Studies on the Structure of Basement Membranes

基底膜结构的研究

• 批准号: 8717632
• 负责人: BILLY GERALD HUDSON
• 金额: $ 56.72万
• 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/8717632
• 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.

描述（由申请人提供）：肾脏过滤单元的肾小球疾病占所有终末期肾脏疾病病例的60％以上，这是全球主要的健康问题。其中包括糖尿病性肾病，Goodpasture（GP）综合征和Alport综合征，它们影响肾小球基底膜的胶原蛋白IV网络。五个特定目的针对有关胶原蛋白IV结构和组装的基本问题，以及在Goodpasture自身免疫性疾病中的作用。总体目标是在原子水平上洞悉分子识别的机制，这些机制是结构功能关系的基础。目标1：表征硫亚胺键的形成。我们在胶原蛋白IV的NC1结构域中发现了蛋氨酸和羟基胺之间的新型亚硫胺键（S = N）。这种纽带稳定了网络，并在赋予对良好的自身免疫性疾病反应性的免疫特权的免疫特权中起关键作用。我们假设S = N键形成是由细胞外基质中的酶催化的。目标2：确定通过NC1域识别模块的胶原蛋白IV网络的链特异性组装机制。我们已经确定NC1域在识别模块中起关键作用。我们假设组装代码主要驻留在两个不同的NC1图案中。目标3：表征胶原蛋白IV网络7S域的第四纪结构。位于原始人N端的7S域对于网络组装至关重要。它包含Usherin的结合位点，Usherin，Usher综合征中有缺陷的蛋白质，用于慢性肾盂肾炎中的细菌DR粘附素和整联蛋白。我们假设7S十二焦点的四元结构均被非二硫化物（硫亚胺和赖赛醛衍生的交叉链接）和二硫键稳定。MAIM4：以表征13 GP和15 GP自动抗体的异质性。我们最近确定，除了众所周知的A3 GP自身抗体外，A5 GP自身抗体是致病性的。当前有关其分子多样性和GP自动抗体结构的知识非常有限。我们假设这些致病性GP自身抗体是克隆限制的（寡聚）。 AIM 5：确定13NC1单体中完整GP NEPITOPES的分子结构。我们确定了13NC1和15NC1单体内的四个同源热点区域，这对于GP抗体结合很重要。我们假设13NC1特异性GP自身抗体包含EA/EB区域，但在这些区域之外延伸，并在六聚体构象的变化之外形成。这些目标的实现将导致对机制的新见解，该机制可以作为GP综合征，糖尿病和其他肾小球疾病的患者开发治疗肾小球硬化治疗的平台，从而积极影响数百万人的生活质量。