Abstract
The redox states of NAD and NADP are linked to each other in the mitochondria thanks to the enzyme nicotinamide nucleotide transhydrogenase (NNT) which, by utilizing the mitochondrial membrane potential (mΔΨ), catalyzes the transfer of redox potential between these two coenzymes, reducing one at the expense of the oxidation of the other. In order to define NNT reaction direction in CF cells, NNT activity under different redox states of cell has been investigated. Using spectrophotometric and western blotting techniques, the presence, abundance and activity level of NNT were determined. In parallel, the levels of NADPH and NADH as well as of mitochondrial and cellular ROS were also quantified. CF cells showed a 70% increase in protein expression compared to the Wt sample; however, regarding NNT activity, it was surprisingly lower in CF cells than healthy cells (about 30%). The cellular redox state, together with the low mΔΨ, pushes to drive NNT reverse reaction, at the expense of its antioxidant potential, thus consuming NADPH to support NADH production. At the same time, the reduced NNT activity prevents the NADH, produced by the reaction, from causing an explosion of ROS by the damaged respiratory chain, in accordance with the reduced level of mitochondrial ROS in NNT-loss cells. This new information on cellular bioenergetics represents an important building block for further understanding the molecular mechanisms responsible for cellular dysfunction in cystic fibrosis.
Highlights
Nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP) act as “electron carriers”
We found that CF cells have increased NADPH oxidase (NOX) activity and decreased glutathione reductase (GR) activity, both NADPH consuming enzymes (NCE), but with opposite role: if NOX utilizes NADPH to produce ROS, NADPH serves to recover the GSH level from GSSG in the GR reaction
We proved that both defective CFTR and NOX/GR activity imbalance contribute to NADPH and GSH level decrease and ROS overproduction in CF cells
Summary
Nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP) act as “electron carriers”. In this regard, it should be borne in mind that neither NAD(H) nor NADP(H) are transported across intracellular membranes [10,11], and multistep shuttles involving compartmentalized redox reactions are used to transfer electrons between the mitochondria and cytosol [12]. It should be borne in mind that neither NAD(H) nor NADP(H) are transported across intracellular membranes [10,11], and multistep shuttles involving compartmentalized redox reactions are used to transfer electrons between the mitochondria and cytosol [12] This organization facilitates the maintenance of different NADH/NAD and NADPH/NADP+ ratios in each subcellular location and allows for the execution of compartment-specific metabolic processes
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