Kovarova 2015 Abstract MiP2015

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Tissue- and species-specific differences in cytochrome c oxidase assembly induced by SURF1 defects.


Kovarova N, Pecina P, Nuskova H, Vrbacky M, Zeviani M, Mracek T, Viscomi C, Houstek J (2015)

Event: MiP2015

In this study we focused on distinct biochemical phenotype of cytochrome c oxidase (COX) deficiency in mouse and humans due to the absence of SURF1 protein, an important ancillary factor of COX biogenesis, which exact function is not known yet. While mutations in SURF1 gene lead to a fatal neurodegenerative mitochondrial disorder in humans, the Leigh syndrome, SURF1-/--/- knockout in mouse results in surprisingly mild COX deficiency and no neurodegenerative disorder [1,2]. The aim of our study was to find out interspecies differences in the impaired process of COX biogenesis, from early assembly intermediates to formation of COX supercomplexes with other respiratory enzymes. This was achieved by investigating SURF1-/- mouse tissues and fibroblasts in comparison with patient fibroblasts lacking SURF1 protein due to SURF1 gene mutations.

Isolated mitochondria from control (SURF1+/+) and SURF1-/- mouse tissues and fibroblasts and from human control and SURF1 patient fibroblasts were analyzed using 2D BNE/SDS PAGE, activities of COX and citrate synthase were measured. Doxycycline reversible inhibition and pulse-chase metabolic labeling of mitochondrial DNA encoded subunits were used for investigating of COX biogenesis in SURF1+/+ and SURF1-/- mouse fibroblasts and in control and SURF1 patient fibroblasts.

Our study revealed considerably decreased COX monomer and COX activity in SURF1 patient fibroblasts compared to SURF1-/- mouse tissues/fibroblasts. SURF1-/- mouse tissues/fibroblasts also showed much lower accumulation of COX assembly intermediates on one hand and very low amount of I-III2-IVn COX supercomplex on the other. In contrast, assembled COX was present mainly in I-III2-IVn supercomplex in SURF1 patient fibroblasts where the prominence of COX assembly defect was also apparent from accumulation of incomplete COX assembly intermediates. We subsequently characterized kinetics of COX biogenesis in SURF1 patient and SURF1-/- mouse fibroblasts by doxycycline reversible arrest of mitochondrial translation and 35S-labeling of mtDNA encoded proteins. Doxycycline inhibition and gradual recovery to steady state revealed rather stable proportion between COX monomer and supercomplexes in human control cells, while in SURF1 patient cells COX monomer markedly decreased and formation of supercomplexes was preferred. In SURF1+/+ and SURF1-/- mouse cells, however, the recovery proceeded mainly to the level of COX monomer. Pulse-chase metabolic labeling clearly showed higher stability of COX monomer and faster proteolytic degradation/depletion of accumulated COX assembly intermediates in SURF1-/- mouse fibroblasts, while more persistent COX assembly intermediates prevailed over the gradually decreasing signal of COX monomer in SURF1 patient cells.

Our experiments clearly demonstrate crucial importance of the SURF1 protein for effective COX biogenesis in human cells, whereas its absence is much better tolerated in mouse cells and tissues with faster COX turnover.

O2k-Network Lab: CZ Prague Houstek J

Labels: MiParea: mtDNA;mt-genetics, Comparative MiP;environmental MiP, Patients  Pathology: Neurodegenerative 

Organism: Human, Mouse  Tissue;cell: Fibroblast  Preparation: Isolated mitochondria  Enzyme: Complex II;succinate dehydrogenase 

Event: B1, Poster  MiP2015 


1-Dept Bioenergetics, Inst Physiology Czech Acad Sc, Prague, Czech Republic; 2-Molecular Neurogenetics Unit, Inst Neurologico “C. Besta”, Milan, Italy; 3-RC-Mitochondrial Biol Unit, Addenbrookes Hospital Hills Rd, Cambridge, UK. - [email protected]

References and acknowledgements

  1. Kovarova N, Cizkova Vrbacka A, Pecina P, Stranecky V, Pronicka E, Kmoch S, Houstek J (2012) Adaptation of respiratory chain biogenesis to cytochrome c oxidase deficiency caused by SURF1 gene mutations. Biochim Biophys Acta 1822:1114-24.
  2. Dell'Agnello C, Leo S, Agostino A, Szabadkai G, Tiveron C, Zulian A, Prelle A, Roubertoux P, Rizzuto R, Zeviani M (2007) Increased longevity and refractoriness to Ca(2+)-dependent neurodegeneration in Surf1 knockout mice. Hum Mol Genet 16:431-44.

This work was supported by the Grant Agency of the Czech Republic (14-36804G), Ministry of Education, Youth and Sports of the Czech Republic (ERC CZ: LL1204, RVO:67985823), the Grant Agency of the Ministry of Health of the Czech Republic (NT12370-5) and ERC Advanced Grant FP7-322424.