Activity Probes to Monitor ATP-Dependent Proteolysis

通过这一奖项，化学部门的生命过程化学项目资助凯斯西大学的Irene Lee博士开发和利用化学工具，在翻译后水平上研究线粒体atp依赖性蛋白酶的生理功能。线粒体被称为细胞的发电站，因为它们产生维持生命的三磷酸腺苷（ATP）化学能。这些能量发电站的功能是利用分子氧的化学势，通过呼吸作用产生ATP。人类线粒体功能障碍常与疾病有关。当氧气在线粒体中分解产生ATP时，会产生被称为活性氧（ROS）的活性中间体。当线粒体内的蛋白质、脂质或核酸与活性氧随机反应时，线粒体的完整性就会受到损害。本提案的总体目标是研究与ROS反应受损的蛋白质通过atp依赖性蛋白酶（称为Lon和ClpXP）从线粒体中去除的机制。在本研究中，我们将开发化学试剂来追踪线粒体中Lon和ClpXP活性的变化，以了解这两种蛋白酶在保护线粒体免受ros诱导的蛋白质损伤中的作用。获得这些信息将增强我们对线粒体生物学的理解。参与该项目的学生和博士后将在一个高度跨学科的环境中进行培养。他们还将接受科学教学方面的培训，并通过参加一个名为“全国青少年体育项目”（NYSP）的推广项目，更多地参与社区活动。NYSP是一个面向大克利夫兰地区10-16岁低收入家庭儿童的暑期项目，旨在让孩子们接触各种体育和学术丰富活动。现在有大量的文献数据表明，atp依赖的蛋白质水解在线粒体基质中氧化修饰蛋白的周转中起着至关重要的作用。在分离的哺乳动物线粒体基质中，lon蛋白酶的活性评估一直是在ClpXP存在的情况下进行的，使用标记酪蛋白作为底物，该底物被两种蛋白酶降解。鉴于这两种蛋白酶在某些蛋白质的降解中表现出重叠的特异性，开发出专门检测线粒体代谢过程中各自蛋白酶生理功能的化学试剂将有助于了解它们对保持细胞器完整性的贡献。在本研究中，实验将通过修改肽底物的序列来提高现有的Lon和ClpXP活性探针的选择性、线粒体通透性和敏感性；并将荧光标记的肽基氯甲基酮（Lon和ClpXP的不可逆抑制剂）开发成基于活性的探针，以监测细胞中两种蛋白酶的激活。

With this award, the Chemistry of Life Processes Program in the Chemistry Division is funding Dr. Irene Lee of Case Western University to develop and utilize chemical tools to interrogate the physiological functions of mitochondrial ATP-dependent proteases at a posttranslational level. Mitochondria are known as the powerhouse of cells because they generate chemical energy known as adenosine triphosphate (ATP) that sustains life. These powerhouses function by harnessing the chemical potential of molecular oxygen to generate ATP through respiration. Dysfunctions in human mitochondria are often associated with diseases. As oxygen is broken down in mitochondria to produce ATP, reactive intermediates known as reactive oxygen species (ROS) are generated. The integrity of mitochondria is compromised when proteins, lipids or nucleic acids housed within the mitochondria react randomly with ROS. The overarching goal of this proposal is to study the mechanism by which proteins damaged by reaction with ROS are removed from mitochondria by the ATP-dependent proteases known as Lon and ClpXP. In this proposal, chemical agents will be developed to track the changes in Lon and ClpXP activities individually in mitochondria in order to understand the involvement of the two proteases in protecting mitochondria from ROS-induced protein damage. Obtaining such information will enhance our understanding of the biology of mitochondria. The students and a post-doctoral fellow participating in this project will be trained in a highly multidisciplinary environment. They will also be trained to teach science and become more involved in the community by participating in an outreach program known as the National Youth Sports Program (NYSP). NYSP is a summer program offered to children of low-income families in the greater Cleveland area between ages 10-16, designed to expose the children to a variety of sports and academic enrichment activities.There is now a wealth of data in the literature to suggest that ATP-dependent proteolysis plays a crucial role in the turnover of oxidatively modified proteins in the mitochondrial matrix. The evaluation of Lon protease activity in isolated mammalian mitochondrial matrix has always been done in the presence of ClpXP using labeled casein as substrate, which is degraded by both proteases. Given that the two proteases exhibit overlapping specificity in the degradation of certain proteins, chemical reagents developed to specifically detect the physiological functions of the respective proteases during mitochondrial metabolism will provide insights into their contributions to the preservation of the integrity of the organelles. In this proposal, experiments will be conducted to improve the selectivity, mitochondria-permeability and sensitivity of existing activity probes of Lon and ClpXP by modifying the sequences of the peptide substrates; and to develop fluorescently labeled peptidyl chloromethyl ketones, which are irreversible inhibitors of Lon and ClpXP, into activity-based probes to monitor the activation of the two proteases in cells.