Toward a unified nomenclature for mammalian ADP-ribosyltransferases

Review

Toward a unified nomenclature for mammalian ADP-ribosyltransferases Michael O. Hottiger1*, Paul O. Hassa1*, Bernhard Lu¨scher2*, Herwig Schu¨ler3* and Friedrich Koch-Nolte4* 1

Institute of Veterinary Biochemistry and Molecular Biology, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland 2 Institute of Biochemistry and Molecular Biology, Medical School, RWTH Aachen University, Pauwelsstrasse 30, 52057 Aachen, Germany 3 Structural Genomics Consortium, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Scheeles va¨g 2, 17177 Stockholm, Sweden 4 Institute of Immunology, University Medical Center Hamburg-Eppendorf, Martinistr. 52, 20246 Hamburg, Germany

ADP-ribosylation is a post-translational modification of proteins catalyzed by ADP-ribosyltransferases. It comprises the transfer of the ADP-ribose moiety from NAD+ to specific amino acid residues on substrate proteins or to ADP-ribose itself. Currently, 22 human genes encoding proteins that possess an ADP-ribosyltransferase catalytic domain are known. Recent structural and enzymological evidence of poly(ADPribose)polymerase (PARP) family members demonstrate that earlier proposed names and classifications of these proteins are no longer accurate. Here we summarize these new findings and propose a new consensus nomenclature for all ADP-ribosyltransferases (ARTs) based on the catalyzed reaction and on structural features. A unified nomenclature would facilitate communication between researchers both inside and outside the ADP-ribosylation field. ADP-ribosylation of proteins Nicotinamide adenine dinucleotide (NAD+) is best known for its role as a conenyzme in redox reactions, in which oxidoreductases interconvert NAD+ and NADH to reduce or oxidize small molecule metabolites [1,2]. Oxidoreductases typically bind NAD+ via a pair of Rossmann folds that are conserved nucleotide-binding domains. Nature has invented at least three other NAD+-binding folds for three classes of NAD+-consuming enzymes: ADP-ribosyltransferases (ARTs), ADP-ribosylcyclases, and sirtuins [1,3]. These enzymes harness the high-energy bond between the nicotinamide and ADP-ribose moieties in NAD+ to catalyze ADP-ribose transfer, dephosphorylation or deacetylation reactions, and/or the synthesis of ADPribose and derivatives of ADP-ribose. Here we focus on the ART family, whose members catalyze ADP-ribosylation of amino acids, nucleotides, antibiotics, and the dephosphorylation of a tRNA splicing intermediate [4– 11]. For a more detailed description of ADP-ribosylcyclases, sirtuins and other members of the NAD+ and Corresponding authors: Hottiger, M.O. ([email protected]); Hassa, P.O. ([email protected]); Lu¨scher, B. ([email protected]); Schu¨ler, H. ([email protected]); Koch-Nolte, F. ([email protected]) * All authors contributed equally to this manuscript.

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ADP-ribose proteomes, the reader is referred to other reviews [1–3,12,13]. Mono- and poly-ADP-ribosylation of proteins are phylogenetically ancient, reversible, post-translational modifications implicated in a wide range of processes. These include maintenance of genomic stability, transcriptional regulation, energy metabolism and cell death, although in many instances the precise molecular consequences are not known. The modification is catalyzed by enzymes that transfer an ADP-ribose unit from the co-substrate NAD+ onto side chains of specific amino acid residues of substrate proteins. Known acceptors in eukaryotic cells are lysine (K), arginine (R), glutamate (E), aspartate (D), cysteine (C), diphthamide (Dph), phospho-serine (pS) and asparagine (N) residues. Although ADP-ribosylation is best known as a posttranslational protein modification, some members of the ART family can catalyze ADP-ribose transfer to rifampin (an antibiotic), water (resulting in the hydrolysis of NAD+), guanosine residues in DNA, or phosphate at the splice junction of tRNA (resulting in dephosphorylation of this splicing intermediate and the release of mature tRNA and ADP-ribose 10 -20 cyclic phosphate) [6]. Protein-linked ADP-ribose (ADPr) can serve as an acceptor for further ADP-ribosylation. The extent of ADP-ribosylation of proteins within a cell depends not only on the enzymes catalyzing this modification, but also on the activity of cellular ADP-ribose-protein hydrolases, that reverse the reaction by hydrolyzing the protein–ADP-ribose bond and/ or the bonds between different ADP-ribose units of pADPr [4–10]. Mono-ADP-ribosylation reactions were originally identified as an important aspect of bacterial pathophysiology, catalyzed by several toxins including diphtheria (Box 1), pertussis, cholera, and certain clostridial toxins [9–11,14,15]. Subsequently, mono-ADP-ribosylation was also discovered in bacteriophages and in eukaryotic cells. Intracellular mono-ADP-ribosylation has been suggested to play important roles in the regulation of intracellular signaling cascades, gene expression, as well as cell differentiation and proliferation [4,6,9,10]. Poly-ADPribosylation was discovered in multicellular eukaryotes and appears to be less widely used compared to mono-

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Review

Trends in Biochemical Sciences

Box 1. Bacterial diphtheria toxin-like ARTs (ARTD, H-Y-E class) ADP-ribosylation was originally discovered as the pathogenic mechanism of diphtheria toxin (DT), a secreted protein encoded by a phage of Corynebacterium diphtheriae [56]. The DT holotoxin encompasses three distinct domains: an N-terminal catalytic domain, a central translocation domain, and a C-terminal receptorbinding domain. The DT-receptor, the membrane anchored form of heparin binding epidermal growth factor (HB-EGF), is expressed by many human cell types. DT is a mono-specific ART that targets elongation factor 2 (eEF2), which is essential for protein synthesis. DT catalyzes mono-ADP-ribosylation of eEF2 at the diphthamide residue 699 (in yeast), thereby blocking eEF2 association with other proteins of the translation machinery, and effectively blocking host cell protein synthesis. Two other toxins catalyze ADP-ribosylation of eEF2 at the same residue: exotoxin A (ExoA), one of four ARTs secreted by Pseuodomonas aeruginosa, and cholix toxin (ChT), one of two ARTs secreted by Vibrio cholera [32]. ExoA and ChT share a similar domain architecture with DT, albeit in reversed order (an Nterminal receptor binding domain and a C-terminal catalytic domain). The catalytic domains of DT, ExoA and ChT belong to the H-Y-E class of ARTs. These bacterial ARTs share greater structural similarity with the mammalian PARP and TpT families than with the R-S-E class of ARTs (Box 2) [18,30]. Recently a small molecule ART was identified as a member of the H-Y-E class of ARTs: Arr of Mycobacterium smegmatis catalyzes ADP-ribosylation of the antibiotic rifampin [52]. Arr lacks the catalytic glutamate at the beginning of b5, but carries an aspartate residue in b4 that evidently has catalytic activities (see red arrow in Figure 1a). The recent cocrystals of Arr and ExoA with their respective targets, rifampin and eEF2, substantiate the predicted role of the loop connecting b4 and b5 for substrate recognition [50,52].

