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

蛋白水解蛋白加工改变和蛋白结构变化在足细胞功能障碍、消失和蛋白尿肾病中的作用

• 批准号: 517812866
• 负责人: Professor Dr. Thomas Benzing
• 金额: --
• 依托单位: Arbeitsgruppe Functional Proteomics
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/517812866?language=en
• 关键词: role; altered; proteolytic; protein; processing

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.

进展为慢性肾病的大多数肾病始于肾小球（肾过滤单位），这是由于肾小球再生能力有限以及终末分化的肾小球足细胞自我更新能力有限。当足细胞受损时，它们呈现简化的形态。这种结构变化被称为消失，其特征是过滤缝缩短和足突增宽。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中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核心目标。我们实验室的最新数据表明，足细胞疾病的早期功能和超微形态学变化可能不能充分解释单独的转录或翻译的变化。相反，我们意识到细胞应激诱导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型的表达，而不一定影响整体蛋白质丰度（Rinschen，...，Huesgen，Benzing（2017）J Am Soc Nephrol 28，2867-2878）。在这里，我们使用创新的技术来研究损伤诱导的整体蛋白质结构变化和蛋白质切割的精确度损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水解-质谱（LiP-MS）转化为全局蛋白质结构变化，以及如何使用基于相关性的功能ProteoForm评估技术（COPF）转化为Proteoform表示。此外，我们将（2）评估体内受调节的蛋白质切割和足细胞蛋白质的蛋白质型代表性的变化（HUNTER N-降解组学质谱法），以及（3）使用创新的小鼠模型阐明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。