Exosites in Matrix Metalloproteinase Localization and Activity

描述(由申请人提供)：最近发现了基质金属蛋白酶(MMPs)的组织和细胞特异性作用部位。然而，MMP如何与这些战略地点关联仍不清楚，削弱了剥削。基质金属蛋白酶-12位于炎症肺中活化的巨噬细胞表面，包埋在主动脉形成的动脉瘤中正在被消化的弹性蛋白纤维中。在肿瘤细胞、肠上皮细胞及其正在消化的巨噬细胞和弹性蛋白纤维之间的表面发现了基质金属蛋白酶-7。在细胞上，基质金属蛋白酶-7与胆固醇硫酸盐和蛋白多糖链的大量硫酸盐糖胺多聚糖(GAG)相联系。MT1-MMP位于肿瘤细胞侵袭前沿，内皮细胞通过基质或基底膜迁移。这个项目将测试支持(1)弹性蛋白的亲和力和活性，(2)与膜脂结合，以及(3)与硫酸盐糖结合和激活的各种外生体的新假说。核磁共振结构和动力学方法将应用于溶液中具有代表性的组装体。定向突变将被用来测试催化效率和相关性中的功能作用。目标1将评估外显子(一些遥远的)和稳定性调节残基在基质金属蛋白酶-12和-7降解弹性蛋白中的作用和范围。对基质金属蛋白酶-7的弹性蛋白酶活性的研究将使用最近用基质金属蛋白酶-12和-3演示的分子识别和生物物理方法进行。探索基质金属蛋白酶-7对新的1-弹性蛋白衍生物的识别将由BINDSIight指导，该公司将生物信息学和核磁共振相结合，以发现相互作用的特异性。在活性(基于弹性蛋白)、折叠稳定性和毫秒动力学之间的权衡中，将比较基质金属蛋白酶-7在活性(弹性蛋白)、折叠稳定性和毫秒动力学之间的平衡，并将其与前一时期表征的55%相同的基质金属蛋白酶-12和-3相比较。在目标2中，新的生物信息学预测将通过对基质金属蛋白酶-胶束界面的核磁共振图谱和两种已知的与基质金属蛋白酶结合的胶束复合体的核磁共振结构测定来检验与脂膜相关的基质金属蛋白酶催化结构域。目的3将研究GAG如何加速原基质金属蛋白酶-7的激活，验证三分子激活与双分子“变构”激活共定位的假说。硫酸糖竞争分析(通过表面等离子激元共振)和活化分析将为此铺平道路。原子力显微镜和流体力学将阐明糖与原基质金属蛋白酶-7和基质金属蛋白酶-7的络合物的性质。将选择一个配合物通过核磁共振波谱进行详细的结构表征。该项目将深刻地拓宽MMPs分子识别的视野，以及新的相互作用如何详细地将MMPs的活性导向心血管疾病、肺部疾病和癌症中的细胞表面和弹性蛋白纤维。结合模式将揭示独特的功能表位和位置，未来的基质金属蛋白酶特异性抗体可以在诊断、研究和治疗应用中针对这些关联进行干扰。

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.