ADP-Ribosylation of Highly Purified Rat Brain Mitochondria

Highly purified synaptic and nonsynaptic mitochondria were prepared from rat brain, and their ADP-ribosyl transferase and NAD glycohydrolase activities were investigated. Data show that there is no significant difference in ADP-ribosyl transferase activity between these two types of subcellular preparations. However, NAD glycohydrolase activity appeared to be much higher in nonsynaptic mitochondria. The specific activity of both enzymes was investigated in the presence of the inhibitor nicotinamide or its analogue 3-aminobenzamide or other adenine nucleotides, such as ATP or ADP-ribose. The inhibitory effect of nicotinamide or 3-aminobenzamide on ADP-ribosyl transferase appears rather weak compared with their effect on NAD glycohydrolase activity. However, ADP-ribose and ATP appeared more effective in inhibiting ADP-ribosyl transferase. Our results provide evidence for the existence of ADP-ribosyl transferase activity in rat brain mitochondria. When NAD glycohydrolase was inhibited totally by nicotinamide, the transfer of ADP-ribose from NAD to mitochondrial proteins still occurred. The chain length determinations show that the linkage of ADP-ribose to mitochondrial proteins is oligomeric.     

Membrane-bound form of ADP-ribosyl cyclase in rat cortical astrocytes in culture

ADP-ribosyl cyclase activities in cultured rat astrocytes were examined by using TLC for separation of enzymatic products. A relatively high rate of [3H]cyclic ADP-ribose production converted from [3H]NAD+ by ADP-ribosyl cyclase (2.015+/-0.554 nmol/min/mg of protein) was detected in the crude membrane fraction of astrocytes, which contained approximately 50% of the total cyclase activity in astrocytes. The formation rate of [3H]ADP-ribose from cyclic ADP-ribose by cyclic ADP-ribose hydrolase and/or from NAD+ by NAD glycohydrolase was low and enriched in the cytosolic fraction. Although NAD+ in the extracellular medium was metabolized to cyclic ADP-ribose by incubating cultures of intact astrocytes, the presence of Triton X-100 in the medium for permeabilizing cells increased cyclic ADP-ribose production three times as much. Isoproterenol and GTP increased [3H]cyclic ADP-ribose formation in crude membrane-associated cyclase activity. This isoproterenol-induced stimulation of membrane-associated ADP-ribosyl cyclase activity was confirmed by cyclic GDP-ribose formation fluorometrically. This stimulatory action was blocked by prior treatment of cells with cholera toxin but not with pertussis toxin. These results suggest that ADP-ribosyl cyclase in astrocytes has both extracellular and intracellular actions and that signals of beta-adrenergic stimulation are transduced to membrane-bound ADP-ribosyl cyclase via G proteins within cell surface membranes of astrocytes.     

Phospho adenylylation and phospho ADP-ribosylation, types of covalent protein modification derived from NADP

The structure of NADP implies, in addition to the hydrogen transfer potential, two activated groups: 2'-phospho AMP and 2'-phospho ADP-ribose. Recent findings demonstrate that both can be used to modify covalently eukaryotic proteins. 2'-Phospho adenylylation appears to be an important route of post-translational modification involving various acceptor polypeptides in different subcellular compartments of rat liver. The true substrate of the transferases involved, however, is free 2'-phospho ADP-ribose derived from NADP by the action of NADP glycohydrolase, conferring a new function to the glycohydrolase beyond its purely catabolic action. The second type of modification, 2'-phospho ADP-ribosylation, was detected as an activity of the arginine specific ADP-ribosyl transferase from erythrocytes (Moss and Vaughan, 1978) which in the presence of H1 used NADP in preference to NAD. These findings show that both pyridine nucleotides represent versatile, multifunctional co-factors, serving as hydrogen-transferring as well as group-transferring co-enzymes.     

