apparatus; Health; Homeostasis; Hypertension; Ion Transport; Kidney; Kidney Diseases; Laboratories; Lead; Life; Link; Lipids; Lysosomal Storage Diseases; Lysosomes; MDCK cell; Maintenance; Membrane; Membrane Microdomains; Membrane Protein Traffic; Membrane Proteins; Modeling; Pathway interactions; Patients; Peptides; Proteins; Proteomics; Recycling; Renal function; Renal tubule structure; Research; Route; Signal Transduction; Sorting - Cell Movement; Surface; apical membrane; base; basolateral membrane; cellular imaging; glycosylation; insight; kidney cell; novel; protein transport; research study; secretory protein; targeted delivery; trafficking

DESCRIPTION (provided by applicant): Maintenance of epithelial polarity is essential for proper function and homeostasis the kidney. This requires the targeted delivery of newly synthesized and recycled proteins to the apical and basolateral surfaces. Disregulation of protein delivery can lead to serious diseases such as hypertension, diabetes, and lysosomal storage diseases. The long term objective of my project is to examine the signals and mechanisms that regulate the apical delivery of secreted proteins. Recent studies from our lab and others have demonstrated that there are multiple routes to the apical surface of polarized renal cells. The aims of my proposal are: 1) to determine whether secreted proteins traverse endocytic compartments en route to the apical surface of polarized epithelial cells; and 2) to characterize post-Golgi compartments involved in biosynthetic delivery of apically-destined proteins. I will use the Madin-Darby canine kidney (MDCK) cell line to investigate polarized delivery of distinct classes of secreted apical proteins. MDCK cells are a valuable model because they emulate the structural and functional polarity of the kidney tubule. In aim 1 I will examine the biosynthetic route of two apically secreted proteins that are sorted via distinct mechanisms in MDCK cells using biochemical and live-cell imaging approaches. In aim 2 I will use a novel flow-cytometry approach to selectively collect post-Golgi carriers containing fluorescent protein-tagged versions of these markers and employ proteomic approaches to identify other protein components enriched in carriers that ferry secreted proteins. The results of my studies will provide new insights into the mechanisms that regulate sorting and trafficking of this important class of proteins. The disruption of normal kidney function can be related to diseases such as, hyper- and hypo-tension, and diabetes. The goal of our research is to study kidney cells and how they process and produce proteins that are critical for maintaining kidney health. Ultimately, our research will provide the basis for development of highly specific treatments for patients with kidney diseases.

描述（由申请人提供）：上皮极性的维持对于肾脏的正常功能和稳态至关重要。这需要将新合成和回收的蛋白质有针对性地输送到顶端和基底外侧表面。蛋白质输送失调可导致严重疾病，如高血压、糖尿病和溶酶体贮积病。我的项目的长期目标是检查调节分泌蛋白顶端递送的信号和机制。我们实验室和其他实验室的最新研究表明，到达极化肾细胞顶端表面的途径有多种。我的建议的目的是：1）确定分泌的蛋白质是否穿过内吞区室到达极化上皮细胞的顶端表面； 2) 表征参与顶端蛋白质生物合成传递的后高尔基体区室。我将使用 Madin-Darby 犬肾 (MDCK) 细胞系来研究不同类别的分泌性顶端蛋白的极化传递。 MDCK 细胞是一种有价值的模型，因为它们模拟肾小管的结构和功能极性。在目标 1 中，我将使用生化和活细胞成像方法检查 MDCK 细胞中通过不同机制分选的两种顶端分泌蛋白的生物合成途径。在目标 2 中，我将使用一种新颖的流式细胞术方法选择性地收集含有这些标记的荧光蛋白标记版本的后高尔基体载体，并采用蛋白质组学方法来鉴定富含转运分泌蛋白的载体中的其他蛋白质成分。我的研究结果将为调节这一类重要蛋白质的分类和运输的机制提供新的见解。正常肾功能的破坏可能与高血压、低血压以及糖尿病等疾病有关。我们研究的目标是研究肾细胞以及它们如何处理和产生对维持肾脏健康至关重要的蛋白质。最终，我们的研究将为开发针对肾脏疾病患者的高度特异性治疗方法奠定基础。