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: 8344909
• 负责人: Keir Neuman
• 金额: $ 10.47万
• 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/8344909
• 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; Quantum Dots; 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 quantum dot labels, or nitrogen vacancy nano-diamond labels.

在这个项目中，与华盛顿大学圣路易斯的Gregory Goldberg合作，我们采用了单分子TIRF来研究胶原蛋白消化过程中单基质金属蛋白酶（MMP）的运动。 MMP在生理胶原蛋白加工途径中起重要作用，包括组织重塑，伤口愈合和细胞迁移。然而，由于胶原蛋白底物的复杂性质和MMP的运动，MMP与胶原蛋白相互作用的机械细节已难以理解。通过在具有高空间和时间分辨率的孤立天然胶原纤维上跟踪单个MMP，我们可以表征MMP在底物上的运动，以及该运动如何与蛋白水解活性耦合。这种方法为这类酶提供了详细的机械信息。我们首次观察到单个MMP在胶原蛋白纤维上的复杂运动，并开发了一个全面的定量模型，描述了该运动如何与胶原纤维的蛋白水解结合在一起。 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. 逃离长暂停状态与动力学途径一致，该途径包括10个或更多的动力学步骤，每个动力学步骤的正向速率为10/s。 长时间的一小部分（5％）导致胶原蛋白降解的启动，随后是纤维中15个胶原蛋白单体的快速和过程降解。 这些结果是意想不到的，并提供了对MMP与胶原蛋白相互作用的前所未有的见解，同时突出了单分子方法的独特能力来测量复杂的生物分子过程。最初的测量和全面的建模已经完成，我们提交了第一个手稿。 此外，我们开发了新的方法来分析单分子痕迹的扩散，这些痕迹适用于任何扩散轨迹的单分子分析。除了通过使用量子点标签或氮空位纳米二角标签延长单个轨迹的持续时间外，未来在MMP跟踪方面的未来工作将集中于改善跟踪的时间和空间分辨率。