Studies on the Structure of Basement Membranes

基底膜结构的研究

基本信息

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
项目类别：
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

项目摘要

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 基因中的数百种变异可导致广泛的肾小球病这些基因编码影响肾小球基底膜（GBM）的功能。 IV 型胶原蛋白 α345 支架，GBM 的主要成分，Goodpasture 的自身抗原 (GP) 自身免疫性疾病，以及阿尔波特综合征和其他遗传性肾小球病中的蛋白质突变。疾病已经并将继续充当解开分子结构之谜的先锋 α345 支架和获得性和遗传性肾小球病的致病机制。总体假设是：IV 型胶原蛋白 α345 支架束缚大分子形成超分子复合物和支架的扰动导致肾小球病的四个具体目标解决了关键问题。根据我们之前和最近的发现定义的未解答的问题。目标 1：α345NC1。确定α345NC1六聚体的原子结构和GP表位的机制。 GP 自身抗原 α345NC1 六聚体的结构尚不清楚。非免疫原性 345NC1 六聚体、EA 和 EB 区域四级结构的扰动发生构象变化，形成致病性 GP 新表位目标 2：α3 Zurich 突变。确定 α3 Zurich 突变对 GP 表位形成的影响我们发现 α3NC1 中存在突变。与第一例家族性 GP 疾病相关的域，为触发提供遗传证据我们认为突变会导致 EA 和 EB 区域的结构扰动。 α3NC1，有助于 GP 表位的呈现。目的 3. 确定氯环的作用。 IV 型胶原蛋白 α345 支架的组装和 GP 表位的形成中的氯化物。 α121支架的组装和功能已经成功研究了40多年，我们的知识关于 α345 支架的情况仍然不清楚。我们证明氯化物浓度是 GP 的关键因素。我们追求 α345NC1 六聚体的组装，其稳定性和 GP 反应性是目标 4：α121 超分子复合物的表征。我们发现了基底膜内 α121 IV 型胶原蛋白的复合物。涂有蛋白聚糖的α121支架是基底膜的潜在核心特征。我们寻求 IV 型胶原蛋白 α121 支架束缚大分子，形成独特的超分子实现基底膜组装的复合物将产生新的见解。 GP 疾病的病因学以及 IV 型胶原蛋白支架的结构和组装，形成了一个框架开发 GBM 疾病的新治疗策略。