Mono ADP-ribosylated proteins

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. 

Hilz H, Bredehorst R, Adamietz P, Wielckens K (1982) Subfractions and subcellular distribution of mono(ADP-ribosyl) proteins in eukaryotic cells. In ADP-ribosylation reactions, chap 11. Academic Press, London New York... 

Fig. 1. Release of cysteine-linked ADP-ribose by mercuric ion. The acid insoluble fraction of rat liver (15) was dissolved in 98% ice cold formic acid and radiolabeled mono-ADP-ribosylated protein was added. The solution was diluted with five volumes of ice cold H2O and precipitated by addition of 100% (w/v) ice cold trichloroacetic acid to a final concentration of 20% (w/v). The sample was held on ice for 10 min and subjected to centrifugation. The precipitate was resuspended in ice cold, 98% formic acid and stored at -20° C for subsequent use. To release cysteine-linked ADP-ribose, the sample in ice cold 98% formic acid was diluted with an equal volume of ice cold H2O or a freshly prepared solution of 20 mM mercuric acetate and the resulting solution was incubat at 37°C 10 min. The samples were then placed on ice, 5 volumes of ice cold H2O were added followed by 100% (w/v) trichloroacetic acid to a final concentration of 20%. After 10 min on ice, the samples were collected by centrifugation and the supernatant was removed. A sample was taken to determine released radiolabeled mono-ADP-ribose. The pellet containing the remaining protein-bound mono-ADP-ribose was dissolved in 250 mM ammonium acetate, 10 EDTA and 6 M guanidine before sampling for radioactivity. ( ) presence, (O) absence of mercuric ion. Panel A shows a time course at 10 mM mercuric ion and Panel B shows a 10 min incubation at the indicated concentrations of mercuric ion.

Comparison of WEHI Mono-ADP-Ribosylated Proteins to ADP-Ribose Protein Conjugates of Known Linkage... 

In rat liver most of the cellular mono(ADP-ribosylated) proteins are associated with the mitochondrial fraction (1). Two mono(ADP-ribosyl)ating systems have been described in mitochondria, one in die soluble (matrix) fraction (2, 3), the other in submitochondrial particles (SMP, inverted inner membrane vesicles) (3, 4). The ADP-ribosylated matrix protein has a molecular mass of 100 kDa and appears to consist of two major subunits of equal mass. In SMP of both rat liver (4) and beef heart (3), there is one major acceptor protein for mono(ADP-ribose), which migrates with an apparent molecular mass of 30 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Mono(ADP-ribosylation) of the acceptor protein of beef heart SMP was suggested to occur non-enzymicaUy (3). In rat liver SMP, ADP-ribosylation of the 30 kDa protein most probably occurs at an arginine residue, and is readily reversible in the presence of ATP (4). The characteristics of this ADP-ribosylation reaction, i.e. protein specificity and sensitivity to ATP, together widi the observation that intramitochondrial hydrolysis of NAD(P)+ is accompanied by release of Ca + from mitochondria suggests a functional link between mitochondrial protein ADP-ribosylation and Ca2+ release (5,6). 

ADP-ribose-protein conjugate proved stable for at least 12 hr at pH 4.0 and 4°C, as well as in the presence of 1 M ammoniiun chloride at neutral pH and 37°C (the latter condition henceforth will be referred to as "in the absence of hydroxylamine"). The linkage is moderately labile to 1 M and 3 M neutral hydroxylamine at 37°C, with a half-life in 3 M hydroxylamine of 6.0 hr. In 4 M hydroxylamine the half-life of the linkage is 4.0 hr. At basic pH, radioactivity is released very fast from the protein, with a half-life at pH 9.3 of 50 min, and of 20 min in 1 M sodium hydroxide. This is in agreement with and extends our previous findings (4). The release of protein-boimd radioactivity follows single first-order kinetics under aU conditions. This indicates that only one class of mono(ADP-ribosylated) proteins has been formed by incubation of SMP with NAD+. 

Figure 2 The actin-ADP-ribosylating toxins, (a) Molecular mode of action. The actin-ADP-ribosylating toxins covalently transfer an ADP-ribose moiety from NAD+ onto Arg177 of G-actin in the cytosol of targeted cells. Mono-ADP-ribosylated G-actin acts as a capping protein and inhibits the assembly of nonmodified actin into filaments. Thus, actin polymerization is blocked at the fast-growing ends of actin filaments (plus or barbed ends) but not at the slow growing ends (minus or pointed ends). This effect ultimately increases the critical concentration necessary for actin polymerization and tends to depolymerize F-actin. Finally, all actin within an intoxicated cell becomes trapped as ADP-ribosylated G-actin.