Identification of regulatory domains in ADP-ribosyltransferase-1 that determine transferase and NAD glycohydrolase activities

Mono-ADP-ribosyltransferases (ART1-7) transfer ADP-ribose from NAD+ to proteins (transferase activity) or water (NAD glycohydrolase activity). The mature proteins contain two domains, an alpha-helical amino terminus and a beta-sheet-rich carboxyl terminus. A basic region in the carboxyl termini is encoded in a separate exon in ART1 and ART5. Structural motifs are conserved among ART molecules. Successive amino- or carboxyl-terminal truncations of ART1, an arginine-specific transferase, identified regions that regulated transferase and NAD glycohydrolase activities. In mouse ART1, amino acids 24-38 (ART-specific extension) were needed to inhibit both activities; amino acids 39-45 (common ART coil) were required for both. Successive truncations of the alpha-helical region reduced transferase and NAD glycohydrolase activities; however, truncation to residue 106 enhanced both. Removal of the carboxyl-terminal basic domain decreased transferase, but enhanced NAD glycohydrolase, activity. Thus, amino- and carboxyl-terminal regions of ART1 are required for transferase activity. The enhanced glycohydrolase activity of the shorter mutants indicates that sequences, which are not part of the NAD binding, core catalytic site, exert structural constraints, modulating substrate specificity and catalytic activity. These functional domains, defined by discrete exons or structural motifs, are found in ART1 and other ARTs, consistent with conservation of structure and function across the ART family.     

Quantitative studies of inhibitors of ADP-ribosylation in vitro and in vivo

The ADP-ribosyl moiety of NAD+ is consumed in reactions catalyzed by three classes of enzymes: poly(ADP-ribose) polymerase, protein mono(ADP-ribosyl)transferases, and NAD+ glycohydrolases. In this study, we have evaluated the selectivity of compounds originally identified as inhibitors of poly(ADP-ribose) polymerase on members of the three classes of enzymes. The 50% inhibitory concentration (IC50) of more than 20 compounds was determined in vitro for both poly(ADP-ribose) polymerase and mono(ADP-ribosyl)transferase A in an assay containing 300 microM NAD+. Of the compounds tested, benzamide was the most potent inhibitor of poly(ADP-ribose) polymerase with an IC50 of 3.3 microM. The IC50 for benzamide for mono(ADP-ribosyl)transferase A was 4.1 mM, and similar values were observed for four additional cellular mono(ADP-ribosyl)transferases. The IC50 for NAD+ glycohydrolase for benzamide was approximately 40 mM. For seven of the best inhibitors, inhibition of poly(ADP-ribose) polymerase in intact C3H1OT1/2 cells was studied as a function of the inhibitor concentration of the culture medium, and the concentration for 50% inhibition (culture medium IC50) was determined. Culture medium IC50 values for benzamide and its derivatives were very similar to in vitro IC50 values. For other inhibitors, such as nicotinamide, 5-methyl-nicotinamide, and 5-bromodeoxyuridine, culture medium IC50 values were 3-5-fold higher than in vitro IC50 values. These results suggest that micromolar levels of the benzamides in the culture medium should allow selective inhibition of poly(ADP-ribose) metabolism in intact cells. Furthermore, comparative quantitative inhibition studies should prove useful for assigning the biological effects of these inhibitors as an effect on either poly(ADP-ribose) or mono(ADP-ribose) metabolism.     

Purification and characterization of an (ADP-ribose)n glycohydrolase from human erythrocytes

