Combined high-resolution structural and functional characterization of the multi-layered bipartite NEPH-NEPHRIN based slit diaphragm

基于狭缝隔膜的多层二分 NEPH-NEPHRIN 的高分辨率结构和功能综合表征

• 批准号: 429077705
• 负责人: Professor Dr. Tobias B. Huber
• 金额: --
• 依托单位: III. Medizinische Klinik und Poliklinik (Nephrologie, Rheumatologie - Nierentransplantation - Sektion Endokrinologie/Diabetologie)
• 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/429077705?language=en
• 关键词: Combined; resolution; structural; functional; characterization

Ultrafiltration is a key function to maintain body homeostasis, and a major target of hereditary kidney diseases. While the structural components of the kidney filter - endothelial cells, glomerular basement membranes and slit diaphragms (SD) - have been described many years ago, the complex ultrastructure of the SD was only recently further elucidated by our multispecies platform, combining genetic modeling and cryo-EM analysis. Contrary to current concepts, we discovered that NEPH1 and NEPHRIN form a multi-layered bipartite scaffold between adjacent foot processes with only very few NEPH1/NEPHRIN trans-interactions. Structural modeling and alignment with other known immunoglobulin superfamily proteins such as TITIN suggests that the SD acts as a highly flexible, non-clogging mesh. Paradigm shifting, our model does not view the SD as a critical barrier for albumin, but rather as the gatekeeper for foot process formation, maintenance and function. Since our working hypothesis has extensive implications for hereditary and acquired kidney diseases affecting the SD, we propose to refine the current SD model by 1) functional analysis of SD permeability, combining inducible adult NEPH1/NEPHRIN KO models and single-glomeruli micro-puncture for primary urine proteome analysis, 2) high-resolution structural analysis of NEPH1/NEPHRIN using an integrative structural biology approach, by combining X-ray crystallography, electron microscopy and complementary methods, 3) genetic analysis, by modeling of human disease-causing NEPHRIN mutations based on the structural data and by screening the functionality of mutated molecules in 3D cell culture models. Collectively, these studies will provide novel insight into the structure and function of the SD, thereby facilitating therapeutic approaches to ameliorate hereditary and acquired glomerular diseases.

超滤是维持机体内环境稳定的重要功能，也是遗传性肾病的主要靶点。虽然肾脏滤过器的结构成分-内皮细胞，肾小球基底膜和狭缝隔膜（SD）-多年前就已被描述，但SD的复杂超微结构最近才通过我们的多物种平台，结合遗传建模和cryo-EM分析进一步阐明。与目前的概念相反，我们发现NEPH 1和NEPHRIN在相邻的足突之间形成多层二分支架，只有很少的NEPH 1/NEPHRIN反式相互作用。结构建模和与其他已知的免疫球蛋白超家族蛋白如TITIN的比对表明，SD作为一个高度灵活的，不堵塞的网。范式转变，我们的模型不认为SD是白蛋白的关键屏障，而是作为足突形成，维持和功能的守门人。由于我们的工作假设对影响SD的遗传性和获得性肾脏疾病具有广泛的影响，我们建议通过1）SD渗透性的功能分析，结合诱导型成人NEPH 1/NEPHRIN KO模型和单肾小球微穿刺进行初级尿蛋白质组分析，2）使用整合结构生物学方法对NEPH 1/NEPHRIN进行高分辨率结构分析，通过结合X射线晶体学、电子显微镜和互补方法，3）遗传分析，通过基于结构数据的人类致病性NEPHRIN突变的建模和通过在3D细胞培养模型中筛选突变分子的功能。总的来说，这些研究将为SD的结构和功能提供新的见解，从而促进改善遗传性和获得性肾小球疾病的治疗方法。