Mechanism & Inhibition of Collagenolytic Activity

机制

• 批准号: 6850791
• 负责人: GREGG B FIELDS
• 金额: $ 29.34万
• 依托单位: FLORIDA ATLANTIC UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-03-05 至 2008-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6850791
• 关键词: SDS polyacrylamide gel electrophoresis; collagen; collagenase; cysteine endopeptidases; enzyme activity; enzyme inhibitors; enzyme mechanism; fluorescent dye /probe; high performance liquid chromatography; ionophores; mass spectrometry; metalloendopeptidases; nuclear magnetic resonance spectroscopy; stromelysin; triple helix; trypsin

DESCRIPTION (provided by applicant): The dissolution of the collagen triple-helix has been implicated in a variety of diseases that effect the structural integrity of various components of the body. Collagen also provides a barrier between tissues and cells; destruction of this barrier plays a role in tumor cell invasion and metastasis. The matrix metalloproteinase (MMP) family has been recognized for their collagenolytic activities, and has thus been the subject of intense research efforts, in order to elucidate their mechanisms of action and allow for rational design of inhibitors. We have developed methodology for constructing triple-helical peptides (THPs) and have applied these synthetic proteins for the study of enzyme interactions with collagens. "Triple-helical peptidase" activity was found to be more widespread amongst proteases than previously believed. It appears that the unique aspect of collagenolytic activity may not be the ability to cleave a triple-helix, but rather the ability to bind and orient the native collagen molecule properly. This paradigm shift could have significant effects on the design of inhibitors against collagenolytic activity. To further explore the nature of triple-helical peptidase activity, a series of THP substrates will be assembled, incorporating known sites of collagen hydrolysis and varying over a range of Tm values. Substrate thermal stability will be correlated to enzyme activity. In tandem, 2D NMR experiments using 15N-labeled amino acids will examine the mobilities of the peptide backbone in these substrates. Individual kinetic parameters and activation energies will be evaluated for MMP, trypsin, thermolysin, cathepsin K, and aggrecanase hydrolysis of THPs. MMP-1, MMP-2, MMP-8, and cathepsin K mutants will be utilized to determine the regions within these enzymes critical for triple-helical peptidase activity. Finally, selective THP substrates and novel THP inhibitors will be designed and tested. Overall, triple-helical peptidase activity will be systematically evaluated for a variety of proteases as a function of substrate sequence and conformational flexibility, and the mechanism of collagenolytic activity will better understood.

描述(由申请人提供)：胶原蛋白三螺旋的溶解与多种疾病有关，这些疾病影响身体不同组成部分的结构完整性。胶原蛋白还提供了组织和细胞之间的屏障；这种屏障的破坏在肿瘤细胞的侵袭和转移中发挥了作用。基质金属蛋白酶(MMPs)家族具有良好的胶原酶活性，为阐明其作用机制和合理设计抑制剂提供了有力的研究基础。我们开发了构建三螺旋多肽(THPS)的方法，并将这些合成的蛋白质用于研究酶与胶原蛋白的相互作用。“三螺旋多肽酶”的活性被发现比之前认为的更广泛地存在于蛋白酶中。看来，胶原酶活性的独特方面可能不是切割三螺旋的能力，而是正确结合和定位天然胶原分子的能力。这种范式的转变可能会对抗胶原溶解活性的抑制剂的设计产生重大影响。为了进一步探索三螺旋多肽酶活性的本质，将组装一系列THP底物，结合已知的胶原蛋白水解点，并在Tm值范围内变化。底物的热稳定性与酶的活性有关。同时，使用15N标记氨基酸的2D核磁共振实验将检查这些底物中肽骨架的迁移率。单独的动力学参数和活化能将被评估的基质金属蛋白酶，胰酶，热裂解酶，组织蛋白酶K，和聚集聚糖酶的THPS的水解。将利用基质金属蛋白酶-1、基质金属蛋白酶-2、基质金属蛋白酶-8和组织蛋白酶K突变体来确定这些酶中对三螺旋多肽酶活性至关重要的区域。最后，将设计和测试选择性THP底物和新型THP抑制剂。总体而言，作为底物顺序和构象灵活性的函数的各种蛋白酶的三螺旋多肽酶活性将被系统地评估，并且将更好地理解胶原酶分解活性的机制。