An (ADP-ribose)n glycohydrolase from human erythrocytes was purified approximately 13,000-fold and characterized. On sodium dodecyl sulfate/polyacrylamide gel the purified enzyme appeared homogeneous and had an estimated relative molecular mass (Mr) of 59,000. Amino acid analysis showed that the enzyme had a relatively high content of acidic amino acid residues and low content of basic amino acid residues. Isoelectrofocusing showed that the enzyme was an acidic protein with pI value of 5.9. The mode of hydrolysis of (ADP-ribose)n by this enzyme was exoglycosidic, yielding ADP-ribose as the final product. The Km value for (ADP-ribose)n (average chain length, n = 15) was 5.8 microM and the maximal velocity of its hydrolysis was 21 mumol.min-1.mg protein-1. The optimum pH for enzyme activity was 7.4 KCl was more inhibitory than NaCl. The enzyme activity was inhibited by ADP-ribose and cAMP but not the dibutyryl-derivative (Bt2-cAMP), cGMP or AMP. These physical and catalytic properties are similar to those of cytosolic (ADP-ribose)n glycohydrolase II, but not to those of nuclear (ADP-ribose)n glycohydrolase I purified from guinea pig liver [Tanuma, S., Kawashima, K. & Endo, H. (1986) J. Biol. Chem. 261, 965-969]. Thus, human erythrocytes contain (ADP-ribose)n glycohydrolase II. The kinetics of degradation of poly(ADP-ribose) bound to histone H1 by purified erythrocyte (ADP-ribose)n glycohydrolase was essentially the same as that of the corresponding free poly(ADP-ribose). In contrast, the glycohydrolase showed appreciable activity of free oligo(ADP-ribose), much less activity on the corresponding oligo(ADP-ribose) bound to histone H1. The enzyme had more activity on oligo(ADP-ribose) bound to mitochondrial and cytosolic free mRNA ribonucleoprotein particle (mRNP) proteins than on oligo(ADP-ribose) bound to histone H1. It did not degrade mono(ADP-ribosyl)-stimulatory guanine-nucleotide-binding protein (Gs) and -inhibitory guanine-nucleotide-binding protein (Gi) prepared with cholera and pertussis toxins, respectively. These results suggest that cytosolic (ADP-ribose)n glycohydrolase II may be involved in extranuclear de(ADP-ribosyl)n-ation, but not in membrane de-mono(ADP-ribosyl)ation.     

Cellular regulation of ADP-ribosylation of proteins. IV. Conversion of poly(ADP-ribose) polymerase activity to NAD-glycohydrolase during retinoic acid-induced differentiation of HL60 cells.

Two enzymatic activities of the nuclear enzyme poly(ADP-ribose) polymerase or transferase (ADPRT, EC 2.4.2.30), a DNA-associating abundant nuclear protein with multiple molecular activities, have been determined in HL60 cells prior to and after their exposure to 1 microM retinoic acid, which results in the induction of differentiation to mature granulocytes in 4-5 days. The cellular concentration of immunoreactive ADPRT protein molecules in differentiated granulocytes remained unchanged compared to that in HL60 cells prior to retinoic acid addition (3.17 +/- 1.05 ng/10(5) cells), as did the apparent activity of poly(ADP-ribose) glycohydrolase of nuclei. On the other hand, the poly(ADP-ribose) synthesizing capacity of permeabilized cells or isolated nuclei decreased precipitously upon retinoic acid-induced differentiation, whereas the NAD glycohydrolase activity of nuclei significantly increased. The nuclear NAD glycohydrolase activity was identified as an ADPRT-catalyzed enzymatic activity by its unreactivity toward ethenoadenine NAD as a substrate added to nuclei or to purified ADPRT. During the decrease in in vitro poly(ADP-ribose) polymerase activity of nuclei following retinoic acid treatment, the quantity of endogenously poly(ADP-ribosylated) ADPRT significantly increased, as determined by chromatographic isolation of this modified protein by the boronate affinity technique, followed by gel electrophoresis and immunotransblot. When homogenous isolated ADPRT was first ADP-ribosylated in vitro, it lost its capacity to catalyze further polymer synthesis, whereas the NAD glycohydrolase function of the automodified enzyme was greatly augmented. Since results of in vivo and in vitro experiments coincide, it appears that in retinoic acid-induced differentiated cells (granulocytes) the autopoly(ADP-ribosylated) ADPRT performs a predominantly, if not exclusively, NAD glycohydrolase function.     

NAD Glycohydrolase Activities and ADP-Ribose Uptake in Erythrocytes From Normal Subjects and Cancer Patients

