Proteolytic Regulation of Podocyte Function by Cathepsin L.

组织蛋白酶 L 对足细胞功能的蛋白水解调节。

• 批准号: 7145238
• 负责人: Jochen Reiser
• 金额: $ 31.62万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-08-15 至 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7145238
• 关键词: actins; cysteine endopeptidases; cytoprotection; intercellular connection; laboratory mouse; lysosomes; membrane transport proteins; nephrotic syndrome; podocyte; protein localization; protein protein interaction; proteinuria; proteolysis

DESCRIPTION (provided by applicant): About 20 million Americans have kidney disease. The number of people diagnosed with kidney disease has doubled each decade for the last two decades impacting on human suffering and enormous Medicare costs imposed by end-stage renal failure. Renal ultrafiltration is located within the renal glomerulus and performed by highly specialized podocyte cells. Podocyte foot processes (FPs) and the interposed slit diaphragms (SDs) cover the outer aspect of the glomerular filtration barrier and form a final barrier to protein loss. Damage of podocytes results in proteinuria and may lead to progressive decline of renal function. It is therefore in the interest of the public health to define the regulation of podocyte structure and function at the cellular and molecular levels and identify molecular targets involved in early structural changes leading to podocyte damage and the development of proteinuria. We have made the novel finding that cathepsin L enzyme is present within the cytoplasm of podocytes during nephrotic syndrome and cleaves CD2AP, an important SD protein. Cathepsin L is a lysosomal protease that has a broad biological significance such as intracellular protein degradation, activation of enzyme precursors, and tumor invasion. Normally located in lysosomes, cathepsin L can be found in the cytoplasm or can be secreted under certain conditions. We have developed a novel murine model of transient nephrotic syndrome that shares key features with human nephrotic syndrome which allows us to delineate proteolytic processes in podocytes resulting from the induction of cathepsin L. We propose 3 Specific Aims to unravel the function of extralysosomal cathepsin L expression and activity in podocytes. In the first Specific Aim, we propose to analyze the course and severity of proteinuria in mice lacking cathepsin L and how the lack of this enzyme affects CD2AP binding interactions with other SD proteins. We will then test the hypothesis in detail that the SD protein CD2AP is a proteolytic target protein of cathepsin L and explore the cell-junction stability of cathepsin L cleavage resistant CD2AP mutants (Specific Aim 2). In Specifc Aim 3, we will address the biological significance of cleaved CD2AP fragments and analyse the effects of such peptides on the actin based lysosomal degradome using organellar proteomics. The proposed role of proteolytic processing of podocyte structural and regulatory proteins during nephrotic syndrome represents a novel concept in the molecular work-up of proteinuria. If our hypothesis is correct, our work will have broad significance for the basic understanding of glomerular pathology e.g. the mechanism of podocyte FP effacement. Uncovering the role of podocyte proteolysis will help to develop novel pharmaco-therapeutics (such as cathepsin L resistant CD2AP mutants or small molecules blocking CD2AP cleavage) to tackle proteinuria and progression of glomerular disease.

描述（由申请人提供）：大约有2000万美国人患有肾脏疾病。在过去的二十年里，被诊断患有肾脏疾病的人数每十年翻一番，这对人类的痛苦和终末期肾衰竭造成的巨大医疗费用产生了影响。肾超滤位于肾小球内，由高度特化的足细胞执行。足细胞足突（FP）和插入的狭缝隔膜（SD）覆盖肾小球滤过屏障的外部，并形成蛋白质损失的最终屏障。足细胞损伤导致蛋白尿，并可能导致肾功能进行性下降。因此，在细胞和分子水平上确定足细胞结构和功能的调节，并确定参与导致足细胞损伤和蛋白尿发展的早期结构变化的分子靶点，符合公众健康的利益。我们已经取得了新的发现，组织蛋白酶L酶存在于肾病综合征期间的足细胞的细胞质内，并切割CD 2AP，一种重要的SD蛋白。组织蛋白酶L是一种溶酶体蛋白酶，具有广泛的生物学意义，如细胞内蛋白质降解，酶前体的活化和肿瘤侵袭。组织蛋白酶L通常位于溶酶体中，可以在细胞质中发现或在某些条件下分泌。我们已经开发了一种新的短暂性肾病综合征的小鼠模型，该模型与人类肾病综合征具有关键特征，这使我们能够描述由组织蛋白酶L诱导引起的足细胞中的蛋白水解过程。我们提出了3个具体的目的，以阐明功能的溶酶体外组织蛋白酶L的表达和活性的足细胞。在第一个具体目标中，我们建议分析缺乏组织蛋白酶L的小鼠蛋白尿的过程和严重程度，以及缺乏这种酶如何影响CD2AP与其他SD蛋白的结合相互作用。然后，我们将详细检验SD蛋白CD2AP是组织蛋白酶L的蛋白水解靶蛋白的假设，并探索组织蛋白酶L切割抗性CD2AP突变体的细胞连接稳定性（具体目标2）。在第3章中，我们将讨论切割的CD2AP片段的生物学意义，并使用细胞器蛋白质组学分析这些肽对基于肌动蛋白的溶酶体降解的影响。肾病综合征期间足细胞结构和调节蛋白的蛋白水解加工的作用代表了蛋白尿分子后处理的一个新概念。如果我们的假设是正确的，我们的工作将有广泛的意义，为肾小球病理学的基本理解，如足细胞FP消失的机制。揭示足细胞蛋白水解的作用将有助于开发新的药物治疗（如组织蛋白酶L抗性CD2AP突变体或阻断CD2AP裂解的小分子），以解决蛋白尿和肾小球疾病的进展。