Cellular Compartmentation and the Redox/Nonredox Functions of NAD

Abstract

Significance: Nicotinamide adenine dinucleotide (NAD⁺) spans diverse roles in biology, serving as both an important redox cofactor in metabolism and a substrate for signaling enzymes that regulate protein post-translational modifications (PTMs). Critical Issues: Although the interactions between these different roles of NAD⁺ (and its reduced form NADH) have been considered, little attention has been paid to the role of compartmentation in these processes. Specifically, the role of NAD⁺ in metabolism is compartment specific (e.g., mitochondrial vs. cytosolic), affording a very different redox landscape for PTM-modulating enzymes such as sirtuins and poly(ADP-ribose) polymerases in different cell compartments. In addition, the orders of magnitude differences in expression levels between NAD⁺-dependent enzymes are often not considered when assuming the effects of bulk changes in NAD⁺ levels on their relative activities. Recent Advances: In this review, we discuss the metabolic, nonmetabolic, redox, and enzyme substrate roles of cellular NAD⁺, and the recent discoveries regarding the interplay between these roles in different cell compartments. Future Directions: Therapeutic implications for the compartmentation and manipulation of NAD⁺ biology are discussed. Antioxid. Redox Signal. 31, 623-642.

Keywords: compartmentation; glycolysis; metabolism; mitochondria; nicotinamide adenine dinucleotide; redox.

FIG. 1.

Biosynthesis of NAD⁺. Five biosynthetic routes to NAD⁺ in mammalian cells are shown, with NAD⁺ itself in the gray box at lower right. (A) The Priess–Handler pathway is the main synthetic route to NAD from precursors such as NA. (B) NAD⁺ can also be salvaged from NAM, the product of many NAD⁺-consuming enzymes. (C) NMN can be synthesized from NR, which is orally bioavailable. (D) NAMN in the Priess–Handler pathway (A) can be synthesized de novo from the amino acid tryptophan. (E) NA or NAMN can be synthesized from NAR. For full discussion, see text (the NAD⁺ Synthesis and Compartmentation section). NA, nicotinic acid; NAD⁺, nicotinamide adenine dinucleotide; NAM, nicotinamide; NAMN, nicotinic acid mononucleotide; NAR, nicotinic acid riboside; NMN, nicotinamide mononucleotide; NR, nicotinamide riboside.

FIG. 2.

Compartmentation of NAD⁺ biology. The cellular compartments considered herein are cytosol, nucleus, mitochondrion, and extracellular space. The major routes for import, biosynthesis, and consumption of NAD⁺ in each of these compartments are shown. Dotted lines and question marks denote pathways for which limited evidence is available. ATP, adenosine triphosphate; ENT, equilibrative nucleoside transporter; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; LDH, lactate dehydrogenase; MAS, malate-aspartate shuttle; NAAD, nicotinic acid adenine; NADH, nicotinamide adenine dinucleotide reduced form; NADK, NAD⁺ kinase; NADP⁺, nicotinamide adenine; NADPH, nicotinamide adenine dinucleotide phosphate reduced form; NAMPT, nicotinamide phosphoribosyltransferase; NMNAT, nicotinamide mononucleotide adenylyltransferase; NNT, nicotinamide nucleotide transhydrogenase; NRK, nicotinamide riboside kinase; OX-PHOS, oxidative phosphorylation; PARP, poly(ADP-ribose) polymerase; PPP, pentose phosphate pathway; SARM1, sterile alpha and TIR motif-containing protein 1; SIRT, sirtuin. Color images are available online.

FIG. 3.

Cellular fates of NAD⁺. NAD⁺ has numerous fates in the cell; clockwise from top. (1) Numerous metabolic enzymes use NAD⁺ as redox cofactor, with the reduced form (NADH) being a cellular “reducing equivalent” used to drive biosynthetic and other reactions. (2) mARTs and PARPs catalyze the adduction of ADP-ribosyl groups onto proteins, either singly or in linear or branched chains. This is an important protein post-translational modification that regulates protein function. (3) NAD⁺ can be phosphorylated to make NADP⁺, which is used as a redox cofactor like NAD⁺. Its reduced form (NADPH) is used to drive many antioxidant enzymes important for defense against oxidative stress. (4) Cell surface CD73 and mitochondrial nudix hydrolase 13 (NUDT13) enzymes have the pyrophosphohydrolase activity, converting NAD⁺ to NMN. CD73 further converts NMN into NR and plays an important role in cellular uptake of NAD⁺ and NMN. (5) CD38/CD57/SARM1 enzymes can perform either (i) a cyclase activity that generates NAM and cyclic ADPR (cADPR), the latter being an important cell signaling second messenger, or (ii) a hydrolase activity that generates NAM and ADPR. CD hydrolase is considered an NAD⁺ degradation pathway. (6) The SIRT family of lysine deacylases use NAD⁺ as a substrate, generating NAM while removing an acyl group (e.g., acetyl, succinyl, malonyl) from a protein lysine residue to form 2′-O-acyl-ADPR. ADPR, adenosine diphosphate ribose; mARTs, mono-ADPR transferases.