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ADP-ribosylation. However recent evidence suggests that it also exists in dinoflagellates and archaebacteria. ARTencoding genes are found in many eukaryotic species of the animal, plant, fungi, and protist kingdoms (Table 1). Lower eukaryotes generally contain fewer ART genes, and based on their sequence similarity and conserved domain structures, all vertebrate ART genes can be assigned to a particular orthologue. By contrast, ART genes of lower eukaryotes can be assigned to a subgroup, but not to a particular vertebrate ART gene. It should be noted that there is no correlation between genome size, chromosome or gene numbers and the total number of ART genes in a genome. Sequence and structure homology searches have identified 22 human genes encoding proteins that encompass an ADP-ribosyltransferase fold and are thus potentially associated with distinct ADP-ribosylation activities. These proteins have been grouped into three major families: (i) the extracellular membrane-associated ADP-ribosyltransferases (ecto-ARTs) [6]; (ii) a single member family of NAD+dependent tRNA 2’-phosphotransferases [16,17]; and (iii) the mammalian poly-ADP-ribose polymerases (PARPs) [4,18,19]. Mammalian ecto-ARTs constitute a family of structurally related proteins expressed at the cell surface or secreted into the extracellular compartment [6,20]. Using NAD+ as a substrate, they transfer mono-ADP-ribose onto target proteins. ART activity in the extracellular compartment provides sophisticated regulatory mechanisms for cell communication (Box 2). Over the past 10 years, 17 mam-

Table 1. Distinct ART genes in distantly related speciesa Species Homo sapiens Pan troglodytes Equus caballus Bos taurus Canis familiaris Felis catus Mus musculus Rattus norvegicus Gallus gallus Xenopus laevis Danio rerio Oryzias latipes Anopheles gambiae Drosophila melanogaster Caenorhabditis elegans Arabidopsis thaliana Oryza sativa Trypanosoma brucei Paramecium tetraurelia Dictyostelium discoideum Entamoeba histolytica Schizosaccharomyces pombe Saccharomyces cerevisiae Aspergillus fumigatus Magnaporte grisea Neurospora crassa

Genome size (Mbp) 3038 3300 2200 3000 2400 3000 3000 2750 1200 3100 1700 700 278 170 97 119.2 450 26 120 and 100 f 34 23.8 14 12.8 30 40 39.23

Gene count  30000  30000  21000  22000  20000  20000  25000  21000  22000  20000  25000  21000  14000  15000  21000  28000  45000  10000  40000  12500  10000  5000  6500  10000  12000  10000

Haploid chromosome count 23 24 32 30 39 14 20 21 40 18 25 24 3 4 6 5 12 11 50 and 350 f 6 14 3 16 8 7 7

Trpt gene count 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

Parp gene countb,d 17 17 17 16 17 >16 e 16 16 >15 e 15 16 16 >3 e 2 3 >10 e >3 e >1 e >7 e >9 e >6 e none none >1 e >1 e >1 e

e-Mart gene countc,d 4 (5) 4 (5) >3 e >4 e >4 e >4 e 6 5 >5 e >2 e >3 e >3 e none none none none none none none none none none none none none none

a

Summary of the matches of ART genes found in model organisms with complete or near-complete genome sequences. ART genes were identified by BLAST searches of the genome and cDNA databases using as queries the DNA sequences of each catalytic domains of the 17 human polyADP-ribose polymerases (PARP1-17) and each of the 6 mouse ecto-mono-ADP-ribosyltransferases (ART1-5). d Note: the exact number will most likely change as genome sequences are being fully validated. e >: at least. f Ciliates possess two nuclei, a germinal nucleus (micronucleus) and a somatic nucleus (macronucleus). b,c

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Box 2. Bacterial and mammalian C2 and C3-like ARTs (ARTC, R-S-E class) The R-S-E class of ARTs encompasses a large number of virulence factors from bacteria and bacteriophages, key regulatory enzymes of bacterial nitrogen fixation, the family of mammalian ecto-ARTs, and two small families of DNA-specific ARTs from shellfish and butterflies [6,29,30]. The best-known subfamily of R-S-E ARTs encompasses the AB5 toxins, E.coli heat-labile enterotoxin (LT), cholera toxin (CT) and pertussis toxins (PT), all of which mono-ADP-ribosylate the alpha subunit of heterotrimeric G-proteins at a specific arginine (LT, CT) or cysteine (PT) residue. MTX, an insecticidal toxin of Bacillus sphaericus, ADP-ribosylates numerous proteins at arginine residues, whereas the structurally related Pierisin from cabbage butterflies ADP-ribosylates guanine bases in DNA [57,58]. The 3D structures of the CT/PT/MTX subfamily of R-S-E ARTs, like all available structures of H-Y-E ARTs, exhibit a catalytic core of six anti-parallel b-strands. By contrast, the structures of the C2/C3/VIP2/ART2 subfamily of R-S-E ARTs carry an additional b-strand (in parallel to ß-strand 5) before bstrand 6 (Figure 1b,c) [18]. Several binary AB toxins (Clostridial C2 and iota toxins and VIP2 of Bacillus cereus) catalyze ADP-ribosylation of actin at R177, as does the Salmonella virulence protein SpvB [34,51,59]. Clostridial C3 and

malian genes with distant sequence homology to bacterial diphtheria toxin have been described [4,5,18]. The first and best characterized of these proteins was originally noted for its ability to synthesize free ADP-ribose polymers [21–23] and was thus termed poly-ADP-ribose synthase or polyADP-ribose polymerase 1 (PARS or PARP1) [19]. Based on the high sequence similarities within their catalytic domains, the 17 mammalian proteins were named PARPs 1–17 [5,18]. Some members of the sirtuin family were also reported to catalyze ADP-ribosylation reactions under certain conditions [24–27]; however, sirtuins are structurally distinct and their predominant enzymatic activity is NAD+dependent protein deacetylation. The current PARP nomenclature is not accurate Recent enzymatic data support the view that the earlier proposed name ‘‘PARP’’ and the provided numbering are no longer accurate. The term polymerase is commonly used for template-dependent DNA or RNA synthesizing enzymes, but not for proteins such as glycosyltransferases that modify proteins at a defined amino acid [18]. Some of the recently identified PARP members were reported to catalyze monoADP-ribosylation, and thus do not comply with the name polymerase. Furthermore, the currently assigned numbers do not reflect the fact that some of the PARP family members form phylogenetic subgroups. In addition, PARP5a and PARP5b are two distinct proteins encoded by different genes, rather than splice variants, and therefore should be numbered individually. Several new classifications have already been proposed for the PARP-like genes [4,5,18]. In the following sections we present the concept for a unified nomenclature of ART proteins that can reconcile recent findings and facilitate communication within the ADP-ribosylation field. Structural insights Crystal structures of diphtheria toxin have long provided the gold standard for understanding ADP-ribosyltransferases, partly because the toxin was crystallized years ago in complex with NAD+, whereas structural data on NAD+ complexes of many ARTs are still missing today [28]. 210

