Mechanistic studies of E3 Ub ligase in peroxisomes and mitochondria

About This Grant

Summary Given the central role of mitochondrial dysfunction in disease and accumulating data linking peroxisomal dysfunction with disease development, understanding the mitochondrial arm of the UPS and its crosstalk with other organelles, such as peroxisomes, is critical. With current trends toward the aging population, developing therapies for these diseases is a critical need. Strategies primarily aimed at neutralizing toxic reactive oxygen species (ROS) have only partially succeeded. Thus, new treatment options with independent modes of action are needed to improve the success rate of current therapies. The proposed work directly responds to this need and will provide critical insights into the regulation and function of mitochondrial and peroxisomal quality control and biogenesis pathways. Extensive evidence supports the vital role of the ubiquitin (Ub) proteasome system (UPS) in mitochondrial function. Mitochondrial ubiquitination requires factors, such as the prototypical outer mitochondrial membrane (OMM) E3 Ub ligase MARCH5, that uniquely target many mitochondrial pathways. Our new data indicate that MARCH5 activity is also vital for peroxisomes. These organelles host diverse oxidative reactions and play an essential role in fatty acid β-oxidation to generate energy intermediates for ATP production by oxidative phosphorylation (OXPHOS). The preliminary data also show that a subset of MARCH5 localizes to the peroxisomes and controls the abundance of peroxisomal proteins, peroxisome size, and maturation. We will investigate the role of MARCH5 in the biogenesis of mitochondria-derived pre-peroxisomes and in protecting mitochondria against the toxic effects of abnormal accumulation of peroxisomal proteins in the mitochondria when peroxisome biogenesis is defective. A model that we recently developed in which cells exclusively rely on oxidative phosphorylation (OXPHOS) for ATP generation shows that bioenergetic shift from glycolysis to OXPHOS induces (i) degradation of MARCH5 client proteins associated with (ii) increased expression of the OXPHOS proteins, (iii) enhanced capacity of mitochondria to generate ATP, and (iv) proliferation of peroxisomes. These changes were reduced in MARCH5-/- cells and differently affected by distinct MARCH5 mutants. We will test the central hypothesis that, through its mitochondrial and peroxisomal functions, MARCH5 controls the metabolic adaptation of the cell to varying energy substrates. We combine biochemical, state-of- the-art imaging, and gene editing methods to address the following questions: (1) What is the role, cofactors, and mechanism of MARCH5 in peroxisomal biogenesis? (2) What domains and cofactors control MARCH5 specificity toward distinct mitochondrial and peroxisomal proteins and modes of protein degradation? (3) How, through MARCH5 activity, does the bioenergetic status of the mitochondria control cellular energy balance and sensitivity to cell death in healthy cells and cells from "peroxisome-biogenesis" disease-affected patients?