Poly acceptor proteins

NAD is used in post translational modification of a variety of proteins, nc>tab y some of the proteins of the chromosomes. The chromosomes are composed of DNA, histones, and nonhistone proteins. The histones, which are distinguished by their high content of basic amino acids, serve as a scaffold and maintain the coiled and folded structure of the DNA. The other proteins are used in regulating the expression of specific genes. Poly(ADP-ribose) polymerase catalyzes the attachment of ADP-rlbose to various chromostimal pniteins. This modification, shown in Figure 9.65A, is more dramatic than a simple methylalion or phosphorj lation. The enzyme uses NAD as a substrate. Here, NAD docs not serve its usual role as an Oxidant or reductant. The ADP-ribosyl moiety of NAD is donated to the acceptor protein. A molecule of nicotinamide is discharged with each event of... 

Figure 1. Structure of poly(ADP-ribose) showing a single branch link. Points to note are (1) the shaded areas show respective ADP-ribose moieties in the polymer, (2) the polymer is shown attached to the acceptor protein via an ester linkage, (3) in each ADP-ribose moiety, the carbon atoms on the ribose nearest the adenine residues are numbered 1 to 5, and those on the distal ribose from 1 "to 5". The two ribose residues are oriented with their respective 5 to 5" C atoms through the two phosphates, (4) polymer chains are joined by a-glycosidic links from the 1" position of a ribose furthest from the adenine to a 2 position of a ribose nearest to the adenine in the neighboring ADP-ribose moiety (i.e., the polymer has a ribose (1" 2 ) ribose-phosphate-phos-...

The chemical structure of poly(ADP-ribose) su ests not only that its modification of acceptor proteins should modify the struaure and function of the acceptor proteins, but also that the poly(ADP-ribose) molecule itself should possess an intrinsic structural information that can alter cellular fimction(s). 

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.

In work with the bovine thymus synthetase, Tanaka et al. 212) demonstrated that the enzyme was completely dependent on histone when Mg + was omitted from the assay histone HI was ADP-ribosylated under these reaction conditions. Maximum stimulation and ADP-ribosylation occurred when the ratio of DNA to histone HI was 1 to 10 on a weight basis stimulation was lost when the amount of DNA was increased to 50% of histone HI. All other histone fractions were efiective in stimulating the reaction but none was as active as histone HI. Kawaichi et al. 109) and Ueda and co-workers 222) also observed synthesis of poly(ADP-ribosyl) histone using an apparently homogeneous preparation of rat liver poly( ADP-ribose) synthetase. As opposed to the requirement for a large excess of histone over DNA used by Tanaka et al. 212) to demonstrate modification of Hi, a ratio of DNA to histone of 1 1 (on a weight basis) appeared to be optimal. The amount of ADP-ribose incorporated into histone Hi increased linearly as the DNA to histone Hi ratio was fixed at unity and their concentrations increased from 25 to 150 jug/ml. The ADP-ribose incorporated into histone HI represented, however, only about 50% of the total poly ADP-ribosylation the remainder was polymer associated with the synthetase itself. In these studies 212), Mg + was present at a concentration of 10 mAf. All histone subfractions were tested as acceptor proteins Hi was best, followed by H2B H2A, H3, and H4 were poor acceptors. This order of effectiveness is nearly identical to that found in experiments with intact nuclei (2,28,30, 64, 76,102,103,149,162,164,178-180, 200, 215, 229). 

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. 

The most interesting new finding to emerge is the arrest of cells in Gi and G2 by high concentrations of 3-acetamidobenzamide, but not 3-nitrobenzamide. This opens the possibility of synchronizing cells at a poly(ADP-ribose)-mediated restriction point. Release of such a population would allow one to define which events are dependent on release. It should also make isolating cell-cycle specific acceptor proteins from cells considerably easier. 

Our previous studies [1,2] revealed that the degradation of poly(ADP-ribosyl) proteins is carried out by consecutive actions of two enzymes, poly(ADP-ribose) giycohydrolase [3, 4] and ADP-ribosyl protein lyase (formerly termed ADP-ribosyl histone splitting enzyme) [5] (Fig. 1). The latter enzyme catalyzes removal of the last proximal ADP-ribosyl residue from acceptor protein. This report presents, after a brief review of ADP-ribosyl histones and the lyase, the identification of the enzymatic split product... 

Furthermore, the acceptor proteins were analyzed by acid urea polyacrylamide gel electrophoresis (Fig. 2). Histones H2B, HI and protein A24 were found to be poly-(ADP-ribosyl)ated in native chromatin while in histone Hl-depleted chromatin and core particles it was found that histone H2B and protein A24 were poly(ADP-ribosyl)-ated. The presence of the hyper(ADP-ribosyl)ated forms of histone H2B on each of these various levels of chromatin structure have been confirmed by western blot analysis and by two-dimensional polyacrylamide gel electrophoresis (data not shown). The poly(ADP-ribosyl)ation of protein A24 have also been demonstrated by this last technique. 

Figure 1 shows the proteins contained in various subnuclear fractions. Histones were not detectable in the isolated nuclear matrix by Coomassie blue staining. But these known acceptor proteins for ADP-ribose were found in the ammonium sulfate extract. Autoradiography revealed that great amounts of the self-modified poly(ADP-ribose) synthetase - in accordance with the results of Table 1 - were released by DNase, RNase digestion of the isolated nuclei (not shown). 

