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

• Applicants: Professor Dr. Bernd Fakler; Privatdozent Dr. Florian Grahammer
• Subject Area: Anatomy and Physiology
• Term: since 2020
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 442759790

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.

• DFG Programme: Research Grants