Erythrocytes from cancer patients exhibited up to fivefold higher NAD glycohydrolase activities than control erythrocytes from normal subjects and also similarly increased [14C] ADP-ribose uptake values. When [adenosine-14C] NAD was used instead of free [14C] ADP-ribose, the uptake was dependent on ecto-NAD glycohydrolase activity. This was reflected in the inhibition of ADP-ribose uptake from [adenosine-14C] NAD by Cibacron Blue. ADP-ribose uptake in erythrocytes appeared to be complex: upon incubation with free [14C] ADP-ribose, the radiolabel associated with erythrocytes was located in nearly equal parts in cytoplasm and plasma membrane. Part of [14C] ADP-ribose binding to the membrane was covalent, as indicated by its resistance to trichloroacetic acid-treatment. A preincubation with unlabeled ADP-ribose depressed subsequent erythrocyte NAD glycohydrolase activity and binding of [14C] ADP-ribose to erythrocyte membrane; but it failed to inhibit the transfer of labeled ADP-ribose to erythrocyte cytoplasm. On the other hand, incubation with [adenosine-14C] NAD did not result in a similar covalent binding of radiolabel to erythrocyte membrane. In line with this finding, a preincubation with unlabeled NAD was not inhibitory on subsequent NAD glycohydrolase reaction and ADP-ribose binding. ADP-ribose binding and NAD glycohydrolase activities were found also in solubilized erythrocyte membrane proteins and, after size fractionation, mainly in a protein fraction of around 45kDa-molecular weight.     

Modification of the ADP-ribosyltransferase and NAD glycohydrolase activities of a mammalian transferase (ADP-ribosyltransferase 5) by auto-ADP-ribosylation.

Mono-ADP-ribosylation, a post-translational modification in which the ADP-ribose moiety of NAD is transferred to an acceptor protein, is catalyzed by a family of amino acid-specific ADP-ribosyltransferases. ADP-ribosyltransferase 5 (ART5), a murine transferase originally isolated from Yac-1 lymphoma cells, differed in properties from previously identified eukaryotic transferases in that it exhibited significant NAD glycohydrolase (NADase) activity. To investigate the mechanism of regulation of transferase and NADase activities, ART5 was synthesized as a FLAG fusion protein in Escherichia coli. Agmatine was used as the ADP-ribose acceptor to quantify transferase activity. ART5 was found to be primarily an NADase at 10 microM NAD, whereas at higher NAD concentrations (1 mM), after some delay, transferase activity increased, whereas NADase activity fell. This change in catalytic activity was correlated with auto-ADP-ribosylation and occurred in a time- and NAD concentration-dependent manner. Based on the change in mobility of auto-ADP-ribosylated ART5 by SDS-polyacrylamide gel electrophoresis, the modification appeared to be stoichiometric and resulted in the addition of at least two ADP-ribose moieties. Auto-ADP-ribosylated ART5 isolated after incubation with NAD was primarily a transferase. These findings suggest that auto-ADP-ribosylation of ART5 was stoichiometric, resulted in at least two modifications and converted ART5 from an NADase to a transferase, and could be one mechanism for regulating enzyme activity.     

Inhibition of DNA polymerase alpha, DNA polymerase beta, terminal deoxynucleotidyl transferase, and DNA ligase II by poly(ADP-ribosyl)ation reaction in vitro

Incubation of DNA polymerase alpha, DNA polymerase beta, terminal deoxynucleotidyl transferase, or DNA ligase II in a reconstituted poly(ADP-ribosyl)ating enzyme system markedly suppressed the activity of these enzymes. Components required for poly(ADP-ribose) synthesis including poly(ADP-ribose) polymerase, NAD+, DNA, and Mg2+ were all essential for the observed suppression. Purified poly(ADP-ribose) itself, however, was slightly inhibitory to all of these enzymes. Furthermore, the suppressed activities of DNA polymerase alpha, DNA polymerase beta, and terminal deoxynucleotidyl transferase were largely restored (3 to 4-fold stimulation was observed) by a mild alkaline treatment, a procedure known to hydrolyze alkaline-labile ester linkage between poly(ADP-ribose) and an acceptor protein. All of these results strongly suggest that the four nuclear enzymes were inhibited as a result of poly(ADP-ribosyl)ation of either the enzyme molecule itself or some regulatory proteins of these enzymes.     