NAD is the source of ADP-ribose for the modification of proteins by mono-ADP-ribosylation, catalyzed by ADP-ribosyltransferases (Section 8.4.2), and poly(ADP-ribosylation), catalyzed by poly(ADP-ribose) polymerase (Section 8.4.3). It is also the precursor of two second messengers that act to increase the intracellular concentration of calcium, cADP-ribose, and nicotinic acid adenine dinucleotide phosphate (Section 8.4.4). 

Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. In Nature (London) 340 680-685 Laugwitz KL, Spicher K, Schultz G et al. (1994) Identification of receptor-activated G proteins selective immunoprecipitation of photolabeled G-protein a subunits. In Methods Enzymol. 237 283-294 Meyer T, Hilz H (1986) Production of anti-(ADP-ribose) antibodies with the aid of a dinucleotide-pyrophosphatase-resistent hapten and their application for the detection of mono(ADP-ribosyl)ated polypeptides. In Ear. J. Biochem. 155 157-165... 

Simpson LL, Stiles BG, Zepeda H etal. (1989) Production by Clostridium spiroforme of an iotalike toxin that possesses mono(ADP-ribosyl)transferase activity Identification of a novel class of ADP- ribosyltransferases. In Infect. Immun. 57 255-61 Stiles BG, Wilkens TD (1986) Purification and characterization of Clostridium perfringens iota toxin dependence on two nonlinked proteins for biological activity. In Infect. Immun. 54 683-8... 

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). 

Richter, C., Frei, B., Schlegel, J. (1985). Calcium transport and mono(ADP-ribosylation) in mitochondria. In ADP-Ribosylation of Proteins (Althaus, F. R., Hilz, H., Shall, S., Eds.), pp. 530-535. Springer-Verlag, Berlin. 

The NAD -dependent modification of GAPDH originally was thought to be identical to mono-ADP-ribosylation. Although NO-induced GAPDH modification resembles some features of ADP-ribosylation reactions, conditions for optimal protein modification are different from those in the toxin-... 

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). 

The transfer of the ADP-ribose moiety from NAD onto a biological macromolecule (in almost all cases an amino acid side chain of a protein) is referred to as ADP-ribosylation. Subsequently, another ADP-ribose unit can be attached to the protein-bound ADP-ribose. Further elongation of the ADP-ribose chain will result in poly-ADP-ribosylation. This process will not be further addressed in this chapter. However, mono-ADP-ribosylation, i.e., attachment of a single ADP-ribose unit to a protein, has also been established as a specific protein modification with important r ulatory functions. 

Corda D, Di Girolamo M. Functional aspects of protein mono-ADP-ribosylation. EMBO J 2003 22 1953-1958. 

Tanigawa Y, Tsuchiya M, Imai Y, Shimoyama M (1984) Mono(ADP-ribosyl)ation of hen liver nuclear proteins suppresses phosphorylation. Biochem Biophys Res Commun 113 135-141... 

SDS-polyacrylamide gel electrophoresis. The substrate specificity was rather loose with the protein portion ADP-ribosyl histones HI and H2B, peptide fragments of H2B, and nonhistone proteins (a mixture) served as substrates. In contrast, the specificity was very tight with the mono(ADP-ribosyl) portion and the carboxyl ester bond poly- or oligo(ADP-ribosyl) histones were hardly split, and ADP-ribose histone adducts formed chemically through Schiff base reduction [3] or ADP-ribosyl arginine bond formed by avian erythrocyte ADP-ribosyltransferase [14] did not serve as substrate. 

Wielckens K, Schmidt A, George E, Bredehorst R, Hilz H (1982) DNA fragmentation and NAD depletion. Their relation to the turnover of endogenous mono(ADP-ribosyl) and poly(ADP-ribosyl) proteins. J Biol Chem 257 12872-12877... 

Isolation and Identification of Mono- and Poly(ADP-Ribosyl) Proteins Formed in Intact Cells in Association with DNA Repair... 

Effect of DMS on Endogenous Mono(ADP-Ribosyl)ation of Proteins... 

In alkylated cells considerable amounts of free ADPR must be formed in consequence of the highly stimulated turnover of (ADPR)j, proteins [3,4]. Since free ADPR can form acid-insoluble protein conjugates [7, 8], mono(ADP-ribosyl) histone HI could have been arisen by such a reaction. We, therefore, analyzed endogenous conjugates with respect to chemical stability and compared it with other conjugates of known structure as well as with nonenzymic ADPR-Hl adducts. In addition, location of the ADPR groups on the histone HI molecule was investigated. 

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