The role of altered proteolytic protein processing and protein structural changes in podocyte dysfunction, effacement, and albuminuric kidney disease

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Most kidney diseases that progress to chronic kidney disease start in the glomerulus, the renal filtration unit, due to a limited capacity of glomeruli for regeneration and the limited ability of terminally differentiated glomerular podocytes for self-renewal. When podocytes are injured, they take on a simplified morphology. This structural change is called effacement and is characterized by the shortening of the filtration slit and widening of the foot processes. Although our recent data revealed the first experimentally validated model of glomerular ultrafiltration (Butt et al. (2020) Nat Metab 2, 461-474), the molecular cues that trigger podocyte cell shape changes in disease on the molecular level are still elusive. Closing this gap is the central aim of this project. Recent data from our lab showed that early functional and ultramorphological changes in podocyte disease might not be sufficiently explained by transcriptional or translational changes alone. Instead, we realized that cell stress induced a change in the fidelity of protein cleavage and altered the representation of proteoforms of cytoskeletal and cell junctional proteins without necessarily affecting overall protein abundance (Rinschen, …, Huesgen, Benzing (2017) J Am Soc Nephrol 28, 2867-2878). Here we use innovative technologies to study the contribution of damage-induced overall protein structural changes and loss of precision in regulated protein cleavage to podocyte dysfunction and effacement. We will (1) analyze how podocyte damage translates to global protein structural changes using limited proteolysis-mass spectrometry (LiP-MS) and to proteoform representation using COrrelation-based functional ProteoForm assessment technology (COPF). Moreover, we will (2) assess resulting changes in regulated protein cleavage and the representation of proteoforms of podocyte proteins in vivo (HUNTER N-degradomics mass spectrometry), and (3) unravel the mechanistic consequences of altered protein structure and protein cleavage for protein complex assembly in vitro and kidney disease in vivo using innovative mouse models.