The second enzyme that could be identified among the endogenous acceptors for poly(ADPR) was topoisomerase I. Figure 4 shows the relaxation of supercoiled form I DNA (of phage phi x 174) catalyzed by a renatured sample of the isolated poly(ADPR) proteins. Although this assay was not performed with material extracted from the gel but with the whole conjugate fraction, it is very likely that the topoisomerase I is represented by the 100 kD band as this value agrees very well with the molecular size of eukaryotic type I topoisomerases. 

Since it is difficult to draw conclusions about the possible mechanism of poly(ADPR) function in association with DNA repair before having identified all acceptor proteins. 

Thus, any model designed to explain the biological role of poly(ADPR) has to consider that both structural constituents like histone H2B as well as DNA-binding enzymes like topoisomerase I and poly(ADPR) synthase are modified by poly(ADPR). Assuming that poly(ADPR) synthase remains attached to the chromatin when activated by a DNA break, I favor the idea that poly(ADP-ribosyl)ation of the isolated acceptors is primarily a function of their accessibility to the synthase. The acceptor proteins may merely function as matrix to permit the accumulation of relatively large amounts of poly(ADPR) at distinct sites of the chromatin adjacent to the stimulating event. Thus, poly(ADPR), probably in the form of a three-dimensional network, may represent a specific tool to introduce changes into the chromatin structure. 

Poly(AIH -Ribose) Modified Proteins. Poly(ADP-ribose) appears to be involved as a modifier of chromatin-associated proteins in several cellular processes (reviewed in [12]). To determine the role of poly(ADP-ribose) in myogenesis we attempted to identify the acceptor proteins which undergo this modification, and to de e the specificity of such modification. 

During the limited period in which prefusion myoblasts are sensitive to inhibitors of poly(ADP-ribose) synthetase, two significant changes involving modification of acceptor proteins were observed. A modified protein of 116 kD disappears and a slower migrating component becomes increasingly modified and abundant. That the disappearance of the poly(ADP-ribose)modified 116 kD protein accompanies differentiation is also indicated by our results with a nonfusing myoblast variant. 

Poly(ADP-ribosylation) of nuclear proteins is one of a number of post-translational events which has been demonstrated in eukaryotic cells. The reaction is catalysed by poly(ADP-ribose) synthetase which transfers ADP-ribose from NAD to a suitable acceptor protein. Histone HI and HMG proteins 1, 2, 14 and 17 have been reported as acceptors in a variety of tissues ranging from trout testis [1] to mammary carcinoma cells [2]. Poly(ADP-ribosylation) of HMG proteins is of particular interest because of the reported association of these proteins with actively transcribed genes [3]. Functionally, poly(ADP-ribosylation) has been implicated in a variety of regulatory events such as DNA synthesis [4], DNA excision repair [5, 1], gene expression [6] and cell differentiation [7]. 

From these results, we conclude that the ADP-ribosylation of Ca2+-dependent ATPase by endogenous ADP-ribosyltransferase occurs in rabbit skeletal muscle SR vesicles and that this reaction seems to be important for regulating Ca transport in skeletal muscle. The basic peptides poly L-omithine and poly L-lysine are proving to be useful tools for identifying acceptor proteins in the sarcoplasmic reticulum. 

To evaluate the effect of poly(ADP-ribose) glycohydrolase activity on the accumulation of poly(ADP-ribose) on the different acceptor proteins, we... 

Fig. 3. Inhibition of poly(ADP-ribose) glycohydrolase effect on poly ADP-ribosylation of proteins. Incubation and determination of the levels of poly(ADP-ribose) were done as described in Fig. 1 but with the presence of 100 pM Tilorone R10,556 Da to inhibit poly(ADP-ribose) glycohydrolase. Gel electrophoresis was carried out as described in the legend of Fig. 2. A Time course of poly ADP-ribosylation. ( ) poly(ADP-ribose) polymerase alone (2 units) (g) poly(ADP-ribose) polymerase and glycohydrolase (13) poly(ADP-ribose) polymerase and glycohydrolase in the presence of Tilorone. B Specific determination of poly(ADP-ribose) levels on each acceptor protein, (s) acceptor of MW >116 kDa (c) acceptor of 116 kDa (t) acceptor of 30-40 ld)a. Inset autoradiogram of the stained gel, 1 to 8 represents the different time points. Molecular weight standards in kDa are shown on the left part of the gel.

Previous experimental approaches have suggested that poly(ADP-ribose) synthesis begins with the ADP-ribosylation of an "acceptor" protein, and that the poly(ADP-ribose) chain is elongated by the repetitive "distal" addition of further ADP-ribose residues to the 2 OH end (1). However the first product of poly(ADP-ribose) syndiesis is an auto-modified polymerase (1-3), which is ficult to reconcile with distal addition. For fliis and other reasons we have considered the alternative possibility that a polymerase molecule would build a polymer of ADP-ribose upon itself (proximal addition). We describe here two tests of the direction of elongation of poly(ADP-ribose) both of which support proximal over distal addition. 

The cellular content of NAD the substrate for poly(ADP-ribose) polymerase is probably one of the important factors regulating the enzyme activity. Several studies relate the significance of NAD metabolism to the poly(ADP-ribosyl)ation status of the acceptor proteins under a variety of experimental conditions (13). We find here (Table 3) that there is an inverse relationship between the levels of poly(ADP-ribose) polymerase activity and NAD levels of Ewing s sarcoma and laryngeal squamous cell carcinoma and lung carcinoma as compared to normal human fibroblasts. 

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