Effects of nucleotides on activity of a purified ADP-ribosyltransferase from turkey erythrocytes

A transferase purified from turkey erythrocytes catalyzed the NAD-dependent ADP-ribosylation of proteins in the supernatant, particulate, and detergent-solubilized fractions of bovine thymus as well as several purified proteins. Nucleoside triphosphates increased the rate of ADP-ribosylation of multiple soluble proteins from thymus and several purified proteins by about twofold. With lysozyme as substrate and 10 mm nucleotide, the order of effectiveness was ATP > ITP = GTP > CTP = UTP. Half-maximal stimulation of ADP-ribose incorporation into lysozyme was observed with 2.5 mm ATP. App(NH)p and inorganic tri- and tetrapolyphosphate were less effective than ATP; ADP, AMP, cAMP, and inorganic pyrophosphate were ineffective. Enhancement of transferase-catalyzed ADP-ribosylation by ATP was observed only at low (20–200 μm) NAD concentrations; with lysozyme as substrate, however, the effect of ATP was not due to prevention of NAD hydrolysis during the assay, nor was it due to an effect on ionic strength. The transferase catalyzed the ADP-ribosylation of several purified proteins and, depending on the protein substrate, ATP either increased, decreased, or did not alter the rate of ADP-ribosylation. It appears that ADP-ribosylation of cellular proteins by endogenous ADP-ribosyltransferases may be subject to regulation by nucleoside triphosphates.     

Lithium activates the c-Jun NH2-terminal kinases in vitro and in the CNS in vivo

The therapeutic efficacy of lithium in the treatment of mood disorders is delayed and only observed after chronic administration, a temporal profile that suggests alterations at the genomic level. Lithium has been demonstrated to modulate AP-1 DNA binding activity as well as the expression of genes regulated by AP-1, but the mechanisms underlying these effects have not been fully elucidated. In the present study, we found that the lithium-induced increases in AP-1 DNA binding activity were accompanied by increases in p-cJun and cJun levels in SH-SY5Y cells. Lithium also increased cJun-mediated reporter gene expression in a dose-dependent manner, with significant effects observed at therapeutically relevant concentrations. Lithium's effects on cJun-mediated reporter gene expression in SH-SY5Y cells were more pronounced in the absence of myo-inositol and were blocked by protein kinase C (PKC) inhibitors and by cotransfection with a PKCalpha dominant-negative mutant. Chronic in vivo lithium administration increased AP-1 DNA binding activity in frontal cortex and hippocampus and also increased the levels of the phosphorylated, active forms of c-Jun NH2-terminal kinases (JNKs) in both brain regions. These results demonstrate that lithium activates the JNK signaling pathway in rat brain during chronic in vivo administration and in human cells of neuronal origin in vitro; in view of the role of JNKs in regulating various aspects of neuronal function and their well-documented role in regulating gene expression, these effects may play a major role in lithium's long-term therapeutic effects.     

Identification of a botulinum C3-like enzyme in bovine brain that catalyzes ADP-ribosylation of GTP-binding proteins.

A novel enzyme activity was found in bovine brain cytosol that transfers the ADP-ribosyl moiety of NAD to proteins with Mr values of 22,000 and 25,000. The substrates were the same GTP-binding proteins serving as the substrate of an ADP-ribosyltransferase C3 which was produced by a type C strain of Clostridium botulinum. The brain enzyme was partially purified from the cytosol and had a molecular mass of approximately 20,000 on a gel filtration column. The brain endogenous enzyme displayed unique properties similar to those observed with botulinum C3 enzyme. The enzyme activity was markedly stimulated by a protein factor that had been initially found in the cytosol as an activator for botulinum C3-catalyzed ADP-ribosylation (Ohtsuka, T., Nagata, K., Iiri, T., Nozawa, Y., Ueno, K., Ui, M., and Katada, T. (1989) J. Biol. Chem. 264, 15000-15005). The activity of the brain enzyme was also affected by certain types of detergents or phospholipids. The substrate of the brain enzyme was specific for GTP-binding proteins serving as the substrate of botulinum C3 enzyme; the alpha-subunits of trimeric GTP-binding proteins which served as the substrate of cholera or pertussis toxin were not ADP-ribosylated by the endogenous enzyme. Thus, this is the first report showing an endogenous enzyme in mammalian cells that catalyzes ADP-ribosylation of small molecular weight GTP-binding proteins.     

