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chemi.vrn, 14. .?Y I5

Poly ADP-ribose and ADP-ribosylation of proteins

Osamu Hayaishi

Covalent modification of proteins, by attachment of ADP-ribose, appears to regulate cell growvth, protein metabolism, DNA and RNA metabolism.

Several new examples of an unusual enzy- mic modification of proteins have recently been reported. In these reactions, the adenosine diphosphate ribose (ADP- ribose) moiety of NAD is transferred and covalently attached to an acceptor protein in either a polymeric or a monomeric form (Fig. 1). Nicotinamide and proton(s) are concomitantly released. Poly ADP-ribose has so far been found in nuclei of eucar- yotes, whereas mono ADP-ribosylation of proteins has been found in procaryotes as well. These protein modifications appear to participate in the regulation of various biological functions, including cell growth and syntheses of protein, DNA and RNA.

Poly ADP-ribosylation

Since the original discovery of NAD by von Euler in 1936, this coenzyme has been found to be widely distributed in nature and to function as an electron carrier in various dehydrogenase systems. Although the nicotinamide-riboside linkage of NAD is a so-called high energy bond with a free energy of hydrolysis of about -8.2 kcal/mol at pH 7 and 25 “C [1], the biological significance of this bond energy has not been fully appreciated until recently. About ten years ago, three groups of investigators concurrently and indepen- dently, reported that mammalian nuclei can catalyze the polymerization of the ADP-ribose moiety of NAD into a novel homopolymer composed of repeating ADP-ribose units linked by ribose to ribose (1’ --, 2’) bonds as shown in Fig. 2 [2-41. The average chain length of this polymer, which is referred to as poly ADP- ribose, ranges from several up to 50. Sub-

O.H. is Professor of Medical Chemistry at the Kyoto University Faculty qf Medicine. Sakyo-ku. Kyoto 606, Japan

matin of rat liver and calf thymus nuclei. It cleaves specifically the 1’,2’-glycosidic bond of poly ADP-ribose resulting in the formation of ADP-ribose as shown in Fig. 2. On the other hand, snake venom phos- phodiesterase and liver phosphodiesterase cleave the pyrophosphate linkage yielding 2’-(5”-phosphoribosyl)-5’-AMP and the terminal AMP.

Although the natural existence of this unique polymer in vivo has been described from several laboratories, its biological function has not yet been clearly under- stood. Recently there have been a number of reports indicating a close relationship between poly ADP-ribose synthesis and DNA metabolism. For example, Kidwell showed, using nuclei from synchronized mouse L-929 and human HeLa S3 cell lines, that [ ‘HJNAD incorporation into oligo ADP-ribose in vitro is the highest at the Gz phase [8], suggesting a crucial

ADPribose r-----l

i ($$ i wNH2

I I

1 1- _T7

I + Protein

YhlylADRribosyllprotein + f$‘oNH2

I I \ m CONH2 I AWriboryl protein + I I I --___--____J

NAD

Fig. I. Poly- and mono-ADP ribosylation of proteins.

sequently it was shown to be covalently attached to nuclear proteins, probably his- tones [5,6].

The poly ADP-ribose synthetase is pre- sent in a variety of vertebrate tissues, as well as in Tetrahymena and Physarum, a slime mold, but this activity has not yet been found either in higher plants or in procaryotic organisms. The enzyme acti- vity is exclusively localized in the nucleus and more than 90’4 of the total activity is associated with chromatin. It has been solubilized from rat liver chromatin and purified about 5000 fold [7]. As the extent of purification increased, shorter chains were synthesized suggesting the possibility that the initial attachment of poly ADP- ribose to the acceptor protein and the sub- sequent chain elongation might be cata- lyzed by two different enzymes. The highly purified enzyme exhibits an absolute requirement for DNA or poly dAdT for activity. Histone appears to stimulate chain elongation.

The degradation of this polymer in vivo is mainly catalyzed by poly ADP-ribose glycohydrolase, which has been solubi- lized and partially purified from chro-

role of this polymer in the regulation of growth cycle of cultured cells. Burzio and Koide [9] reported that treatment of rat liver nuclei with NAD markedly reduced their capacity to incorporate [3H]TTP into acid insoluble material. Subsequently Yos- hihara et al. reported the ADP-ribosyla- tion of a Ca2 +, Mg* + dependent endonuc- lease and the concomitant inhibition of the enzyme activity [lo]. The ADP-ribose bound to the endonuclease was in the form of monomers and oligomers and not long chain polymers. It was inferred that the ADP-ribosylation of the endonuclease led to inactivation of the enzyme and its ability to generate primer sites on DNA, thereby causing significant inhibition of the tem- plate capacity of the resultant DNA nuc- lear protein matrix of chromatin for DNA polymerase. In HeLa cells, however, Smul- son et al. demonstrated that ADP-ribosy- lation led to an enhancement rather than to an inhibition in the number of primer sites for DNA polymerase [ 1 I].

