Mitochondrial Biogenesis in Health and Disease

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 DESCRIPTION (provided by applicant): Mitochondria allow our cells to use oxidative phosphorylation (OXPHOS) as a highly efficient way to generate ATP. The inner membrane-embedded OXPHOS system enzymes are multimeric complexes composed of proteins from two different genetic origins, namely the nuclear and the mitochondrial DNA. Nucleus-encoded proteins are synthesized in cytoplasmic ribosomes and imported into mitochondria. The mitochondrion-encoded proteins, usually catalytic core subunits of the complexes, are synthesized into distinct mitochondrial ribosomes. We have developed a scientific research program aiming at understanding the molecular mechanisms underlying the assembly of OXPHOS complexes and mitochondrial ribosomes. Our studies focus on the assembly of OXPHOS enzymes as individual complexes, with a focus on cytochrome c oxidase, the terminal oxidase of the mitochondrial respiratory chain. We have uncovered translational regulation, heme and redox sensing processes ruling COX biogenesis in yeast and/or human cells. Additionally, we aim to understand how OXPHOS complexes form higher order assemblies known as supercomplexes or respirasomes. We have already reported the first respirasome assembly pathway in human cells. Dedicated chaperone-like factors are required to assist and regulate complex and supercomplex assembly in mitochondria. While many have been already identified, their specific functions remain to be precisely characterized. In another aspect of our work we address the question of how mitochondrial ribosomes assemble into functional protein synthesis machineries. We have recently identified the first two DEAD-box RNA helicases acting on the assembly of the large mitoribosome subunit in yeast and human cells. Also, we have gained insight into the compartmentalization of the ribosome assembly process by identifying matrix RNA granules as the "mitochondriolus" (per equivalence to the nucleoulus). Despite high-resolution cryo-EM structures of yeast and human ribosomes have been recently reported, the pathway of mitoribosome biogenesis and the factors involved are poorly characterized. Studies outlined in this proposal will involve yeast genetics, gene disruption in human cells using new gene-editing techniques (TALENs and CRISPRs) and mechanistic biochemistry in yeast, human cell lines, isolated mitochondria and purified native and recombinant proteins to gain insight into the role/s of OXPHOS complex, supercomplex and mitoribosome assembly factors. To further fill the gaps, we are implementing innovative strategies to identify new assembly factors and to define the biosynthetic pathways under study, by applying quantitative mass spectrometry and structural approaches. The analysis of the principles of the biogenesis process and the activities of the assembly factors is of central importance for our understanding of the molecular basis of human mitochondrial disorders.