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...

No abstract

N-terminal acetylation is a major and vital protein modification catalyzed by N-terminal acetyltransferases (NATs). NatF, or Nα-acetyltransferase 60 (Naa60), was recently identified as a NAT in multicellular eukaryotes. Here, we find that Naa60 differs from all other known NATs by its Golgi localization. A new membrane topology assay named PROMPT and a selective membrane permeabilization assay established that Naa60 faces the cytosolic side of intracellular membranes. An Nt-acetylome analysis of NAA60-knockdown cells revealed that Naa60, as opposed to other NATs, specifically acetylates transmembrane proteins and has a preference for N termini facing the cytosol. Moreover, NAA60 knockdown causes Golgi fragmentation, indicating an important role in the maintenance of the Golgi's structural integrity. This work identifies a NAT associated with membranous compartments and establishes N-terminal acetylation as a common modification among transmembrane proteins, a thus-far poorly characterized part of the N-terminal acetylome.

NAD is a vital redox carrier, and its degradation is a key element of important regulatory pathways. NAD-mediated functions are compartmentalized and have to be fueled by specific biosynthetic routes. However, little is known about the different pathways, their subcellular distribution, and regulation in human cells. In particular, the route(s) to generate mitochondrial NAD, the largest subcellular pool, is still unknown. To visualize organellar NAD changes in cells, we targeted poly-(ADP-ribose) polymerase activity into the mitochondrial matrix. This activity synthesized immunodetectable poly(ADPribose) depending on mitochondrial NAD availability. Based on this novel detector system, detailed subcellular enzyme localizations, and pharmacological inhibitors, we identified extracellular NAD precursors, their cytosolic conversions, and the pathway of mitochondrial NAD generation. Our results demonstrate that, besides nicotinamide and nicotinic acid, only the corresponding nucleosides readily enter the cells. Nucleotides (e.g. NAD and NMN) undergo extracellular degradation resulting in the formation of permeable precursors. These precursors can all be converted to cytosolic and mitochondrial NAD. For mitochondrial NAD synthesis, precursors are converted to NMN in the cytosol. When taken up into the organelles, NMN (together with ATP) serves as substrate of NMNAT3 to form NAD. NMNAT3 was conclusively localized to the mitochondrial matrix and is the only known enzyme of NAD synthesis residing within these organelles. We thus present a comprehensive dissection of mammalian NAD biosynthesis, the groundwork to understand regulation of NAD-mediated processes, and the organismal homeostasis of this fundamental molecule.NAD is an essential electron carrier and a key molecule of signaling pathways (1-4). In bioenergetic pathways, NAD is reversibly converted between its oxidized (NAD ϩ ) and reduced (NADH) states, which would not require continuous regeneration. Indeed, when the principal pathway of NAD ϩ synthesis from nicotinic acid (NA) 2 had been established (5), the "case" was nearly closed, because neither additional roles of NAD nor a regulatory importance of its synthesis were suspected. Meanwhile, discoveries of signaling processes in which NAD ϩ is degraded have dramatically changed this view. Signaling conversions of NAD ϩ include the cleavage to nicotinamide (Nam), which is recycled into NAD ϩ synthesis, and a concomitant reaction of the remaining ADP-ribose moiety. NAD ϩ -dependent deacetylases (members of the sirtuin family) and mono-ADP-ribosyltransferases control life span, the biological clock, insulin secretion, and key metabolic enzymes (6 -9). In addition, NAD ϩ represents the substrate for poly(ADP-ribosylation) to regulate DNA repair, transcription, telomerase activity, and chromatin dynamics (10 -12). NAD ϩ is also the precursor of cyclic ADP-ribose and NAADP, potent agents to mobilize calcium from intracellular stores (13). This multitude of NAD ϩ -degrading reactions clearly necessitates permanent re...