Poly(ADP-ribose) accessibility to poly(ADP-ribose) glycohydrolase activity on poly(ADP-ribosyl)ated nucleosomal proteins

Hydrolysis of protein-bound 32P-labelled poly(ADP-ribose) by poly(ADP-ribose) glycohydrolase shows that there is differential accessibility of poly(ADP-ribosyl)ated proteins in chromatin to poly(ADP-ribose) glycohydrolase. The rapid hydrolysis of hyper(ADP-ribosyl)ated forms of histone H1 indicates the absence of an H1 dimer complex of histone molecules. When the pattern of hydrolysis of poly(ADP-ribosyl)ated histones was analyzed it was found that poly(ADP-ribose) attached to histone H2B is more resistant than the polymer attached to histone H1 or H2A or protein A24. Polymer hydrolysis of the acceptors, which had been labelled at high substrate concentrations (greater than or equal to 10 microM), indicate that the only high molecular weight acceptor protein is poly(ADP-ribose) polymerase and that little processing of the enzyme occurs. Finally, electron microscopic evidence shows that hyper(ADP-ribosyl)ated poly(ADP-ribose) polymerase, which is dissociated from its DNA-enzyme complex, binds again to DNA after poly(ADP-ribose) glycohydrolase action.     

ADP-ribosylation of proteins in nuclei and mitochondria from tissues rats of various age exposed gamma-radiation

Constitutive and gamma-induced ADP-ribosylation of nuclei and mitochondrial proteins in 2- and 29-month-old rats was studied. ADP-ribosylation was determined by binding of [3H]-adenin with the proteins after incubation of cellular organells in reaction mixture supplemented with [adenin-2,8-3H]-NAD. It was detected that the level of total protein ADP-ribosylation in the nuclei is 4.5-6.2 times higher than in the mitochondria. By inhibition of poly(ADP-ribose) polymerase (PARP) with 3-aminobenzamidine and treatment of ADP-ribosylated proteins with phosphodiesterase I, it was demonstrated that about 90% of [3H]-adenin bound by proteins in the nuclei and 70% in the mitochondria was the result of PARP activity. The level of total ADP-ribosylation of nuclear and mitochondrial proteins in the tissues of old rats was reliably lower than in young animals. This reduction of ADP-ribosylation in old animals is the result of the lower activity of PARP, not of mono(ADP-ribosyl) transferase (MART). The level of ADP-ribosylation of proteins in the nuclei of brain and spleen cells of 2-month-old rats irradiated with of 5 and 10 Gy was by 49-109% higher than in the control. At the same doses of radiation, the level of ADP-ribosylation of nuclear proteins in brain and spleen of old rats increased only by 29-65% compared to the control. Unlike cell nuclei, the radiation-induced activation of ADP-ribosylation in mitochondria was less expressed: the level of ADP-ribosylation increased by 34-37% in young rats and by 11-27% in old animals. This increased binding of ADP-ribose residues by the proteins of nuclei and mitochondria from tissues of gamma-irradiated rats is exceptionally conditioned by activation of poly(ADP-ribosyl)ation because the level of mono(ADP-ribosyl)ation remains constant. The results of this study enable the suggestion that poly(ADP-ribosyl)ation also occurs in the mitochondria of brain and spleen cells of the gamma-irradiated rats, though less pronounced than in cell the cell nuclei of these tissues. Thus, one of the probable causes of the less efficient repair of radiation-induced DNA damage in old organisms is a decline of both constitutive and induced poly(ADP-ribosyl)ation of proteins in cell nucleus and mitochondria.     

Functional localization of two poly(ADP-ribose)-degrading enzymes to the mitochondrial matrix

Recent discoveries of NAD-mediated regulatory processes in mitochondria have documented important roles of this compartmentalized nucleotide pool in addition to energy transduction. Moreover, mitochondria respond to excessive nuclear NAD consumption arising from DNA damage-induced poly-ADP-ribosylation because poly(ADP-ribose) (PAR) can trigger the release of apoptosis-inducing factor from the organelles. To functionally assess mitochondrial NAD metabolism, we overexpressed the catalytic domain of nuclear PAR polymerase 1 (PARP1) and targeted it to the matrix, which resulted in the constitutive presence of PAR within the organelles. As a result, stably transfected HEK293 cells exhibited a decrease in NAD content and typical features of respiratory deficiency. Remarkably, inhibiting PARP activity revealed PAR degradation within mitochondria. Two enzymes, PAR glycohydrolase (PARG) and ADP-ribosylhydrolase 3 (ARH3), are known to cleave PAR. Both full-length ARH3 and a PARG isoform, which arises from alternative splicing, localized to the mitochondrial matrix. This conclusion was based on the direct demonstration of their PAR-degrading activity within mitochondria of living cells. The visualization of catalytic activity establishes a new approach to identify submitochondrial localization of proteins involved in the metabolism of NAD derivatives. In addition, targeted PARP expression may serve as a compartment-specific “knock-down” of the NAD content which is readily detectable by PAR formation.     

