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）的最常见原因，其特征在于肾小球硬化，即肾小球细胞外基质中IV型胶原蛋白的早期和进行性异常积聚。尽管其对公共卫生具有重要意义，但导致肾小球纤维化的分子机制尚未得到很好的理解。本提案的总体目标是研究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中的病理机制至关重要。胶原蛋白IV网络通过三螺旋原聚体的寡聚化自组装，所述三螺旋原聚体通过C末端结构域的二聚化形成NC 1六聚体，和N末端结构域的四聚化形成7S十二聚体。我们最近发现，NC 1六聚体是共价交联的新型硫亚胺（-S=N-）的化学键，其在生物分子中的第一种。随后，我们发现，交联是必不可少的功能，它是由过氧化物酶，BM嵌入的过氧化物酶。在7S十二聚体中，在三螺旋原聚体的相对端，非二硫键交联的性质和位置以及催化其形成的酶的身份是未知的。我们假设在由赖氨酰氧化酶家族的成员形成的7S十二聚体中存在赖氨酰衍生的交联。这三个具体目标针对胶原IV结构和组装的基本问题，以及赖氨酰氧化酶和可能的其他胞外酶在此过程中的贡献。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十二聚体中非二硫键交联的性质和位置。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，我们将鉴定在7S十二聚体中形成非二硫键交联的酶。我们将采用我们已经开发的重组系统，以便在网络组装过程中轻松监测IV型胶原交联。在我们的最后一个目标3中，我们将确定赖氨酰氧化酶催化的肾小球IV型胶原网络的交联。这三个目标的实现将促进对健康和疾病中IV型胶原蛋白网络的组装、功能和功能障碍的理解。这一新的信息可能导致发现新的分子靶点，用于开发治疗DN的新的替代疗法。

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.