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.

肾脏裂缝膜片（SD）是一个蛋白质层，在相邻的足细胞的脚步之间延伸，并含有蛋白质肾蛋白和podocin。两种蛋白质的突变引起遗传性肾病综合征。从功能上讲，SD被认为是肾脏过滤过程中的关键屏障和/或作为“分子传感器”，以动态控制足小胞胎的形成和功能。为了定义SD的结构和动力学，我们对肾素，Neph1和Podocin进行了高分辨率蛋白质组学分析，该分析提供了SD构建块（蛋白质组织或相互作用体）的第一个综合数据，并阐明了几个关键发现。三个相互作用（15-26个组成）是（i）不同的目的地一些相互重叠，（ii）完成一系列信号蛋白以及结构/基质蛋白，并且（iii）包含许多构成，这些构成缺乏公共数据库中主要功能的注释。了解新确定的蛋白质组织对SD的建立，组织和功能的重要性需要进一步的无偏生化/蛋白质组学分析和功能研究。利用亲和力化蛋白质组学，通过基于蓝色页面的蓝色页面质谱法（CS-BN/MS）进行复合体分析，有机蛋白质组学，SDS-Freeze裂缝复制中的免疫 - EM，免疫 - EM，并在使用新的“流动性”共同培训中，详细的功能分析，详细的功能分析，并使用“流动性”的构建方式，我们的友好型杂物均与妈妈的组合型，妈妈，目标：（1）阐明三种蛋白质框架中SD的建筑物的建筑物和多样性的蛋白质 - 蛋白质相互作用，（2）阐明了在不同的膜隔室中SD的组装过程和动态，（3）在体外和体外研究蛋白质组的功能，包括蛋白质组和4个蛋白质的功能，包括蛋白质群体和四个序列均为2次分析序列，（4这些这些基因敲除这些构成在定义的疾病条件下构成SD功能的影响。