The pyridine nucleotides NAD and NADP play vital roles in metabolic conversions as signal transducers and in cellular defence systems. Both coenzymes participate as electron carriers in energy transduction and biosynthetic processes. Their oxidized forms, NAD + and NADP + , have been identified as important elements of regulatory pathways. In particular, NAD + serves as a substrate for ADP-ribosylation reactions and for the Sir2 family of NAD + -dependent protein deacetylases as well as a precursor of the calcium mobilizing molecule cADPr (cyclic ADP-ribose). The conversions of NADP + into the 2 -phosphorylated form of cADPr or to its nicotinic acid derivative, NAADP, also result in the formation of potent intracellular calcium-signalling agents. Perhaps, the most critical function of NADP is in the maintenance of a pool of reducing equivalents which is essential to counteract oxidative damage and for other detoxifying reactions. It is well known that the NADPH/NADP + ratio is usually kept high, in favour of the reduced form. Research within the past few years has revealed important insights into how the NADPH pool is generated and maintained in different subcellular compartments. Moreover, tremendous progress in the molecular characterization of NAD kinases has established these enzymes as vital factors for cell survival. In the present review, we summarize recent advances in the understanding of the biosynthesis and signalling functions of NAD(P) and highlight the new insights into the molecular mechanisms of NADPH generation and their roles in cell physiology.

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...

Although targeting of the death receptors (DRs) DR4 and DR5 still appears a suitable antitumoral strategy, the limited clinical responses to recombinant soluble TNF-related apoptosis inducing ligand (TRAIL) necessitate novel reagents with improved apoptotic activity/tumor selectivity. Apoptosis induction by a single-chain TRAIL (scTRAIL) molecule could be enhanced >10-fold by generation of epidermal growth factor receptor (EGFR)-specific scFv-scTRAIL fusion proteins. By forcing dimerization of scFv-scTRAIL based on scFv linker modification, we obtained a targeted scTRAIL composed predominantly of dimers (Db-scTRAIL), exceeding the activity of nontargeted scTRAIL ∼100-fold on Huh-7 hepatocellular and Colo205 colon carcinoma cells. Increased activity of Db-scTRAIL was also demonstrated on target-negative cells, suggesting that, in addition to targeting, oligomerization equivalent to an at least dimeric assembly of standard TRAIL per se enhances apoptosis signaling. In the presence of apoptosis sensitizers, such as the proteasomal inhibitor bortezomib, Db-scTRAIL was effective at picomolar concentrations in vitro (EC50 ∼2 × 10−12 M). Importantly, in vivo, Db-scTRAIL was well tolerated and displayed superior antitumoral activity in mouse xenograft (Colo205) tumor models. Our results show that both targeting and controlled dimerization of scTRAIL fusion proteins provides a strategy to enforce apoptosis induction, together with retained tumor selectivity and good in vivo tolerance.

The TNF-related apoptosis-inducing ligand (TRAIL) is a powerful inducer of apoptosis in tumor cells; however, clinical studies with recombinant soluble TRAIL were rather disappointing. Here, we developed TRAIL-functionalized liposomes (LipoTRAIL, LT) to mimic membrane-displayed TRAIL for efficient activation of death receptors DR4 and DR5 and enhanced induction of apoptosis, which were combined with an anti-EGFR single-chain Fv fragment (scFv) for targeted delivery to EGFR-positive tumor cells. These immuno-LipoTRAILs (ILTs) bound specifically to EGFR-expressing cells (Colo205) and exhibited increased cytotoxicity compared with that of nontargeted LTs. Compared to that of the soluble TRAIL, the plasma half-life of the functionalized liposomes was strongly extended, and increased antitumor activity of LT and ILT was demonstrated in a xenograft tumor model. Thus, we established a multifunctional liposomal TRAIL formulation (ILT) with improved pharmacokinetic and pharmacodynamic behavior, characterized by targeted delivery and increased induction of apoptosis due to multivalent TRAIL presentation.