related exoenzymes from Bacillus cereus and Staphylococcus aureus catalyze ADP-ribosylation of Rho proteins at N14 [14,33]. Exoenzymes S and T of Pseudomonas aeruginosa are promiscuous enzymes that ADP-ribosylate numerous target proteins on arginine residues, akin to the structurally related mammalian ART1, ART2 (both GPI-anchored membrane proteins), and ART5 (secreted) ectoenzymes [20,36]. DRAT (dinitrogenase reductase ADP-ribosyltransferase) is a cytosolic R-S-E class ART that plays an important role in regulating nitrogen fixation in phototrophic bacteria by reversible ADP-ribosylation of R101 of the key enzyme nifA [60]. Alt and ModA are R-S-E class ARTs of T-even phages that ADP-ribosylate numerous target proteins at arginine residues in their E.coli hosts [61]. Common features of the R-S-E and H-Y-E classes of ARTs include the same linear order of five conserved b-strands, a similar mode of NAD+-binding, a role for the loop between b4 and b5 in substrate binding, and a widely conserved catalytic glutamate residue at the Nterminus of b5 [6]. The H-Y-E and R-S-E classes of ARTs can be distinguished on the basis of additional secondary structure units that are unique to each particular class [18].

However, in addition to the previously determined crystal structures of bacterial toxins and mono-ARTs in complex with NAD+ or NAD+ analogues, recent determination of PARP family members in complex with compounds that mimic either NAD+ or its nicotinamide moiety have facilitated the interpretation of the role of conserved sequence motifs across the different ART families (Table 2). Prokaryotic and eukaryotic ARTs are characterized by a conserved NAD+ binding core with a central 6-stranded bsheet [14,18,20,29–32]. X-ray crystallography of the NAD+binding core of ARTs has identified 3 motifs (in b-strands 1 and 2, and at the N-terminal end of b-strand 5) that are conserved across the kingdoms of life [18,20,29,30,33–35] (Figure 1a-c and Table 3): (i) The R/H-G-T/S (histidine-glycine-threonine, or R/H) motif located in the first b-strand is involved in NAD+ binding. The backbone carbonyl and amide groups of the glycine residue coordinate the nicotinamide moiety of NAD+. (ii) The S-T-S (serine-threonine-serine) motif, located in the second b-strand, is also involved in NAD+ binding. The S-T-S motif is found in all bacterial arginine-, cysteine-, and asparagine-specific monoARTs, and eukaryotic arginine-specific ecto-ARTs. In eukaryotic diphtheria toxin-related ARTs and bacterial diphthamide-specific ARTs, the S-T-S motif is either partially lost (Y-X-T/S) or entirely replaced (YF/X-A/X). (iii) The ARTT loop (ADP-ribosylating turn-turn) and the following b-strand 5 contain the catalytic glutamate and the Q/E-X-E motif, that is found in all known bacterial arginine-, cysteine-, asparagine-, and guanosine-specific ARTs, including the eukaryotic arginine-specific ecto-ARTs. The catalytic glutamate in the first position of b-strand 5 is conserved in bacterial diphthamide-specific ARTs such as diphtheria toxin, cholix toxin, Pseudomonas exotoxin A and a subgroup of eukaryotic diphtheria toxinrelated ARTs; the turn-turn motif, however, is not. The residues in this motif probably are involved in substrate recognition [36].

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Figure 1. Sequence alignments of the conserved structural elements and summary of structural features of ADP-ribose transferase catalytic domains. The nomenclature proposed here is based on three sequence motifs (H-Y-E, variant H-Y-E, and R-S-E), their documented placement in a 3D-structural context, and their consequence for enzymatic properties of ARTs. (a) Red stars indicate the three positions of the H-Y-E, variant H-Y-E, and R-S-E triads: the first position of the R/H motif; the first position of the S-T-S motif; and the position of the catalytic glutamate immediately following the ARTT loop. A blue star indicates the position of the small hydrophobic side chain that stacks with the nicotinamide moiety. The red arrowhead marks the position of the alternative catalytic aspartate in Arr, which could be present also in certain of the variant H-Y-E PARPs. bsheets 1–6 are indicated above the alignment using the same color scheme as in panels b and c. Sequences shown are those of Diphtheria toxin (gi:118949); chicken PARP1 (gi:3220000); human tankyrase-1 (gi:71052184); human PARP10 (gi:157738665); Aeropyrum pernix RNA 2’-phosphotransferase like protein (gi:118430933); Mycobacterium smegmatis rifampin ADP-ribosyltransferase (gi:118469048); Bacillus cereus Vip2 (gi:6730533); Staphylococcus aureus C3 toxin (gi:24636605); Salmonella typhimurium SpvB (gi:114794128); and rat mART2.2 (gi:40254800). The numbering of the first and last residue shown for each sequence is indicated. (b) Comparison of the key structural features and details of the active sites of the R-S-E ARTs (bacterial exotoxins and mammalian ecto-mART), H-Y-E ARTs (bacterial toxins and bona fide PARPs), and H-Y-E variant ARTs (bacterial transferases and novel ‘‘mono PARP’’ enzymes). Structures shown are those of Diphtheria toxin (pdb entry 1tox), chicken PARP1 (1a26; 3pax), human PARP10 (3hkv), tRNA phosphotransferase (1wfx), C3 exotoxin (1ojz), and rat ART2 (1og3). All structures are shown in a similar orientation with respect to their cofactor binding (NAD+ donor) sites. Six consecutive b-strands form the scaffold of the ART catalytic domain; these b-strands are shown in rainbow colors from the first N-terminal strand in blue to the 6th strand in red. The variable ARTT loop connecting b-strands 4 and 5 is shown in purple. Ligands and conserved motif side chains are shown as sticks of the same structures, highlighting the sequence motifs discussed in the text. In the PARP10 structure, the ligand 3-aminobenzamide (3AB) marks the nicotinamide-binding pocket. The active site of PARP1 (1a26) contains ADP, presumably the hydrolysis product of the added ligand carba-NAD+, in the acceptor site, marking the putative position of the poly-ADP-ribose chain. Here the ligand 3-methoxybenzamide (3MB; cyan) from crystal structure 3pax was overlaid to illustrate the position of the nicotinamide binding pocket. Likewise the crystal structure of TpT is ligand free; here, the structure of PARP10 was overlaid, and its ligand 3AB is shown in green to indicate the nicotinamide site. Note also the small aromatic side chains that stack with the nicotinamide moiety of NAD+ or the nicotinamide mimicking ligand, respectively. (c) Schematic representation of the interactions between the NAD+ co-substrate bound to the donor site and the residues of the H-Y-E and R-S-E motifs. Red and blue letters refer to the positions marked by red and blue stars in panel a.

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Table 2. Examples of crystal structures of ART catalytic domains published in the Protein Data Banka,b Triad motif HYE

Protein

Res.

