ADP-ribosylation Reaction

Ame J-C, Jacobson EL, Jacobson MK (2000) ADP-ribose polymer metabolism. In de Murcia G, Shall S (eds) From DNA damage and stress signalhng to cell death poly ADP-ribosylation reactions. Oxford University Press, New York, ppl—34... 

The heat-labile E. coli enterotoxin, whose gene is carried on a plasmid, is a close relative of cholera toxin11 0 and also catalyzes ADP ribosylation of arginine 201 of the Gsa subunit.111 Bordetella pertussis, which causes whooping cough, forms a similar toxin that attacks the inhibitory regulatory protein G v as well as transducin and inactivates them by ADP ribosylation. Diphtheria toxin (Box 29-A), the exotoxin from Pseudomonas aeruginosa, and the toxin from Clostridium botulinum also catalyze ADP-ribosylation reactions.k/1 ... 

Why do our cells obligingly provide both initial receptors and means of uptake for these dangerous toxic proteins Some ADP-ribosylation reactions are a natural part of cell function and some hormones, for example thyrotropin, seem to stimulate their activity. It may be that the toxins use mechanisms designed to respond to normal hormonal stimulation. 

Archaea are systematically susceptible to ADP-ribosylation from NAD by the diphtheria toxin [118,166-168] although the rate of the ADP-ribosylation reaction is three orders of magnitude slower than that typically observed with eucaryal EF-2 [164,168]. Furthermore, in accordance with the sensitivity data, diphthamide occurs in archaea (//. halobium) but not in bacteria ( . coli) [164]. The archaeal EF-G-equivalent factor, therefore, resembles the eucaryal factor in having both the diphthamide that acts as the receptor for the ADP-ribose moiety of NAD, and the enzyme system which assists the post-translational conversion of histidine to diphthamide. 

FIGURE 9.65 A (facing page). InvolvcinctU of NAD for ADP-ribosylation reactions. Four molecules of NAD arc shown for the synthesis of a polymer consisting uf four residues of ADP-ribose. P (above). Cyclic ADP-ribose, a molecule thought to be used for cell-signaling, is made from NAD. 

Like other bacterial ADP-ribosylating toxins (e.g. diphtheria toxin. Pseudomonas aeruginosa exotoxin A, cholera toxin, pertussis toxin, and C. botulinum C2 toxin (Aktories and Just, 1993)), C3 is a mono-ADP-ribosyltransferase (Aktories et ai, 1988b). Treatment of ADP-ribosylated Rho with phosphodiesterase releases 5 -AMP and not phosphoribosyl-AIVtP, a cleavage product of poly(ADP-ribose) (Aktories et ai, 1988b Rubin ef a/., 1988). Accordingly, thymidine, an inhibitor of poly(ADP-ribose)polymerase, does not block C3-like ADP-ribosyltransferases, and can be included in C3 ADP-ribosylation assays to block poly-ADP-ribosylation reactions. 

ADP-ribosylation by C3 and its isoenzymes is a reversible reaction, i.e. in the presence of high concentrations of nicotinamide (lOm/Vl) and at low pH (pH <7) it can be reversed (Habermann et ai, 1991). The de-ADP-ribosylation reaction has been exploited to identify the acceptor amino acid of ADP-ribosylation by C3-like isoforms. Like other ADP-ribosyltransferases, C3-like exoenzymes exhibit NAD gly-cohydrolase activity (Aktories et ai, 1988b). However, this enzyme activity is at least 100-fold lower than the transferase activity and most likely has no physiological significance. 

Aktories K, RbsenerS, Blaschke U et al. (1988b) Botulinum ADP-ribosyltransferase C3. Purificafion of the enzyme and characterization of the ADP-ribosylation reaction in platelet membranes. In Eur. J. Biochem. 172 445-50... 

The ADP-ribosylation reaction is utilized to determine the amount of Rho in cells and tissues. When [ P]-NAD with a higher specific activity is used, the ADP-ribosylation reaction is an extremely sensitive method for detecting a small amount of Rho. 