FIG. 4.

NADH/NAD⁺ redox in metabolism. The enzyme GAPDH, a key step in glycolysis, reduces NAD⁺ to NADH. Depending on the functionality of mitochondria, glycolytic NADH has two different routes for reoxidation. In the presence of oxygen (the so called “aerobic glycolysis”), the reducing equivalency of NADH is transmitted into mitochondria via the MAS, comprising cytosolic plus mitochondrial isoforms of MDH working in opposing directions, and cytosolic plus mitochondrial isoforms of AST working in opposing directions. In addition the G3PDH shuttle can transfer NADH reducing equivalents into mitochondria. The mitochondrial import/export of the relevant α-keto acids and amino acids is handled by a pair of electrophoretically driven membrane exchange proteins. The end product of glycolysis, pyruvate, is imported to mitochondria and further oxidized by the Krebs' cycle. In the absence of adequate mitochondrial function (e.g., hypoxia), glycolytic NADH is instead reoxidized by LDH, generating lactic acid as a terminal product (i.e., “anaerobic glycolysis”). In either scenario, the reoxidation of NADH is an absolute requirement for glycolysis to function. AST, aspartate aminotransferase; G3PDH, glycerol-3-phosphate dehydrogenase. Color images are available online.

FIG. 5.

Noncanonical functions of NAD(P)-dependent dehydrogenases. Several pathophysiological conditions can manifest neomorphic enzyme activities from NAD(H)- and NADP(H)-dependent dehydrogenases such as those found in the Krebs' cycle. (A) Normal (canonical) function of dehydrogenases: MDH oxidizes malate to OAA, reducing NAD⁺ to NADH. LDH reduces pyruvate to lactate, oxidizing NADH to NAD⁺. IDH isoforms 1 and 2 decarboxylate isocitrate to α-KG in an NADP⁺ dependent manner, whereas IDH isoform 3 performs the same reaction in an NAD⁺-dependent manner. (B) Under conditions of hypoxia and/or acidosis, both MDH and LDH can use NADH to reduce α-KG to the l-enantiomer of 2-hydroxyglutarate (l-2-HG). In addition, IDH 1/2 can operate in reverse, using NADPH to perform reductive carboxylation of α-KG to form isocitrate. The latter is an important biosynthetic pathway in cancer cells, using α-KG derived from the amino acids glutamine or glutamate, to fuel fatty acid synthesis for biomembranes. (C) Mutations in IDH 1/2 are associated with aggressive forms of cancer such as glioma and result in a neomorphic activity, that is, the ability to use NADPH to reduce α-KG to the d-enantiomer of 2-hydroxyglutarate (d-2-HG). For this reason, d-2-HG is commonly referred to as an “oncometabolite.” α-KG, α-ketoglutarate; IDH, isocitrate dehydrogenase; OAA, oxaloacetate. Color images are available online.

FIG. 6.

Lysine acetylation as a metabolic signal in the regulation of fatty acid β-oxidation. It is generally recognized that lysine acetylation of FAO enzymes (FAO Enz′) is inhibitory. In tissues such as the heart that rely heavily on FAO for bioenergetic needs, the ratio of acetyl-CoA (Ac-CoA) to NAD⁺ can serve as a metabolic signal for workload, to adjust FAO accordingly to provide ATP. Under conditions of low work, Krebs' cycle activity is low, leading to build up of Ac-CoA, which results in acetylation and inhibition of FAO enzymes. This may be an important feedback mechanism to prevent acyl-carbon-stress due to too much accumulation of acyl-CoA groups. Contrastingly, under conditions of high workload NADH will be rapidly oxidized by respiratory complex I (CxI) of OX-PHOS, yielding NAD⁺, which stimulates sirtuin activity to deacetylate (and thus activate) FAO enzymes. Resulting stimulation of FAO provides the ATP needed to drive the increased workload. FAO, fatty acid oxidation. Color images are available online.