Ligand

Organism

PDB code

Diphtheria toxin Pseudomonas exotoxin-A Pseudomonas exotoxin-A (eEF2 complex) PARP1 PARP1 PARP1 PARP2 PARP3 TNKS1

G1-S535 F400-R609 A399-G605 K662-W1014 K662-W1014 K664-T1014 E207-Q557 S176-H532 Q1104-E1314

NAD+ AMP + nicotinamide NAD+ 3-MB ADP A861695 Apo DR2313 apo

C. diphtheriae P. aeruginosa P. aeruginosa G. gallus G. gallus H. sapiens M. musculus H. sapiens H. sapiens

1tox c 1dma 2zit 3pax 1a26 2rd6 1gs0 3c4h 2rf5

TpT (tRNA phosphotransferase) ARR (Rifampin ADPr transferase) PARP10 PARP12 PARP14 PARP15

V3-L182 P6-D143 W817-P1008 D495-S689 K1532-K1720 L460-A656

apo Rifampin 3-AB 3-AB 3-AB PJ-34

Aeropyrum pernix Mycobacterium smegmatis H. sapiens H. sapiens H. sapiens H. sapiens

1wfx 2hw2 3hkv 2pqf 3goy 3gey

C3 ectotoxin VIP2 SpvB ART2

A36-K247 K62-N462 S392-S591 P4-S226

NAD+ NAD+ NAD+ NAD+

Staphylococcus aureus Bacillus cereus Salmonella typhimurium R. norvegicus

1ojz 1qs2 2gwl 1og3

HYE Variant

RSE

a

The RCSB Protein Data Bank (www.pdb.org) provides a number of straightforward tools for the analysis of three-dimensional protein structures, including viewer applications, ligand-related links and, via the ‘‘sequence search’’ tool, BLAST searches of the database content. Similar contents can also be accessed via the conserved domains browser provided by NCBI (www.ncbi.nlm.nih.gov/Structure/cdd/), or the UniProt website (www.uniprot.org). b Multiple entries of identical proteins (e.g., different inhibitor complexes) were omitted. c Italics indicate entries shown in Figure 1a, and bold face entries shown in Figure 1b.

Table 3. A comparison of catalytic core motifs and described/suggested enzymatic activities of PARP like enzymes, ecto-ARTs and bacterial toxinsa Triad motif b

R/H-G-T/S motif in b-strand 1

S-X-S/Y-X-X motif in b-strand 2

X-X-E motif at front edge of b-strand 5

Loop length (b-strands 4 and 5) c

ADP-ribosylation activity: mono (M), oligo (O), poly (P) and branching (B)

Automodification c

Refs.

PARPs: hPARP1 mPARP1 hPARP2

H-Y-E H-Y-E H-Y-E

HGS HGS HGS

YFA YFA YFA

YNE YNE YNE

+++ (37) +++ (37) +++ (40)

+ + +

[42] [39] [42]

mPARP2

H-Y-E

HGS

YFA

YNE

+++ (40)

+

[42]

hPARP3

H-Y-E

HGT

YFA

QSE

+++ (42)

+

[42]

mPARP3

H-Y-E

HGT

YFA

QSE

+++ (42)

+

[42]

hPARP4 mPARP4 hPARP5a mPARP5a hPARP5b mPARP5b hPARP9 mPARP9 hPARP10 mPARP10 hPARP14 mPARP14 hPARP15 e hPARP7 mPARP7 hPARP11 mPARP11 hPARP12 mPARP12 hPARP13 mPARP13 hPARP6 mPARP6

H-Y-E H-Y-E H-Y-E H-Y-E H-Y-E H-Y-E Q-Y-T Q-Y-T H-Y-I H-Y-I H-Y-L H-Y-I H-Y-L H-Y-I H-Y-I H-Y-I H-Y-I H-Y-I H-Y-I Y-Y-V H-Y-V H-Y-I H-Y-I

HGS HGS HGS HGS HGS HGS QQV QQV HGT HGT HGT HGT HGT HGT HGT HGT HGT HGT HGT YAT HAV HGS HGS

YFS YFS YFA YFA YFA YFA YFT YFT YFA YFA YFA YFA YFA YFA YFA YFA YFA YFA YFA YFA YFA YLS YLS

DDE DDE YAE YAE LAE LAE PET PET PSI PRI PSL PSI PKL PQI PQI PKI PKI PSI PTI PSV PSV GEI GEI

++ (15) ++ (15) ++ (11) ++ (11) ++ (11) ++ (11) + (6) + (6) + (7) + (7) + (7) + (7) + (7) + (6) + (6) + (6) + (6) + (9) + (9) + (6) + (6) + (2) + (2)

P, B (confirmed) P, B (confirmed) P (confirmed), B (postulated) P (confirmed), B (postulated) M (confirmed) O/P (postulated) M (confirmed) O/P (postulated) P/O (postulated) P/O (postulated) O (confirmed) O (postulated) O (postulated) O (postulated) ND d ND M (confirmed) M (postulated) M (postulated) M (confirmed) M (postulated) M (postulated) M (postulated) M (postulated) M (postulated) M (postulated) M (postulated) ND d M (postulated) M (postulated) M (postulated)

+ ND h + ND h + ND h NDd,h ND h + ND h + + + + ND h ND h ND h ND h ND h NDd,h ND h ND h ND h

[41] [44] [69] [44] [46] [45] [45] [46] [4,18] [4,18] [4,18] [4,18] [4,18] [4,18] [4,18] [4,18] [4,18] [4,18]

Current ART family and protein names

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Table 3 (Continued ) ADP-ribosylation activity: mono (M), oligo (O), poly (P) and branching (B) M (postulated) M (postulated) M (postulated) M (postulated)

Automodification c

Refs.

GNI GNI PKY PKY

Loop length (b-strands 4 and 5) c + (2) + (2) ++ (13) ++ (13)

ND h ND h ND h ND h

[4,18] [4,18] [4,18] [4,18]

HLA HLA

NGV NGV

+ (6) + (6)

M (p-RNA postulated) M (p-RNA postulated)

none none

[16] [16]

RGV RGV

SAS SAS

EEE EEE

11 11

M (arg, confirmed) M (arg,confirmed)

ND h +

[70] [71] [72]

RGT RGS RTS STS YRT HGM RGV RGV

SSS SSS SAK SAK STS SAS SSS SSS

EEE EEE ERI DSV KKE RKE ERE ERE

11 11 11 11 9 9 11 11

M (arg, confirmed) M/(O) (arg, confirmed) g M (postulated) M (postulated) M (postulated) M (postulated) M (arg, confirmed) M (arg, confirmed)

+ ND h ND h ND h ND h ND h +

[73,74] [20] [20] [20] [20] [20] [75]

ARTs HGT HGT HGT

YST YIA YVA

SVE RLE EDE

7 8 8

M (dph, confirmed) M (dph, confirmed) M (dph, confirmed)

none none none

[28] [76] [77]

Cholera toxin-like bacterial ARTs R-S-E RYD PT R-S-E RAD CT R-S-E RWC VIP2 R-S-E RGT exoS R-S-E RRV C2 R-S-E RGD C3 bot R-S-E RLL C3 stau

STS STS STS STS STS STS STS

QSE EQE EKE EKE EQE QLE QQE

29 15 11 11 11 13 11

M M M M M M M

ND h + ND h + + + +

[78] [79] [34] [80] [49] [49] [49]

