High-resolution proteomics and functional analyses of the podocyte slit diaphragm and its disease-induced dynamics

足细胞裂隙隔膜及其疾病诱导动力学的高分辨率蛋白质组学和功能分析

• 批准号: 442759790
• 负责人: Professor Dr. Bernd Fakler
• 金额: --
• 依托单位: Lehrstuhl für Physiologie II - Molecular Physiology
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/442759790?language=en
• 关键词: resolution; proteomics; functional; analyses; podocyte

The renal slit diaphragm (SD) is a proteinaceous layer that extends between the foot processes of neighboring podocytes and contains proteins Nephrin and Podocin; mutations in either protein cause hereditary nephrotic syndrome. Functionally, the SD is thought to either serve as the crucial barrier in the renal filtration process and/or as a ‘molecular sensor’ for dynamic control of formation and function of podocyte foot processes. To define the structure and dynamics of the SD, we performed high-resolution proteomic analyses of Nephrin, Neph1 and Podocin, which provided the first comprehensive data of the SD building blocks (proteomes or interactomes) and elucidated several key findings. The three interactomes (15-26 constituents) are (i) distinct despite some mutual overlap, (ii) comprise a series of signaling proteins as well as structural/matrix proteins, and (iii) contain a number of constituents that lack annotation of primary function(s) in public databases. Understanding the significance of the newly identified proteomes for establishment, organization and function of the SD requires further unbiased biochemical/proteomic analyses and functional studies. Utilizing affinity-purification proteomics, complexome profiling via cryo-slicing blue-native PAGE-based mass spectrometry (cs-BN/MS), organellar proteomics, immuno-EM in SDS-freeze fracture replicas and detailed functional analyses employing a newly developed “flow-through” co-culture system in combination with mammalian and non-mammalian model organisms, we will pursue the following goals: (1) unravel the protein-protein interactions reconstituting the building and diversity of the SD in the framework of the three proteomes, (2) elucidate the assembly processes and dynamics of the SD in distinct membrane compartments, (3) investigate in vitro and in vivo the functional significance of the proteome constituents, including the proteins MERTK, ITM2B and ANPRC, and (4) analyze the effect of mammalian gene knock-out of these constituents on SD function in defined disease conditions.

肾裂隔膜 (SD) 是在邻近足细胞的足突之间延伸的蛋白质层，含有 Nephrin 和 Podocin 蛋白；从功能上讲，SD 被认为是遗传性肾病综合征的关键屏障。肾滤过过程和/或作为动态控制足细胞足突的形成和功能的“分子传感器”为了定义 SD 的结构和动力学，我们进行了研究。 Nephrin、Neph1 和 Podocin 的高分辨率蛋白质组学分析，提供了 SD 构建模块（蛋白质组或相互作用组）的第一个全面数据，并阐明了三个相互作用组（15-26 个成分）的几个关键发现：(i) 尽管存在一些差异。相互重叠，(ii) 包含一系列信号蛋白以及结构/基质蛋白，并且 (iii) 包含许多缺乏公共主要功能注释的成分了解新发现的蛋白质组对于 SD 的建立、组织和功能的重要性需要进一步利用亲和纯化蛋白质组学、通过基于蓝色天然 PAGE 的冷冻切片质谱分析进行复合体分析。 (cs-BN/MS)、细胞器蛋白质组学、SDS 冷冻断裂复制品中的免疫电镜以及采用新开发的“流通”的详细功能分析通过与哺乳动物和非哺乳动物模型生物相结合的共培养系统，我们将追求以下目标：（1）揭示蛋白质-蛋白质相互作用，在三种蛋白质组的框架中重建SD的构建和多样性，（2）阐明不同膜区室中 SD 的组装过程和动力学，(3) 在体外和体内研究蛋白质组成分（包括蛋白质 MERTK、ITM2B 和 ANPRC）的功能意义，以及(4) 分析哺乳动物基因敲除这些成分对特定疾病条件下 SD 功能的影响。