Mono(ADP-ribosyl)ation Enzymes and NAD+ Metabolism: A Focus on Diseases and Therapeutic Perspectives

Mono(ADP-ribose) transferases and mono(ADP-ribosyl)ating sirtuins use NAD+ to perform the mono(ADP-ribosyl)ation, a simple form of post-translational modification of proteins and, in some cases, of nucleic acids. The availability of NAD+ is a limiting step and an essential requisite for NAD+ consuming enzymes. The synthesis and degradation of NAD+, as well as the transport of its key intermediates among cell compartments, play a vital role in the maintenance of optimal NAD+ levels, which are essential for the regulation of NAD+-utilizing enzymes. In this review, we provide an overview of the current knowledge of NAD+ metabolism, highlighting the functional liaison with mono(ADP-ribosyl)ating enzymes, such as the well-known ARTD10 (also named PARP10), SIRT6, and SIRT7. To this aim, we discuss the link of these enzymes with NAD+ metabolism and chronic diseases, such as cancer, degenerative disorders and aging.


Introduction
Nicotinamide adenine dinucleotide (NAD + ) is an essential pyridine nucleotide cofactor that is crucial for the activity of numerous enzymes involved in fundamental cellular processes, such as cellular energy metabolism and adaptive response to stress conditions. Being NAD + , a limiting factor for the activity of dehydrogenase and NAD + -utilizing enzymes, such as (ADP-ribosyl) transferases (ART), Sirtuins (Sirt) and NAD + -dependent histone deacetylases, the availability of NAD + and an optimal NAD+/NADH ratio govern vital cellular redox and enzymatic reactions, including mitochondrial biology, energy production, metabolism, DNA repair, epigenetic modulation of gene expression, apoptosis and intracellular signaling [1]. Thereby, NAD + metabolism has a key role in the maintenance of cellular physiology and predisposition to a wide range of chronic diseases [2].
ARTs, which include the superfamily of poly(ADP-ribose) polymerase (PARP) enzymes, contribute significantly to the reduction of cellular NAD + supply. Indeed, NAD + depletion easily occurs in response to excessive DNA damage, which is one of the wellknown activation mechanisms of the main PARPs involved in DNA damage repair, namely PARP1 and PARP2 (also named ARTD1 and ARTD2, respectively) [3][4][5]. Specifically, ARTs utilize NAD + as a donor to transfer single or multiple units of ADP-ribose to substrate molecules, which can be proteins and, in some cases, nucleic acids [6][7][8][9][10][11]. Such enzymatic reaction is reversible and takes the name of ADP-ribosylation, a type of posttranslational modification (PTM) [12,13]. Two forms of ADP-ribosylation are described, the poly(ADP-ribosyl)ation (abbreviated as PARylation), determined by polymers of ADPribose covalently linked onto target substrates, and mono(ADP-ribosyl)ation (abbreviated

Kynurenine Pathway
De novo synthesis of NAD + in cells, called the kynurenine pathway, originates from tryptophan and involves quinolinate phosphoribosyltransferase (QPRT). Interestingly, generative disorders and cancer. Cellular NAD + concentrations decrease during aging, as well as the expression of enzymes of the NAD + salvage pathway. NAMPT expression levels decline with aging [53]: NAMPT depletion aggravates, while NAMPT overexpression prevents age-related changes [53]. NAMPT activation requires the deacetylase activity of SIRT6 and modulation by AMPK; thus, a block in any of these modifications may be relevant in NAD + salvage. CD38 is reported to increase in aged individuals [53], with consequences on increased NAD + consumption, which is linked to oxidative stress [27]. Brain hypoperfusion appears to induce oxidative stress (OS), largely due to reactive oxygen species (ROS), and, over time, driving mitochondrial failure, an initiating factor of Alzheimer's disease (AD) [54]. Mitochondria play a critical role in viability and death in neurons and neuroglia since they regulate energy and oxygen metabolism as well as cell death pathways [55]. Thus, defects in NAD + metabolism has been proposed as a hallmark of metabolic diseases (obesity, diabetes, dyslipidemia, non-alcoholic fatty liver disease) as well as for neurodegeneration [55][56][57]. It has been proposed that the modulation of NAD + levels is an important element to control metabolism, either in health or in disease [18]. There is an axis linking NAD + with Sirt in aging and disease [17], as well as linking NAD + and ARTDs/PARPs, showing the beneficial effects of using PARP inhibitors (relevant ART inhibitors are discussed in Section 3.1.2 and listed in Table 1) [58][59][60][61][62].  [78]. Indeed, the increased NAD + levels may improve the immune response to the virus, as reported [79]. It may be important to investigate the link between mART enzyme activity and NAD + levels and test mART inhibitors in disease models.
