COLLAGENOLYSIS-DRIVEN MOLECULAR MOTORS IN CELL MIGRATION AND MATRIX REMODELING

描述(申请人提供)：金属蛋白酶(MMPs)在形态形成、伤口愈合、血管生成、子宫复旧和骨吸收的组织重塑过程中发挥关键作用。MMPs催化的细胞周围蛋白分解是决定正常组织和肿瘤组织驻留细胞微环境的重要因素。恶性细胞利用MMPs促进肿瘤的侵袭和转移。自上世纪90年代我们实验室首次报道了细胞表面的基质金属蛋白酶-2激活复合体(MT1-MMP2/TIMP-2/MMP2)以来，我们对该复合体催化的空间调控的分子机制进行了研究，以阐明MMPs在细胞-ECM相互作用中的作用。我们最近发现了MMP的一些显著特征。我们发现基质金属蛋白酶通过底物表面扩散机制与胶原蛋白相互作用。跨膜的MT1-MMP1和分泌的MMP一1、MMP2和MMP9均能在天然胶原纤维表面扩散；3.基质金属蛋白酶-1是一种独特的、以扩散为基础的、不依赖于ATP的运动酶，由胶原蛋白分解所驱动；MT1-MMP胞外部分通过类似的偏向扩散机制与胶原蛋白相互作用；基质金属蛋白酶-2 C末端结构域与抑制剂TIMP-2的复合形成不影响扩散速度。因此，整个膜连接的胶原酶分解复合体(MT1-MMP2/TIMP-2/MMP2)相对于底层的胶原底物是可移动的。这些发现对于从机制上理解MMPs在富含胶原蛋白的微环境中的细胞运动中的作用具有深远的意义。因此，我们提出了一个移动细胞表面胶原酶分解界面的模型。在这个模型中，我们假设膜系带的胶原酶分解复合体通过其沿着下面的胶原纤维定向滑动的能力来帮助细胞移动。这一机制很可能在细胞运动过程中与细胞骨架-整合素黏附装置协同协调细胞黏附-解吸事件中起重要作用。为了支持这一概念，我们现在提出证据表明，细胞表面胶原酶溶解复合体的活性有助于细胞在3D胶原组织结构中施加的力量的产生。这促使我们进一步研究细胞周围的胶原酶分解是否有助于细胞在富含胶原蛋白的微环境中产生力量。因此，我们建议：1.完成MT1-基质金属蛋白酶作为蛋白水解酶驱动的布朗棘轮的研究；2.完成MT1-基质金属蛋白酶作为蛋白分解驱动的布朗棘轮的研究。明确基质金属蛋白酶-1和/或MT1-基质金属蛋白酶中与底物表面扩散机制和III有关的结构-功能关系。为了确定细胞周围的胶原酶分解对细胞在富含胶原蛋白的微环境中强制生成的贡献，使用几种实验方法，包括2D培养中的牵引力显微镜、3D组织结构中的力测量，以及最终测量细胞可以利用含有消失酶基因的细胞(S)和胶原酶裂解位点突变的胶原蛋白对单个胶原纤维施加的力。这些研究将为进一步研究细胞如何利用细胞周围蛋白分解来协调细胞运动和组织重塑中的黏附-脱落事件提供必要的背景，而细胞运动和组织重塑是转移性肿瘤侵袭的关键过程。 与公共健康相关：基质金属蛋白酶(MMPs)是一组特殊的酶，具有独特的催化细胞外基质成分(如胶原)周转的能力。在形态发生、伤口愈合、血管生成、子宫复旧和骨吸收等正常过程中，这些酶在组织重塑中起着关键作用。恶性细胞利用MMPs促进肿瘤的侵袭和转移。这一建议的目的是为了更好地理解MMPs催化的空间调控的细胞周围蛋白分解的分子机制及其在许多正常和病理条件下的功能作用。

DESCRIPTION (provided by applicant): Metalloproteases (MMPs) play a pivotal role in tissue remodeling during morphogenesis, wound healing, angiogenesis, uterine involution and bone resorption. Pericellular proteolysis catalyzed by MMPs is an important factor in defining the microenvironment of the resident cells of normal and neoplastic tissues. Malignant cells exploit MMPs to promote tumor invasion and metastasis. Since the cell surface MMP-2 activation complex ((MT1-MMP)2/TIMP-2/MMP-2) was described in our lab in the 1990s, we have studied molecular mechanisms of the spatial regulation catalyzed by this complex to elucidate the role of MMPs in cell - ECM interactions. We have recently identified some remarkable features of MMPs. We showed that i. MMPs interact with collagen via a substrate surface diffusion mechanism. Both trans-membrane MT1-MMP and secreted MMP-1, -2 and 9 can diffuse on the surface of native collagen fibrils; ii. MMP-1 acts as a unique, diffusion-based, ATP- independent motor enzyme driven by collagen proteolysis; iii. The extra-cellular portion of MT1-MMP interacts with collagen through a similar biased-diffusion mechanism; iv. Complex formation of MMP-2 C-terminal domain with the inhibitor TIMP-2 does not affect the rate of diffusion. Thus the entire membrane tethered collagenolytic complex (MT1-MMP)2/TIMP-2/MMP-2 is mobile relative to the underlying collagen substratum. These findings have profound implications for a mechanistic understanding of the role of MMPs in cell locomotion in a collagen rich microenvironment. We thus propose a model for a Mobile Cell Surface Collagenolytic Interface. In this model we hypothesize that the membrane tethered Collagenolytic Complex assists cell locomotion by virtue of its ability to slide directionally along the underlying collagen fibril. This mechanism is likely to be instrumental in orchestration of cell adhesion-desorption events during cell locomotion in collaboration with the cytoskeleton-integrin adhesion apparatus. To support this notion we now present evidence that the activity of the cell surface collagenolytic complex aids in force generation that cells exert in 3D collagen tissue constructs. This motivates us to further examine whether pericellular collagenolysis contributes to force generation by cells in a collagen- rich microenvironment. Thus here we propose i. To complete the investigation of the MT1-MMP as a proteolysis driven Brownian ratchet; ii. To define the structure-function relationship in MMP-1 and/or MT1-MMP relevant to the mechanism of substrate surface diffusion and iii. To determine the contribution of pericellular collagenolysis to force generation by cells in a collagen rich microenvironment using several experimental approaches including traction force microscopy in 2D cultures, the force measurements in 3D tissue constructs, and finally to measure the force that cells can exert on an individual collagen fibril utilizing cells with ablated enzyme gene(s) and collagen with mutated collagenase cleavage site. These studies will provide the necessary background for further investigation of how cells utilize pericellular proteolysis to orchestrate the adhesion-detachment events in cell locomotion and tissue remodeling that is a key process in metastatic tumor invasion. Public Health Relevance: Matrix Metalloproteases (MMPs) are the specialized group of enzyme with unique ability to catalyze turnover of extracellular matrix components such as collagen. These enzymes play a pivotal role in tissue remodeling during normal processes of morphogenesis, wound healing, angiogenesis, uterine involution and bone resorption. Malignant cells exploit MMPs to promote tumor invasion and metastasis. The goal of this proposal is to provide for a better understanding of the molecular mechanisms of spatially regulated peri-cellular proteolysis catalyzed by MMPs and its functional role in numerous normal and pathological conditions.