Activity Probes to Monitor ATP-Dependent Proteolysis

获得该奖项后，化学部的生命过程化学项目将资助凯斯西大学的艾琳·李博士开发和利用化学工具，在翻译后水平上研究线粒体ATP依赖的蛋白水解酶的生理功能。线粒体被称为细胞的动力源，因为它们产生的化学能量被称为三磷酸腺苷(ATP)，可以维持生命。这些发电站的功能是利用分子氧的化学势通过呼吸产生三磷酸腺苷。人类线粒体功能障碍通常与疾病有关。当线粒体中的氧被分解以产生ATP时，就会产生被称为活性氧物种(ROS)的活性中间体。当线粒体中的蛋白质、脂类或核酸与ROS随机反应时，线粒体的完整性就会受到损害。这项提议的首要目标是研究通过与ROS反应而受损的蛋白质被被称为Lon和ClpXP的ATP依赖的蛋白酶从线粒体中移除的机制。在这项建议中，将开发化学试剂来跟踪线粒体中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研究员将在高度多学科的环境中接受培训。他们还将接受教授科学的培训，并通过参加名为国家青年体育计划(NYSP)的外联计划更多地参与社区。NYSP是为大克利夫兰地区10-16岁低收入家庭的儿童提供的暑期计划，旨在让孩子们接触到各种运动和学术充实活动。现在有大量文献数据表明，依赖于ATP的蛋白分解在线粒体基质中氧化修饰蛋白的周转中起着关键作用。在以酪蛋白为底物的ClpXP存在下，对分离的哺乳动物线粒体基质中Lon蛋白酶活性的评估一直是以两种酶都能降解的方式进行的。鉴于这两种蛋白酶在某些蛋白质的降解过程中表现出重叠的特异性，开发专门检测各自在线粒体代谢过程中的生理功能的化学试剂将有助于深入了解它们对保持细胞器完整性的贡献。在这项建议中，将进行实验，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，以监测细胞中这两种蛋白酶的激活情况。

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.