In the past years, the major contribution to the understanding of ADP-ribosylation reactions has been provided by studies on ARTD1/PARP1-and ARTD2/PARP2-dependent PARylation performed under stress conditions [3,4], considered to be the major source of NAD + consumption. By contrast, the investigation of MARylation reactions has suffered major technical limitations, such as the lack of antibodies to visualize this modification. It is important to determine whether the activity of ARTs that catalyze MARylation are regulated by changes in the levels of free NAD + in the subcellular compartment in which they are restricted; equally important is to understand how NAD + consumption activities by ART localized in different cellular compartments may influence the activity of other NAD + consumers and redox enzymes in a different compartment. In order to address these issues, an in-depth understanding of ART biochemical and cellular features, as well as of proteins modified by ADP-ribosylation, is needed.
A large number of target proteins are known as acceptors of ARTD enzymes with MARylating activity, which under certain circumstances could become dominant NAD + consumers.
ARTD3/PARP3 has been involved in cellular response to DNA damage and mitotic progression, for instance, through the modification of the mitotic spindle components NuMa1 and ARTD5/PARP5a, the DNA repair proteins Ku80 and ARTD1/PARP1, and the histone H2B [82,108,109]. ARTD8 MARylates HDAC2 and HDAC3, the histone deacetylases involved in the epigenetic regulation of chromatin, TBK-1, an inhibitor of interferon synthesis, and STAT1 [110], consequently reducing STAT1 phosphorylation and suppressing the IFNγ-STAT1 signaling and the TNF-α/IL1-β proinflammatory pathway in macrophages. ARTD8 enhances histone acetylation to promote transcription of IFN-I genes [110].
N-(2(-9H-carbazol-1-yl)phenyl)acetamide (GeA-69) was identified as a novel allosteric ARTD8 inhibitor [111]. Recently, a role for the ADP-ribosyltransferase 8 (ARTD8) has been identified in the dynamics of the DNA replication controlled by ATR [112], as it modulates the response to ATR-CHK1 pathway inhibitors. ARTD8 interacts with PCNA, a DNA replication machinery component, promoting replication of DNA lesions and common fragile sites [113]. By using an engineered ARTD8/PARP14 variant, Carter-O'Connell and colleagues [114] identified 114 specific MARylation targets, several of which are RNA regulatory proteins. Interestingly, one of these targets is a catalytic inactive ARTD, namely ARTD13/PARP13, which is known to play a role in regulating RNA stability [8,84,[115][116][117]. Then, ARTD8 MARylates ARTD13 on several acidic amino acids [114], whose biological outcome still must be addressed. Indeed, mutation of ARTD13 amino acids modified by ARTD8 does not affect RNA-binding functions. Interestingly, ARTD8 automodification sites have been mapped on tyrosine and histidine sites [102], thus raising questions about the real amino acid specificity of this enzyme. ARTD17/PARP6, considered as a tumor suppressor, limits the proliferation and spreading ability of the hepatocellular carcinoma cells by degrading XRCC6/Ku70 and by regulating the Wnt/ß-catenin pathway [118]. The ubiquitin ligase HDM2 can interact with ARTD17 and XRCC6. The recent identification of ARTD17 as a regulator of dendrite morphogenesis supports a role for MARylation in neuron development [94]. Expression of wild-type ARTD17 increased dendritic complexity; conversely, the expression of a catalytically inactive ARTD17 mutant or a cysteine-rich domain deletion mutant with a reduced catalytic activity decreased dendritic complexity [94].
Membrane-anchored ARTD15/PARP16 MARylates karyopherin-1-β (Kapβ1), which interacts with importin-α/karyopherin-α (Kapα) [122]. Kapβ1 molecules can also transport cargoes independently of Kapα. Both exportin1, also known as Chromosomal maintenance 1 (Crm1), and Kapß1 have the potential to be regarded as biomarkers and therapeutic targets, as the inhibition of Kapß1 expression in cervical cancer cells leads to apoptotic cell death, suggesting a functional dependency on Kapß1 overexpression for cervical cancer cells transformation ability. Thus, the upregulation of either the importer or exporter components of nucleo-cytoplasmic trafficking may result in efficient transport, which can sustain the high proliferation rate of the cancer cells. Whereas normal epithelial and fibroblast cells are unaffected by Kapß1 inhibition, cancer cells die as a consequence of Kapß1 inhibition. Moreover, Kapß1 has a role in ER stress and unfolded protein response (UPR). ARTD15 MARylates PKR-like endoplasmic reticulum kinase (PERK) and inositolrequiring enzyme 1 α (IRE1α), two key stress sensors in the UPR in the endoplasmic reticulum. These findings link ARTD15 to inflammation showing that the UPR-linked inflammation is involved in the pathogenesis of inflammatory diseases [91]. Through Kapß1 MARylation, ARTD15 may represent a novel, crucial element in the regulatory mechanism of nucleo-cytoplasmic trafficking. In this respect, both the site of ADP ribosylation on Kapß1 and the ARTD15 catalytic site can be envisaged as potential targets for innovative therapeutic strategies.
