Exosites in Matrix Metalloproteinase Localization and Activity

描述（由申请人提供）：基质金属蛋白酶（MMPs）的组织和细胞特异性作用位点最近已被确定。然而，MMPs如何与这些战略位点相关联仍不清楚，阻碍了开发。MMP-12位于来自发炎的肺的活化的巨噬细胞的表面上，并且嵌入在主动脉中形成的动脉瘤中被消化的弹性蛋白原纤维中。MMP-7被发现在肿瘤细胞、肠上皮细胞的表面上，以及它们正在消化的巨噬细胞和弹性蛋白原纤维之间。在细胞上，MMP- 7与胆固醇硫酸盐和蛋白聚糖的重度硫酸化糖胺聚糖（GAG）链缔合。MT 1-MMP位于肿瘤细胞的侵袭前沿，内皮细胞通过基质或基底膜迁移。该项目将测试各种外位点的新假设，这些外位点支持（1）对弹性蛋白的亲和力和活性，（2）与膜脂质的结合，以及（3）与硫酸化的磷脂酶的结合和活化。NMR结构和动力学方法将适用于解决方案中的代表性组件。定向诱变将用于测试在催化效率和关联中的功能作用。目的1将评估MMP-12和-7在弹性蛋白降解中外切位点（一些远程）和稳定性调节残基的作用和范围。MMP-7的弹性蛋白酶活性的研究将使用最近用MMP-12和-3证明的分子识别和生物物理方法进行。探索MMP-7识别新的1-弹性蛋白衍生物将由BINDSight指导，该软件结合了生物信息学和NMR来发现相互作用的特异性。MMP-7在活性（弹性蛋白）、折叠稳定性和毫秒动力学之间的平衡将与前一时期表征的55%相同的MMP-12和-3对应物进行比较。在目标2中，新的生物信息学预测，MMP催化结构域与脂质膜将进行测试与NMR映射MMP胶束界面和NMR结构测定的两个胶束复合物与MMP现在已知的结合细胞表面。目的3将研究GAG如何加速proMMP-7的活化，测试共定位三分子活化与双分子“变构”活化的假设。硫酸化糖竞争测定（通过表面等离子体共振）和活化测定将铺平道路。原子力显微镜和流体力学将澄清与proMMP-7和MMP-7的糖复合物的性质。将选择复合物，通过NMR光谱进行详细的结构表征。该项目将深刻拓宽MMPs分子识别的观点，以及新的相互作用如何详细地将MMP活性引导到心血管和肺部疾病以及癌症中的细胞表面和弹性蛋白原纤维。结合模式将揭示独特的功能性表位和位点，未来MMP特异性抗体可以靶向这些位点以干扰诊断、研究和治疗用途中的这些关联。

DESCRIPTION (provided by applicant): Tissue- and cell-specific sites of action of matrix metalloproteinases (MMPs) have recently been identified. However, how MMPs associate with these strategic sites remains unclear, blunting exploitation. MMP-12 was located on the surface of activated macrophages from inflamed lungs and embedded in the elastin fibrils being digested in aneurysms forming in the aorta. MMP-7 was found on the surfaces of tumor cells, intestinal epithelial cells, and between macrophages and elastin fibrils they are digesting. On cells, MMP- 7 associates with cholesterol sulfate and heavily sulfated glycosaminoglycan (GAG) chains of proteoglycans. MT1-MMP is found on the invading front of tumor cells and endothelial cells migrating through the matrix or basement membranes. This project will test new hypotheses of various exosites that support (1) affinity for and activity upon elastin, (2) association with membrane lipids, and (3) association with and activation by sulfated saccharides. NMR structural and dynamics approaches will be applied to representative assemblies in solution. Directed mutagenesis will be used to test functional roles in catalytic efficiency and associations. Aim 1 will evaluate role and scope of exosites (some remote) and stability-modulating residues in elastin degradation by MMP-12 and -7. Investigation of the elastase activity of MMP-7 will proceed using the molecular recognition and biophysical approaches recently demonstrated with MMP-12 and -3. Probing of MMP-7 recognition of new 1-elastin derivatives will be guided by BINDSIght, which combines bioinformatics and NMR to discover specificity of interactions. The balance that MMP-7 strikes in tradeoffs among activity (upon elastin), folding stability, and millisec dynamics will be compared with its 55% identical MMP-12 and -3 counterparts characterized in the previous period. In Aim 2, new bioinformatics predictions that MMP catalytic domains associate with lipid membranes will be tested with NMR mapping of MMP-micelle interfaces and NMR structure determination of two micelle complexes with MMPs now known to bind cell surfaces. Aim 3 will investigate how GAGs accelerate activation of proMMP-7, testing hypotheses of co-localizing trimolecular activation vs. bimolecular "allosteric" activation. Sulfated saccharide competition assays (by surface plasmon resonance) and activation assays will pave the way. Atomic force microscopy and hydrodynamics will clarify the nature of the saccharide complexes with proMMP-7 and MMP-7. A complex will be selected for detailed structural characterization by NMR spectroscopy. The project will profoundly broaden views of molecular recognition by MMPs and how in detail the novel interactions may direct MMP activity to cell surfaces and elastin fibrils in cardiovascular and pulmonary disease and cancer. The binding modes will reveal unique functional epitopes and sites where future MMP-specific antibodies can be targeted to interfere in these associations in diagnostic, research, and therapeutic uses.