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、伤口愈合、血管生成、子宫复旧和骨吸收过程中起着关键作用。恶性细胞利用MMPs促进肿瘤侵袭和转移。本实验室的长期目标是：（1）了解MMPs催化的细胞外蛋白水解的空间调节的分子机制;（2）阐明MMPs在ECM的生理学和病理学中的特定作用。组织中酶活性的空间控制机制涉及MMP与驻留细胞表面的结合、原位活化以及与下层ECM基质的独特相互作用模式。我们最近的结果代表了这些领域的重大进展，即。我们的实验坚定地建立，基质金属蛋白酶利用一个显着的表面扩散机制基板相互作用。MMP-1、MMP-2和MMP-9可以在基底表面上横向扩散而没有明显的解离。MMP-2的侧向扩散需要血红素结合蛋白C-末端结构域。pro-MMP-2和-9分别与抑制剂ITMP-2和-1的复合物形成不干扰扩散过程，ii.最有趣的是，我们已经表明，激活MMP-1是一种新型的扩散为基础的，ATP-独立的马达酶，是由其底物，胶原蛋白，iii.我们证明了MMP-1原，而不是活化的MMP-1，通过引起原位活化的前肽结构域特异性结合细胞表面; iv. MMP-9原与明胶或IV型胶原底物的结合足以使酶获得部分活性，其前肽仍然完整。这些发现对于理解MMPs在受控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，移动的细胞表面基质接口，允许控制ECM重塑的移动细胞。我们已经确定了这个接口中的几个关键交互。在这里，我们建议检查的机制，横向扩散和原位激活，以及两者之间可能存在的联系。这些研究将为阐明细胞表面-基质界面的功能组分和MMP-1分子马达的工作机制的生物物理研究奠定基础。具体 目的1提出通过生物化学定义细胞表面相互作用来扩展MMP-1细胞表面结合和活化研究。具体目标2和3建议从明胶酶模型开始，以定义明胶酶与底物的生物化学相互作用，并研究这些相互作用如何使进行性扩散和酶激活成为可能。

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.