ARTD9/PARP9 recently joined the family of MARylating enzymes. Indeed, it has been listed as an inactive ARTD until recently. Yang and colleague [123] reported ARTD9 to heterodimerize with DTX3L, a histone E3 ligase involved in DNA damage repair catalyzing the NAD + -dependent MARylation of ubiquitin molecules, at the carboxyl group of ubiquitin Gly76. As Gly76 is normally used for ubiquitin conjugation to substrates, ADPribosylation of the ubiquitin precludes ubiquitylation reactions [123]. The DTX3L/ARTD9 complex has also been found involved in the regulation of numerous processes, such as the ubiquitination of histones (such as histone H2BJ) and viral proteases (specifically viral 3C proteases), and the interferon-driven ubiquitination signaling able to control viral infections [124]. It also has been regarded as a component of a ubiquitinating LPS-responsive protein complex suggesting a role in LPS-mediated macrophage activation [125]. Surprisingly, Chatrin and colleagues [126] showed that the ART activity on ubiquitin's Gly76 is not provided by ARTD9 but by the conserved carboxyl-terminal RING and DTC (deltex carboxyl-terminal) domains of DTX3L and other human deltex proteins (DTX1 to DTX4). Indeed, Yang and colleagues [123] made their observation in the context of DTX3L/ARTD9 heterodimer, but not of DTX3L on its own. Thus, this milestone finding first suggests that ARTD9 putative enzymatic activity remains elusive and further add Deltex proteins as novel NAD + -dependent transferases.
In addition to protein substrates of mART, several observations suggest that nucleic acids, both DNA and RNAs, can be ADP-ribosylated. In this regard, ARTD10/PARP10, as well as ARTD11/PARP11, ARTD7/PARP15, and the divergent PARP homolog TRPT1, also named PARP18, can ADP-ribosylate phosphorylated ends of RNA [8,127]. Originally, MAR/PAR modification of nucleic acids was studied for DNA. ARTD1, ARTD2 and ARTD3 modify the 5'-phosphate group of DNA ends to repair damaged DNA ends [83,128]. The phosphate ends of DNA duplexes or the single-stranded oligonucleotides can be PARylated. Eukaryotic RNA 5'-end sustains various forms of capping. NAD + capping and de-NADding is one of the modifications related to NAD + homeostasis [129]. NAD + consumption in nuclear compartments may affect NAD + availability for all the enzymes dependent on NAD + for modification of proteins and of nucleic acids, including both MARylation of proteins and RNAs and RNA NADdylation. Nicotinamide (NAM) is recycled in the NAD + salvage pathway in cells under physiological conditions, while during aging and in chronic conditions, most of the NAD + salvage pathway enzymes are decreased or downregulated. Reduced NAD + availability under these conditions may affect both the NAD + dependent enzymes operating in mitochondria as well as those residing in the other cell compartments.

ARTD10 Functions
ARTD10/PARP10 is the best-studied MARylating enzyme so far. ARTD10 shuttles from nuclei to the cytoplasm, thus targeting a wide array of proteins in the different compartments. ARTD10 has been involved in the modulation of mitochondrial function by means of silencing studies showing enhancement of mitochondrial oxidative capacity [130]. ARTD10 catalytic domain, a site for automodification, recruits GAPDH into intracellular compartments such as stress granules [131]. Two macrodomains in ARTD8 can selectively interact with ADP-ribosylated ARTD10 [132,133]. Human ARTD8 interacts with ARTD10 independent of automodification activity, while murine Artd8 macrodomains interaction with ARTD10 was found dependent on MARylation [134]. ARTD10 MARylates SRPK2, exportin-5 (XPO5), tubulin-β chain, pyruvate kinase (PKM), elongation factor 1-α1, UBEC3, and NF-κB essential modulator (NEMO, IKK-γ), a subunit of NF-κB transcription factor complex [133,135]: for these interactions, ARTD10 is considered to contribute to neurodegenerative disorders. Still, ARTD10 ADP-ribosylates PLK1, significantly inhibiting its kinase activity and oncogenic function in hepatocellular carcinoma (HCC) [121]. ARTD10 interacts with and MARylates aurora A, inhibiting its kinase activity [136,137]. Moreover, ARTD10 promotes cellular proliferation and alleviates replication stress [136]. Based on the chemical analysis of the reaction products, it was proposed that ARTD10 selectively modifies acidic amino acids. It was proposed that ARTD family members may also be capable of modifying serine residues proximal to lysine on account of the KS recognition motif [10,11]. ARTD10 contains several additional domains and motifs, including a RNA recognition motif (RRM), two functional ubiquitin interaction motifs (UIM), sequences capable of promoting nuclear targeting and nuclear export, and a small motif that mediates interaction with PCNA [138] and with ubiquitin receptor p62/SQSTM1 [133,139,140]. It is conceivable that some of these domains orient ARTD10 ADP-ribosylation toward specific substrates in different compartments. Furthermore, the ARTD10-mediated modification of proteins can regulate substrate function directly, as exemplified by glycogen synthase kinase 3ß (GSK3ß). GSK3ß is a well-investigated enzyme with established functions in WNT signaling, apoptosis, metabolism, neuronal development, immunity, and tumorigenesis [141][142][143][144][145][146][147][148]. ARTD10 ADP-ribosylates GSK3ß in vitro, reducing its kinase activity. This inhibition could not be overcome by increasing substrate concentration, implying that MARylation functions as an Cells 2021, 10, 128 9 of 25 allosteric inhibitor of GSK3ß. ARTD10-modified GSK3β [87,149] has a regulatory role in type I IFN antiviral innate immune response, affecting the activation of IRF3 [150]. The nuclear transport protein RAN is also an ARTD10 substrate [87]. WRIP1 protein was used as a dual ARTD10/ARTD11 MARylation target in cell extracts [119,120].