Current ART family and protein names

Triad motif b

R/H-G-T/S motif in b-strand 1

S-X-S/Y-X-X motif in b-strand 2

X-X-E motif at front edge of b-strand 5

hPARP8 mPARP8 hPARP16 mPARP16

H-Y-I H-Y-L H-Y-Y H-Y-Y

HGS HGS HGS HGS

YLS YLS YLT YLT

TpT hTpT1 mTpT1

H-H-V H-H-V

HGT HGT

R-S-E R-S-E R-S-E R-S-E K-L-V S-L-I G-S-E K-S-E R-S-E R-S-E

Ecto mARTs: hART1 mART1 hART2P f mART2.1 f mART2.2 f hART3 mART3 hART4 mART4 hART5 mART5

Diphteria toxin-like bacterial H-Y-E DT H-Y-E ExoA H-Y-E ChT

(cys, confirmed) (arg, confirmed) (arg, confirmed) (arg, confirmed) (arg, confirmed) (asn, confirmed) (asn, confirmed)

a

The catalytic domains of different ART enzymes from human, mice, rat and bacteria were compared according to various published structural models. For eukaryotic enzymes only human and mouse proteins are listed. The catalytic core motifs and the loop length between b sheets 4 and 5, as well as confirmed enzymatic activities are indicated. Enzymatic activities of remaining family members are postulated according to the catalytic core motif and the loop length. b Three residues relevant for catalytic activities of ART enzymes (see also Figure 1b,c). c +++, ++, and + indicate long, intermediate and short loops, respectively. d ADP-ribosylation assays were performed only with the catalytic domain in isolation, and/or in the absence of a proven substrate. e BAL3 (PARP15) does not exist in rat and mouse. f Human ART2 does not encode a functional protein (P: pseudogene), whereas the mouse contains two functional Art2 genes (ART2.1 and ART2.2) and the rat two allelic variants. g Oligo-ADP-ribosylation has been observed as an auto-modification for rat ART2.2 incubated with high concentrations of NAD+. h ND: not determined.

Three amino acid residues from these motifs together form an evolutionarily conserved characteristic triad (Table 3). Arginine-, cysteine-, and asparagine-specific ARTs are characterized by a conserved R-S-E triad motif. In diphtheria toxin, exotoxin A, cholix toxin and all eukaryotic ARTs catalyzing poly-ADP-ribosylation the R-S-E triad is replaced by an H-Y-E triad motif. Inspection of crystal structures shows that the side chains of H-Y-E motifs interact with the NAD+ donor (Figure 1b,c). In diphtheria toxin (pdb entry 1tox), Ne of H21 (the first residue of the H-Y-E motif) hydrogen bonds with the 2’hydroxyl of the A-ribose. The Y54 side chain stacks with the N-ribose, whereas the carboxyl of the catalytic glutamate E148 coordinates the 200 -hydroxyl of the same ribose. Exotoxin-A residues H440, Y470 and E553, and cholix toxin residues H460, Y504, and E581 have corresponding roles. Crystal complexes of e.g. PARP3 with different inhibitors suggest that the H-Y-E motif functions similarly in the ARTs of higher eukaryotes ([37], pdb entry 3c4h).

Examination of the crystal structures of ART catalytic domains has led us to suggest that the PARP family can be subdivided into two groups: one containing 6 diphtheria toxin-like mammalian enzymes characterized by the presence of a glutamate in the H-Y-E triad motif; and a second group containing the remaining 11 members characterized by lack of the glutamate residue, that is replaced by I, L, T, V or Y (Table 3). This glutamate (E988 in hPARP1) is required for the elongation reaction in PARP1 and thus for the formation of poly-ADP-ribose chains [38,39]. The carboxyl group of this glutamate is thought to be critical for NAD+ activation by hydrogen bonding with the 200 hydroxyl of the N-ribose (Figure 1b,c). Additional residues thought to be important for poly-ADP-ribose chain elongation and branching activities are also not conserved in the 11 novel PARP-like proteins [18,40]. A phylogenetic tree of PARP1-like ARTs derived from structure-based sequence alignments also supports subdivision into two groups, and allows further division of PARPs 6 to 17 into 213

Review three subgroups, where members of each subgroup likely descended from a common metazoan ancestor [18]. Furthermore, with the exception of PARP16, the PARP members 6 to 17 do not contain the extensive loop region immediately upstream of b5 found in bona fide PARP1like ARTs. These features clearly distinguish the structures of these 11 mammalian diphtheria toxin-related enzymes from those of the diphtheria toxin-related PARP1-like ARTs and C2- and C3-toxin-related ectoARTs. Enzymological characterization of ARTs Classification of ARTs as either mono- or PARP1-like ARTs requires a set of criteria to be met. Different tools have been developed to achieve a characterization according to these criteria (Box 3). One important complication is that, due to the lack of suitable substrates, many studies have relied on the analysis of ART automodification. This has been observed for PARPs 1–3, v-PARP, and tankyrases-1 and -2. Whereas PARP1 and PARP2 synthesize large linear or branched ADP-ribose polymers, tankyrases-1 and -2 synthesize oligomers of an average chain length of 20 units without detectable chain branching [41– 44]. The enzymatic activities of PARP3 and vPARP are not well defined. PARP3 has only weak catalytic activity in vitro and so far no evidence has been published that supports polymer formation [37,42,43]. The recent generation of chimeric proteins revealed that the N-terminal domains of PARP1, PARP2 and PARP3 cooperate with their corresponding catalytic domains [42]. The DNA-dependent interaction between the N-terminal DNA binding domain and the catalytic domain of PARP1 increased Vmax and decreased the Km for NAD+, thus resulting in enhanced auto-poly-ADPribosylation [42]. Thus, analysis of the separated catalytic domains of different PARP family members alone, as presented in many published

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studies, is not sufficient to elucidate their enzymatic characteristics. The enzymatic activities of the remaining 11 mammalian diphtheria toxin-related enzymes are only partially investigated. However, two of these enzymes (PARP10 and 14) were recently characterized as ARTs that catalyze mono-ADP-ribosylation [45]. Whether additional members of the PARP family catalyze only mono-ADP-ribosylation remains to be determined. There is preliminary experimental evidence that PARP9 (also called BAL1) and PARP13 (also called ZAP or ZC3HAV1) lack auto-ADPribosylation activity, at least for the catalytic domain alone [45,46]. As both proteins harbor substitutions in the three motifs mentioned above, they could lack enzymatic activity altogether. Specificity of ADP-ribosylation Little is known presently about the mechanism of substrate recognition by ARTs; indeed, substrate specificity is probably determined by multiple parameters. The predicted central role of the loop between b-strands 4 and 5 upstream of the catalytic glutamate has been confirmed by site-directed mutagenesis, loop grafting and co-crystallization studies [31,33]. Exchanging the E/Q residue, two residues upstream of the catalytic E can convert an NAD+-hydrolase into an arginine-specific transferase (and vice versa) [47,48] or an asparagine-specific ART into an arginine-specific ART [49]. Grafting of the entire ARTTloop between exoenzymes S and T (both arginine-specific ARTs) largely transferred the preferred substrate specificity of the respective enzymes [36]. The crystal structure of chicken PARP1 showed a co-crystallized carba-NAD+ molecule cradled in the corresponding large loop, suggesting that this loop is involved in recognizing polyADP-ribose as a PARP-substrate for further addition of ADP-ribose [40]. Recently, the first structures of two ARTs