Endogenous ADP-Ribosylation in Brain: Initial Characterization of Substrate Proteins

Cholera and pertussis toxin-mediated ADP-ribosylation has been used extensively to study regulation of guanine nucleotide binding proteins (G proteins) in the nervous system, but much less is known about possible endogenous ADP-ribosylation of G proteins in brain. The present study demonstrates endogenous ADP-ribosylation, in the absence of cholera and pertussis toxins, of four predominate proteins in homogenates of rat cerebral cortex. These proteins showed apparent molecular masses of 20, 42, 45, and 50 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The 42- and 45-kDa proteins comigrated precisely with the major cholera toxin-labeled bands. Furthermore, the endogenous ADP-ribosylated and cholera toxin-ADP-ribosylated bands yielded identical 32P-labeled peptide fragments by one-dimensional peptide mapping, indicating that they are probably the same proteins, presumably the alpha-subunits of Gs. In contrast, peptide maps of the 50-kDa protein, which migrated close to a 48-kDa cholera toxin-labeled band, demonstrated that this protein is distinct from the toxin-labeled band and from Gs alpha. Levels of endogenous ADP-ribosylation activity showed regional heterogeneity in brain, with a nearly threefold variation observed among the brain regions examined. Chronic administration (7 days) of corticosterone significantly increased overall levels of endogenous ADP-ribosylation, indicating that components of this system may be under hormonal control in vivo. Attempts to identify neurotransmitters or second messenger systems that regulate endogenous ADP-ribosylation activity in brain have so far been unsuccessful with one exception.(ABSTRACT TRUNCATED AT 250 WORDS)     

Exchange of glutamine-217 to glutamate of Clostridium limosum exoenzyme C3 turns the asparagine-specific ADP-ribosyltransferase into an arginine-modifying enzyme

C3-like ADP-ribosyltransferaseses are produced by Clostridium species, Bacillus cereus, and various Staphylococcus aureus strains. The exoenzymes modify the low-molecular-mass GTPases RhoA, B, and C. In structural studies of C3-like exoenzymes, an ARTT-motif (ADP-ribosylating turn-turn motif) was identified that appears to be involved in substrate specificity and recognition (Han, S., Arvai, A. S., Clancy, S. B., Tainer, J. A. (2001) J. Mol. Biol. 305, 95-107). Exchange of Gln217, which is a key residue of the ARTT-motif, to Glu in C3 from Clostridium limosum results in inhibition of ADP-ribosyltransferase activity toward RhoA. The mutant protein is still capable of NAD-binding and possesses NAD+ glycohydrolase activity. Whereas recombinant wild-type C3 modifies Rho proteins specifically at an asparagine residue (Asn41), Gln217Glu-C3 is capable of ADP-ribosylation of poly-arginine but not poly-asparagine. Soybean trypsin inhibitor, a model substrate for many arginine-specific ADP-ribosyltransferases, is modified by the Gln217Glu-C3 transferase. Also in C3 ADP-ribosyltransferases from Clostridium botulinum and B. cereus, the exchange of the equivalent Gln residue to Glu blocked asparagine modification of RhoA but elicited arginine-specific ADP-ribosylation. Moreover, the Gln217Glu-C3lim transferase was able to ADP-ribosylate recombinant wild-type C3lim at Arg86, resulting in decrease in ADP-ribosyltransferase activity of the wild-type enzyme. The data indicate that the exchange of one amino acid residue in the ARTT-motif turns the asparagine-modifying ADP-ribosyltransferases of the C3 family into arginine-ADP-ribosylating transferases.     

CTP-dependent endogenous ADP-ribosylation of a 38 kDa protein in HL-60 cell membranes

Incubation of membranes of human promyelocytic leukemia HL-60 cells with [32P]NAD led to ADP-ribosylation of several proteins including a 38 kDa protein by endogenous ADP-ribosyltransferases. The ADP-ribosylation of the 38 kDa protein was distinctly different from others on the basis of pH dependency and heat stability at 50 degrees C, suggesting that there are at least two endogenous ADP-ribosyltransferases. It was enhanced by CTP, but not affected by ATP, GTP and UTP, whereas it was inhibited by GTP gamma S. [alpha-32P]CTP bound to the 38 kDa protein immobilized on a nitrocellulose sheet, indicating that the 38 kDa protein which bound CTP is strongly ADP-ribosylated by an endogenous ADP-ribosyltransferase.     

