Single-molecule measurements of collagen processing by Matrix Metalloproteinases

基质金属蛋白酶对胶原蛋白加工的单分子测量

• 批准号: 8939875
• 负责人: Keir Neuman
• 金额: $ 6.27万
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8939875
• 关键词: Atherosclerosis; Binding; Bone Resorption; Cleaved cell; Collaborations; Collagen; Collagen Fiber; Collagen Fibril; Collagen Type I; Complex; Coupled; Diamond; Diffusion; Digestion; Enzymes; Extracellular Matrix; Fiber; Fibrillar Collagen; Future; Goals; Hot Spot; Human; Individual; Kinetics; Label; MMP8 gene; Manuscripts; Matrix Metalloproteinases; Measurement; Measures; Methodology; Methods; Modeling; Morphogenesis; Motion; N-terminal; Nature; Nitrogen; Pathway interactions; Peptide Hydrolases; Physiological; Physiological Processes; Play; Process; Property; Proteins; Proteolysis; Refractory; Resolution; Rheumatoid Arthritis; Role; Site; System; Time; Tissues; Universities; Vertebrates; Washington; Work; Wound Healing; angiogenesis; cancer cell; cell motility; collagenase; human MMP14 protein; improved; insight; monomer; nano; polypeptide; scaffold; single molecule

in this project, a collaboration with Gregory Goldberg at Washington University St. Louis, we employed single-molecule TIRF to study the motion of single matrix metalloproteinases (MMPs) during the digestion of collagen. MMPs play an important role in physiological collagen processing pathways including tissue remodeling, wound healing and cell migration. However, the mechanistic details of MMP interactions with collagen have been refractory to study due to the complex nature of the collagen substrate and the motion of the MMPs. By tracking individual MMPs on isolated native collagen fibers with high spatial and temporal resolution we could characterize the motion of the MMP on the substrate, and how this motion is coupled to proteolytic activity. This approach has provided detailed mechanistic information for this important class of enzymes. We have, for the first time, observed the complex motion of individual MMPs on collagen fibers and have developed a comprehensive quantitative model describing how this motion is coupled to proteolysis of the collagen fiber. We found that the motion of MMPs on collagen is both biased and hindered diffusion, that there are binding hot-spots for MMPs on collagen periodically spaced 1.3 and 1.5 microns apart, and that the motion of MMPs on collagen is interrupted by two classes of pauses: short exponentially distributed pause of duration 0.4s and long non-exponentially distributed pauses of duration 1 second. Escape from the long pause state is consistent with a kinetic pathway that includes 10 or more kinetic steps each with a forward rate of 10/s. A small fraction (5%) of the long pauses result in the initiation of collagen degradation, which is followed by the rapid and processive degradation of 15 collagen monomers in the fiber. These results were unanticipated and provide unprecedented insight into the interaction of MMPs with collagen while highlighting the unique capabilities of single-molecule methods to measure complex biomolecular processes. The initial measurements and comprehensive modeling are complete and we have submitted the first manuscript. Furthermore, we developed new methodologies to analyze diffusion in single-molecule traces, which are applicable to any single-molecule analysis of diffusion trajectories. Future work on MMP tracking will be focused on improving the temporal and spatial resolution of the tracking in addition to extending the duration of individual trajectories through the use of nitrogen vacancy nano-diamond labels.

在这个与圣路易斯华盛顿大学的 Gregory Goldberg 合作的项目中，我们采用单分子 TIRF 来研究单基质金属蛋白酶 (MMP) 在胶原蛋白消化过程中的运动。 MMP 在生理胶原蛋白加工途径中发挥着重要作用，包括组织重塑、伤口愈合和细胞迁移。然而，由于胶原蛋白基质和 MMP 运动的复杂性，MMP 与胶原蛋白相互作用的机制细节一直难以研究。通过以高空间和时间分辨率跟踪分离的天然胶原纤维上的单个 MMP，我们可以表征基质上 MMP 的运动，以及这种运动如何与蛋白水解活性耦合。这种方法为这一类重要的酶提供了详细的机制信息。我们首次观察到胶原纤维上单个 MMP 的复杂运动，并开发了一个全面的定量模型，描述这种运动如何与胶原纤维的蛋白水解耦合。我们发现，MMP 在胶原蛋白上的运动既存在偏差又受阻碍扩散，胶原蛋白上的 MMP 存在周期性间隔 1.3 和 1.5 微米的结合热点，并且 MMP 在胶原蛋白上的运动被两类暂停中断：持续时间为 0.4 秒的短指数分布暂停和持续时间为 1 秒的长非指数分布暂停。逃离长暂停状态与包含 10 个或更多动力学步骤的动力学路径一致，每个动力学步骤的前进速率为 10/s。一小部分 (5%) 的长暂停会导致胶原蛋白降解的开始，随后纤维中 15 种胶原蛋白单体会快速、持续地降解。 这些结果是出乎意料的，为 MMP 与胶原蛋白的相互作用提供了前所未有的见解，同时凸显了单分子方法测量复杂生物分子过程的独特能力。初步测量和综合建模已经完成，我们已经提交了第一份手稿。此外，我们开发了新的方法来分析单分子痕迹中的扩散，该方法适用于扩散轨迹的任何单分子分析。 MMP 跟踪的未来工作将集中于提高跟踪的时间和空间分辨率，以及通过使用氮空位纳米金刚石标签来延长单个轨迹的持续时间。