Importantly, downregulation of ARTD10 induces glycolysis and mitochondrial fatty acid oxidation, which associates with a hypermetabolic cellular state [151]. Lastly, targeting ARTD10 may reduce the proliferation of cancer cells, as shown using antisense constructs or analyzing ARTD10 downregulated cell systems [137,152,153]. PARP10 deficiency produced severe developmental delay and DNA repair defect [152]. PLK1 inhibitors, alone or with NF-κB antagonists, were suggested as potential effective therapeutics for PARP10expressing HCC [153]. Finally, ARTD10 can modify all four histones [140]. In Table 2 are described the ARTD10 interaction partners and MARylation effects. Table 2. Known substrates and biological roles of ARTD10.

mART Inhibitors
Numerous research groups and drug discovery programs have been dedicated to inhibitors of ART enzymes [155]. Targeting ARTs has proven to be efficacious clinically, but the exploration of the therapeutic potential of ART inhibiting molecules has been largely limited by targeting the poly(ADP-ribose) generating PARP, including ARTD1 and ARTD2, as well as tankyrases [4]. Less attention has been put on the identification of selective inhibitors of mART. Nevertheless, considerable efforts have been made in order to deliver structure-based selective and potent drugs, which may be exploited for the treatment of pathological conditions, such as cancer, inflammatory diseases, as well as in buffering pathological NAD + consumption.
For instance, the ARTD3 selective and cell-permeable ME0328, which displays >7-fold selectivity over ARTD1 and its nearest homologs [64], has been developed in Schuler laboratory, providing both a valuable tool to investigate ARTD3 functions in DNA damage repair and in delaying DNA repair in irradiated cancer cells. Similar approaches have led to the identification of the potent ARTD11/PARP11 inhibitor, which is greater than 200-fold selective over other mARTD family members [68]. By screening a collection of compounds for their ability to induce mitotic defects, AZ0108 was identified as a potent ARTD17/PARP6 inhibitor, which has been proved, leading to apoptosis in a subset of breast cancer cells in vitro and antitumor effects in vivo [69]. Several groups have contributed to identifying molecules with the potential to selectively inhibit ARTD10 [65,76]; of these, the most employed is the cell-permeable OUL35 [66]. However, additional compounds with submicromolar cellular potency have been additionally developed, such as a 3,4dihydroisoquinolin-1(2H)-one that contains a methyl group at the C-5 position and a substituted pyridine at the C-6 position [67], and 4-benzyloxybenzimide derivatives [156].
Similarly, structure-based drug designing has led to the development of potent ARTD8/PARP14 inhibitors, such as the H10 showing >20-fold selectivity over ARTD1 [73] and a series of (Z)-4-(3-carbamoylphenylamino)-4-oxobut-2-enyl amides, the most potent of which was the compound 4t, that lacks selectivity against ARTD1, but displays >10-fold selectivity over ARTD5/PARP5a and >5-fold selectivity over closely related ARTD10 [74]. In addition, a series of diaryl ethers have been identified for their ability to discern between two closely related mARTDs, namely ARTD10 and ARTD8. Structure-activity studies identified compound 8b as a sub-micromolar inhibitor of ARTD10 with~15-fold selectivity over ARTD8. By contrast, compounds 8k and 8m were discovered to have sub-micromolar potency against ARTD8 and demonstrated moderate selectivity over ARTD10. Importantly, all such compounds demonstrate selectivity over ARTD1 [77].
Finally, a first-in-class ARTD14/PARP7 inhibitor (RBN-2397) has been developed by Ribon Therapeutics, which already entered a phase 1 clinical trial (Identifier: NCT04053673) for patients with advanced or metastatic solid tumors. The rationale of this trial is based on ARTD14/PARP7 dependency of several cancer cells (such as lung cancer cells) for proliferation, especially of those cell lines with higher baseline expression of interferon (IFN)stimulated genes. In particular, RBN-2397 appears to induce both cancer cell-autonomous and immune-stimulatory effects via enhanced IFN signaling [72].
In Table 1 are listed the known effects of mART inhibitors with a specific selectivity for members of the ARTD family, letting envisage their potential therapeutic application in human diseases.

