Cox17 is an assembly factor that participates in early cytochrome c oxidase (COX, CcO) assembly stages. Cox17 shuttles copper ions from the cytosol to the mitochondria and, together with Sco1 and Sco2, provides copper ions to the Cox1 and Cox2 mitochondrially encoded subunits. In Saccharomyces cerevisiae, Cox17 also modulates mitochondrial membrane architecture due to the interaction of Cox17 with proteins of the MICOS complex (mitochondrial contact site and cristae organizing system). There is currently no data regarding the impact of long-term Cox17 deficiency in human cells. Here, we present construction and characterization of three stable COX17 shRNA-downregulated HEK293 cell lines that have less than 10 % of the residual Cox17 protein level. Cox17-depleted cell lines exhibited decreased intramitochondrial copper content, decreased CcO subunit levels (Cox1, Cox4 and Cox5a) and accumulation of CcO subcomplexes. Similarly to yeast cells, mitochondria in Cox17-downregulated HEK293 cell lines exhibited ultrastructural changes including cristae reduction and mitochondrial swelling. Characterization of the molecular pathogenesis of long-term Cox17 deficiency complements our knowledge of the mitochondrial copper metabolism and assembly of cytochrome c oxidase in human cells.
- MeSH
- HEK293 buňky MeSH
- lidé MeSH
- malá interferující RNA genetika MeSH
- měď metabolismus MeSH
- mitochondriální proteiny genetika metabolismus MeSH
- mitochondrie metabolismus ultrastruktura MeSH
- respirační komplex IV genetika metabolismus MeSH
- transportéry mědi genetika metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Publikační typ
- abstrakt z konference MeSH
Mammalian CcO (cytochrome c oxidase) is a hetero-oligomeric protein complex composed of 13 structural subunits encoded by both the mitochondrial and nuclear genomes. To study the role of nuclear-encoded CcO subunits in the assembly and function of the human complex, we used stable RNA interference of COX4, COX5A and COX6A1, as well as expression of epitope-tagged Cox6a, Cox7a and Cox7b, in HEK (human embryonic kidney)-293 cells. Knockdown of Cox4, Cox5a and Cox6a resulted in reduced CcO activity, diminished affinity of the residual enzyme for oxygen, decreased holoCcO and CcO dimer levels, increased accumulation of CcO subcomplexes and gave rise to an altered pattern of respiratory supercomplexes. An analysis of the patterns of CcO subcomplexes found in both knockdown and overexpressing cells identified a novel CcO assembly intermediate, identified the entry points of three late-assembled subunits and demonstrated directly the essential character as well as the interdependence of the assembly of Cox4 and Cox5a. The ectopic expression of the heart/muscle-specific isoform of the Cox6 subunit (COX6A2) resulted in restoration of both CcO holoenzyme and activity in COX6A1-knockdown cells. This was in sharp contrast with the unaltered levels of COX6A2 mRNA in these cells, suggesting the existence of a fixed expression programme. The normal amount and function of respiratory complex I in all of our CcO-deficient knockdown cell lines suggest that, unlike non-human CcO-deficient models, even relatively small amounts of CcO can maintain the normal biogenesis of this respiratory complex in cultured human cells.
The impact of point mutations in mitochondrial tRNA genes on the amount and stability of respiratory chain complexes and ATP synthase (OXPHOS) has been broadly characterized in cultured skin fibroblasts, skeletal muscle samples, and mitochondrial cybrids. However, less is known about how these mutations affect other tissues, especially the brain. We have compared OXPHOS protein deficiency patterns in skeletal muscle mitochondria of patients with Leigh (8363G>A), MERRF (8344A>G), and MELAS (3243A>G) syndromes. Both mutations that affect mt-tRNA(Lys) (8363G>A, 8344A>G) resulted in severe combined deficiency of complexes I and IV, compared to an isolated severe defect of complex I in the 3243A>G sample (mt-tRNA(LeuUUR). Furthermore, we compared obtained patterns with those found in the heart, frontal cortex, and liver of 8363G>A and 3243A>G patients. In the frontal cortex mitochondria of both patients, the patterns of OXPHOS deficiencies differed substantially from those observed in other tissues, and this difference was particularly striking for ATP synthase. Surprisingly, in the frontal cortex of the 3243A>G patient, whose ATP synthase level was below the detection limit, the assembly of complex IV, as inferred from 2D-PAGE immunoblotting, appeared to be hindered by some factor other than the availability of mtDNA-encoded subunits.
