ADP-ribosylated proteins

Scheme 16 Catalytic mechanism of ADP-ribosylation. The nucleophilic side chain of the amino acid attacks the anomeric carbon of ribose carrying nicotinamide to create ADP-ribosylated proteins.

The enzyme catalyzing the addition of ADP-ribose units onto the histones and itself is poly(ADP-ribose) polymerase or synthetase. Poly(ADP-ribose) polymerase is a nuclear, DNA-dependent enzyme that is stimulated by DNA breaks [302]. This property of the enzyme would target its action to sites that have DNA strand breaks (regions of the genome involved in replication, repair, recombination). The enzyme is associated with chromatin areas and perichromatin regions in interphase Chinese hamster ovary cells [312]. Degradation of the ADP-ribose polymer is catalyzed by the nuclear enzyme poly(ADP-ribose) glycohydrolase and ADP-ribosyl protein lyase. 

Exotoxin A is an NAD -dependent ADP-ribosylating protein from Pseudomonas aeruginosa. 

Meyer T, Koch R, Fanick W et al. (1988) ADP-ribosyl proteins formed by pertussis toxin are specifically cleaved by mercury ions. In Biol. Chem. Hoppe Seyler 369 579-583... 

Cellular actin is also modified by non-enzymatic ADP-ribosylation. This modification occurs at cysteine residues and depends on the presence of free ADP-ribose. Thus, cleavage of NAD by endogenous NAD glycohydrolases or release of ADP-ribose from poly-ADP-ribosylated proteins can increase the amount of ADP-ribose and may induce non-enzymatic modification of actin. Non-enzymatic labeling of actin in the presence of [ " C]/[ P]NAD can be identified because this reaction is quenched by unlabelled ADP-ribose at rather high concentrations (1 mM), whereas enzymatically-catalyzed [ C]/pP]-ADP-ribosylation is not influenced by ADP-ribose. 

PARP contains two zinc fingers that bind the two broken stands of DNA and stabilize the structure. PARP uses NAD as a substrate to poly(ADP)-ribosylate proteins, including itself. Early on, there is a small amount of self-poly(ADP)-ribosylation that activates PARP by causing it to homodimerize. This dimerization allows the activated PARP to bind to four strands of fragmented DNA, stabilize the structure, and facilitate repair. PARP also poly(ADP-ribosylates) endonuclease, which deactivates endonuclease and protects DNA from cleavage. 

ADP-protein lyase. The single ADP-ribose residue still attached to protein after glycohydrolase action on the polymer can be cleaved by ADP-ribosyl protein lyase. The enzyme is specific for ester linkages to the protein. [Clinical note Williams and collaborators at the University of Texas Health Center have described the case... 

Figure 2. Enzymatic degradation of poly(ADP-ribose).The arrows indicate the covalent bonds that are cleaved by poly(ADP-ribose) glycohydrolase ( ), ADP-ribosyl protein lyase (-- ), and phosphodiesterase (Jl). The reader can work out what the respective reaction products would be in each case (refer also to Figure 1).

Mitochondrial ADP-ribosylation. Other protein substrates for mono(ADP-ribosyl) transferases continue to be reported, but the best characterized reaction is that of mammalian cell mitochondria. Most mono-ADP-ribosyl-protein conjugates in eukaryotic cells are associated with mitochondria. A specific function, namely, stimulation of calcium release from mitochondria, has been ascribed to ADP-ribosylation activity in this organelle. This could, therefore, be an important cell regulatory mechanism, since numerous calcium-dependent enzymes play an important role in cell functioning. 

P]NAD is employed as substrate in this reaction, the ADP-ribosylated protein may be identified by the method of polyacrylamide gel electrophoresis followed by autoradiography of the gel. This method was employed in an examination of the membrane proteins of control and desensitized platelets. Coomassie-blue staining of the gels revealed no differences following desensitization, but there was a > 80% loss of the 45 000 Da protein as revealed by [ P]ADP-ribosylation. The autoradiograph and scans are shown in Figure 8.26. 

A third enzyme, ADP-ribosyl protein lyase, catalyzes the nonhydrolytic cleavage of protein proximal ADPR residues yielding the unique nucleotide ADP-3"-deoxy-pentos-2"-ulose (ADP-DP). In addition to the basic enzymology shown in Figure 1, related enzyme activities... 

Oka J, Ueda K, Hayaishi O et al. ADP-ribosyl protein lyase. Purification, properties and identification of the product. J Biol Chem 1984 259(2) 986-95. 

PolyfADP-ribose) is synthesized by PARP-1 and hydrolyzed by enzymes known as polyfADP-ribose) glycohydrolase (PARC), phosphodiesterases (PDases) and ADP-ribosyl protein lyase. Among these, PARC serves as an enzyme that hydrolyzes poly(ADP-ribose) chains quite efficiendy, including the branched pordon, and finally leaves the protein-proximal mono-ADP-ribose molecule, which might be removed by ADP-ribosyl protein lyase or released spontaneously at neutral pH. Such de-modificadon would enable PARP-1 to use the same acceptor protein in a new cycle of poly(ADP-ribos )adon (F 1). 