Cholera-Toxin-Like Mono(ADP-Ribosyl) Transferases
ARTCs are able to MARylate protein substrates on arginine residues through Nglycoside bonds. Four ARTCs are expressed in humans (namely ARTC1, ARTC3, ARTC4, ARTC5), and six in mice (Artc1, Artc2.1, Artc2.2, Artc3, Artc4, and Artc5) [157]. The majority of mammalian ARTCs are glycosylphosphatidylinositol (GPI)-anchored proteins, with the exception of Artc5 that is a secreted enzyme [15,158]. ARTC1 is a GPI anchored protein facing the extracellular space, which modifies T-cell co-receptors and circulating hemopexin, a heme transport protein [158,159]. ARTC1 is also present on membranes of intracellular compartments and modifies Grp78/BiP in ER, which dissociates from stress sensors [160,161]. Among heat shock proteins, Hsp70-5 (HSPA5/BiP/GRP78) is localized at the endoplasmic reticulum facilitating transport and folding of nascent polypeptides into the ER lumen. Hsp70-8 (HSPA8, Hsc70, Hsp73, Hsc72) is the cognate Hsp70 family member that exhibits essential housekeeping functions, i.e., the folding of nascent polypeptides and misfolded proteins. Hsp70-9 (HSPA9, mortalin, GRP75, mtHsp70) is a mitochondrial Hsp70 isoform that bears a 46-amino acid target signal responsible for localization to the mitochondrial lumen. In CHO cells and human HEK293T and HeLa cells, BiP is ADPribosylated by hamster ARTC2.1 and by human ARTC1. BiP is ADP-ribosylated during ER stress, leading to a block in protein translation. In quiescent Swiss 3T3, Rat-1 cells, and mouse embryonic fibroblasts, BiP is ADP-ribosylated; however, when proliferation is induced, ADP-ribosylation is reduced. Mass spectrometry studies have led to identification of ADP-ribosylated residues, such as D78 and K81 of BiP, as well as D53 of Hsc70 in HeLa cells, and residue R50 of BiP and R346 of HSPA13 in murine skeletal muscle. R470 or R492 may be either ADP-ribosylated or may be AMPylated. ADP-ribosylated BiP is present in the lower-molecular weight fractions, indicating that ADP-ribosylation prevents BiP participation in the multi-chaperone complexes. Additionally, ADP-ribosylation is found only on the oligomeric form of BiP, which is the predominant form under the low protein-folding burden. Then, BiP is ADP-ribosylated during low protein production/low unprocessed protein states [162]. ARTC1 ADP-ribosylates integrin α7 (ITGA7) and regulates the binding of integrin α7β1 to laminin [159]. ARTC5 is found expressed as a secreted enzyme and probably MARylates itself on arginine; defensin HNP-1 inhibits hARTC5 auto-ADP-ribosylation and is not a substrate for ARTC5 activity [162].

Sirtuins with MARylating Activity
Sirt uses NAD + to remove acyl groups (including acetyl, glutaryl, malonyl, succinyl, and lipoyl groups) from lysine residues to form 2 -O-acyl-ADP-ribose in protein deacylation, and, in the case of SIRT4, SIRT6 and SIRT7, in MARylation of protein targets, transferring ADP-ribose from NAD + to Arg, Cys, Ser, or Thr residues of proteins [163]. SIRT4 ADP-ribosylates glutamate dehydrogenase (GDH) [164] in mitochondria. This activity opposes the effects of calorie restriction; thus, SIRT4 effectively antagonizes SIRT1. Furthermore, researchers ascribed to SIRT4 a negative effect on mitochondrial quality. The targeting of GDH inhibits the conversion of glutamate to α-ketoglutarate (α-KG), decreasing glutamine uptake, inhibiting cancer cell growth and interfering with epithelial to mesenchymal transition (EMT) in gastric cancer. Therefore, SIRT4 is considered a tumor suppressor [165,166]. SIRT4 was also associated with negative impacts on the mitochondrial quality and with aging [15]. It is possible that the antagonism with SIRT1 may increase cellular oxidative stress. α-KG upregulates H3/H4 histone acetylases and is required for α-KG-dependent N6-methyladenine demethylase ALKBH5, an RNA modifying enzyme; other Krebs cycle intermediates also show an inhibitory effect on epigenetic enzymes [167]. Importantly, the concentration of NAD + in mitochondria needs to sustain SIRT4 activity; thus, NAMPT downregulation during aging may affect SIRT4 regulatory function.