- MeSH
- 2D gelová elektroforéza MeSH
- dítě MeSH
- fatální výsledek MeSH
- financování organizované MeSH
- imunoblotting MeSH
- kinetika MeSH
- kosterní svalová vlákna enzymologie patologie MeSH
- kosterní svaly enzymologie patologie MeSH
- lidé MeSH
- mitochondriální protonové ATPasy metabolismus MeSH
- mitochondrie enzymologie genetika MeSH
- mladiství MeSH
- mozek enzymologie MeSH
- mutace genetika MeSH
- novorozenec MeSH
- orgánová specificita MeSH
- oxidativní fosforylace MeSH
- podjednotky proteinů metabolismus MeSH
- RNA transferová Lys genetika MeSH
- spotřeba kyslíku MeSH
- transport elektronů genetika MeSH
- Check Tag
- dítě MeSH
- lidé MeSH
- mladiství MeSH
- mužské pohlaví MeSH
- novorozenec MeSH
- ženské pohlaví MeSH
- Publikační typ
- kazuistiky MeSH
- srovnávací studie MeSH
The Oxa1 protein is a founding member of the evolutionarily conserved Oxa1/Alb3/YidC protein family, which is involved in the biogenesis of membrane proteins in mitochondria, chloroplasts and bacteria. The predicted human homologue, Oxa1l, was originally identified by partial functional complementation of the respiratory growth defect of the yeast oxa1 mutant. Here we demonstrate that both the endogenous human Oxa1l, with an apparent molecular mass of 42 kDa, and the Oxa1l-FLAG chimeric protein localize exclusively to mitochondria in HEK293 cells. Furthermore, human Oxa1l was found to be an integral membrane protein, and, using two-dimensional blue native/denaturing PAGE, the majority of the protein was identified as part of a 600-700 kDa complex. The stable short hairpin (sh)RNA-mediated knockdown of Oxa1l in HEK293 cells resulted in markedly decreased steady-state levels and ATP hydrolytic activity of the F(1)F(o)-ATP synthase and moderately reduced levels and activity of NADH:ubiquinone oxidoreductase (complex I). However, no significant accumulation of corresponding sub-complexes could be detected on blue native immunoblots. Intriguingly, the achieved depletion of Oxa1l protein did not adversely affect the assembly or activity of cytochrome c oxidase or the cytochrome bc(1) complex. Taken together, our results indicate that human Oxa1l represents a mitochondrial integral membrane protein required for the correct biogenesis of F(1)F(o)-ATP synthase and NADH:ubiquinone oxidoreductase.
- MeSH
- 2D gelová elektroforéza MeSH
- adenosintrifosfát metabolismus MeSH
- financování organizované MeSH
- fluorescenční protilátková technika MeSH
- hydrolýza MeSH
- imunoblotting MeSH
- imunoglobulin G MeSH
- imunoprecipitace MeSH
- jaderné proteiny MeSH
- kultivované buňky MeSH
- kur domácí MeSH
- lidé MeSH
- malá interferující RNA farmakologie MeSH
- mitochondriální proteiny antagonisté a inhibitory genetika imunologie MeSH
- mitochondriální protonové ATPasy antagonisté a inhibitory biosyntéza MeSH
- mitochondrie metabolismus MeSH
- respirační komplex I antagonisté a inhibitory biosyntéza MeSH
- respirační komplex III metabolismus MeSH
- respirační komplex IV MeSH
- subcelulární frakce MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- MeSH
- dítě MeSH
- hypertrofická kardiomyopatie MeSH
- kardiomyopatie MeSH
- lidé MeSH
- mitochondriální DNA MeSH
- mitochondriální myopatie MeSH
- mitochondriální nemoci MeSH
- Check Tag
- dítě MeSH
- lidé MeSH
- mužské pohlaví MeSH
- Publikační typ
- kazuistiky MeSH
- MeSH
- acidóza laktátová MeSH
- biochemie MeSH
- chemické techniky analytické MeSH
- elektroforéza MeSH
- kojenec MeSH
- lidé MeSH
- mitochondriální DNA MeSH
- mitochondriální nemoci MeSH
- mitochondriální protonové ATPasy MeSH
- mutace genetika MeSH
- novorozenec MeSH
- předškolní dítě MeSH
- statistika jako téma MeSH
- Check Tag
- kojenec MeSH
- lidé MeSH
- mužské pohlaví MeSH
- novorozenec MeSH
- předškolní dítě MeSH
- Publikační typ
- kazuistiky MeSH
