The ADP/ATP carrier (AAC in yeast, ANT in mammals) plays a key role in OXPHOS, the main energy-generating process in the cell. We showed that the major AAC isoform in yeast, Aac2p, associates with respiratory supercomplexes but only when cardiolipin (CL) is around. Also, we demonstrated that the ability to interact with heterologous binding partners is evolutionarily conserved in human ANTs (hANTs), suggesting that these protein-protein interactions are physiologically relevant. ANT1 deficiencies are linked with diseases like Sengers syndrome, which presents with hypertrophic cardiomyopathy, skeletal myopathy, among other symptoms. Our unbiased proteomics analysis revealed that acylglycerol kinase (AGK), whose gene mutations are the cause of Sengers syndrome, is part of the hANT interactome. This proposal will address the hypothesis that AGK plays a role in OXPHOS regulation and mitochondrial maintenance by influencing ANT functionality. In Aim 1, we will define AGK's molecular function. We will knock-out AGK in HEK293 Flp-In and induced pluripotent stem cell (IPSC)-derived cardiomyocytes thru the CRISPR-Cas9 system and assess the effects of agk deletion on metabolite levels, energy production, and steady state levels of ANTs and OXPHOS components. Completion of this aim will establish a novel cellular model of Sengers syndrome. Patients with the said disease have low levels of ANT1. In Aim 2, we will investigate the role of AGK in hANT regulation. Two probable mechanisms will be looked at. First is whether the capacity of AGK to produce lysophosphatidic and phosphatidic acid significantly contributes to CL biosynthesis to impact hANT stability. Phospholipid analysis will be done using AGK-deficient HEK293 and cardiomyocytes. Since previous reports showed that ANT1 levels were decreased in patient myotubes but not in undifferentiated myoblasts, another possible mechanism is that AGK post-translationally regulates ANT. This will be tested by looking at hANT phosphorylation with or without AGK in HEK293 thru an unbiased proteomics approach, and by measuring hANT half-lives in agk null HEK293, undifferentiated pluripotent stem cells, and differentiated cardiomyocytes in comparison to isogenic wild-type controls. Accomplishing Aim 2 will illuminate how AGK modulates ANT expression. This proposal would provide functional and mechanistic insights for AGK and the AGK-ANT1 interaction that we speculate to be involved in the pathogenic basis of Sengers syndrome.
|Program type||Predoctoral Fellowship|
|Effective start/end date||07/01/2016 → 06/30/2018|