COLLAGENOLYSIS-DRIVEN MOLECULAR MOTORS IN CELL MIGRATION AND MATRIX REMODELING

细胞迁移和基质重塑中胶原蛋白溶解驱动的分子马达

• 批准号: 7464881
• 负责人: GREGORY I GOLDBERG
• 金额: $ 25.23万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-06-01 至 2012-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7464881
• 关键词: Adhesions; Affect; Atomic Force Microscopy; Attention; Biological; Biology; Bone Resorption; C-terminal; Cell Adhesion; Cell surface; Cells; Characteristics; Collaborations; Collagen; Collagen Fibril; Complex; Condition; Cytoskeleton; Diffuse; Diffusion; Dissociation; Endopeptidases; Enzyme Gene; Enzymes; Event; Extracellular Matrix; Fluorescence; Gelatinase A; Generations; Goals; Individual; Integrins; Interstitial Collagenase; Investigation; Laboratories; MMP14 gene; Matrix Metalloproteinases; Measurement; Measures; Membrane; Metalloproteases; Methods; Microscopic; Modeling; Molecular; Molecular Motors; Morphogenesis; Motor; Mutagenesis; Mutate; Neoplasm Metastasis; Normal Cell; Peptide Hydrolases; Play; Process; Proteolysis; Public Health; Rate; Reader; Regulation; Relative (related person); Reporting; Research; Role; Site; Slide; Spectrum Analysis; Staging; Stimulus; Structure; Structure-Activity Relationship; Substrate Interaction; Surface; System; Technology; Tissues; Traction; Translating; Tumor Cell Invasion; Wound Healing; angiogenesis; base; cancer cell; cell motility; collagenase; editorial; extracellular; grasp; human MMP14 protein; inhibitor/antagonist; mutant; neoplastic; novel; research study; single molecule; tool; two-photon

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.

描述（由申请人提供）：金属蛋白酶（MMPs）在形态发生、伤口愈合、血管生成、子宫复旧和骨吸收过程中的组织重塑中起关键作用。MMPs催化的细胞外蛋白水解是决定正常和肿瘤组织驻留细胞微环境的重要因素。恶性细胞利用MMPs促进肿瘤侵袭和转移。自从我们的实验室在20世纪90年代描述了细胞表面MMP-2激活复合物（(MT1-MMP)2/TIMP-2/MMP-2）以来，我们研究了由该复合物催化的空间调节的分子机制，以阐明MMPs在细胞- ECM相互作用中的作用。我们最近发现了MMPs的一些显著特征。我们发现i. MMPs通过底物表面扩散机制与胶原相互作用。跨膜MT1-MMP和分泌的MMP-1、-2和9均可在天然胶原原纤维表面扩散；2。MMP-1是一种独特的、基于扩散的、不依赖ATP的运动酶，由胶原蛋白水解驱动；3。MT1-MMP的细胞外部分通过类似的偏倚扩散机制与胶原蛋白相互作用；iv. MMP-2 c -末端结构域与抑制剂TIMP-2形成复合物不影响扩散速率。因此，整个膜系系的胶原溶解复合物（MT1-MMP）2/TIMP-2/MMP-2相对于胶原基质是可移动的。这些发现对在富含胶原的微环境中MMPs在细胞运动中的作用的机制理解具有深远的意义。因此，我们提出了一个移动细胞表面胶原溶解界面的模型。在这个模型中，我们假设膜系结的胶原溶解复合体通过其沿着胶原原纤维定向滑动的能力来帮助细胞运动。这一机制很可能与细胞骨架-整合素粘附装置合作，在细胞运动过程中协调细胞粘附-解吸事件。为了支持这一观点，我们现在提出的证据表明，细胞表面胶原溶解复合物的活性有助于细胞在三维胶原组织结构中施加的力的产生。这促使我们进一步研究细胞周围胶原溶解是否有助于细胞在富含胶原的微环境中产生力。因此，我们提出i.完成MT1-MMP作为蛋白水解驱动的布朗棘轮的研究；2。定义MMP-1和/或MT1-MMP中与底物表面扩散机制相关的结构-功能关系；为了确定细胞周围胶原溶解对富含胶原的微环境中细胞产生力的贡献，使用了几种实验方法，包括二维培养中的牵引力显微镜，三维组织结构中的力测量，最后利用酶基因消融的细胞和胶原酶裂解位点突变的胶原蛋白来测量细胞对单个胶原纤维的力。这些研究将为进一步研究细胞如何利用细胞周蛋白水解来协调细胞运动和组织重塑中的粘附-脱离事件提供必要的背景，这是转移性肿瘤侵袭的关键过程。