Discovering New Proteins and Pathways Driving Mitochondrial Bioenergetics
Our research is focused on identifying critical regulators of the mitochondrial respiratory chain (MRC) to understand the molecular basis of mitochondrial dysfunction in human disorders. Contrary to general belief, many aspects of mitochondrial energy metabolism are poorly understood. For example, what factors are required for the formation and stabilization of MRC complexes and supercomplexes? What role does the lipid milieu play in MRC complex assembly? The following projects will address these important questions using the tools of genomics, genetics, and biochemistry in human cells, zebrafish, and the yeast, Saccharomyces cerevisiae.
Discovery and Characterization of Novel MRC Biogenesis Genes
In order to systematically discover novel MRC biogenesis genes, we have used an integrative approach based on clues from evolutionary history, human genetics, and organelle proteomics to shortlist a subset of uncharacterized mitochondrial proteins that are conserved between yeast and humans. We predict that many of these proteins are involved in fundamental functions of mitochondria, including energy production by the MRC. We will experimentally validate these candidate genes using a nutrient-sensitized screening platform that we have developed. The MRC biogenesis genes identified as screening hits can act at any step in the canonical pathway of MRC biogenesis, including mitochondrial DNA maintenance and expression, addition of cofactors to the MRC apoenzymes, and formation of the supercomplexes. We will assign novel MRC genes to these specific steps using a battery of bioenergetic, biochemical and molecular assays. Identifying novel MRC genes will not only provide a protein parts-list to understand the fundamental pathway of MRC biogenesis, but will also be a valuable database for identifying potential mitochondrial disease-candidate genes.
Phospholipid Requirements for MRC Function and Formation
Because MRC complexes are integral to the inner mitochondrial membrane, it is not surprising that their function and assembly depends on the membrane lipid composition. Indeed, a large body of data has identified a critical role of cardiolipin (CL), a signature mitochondrial phospholipid, in MRC function and formation. However, until recently, it was not clear if MRC function specifically depended on CL because the roles of more abundant mitochondrial phospholipids, phosphatidylcholine (PC) and phosphatidylethanolamine (PE), were not investigated. Our recent work using using yeast mutants of PE and PC biosynthesis suggests a specific requirement for non-bilayer forming phospholipids (PE and CL) in MRC biogenesis. Interestingly, we also found that stimulating the cytosolic pathway of PE biosynthesis by exogenous supplementation of ethanolamine could compensate for the loss of mitochndrial PE biosynthesis, suggesting the existence of a mitochondrial PE-import pathway. Recently, we have discovered that a clinically used compound, meclizine, specifically inhibits the synthesis of non-mitochondrial PE and attenuates MRC activity, raising the intriguing possibility that non-mitochondrial PE can modulate MRC function. Using meclizine in combination with genetic mutants of phospholipid biosynthetic pathways, we will determine the contribution of non-mitochondrial phospholipids in MRC structure and activity. Understanding the biochemical basis of phospholipid:MRC protein interactions will allow manipulation of MRC function, which is clinically important since modulating MRC activity has been shown to be efficacious in the treatment of cardiovascular and neurodegenerative disorders.