Collagen IV Networks of Basement Membranes

描述(申请人提供)：糖尿病肾病(DN)是西方世界终末期肾病(ESRD)最常见的原因，其特征是肾小球硬化，即肾小球细胞外基质中IV型胶原的早期和进行性异常积聚。尽管它对公众健康很重要，但导致肾小球纤维化的分子机制尚不清楚。这项建议的总体目标是研究IV型胶原网络组装中的关键分子事件，这对于了解糖尿病肾病肾小球硬化的发病机制具有重要意义。IV型胶原通过C-末端结构域的二聚化、NC1六聚体的形成和N-末端结构域的四聚形成7S十二聚体，通过三螺旋结构域的寡聚自组装成IV型胶原网络。我们最近发现，NC1六聚体通过新型的磺胺(-S=N-)化学键共价交联，这在生物分子中还是第一次。随后，我们发现这种交联物是功能所必需的，而且它是由BM包埋的过氧化物酶过氧化物酶形成的。在7S十二聚体中，在三螺旋原基的另一端，非二硫交联键的性质和位置以及催化它们形成的酶(S)的身份尚不清楚。我们假设在7S十二聚体中存在赖氨酰衍生的交联物，该交联物由赖氨酰氧化酶家族的一个成员形成。这三个特定的目标针对的是关于IV型胶原的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这一过程中的作用。在目标1中，我们将使用最先进的质谱分析和核磁共振分析来确定7S十二聚体中非二硫交联物的性质和位置。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交联物的酶(S)。我们将使用我们开发的重组系统，在网络组装过程中轻松监控IV型胶原的交联性。在我们的最后一个目标3中，我们将识别存在于肾小球中的IV型胶原网络上赖氨酰氧化酶催化的交联链。这三个目标的实现将促进对IV型胶原网络在健康和疾病中的组装、功能和功能的理解。这一新信息可能导致发现新的分子靶点，用于开发治疗糖尿病肾病的新的替代疗法。

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.