DETERMINANTS OF STABILITY AND INTEGRIN BINDING WITHIN COLLAGEN IV NETWORKS

在目前的赠款期间，作为本次续签的基础的三项发现： A)确定涉及两个稳定的翻译后修饰的新的交联体 IV型胶原网络；b)非胶原蛋白1(Nc1)识别结构域的鉴定 IV型胶原网络的组装；以及c)IV型胶原中整合素的新结合位点的鉴定 A1？1和a2？1整合素在调节肾脏IV型胶原稳态中的网络和关键作用 肾小球与健康和疾病。尤其重要的是，认知域现在为我们提供了 一种新的策略，首次设计组装三螺旋杂三聚体用于研究 IV型胶原蛋白网络中新的整合素结合位点。这个重建项目的中心假设是： IV型胶原网络，存在稳定这些多价配体结构的交联点，以及 与a1？1和a2？1整合素结合的不同位点。在这份续期申请中，我们将澄清 两个肾小球IV型胶原网络在分子和原子水平上的临界相互作用 整合素。为了验证这一假设，我们提出了以下目标：目标1)推导出化学物质 IV型胶原新型羟赖氨酸-蛋氨酸交联剂的结构及形成机理 网络。我们假设蛋氨酸和羟赖氨酸残基通过 该羟化反应由赖氨酰羟基酶的一种异构体催化。目标2) 定位和鉴定a1a1a2网络中新的a1？1和a2？1整合素结合位点。我们 假设IV型胶原的a1a1a2网络具有不同的ALPL和a2p1整合素结合位点。 目的3)定位并鉴定a3a4a5网络中的整合素结合位点。我们假设 A3a4a5网络中a1？1和a2？1整合素的不同结合位点可以介导特定的肾小球 功能。 我们预计，这项研究将对IV型胶原的结构基础产生新的见解 网络结合整合素a1？1和a2？1。这一关键事件对维持 肾小球功能。这些研究需要整合素、胶原、细胞和 结构生物学家。

Three discoveries were made during the current grant that serve as a basis for the present renewal: a) the identification of novel crosslinks which involve two post-translational modifications that stabilize collagen IV networks; b) identification of non-collagenous 1 (NC1) recognition domains governing the assembly of collagen IV networks; and c) identification of novel binding sites for integrins in collagen IV networks and a critical role for a1¿1 and a2¿1 integrins in modulating collagen IV homeostasis in kidney glomerulus in health and disease. Of particular importance, the recognition domains now provide us with a novel strategy to engineer, for the first time, the assembly of triple helical heterotrimers for studies of novel integrin binding sites in collagen IV networks. The central hypothesis of this renewal project is: On collagen IV networks, there are crosslink sites that stabilize the structure of these multivalent ligands, and distinct sites that confer binding of a1¿1 and a2¿1 integrins. In this renewal application, we will elucidate critical interactions at the molecular and atomic level of the two glomerular collagen IV networks and integrins. To test this hypothesis, we propose the following aims: Aim 1) To deduce the chemical structure and the mechanism of formation of the novel hydroxylysine-methionine crosslink of collagen IV networks. We hypothesize that methionine and hydroxylysine residues are covalently connected through a sulfonium structure, and that the hydroxylation is catalyzed by one isoform of lysyl hydroxylase. Aim 2) To map and characterize the novel a1¿1 and a2¿1 integrin binding sites in the a1a1a2 network. We hypothesize that a1a1a2 network of collagen IV possess distinct binding sites for alpl and a2p1 integrin. Aim 3) To map and characterize integrin binding sites in the a3a4a5 network. We hypothesize that distinct binding sites for a1¿1 and a2¿1 integrin within a3a4a5 network could mediate specific glomerular functions. We anticipate that this study will generate novel insights into the structural basis whereby collagen IV networks bind integrins a1¿1 and a2¿1. This critical event is fundamental for the maintenance of glomerular function. These studies require a collaborative effort among integrin, collagen, cell, and structural biologists.