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PARP1 shows that core structural elements such as the NAD+-binding residues are conserved, whereas a catalytic glutamate that defines the enzymatic polymerase activity of PARP1 (Glu988) is missing in PARP10 and PARP12. In addition, PARP10 and PARP12 are characterized by a short β4–β5 loop, which has been implicated in substrate recognition. Using these structural differences, the authors classify PARPs into three subgroups (Fig. 2): PARPs 1–5, which are bona fide PARPs; PARPs 6–8, 10–12 and 14–16, which are likely to be mARTs; and PARPs 9 and 13, which lack a NAD+-binding histidine in addition to the catalytic glutamate and may therefore be inactive.

To identify the PARP10 residues that are post-translationally modified by ADP-ribosylation, they tested GST-tagged protein fragments for automodification and trans-ADP-ribosylation activity. They find that the PARP10818–1025 fragment is catalytically active and contains the automodification site(s). PARP10 does not seem to be capable of modifying cysteine or arginine residues but rather is suggested to mono-ADP- ribosylate glutamates. In keeping with this hypothesis, mutation of Glu882 to alanine greatly reduces automodification activity. Further analysis of PARP10 mutants led the authors to the conclusion that PARP10 may be using a catalytic mechanism that distinguishes PARP10 from the classic PARPs and from other mARTs. Classic polymerases such as PARP1 use a catalytic glutamate at

might exist. This is where a recent article in Molecular Cell provides a new prospect for the PTM field. Kleine et al.7 studied PARP10, an enzyme that, on the basis of homology to PARP1, was expected to function as a poly-ADP-ribose polymerase. Yet the authors find PARP10 to function as a bona fide mART.

PARP10 was first identified as a c-Myc- interacting protein by Yu et al.8. PARP10 has auto-ADP-ribosylation activity and is a ubiquitously expressed protein that shuttles between nuclear and cytoplasmic compartments. In their current report, Kleine et al.7 present a thorough study of the enzymatic properties of PARP10. Using tools that include recombinant protein expression, enzymatic kinetic assays and crystal structure homology models, they conclude that PARP10 and several of the other 16 PARP-like proteins may show mART activity.

PARP10 purified from mammalian cells has auto-ADP-ribosylation activity but, in contrast to PARP1 (Fig. 1), does not show the size shift in PAGE gels that derives from PARP-bound poly-ADP-ribose polymers, suggesting that PARP10 has mono-ADP- ribosyltransferase, but no polymerase, activity. Interestingly, the enzyme is able to trans-ADP-ribosylate nuclear target proteins such as histones. To map and better analyze the molecular basis of the apparent mART activity, the authors generated a structural model for the PARP10 catalytic domain. Comparison of the PARP10 model with

Chris Walsh’s landmark textbook on the post-translational modification (PTM) of proteins1 contains a precious, slender chapter on ADP-ribosylation. For decades, this field has been dominated by two classes of NAD+-metabolizing enzymes, PARP1 and the extracellular mono-ADP- ribosyltransferases (mARTs). Yet, despite a strong suspicion that cellular mARTs having signal- transduction and regulatory functions might exist, their identity remained hard to pin down. Extracellular enzymes include diphtheria, cholera and pertussis toxins2,3, which mono-ADP- ribosylate host proteins such as the regulatory subunits of G proteins4. PARP1, in contrast, is a cellular, nuclear poly-ADP-ribose- polymerase (PARP) that catalyzes the formation of complex, branched oligonucleotides that act as post- translational modifiers of proteins involved in DNA repair, transcription and mitotic spindle formation, among other functions. Substrates include histone proteins, and its ability to recognize nucleic acids, such as those with double-strand breaks, implicates it in regulating cellular apoptosis (reviewed in ref. 5). More recently, Sir2-related enzymes (collectively known as sirtuins and implicated in aging, cancer and heterochromatin formation) have been described as either NAD+-dependent deacetylases or ADP-ribosyltransferases. Notably, human SirT6 does not function as a deacetylase but rather is able to mono-ADP- ribosylate itself 6. It is still unclear what biological role this modification plays, although the known reversibility of protein ADP-ribosylation potentially makes this PTM very interesting for regulation in biology.

However, to date, only extracellular enzymes and bacterial toxins had been thought to mono-ADP-ribosylate protein targets, so it was not clear whether endogenous, cellular proteins capable of trans-ADP-ribosylation

PARP: a transferase by any other nameSusanne Till, Konstantina Diamantara & Andreas G Ladurner

A recent report shows that several ‘poly-ADP-ribose-polymerases’ may function exclusively as a family of endogenous mono-ADP-ribosyltransferases, providing a new, molecularly less complex and broadened cellular role for this elusive post-translational modification.

