Exosites in Matrix Metalloproteinase Localization and Activity

描述（由申请人提供）：最近已经确定了基质金属蛋白酶（MMPs）的组织和细胞特异性作用位点。然而，MMPs如何与这些战略地点联系在一起尚不清楚，这阻碍了开发。MMP-12位于炎症肺的活化巨噬细胞表面，并嵌入在主动脉形成的动脉瘤中被消化的弹性蛋白原纤维中。MMP-7存在于肿瘤细胞表面、肠上皮细胞表面以及巨噬细胞和它们正在消化的弹性蛋白原纤维之间。在细胞上，MMP- 7与胆固醇硫酸盐和重硫酸化糖胺聚糖（GAG）蛋白聚糖链结合。MT1-MMP存在于肿瘤细胞的侵袭前和通过基质或基底膜迁移的内皮细胞上。该项目将测试各种外源的新假设，这些假设支持(1)与弹性蛋白的亲和力和活性，(2)与膜脂的关联，以及(3)与硫酸糖的关联和活化。核磁共振结构和动力学方法将应用于溶液中的代表性组件。定向诱变将用于测试催化效率和关联的功能角色。目的1将评估外源位点（一些远程）和稳定性调节残基在MMP-12和-7降解弹性蛋白中的作用和范围。对MMP-7弹性酶活性的研究将使用最近在MMP-12和-3中证明的分子识别和生物物理方法进行。探索新的1-弹性蛋白衍生物的MMP-7识别将在BINDSIght的指导下进行，BINDSIght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（基于弹性蛋白）、折叠稳定性和毫秒动力学之间取得的平衡将与前一时期55%相同的MMP-12和-3进行比较。在Aim 2中，新的生物信息学预测MMP催化结构域与脂质膜相关，将通过MMP-胶束界面的核磁共振图谱和已知结合细胞表面的MMPs胶束复合物的核磁共振结构测定来测试。目的3将研究GAGs如何加速promp -7的激活，测试共定位三分子激活和双分子“变构”激活的假设。硫酸糖竞争分析（通过表面等离子体共振）和活化分析将铺平道路。原子力显微镜和流体动力学将阐明与proMMP-7和MMP-7的糖配合物的性质。将选择一个配合物，通过核磁共振光谱进行详细的结构表征。该项目将深刻地拓宽mmmp分子识别的观点，以及新的相互作用如何详细地指导MMP活性到心血管、肺部疾病和癌症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表位和位点，未来的mmp特异性抗体可以靶向干预这些关联，用于诊断、研究和治疗。

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. PUBLIC HEALTH RELEVANCE: Cardiovascular diseases, cancer, lung diseases, and arthritis have a destructive excess of inflammation that is fostered by enzymes called MMPs that cut connective tissue and proteins near cell surfaces. This project will identify working surfaces of MMPs that help them (1) attack the elastic fibrils of arteries, lungs, and skin and (2) associate with cell surface membranes and saccharides to attack proteins strategically from there. These discoveries will suggest where to direct future antibodies to interfere in these processes for clinical insight and possible tools for treatment.