Nuskova 2015 Abstract MiPschool London 2015

Insufficient energy provision or increased oxidative stress – what matters more in ATP synthase deficiencies?

Link:

Nuskova Hana, Kovalcikova J, Pecinova A, Pecina P, Houstek J, Mracek T (2015)

Event: MiPschool London 2015

ATP produced by the mitochondrial FoF1-ATP synthase represents a major source of energy for aerobic organisms. Unsurprisingly, ATP synthase deficiencies are associated with severe pathologic phenotypes. To shed light on the functional consequences of ATP synthase deficiencies, we utilised a model of HEK293 cell line and explored the effect of RNAi mediated knockdown of the three subunits (γ, δ and ε) forming the central stalk of the enzyme connecting Fo and F1 domains.

For functional evaluations of ATP synthase deficiencies, 10 stable knockdown clones with down-regulated subunits γ (ATP5C1 gene), δ (ATP5D gene), or ε (ATP5E gene) have been selected. The protein content of ATP synthase subunit α among the knockdown clones covers the range of 40–100 % as compared to controls. Further characterization of these clones revealed 2–78 % oligomycin-sensitive ATPase hydrolytic activity that parallels a decrease in the content of fully assembled ATP synthase complex.

Two aspects of cellular energetics have been examined in detail, specifically respiration and glycolysis, using the Seahorse XFe24 analyser. Our results indicate that the clones with less than 30 % of residual ATPase activity switched their metabolism to enhanced glycolysis. There is a decrease in their basal respiration rate relatively to their respiratory capacity (47 vs 61 % in controls) and in parallel, their basal glycolytic rates utilise by up to 20 % more of their glycolytic capacity. These findings clearly demonstrate metabolic adaptations of these cells. On the other hand, the clones with more than 30 % residual ATPase activity displayed a change neither in the respiration nor in their basal glycolytic rate.

In the case of ATP synthase deficiency, the mitochondrial membrane potential is expected to rise, which would then stimulate the production of reactive oxygen species (ROS). Indeed, the γ knockdown clones with a very low residual ATPase activity exhibit elevated ROS production. With respect to the role of the oxidative stress in ATP synthase deficiencies, we aim to examine oxidative damage of cell structures and the content of antioxidant enzymes.

In conclusion, using these model clones, we are planning on investigating the effect of ATP synthase deficiency on the mitochondrial energetics, oxidative stress, energy state, and cell viability and define the threshold residual activity of ATP synthase for the presentation of pathological phenotype. At this moment, our data suggest that the threshold for metabolic remodelling equals to approximately 30 % of ATPase activity.

• Keywords: ATP synthase deficiencies

• O2k-Network Lab: CZ Prague Houstek J, CZ Prague Kalous M

Labels: MiParea: mt-Membrane 

Organism: Human  Tissue;cell: HEK 

Regulation: ATP production  Coupling state: ROUTINE, OXPHOS 

Affiliations

Dept Bioenergetics, Inst Physiol Czech Acad Sci, Prague, Czech Republic. - [email protected]

Support

This project is supported by the Grant Agency of Charles Univ (grant 1160214) and the Czech Science Foundation (P303/12/1363).