Chronic Administration of Lithium or Other Antidepressants Increases Levels of DARPP-32 in Rat Frontal Cortex

Abstract: We studied the chronic actions of lithium on rat brain by investigating its effects on cyclic AMP-dependent protein phos-phorylation by use of a back-phosphorylation procedure. We identified one heavily regulated phosphoprotein in frontal cortex as the 32-kDa dopamine- and cyclic AMP-regulated phosphoprotein (DARPP-32). Immunoblot experiments demonstrated that chronic lithium regulation of DARPP-32 back-phosphorylation is associated with equivalent increases in levels of DARPP-32 immunoreactivity. Lithium regulation of DARPP-32 immunoreactivity required chronic drug administration and was not observed in several other brain regions examined. Moreover, chronic administration of the antidepressant imipramine or tranylcypromine produced a similar increase in levels of DARPP-32 in frontal cortex, whereas other types of psychotropic drugs, including haloperidol. morphine, and cocaine, did not influence DARPP-32 levels. Increased levels of DARPP-32 could reflect a common functional effect on frontal cortex of long-term exposure to lithium and some other antidepressant medications, an effect possibly related to the clinical actions of these drugs.     

Mono-ADP-Ribosylation in Brain: Purification and Characterization of ADP-Ribosyltransferases Affecting Actin from Rat Brain

Four ADP-ribosyltransferases that acted on non-muscle actin were purified more than 3,000-fold from rat brain extract. The molecular weights of these brain ADP-ribosyltransferases were 66,000 as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration on TSK gel G3000SW. The Km values for NAD were approximately 20 microM. These enzymes were not inhibited by thymidine or nicotinamide, but were inhibited by ADP and ADP-ribose. Two soluble ADP-ribosylation factors purified from rat brain had different effects on the four ADP-ribosyltransferases during the ADP-ribosylation of non-muscle actin. These ADP-ribosyltransferases modified not only actin but also the stimulatory guanine nucleotide-binding protein of adenylate cyclase, Gs, and another guanine nucleotide-binding protein in brain, Go. These findings suggest that the four brain ADP-ribosyltransferases are concerned with nerve functions in the central nervous system.     

Cholinotoxic Effects of Aluminum in Rat Brain

The in vivo and in vitro effects of Al on the cholinergic system of rat brain were studied. The amount of Al accumulated after the chronic, intraperitoneal administration of aluminium gluconate (Al-G) or AlCl3, both at a dose of 1 mg/ml/100 g of body weight, increased in the frontal and parietal cortices, the hippocampus, and the striatum. Significantly decreased choline acetyltransferase activities after chronic Al treatment were measured in the parietal cortex, the hippocampus, and the striatum, but not in the frontal cortex. The acetylcholinesterase activity was not changed significantly in any brain area investigated. Both Al-G and AlCl3 administrations resulted in a general decrease (to 40-70% of the control values) in the specific l-[3H]nicotine binding, involving all brain areas studied. The specific (-)-[3H]quinuclidinyl benzilate binding was reduced (to 40-60% of the control values) only after 25 days of Al treatment. Al-G and AlCl3 were equivalent in eliciting these reductions in vitro studies revealed different alterations of the cholinergic system in response to Al treatment. No changes were observed either in choline acetyltransferase activity or in cholinergic receptor bindings. Both Al-G and Al2(SO4)3 treatments, however, exhibited a biphasic effect on the acetylcholinesterase activity. At low Al concentrations (10(-8)-10(-6) M), the activity was slightly increased, whereas at higher concentrations (10(-6)-10(-4) M), it was inhibited by a maximum of 25% as compared to the controls. Thus, these cholinotoxic effects are probably due not to a direct interaction between the metal and the cholinergic marker proteins, but rather to a manifestation and consequence of its neurodegenerative effects.     

Target protein for eucaryotic arginine-specific ADP-ribosyltransferase

Among ADP-ribosyltransferases reported in eucaryotes, arginine-specific transferases from turkey erythrocytes, chicken heterophils and rabbit skeletal muscle have been purified and extensively studied. They were reported to modify a number of proteinsin vitro. ADP-ribosylation of Ha-ras-p21 and transducin by the turkey erythrocyte transferase inhibits their GTPase and GTP-binding activities. Chicken heterophil enzyme modifies several substrate proteins for protein kinases and decreases the phosphate-acceptor activity. Rabbit skeletal muscle Ca²⁺-ATPase is inhibited by ADP-ribosylation catalyzed by the muscle transferase. Three transferases all ADP-ribosylate small molecular weight guanidino compounds such as arginine, arginine methylester and agmatine and poly-L-arginine and nuclear histones. However, the observation that muscle transferase did not ADP-ribosylate casein or actin, both of which can be modified by the heterophil transferase under the same conditions indicates that substrate specificity of these two enzymes are different. Substrate-dependent effects were observed with polyions of nucleotides such that polyanions stimulate the ADP-ribosylation of possible target protein, p33 by chicken heterophil transferase but has no effect on the modification of casein by the same enzyme.     

