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

由于肾小球再生能力有限，终末分化的肾小球足细胞自我更新能力有限，大多数进展为慢性肾脏疾病的肾脏疾病始于肾小球，即肾滤过单位。当足细胞受到损伤时，它们呈现出简化的形态。这种结构变化被称为消隐，其特征是过滤缝的缩短和足突的扩大。尽管我们最近的数据揭示了第一个实验验证的肾小球超滤模型（Butt et al. (2020) Nat Metab 2,461 -474），但在分子水平上触发疾病足细胞形状变化的分子线索仍然难以捉摸。缩小这一差距是该项目的中心目标。我们实验室最近的数据表明，足细胞疾病的早期功能和超微改变可能无法仅用转录或翻译变化来充分解释。相反，我们意识到细胞应激诱导了蛋白质切割保真度的变化，并改变了细胞骨架和细胞连接蛋白的蛋白质形态的表现，而不一定影响总体蛋白质丰度（Rinschen，…，Huesgen, Benzing (2017) J Am Soc Nephrol 28, 2867-2878）。在这里，我们使用创新技术来研究损伤诱导的整体蛋白质结构变化和调节蛋白切割精度的丧失对足细胞功能障碍和消失的贡献。我们将(1)分析足细胞损伤如何使用有限的蛋白质水解质谱（LiP-MS）转化为整体蛋白质结构变化，并使用基于相关性的功能蛋白质形态评估技术（COPF）来表达蛋白质形态。此外，我们将(2)评估体内调节蛋白切割的变化和足细胞蛋白的蛋白质形态表征（HUNTER n -降解组学质谱法），以及(3)利用创新的小鼠模型揭示蛋白质结构和蛋白质切割改变对体外蛋白质复合物组装和体内肾脏疾病的机制后果。

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.