Mechanism for the selection of undamaged physiological substrates by the ATP-dependent protease Lon

ATP依赖性蛋白酶Lon选择未受损生理底物的机制

• 批准号: 2210869
• 负责人: Irene Lee
• 金额: $ 50万
• 依托单位: Case Western Reserve University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-01 至 2025-06-30
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2210869&HistoricalAwards=false
• 关键词: Mechanism; selection; undamaged; physiological; substrates

This project will determine how the cellular protease Lon, which degrades various proteins, regulates the transcription of a genetic material known as ribonucleic acid (RNA). Since Lon-mediated protein degradation and RNA transcription are conserved in bacteria and humans, findings generated from this work will provide insights into the workings of a universal process that is important for sustaining life. The PI hypothesizes that the competing interaction between Lon and the physiological binding partner(s) for the same cellular protein dictates this cellular protein's fate and, ultimately, function. The project will test this hypothesis by determining how purified Escherichia coli Lon protease (ELon) affects the assembly of the RNA transcription complex constituting the Escherichia coli RNA polymerase (ERNAP), the lambda N protein, the NusA protein, and the nucleic acid containing the nutL sequence. In addition, the proposed research will determine the extent to which the assembly of the RNA transcription process protects the lambda N protein from degradation by ELon. This project will integrate research into education. The broader impact of this project lies in promoting science literacy to underprivileged children in Cleveland, Ohio. Trainees participating in this project will learn to teach and communicate scientific concepts to the general public, which will prepare them for an education career in STEM. Undergraduate students and post-doctoral fellows will be trained to conduct research in organic synthesis, molecular cloning, protein purification and labeling, electron microscopy, and enzymology. The trainees will learn to teach and communicate scientific concepts to the general public. This kind of experience will provide a training ground for future educators in STEM.This project will determine if the Escherichia coli Lon protease (Elon) participates in the regulation of RNA transcription. The project will test the hypothesis that Lon disrupts RNA transcription by selectively degrading the lambda N protein, which is needed for antitermination RNA transcription. A combination of enzyme kinetics, mutagenesis, and electron microscopy imaging approaches will be used to evaluate the impact of Escherichia coli Lon protease (ELon) on the kinetic assembly of the RNA transcription complex constituting the Escherichia coli RNA polymerase (ERNAP), the lambda N protein, the NusA protein, and the nucleic acid containing the nutL sequence. The effect of RNA transcription on Lon-mediated proteolysis of the lambda N protein will also be evaluated. Since Lon is found in most organisms, physiologically relevant mechanistic findings on ELon could be transformed to understand the workings Lon homologs in eukaryotes. The quantitative approach developed for elucidating the mechanism of Lon would benefit the adaptation of the same approach to study other ATP-dependent proteases to reveal mechanistic differences among this family of enzymes.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

该项目将确定降解各种蛋白质的细胞蛋白酶Lon如何调节称为核糖核酸（RNA）的遗传物质的转录。由于Lon介导的蛋白质降解和RNA转录在细菌和人类中是保守的，因此这项工作产生的发现将为维持生命的重要通用过程的运作提供见解。PI假设Lon与同一细胞蛋白的生理结合伴侣之间的竞争性相互作用决定了该细胞蛋白的命运，并最终决定了其功能。该项目将通过确定纯化的大肠杆菌Lon蛋白酶（ELon）如何影响构成大肠杆菌RNA聚合酶（ERNAP）、λ N蛋白、NusA蛋白和含有nutL序列的核酸的RNA转录复合物的组装来验证这一假设。此外，拟议的研究将确定RNA转录过程的组装在多大程度上保护λ N蛋白免受ELon降解。 该项目将把研究纳入教育。该项目更广泛的影响在于促进俄亥俄州克利夫兰贫困儿童的科学素养。参加该项目的学员将学习向公众教授和传播科学概念，这将为他们在STEM的教育生涯做好准备。本科生和博士后研究员将接受培训，进行有机合成，分子克隆，蛋白质纯化和标记，电子显微镜和酶学研究。 学员将学习向公众讲授和传播科学概念。这种经验将为未来的STEM教育工作者提供培训基地。该项目将确定大肠杆菌Lon蛋白酶（Elon）是否参与RNA转录的调控。该项目将测试Lon通过选择性降解抗终止RNA转录所需的λ N蛋白来破坏RNA转录的假设。将使用酶动力学、诱变和电子显微镜成像方法的组合来评估大肠杆菌Lon蛋白酶（ELon）对构成大肠杆菌RNA聚合酶（ERNAP）、λ N蛋白、NusA蛋白和含有nutL序列的核酸的RNA转录复合物的动力学组装的影响。还将评估RNA转录对Lon介导的λ N蛋白的蛋白水解的影响。由于Lon存在于大多数生物体中，因此可以将关于ELon的生理学相关机制研究结果转化为了解Lon同系物在真核生物中的作用。为阐明Lon的机制而开发的定量方法将有利于采用相同的方法来研究其他ATP依赖性蛋白酶，以揭示该家族酶之间的机制差异。该奖项反映了NSF的法定使命，并被认为值得通过使用基金会的知识价值和更广泛的影响审查标准进行评估来支持。