Activation of Melatonin Receptor Sites Retarded the Depletion of Norepinephrine Following Inhibition of Synthesis in the C3H/HeN Mouse Hypothalamus

The aim of the present study was to determine the effect of activation of melatonin receptor sites on the activity of noradrenergic neurons in the C3H/HeN mouse brain. Changes in noradrenergic activity were assessed by measuring norepinephrine (NE) levels in the hypothalamus, frontal cortex, and hippocampus following inhibition of NE synthesis with alpha-methyl-p-tyrosine (alpha-MpT) (300 mg/kg, i.p., 2 h). 6-Chloromelatonin (1-30 mg/kg, i.p.) significantly retarded the alpha-MpT-induced decrease in NE levels in the hypothalamus, but not in hippocampus and frontal cortex. This effect was observed at 30 min and 60 min after 6-chloromelatonin administration and was dose dependent. At noon, when the levels of endogenous melatonin are low, the melatonin receptor antagonist luzindole (30 mg/kg, i.p., 30 min) did not affect the depletion of NE by alpha-MpT; however, it (1-30 mg/kg) completely antagonized the 6-chloromelatonin-induced reduction of NE depletion elicited by alpha-MpT in hypothalamus. These results suggest that activation of melatonin receptor sites in brain of C3H/HeN mouse retarded the depletion of NE elicited by alpha-MpT. At midnight, when the levels of melatonin are high, luzindole (30 mg/kg) significantly accelerated the depletion of NE by alpha-MpT in hypothalamus, but not in frontal cortex or hippocampus, suggesting activation of melatonin receptor sites by endogenous melatonin. We conclude that activation of melatonin receptor sites in C3H/HeN mouse brain by endogenous melatonin inhibits the activity of noradrenergic neurons innervating the hypothalamus.     

Enzymatic and nonenzymatic ADP-ribosylation of cysteine

Mono-ADP-ribosylation is a protein modification that occurs at a number of different amino acids, dictated by the specificity of the individual ADP-ribosyltransferases. A specific cysteine in several guanine nucleotide-binding regulatory proteins is ADP-ribosylated by the bacterial protein pertussis toxin. Recent purification of an ADP-ribosylcysteine hydrolase and NAD:cysteine ADP-ribosyltransferase, and detection of ADP-ribose-cysteine linkages in tissue samples has raised hope that an endogenous regulatory cysteine-specific ADP-ribosylation pathway exists. A current goal is the identification of such a pathway for ADP-ribosylation of cysteine within animal cells. Interpretation of the data in this field has been complicated by recent reports that revealed several unforeseen chemical reactions of NAD and its metabolites with free cysteine and cysteine in proteins. This mini-review covers the latest understanding of the ADP-ribosylation reactions associated with cysteine, and provides a set of criteria for future research to establish positively the existence of an endogenous cysteine-specific mono-ADP-ribosyltransferase.     

ADP-Ribosylation of the Neuronal Phosphoprotein B-50/GAP-43

Abstract: The neuronal phosphoprotein B-50/GAP-43 is associated with growth and regeneration within the nervous system and its posttranslational status can be correlated with its cellular localization during growth and regeneration. Recently, B-50 has been shown to interact with certain G protein subunits. Regulation of G protein-mediated signal transduction may involve ADP-ribosylation in vivo. In the present study we have demonstrated that B-50 is a substrate for endogenous ADP-ribosyltransferases. The results are discussed with respect to the possible interaction of B-50 with G proteins, but also with regard to the posttranslational modification of B-50 by all major regulatory mechanisms that act at, or through, the neuronal membrane.     