Susanne Till, Konstantina Diamantara and Andreas G. Ladurner are in the Gene Expression Unit and Structural & Computational Biology Unit, European Molecular Biology Laboratory, 69117 Heidelberg, Germany. e-mail: ladurner@embl.de

10 110 114 212 280 386 549 663 796333 464 634 1008

1 1014Zn I Zn II Zn III BRCT WGR PRD PARP

DNA bindingdomain

Catalytic core domain

1 1025GRDRRM mART

11 85 281 399 650 673 1007820

PARP1

PARP10

UIM domains Glu882

Figure 1 Domain architecture of the classic poly-ADP-ribose-polymerase PARP1 and the newly characterized mono-ADP-ribosyltransferase PARP10. PARP1 contains three zinc-finger domains (Zn I, II and III), a breast cancer suppressor protein-1 domain (BRCT), a WGR domain (named after a conserved sequence motif), a PARP regulatory domain (PRD) and the catalytic PARP domain. PARP10 consists of a RNA recognition motif (RRM), a glycine-rich region (GRD) and three ubiquitin-interaction motifs (UIM), in addition to its catalytic mono-ADP-ribosyltransferase (mART) domain.

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1244 volume 15 number 12 DeCember 2008 nature structural & molecular biology

It could therefore be that these enzymes are effectors for signaling cascades that remain to be determined.

Whereas poly-ADP-ribosylation generates a PTM that is highly dynamic, of variable length and with a branched, complex structure, the mono-ADP-ribosylation of specific side chains in target proteins represents a more conventional, one could say ‘simpler’, molecularly specific PTM. The opportunities and goals in the coming years will thus be to (i) identify the cellular proteins that are mono-ADP-ribosylated by endogenous intracellular enzymes, (ii) check whether specific domains or modules may be able to recognize this PTM with high specificity and selectivity (perhaps including the recognition of the context in which the mono-ADP- ribosylation PTM occurs), (iii) determine which enzymes may be able to reverse this PTM (poly-ADP-ribose glycohydrolase and the ADP-ribose-hydrolase family of proteins are good candidates) and (iv) identify how the activity of these new mARTs may be regulated (for example, by cellular signal transduction cascades or by nucleic acids). In short, now that several so-called PARPs are likely to be (re-)assigned in enzyme function to the simpler category of mART enzymes, the relevant chapter in Chris Walsh’s tribute to post-translational modification may well be significantly expanded.

ACKNOWLEDGMENTSK.D. is funded by the European Union FP6 Marie Curie Research and Training Network “Chromatin Plasticity”. S.T. and K.D. are students in the European Molecular Biology Laboratory’s International PhD Programme. A.G.L. acknowledges the financial support of the European Molecular Biology Laboratory.

1. Walsh, C.T. Posttranslational Modification of Proteins: Expanding Nature’s Inventory (Roberts, Greenwood Village, Colorado, USA, 2006).

2. Krueger, K.M. & Barbieri, J.T. Clin. Microbiol. Rev. 8, 34–47 (1995).

3. Ueda, K. & Hayaishi, O. Annu. Rev. Biochem. 54, 73–100 (1985).

4. Lupi, R., Corda, D. & Di Girolamo, M. J. Biol. Chem. 275, 9418–9424 (2000).

5. Hassa, P.O. & Hottiger, M.O. Front. Biosci. 13, 3046–3082 (2008).

6. Liszt, G., Ford, E., Kurtev, M. & Guarente, L. J. Biol. Chem. 280, 21313–21320 (2005).

7. Kleine, H. et al. Mol. Cell 32, 57–69 (2008).8. Yu, M. et al. Oncogene 24, 1982–1993 (2005).9. Allis, C.D., Jenuwein, T. & Reinberg, D. Epigenetics (Cold

Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA, 2007).

10. Liou, G.G., Tanny, J.C., Kruger, R.G., Walz, T. & Moazed, D. Cell 121, 515–527 (2005).

11. Huse, M. & Kuriyan, J. Cell 109, 275–282 (2002).12. Karras, G.I. et al. EMBO J. 24, 1911–1920 (2005).13. Kustatscher, G., Hothorn, M., Pugieux, C., Scheffzek, K.

& Ladurner, A.G. Nat. Struct. Mol. Biol. 12, 624–625 (2005).