Background:The mechanisms through which TRAILR4 interferes with proapoptotic signaling are not conclusively elucidated. Results: TRAILR4 forms ligand-independent heterodimers with TRAIL death receptors, thereby inhibiting both pro-and anti-apoptotic signaling. Conclusion: TRAILR4 exerts a dominant negative effect on TRAILR1 through the PLAD-mediated formation of mixed receptor complexes. Significance: Understanding the mechanism of TRAILR4-mediated apoptosis-inhibition can be advantageous for the development of new TRAIL receptor agonists for cancer therapy.

Nicotinamide adenine dinucleotide phosphate (NADP) is a critical cofactor during metabolism, calcium signaling, and oxidative defense, yet how animals regulate their NADP pools in vivo and how NADP-synthesizing enzymes are regulated have long remained unknown. Here we show that expression of Nadk, an NAD + kinase-encoding gene, governs NADP biosynthesis in vivo and is essential for development in Xenopus frog embryos. Unexpectedly, we found that embryonic Nadk expression is dynamic, showing cell type-specific up-regulation during both frog and sea urchin embryogenesis. We analyzed the NAD kinases (NADKs) of a variety of deuterostome animals, finding two conserved internal domains forming a catalytic core but a highly divergent N terminus. One type of N terminus (found in basal species such as the sea urchin) mediates direct catalytic activation of NADK by Ca 2+ /calmodulin (CaM), whereas the other (typical for vertebrates) is phosphorylated by a CaM kinase-dependent mechanism. This work indicates that animal NADKs govern NADP biosynthesis in vivo and are regulated by evolutionarily divergent and conserved CaM-dependent mechanisms. + and reduced form NADPH) is one of the most fundamental metabolic molecules inside cells. NADPH serves essential functions in reductive biosyntheses, detoxification processes (mediated by cytochrome P450), and the regeneration of oxidative defense systems. Moreover, it acts as an electron donor for NADPH oxidases, which generate reactive oxygen species for immune cell respiratory bursts and for redox signaling (1). NADP + can also be converted to NAADP, a potent intracellular calcium messenger (2). However, despite its importance for cellular function, surprisingly little is known about how animals regulate their cellular NADP pools.Earlier work has shown that animals use vitamin B3 to produce NAD, and in vitro studies have established that NAD can be phosphorylated to yield NADP by NAD kinase (NADK) enzymes using ATP as a phosphoryl donor (3-5) (Fig. 1A). However, there have been no in vivo studies examining the function of animal NADK genes, and thus whether NADK is required for sustaining NADP levels in vivo has remained unknown.In addition, the mechanisms governing the regulation of the NADK enzyme are not well-understood. Previous studies, however, have demonstrated a species-dependent differential response of NADK activity following exposure to calcium/calmodulin (Ca 2+ /CaM). For instance, NADK activity in plant extracts is increased by the addition of Ca 2+ /CaM (6). However, none of the three recombinantly expressed NADK isoforms from Arabidopsis thaliana were responsive to Ca 2+ /CaM (7,8). In animals, a Ca 2+ / CaM-dependent rise of NADP following sea urchin egg fertilization was observed more than 30 y ago, although the molecular mechanisms underlying this phenomenon have not been elucidated.We previously identified and characterized the human NADK encoded by the gene Nadk (1, 9), finding its enzymatic activity insensitive to Ca 2+ /CaM. Thus, the interaction between the N...

No abstract

The influence of 3D microenvironments on apoptosis susceptibility remains poorly understood. Here, we studied the susceptibility of cancer cell spheroids, grown to the size of micrometastases, to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Interestingly, pronounced, spatially coordinated response heterogeneities manifest within spheroidal microenvironments: In spheroids grown from genetically identical cells, TRAIL-resistant subpopulations enclose, and protect TRAIL-hypersensitive cells, thereby increasing overall treatment resistance. TRAIL-resistant layers form at the interface of proliferating and quiescent cells and lack both TRAILR1 and TRAILR2 protein expression. In contrast, oxygen, and nutrient deprivation promote high amounts of TRAILR2 expression in TRAIL-hypersensitive cells in inner spheroid layers. COX-II inhibitor celecoxib further enhanced TRAILR2 expression in spheroids, likely resulting from increased ER stress, and thereby re-sensitized TRAIL-resistant cell layers to treatment. Our analyses explain how TRAIL response heterogeneities manifest within well-defined multicellular environments, and how spatial barriers of TRAIL resistance can be minimized and eliminated.