Box 3. Experimental possibilities to characterize ADP-ribosyltransferases Different experimental methods have been developed to detect ARTs and their ADP-ribosylated targets in vitro and in vivo. Although antibodies have been raised against different ARTD and ARTC family members, only a few antibodies are available for detecting ADPribosylated targets. Currently, two different antibodies against pADPr are used widely: mAb 10H that recognizes pADPr chains of at least 20 ADP-ribose units and pAb 96-10-04, that is described as recognizing oligomers of six or more ADPr units ([62,63] and Alexis Biochemicals). Unfortunately, only polyclonal antibodies have been raised against mono-ADP-ribosylated proteins and these are not available widely [64,65]. Depending on the specificity and sensitivity of the antibodies and the conditions for sample preparation (given the unstable nature of pADPr), antibody-based analyses might lead to conflicting conclusions. Furthermore, auto-modification of ARTs can lead to a retardation of protein migration on SDS-PAGE, especially in cases of poly-ADP-ribosylation of proteins with polymers of considerable length, thus providing additional evidence for pADPr modification. Recently, a new method for detecting and purifying mono- and poly-ADP-ribosylated proteins has been reported that utilizes the intrinsic capacity of the macro domain to specifically bind ADP-ribose [66]. Modification of NAD+ with radio-isotopes or chemical groups (e.g. 32 P-NAD+, etheno-NAD+ [67], or biotin-NAD+), provides additional possibilities to detect ADP-ribosylated proteins or, after appropriate

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release from the protein, to analyze the ADP-ribose products. NAD+ metabolic labeling followed by high resolution PAGE and fluorography of isolated ADP-ribose polymers has also been described as an efficient method to characterize polymers (as short as 3-mers) produced in living cells [68]. Furthermore, the type of protein ribosylation (mono- versus poly-ADP-ribosylation) can be analyzed by phosphodiesterase digestion of products generated by recombinant, purified proteins. Digestion of a pADPr-modified protein by phosphodiesterase leads to the generation of phosphoribosyl-AMP (PR-AMP), whereas only AMP is generated from mono-ADP-ribosylated proteins. Both molecules can be detected using radiolabelled ADP-ribose and the digestion products can be analyzed by 2 dimensional thin layer chromatography (2D-TLC) followed by autoradiography. Alternatively, PR-AMP can also be identified by high performance liquid chromatography (HPLC) and mass spectrometry (MS). The most robust analysis to investigate products is the separation of ADP-ribose on a sequencing gel; reaction products are released from proteins by alkali treatment and separated, owing to their negative charge. Products can be visualized either by silver staining or by autoradiography of radiolabelled NAD+. As silver only stains pADPr chains of at least 4-10 ADP-ribose units, the use of radiolabelled NAD+ is certainly recommended. Defined standards (e.g. ADP-ribose) are required to determine the number of isolated ADP-ribose units.

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co-crystallized with their target proteins (ExoA with elongation factor 2, iota-toxin with actin) uncovered a central role of the ARTT loop in target protein binding, but also revealed important roles for at least two other loops in target binding [50,51]. Similarly, the recently described crystal structure of the diphtheria toxin-related rifampin ART (Arr, pdb entry 2hw2) in complex with its substrate, the antibiotic rifampin, revealed a central role for this loop in substrate binding [52]. Interestingly, whereas Arr contains the H and Y residues in b-strands 1 and 2, it harbors a serine in place of the catalytic glutamate at the beginning of b5. Strikingly, an aspartate residue at the end of the neighboring b-strand (b4) has likely acquired the corresponding catalytic role. Thus, it is conceivable that, for catalysis, some of the PARP-like ARTs that lack the catalytic glutamate, might employ alternative side chains, with a slightly different geometry. Notably PARPs 14, 9, 10, 11, 13, and 7 contain an aspartate at the position corresponding to the catalytic aspartate of Arr (Figure 1; red arrowhead). Together, these experiments have not led to an easily recognizable substrate recognition motif, and at present the substrate specificity for a given ADP-ribosyltransferase can only be elucidated experimentally. X-ray crystallography, the confirmation of predicted acceptor sites within a protein by site-directed mutagenesis, and/or mass spectrometric analysis of modified proteins will be required to define substrate specificity. Toward a new nomenclature A protein nomenclature can be based on properties that include the molecular weights of proteins, the products generated, the enzymatic reactions catalyzed, or on struc-

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tural characteristics. As some PARP family members remain poorly characterized with regard to the products of catalysis (mono-ADP-ribose vs. poly-ADP-ribose), we propose a nomenclature based on the type of enzymatic reaction and on structural features. Importantly, omitting the prefix ‘‘poly’’ and ‘‘mono’’ in the name avoids the necessity of renaming individual family members if subsequent evidence clarifies their catalytic activities in the future. Moreover, poly-ADP-ribose polymerase is a pleonasm. As mentioned above, all 17 mammalian PARPs are currently referred to as PARP enzymes, in accordance with the original characterization of the family’s founding member PARP1. Based on the enzymatic reaction catalyzed by these proteins (post-translational modification of amino acid residues), and on the rules for biochemical classifications, ‘‘transferase’’ is a more appropriate and accurate name for these enzymes. Thus, we propose to rename the mammalian PARPs, according to the recommendations of the International Union of Biochemistry and Molecular Biology (IUBMB), as ADP-ribosyltransferases (or ARTs). Before the name PARP came into use, some laboratories had already named the protein ADP-ribosyltransferase, although with another acronym (ADPRT) [53,54]. To distinguish diphtheria toxin, the first structurally characterized protein mono-ADP-ribosyltransferase, and PARP-like ARTs from the structurally distinct C2 and C3 toxin-like ARTs, the names of the former are extended with a D for diphtheria toxin-like (ARTD). Accordingly, the names for the C2 and C3 toxin-like proteins are extended with a C (ARTC) [55] (Table 4 and Figure 2). We propose ARTD and ARTC rather than DART or CART, as the latter two names are currently in use for other proteins. Family member numbering (ARTD1 or ARTC1) are assigned based on the H-Y-E triad

