Mechanism of ATP-Dependent Proteolysis by Lon Protease

本项目的总体目标是阐明ATP（三磷酸腺苷）依赖性蛋白水解Lon蛋白酶的机制。lon蛋白酶是一种atp依赖性丝氨酸蛋白酶复合物，具有维持细胞内稳态和缓解细胞应激的功能。这种蛋白酶具有内在的三磷酸腺苷酶活性，在蛋白质降解过程中升高。该项目的具体目标是确定化学能量来源ATP是如何被Lon用来催化蛋白质降解的，并从这里获得洞察为什么大自然设计蛋白酶利用ATP作为激活剂。在这个项目中，PI将进行动力学实验，以确定酶中间体，其形成依赖于ATP水解的非结构化蛋白质称为lambda N的降解途径，并确定相应的功能。通过快速化学淬火技术评估了lambda N降解过程中ATP结合和随后水解的动力学机制。通过荧光停止流动技术评估了ATP-与amppnp -依赖的λ N内特定位点裂解的动力学机制。通过停止流动技术监测荧光共振能量转移信号，评估了ATP-与amppnp -依赖于lambda N中定义的可剪切位点到细菌Lon蛋白水解位点的易位的动力学机制。这项研究的结果将有助于深入了解Lon结合其atp酶和肽酶活性来降解缺乏明确结构的内源性蛋白质底物的机制。这项工作将代表第一个定量研究，旨在确定atp酶活性对蛋白质降解的贡献。更广泛的影响本项目采用多学科的方法来确定atp依赖性蛋白水解的机制。因此，参与该项目的学生在酶动力学、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方面获得了多学科的培训。这些研究活动为生物化学的本科生和研究生提供了极好的训练机会。此外，在PI实验室工作的所有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岁的低收入家庭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中的化学知识。这项一年一度的外展活动已经成为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为弱势群体提供教育。

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.