Mechanism of ATP-Dependent Proteolysis by Lon Protease

Lon 蛋白酶的 ATP 依赖性蛋白水解机制

• 批准号: 0919631
• 负责人: Irene Lee
• 金额: $ 59.19万
• 依托单位: Case Western Reserve University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-01 至 2013-06-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0919631&HistoricalAwards=false
• 关键词: Mechanism; ATP; Dependent; Proteolysis; Lon

The overall goal of this project is the elucidation of the mechanism of ATP (adenosine triphosphate-dependent proteolysis by Lon protease. Lon protease is an ATP-dependent serine protease complex functioning to maintain cellular homeostasis and relieve cell stress. This protease possesses an intrinsic ATPase activity that is elevated during protein degradation. The specific goal of this project is to determine how the chemical energy source ATP is used by Lon to catalyze protein degradation, and from here, obtain insights into why nature design proteases that utilize ATP as activators. In this project, the PI will perform kinetic experiments to identify enzyme intermediates, whose formations are dependent on ATP hydrolysis in the degradation pathway of an unstructured protein called lambda N, and determine their functions accordingly. The kinetic mechanisms of ATP binding and subsequent hydrolysis during lambda N degradation are evaluated by rapid chemical quench techniques. The kinetic mechanisms of ATP- versus AMPPNP-dependent cleavage of specific sites within lambda N are evaluated by fluorescence stopped flow technique. The kinetic mechanisms of ATP- versus AMPPNP-dependent translocation of a defined scissile site in lambda N to the proteolytic site of a bacterial Lon are evaluated through monitoring the fluorescence resonance energy transfer signal by stopped flow techniques. Results generated from this study will provide insight into the mechanism by which Lon couples its ATPase and peptidase activities to degrade an endogenous protein substrate lacking defined structure. This work will represent the first quantitative study designed to determine the contribution of the ATPase activity of Lon to protein degradation. Broader ImpactsThis project employs a multi-disciplinary approach to determine the mechanism of ATP-dependent proteolysis. As such, students participating in this project acquire a multidisciplinary training in enzyme kinetics, protein chemistry, liquid chromatography, mass spectrometry, chemical synthesis, solid phase peptide synthesis, fluorescence spectroscopy, protein purification and molecular cloning. These research activities provide excellent training opportunities to undergraduate and graduate students in biochemistry. Additionally, all personnel working in the PI's laboratory serve as volunteers to the National Youth Sports Program (NYSP). NYSP is summer program offered to children of low-income families in the Greater Cleveland area between the ages of 10 and 16. Participating students teach these children chemistry of daily life through experimentation with household chemicals and dry ice. This annual outreach activity has become a tradition in the PI's laboratory, where undergraduate and graduate students learn to become more involved in the community and education for the underprivileged.

该项目的总体目标是阐明 ATP（Lon 蛋白酶依赖的三磷酸腺苷蛋白水解）的机制。Lon 蛋白酶是一种 ATP 依赖的丝氨酸蛋白酶复合物，其功能是维持细胞稳态和缓解细胞应激。该蛋白酶具有内在的 ATP 酶该项目的具体目标是确定 Lon 如何利用化学能源 ATP 来催化蛋白质降解。在这里，深入了解为什么大自然设计利用 ATP 作为激活剂的蛋白酶。在这个项目中，PI 将进行动力学实验来识别酶中间体，其形成依赖于一种称为 lambda N 的非结构化蛋白质的降解途径中的 ATP 水解。通过快速化学猝灭技术评估 ATP 结合和随后的 lambda N 降解过程中的动力学机制。通过荧光停流技术。 通过停流技术监测荧光共振能量转移信号，评估 ATP 与 AMPPNP 依赖的 lambda N 中确定的易位点到细菌 Lon 的蛋白水解位点的易位的动力学机制。这项研究产生的结果将深入了解 Lon 结合其 ATP 酶和肽酶活性来降解缺乏确定结构的内源蛋白质底物的机制。 这项工作将代表第一项旨在确定 Lon ATP 酶活性对蛋白质降解的贡献的定量研究。更广泛的影响该项目采用多学科方法来确定 ATP 依赖性蛋白水解机制。因此，参与该项目的学生获得了酶动力学、蛋白质化学、液相色谱、质谱、化学合成、固相肽合成、荧光光谱、蛋白质纯化和分子克隆等多学科培训。这些研究活动为生物化学领域的本科生和研究生提供了极好的培训机会。 此外，PI 实验室的所有工作人员都是国家青少年体育计划 (NYSP) 的志愿者。 NYSP 是为大克利夫兰地区 10 至 16 岁低收入家庭的儿童提供的暑期项目。参与的学生通过家用化学品和干冰的实验向这些孩子传授日常生活化学知识。 这项一年一度的外展活动已成为 PI 实验室的传统，本科生和研究生在这里学习如何更多地参与社区和弱势群体的教育。