Recent research has unraveled a number of unexpected functions of the pyridine nucleotides. In this review, we will highlight the variety of known physiological roles of NADP. In its reduced form (NADPH), this molecule represents a universal electron donor, not only to drive biosynthetic pathways. Perhaps even more importantly, NADPH is the unique provider of reducing equivalents to maintain or regenerate the cellular detoxifying and antioxidative defense systems. The roles of NADPH in redox sensing and as substrate for NADPH oxidases to generate reactive oxygen species further extend its scope of functions. NADP(+), on the other hand, has acquired signaling functions. Its conversion to second messengers in calcium signaling may have critical impact on important cellular processes. The generation of NADP by NAD kinases is a key determinant of the cellular NADP concentration. The regulation of these enzymes may, therefore, be critical to feed the diversity of NADP-dependent processes adequately. The increasing recognition of the multiple roles of NADP has thus led to exciting new insights in this expanding field.

Summary Mitochondria require nicotinamide adenine dinucleotide (NAD + ) in order to carry out the fundamental processes that fuel respiration and mediate cellular energy transduction. Mitochondrial NAD + transporters have been identified in yeast and plants 1 , 2 but their very existence is controversial in mammals 3 – 5 . Here we demonstrate that mammalian mitochondria are capable of taking up intact NAD + and identify SLC25A51 (an essential 6 , 7 mitochondrial protein of previously unknown function, also known as MCART1) as a mammalian mitochondrial NAD + transporter. Loss of SLC25A51 decreases mitochondrial but not whole-cell NAD + content, impairs mitochondrial respiration, and blocks the uptake of NAD + into isolated mitochondria. Conversely, overexpression of SLC25A51 or a nearly identical paralog, SLC25A52, increases mitochondrial NAD + levels and restores NAD + uptake into yeast mitochondria lacking endogenous NAD + transporters. Together, these findings identify SLC25A51 as the first transporter capable of importing NAD + into mammalian mitochondria.

Nicotinamide mononucleotide adenylyl transferase (NMNAT) is an essential enzyme in all organisms, because it catalyzes a key step of NAD synthesis. However, little is known about the structure and regulation of this enzyme. In this study we established the primary structure of human NMNAT. The human sequence represents the first report of the primary structure of this enzyme for an organism higher than yeast. The enzyme was purified from human placenta and internal peptide sequences determined. Analysis of human DNA sequence data then permitted the cloning of a cDNA encoding this enzyme. Recombinant NMNAT exhibited catalytic properties similar to the originally purified enzyme. Human NMNAT (molecular weight 31 932) consists of 279 amino acids and exhibits substantial structural differences to the enzymes from lower organisms. A putative nuclear localization signal was confirmed by immunofluorescence studies. NMNAT strongly inhibited recombinant human poly(ADP-ribose) polymerase 1, however, NMNAT was not modified by poly(ADP-ribose). NMNAT appears to be a substrate of nuclear kinases and contains at least three potential phosphorylation sites. Endogenous and recombinant NMNAT were phosphorylated in nuclear extracts in the presence of [Q Q-32 P]ATP. We propose that NMNAT's activity or interaction with nuclear proteins are likely to be modulated by phosphorylation. ß 2001 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.

NAD biosynthesis has become of considerable interest owing to the important signaling functions of the pyridine nucleotides which have been recognized over the past years. The formation of the dinucleotides from ATP and the mononucleotide of niacin (either nicotinamide or nicotinic acid) constitute the critical step in NAD generation which is catalyzed by NMN/NaMN adenylyltransferases, NMNATs. Recent research has established the molecular, catalytic and structural properties of NMNATs from many organisms. Detailed studies, particularly of the human NMNATs, have revealed distinct isoform-specific characteristics relating to enzyme kinetics and substrate specificity, oligomeric assembly as well as subcellular and tissue distribution. Moreover, direct functional relationships between NMNATs and major NAD-mediated signaling processes have been discovered suggesting that at least some of these proteins might play more than just an enzymatic role. Several investigations have also pointed to a critical role of NMNATs in pathological states such as cancer and neurodegeneration. This article intends to provide a comprehensive overview of the family of NMNATs and highlights some of the recently identified functional roles of these enzymes.