NAD Kinase Levels Control the NADPH Concentration in Human Cells

Abstract

NAD kinases (NADKs) are vital, as they generate the cellular NADP pool. As opposed to three compartment-specific isoforms in plants and yeast, only a single NADK has been identified in mammals whose cytoplasmic localization we established by immunocytochemistry. To understand the physiological roles of the human enzyme, we generated and analyzed cell lines stably deficient in or overexpressing NADK. Short hairpin RNAmediated down-regulation led to similar (about 70%) decrease of both NADK expression, activity, and the NADPH concentration and was accompanied by increased sensitivity toward H 2 O 2 . Overexpression of NADK resulted in a 4 -5-fold increase in the NADPH, but not NADP ؉ , concentration, although the recombinant enzyme phosphorylated preferentially NAD ؉ . Surprisingly, NADK overexpression and the ensuing increase of the NADPH level only moderately enhanced protection against oxidant treatment. Apparently, to maintain the NADPH level for the regeneration of oxidative defense systems human cells depend primarily on NADP-dependent dehydrogenases (which re-reduce NADP ؉ ), rather than on a net increase of NADP. The stable shifts of the NADPH level in the generated cell lines were also accompanied by alterations in the expression of peroxiredoxin 5 and Nrf2. Because the basal oxygen radical level in the cell lines was only slightly changed, the redox state of NADP may be a major transmitter of oxidative stress.Recent investigations have established the pyridine nucleotides not only as key molecules for metabolic conversions, but also as critical regulators of major cellular events. In particular, NAD ϩ appears to act as a versatile molecule with both messenger and bioenergetic functions (1). Whereas NAD is largely present in its oxidized state (NAD ϩ ), its phosphorylated counterpart, NADP, is predominantly found in its reduced form, as NADPH (2, 3). Indeed, the most prominent function of NADP appears to be the maintenance of a pool of reducing equivalents for metabolic systems that in one way or another protect the cell from damage. Most prominently, NADPH is essential to the regeneration of all known oxidative defense systems, such as glutathione, thioredoxin, and peroxiredoxins. Moreover, detoxifying pathways (for example, cytochromes P450 and catalase) as well as the NADPH oxidase, which catalyzes the "oxidative burst" as part of the immune response, depend on NADPH. Interestingly, the redox properties of the NAD ϩ / NADH and NADP ϩ /NADPH couples are similar, but their functions are largely divergent. Apparently, a major reason for this separation is the possibility to maintain one pool, namely NADP, in its reduced form to assure an immediate regeneration of the defense systems following oxidative assaults. In this role, NADPH is of vital importance, because survival following oxidative stress, which accompanies a multitude of pathological states such as inflammatory processes or ischemia/reperfusion injury, depends primarily on the capacity of the defense systems. Nevertheless, surprisingly little i...

E. coli NfsA: an alternative nitroreductase for prodrug activation gene therapy in combination with CB1954

“…The maximum benefit of NfsA would occur if the intracellular NADPH concentration greatly exceeded that of NADH; conversely as indicated by Figure 3A, a preponderance of NADH would favour NfsB. We could find very few reports of cofactor concentrations in cells or tissues; one study measured approximately 15 -44 mM NADPH in three B-cell lines (Hancock et al, 1989), and HPLC analysis of HEK293 cells (adenovirustransformed human embryonic kidney cells) showed NADH levels that appeared approximately 1 -2 times greater than NADPH (Pollak et al, 2007). To compare cancer cell sensitisation with CB1954 by the enzymes after gene transfer, we used replicationdefective adenoviruses AdSV042 (expressing NfsA) compared with the similar adenovirus, CTL102 (expressing NfsB).…”

Structure and Function of NAD Kinase and NADP Phosphatase: Key Enzymes That Regulate the Intracellular Balance of NAD(H) and NADP(H)

“…So far, it has been reported that exogenous oxidative stress induces transcription of NADK1 in A. thaliana and POS5 in S. cerevisiae, whereas oxidative stress has no influence on the activity of NAD kinase in human cells. 37,39,46) This might reflect the fact that A. thaliana and S. cerevisiae contain three NAD kinase homolog genes while humans have only one gene. 46) In other words, the regulation of NAD kinase in humans might differ from that of NADK1 in A. thaliana and POS5 in S. cerevisiae.…”

“…46) Briefly, in NADK(+) cells that show approximately 180-foldhigher NAD kinase activity than control cells, the NADP þ level is not prominently altered, and the NADPH level increases by 4-to 5-fold, not 180-fold, suggesting that NADP þ is immediately reduced to NADPH by specific dehydrogenases. The authors suggested that one possible reason for the increase in the NADPH level by only 4-to 5-fold is that human NAD kinase activity is inhibited by NADPH.…”

“…Furthermore, human cell lines deficient in or overexpressing human NAD kinase have been established, and these have provided insight into the in vivo role of human NAD kinase. 46) 1. Physiological functions of NAD kinases in S. cerevisiae In S. cerevisiae, Pos5p is located in the mitochondria, 3) and Utr1p is localized in the cytosol.…”

Nicotinic acid inducing G0/G1 cell cycle arrest and apoptosis in Ehrlich ascites carcinoma cells in vivo

“…Niacin also called nicotinic acid is a precursor for the synthesis of important coenzymes nicotinamide adenine dinucleotide [NAD (H)] and nicotinamide adenine dinucleotide phosphate [NADP (H)] (Pollak et al, 2007). In addition to its role as a co-factor in redox reactions and as a regulator of the redox state (NAD+/NADH), NAD+ is a substrate for the nuclear enzyme poly (ADP-ribose) polymerase (PARP) which play a critical role in genomic stability (Kim et al, 2004).…”