Although the poly ADP-ribosylation of proteins was originally discovered in eucaryotic cells and poly ADP-ribose syn- thesis seems to be restricted to the nuclei,

TIBS - Januan, I576 10

2’-(S’LPhorphoribosyI)-CS’-AMP

I I

ADP-ribose r------

dicrteke diestemse

Fig. 2. Structure qfpoly ADP-ribosylprorein.

more recently mono ADP-ribosylation of proteins has been observed in a number of other systems including procaryotes.

ADP-ribosylation of EF2 Collier and Pappenheimer [12] demon-

strated that NAD is required for the inhi- bition by diphtheria toxin of polypeptide synthesis in mammals. Subsequently, Col- lier [ 131 reported that elongation factor 2 (EF2), an enzyme involved in protein syn- thesis, was inactivated by the action of diphtheria toxin in the presence of NAD. However, the molecular basis of this inac- tivation was not understood until 1968 when Honjo et al. were able to demon- strate that diphtheria toxin catalyzes the transfer of the ADP-ribose moiety of NAD to EF2, resulting in the concomitant inactivation of this enzyme [14]. This reac- tion, unlike the poly ADP-ribosylation reaction mentioned above, is reversible since the free energy of hydrolysis of the ADP-ribose EF2 linkage is approximately - 4.0 kcal/mol at pH 7 and 25 “C. Despite extensive efforts in two laboratories [ 15,161, the exact site of attachment of the ADP-ribose moiety to EF2 has not been identified.

Recently, Pseudomonas aeruginosa exo- toxin (PA toxin) was reported to catalyze essentihlly the same reaction as diphtheria toxin [17]. Although PA and diphtheria toxins have different molecular and im- munological properties and produce dif- ferent clinical symptoms, the molecular basis of their mechanisms of action appears to be identical.

It is interesting to note that NAD is a necessary cofactor for the activation of adenylate cyclase by cholera toxin. Gill [18] suggested that NAD might transfer its ADP-ribosyl moiety to a target in a manner similar to that by which diphtheria toxin catalyzes transfer of ADP-ribose to EF2. Alternatively,NAD could be reduced to NADH while the target molecule is

dehydrogenated. The exact role of NAD in this process, however, remains to be elucidated.

ADP-riboeylation of E. coli RNA polymerase

The a-polypeptides of Escherichia coli RNA polymerase are known to be chemi- cally modified within four min after infec- tion by bacteriophage T4. it has been reported from two laboratories [ 19,201 that NAD is utilized in this process and that ADP-ribosylation involves a specific arginine in the a polypeptide at the sequence of Thr-Val-Arg. ADP-ribose appears to be linked through its terminal ribose to a guanido nitrogen of arginine. The RNA polymerase activity is not signi- ficantly affected by this modification. This reaction as well as the diphtheria toxin catalyzed ADP-ribosylation of EF2 are both related to phage-induced enzymes, because diphtheria toxin, excreted by Cor- ynebacterium diphtheriae strains lysogenic for phage /II, is known to be the product of a phage gene.

ADP-ribo~ylstioa in rat liver mitocbondria

Rat liver mitochondria contain a MgZ+- dependent enzyme system that transfers the ADP-ribose moiety of NAD to an acceptor protein [21]. The ADP-ribosy- lated protein has been isolated and was shown to have a molecular weight of 100,OOOconsisting of two subunits. A frac- tion of the protein-bound ADP-ribose was reported to be present as an oligomer, although the reaction was said to be revers- ible. Further investigation appears neces- sary for better understanding of this reac- tion and its biological implication.

In summary, enzymic poly- and mono- ADP ribosylation of proteins and enzymes have recently been reported in increasing numbers. Available evidence suggests that these covalent modification reactions

might play an important role in the regula- tion of protein and nucleic acid metabo- lism. Three symposia exclusively devoted to this topic were held in Hamburg (1972), Bethesda (1973) and Tomakomai (1974) and their proceedings were published [22- 241. The early phase of the research on poly ADP-ribose and ADP-ribosylation has been reviewed previously [25,26].

References 1

2

3

4

II

12

13 14

15

16

17

18

19 20

21

22

23

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