Opening of the mitochondrial permeability transition pore causes depletion of mitochondrial and cytosolic NAD+ and is a causative event in the death of myocytes in postischemic reperfusion of the heart

The opening of the mitochondrial permeability transition pore (PTP) has been suggested to play a key role in various forms of cell death, but direct evidence in intact tissues is still lacking. We found that in the rat heart, 92% of NAD(+) glycohydrolase activity is associated with mitochondria. This activity was not modified by the addition of Triton X-100, although it was abolished by mild treatment with the protease Nagarse, a condition that did not affect the energy-linked properties of mitochondria. The addition of Ca(2+) to isolated rat heart mitochondria resulted in a profound decrease in their NAD(+) content, which followed mitochondrial swelling. Cyclosporin A(CsA), a PTP inhibitor, completely prevented NAD(+) depletion but had no effect on the glycohydrolase activity. Thus, in isolated mitochondria PTP opening makes NAD(+) available for its enzymatic hydrolysis. Perfused rat hearts subjected to global ischemia for 30 min displayed a 30% decrease in tissue NAD(+) content, which was not modified by extending the duration of ischemia. Reperfusion resulted in a more severe reduction of both total and mitochondrial contents of NAD(+), which could be measured in the coronary effluent together with lactate dehydrogenase. The addition of 0.2 microm CsA or of its analogue MeVal-4-Cs (which does not inhibit calcineurin) maintained higher NAD(+) contents, especially in mitochondria, and significantly protected the heart from reperfusion damage, as shown by the reduction in lactate dehydrogenase release. Thus, upon reperfusion after prolonged ischemia, PTP opening in the heart can be documented as a CsA-sensitive release of NAD(+), which is then partly degraded by glycohydrolase and partly released when sarcolemmal integrity is compromised. These results demonstrate that PTP opening is a causative event in reperfusion damage of the heart.     

Metabolism of cyclic ADP-ribose: Zinc is an endogenous modulator of the cyclase/NAD glycohydrolase ratio of a CD38-like enzyme from human seminal fluid

CD38, a bifunctional enzyme capable of both synthesis and hydrolysis of the second messenger cyclic ADP-ribose (cADPR). Using the natural substrate of the enzyme, NAD+, the ratio of ADP-ribosyl cyclase/NAD glycohydrolase of CD38 is about 1/100. Here we describe that human seminal fluid contain a soluble CD38 like enzyme with an apparent M.W. of 49 kDa. When purified this enzyme has a cyclase/NAD glycohydrolase ratio of about 1/120. However, the in situ cyclase/NAD glycohydrolase ratio measured in seminal plasma approaches 1/1. We also found that physiological concentrations of zinc present in the seminal fluid, in the range of 0.6 to 4 mM, are responsible for the modulation of the cyclase/NAD glycohydrolase ratio. This new information indicates that the cyclase/NAD glycohydrolase ratio can be modified in vivo.     

Functional interaction of poly(ADP-ribose) with the 20S proteasome in vitro.

The nuclear enzyme poly(ADP-ribosyl) transferase (pADPRT) catalyzes the formation of poly(ADP-ribose) from NAD+. Several nuclear proteins and pADPRT itself are targets for the modification by poly(ADP-ribosyl)ation. It is demonstrated here that poly(ADP-ribose) or pADPRT automodified with poly(ADP-ribose) interacts noncovalently with the 20S proteasome in vitro. The interaction of pADPRT with the 20S proteasome requires the long ADP-ribose polymers formed by automodification of the pADPRT with poly(ADP-ribose). As a result pADPRT automodified with short ADP-ribose oligomers is unable to associate with the 20S proteasome. The interaction with poly(ADP-ribose) causes a specific stimulation of the peptidase activity of the 20S proteasome. Modified pADPRT does not serve as a substrate for the degradation by the 20S proteasome. No covalent modification of the 20S proteasome by ADP-ribosylation was observed. The results may point to a functional relationship between pADPRT and the 20S proteasome in a pathway protecting the cell from oxidative damage.