14. Dhalluin, C. et al. Nature 399, 491–496 (1999).15. Jacobson, R.H., Ladurner, A.G., King, D.S. & Tjian, R.

Science 288, 1422–1425 (2000).

for assuming that proteins such as SirT6 may have evolved trans-ADP- ribosylation activities that remain to be characterized. The enzymatic activity of many proteins can in fact be profoundly switched or altered by the interaction of an enzyme with regulatory cofactors, such as proteins or other ligands. A good example of this is yeast Sir2, where its metabolite O-acetyl-ADP-ribose alters the structure and heterochromatic functions of the Sir2-containing SIR silencing complex10. SirT6 and other sirtuins may thus join a family of ‘PARP’ enzymes in functioning as endogenous, cellular mARTs. It is also possible that the auto-ADP-ribosylation observed in sirtuins, in PARP10 and in the new ‘PARP’ mARTs, might lead to autoinhibited structures, as is the case for the non-receptor tyrosine kinase Src11. The predicted mARTs PARP14 and PARP15, for example, contain ‘macro’ domains that bind ADP-ribose with high specificity12,13, so auto-ADP-ribosylation could lead to an altered conformation of the protein. Alternatively, these macro domains may serve or aid the binding of the PARP14 or PARP15 mART to ADP-ribosylated targets in trans, perhaps allowing these enzymes to work processively. Examples of this exist in the chromatin field, where many proteins (for example, P/CAF and Taf1)14,15 contain a ‘writer’ and ‘reader’ module in the same polypeptide, such as an acetyltransferase module coupled to an acetyl- lysine binding bromodomain.

Prompted by the current study, it will now be interesting to identify the cellular targets of mART-catalyzed reactions and to determine how the activity of these enzymes is regulated. In contrast to PARP1 and PARP2, neither PARP10 nor the predicted PARP-family mART enzymes are thought to be activated by DNA damage. In fact—at least in vitro—DNA reduces PARP10 activity7.

position 988, which is missing in PARP10 (replaced by isoleucine) and in several other PARP-family members. Instead, the authors can model Glu882 as the catalytic PARP10 ‘substrate’ in a reaction carried out by a second PARP10 enzyme. Such substrate- assisted catalysis might explain why PARP10 is not able to catalyze poly-ADP- polymerization.

In conclusion, Kleine et al.7 report that PARP10, a member of the PARP family, lacks polymerase activity but is a new type of intracellular mono-ADP- ribosyltransferase. Using sequence alignments, structural models, engineered point mutants and enzymatic assays, the authors are able to make the interesting prediction that many more of the ~16 PARP-like enzymes in the human genome may function as mARTs (Fig. 2). Assuming that most of these enzymes mediate PTMs in trans, the authors’ evidence suggests that a variety of mono-ADP- ribosylated targets might be identified in the coming years.

The ability of PARP10 to ADP-ribosylate histones in vitro raises the interesting possibility that histones may be physiological targets of PARP10. As at least a fraction of cellular PARP10 is located in the nucleus, histones could indeed be the target of PARP10 or of other mARTs, adding to the already complex network of histone modifications. Historically, histone PTMs have often preceded the discovery of related PTMs in non-histone proteins. And notably, in most cases, a specific PTM of both histone and non-histone substrates serves regulatory purposes, this clearly being the case for serine/threonine phosphorylation (one of the earliest PTMs described), lysine acetylation and lysine/ arginine methylation9.

It is not yet known whether any of the sirtuins might mono-ADP-ribosylate target proteins in trans, rather than solely being capable of auto-ADP-ribosylation, as is the case for SirT6 (ref. 6). But there is scope

Class I: bona fide PARPs Class II: mARTs Class III: inactive PARPs?

PARP1–PARP5 PARP9 and PARP13PARP6–8, PARP10–12, PARP14–16

PARP1 PARP10PARP9

Targetprotein

n

Targetprotein

Targetprotein

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LinearBranched

Figure 2 Classification of the poly-ADP-ribose-polymerase (PARP) family according to the structural model proposed by Kleine et al.7. Class I contains the classical, bona fide poly-ADP-ribose polymerases PARP1–PARP5. Members of class II, including the newly identified mART PARP10, are known or predicted to function as mono-ADP-ribosyltransferases (mARTs) rather than PARPs. A third class may include PARP9 and PARP13, whose catalytic activity is in doubt as these proteins lack residues shown to be important for catalysis in PARP enzymes studied.

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