Structural dynamics of the human mitochondrial pyruvate carrier associated with transport and inhibition - Abstract Pyruvate is one of the most important carbon inputs for mitochondria, not only for energy production but also as an anaplerotic and biosynthetic carbon source. Since pyruvate is formed in the cytosol from either glycolysis or lactate and the pyruvate metabolizing enzymes (i.e., pyruvate dehydrogenase and pyruvate carboxylase) reside in the mitochondrial matrix, the transport of pyruvate across the inner mitochondrial membrane represents a critical step in intermediary metabolism. The McCommis laboratory uses a wide range of model systems from in vitro biophysical techniques to genetic mouse models to investigate the importance of mitochondrial metabolism in pathophysiology. Our primary focus has been the mitochondrial pyruvate carrier (MPC). We and others have previously described the importance of the MPC in a variety of metabolic tissues with the use of tissue-specific knockout mice. In some instances, deletion or inhibition of the MPC is beneficial. For example, inhibition of the MPC in the liver can improve diabetes by decreasing hepatic glucose production, enhancing fatty acid and amino acid catabolism, and activating cellular nutrient sensors. Despite this importance of the MPC, there is no experimental knowledge of the protein structure or conformational dynamics during pyruvate transport or inhibition. Additionally, beyond the identification of several synthetic inhibitors, almost nothing is known about factors that regulate MPC activity. The proposed studies will use DEER spectroscopy as a powerful Pulse EPR technique to uncover the conformational changes of the MPC during transport or inhibition. Additionally, using a combination of DEER spectroscopy, biochemical techniques, and bioenergetics measures, we will determine endogenous factor(s) that regulate MPC activity. The branched chain ketoacid of leucine, a-ketoisocaproate (KIC), has been shown to inhibit pyruvate oxidation without affecting pyruvate dehydrogenase activity. We present data herein that indeed KIC inhibits mitochondrial pyruvate oxidation, however this effect is completely lost in PPM1K-/- mitochondria that have reduced oxidation of branched chain ketoacids. KIC also has little-to-no effect in preliminary DEER analyses. This suggests that a downstream metabolite of KIC produces the inhibition of pyruvate metabolism. We will also evaluate how altered cardiolipin composition and content, as commonly observed in cardiometabolic diseases, dysregulates MPC transport function. This mechanistic information will shed light on MPC function and how it is regulated, which is a critical process for nearly all cell types under various physiologic contexts. Ultimately, the overarching goal of our research program is to employ this multidisciplinary approach to mechanistically study orphan mitochondrial transporters with potential pathophysiological significance, aiming to determine the functional dynamics of their regulation by potential substrates and modulators.