SIRT6 and SIRT7 are localized in the nuclei. Overexpression of SIRT6 is found in skin cancer and in non-small cell lung cancer (NSCLC) with poor prognostic value, but in other types of cancers, it may be considered a tumor suppressor. SIRT6 is regulated by deacetylation nicotinamide phosphoribosyltransferase (NAMPT) activity and restores NAD(P)(H) pools in cancer cells [168]. SIRT6 has a role in the stabilization and phosphorylation of tau protein [169]. SIRT6 has been involved in genome integrity, DNA repair, energy metabolism and inflammation, and is found decreased during aging and cell senescence. SIRT6 was found to auto-ADP-ribosylate [170]. SIRT6 has ADP-ribosylation activity on ARTD1 on K521 [170], enhancing DNA repair, especially when phosphorylated by JNK on Ser10. SIRT6 ADP-ribosylates epigenetic enzymes [171] such as lysine demethylase JHDM1A/KDM2A, chromatin silencing factors such as nuclear co-repressor protein KAP1, regulating KAP1 interaction with HP1α and silencing of LINE1 retrotransposons [172,173]. SIRT6 ADP-ribosylates BAF170, activating the transcription of a subset of Nrf2 target genes, and this activity may sustain Nrf2-dependent boost of mitochondrial function [174]. Lamin A binds SIRT6 and promotes histone deacetylation [175], as well as SIRT6-mediated functions upon DNA damage, forming a multiprotein complex with RPA, Ku70, Ku80, proteins that bring together BRCA1, 53BP1, CDH4, and ARTD1. SIRT6 ADP-ribosylating activity induces activation of the p53-and p73-dependent apoptosis induction in cancer cells [176]. SIRT6, in particular the catalytically active form, associates in a phosphorylationdependent mode with Ras-GTPase activator G3BP1 [177], with transcription factors NKRF, BCLAF1 and THRAP3, the telomerase regulator YLPM1, and the RNA polymerase complex factors XRN2 and COIL. SIRT6-deficient cell lines showed increased NF-kB activation and premature aging, linked to high H3 acetylation levels [178]. Signaling converging to NF-kB activation or inhibition [179], showing SIRT1 and SIRT6 regulatory roles interconnected with regulation by ARTD1 and ARTD10, has been schematically drawn, as shown in Figure 2. mode with Ras-GTPase activator G3BP1 [177], with transcription factors NKRF, BCLAF1 and THRAP3, the telomerase regulator YLPM1, and the RNA polymerase complex factors XRN2 and COIL. SIRT6-deficient cell lines showed increased NF-kB activation and premature aging, linked to high H3 acetylation levels [178]. Signaling converging to NF-kB activation or inhibition [179], showing SIRT1 and SIRT6 regulatory roles interconnected with regulation by ARTD1 and ARTD10, has been schematically drawn, as shown in Figure 2. Functional knockdown of SIRT6 results in tumor cell proliferation, invasive profile, and antiapoptotic effect [180]. When SIRT6 was overexpressed, reports observed suppression of NF-κB-mediated inflammatory responses, delaying cellular senescence [181]. SIRT6 promotes heterochromatin silencing at specific genomic loci, prevents genomic instability and telomeres dysfunction [175,180]. Reduced SIRT6 activity was linked to Hutchinson-Gilford progeria syndrome (HGPS), a human premature aging disorder, due to disrupted interaction with lamin A [182]. SIRT6 has been shown involved in liver disease, inflammation, and bone-related issue. Inhibition of SIRT6 by OSS-128167 blocked the expression of thermogenic genes and activation of white fat breakdown, showing that SIRT6/AMPK pathway increase energy consumption, insulin sensitivity and heat production, thereby alleviating metabolic disorders [183]. Treatment of osteosarcoma with SIRT6 inhibitors increased sensitivity to doxorubicin through increased damaged DNA [184]. Finally, the development of small molecules inhibiting specifically either deacetylation or MARylation may provide new clues on SIRT6 functions [185]. SIRT6 deacetylase activity has important effects in cells; therefore, it is difficult to assign the phenotype observed in SIRT6 silenced cells to a defect in MARylation or in deacetylation activity. Deacetylation of histone H3 on K9 and K56 by SIRT6 can block the transcription of GLUT1 and LDH by Hif-1α, inhibits the transcription activity of Myc on Lin28b, and that of NF-κB on survivin, while decreasing transcription of the pro-apoptotic Bax gene in HCC development; a similar regulation of histone H3 leads to block FoxO3 transcription and binding to SREBP1/2 and PCSK9 promoters, leading to metabolic regulation of lipogenesis [182]. Deacetylation of NF-kB and FoxO1 leads to their delocalization from nuclei to cytoplasm. Non-histone substrates Functional knockdown of SIRT6 results in tumor cell proliferation, invasive profile, and antiapoptotic effect [180]. When SIRT6 was overexpressed, reports observed suppression of NF-κB-mediated inflammatory responses, delaying cellular senescence [181]. SIRT6 promotes heterochromatin silencing at specific genomic loci, prevents genomic instability and telomeres dysfunction [175,180]. Reduced SIRT6 activity was linked to Hutchinson-Gilford progeria syndrome (HGPS), a human premature aging disorder, due to disrupted interaction with lamin A [182]. SIRT6 has been shown involved in liver disease, inflammation, and bone-related issue. Inhibition of SIRT6 by OSS-128167 blocked the expression of thermogenic genes and activation of white fat breakdown, showing that SIRT6/AMPK pathway increase energy consumption, insulin sensitivity and heat production, thereby alleviating metabolic disorders [183]. Treatment of osteosarcoma with SIRT6 inhibitors increased sensitivity to doxorubicin through increased damaged DNA [184]. Finally, the development of small molecules inhibiting specifically either deacetylation or MARylation may provide new clues on SIRT6 functions [185]. SIRT6 deacetylase activity has important effects in cells; therefore, it is difficult to assign the phenotype observed in SIRT6 silenced cells to a defect in MARylation or in deacetylation activity. Deacetylation of histone H3 on K9 and K56 by SIRT6 can block the transcription of GLUT1 and LDH by Hif-1α, inhibits the transcription activity of Myc on Lin28b, and that of NF-κB on survivin, while decreasing transcription of the pro-apoptotic Bax gene in HCC development; a similar regulation of histone H3 leads to block FoxO3 transcription and binding to SREBP1/2 and PCSK9 promoters, leading to metabolic regulation of lipogenesis [182]. Deacetylation of NF-kB and FoxO1 leads to their delocalization from nuclei to cytoplasm. Non-histone substrates and additional catalytic activities of SIRT6 have been reported, but these noncanonical roles remain enigmatic. Genetic studies showed critical SIRT6 homeostatic cellular functions and the need to find molecular pathways driving SIRT6-associated phenotypes. As for the physiological role, SIRT6 activity promotes increased longevity by regulating metabolism and DNA repair. In Table 3 are reported the known functions of SIRT6.  Interaction with RELA /p65 NF-κB subunit Attenuates NF-κB on promoters Cell senescence, anti-apoptotic, insulin sensitivity [178] H3 de-acetylation Inhibition GLUT1/LDH transcription by Hif-1α in White and brown fat cells N.D. [182] N.D.
