Exosites in Matrix Metalloproteinase Localization and Activity

基质金属蛋白酶定位和活性中的外部位点

• 批准号: 8323317
• 负责人: Steven R Van Doren
• 金额: $ 36.21万
• 依托单位: UNIVERSITY OF MISSOURI-COLUMBIA
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-06-01 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8323317
• 关键词: Active Sites; Adverse effects; Affinity; Aneurysm; Antibodies; Aorta; Arterial Fatty Streak; Arteries; Arthritis; Asthma; Atomic Force Microscopy; Basement membrane; Binding; Bioinformatics; Biological Assay; Blood capillaries; Breast; Carbohydrates; Cardiovascular Diseases; Catalytic Domain; Cell surface; Cells; Chronic Obstructive Airway Disease; Clinical; Colon; Complex; Connective Tissue; Consult; Development; Diagnostics Research; Disease; Disease Progression; Elastases; Elastin; Employee Strikes; Endothelial Cells; Enzymes; Epithelial Cells; Epitopes; Equilibrium; Foam Cells; Fostering; Future; Glycosaminoglycans; Goals; Head; Inflammation; Inorganic Sulfates; Intestines; Invaded; Investigation; Location; Lung; Lung diseases; Malignant Neoplasms; Malignant neoplasm of lung; Maps; Matrilysin; Matrix Metalloproteinases; Membrane; Membrane Lipids; Membrane Proteins; Micelles; Mutagenesis; Myocardial Infarction; NMR Spectroscopy; Natural Immunity; Nature; Pancreas; Pathologist; Peptide Hydrolases; Peripheral; Pharmaceutical Preparations; Phospholipids; Process; Proteins; Proteoglycan; Reporting; Role; Site; Skin; Small Intestines; Solutions; Specificity; Stomach; Stroke; Structure; Surface; Surface Plasmon Resonance; Testing; Therapeutic; Therapeutic Uses; Tissues; Tube; Twin Multiple Birth; Unspecified or Sulfate Ion Sulfates; Work; antimicrobial peptide; capillary; cholesteryl sulfate; crosslink; human MMP14 protein; insight; macrophage; matrix metalloproteinase 12; molecular recognition; neoplastic cell; novel; polysulfated glycosaminoglycan; prevent; promatrilysin; tool; tumor

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.

描述（由申请人提供）：最近已经确定了基质金属蛋白酶（MMP）的组织和细胞特异性作用位点。但是，MMP如何与这些战略站点相关联，尚不清楚剥削。 MMP-12位于发炎肺激活的巨噬细胞的表面上，并嵌入在弹性蛋白原纤维中被消化在主动脉中形成的动脉瘤中。在肿瘤细胞，肠上皮细胞的表面以及巨噬细胞和弹性蛋白原纤维之间发现了MMP-7。在细胞上，MMP-7与硫酸胆固醇和蛋白聚糖的硫酸胆固醇和重度硫酸糖胺聚糖（GAG）链。 MT1-MMP在肿瘤细胞的入侵前面发现，内皮细胞通过基质或基底膜迁移。该项目将测试支持（1）对弹性蛋白的亲和力和活动的各种外部材料的新假设，（2）与膜脂质的关联，以及（3）与硫酸化糖的关联和激活。 NMR结构和动力学方法将应用于解决方案中的代表性组件。定向诱变将用于测试催化效率和关联中的功能作用。 AIM 1将评估外粒材料（一些远程）和稳定性调节残基在弹性蛋白降解中的作用和范围，MMP-12和-7。 MMP-7的弹性酶活性的研究将使用MMP-12和-3最近证明的分子识别和生物物理方法进行。 MMP-7识别新1-弹性衍生物的识别将由Bindsight指导，Bindsight结合了生物信息学和NMR，以发现相互作用的特异性。将MMP-7在活动（弹性蛋白），折叠稳定性和MilliSec动力学之间取消的平衡与其上一时期表征的55％相同的MMP-12和-3对应物的平衡将进行比较。在AIM 2中，将通过MMP-Micelle界面的NMR映射和NMR结构测定两个胶束复合物与现在已知已知结合细胞表面的MMP的NMR结构测定，将测试与脂质膜相关的MMP催化结构域的新生物信息学预测。 AIM 3将研究插孔如何加速PROMMP-7的激活，测试共定位的三分子激活与双分子“变构”激活的假设。硫酸化的糖竞争测定法（按表面等离子体共振）和激活测定法将铺平道路。原子力显微镜和流体动力学将阐明使用PROMMP-7和MMP-7的糖含量的性质。通过NMR光谱法，将选择一个复合物进行详细的结构表征。该项目将深刻扩展MMP对分子识别的看法，以及新型相互作用如何将MMP活性引导到心血管和肺部疾病和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表位和位点，其中可以将未来MMP特异性抗体的目标靶向在诊断，研究和治疗用途中干扰这些关联。