Proteolytic Regulation of Podocyte Function by Cathepsin L.

描述（由申请人提供）：大约2000万美国人患有肾脏疾病。在过去的二十年中，被诊断患有肾脏疾病的人数每十年翻一番，影响了人类的痛苦和终末期肾衰竭所带来的巨大医疗费用。肾超滤位于肾小球内，由高度特化的足细胞完成。足细胞足突（FPs）和间隙横膈膜（SDs）覆盖了肾小球滤过屏障的外部，形成了阻止蛋白质流失的最终屏障。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，并确定导致足细胞损伤和蛋白尿发展的早期结构变化所涉及的分子靶点，符合公共卫生的利益。我们发现组织蛋白酶L酶在肾病综合征期间存在于足细胞的细胞质中并切割CD2AP，这是一种重要的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，如细胞内蛋白降解、酶前体活化、肿瘤侵袭等。组织蛋白酶L通常位于溶酶体中，可在细胞质中发现或在一定条件下分泌。我们开发了一种新的小鼠短暂性肾病综合征模型，该模型与人类肾病综合征具有相同的关键特征，使我们能够描述由组织蛋白酶L诱导引起的足细胞蛋白水解过程。我们提出了3个具体目标来阐明外溶酶体组织蛋白酶L在足细胞中的表达和活性的功能。在第一个特异性目标中，我们建议分析缺乏组织蛋白酶L的小鼠蛋白尿的病程和严重程度，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，我们将详细验证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抗切割CD2AP突变体的细胞连接稳定性（Specific Aim 2）。在特异性目标3中，我们将讨论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。在肾病综合征过程中足细胞结构蛋白和调节蛋白的蛋白水解过程所提出的作用代表了蛋白尿分子积累中的一个新概念。如果我们的假设是正确的，我们的工作将对肾小球病理的基本认识，如足细胞FP消除的机制具有广泛的意义。揭示足细胞蛋白水解的作用将有助于开发新的药物治疗方法（如组织蛋白酶L耐药CD2AP突变体或阻断CD2AP切割的小分子）来解决蛋白尿和肾小球疾病的进展。

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.