Inhibition of NF-κB transcription activity and suppression of Survivin gene expression in HCC N.D. [179] Lamin A interaction Induces Sir6 chromatin localisation and Histone deacetylation N.D. [175] SIRT7 plays a key role in mitochondrial function and, in the liver, it regulates autophagy and the physiological response to calorie restriction [186]. SIRT7 has been involved in genome integrity and Non-homologous end-joining (NHEJ) DNA repair [187]. SIRT7 has auto-modification MARylation activity [181]. SIRT7 auto-modification occurs on several sites, as proteomic studies identified 7-8 MARylated peptides, modifying SIRT7 chromatin distribution. In the ELHGN catalytic motif, conserved among sirtuins, H187 recognizes acetylated substrates and is involved in deacetylation activity. H 187 is oriented toward the NAD + -binding pocket and the main catalytic site, as the flanking residues E185 and N189. In SIRT6 and SIRT7, these flanking residues are faced in the opposite direction, toward the surface of the cavity, and both residues interact to form a loop. These residues are important for their role in the ADP-ribosylation reaction: E185 is the catalytic residue that initiates the reaction, whereas N189 acts as the first acceptor of the ADP-ribosyl moiety. SIRT7 shows nucleolar enrichment, and SIRT7 auto-modification attracts the ADP-ribose-binding macrodomain of histone H2A1.1 (mH2A1) and promotes the enrichment of mH2A1 in loci associated with metabolic genes [186].
A structure of bacterial Sirt bound to the acetylated +2 arginine peptide shows how this arginine could enter the active site and react with a deacetylation reaction intermediate to yield an ADP-ribosylated peptide [188]. These studies may allow differentiating the residues and structures performing deacetylation from those involved in MARylation.

Additional NAD + -Consuming Reactions
NAD + is also consumed by NAD + glycohydrolases [189], such as the NAD + glycohydrolase/cluster of differentiation 38 (CD38), which catalyzes the hydrolysis of NAD + and cyclic ADP-ribose (cADPR), thus affecting the pool of cellular NAD + [190]. CD38 and sterile alpha and TIR motif-containing 1 (SARM1) are two ectoenzymes on plasma membranes with ADP-ribosyl cyclase/cyclic ADP ribose hydrolase activity [191]. CD38 is also inserted into intracellular membranes, facilitates autophagy, and has a role in autophagic fusion with lysosomes [192]. In the CD38 knockout mouse, NADase activity was absent in all compartments, from plasma membranes to nuclei [193]. CD38 was shown to process NAD + -capped RNA in vitro into ADP-ribose-modified-RNA and nicotinamide [194]. CD38 degrades NAD + and also NMN and NADP, generating second messengers such as ADP ribose (ADPR), cADPR, and nicotinic acid adenine dinucleotide phosphate (NAADP). SARM1 is required for activation of injury-induced axon degeneration and facilitates mitophagy in depolarized mitochondria; thus, SARM1 may be involved in neuroprotection. NAD + depletion can be rescued by increasing NMNAT activity. To protect from NAD + consumption by SARM1 activity, cytosolic NMNAT1 was overexpressed, producing a beneficial effect that was dependent on NMNAT1 activity [195]. CD38 inhibitor 78c was administered to slow down the age-related NAD + decline [196]: a therapy with 78c improved physiological parameters, such as glucose homeostasis, cardiac function, muscle architecture, and exercise capacity. The mechanisms of these antiaging effects are still to be identified. Table 4 shows the effects of NAD + boosters in disease treatment and the therapeutic applications of various drugs influencing NAD + levels and regulating NADdependent enzymes, and their beneficial effects. In Table 5 are presented several drugs involved in the regulation of NAD + consuming enzymes such as CD38, in the activation of Sirt, and in the enhancement of NAD + synthesis and NAM reutilization. NAM levels should be kept under a certain threshold in order to avoid an inhibitory effect of NAD +dependent enzymes when NAMPT and other enzymes in the NAD + salvage pathway are downregulated. Table 4. NAD+ precursors, NAD+ salvage enzyme activators, and Sirt regulators and inhibitors with therapeutic potential.

