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.
Diabetes is the leading cause of end-stage renal disease (ESRD) in the U.S. This work will improve our knowledge of how patients with diabetes develop kidney fibrosis and generate new targets for its treatment.
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