Downloaded from genesdev.cshlp.org on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press SPECIAL SECTION: REVIEW Interplay between compartmentalized NAD+ synthesis and consumption: a focus on the PARP family Michael S. Cohen Department of Chemical Physiology and Biochemistry, Oregon Health and Science University, Portland, Oregon 97210, USA Nicotinamide adenine dinucleotide (NAD+) is an essential erate a polymer of ADP-ribose (ADPr), a process known as cofactor for redox enzymes, but also moonlights as a sub- poly-ADP-ribosylation or PARylation (more on this below) strate for signaling enzymes. When used as a substrate by (Fig. 1; Chambon et al. 1963, 1966; Fujimura et al. 1967a,b). signaling enzymes, it is consumed, necessitating the recy- Unlike NAD+-mediated redox reactions, this glycosidic cling of NAD+ consumption products (i.e., nicotinamide) cleavage reaction is irreversible and leads to the consump- via a salvage pathway in order to maintain NAD+ homeo- tion of NAD+. Consistent with this notion, in the 1970s it stasis. A major family of NAD+ consumers in mammalian was shown that NAD+ exhibits a high turnover in human cells are poly-ADP-ribose-polymerases (PARPs). PARPs cells (Rechsteiner et al. 1976). We now know that there are comprise a family of 17 enzymes in humans, 16 of which many “NAD+ consumers” (e.g., other PARP family mem- catalyze the transfer of ADP-ribose from NAD+ to macro- ber, sirtuins, etc.) beyond PARP1, which are found in molecular targets (namely, proteins, but also DNA and nearly all subcellular compartments, including the nucle- RNA). Because PARPs and the NAD+ biosynthetic en- us, cytoplasm, and mitochondria (Fig. 1; Verdin 2015). zymes are subcellularly localized, an emerging concept Because of these NAD+ consumers, continuous synthesis is that the activity of PARPs and other NAD+ consumers of NAD+ is required for maintaining NAD+ levels. are regulated in a compartmentalized manner. In this re- Curiously, enzymes involved in NAD+ synthesis are lo- view, I discuss NAD+ metabolism, how different subcellu- calized to distinct subcellular compartments—like the lar pools of NAD+ are established and regulated, and how NAD+ consumers themselves—suggesting that NAD+-de- free NAD+ levels can control signaling by PARPs and re- pendent signaling is regulated in a compartmentalized dox metabolism. manner. In this review I discuss the interplay between NAD+ synthesis and consumption by NAD+ consumers, with a particular focus on PARPs because they are the largest family of NAD+ consumers in cells. I first discuss + + NAD metabolism (i.e., its synthesis and consumption). Nicotinamide adenine dinucleotide (NAD ): beyond I then discuss how NAD+ metabolism is compartmental- redox reactions ized. Last, I discuss how PARPs regulate—and are regulat- — + Nicotinamide adenine dinucleotide (NAD+) is found in all ed by changes in free NAD levels within subcellular living organisms and is essential for life. NAD+ was discov- compartments. ered by Sir Arthur Harden in 1906 and for the first half of the 20th century the only known role for NAD+ was as a coenzyme for redox reactions in metabolic processes How is NAD+ synthesized in cells? (e.g., glycolysis). In this capacity, NAD+ binds to oxidore- + ductase enzymes where it undergoes a two-electron reduc- NAD can be synthesized in mammalian cells from pre- tion to generate NADH and an oxidized substrate (Fig. 1). cursors via three major pathways: (1) synthesis from tryp- This reaction is catalytic and reversible (NADH is reoxi- tophan (the de novo pathway), (2) synthesis from nicotinic dized to NAD+); therefore, NAD+ is not consumed by re- acid (NA; Preiss-Handler pathway), and (3) synthesis from dox reactions in cells. However, in the mid-1960s NAD+ nicotinamide (NAM; salvage pathway) (Fig. 1). In humans, + was shown to be a substrate for a nuclear enzyme (now the major source of NAD is from NA and NAM, collec- known as poly-ADP-ribose-polymerase 1, PARP1) that tively referred to as niacin (vitamin B3). Niacin is essential cleaves the nicotinamide-glycosidic bond of NAD+ to gen- © 2020 Cohen This article is distributed exclusively by Cold Spring Har- bor Laboratory Press for the first six months after the full-issue publication [Keywords: ADP-ribosylation; biosensor; NAD; NAD consumer; PARP] date (see http://genesdev.cshlp.org/site/misc/terms.xhtml). After six Corresponding author: [email protected] months, it is available under a Creative Commons License (Attribution- Article published online ahead of print. Article and publication date are NonCommercial 4.0 International), as described at http://creativecom- online at http://www.genesdev.org/cgi/doi/10.1101/gad.335109.119. mons.org/licenses/by-nc/4.0/. 