Thursday, November 21, 2024
11/21/2024
PROJECT SUMMARY/ABSTRACT Mitochondria are centers of metabolism whose activities need to be calibrated to meet changing cellular needs. General dysfunction of these organelles is implicated in many common human disorders, including metabolic syndrome, type 2 diabetes, obesity, non-alcoholic fatty liver disease, heart failure, various cancers and neurodegenerative diseases, and general metabolic inflexibility, most often through unclear means. Defining the pathogenic mitochondrial alterations that contribute to these metabolic disorders and devising new therapeutic strategies to rectify them represent principal challenges in mitochondrial medicine. A potential contributor to this dysfunction is aberrant intra-mitochondrial protein phosphorylation—a process recognized as critical for pyruvate dehydrogenase regulation for more than 50 years, but relatively unexplored otherwise. Recent efforts from our laboratory and others have now revealed that mitochondrial proteins are replete with dynamic phosphorylation that changes reproducibly between healthy and diseased states, and that phosphorylation can alter the activities of proteins involved in core metabolic pathways. Furthermore, we have connected select protein dephosphorylation events to the poorly characterized matrix protein phosphatase PPTC7 and discovered that PPTC7 disruption in mice causes profound metabolic defects and neonatal death. Given these emerging findings, the premise of this project is that reversible phosphorylation may be widely important in calibrating mitochondrial metabolism, and that its mismanagement could contribute to the pathophysiology of mitochondria- related disorders. Rigorous new efforts to reveal how phosphorylation affects mitochondrial protein function and to define the phosphatases that target each site may ultimately enable a new therapeutic strategy focused on manipulation of the mitochondrial phosphorylation network. To this end, we have now extended our phosphoproteomic analyses to 10 mitochondrial phosphatase knockdown lines using a CRISPRi system. This rich dataset forges many unique connections between phosphatases and phosphoproteins and forms the foundation for the further work proposed here. Based on these findings, we propose 1) to establish the functional impact of phosphorylation on putative PPTC7 and HDHD5 substrates involved in core mitochondrial catabolic pathways, including branched chain amino acid and fatty acid catabolism, and 2) to define the role of PGAM5 in regulating mitochondrial cristae architecture by managing an extensive set of phosphorylation events on subunits of the mitochondrial contact site and cristae organizing system (MICOS). Altogether, through a comprehensive approach that combines mammalian physiology, omics-level analyses, and rigorous biochemistry, we aim to make definitive connections between mitochondrial phosphatases and their substrates, establish a broad framework for understanding the role of this post-translation modification in calibrating mitochondrial activities, and ultimately pave the way for a new therapeutic strategy to rectify mitochondrial dysfunction.
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