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.

本项目的总体目标是阐明Lon蛋白酶对三磷酸腺苷依赖的蛋白分解的机制。LON蛋白水解酶是一种依赖于ATP的丝氨酸蛋白酶复合体，其功能是维持细胞内环境的稳定和缓解细胞应激。这种酶具有固有的ATPase活性，这种活性在蛋白质降解过程中会升高。这个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，并从这里获得关于为什么大自然设计利用ATP作为激活剂的蛋白酶的见解。在这个项目中，PI将进行动力学实验，以确定酶中间体的形成依赖于名为lambda N的非结构蛋白降解途径中的ATP水解，并相应地确定它们的功能。用快速化学猝灭技术研究了波长N降解过程中ATP结合和随后的水解的动力学机制。用荧光停流技术研究了依赖于ATP和AMPPNP的Lambda N内特定位点切割的动力学机制。通过停止流技术监测荧光共振能量转移信号，评价了依赖于ATP和AMPPNP的Lambda N中特定剪切点到细菌Lon蛋白水解点易位的动力学机制。这项研究的结果将为深入了解Lon将其ATPase和多肽酶活性偶联以降解缺乏明确结构的内源蛋白质底物的机制。这项工作将是第一次定量研究，旨在确定LON的ATPase活性对蛋白质降解的贡献。更广泛的影响这个项目采用多学科方法来确定依赖于ATP的蛋白分解的机制。因此，参与该项目的学生将获得酶动力学、蛋白质化学、液相色谱、质谱学、化学合成、固相多肽合成、荧光光谱学、蛋白质纯化和分子克隆等多学科培训。这些研究活动为本科生和研究生提供了极好的生物化学培训机会。此外，国际体育协会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计划。参与计划的学生通过实验家用化学品和干冰来教授这些孩子日常生活中的化学。这项一年一度的外展活动已成为国际扶轮实验室的传统，在那里，本科生和研究生学习更多地参与社区和对弱势群体的教育。