MMP1 & MMP9: Mechanism of Activation & Substrate Binding

描述(由申请人提供)： 金属蛋白酶(MMPs)在组织形态发生、创面愈合、血管生成、子宫退缩和骨吸收等过程中发挥着重要作用。恶性细胞利用MMPs促进肿瘤的侵袭和转移。本实验室的长期目标是(1)了解MMPs催化细胞外蛋白分解的空间调控的分子机制；(2)阐明MMPs在ECM的生理和病理中的具体作用。组织中酶活性的空间调控机制包括基质金属蛋白酶与细胞表面的结合、原位激活以及与细胞外基质底物的独特相互作用方式。我们的最新结果代表了这些领域的重大进展，即我们的实验坚定地证明了MMPs利用显著的表面扩散机制来进行衬底相互作用。基质金属蛋白酶-1、-2和9可在基质表面横向扩散，无明显解离现象。基质金属蛋白酶-2的侧向扩散需要血凝蛋白的C-末端结构域。前体-基质金属蛋白酶-2和-9分别与抑制剂1TMP-2和-1形成络合物不干扰扩散过程，II。最有趣的是，我们已经证明，激活的基质金属蛋白酶-1是一种新型的基于扩散的、不依赖于ATP的马动酶，它是由其底物胶原蛋白III的蛋白分解驱动的。我们证明，原-基质金属蛋白酶-1，而不是激活的基质金属蛋白酶-1，通过原多肽结构域特异性地结合到细胞表面，导致原位激活；前-基质金属蛋白酶-9与明胶或IV型胶原底物的结合足以使酶获得部分活性，而其前肽保持不变。这些发现对于从机制上理解MMPs在控制细胞扩散和运动中的作用具有深远的意义。我们提出了一种灵活的、可移动的细胞表面-基质界面，允许移动的细胞控制ECM重塑。我们已经确定了此界面中的几个关键交互。在这里，我们建议研究横向扩散和原位激活的机制，以及两者之间可能的联系。这些研究将为阐明细胞表面-底物界面的功能成分和对基质金属蛋白酶-1分子马达工作方式的生物物理研究奠定基础。特定的 目的1建议通过生物化学定义细胞表面相互作用来扩展对基质金属蛋白酶-1细胞表面结合和激活的研究。特殊目标2和3建议从明胶酶模型开始，定义明胶酶与底物的生化相互作用，并研究这些相互作用如何实现过程扩散和酶激活。

DESCRIPTION (provided by applicant): Metalloproteases (MMPs) play a pivotal role in tissue remodeling during morphogenesis, wound healing, angiogenesis, uterine involution and bone resorption. Malignant cells exploit MMPs to promote tumor invasion and metastasis. The long-term goals of this laboratory are (1) to understand the molecular mechanisms of spatial regulation of extracellular proteolysis catalyzed by MMPs and (2) to elucidate the specific role of MMPs in the physiology and pathology of the ECM. The mechanisms of spatial control of enzymatic activity in tissues involve MMP binding to the surface of resident cells, in situ activation, and a unique mode of interaction with the underling ECM substrata. Our most recent results represent a significant progress in these areas, i. Our experiments firmly establish that MMPs utilize a remarkable surface diffusion mechanism for substrate interaction. MMP-1, -2 and 9 can diffuse laterally on the substrate surface without noticeable dissociation. The lateral diffusion of MMP-2 requires the hemopexin C-terminal domain. Complex formation of pro-MMP-2 and -9 with inhibitors 1TMP-2 and -1 respectively does not interfere with the diffusion process, ii. Most interestingly, we have shown that activated MMP-1 is a novel type of diffusion-based, ATP-independent motor enzyme that is driven by proteolysis of its substrate, collagen, iii. we demonstrate that pro- MMP-1, but not activated MMP-1, specifically binds to the cell surface via the pro-peptide domain causing activation in situ; iv. Binding of pro-MMP-9 to gelatin or type IV collagen substrates is sufficient for the enzyme to acquire partial activity with its pro-peptide still intact. These findings have profound implications for mechanistic understanding of the role of MMPs in controlled cell spreading and motility. We propose a flexible, mobile cell surface-substrata interface that allows for controlled ECM remodeling by the moving cells. We have already identified several of the key interactions in this interface. Here we propose to examine the mechanisms of lateral diffusion and in situ activation, and the possible linkage between the two. These studies will form the basis for elucidating the functional components of the cell surface-substrata interface and for biophysical studies on workings of the MMP-1 molecular motor. Specific Aim 1 proposes to extend the MMP-1 cell surface binding and activation studies by biochemically defining the cell surface interaction. Specific Aims 2 and 3 propose to start with the gelatinase model to define biochemical interactions of gelatinases with substrate and to examine how these can enable processive diffusion and enzyme activation.