Studies on the Structure of Basement Membranes

基底膜结构的研究

• 批准号: 10379924
• 负责人: Sergey Petrovich Budko
• 金额: $ 74.71万
• 依托单位: VANDERBILT UNIVERSITY MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-09-01 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10379924
• 关键词: Achievement; Address; Affect; Animal Experiments; Antibodies; Architecture; Atomic Force Microscopy; Autoantigens; Autoimmune Diseases; Basement membrane; Binding; Biological Assay; COL4A3 gene; CRISPR/Cas technology; Cell Culture Techniques; Chloride Ion; Chlorides; Circular Dichroism Spectroscopy; Collagen Type IV; Complex; Deuterium; Development; Diabetic Nephropathy; Disease; Enzyme-Linked Immunosorbent Assay; Epitopes; Etiology; Genes; Genetic; Hereditary nephritis; Hydrogen; Interferometry; Kidney Diseases; Kidney Failure; Knowledge; Mass Spectrum Analysis; Modification; Molecular; Molecular Conformation; Molecular Structure; Mutate; Mutation; Other Genetics; Pathogenicity; Production; Proteins; Proteoglycan; Recombinant Proteins; Role; Scanning; Structure; Variant; X-Ray Crystallography; base; collagen scaffold; glomerular basement membrane; glomerular function; insight; macromolecule; membrane assembly; neoantigens; novel therapeutic intervention; protein expression; scaffold; simulation; transmission process

Hundreds of variants in the COL4A3, COL4A4 and COL4A5 genes cause a broad range of glomerulopathies affecting the function of the glomerular basement membrane (GBM). These genes encode the assembly of collagen IV α345 scaffolds, the major constituent of the GBM, the autoantigen in Goodpasture’s (GP) autoimmune disease, and the protein mutated in Alport syndrome and other genetic glomerulopathies. GP disease has and continues to serve as the vanguard for unlocking mysteries of the molecular structure of the α345 scaffold and pathogenic mechanisms underlying both acquired and genetic glomerulopathies. Our overarching hypothesis is: Collagen IV α345 scaffold tethers macromolecules forming supramolecular complexes and perturbation of scaffold causes glomerulopathies. Four specific aims address key unanswered questions that are defined based on our previous and recent discoveries. Aim 1: α345NC1 Hexamer. To determine the atomic structure of the α345NC1 hexamer and mechanism of GP epitopes formation. The structure of the 345NC1 hexamer, GP autoantigen, is unknown. We hypothesize that upon perturbation of quaternary structure of the non-immunogenic 345NC1 hexamer, EA and EB regions undergo conformational changes forming pathogenic GP neoepitopes. Aim 2: α3 Zurich Mutation. To determine the impact of α3 Zurich mutation on GP epitopes formation. We found a mutation in α3NC1 domain associated with the first case of familial GP disease, providing genetic evidence for a triggering mechanism. We hypothesize that the mutation causes structural perturbation of the EA and EB regions of α3NC1, which can contribute to GP epitopes presentation. Aim 3. Chloride ring. To determine role of chloride in assembly of the collagen IV α345 scaffold and formation of GP epitopes. Whereas structure, assembly and functions of the α121 scaffold has been successfully studied for over 40 years, our knowledge about the α345 scaffold remains obscure. We demonstrated that chloride concentration is a critical factor in GP antibody binding. We hypothesize that assembly of the 345NC1 hexamer, its stability and GP-reactivity is dependent on chloride ions. Aim 4: α121 Supramolecular complexes. To characterize the supramolecular complexes of α121 collagen IV within a basement membrane. We discovered a garland architecture of the α121 scaffold coated with proteoglycans. This suprastructure is a potential core feature of basement membrane. We hypothesize that collagen IV α121 scaffold tethers macromolecules forming distinct supramolecular complexes which enable basement membrane assembly. The achievement of the aims will yield new insights to the etiology of GP disease and the structure and assembly of collagen IV scaffolds, leading to a framework for development of novel therapeutic strategies for GBM diseases.

COL4A3，COL4A4和COL4A5基因的数百种变体引起广泛的肾小球病变 影响肾小球基底膜（GBM）的功能。这些基因编码组装 胶原蛋白IVα345支架，主要构成GBM，goodpasture的自动抗原（GP） 自身免疫性疾病，蛋白质在Alport综合征和其他遗传肾小球病中突变。 GP 疾病已经并且继续充当先锋，以解锁该分子结构的奥秘 α345支架和遗传性肾小球病的基础的脚手架和致病机制。我们的 总体假设是：胶原蛋白IVα345脚手架Tethers大分子形成超分子 脚手架的复合物和扰动会导致肾小球病。四个特定目标地址密钥 根据我们以前的发现和最近发现定义的未解决问题。目标1：α345NC1 六聚体。确定α345NC1六聚体的原子结构和GP表位的机理 形成。 345NC1六聚体GP自动抗原的结构尚不清楚。我们假设这是 非免疫原性345NC1六聚体，EA和EB区域的第四纪结构的扰动 经历构象变化，形成致病性GP Neoeppitopes。目标2：α3苏黎世突变。到 确定α3苏黎世突变对GP表位形成的影响。我们在α3NC1中发现了一个突变 与第一种家族性GP疾病相关的领域，为触发提供了遗传证据 机制。我们假设突变会导致EA和EB区域的结构扰动 α3NC1，可以促进GP表位的表现。瞄准3。氯环。确定 胶原蛋白IVα345支架和GP表位的形成的胶原蛋白组装中的颗粒状。而结构， α121支架的组装和功能已成功研究了40多年，我们的知识 大约α345支架仍然晦涩。我们证明氯化物浓度是GP的关键因素 抗体结合。我们假设组装345NC1六聚体，其稳定性和GP反应性是 取决于氯离子。 AIM 4：α121超分子复合物。表征超分子 基底膜内α121胶原蛋白IV的复合物。我们发现了一个花环建筑 α121支架涂有蛋白聚糖。该上建筑是基底膜的潜在核心特征。 我们假设胶原蛋白IVα121支架Tethers大分子形成不同的超分子 使地下膜组件的复合物。目标的实现将产生新的见解 GP疾病的病因以及胶原蛋白IV支架的结构和组装，导致框架 用于开发GBM疾病的新型治疗策略。