Table 4. Example of the proposed classification of human ARTD and ARTC proteinsa New classification: Protein names Old classifications: protein names [4,5,18] Aliases ARTD: PARP1, pART1 – ARTD1 PARP2, pART2 – ARTD2 PARP3, pART3 – ARTD3 PARP4, pART4 vaultPARP ARTD4 PARP5a, pART5, PARP5 Tankyrase-1 ARTD5 PARP5b, pART6, PARP6 Tankyrase-2 ARTD6 PARP15, pART7, Pl-mART9 BAL3 ARTD7 PARP14, pART8, Pl-mART11 BAL2 ARTD8 PARP9, pART9, Pl-mART10 BAL1 ARTD9 PARP10, pART10, Pl-mART4 – ARTD10 PARP11, pART11, Pl-mART5 – ARTD11 PARP12, pART12, Pl-mART7 ZC3HDC1 ARTD12 PARP13, pART13, Pl-mART8 ZC3HAV1, ZAP1 ARTD13 PARP7, pART14, Pl-mART6 TiPARP, RM1 ARTD14 PARP16, pART15, Pl-mART1 – ARTD15 PARP8, pART16, Pl-mART3 – ARTD16 PARP6, pART17, Pl-mART2 – ARTD17 TRPT1 TPT1 ARTD18 ARTC: ARTC1 ARTC2P b ARTC3 ARTC4 ARTC5

ART1 ART2P ART3 ART4 ART5

CD296 – – CD297 –

Accession numbers NP_001609.2 NP_001036083.1, NP_005475.2 NP_005476.3, NP_001003931.2 NP_006428.2 NP_003738.2 NP_079511.1 NP_001106995.1, NP_689828.1 NP_060024.2 NP_001139574.1, NP_001139575.1 NP_116178.2 NP_065100.2 NP_073587.1 NP_078901.3, NP_064504.2 NP_056323.2 NP_060321.3 2NP_078891.2 NP_064599. NP_001153861 NP_004305.2 – NP_001123488.1, NP_001123489.1, NP_001170.2 NP_066549.2 NP_001073004

a ARTD/ARTC family members are numbered on the basis of similarities in amino acid sequence, structure of the catalytic domain and postulated catalytic reaction as shown in Table 3. Several accession numbers denote individual isoforms. b Pseudogene in humans; Art2 is duplicated in the mouse.

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Figure 2. Schematic comparison of the domain architecture of the human ARTD (PARP) family. The following domains are indicated: The ART domain is the catalytic core required for basal ART activity. The PARP regulatory domain (PRD) might be involved in regulation of the PARP-branching activity. The WGR domain named after a conserved central motif (W-G-R), is also found in a variety of polyA polymerases and in proteins of unknown function. The BRCT domain (BRCA1 carboxy-terminal domain) is found within many DNA damage repair and cell cycle checkpoint proteins. The sterile alpha motif (SAM), a widespread domain found in signaling and nuclear proteins, can mediate homo- or heterodimerization. The ankyrin repeat domains (ARD) mediate protein–protein interactions in diverse families of proteins. The vault protein interalpha-trypsin (VIT) and von Willebrand type A (vWA) domains are conserved domains found in all inter-alpha-trypsin inhibitor (ITI) family members. Both of these domains are presumed to mediate protein–protein interactions. The WWE domain is named after three conserved residues (W-W-E), and is predicted to mediate specific protein– protein interactions in ubiquitin- and ADP-ribose conjugation systems. The Macro or A1pp domains are structurally related to the catalytic domain of enzymes that process ADP-ribose-10 -phosphate, a reaction product derived from ADP-ribose 10 -20 cyclic phosphate generated by TpT. The Macro domain can serve as ADPr or O-acetyl-ADPribose binding module. ZF: zinc finger domains. SAP: SAF/Acinus/PIAS-DNA-binding domain, MVP-ID: Major-vault particle interaction domain, NLS: nuclear localization signal. CLS: centriole-localization signal. HPS: Histidine-proline-serine region. RRM is an RNA-binding/recognition motif. UIM: ubiquitin interaction motif. MVP-ID: M-vault particle interaction domain. TPH: Ti-PARP homologous domain. GRD: glycine-rich domain. Within each ART domain, the region that is homologous to the PARP signature (residues 859–908 of PARP1) as well as the equivalent of the PARP1 catalytic E988 is shaded. The WWE domain is a protein–protein interaction motif. TM: transmembrane domain.

motif, H-Y-E triad motif variants, as well as phylogenetic analyses of the different catalytic domains, and should, wherever possible, match the already assigned numbers in the old classifications. According to IUBMB guidelines, the number follows the acronym without a hyphen. Sirtuins and ADP-ribosylcyclases are not included in the nomenclature, as they each have a NAD+-binding fold that is clearly distinct from that of the ARTs described here. The new systematic and common names are defined according to the recommendations of the IUBMB (Box 4). However, the EC number given by the IUBMB system is composed of only four numbers, and this is not sufficient for categorizing the ART enzymes. In many cases, there is 216

a poor correspondence between the EC number and gene product, i.e. a single EC number corresponds to several genes with distinct enzymatic activities. We therefore propose a modified EC numbering system that defines exactly the reaction catalyzed by ART families, subfamilies and groups. Our modified EC Number is extended to 8 numbers: EC 2.4.2.30.W.X.Y.Z; (W: structure of catalytic domain, X: substrate specificity, Y: mono/oligo/poly and Z: chemical nature of the covalent bond). Together, the unified nomenclature complies with the IUBMB guidelines and the assigned numbers (ARTD1 or ARTC1) are based on phylogenetic analyses of the different catalytic domains.

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Box 4. Modified ADP-ribosyltransferase enzyme classification tree New extended EC number: EC 2.4.2.30.W.X.Y.Z W: Structure of catalytic domain X: Substrate specificity (amino acid, nucleic acid, antibiotic, water, (n)ADP-ribose) Y: Mono, oligo or poly Z: Chemical nature of the covalent bond Transferases (EC2) Glycosyltransferases (EC2.4) Pentosyltransferases (EC2.4.2) CLASS: ADP-ribosyltransferases (EC 2.4.2.30) Systematic name: NAD+: ADP-D-ribosyl-acceptor ADP-D-ribosyltransferase (ADPRT) Common name: ADP-ribosyltransferase (ART) Definition: The ADP-D-ribosyl group of NAD+ is transferred to an acceptor (amino acid, nucleic acid, antibiotic, water). The linkage can be O-, N-, or S-glycosidic. Any additional ADP-ribosyl groups are transferred to the 2’-position of the terminal AMP moiety, and, in case of branching, to the 200 or 300 -positions of the ribose 50 -phosphate moiety (the N-ribose) of preexisting pADPr chains. SUBCLASSES: Diphtheria toxin-like-ADP-ribosyltransferases: EC 2.4.2.30.1.X.Y.Z. Systematic name: Diphtheria toxin-like NAD+: ADP-D-ribosyl-acceptor ADP-D-ribosyltransferase (ADPRT-DL) Recommended common name: Diphtheria toxin-like ADP-ribosyltransferases (ARTD) Cholera toxin-like-ADP-ribosyltransferases: EC 2.4.2.30.2.X.Y.Z. Systematic name: Cholera toxin-like NAD+:ADP-D-ribosyl-acceptor ADP-D-ribosyltransferase (ADPRT-CL) Common name: Cholera toxin-like ADP-ribosyltransferases (ARTC) FAMILIES: (For Diphtheria toxin-like, Cholera toxin-like, etc) ADP-ribosyltransferases catalyzing mono-ADP-ribosylation (EC 2.4.2.30.W.X.1.Z): Systematic name: NAD+:mono-ADP-D-ribosyl-acceptor-ADP-D-ribosyltransferase (MADPRT) Common name: Mono-ADP-ribosyltransferases (MART) ADP-ribosyltransferases catalyzing poly-ADP-ribosylation (EC 2.4.2.30.W.X.2.Z): Systematic Name: NAD+:poly-ADP-D-ribosyl-acceptor ADP-D-ribosyltransferase (PADPRT) Common name: Polymerizing-ADP-ribosyltransferases (PART)

Concluding remarks As observed in other fields for other protein families, new tools and methods or ways of analysis provide novel opportunities to attribute scientifically correct names to proteins (e.g. the fields of lysine acetyl transferases (KATs), and arginine or lysine methyl transferases (RMTs or KMTs)). We believe that the relative simplicity of a new, correct, common nomenclature will expedite functional comparisons in different species, while remaining flexible enough to accommodate additional species-specific ARTs as they are being discovered.

