Studies on the Structure of Basement Membranes

基底膜结构的研究

• 批准号: 8140518
• 负责人: BILLY GERALD HUDSON
• 金额: $ 66.64万
• 依托单位: VANDERBILT UNIVERSITY
• 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 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8140518
• 关键词: Accounting; Achievement; Affect; Affinity; Antibodies; Architecture; Autoantibodies; Autoimmune Diseases; Back; Bacterial Adhesins; Basement membrane; Basic Science; Binding; Binding Sites; Biochemical; Caenorhabditis elegans; Cell Culture System; Cell Culture Techniques; Chemicals; Chromatography; Chronic; Code; Collagen; Collagen Type IV; Complex; Crystallography; Diabetes Mellitus; Diabetic Nephropathy; Disease; Disulfides; Electron Microscopy; End stage renal failure; Enzymes; Epitopes; Extracellular Matrix; Filtration; Goals; Goodpasture Syndrome; Health; Hereditary nephritis; Heterogeneity; High Pressure Liquid Chromatography; Hydroxylysine; Immune; Immunochemistry; Integrins; Interferometry; Kidney; Kidney Diseases; Kinetics; Knowledge; Location; Mass Spectrum Analysis; Methionine; Molecular; Molecular Conformation; Molecular Structure; NMR Spectroscopy; Nature; Pathogenesis; Patients; Play; Production; Proteins; Protomer; Pyelonephritis; Quality of life; RNA Interference; Recombinants; Renal glomerular disease; Resolution; Role; Shadowing (Histology); Site; Site-Directed Mutagenesis; Specificity; Spectrometry; Structure; Structure-Activity Relationship; Substrate Specificity; Technology; Usher Syndrome; X-Ray Crystallography; analytical ultracentrifugation; crosslink; disulfide bond; fast protein liquid chromatography; glomerular basement membrane; glomerulosclerosis; immunogenic; insight; molecular recognition; monomer; novel; public health relevance

DESCRIPTION (provided by applicant): Diseases of the glomerulus, the filtering unit of the kidney, account for over 60% of all cases of end-stage renal disease, a major health problem worldwide. Among them are diabetic nephropathy, Goodpasture (GP) syndrome and Alport syndrome, which affect the collagen IV network of the glomerular basement membrane. The five Specific aims target fundamental questions about collagen IV structure and assembly, and role in Goodpasture autoimmune disease. The overall goal is to gain insight, at the atomic level, into mechanisms of molecular recognition that underlie structure-function relationships. Aim 1: To Characterize the formation of sulfilimine bonds. We discovered a novel sulfilimine bond (S=N) between methionine and hydroxylysine in the NC1 domains of collagen IV. This bond stabilizes the networks and plays a key role in conferring immune privilege to immunogenic sites that become reactive in Goodpasture autoimmune disease. We hypothesize that S=N bond formation is catalyzed by an enzyme in the extracellular matrix. Aim 2: To determine mechanisms for chain-specific assembly of collagen IV networks via NC1 domain recognition modules. We have established that the NC1 domains play a key role as recognition modules. We hypothesize that the code for assembly resides primarily within two distinct NC1 motifs. Aim 3: To characterize the quaternary structure of the 7S domain of collagen IV networks. The 7S domain, located at the N-terminus of protomers is critical for the assembly of networks. It contains binding sites for usherin, a defective protein in Usher syndrome, for bacterial Dr adhesins in chronic pyelonephritis, and for integrins. We hypothesize that the quaternary structure of the 7S dodecamer is stabilized by both non-disulfide (sulfilimine and lysylaldehyde-derived crosslinks) and disulfide bonds.Aim 4: To characterize the heterogeneity of 13 GP and 15 GP autoantibodies. We have recently determined that the a5 Gp autoantibodies, in addition to the well-known a3 GP autoantibodies are pathogenic. The current knowledge about their molecular diversity and the structure of GP-autoantibody is very limited. We hypothesize that these pathogenic GP autoantibodies are clonally restricted (oligoclonal). Aim 5: To determine the molecular structure of complete GP neoepitopes in the 13NC1 monomer. We identified four homologous hotspot regions within 13NC1 and 15NC1 monomers, which are important for GP antibody binding. We hypothesis that complete epitopes for 13NC1-specific GP autoantibodies encompass the EA/EB regions and but extend outside of these regions and are formed upon the change in hexamer conformation. The achievement of these aims will result in novel insights into mechanisms that can serve as a platform for developing treatments against glomerulosclerosis in patients with GP syndrome, diabetes mellitus, and other glomerular diseases, thus positively affecting the quality of life for millions of people. PUBLIC HEALTH RELEVANCE: Kidney diseases such as diabetic nephropathy, Goodpasture syndrome, and Alport syndrome directly affect the normal functioning of glomerular basement membrane (GBM). We study the molecular architecture and functioning of the collagen IV networks, which are the principal components of the renal filtration barrier. The basic science knowledge of the structure and assembly of collagen IV networks allow us to have a better understanding of the pathogenesis of kidney diseases.

