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

肾裂隔膜（SD）是一个蛋白质层，在相邻足细胞的足突之间延伸，含有蛋白质Nephrin和Podocin;任一蛋白质的突变都会导致遗传性肾病综合征。在功能上，SD被认为是肾脏过滤过程中的关键屏障和/或作为动态控制足细胞足突形成和功能的“分子传感器”。为了定义SD的结构和动力学，我们对Nephrin，Neph 1和Podocin进行了高分辨率蛋白质组学分析，这提供了SD构建模块（蛋白质组或相互作用组）的第一个全面数据，并阐明了几个关键发现。这三种相互作用组（15-26种成分）是（i）不同的，尽管有一些相互重叠，（ii）包含一系列信号传导蛋白以及结构/基质蛋白，和（iii）含有许多在公共数据库中缺乏主要功能注释的成分。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。利用亲和纯化蛋白质组学，通过冷冻切片蓝-天然PAGE质谱进行复合体分析（cs-BN/MS）、细胞器蛋白质组学、SDS冷冻断裂复制品中的免疫EM以及采用新开发的“流通”共培养系统结合哺乳动物和非哺乳动物模式生物的详细功能分析，我们将追求以下目标：（1）阐明在三种蛋白质组的框架内重建SD的构建和多样性的蛋白质-蛋白质相互作用，（2）阐明SD在不同膜隔室中的组装过程和动力学，（3）在体外和体内研究蛋白质组组分的功能意义，包括蛋白质MERTK、ITM 2B和ANPRC，和（4）分析哺乳动物基因敲除这些组分对确定的疾病条件下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.