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

肾裂隙横隔膜(SD)是一层蛋白质层，延伸到相邻足细胞的足突之间，含有Newitin和Podocin蛋白质；这两种蛋白质中的任何一种都会导致遗传性肾病综合征。在功能上，SD被认为是肾滤过过程中的关键屏障和/或作为动态控制足细胞足突的形成和功能的分子传感器。为了确定SD的结构和动力学，我们对Nephin、Neph1和Podocin进行了高分辨率的蛋白质组学分析，提供了SD构建单元(蛋白质组或相互作用)的第一个全面数据，并阐明了几个关键发现。这三个相互作用组(15-26个组分)是(I)尽管相互重叠，但却是截然不同的，(Ii)包括一系列信号蛋白以及结构/基质蛋白，以及(Iii)包含一些在公共数据库中缺乏主要功能注释的组分(S)。要了解新发现的蛋白质组对SD的建立、组织和功能的意义，需要进一步的无偏见的生化/蛋白质组学分析和功能研究。我们将利用亲和纯化蛋白质组学、冷冻切片蓝天然PAGE-MS复杂组谱、细胞器蛋白质组学、SDS-冷冻骨折复制品的免疫EM，以及结合哺乳动物和非哺乳动物模式生物新开发的“流通式”共培养系统进行详细的功能分析，我们将追求以下目标：(1)在这三个蛋白质组的框架内，解开蛋白质之间的相互作用，重构SD的构建和多样性；(2)阐明SD在不同的膜间隔中的组装过程和动力学。(3)研究MERTK、ITM2B和ANPRC等蛋白质组分在体外和体内的功能意义；(4)分析在特定疾病条件下哺乳动物基因敲除这些组分对SD功能的影响。

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.