SUBFAMILIES: (For Diphtheria toxin-like, Cholera toxin-like, etc) O-glycosidic-ADP-ribosyltransferases (OADPRT): (EC 2.4.2.30.W. X.Y.1) Systematic Name: NAD+ ADP-D-ribosyl-L-glutamate-ADP-D-ribosyltransferase (EADPRT) Common name: Glutamate-specific ADP-ribosyltransferase (EART) EC 2.4.2.30.1.1 Reaction: NAD+ + L-glutamate = nicotinamide + O(n)ADP-D-ribosyl-L-glutamate N-glycosidic-(n)ADP-ribosyltransferases (NADPRT): (EC 2.4.2.30.W. X.Y.2) Systematic names: NAD+ ADP-D-ribosyl-L-diphthamide-ADP-D-ribosyltransferase (DphADPRT) NAD+ ADP-D-ribosyl-L-lysine-ADP-D-ribosyltransferase (KADPRT) NAD+ ADP-D-ribosyl-L-arginine-ADP-D-ribosyltransferase (RADPRT) NAD + ADP-D-ribosyl-L-asparagine-ADP-D-ribosyltransferase (NADPRT) Common name: Diphthamide-specific ADP-ribosyltransferase (DphART) Lysine-specific ADP-ribosyltransferase (KART) Arginine-specific ADP-ribosylransferase (RART) Asparagine-specific ADP-ribosyltransferase (NART) EC 2.4.2.30.1.2 Reaction: NAD+ + L-diphthamide = nicotinamide + N-ADP-D-ribosyl-L-diphthamide EC 2.4.2.30.1.2 Reaction: NAD+ + L-lysine = nicotinamide + N-ADP-Dribosyl-L-lysine EC 2.4.2.30.2.2 Reaction: NAD+ + L-arginine = nicotinamide + N-(ADPD-ribosyl-L-arginine EC 2.4.2.30.2.2 Reaction: NAD+ + L-asparagine = nicotinamide + NADP-D-ribosyl-L-asparagine S-glycosidic-(n)ADP-ribosyltransferases (SADPRT): (EC 2.4.2.30.W. X.Y.3) Systematic name: NAD+ ADP-D-ribosyl-L-cysteine-ADP-D-ribosyltransferase (CADPRT) Common name: Cysteine-specific ADP-ribosyltransferase (CART) EC 2.4.2.30.2.3 Reaction: NAD+ + L-cysteine = nicotinamide + S-ADPD-ribosyl-L-cysteine GROUPS: (For Diphtheria toxin-like, Cholera toxin-like, etc) Groups are classified based on the conservation of their domain architecture, reaction catalyzed and substrate specificities Example: EC 2.4.2.30.1.2.1.1. Systematic Name: Diphtheria toxin-like NAD+ ADP-D-ribosyl-L-Glutamate-ADP-D-ribosyltransferase (MD-EADPRT-DL) Common name: Diphtheria toxin-like macro-domain-containing Glutamate-specific mono-ADP-ribosyltransferases macro-EARTD).

Proposing new names of proteins always presents a challenge, as established habits must be changed. For instance, compounds that inhibit the family founding member PARP1 are referred to as ‘‘PARP inhibitors’’, an acronym for a class of substances that are presently evaluated in clinical trials for treatment of different diseases. Many of these compounds also inhibit other ARTDs as well as ARTCs from different species. Ultimately, as selectivity and specificity issues are being addressed, the new nomenclature also aids proper labeling of ART inhibitors. Adopting a new nomenclature now is prudent, considering that 217

Review more laboratories can be expected to launch research on PARP-family members that display mono- rather than poly-ADP-ribosyltransferase activity. In summary, we use structural and biochemical arguments to propose a new nomenclature for ADP-ribosyltransferases. We hope that this initiative will help to unify the ART field, simplify and specify the nomenclature, and provide substantial benefits both for researchers working in the ART field and for newcomers. Final classification and nomenclature of the known PARP/ART proteins will need to be discussed and agreed on by the majority of the research community, a process that requires patience and time. Colleagues who prefer to continue using the term PARP are encouraged to use a footnote referring to the comprehensive nomenclature. Overall, we expect that the long-term benefits will outweigh the short-term drawbacks; indeed, we have already received broad support of this proposed nomenclature. Acknowledgements We apologize to those investigators whose work we could not cite due to a reference limit, and gratefully acknowledge their contributions to the field. We are grateful to the following colleagues for their critical and constructive comments on the manuscript (in alphabetic order): N.-W. Chi (California, San Diego, USA), A. Chiarugi (Florence, Italy), M. CohenArmon (Tel-Aviv, Israel), D. Corda (Naples, Italy), M. DiGirolamo (Santa Maria Imbaro, Italy), R. Fieldhouse (Guelph, Canada), S. Goenka (Indianapolis, USA), F. Haag (Hamburg, Germany), H. Kleine (Aachen, Germany), M. Malanga (Naples, Italy), M. Masutani (Tokyo, Japan), R.G. Meyer (Philadelphia, USA), R. Merrill (Guelph, Canada), M. Miwa (Nagahama, Japan), J. Moss (Bethesda, USA), J. Pascal (Philadelphia, USA), C. Thiemermann (London, UK), K. Ueda (Kobe, Japan), Z.-Q. Wang (Jena, Germany). H.S. would like to acknowledge Tobias Karlberg and Lari Lehtio¨ and members of the Structural Genomics Consortium for insightful discussions. M.O.H. would like to acknowledge Peter Richards (University of Zurich, Switzerland) for critical reading of the manuscript. Work on ADPriboslytransferases in the laboratory of M.O.H is supported by the Swiss National Foundation (SNF 31-122421), P.O.H. is supported by the Swiss National Foundation (SNF 31-125190/1), B.L. is supported by the Deutsche Forschungsgemeinschaft (DFG Lu466/15-1) and F.K.N. is supported by the Deutsche Forschungsgemeinschaft (No 310/6-3, No310/7-1). H.S. is supported by The Structural Genomics Consortium, a registered charity (1097737) that receives funds from Sweden, UK and Canada (http://www.thesgc.org/about/sponsors.php/). The authors declare no conflict of interest.

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