Collagen IV Networks of Basement Membranes

描述（由申请人提供）：糖尿病肾病（DN）是西方世界终末期肾病（ESRD）最常见的病因，其特征是肾小球硬化，肾小球细胞外基质中早期和进行性的IV型胶原异常积累。尽管其具有公共卫生重要性，但导致肾小球纤维化的分子机制尚不清楚。本研究的总体目标是研究IV型胶原网络组装中的关键分子事件，这对于理解糖尿病肾病肾小球硬化发展的病理机制至关重要。胶原IV网络通过c端结构域的二聚化，形成NC1六聚体，以及n端结构域的四聚化，形成7S十二聚体，通过三螺旋原聚体的寡聚化进行自组装。我们最近发现，NC1六聚体是由新型亚胺（- s =N-）化学键共价交联的，这在生物分子中是第一次。随后，我们发现交联对功能至关重要，它是由过氧化物酶（一种嵌入bm的过氧化物酶）形成的。在7S十二聚体中，在三螺旋原聚体的另一端，非二硫交联的性质和位置以及催化其形成的酶的身份是未知的。我们假设在赖氨酸氧化酶家族成员形成的7S十二聚体中存在赖氨酸衍生的交联。这三个特定的目标针对的是关于IV型胶原结构和组装的基本问题，以及赖氨酸氧化酶和其他可能的细胞外酶在这一过程中的作用。在目标1中，我们将通过使用最先进的质谱（MS）和核磁共振（NMR）分析来确定7S十二聚体中非二硫交联的性质和位置。这些结果将为构建7S十二聚体的三维模型提供分子框架。在目标2中，我们将确定在7S十二聚体中形成非二硫交联的酶。我们将采用我们开发的重组系统，以方便地监测网络组装过程中的胶原IV交联。在我们最后的目标3中，我们将鉴定肾小球中存在的赖氨酸氧化酶催化的胶原IV网络上的交联。这三个目标的实现将促进对IV型胶原蛋白网络在健康和疾病中的组装、功能和功能障碍的理解。这一新信息可能会导致发现新的分子靶点，以开发新的替代疗法来治疗DN。

DESCRIPTION (provided by applicant): Diabetic nephropathy (DN), the most common cause of end-stage renal disease (ESRD) in the western world, is characterized by glomerulosclerosis, an early and progressive abnormal accumulation of collagen IV in the glomerular extracellular matrix. Despite its public health importance, the molecular mechanism that leads to fibrosis in the glomerulus is not well understood. The overall goal of this proposal is to study key molecular events in collagen IV network assembly important for the understanding of the pathological mechanism implicated in the development of glomerulosclerosis in diabetic nephropathy. Collagen IV networks self-assemble by the oligomerization of triple helical protomers through dimerization of C-terminal domains, forming NC1 hexamers, and tetramerization of N-terminal domains, forming 7S dodecamers. We recently discovered that the NC1 hexamer is covalently crosslinked by novel sulfilimine (-S=N-) chemical bonds, the first of its kind in biomolecules. Subsequently, we discovered that the crosslink is essential for function and that it is formed by peroxidasin, a BM-embedded peroxidase. In the 7S dodecamer, at the opposite end of the triple-helical protomer, the nature and location of non-disulfide crosslinks and identity of the enzyme(s) that catalyzes their formation are unknown. We hypothesize there are lysyl-derived crosslinks in the 7S dodecamer formed by a member of the lysyl oxidase family. The three specific aims target fundamental questions about collagen IV structure and assembly, and the contribution of lysyl oxidases and possibly other extracellular enzymes in this process. In Aim 1, we will determine the nature and location of non-disulfide crosslinks in the 7S dodecamer by using state-of-the-art mass spectrometry (MS) and nuclear magnetic resonance (NMR) analyses. The results will provide molecular framework for constructing a three- dimensional model for the 7S dodecamer. In aim 2 we will identify the enzyme(s) that forms non-disulfide crosslinks in 7S dodecamer. We will employ a recombinant system that we have developed to easily monitor collagen IV crosslinking during network assembly. In our last aim 3, we will identify lysyl oxidase-catalyzed crosslinks on collagen IV networks present in kidney glomeruli. The achievement of these three aims will advance the understanding of the assembly, function and dysfunction of collagen IV networks in health and disease. This new information may lead to the discovery of novel molecular targets for the development of new alternative therapies for the treatment of DN.