描述（由申请人提供）：肾小球（肾脏的过滤单位）疾病占所有终末期肾病病例的 60% 以上，这是世界范围内的一个主要健康问题。其中包括糖尿病肾病、Goodpasture（GP）综合征和Alport综合征，这些综合征会影响肾小球基底膜的IV型胶原蛋白网络。五个具体目标针对有关 IV 型胶原蛋白结构和组装以及在 Goodpasture 自身免疫性疾病中的作用的基本问题。总体目标是在原子水平上深入了解构成结构-功能关系的分子识别机制。目标 1：表征硫亚胺键的形成。我们在 IV 型胶原蛋白的 NC1 结构域中的蛋氨酸和羟赖氨酸之间发现了一种新的硫亚胺键 (S=N)。这种键稳定了网络，并在赋予古德帕斯彻自身免疫性疾病中反应性的免疫原位点免疫特权方面发挥着关键作用。我们假设 S=N 键的形成是由细胞外基质中的酶催化的。目标 2：确定通过 NC1 域识别模块进行胶原 IV 网络链特异性组装的机制。我们已经确定 NC1 域作为识别模块发挥着关键作用。我们假设组装代码主要位于两个不同的 NC1 基序内。目标 3：表征 IV 型胶原蛋白网络 7S 结构域的四级结构。位于原聚体 N 末端的 7S 结构域对于网络的组装至关重要。它包含 Usherin（Usher 综合征中的一种缺陷蛋白）、慢性肾盂肾炎中的细菌 Dr 粘附素以及整合素的结合位点。我们假设 7S 十二聚体的四级结构通过非二硫键（硫亚胺和赖氨醛衍生的交联）和二硫键稳定。目标 4：表征 13 GP 和 15 GP 自身抗体的异质性。我们最近确定，除了众所周知的 a3 GP 自身抗体之外，a5 Gp 自身抗体也具有致病性。目前对其分子多样性和 GP 自身抗体结构的了解非常有限。我们假设这些致病性 GP 自身抗体受到克隆限制（寡克隆）。目标 5：确定 13NC1 单体中完整 GP 新表位的分子结构。我们在 13NC1 和 15NC1 单体内鉴定了四个同源热点区域，这对于 GP 抗体结合很重要。我们假设 13NC1 特异性 GP 自身抗体的完整表位包含 EA/EB 区域，但延伸到这些区域之外，并在六聚体构象发生变化时形成。这些目标的实现将带来对机制的新见解，这些机制可以作为开发针对 GP 综合征、糖尿病和其他肾小球疾病患者肾小球硬化治疗的平台，从而对数百万人的生活质量产生积极影响。 公众健康相关性：糖尿病肾病、古德帕斯彻综合征和阿尔波特综合征等肾脏疾病直接影响肾小球基底膜 (GBM) 的正常功能。我们研究 IV 型胶原蛋白网络的分子结构和功能，它是肾滤过屏障的主要组成部分。 IV型胶原网络的结构和组装的基础科学知识使我们能够更好地了解肾脏疾病的发病机制。