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.