Molecule Activity Cellular Outcomes and Therapeutic Application References
Kynurenine, quinolinic acid NAD + precursor Preserves mitochondrial integrity in mouse models of AD, Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Huntington's disease (HD) [190] NMN NAD + precursors Reverses JNK activity and progression of AD, age-related pathological processes, such as diabetes, ischemia-reperfusion injury, heart failure [197] NR, NRH NAD + precursors Clinical trial of healthy overweight adults, AD protection through the induction of autophagy. They protect mice against a high fat diet, prevent age-related diseases and increase longevity [26,45,198] Nicotinic acid NAD + precursors Treatment of tumor cells deficient in NAPRT [55,62] NAM NAD + precursors Treatment of early AD [199,200]  OSS-128167 SIRT6 inhibitor Potentiates the anti-tumor effect of doxorubicin [183,184] Acetylated lysine-ADP-ribose conjugates SIRT7 inhibitor Histone H3K18 deacetylation and maintenance of oncogenic transformation [202,203] [193] Apigenin Activation of Sirtuin and inhibition of CD38 Decrease global protein acetylation in obese mice [212,213]

NAD + Boosters and Therapeutic Role in the Treatment of Diseases
A large set of information can be found on the beneficial effects of sustaining NAD + levels in disease states with NAD + boosters [214][215][216] and supplementation of NAD + precursors [198,217]. A clinical trial on healthy overweight adults to test the safety of NIAGEN (nicotinamide riboside chloride) was positively concluded [218]. NAD + supplementation increases mitochondrial function, leading to a lifespan extension [198,199,[217][218][219][220]. Boosting NAD + through precursors such as NAM, NMN or nicotinamide riboside (NR) may increase longevity and prevent age-related diseases. For instance, Zhang and colleagues showed that NAD + repletion enhances the life span of mice [220].
De novo NAD + synthesis or increased availability of NAD + precursors may thus support cognitive functions and lead to a decrease of Aβ amyloid fibrils [221][222][223][224][225]. Nicotinamide treatment preserved mitochondrial integrity in mouse models of AD [220] in cells with a functional NAD + salvage pathway. NMN exogenously added to a mouse model of AD substantially decreased multiple AD-associated pathological characteristics [224,225]. NAD + levels can affect inflammation, caloric restriction, exercise, DNA repair, longevity, and healthspan [207]. In a study on a coronavirus-dependent decrease in NAD + levels, with altered expression of ARTD enzymes and NAMPT, authors recommended administration of NAD + precursors to alleviate the inflammatory state of lungs [207,224]. Presently, it is known that NAD + availability affects SIRT1, and this reflects on cellular metabolism through the SIRT1/AMPK/mTOR axis, but it can be speculated that most NAD + -dependent enzymes, including MARylating enzymes, may increase their activity and may better function when NAD + levels are maintained elevated. ARTD family-specific mART inhibitors may pass the requirement for the potential application in therapy of human diseases and may be combined with approved NAD + boosters, especially in conditions known to decrease the function of the NAD + salvage pathway or with predominant glycolysis state or in the presence of mitochondrial dysfunctions. It can be expected that supplementation of NAD + precursors may delay the onset and progression of ADP-ribosylation-linked disease states, as it has been done for metabolic and degenerative diseases with decreased NAD + levels.

Conclusions
In this review, we shed light on the intimate connection between NAD + , NAD +consuming enzymes, and mitochondrial well-functioning, highlighting the central role of these pathways in the development of chronic, metabolic and degenerative diseases. In the balance of produced and consumed NAD + pools, the activity of NAD + utilizing enzymes alters their bioavailability in intracellular and extracellular compartments, causing modifications to energy production and to metabolites that may lead to cell death. NAD +dependent enzymes, including mARTs, may exert their activity only if NAD + levels are maintained elevated. We reviewed the involvement of ARTDs, ARTCs, and MARylating Sirt in important cellular functions, from inflammation to immunity, from epigenetic modulation to chromatin accessibility, which may be affected when NAD + levels are decreased. Various NAD + precursors have shown beneficial effects in supplementing the NAD + requirements, especially in disease states in which there is a major request by NAD + -dependent enzyme systems, while enzyme small molecule activators or regulators may become beneficial in various stresses and inflammatory diseases.
Author Contributions: Conceptualization, P.P. and L.P.; data curation, L.P.; writing-original draft preparation, P.P.-review and editing L.P.; A.C.; supervision. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement:
The study did not require ethical approval.

Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study are available in the article.

Acknowledgments:
We acknowledge all the authors whose work has shaped the ADP-ribosylation and NAD + fields and who were not cited here simply for space constraints. We thank Roberta Visconti (Institute for the Experimental Endocrinology and Oncology, CNR, Naples) for critical reading of the manuscript.

Conflicts of Interest:
The authors declare no conflict of interest.