The reaction mixture consists of 100 mM Tris HCI, pH 8.0, 10 mM nicotinamide, 10 mM thymidine, 10 mM dithiothreitol, 5mM MgCb, 10 nM pP]-NAD (900 cpm/pmol) (ADP-ribosylation buffer), purified C3 exoenzyme, and recombinant Rho or a crude homogenate in a total volume of 100 jil. The mixture is incubated at 30 C. To measure the amount of Rho, the ADP-ribosylation reaction should reach a plateau. When 50 ng of C3 exoenzyme... 

The ADP-ribosylation reaction is stopped by addition of Laemmli sample buffer, or the proteins are precipitated with 1 ml of trichloroacetic acid (20%, w/v). 

Upon entry into the eytoplasm, fragment A catalyzes the ADP ribosylation of the transfer faetor, EF-2, leading to its inaetivation and the interruption of protein synthesis. The ADP-ribose group is donated by NAD+. The ADP ribosylation reaction, catalyzed by the toxin, is specific for EF-2 of eukaryotic cells other proteins of eukaryotic and bacterial eells are not substrates. This speeificity is due to an unusual amino acid residue in EF-2, diphthamide, which is the acceptor of the ADP ribosyl group. Diphthamide derives from the posttranslational modification of histidine. The acute symptoms are treated with antitoxin. The bacteria, which are gram-positive, succumb to a variety of antibiotics, including penicillin. Diphtheria is effectively prevented by immunization with toxoid (inactivated toxin) preparations. 

Enzymology of ADP-ribosylation Reactions Synthesizing Enzymes ADP-ribose Attachment to Protein Nonenzymadc Attachment to Protein Degrading Enzymes... 

An ADP-ribosylation reaction is the cleavage of the oxidized form of nicotinamide adenine dinucleotide (NAD ), the co-enzyme of numerous redox reactions,... 

ADP-ribosylation reactions are quite ubiquitous and are found in animals, plants, microorganisms, and some viruses. In eukaryotic cells, monomeric ADP-ribosylation occurs essentially in the cytoplasm and the plasma membrane, whereas poly(ADP-rlbosyl)ation is found predominantly in the nucleus. Some terminally differentiated cells lack the polymerizing enzyme activity, e.g., mature granulocytes. The polymerase also seems to be absent in prokaryotic organisms. 

Figure 3. Proposed pleiotropic functions carried out by nuclear ADP-ribosylation reactions. Events such as cellular proliferation, differentiation, transformation, and DNA damage caused by external agents (e.g., ionizing radiation, drugs) involve changes in the integrity of DNA and/or chromatin architecture (a) which activate the poly(ADP-ribose) polymerase to catalyze the ADP-ribosylation of nuclear proteins predominantly at the expense of cytoplasmic NAD (b). The consequences of protein ADP-ribosylation are a decrease in cellular NAD content, alterations in chromatin structure, and possibly also the activity of various enzymes involved in chromatin function (c). This tripartite system operates, either wholly or partly, to ameliorate the activation of the polymerase by modulating the repair of DNA strand breaks, thereby affecting those processes which initially triggered the activation of the enzyme (d). Pr, protein NAm, nicotinamide (ADPR) , poly(ADP-ribose). (From Gaal and Pearson, 1986).

The use of bacterial toxins as molecular probes will continue to provide valuable information on the functions of their various substrates. In addition, studies on endogenous cellular mono(ADP-ribosyl) transferases look set to expand. New substrates will be identified and the biochemical consequences of the different modifications will reveal the roles played by mono(ADP-ribosylation) reactions in different cell compartments. For example, the case of cytoskeletal actin has been discussed (see Figure 8). Work in Mandel s laboratory (Mandel, 1992) has revealed that other cytoskeletal proteins are also substrates for endogenous ADP-ribosyl transferase, including components of the microfilaments (tubulin, intermediate filaments, and the neurofilament proteins L, M, and H). 

Mandel, P. (1992). Some aspects of nuclear and cytqilasmic ADP-ribosylation. Biological and pharmacological perspectives. In ADP-Ribosylation Reactions (Poirier, G. G., Moreau, P. eds.), pp. 163-172, Springer Verlag, Berlin. 

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