Figure 1. Metabolism of poly(ADP-ribose). Poly(ADP-ribosyl)ation is a posttranslational modification of acceptor proteins, and poly(ADP-ribose) itself serves as a component of cell struaure. Poly(ADP-ribose) glycohydrolase (PARC) is a main enzyme to degrade poly(ADP-ribose) and forms ADP-ribose as product. Other degrading enzymes ate phosphodiesterase (or pyrophosphatase) and ADP-ribosyl protein lyase.

Several procedures have been developed for the isolation of ADP-rihosylated nuclear protein. In one described by Hayaishi and coworkers (162) ADP-ribosylated nuclear proteins were separated by affinity chromatography in 6 M guanidine- HCl on a dihydroxyhoryl polyacrylamide column (162). A very specific interaction of the borate residue with the cis diol portion of ribose rings of the ADP-ribose permits the selective isolation of ADP-ribosylated proteins. Applying this technique to rat liver nuclear proteins, they observed a distribution of ADP-ribosylation between histone and nonhistone proteins, with histone H2B (67%) and Hi (33%) being preferentially modified. 

Hayaishi and co-workers (149) were the first to observe that when a nuclear preparation from rat liver was incubated with radiolabeled NAD, the resulting newly synthesized poly(ADP-ribose) was associated with histones Hi, H2A, H2B, and H3 (149). As summarized in the previous section, other groups have corroborated this observation and extended it to include nonhistone nucleosomal proteins (2, 64, 84, 90, 164,178,179, 200, 215, 229). Differences in techniques for isolation of poly ADP-ribosylated proteins and the presence of poly(ADP-ribose) degradative enzymes [poly(ADP-ribose) glycohydrolase and various phosphodiesterases] may explain some of the conflicting reports as to primary acceptor protein, extent of modification, and length of polymer. 

In Section IV,F it was shown that the purified poly(ADP-ribose) synthetase was a major acceptor of poly(ADP-ribose). Recently obtained evidence is consistent with the proposal that the synthetase may also be the major acceptor in nuclei. Jump etal. (103), while investigating the msqor ADP-ribosylated proteins in mid-S phase HeLa cell nu-... 

It was shown that the polyribosomal form of mRNP complexes is actively translated, whereas the free form is not. One mi t expect that a covalent chemical modification of some of the mRNA proteins, such as ADP-ribosylation, will render the mRNA available for translation. We characterized the mRNA-associated ADP-iibosyl transferase in plasmac) oma, in Krebs II, ascite tumor cells, and in liver. Several auto(ADP-ribosylated) proteins could be obtained when mRNP particles were incubated with NAD. It is unlikely that we are dealing with a contamination of chromatin since in plasmocytoma the enzymatic activity in mRNP represent 34% of the total cellular activity, while the maximum DNA contamination is only 4%. Moreover, after DNAse hydrolysis the enzymatic activity remains unchanged and addition of DNA is without effect [31]. More information on these mRNP particles will be given by Thomassin et al. (this volume). 

We have used permeabilized cells supplied with [ P]NAD to label ADP-ribosylated proteins followed by gel electrophoresis and autoradiography to identify [ P]ADP-ribosylated bands [9,10]. Using this approach, we have shown that a number of proteins are labeled in resting human lymphocytes, including bands of mol. wt. 116,000,... 

Since the conditions described above, i.e., intermitotic arrest, proliferation, and DNA damage are associated with dramatic changes in nucleotide pools, we conducted a study to determine the effect of nucleotides and their components on protein acceptors for poly(ADP-ribosyl)ation [11, 12]. Figure 1 shows the effect of increasing concentrations of Ap4A on poly(ADP-ribosyl)ated proteins in human lymphocytes. In the absence of Ap4A, the autoradiograph shows that the most prominent ADP-ribosylated proteins are poly(ADP-ribose) polymerase at mol. wt. 116,000, histone H-1 at 32,000,... 

Native protein ADP-ribosylated protein T 1/2 of proteolysis Activators... 

Williams JC, Chambers JP, Liehr JG (1984) Glutamyl ribose 5-phosphate storage disease. A hereditary defect in the degradation of poly(ADP-ribosylated) proteins. J Biol Chem 259 1037-1042... 

Incubation of mRNP particles with NAD, electrophoresis on lithium dodecyl sulfate polyacrylamide gels, and autoradiography reveal several ADP-ribosylated proteins with a predominant acceptor of mol. wt. 115,000 [4]. Using the protein blotting technique, we have observed that the antiserum directed against the nuclear calf thymus poly(ADPR) polymerase reacts with the 115,000 mol. wt. protein, associated with free mRNP particles. 

ADP-3"-Deoxypentos-2"-Ulose. A Novel Product of ADP-Ribosyl Protein Lyase... 

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