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: 7903721
• 负责人: Jochen Reiser
• 金额: $ 10万
• 依托单位: UNIVERSITY OF MIAMI SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-25 至 2011-09-24
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7903721
• 关键词: Actins; Address; Affect; Aftercare; American; Binding; Biological; Biological Process; CD2-associated protein; Cathepsin L; Cathepsins; Cell Nucleus; Cells; Cleaved cell; Cultured Cells; Cytoplasm; Development; Diagnosis; Disease; End stage renal failure; Enzyme Precursors; Enzymes; Figs - dietary; Foot Process; Gene Transfer; Grant; Human; In Vitro; Injury; Intercellular Junctions; Kidney; Kidney Diseases; Kidney Glomerulus; Lead; Link; Lysosomes; Medicare; Messenger RNA; Metabolism; Modeling; Molecular; Molecular Target; Mus; N-terminal; Nephrotic Syndrome; Pathology; Pathway interactions; Peptide Hydrolases; Peptides; Permeability; Process; Proteins; Proteinuria; Proteolysis; Proteolytic Processing; Proteome; Proteomics; Public Health; Puromycin Aminonucleoside; Regulation; Regulation of Proteolysis; Renal function; Renal glomerular disease; Research Personnel; Resistance; Role; Severities; Structure; Testing; Therapeutic; Transcriptional Regulation; Translations; Tumor Cell Invasion; Ultrafiltration; Work; base; cell motility; cost; genetic regulatory protein; glomerular filtration; in vivo; interest; mutant; novel; podocyte; programs; protein degradation; protein protein interaction; slit diaphragm; small molecule

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 2000 万美国人患有肾脏疾病。在过去的二十年里，被诊断患有肾病的人数每十年翻一番，给人类带来痛苦，并导致终末期肾衰竭带来巨额医疗保险费用。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，形成防止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衰退。因此，在细胞和分子水平上定义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生的早期结构变化的分子靶标，符合公众健康的利益。我们的新发现是，在肾病综合征期间，组织蛋白酶 L 酶存在于足细胞的细胞质中，并裂解重要的 SD 蛋白 CD2AP。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，例如细胞内蛋白质降解、酶前体激活和肿瘤侵袭。组织蛋白酶 L 通常位于溶酶体中，也可以在细胞质中找到或在某些条件下可以分泌。我们开发了一种新型短暂性肾病综合征小鼠模型，其与人类肾病综合征具有相同的关键特征，这使我们能够描绘由组织蛋白酶 L 诱导产生的足细胞中的蛋白水解过程。我们提出了 3 个具体目标来阐明足细胞中溶酶体外组织蛋白酶 L 表达和活性的功能。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 的蛋白水解靶蛋白这一假设，并探索组织蛋白酶 L 抗切割 CD2AP 突变体的细胞连接稳定性（具体目标 2）。在具体目标 3 中，我们将探讨切割的 CD2AP 片段的生物学意义，并使用细胞器蛋白质组学分析此类肽对基于肌动蛋白的溶酶体降解组的影响。肾病综合征期间足细胞结构蛋白和调节蛋白的蛋白水解加工的作用代表了蛋白尿分子检查的一个新概念。如果我们的假设是正确的，我们的工作将对肾小球病理学的基本理解具有广泛的意义。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药物（例如组织蛋白酶 L 抗性 CD2AP 突变体或阻断 CD2AP 裂解的小分子）来解决蛋白尿和肾小球疾病的进展。