254 GENES & DEVELOPMENT 34:254–262 Published by Cold Spring Harbor Laboratory Press; ISSN 0890-9369/20; www.genesdev.org Downloaded from genesdev.cshlp.org on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press A focus on the PARP family Figure 1. Pathways of NAD+ synthesis, consumption, and redox chemistry. The dashed red line indicates cleav- age of the glycosidic bond of NAD+ by NAD+ consumers. NAD+ biosynthetic enzymes are shown in blue, NAD+ consumers are shown in red, and NAD+ redox enzymes are shown in green. (−) Inhibition; (+) activation. for maintaining NAD+ levels in vivo. Indeed, deficiencies phosphoribosyltransferase (NAMPT) and nicotinamide in niacin causes Pellagra, a metabolic disease that results mononucleotide adenylyltransferases (NMNAT) (Berger in diarrhea, dermatitis, dementia, and if untreated, death et al. 2005). NAMPT synthesizes nicotinamide mono- (Kirkland and Meyer-Ficca 2018). Hence, niacin is essen- nucleotide (NMN) from NAM and α-D-5-phospho- tial for the human diet. High levels of niacin are found ribosyl-1-pyrophosphate (PRPP) (Fig. 1; Revollo et al. in many foods, including meat, brown rice, and peanuts; 2007). NMNAT, which exists as three distinct genes however, because these niacin-rich foods were not com- (NMNAT1–3) that have nonoverlapping functions (more monplace in the American diet in the early part of the on this below), synthesizes NAD+ directly from NMN 20th century, especially in the South, grains were fortified and ATP (Fig. 1; Berger et al. 2005). Knockdown of with niacin beginning in the 1930s (Kirkland and Meyer- NAMPT, or inhibition of NAMPT activity using a small Ficca 2018). Recently, there has been much interest in us- molecule inhibitor (e.g., FK866, Fig. 1), substantially ing nicotinamide riboside (NR) to boost NAD+ levels in in reduces NAD+ levels in most cells (Liu et al. 2018). Con- vivo (Trammell et al. 2016). NR is a naturally occurring versely, treatment of cells with a small molecule activator NAD+ precursor that feeds into the salvage pathway (e.g., SBI-797812) (Fig. 1) of NAMPT increases NAD+ levels through the action of enzymes known as nicotinamide in cells (Gardell et al. 2019). Therefore, NAMPT is a criti- riboside kinase 1 and 2 (NRK1/2) (Fig. 1; Bieganowski cal regulator of NAD+ levels in many cultured cells. and Brenner 2004). Notwithstanding the importance of While NAMPT appears to be critical for maintaining dietary niacin and related NAD+ precursors, a recently NAD+ levels in many cells, NAD+ can be synthesized in published study found that in human patients the loss- an NAMPT-independent manner via the Preiss-Handler of-function mutations in enzymes in the de novo pathway. A recent study demonstrated that some cancer tryptophan-to-NAD+ pathway results in congenital mal- cells (e.g., OV4 ovarian cancer cells) are refractory to formations (Shi et al. 2017). In these patients, serum changes in NAD+ levels upon knockdown of NAMPT NAD+ levels were significantly lower, demonstrating (Chowdhry et al. 2019). In OV4 cells, enzymes in the Pre- that dietary tryptophan is also an important source of iss-Handler pathway (synthesis of NAD+ from NA) such NAD+ in vivo. Perhaps this result is not surprising consid- as nicotinate phosphoribosyltransferase (NAPRT) are am- ering the essentiality of NAD+ in human health. plified. NAPRT synthesizes nicotinic acid mononucleo- In many cells, the salvage pathway via the precursor tide (NAMN) from NA, and NAMN is subsequently NAM plays an essential role in maintaining physiological converted into nicotinic acid dinucleotide (NAAD+)by NAD+ levels in cells. NAD+ synthesis from NAM in NMNATs. Finally, NAAD+ is converted into NAD+ by mammalian cells requires two enzymes: nicotinamide NAD+ synthetase (NADS) (Fig. 1). Knockdown of NAPRT GENES & DEVELOPMENT 255 Downloaded from genesdev.cshlp.org on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press Cohen in OV4 cells, which were implanted subcutaneously in PARP family members (PARP3, PARP6–12, and nude mice, decreased tumor NAD+ levels and tumor vol- PARP14–16) catalyze the transfer of a single unit of ume. In contrast, knockdown of NAPRT in subcutaneous- ADPr to their targets, a process referred to as mono- ly implanted H460 lung cancer cells, which do not exhibit ADP-ribosylation (MARylation) (Vyas et al. 2014; Yang amplification of enzymes in the Preiss-Handler pathway, et al. 2017). SIRTs are NAD+-dependent deacylases did not alter NAD+ levels; rather, knockdown of NAMPT (Kosciuk et al. 2019), though some SIRT family members (or treatment with FK866) decreased tumor NAD+ levels (e.g., SIRT4,6) have been shown to also catalyze ADP- and tumor volume. Hence, cancer cells that have high lev- ribosylation (Liszt et al. 2005; Haigis et al. 2006). The els of NAPRT depend on the Preiss-Handler pathway for mechanism of sirtuin-mediated deacylation occurs via survival, whereas cancer cells that have low levels of an ADPr-lysine-imidate intermediate generated from the NAPRT depend on the salvage pathway. Cell type-depen- attack of an acyl oxygen of lysine at the anomeric position dent differences in NAD+ synthesis pathways have impor- of the nicotinamide ribose (Sauve et al. 2001). CD38 is a tant implications in cancer therapeutic strategies aimed transmembrane enzyme that cleaves NAD+ to generate at lowering NAD+ levels in cells.