Branched chain amino acid metabolism and citrate homeostasis in disease

About This Grant

Branched chain amino acids (BCAA) and their metabolites are critical molecules in modulating intermediary metabolism, serving as sensors to determine the switch between anabolic and catabolic processes. Almost two dozen BCAA inborn errors of metabolism (IEMs) have been identified and are the most common of the organic acidemias. The central hypothesis of this project is that the pathophysiology of disorders of BCAA metabolism is related to both BCAA metabolite imbalance and disruption of structural integrity of mitochondria related to the enzymes of BCAA metabolism. Our long-term goal is to characterize BCAA metabolism and its deficiencies and to develop novel therapies for them. Our group has previously demonstrated that led to profound derangements cellular homeostasis that exceeds the current limits of understanding of these disorders. This application has 3 specific aims. Specific Aim 1 is to demonstrate the interactions of the enzymes of BCAA metabolism in situ. We have developed a high resolution in situ cryo-electron tomography (cryo-ET) workflow to visualize the impacts of mitochondrial energy dysfunction on mitochondrial ultrastructure and electron transport chain structure. We propose extending this technology to visualize the in situ organization of BCAA metabolism enzymes in fibroblasts from patients with IEMs of BCAA. We hypothesize that proximal enzymes of BCAA metabolism functionally and structurally interact with respiratory chain supercomplexes, and that this interaction is disrupted in patient-derived cells. Specific Aim 2 is the development of a new therapy for propionic acidemia (PA) and methylmalonic acidemia (MMA). We have identified a novel compound (PMA010) that reduces pathogenic lysine propionylation in patient-derived cells from patients with propionic acidemia (PA) and normalizes protein succinylation, thereby improving cellular bioenergetics. We will use targeted metabolomics and high-resolution cryo-EM to determine changes induced by PMA010 on cells from PA and methylmalonic acidemia (MMA) patients, identify effects of PMA010 on mitochondrial protein lysine acylation and mitochondrial structure, and evaluate efficacy of PMA010 and/or analogs in vivo in a mouse model of PA. We hypothesize that these experiments will identify PMA010 as candidate for clinical trials for these disorders. Specific Aim 3 is to understand the physiology of the mitochondrial citrate transporter and its deficiency. The end products of BCAA catabolism enter the TCA cycle to make citrate. Human SLC25A1 encodes the mitochondrial citrate carrier (mCiC), which facilitates transport of citrate between the cytosol and mitochondrial matrix. Genetic defects in SLC25A1 lead to the organic acidemia combined D, L, 2-hydroxyclutaric acidemia (c2HGA). We will express and characterize the function 4 CiC isoforms, characterize their interactions with BCAA metabolic enzymes via cryo-EM, and use a multi-omics approach to assess the effects of SLC25A1 mutations on: (i) pathophysiology in c2HGA patient-derived fibroblasts. We will test phenylbutyrate (PB), a drug shown in our preliminary data to improve cellular bioenergetics and metabolite profile, as a therapy for this disease. We hypothesize that PB will demonstrate a therapeutic effect in cells and an SLC25A1 deficiency mouse model as pre-clinical proof-of-principle for patient clinical trials.