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.

Public Health Relevance

The goal of this MIRA grant application is to develop a program aiming to understand the biogenesis of mitochondrial membranes, with a focus on (1) oxidative phosphorylation (OXPHOS) individual enzymes and supercomplexes, and (2) mitoribosomes and the mitochondrial translation machinery, as they occur in yeast and human cells. Disorders arising from impaired mitochondrial OXPHOS function or mitochondrial gene expression, including protein synthesis, result in a variety of pathologies including encephalomyopathies and cardiomyopathies. Identifying and characterizing factors required for OXPHOS enzyme assembly, mitoribosome biogenesis and translation, and the pathways in which they act is a prerequisite towards understanding the molecular basis of these disorders.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Unknown (R35)
Project #
1R35GM118141-01
Application #
9071590
Study Section
Special Emphasis Panel (ZGM1-TRN-7 (MR))
Program Officer
Anderson, Vernon
Project Start
2016-06-01
Project End
2021-05-31
Budget Start
2016-06-01
Budget End
2017-05-31
Support Year
1
Fiscal Year
2016
Total Cost
$499,557
Indirect Cost
$174,113
Name
University of Miami School of Medicine
Department
Neurology
Type
Schools of Medicine
DUNS #
052780918
City
Coral Gables
State
FL
Country
United States
Zip Code
33146
Bourens, Myriam; Barrientos, Antoni (2017) Human mitochondrial cytochrome c oxidase assembly factor COX18 acts transiently as a membrane insertase within the subunit 2 maturation module. J Biol Chem 292:7774-7783
Bourens, Myriam; Barrientos, Antoni (2017) A CMC1-knockout reveals translation-independent control of human mitochondrial complex IV biogenesis. EMBO Rep 18:477-494
Kim, Hyun-Jung; Maiti, Priyanka; Barrientos, Antoni (2017) Mitochondrial ribosomes in cancer. Semin Cancer Biol 47:67-81
Timón-Gómez, Alba; Nývltová, Eva; Abriata, Luciano A et al. (2017) Mitochondrial cytochrome c oxidase biogenesis: Recent developments. Semin Cell Dev Biol :
Pérez-Pérez, Rafael; Lobo-Jarne, Teresa; Milenkovic, Dusanka et al. (2016) COX7A2L Is a Mitochondrial Complex III Binding Protein that Stabilizes the III2+IV Supercomplex without Affecting Respirasome Formation. Cell Rep 16:2387-98
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