Chronic Lithium Treatment has Antioxidant Properties but does not Prevent Oxidative Damage Induced by Chronic Variate Stress

This study evaluated the effects of chronic stress and lithium treatments on oxidative stress parameters in hippocampus, hypothalamus, and frontal cortex. Adult male Wistar rats were divided into two groups: control and submitted to chronic variate stress, and subdivided into treated or not with LiCl. After 40 days, rats were killed, and lipoperoxidation, production free radicals, total antioxidant reactivity (TAR) levels, and superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities were evaluated. The results showed that stress increased lipoperoxidation and that lithium decreased free radicals production in hippocampus; both treatments increased TAR. In hypothalamus, lithium increased TAR and no effect was observed in the frontal cortex. Stress increased SOD activity in hippocampus; while lithium increased GPx in hippocampus and SOD in hypothalamus. We concluded that lithium presented antioxidant properties, but is not able to prevent oxidative damage induced by chronic variate stress.     

Flow cytometric and immunoblot assays for cell surface ADP-ribosylation using a monoclonal antibody specific for ethenoadenosine

NAD-dependent ADP-ribosylation is one of the posttranslational protein modifications. On mammalian cells, glycosylphosphatidylinositol-anchored cell surface ADP-ribosyltransferases (ARTs) ADP-ribosylate other cell surface proteins and thereby affect important cellular functions. Here we describe convenient flow-cytometric and immunoblot assays for monitoring ADP-ribosylation of cell surface proteins on living cells by exploiting the capacity of ARTs to utilize etheno-NAD as substrate. Etheno-ADP-ribosylation of cell surface proteins can be detected by flow cytometry with 1G4, a monoclonal antibody specific for ethenoadenosine. Labeling of cells with 1G4 is dependent on the expression of cell surface ARTs and occurs only after incubation of ART-expressing cells with etheno-NAD and not with etheno-ADP-ribose. Dose-response analyses show efficient 1G4 staining of ART-expressing cells at micromolar etheno-NAD concentrations. Half-maximal staining is obtained with 1-2 micro M etheno-NAD, saturation is reached at 5-20 micro M etheno-NAD. Immunoblot analyses confirm that ART-expressing cells incorporate ethenoadenosine covalently (i.e., SDS resistant) into several cell surface proteins. The flow-cytometric 1G4 staining assay can be used to identify subpopulations of cells expressing cell surface ART activity and to select ART(hi) cell variants. The immunoblot 1G4 staining assay can also be used to identify etheno-ADP-ribosylated target proteins. These new assays hold promise for many interesting applications in biochemistry and cell biology.     

Posttranslational regulation of nitrogenase in Rhodobacter capsulatus: existence of two independent regulatory effects of ammonium.

In the photosynthetic bacterium Rhodobacter capsulatus, nitrogenase activity is regulated by ADP-ribosylation of component II in response to the addition of ammonium to cultures or to the removal of light. The ammonium stimulus results in a fast and almost complete inhibition of the in vivo acetylene reduction activity, termed switch-off, which is reversed after the ammonium is exhausted. In the present study of the response of cells to ammonium, ADP-ribosylation of component II occurred but could not account for the extent and timing of the inhibition of activity. The presence of an additional response was confirmed with strains expressing mutant component II proteins; although these proteins are not a substrate for ADP-ribosylation, the strains continued to exhibit a switch-off response to ammonium. This second regulatory response of nitrogenase to ammonium was found to be synchronous with ADP-ribosylation and was responsible for the bulk of the observed effects on nitrogenase activity. In comparison, ADP-ribosylation in R. capsulatus was found to be relatively slow and incomplete but responded independently to both known stimuli, darkness and ammonium. Based on the in vitro nitrogenase activity of both the wild type and strains whose component II proteins cannot be ADP-ribosylated, it seems likely that the second response blocks either the ATP or the electron supply to nitrogenase.     

Regulation by chronic clonidine of adenylate cyclase and cyclic AMP-dependent protein kinase in the rat locus coeruleus

Clonidine and morphine are known to produce tolerance and dependence in rat locus coeruleus (LC) neurons after chronic administration based on electrophysiological criteria. Previous studies have shown that morphine tolerance and dependence is associated with increases in levels of adenylate cyclase, pertussis toxin-mediated ADP-ribosylation of G-proteins, and cyclic AMP-dependent protein kinase in this brain region. The present study was aimed at investigating whether clonidine tolerance and dependence is also associated with alterations in these intracellular messengers. It was found that, similar to chronic morphine, chronic (2 weeks) clonidine administration, under conditions that produce electrophysiological evidence of tolerance and dependence in LC neurons, increased levels of adenylate cyclase activity and cyclic AMP-dependent protein kinase activity in this brain region, but not in several other regions studied, which included the frontal cortex, neostriatum, and dorsal raphe. However, the changes induced by chronic clonidine in the LC, at maximal doses and duration of treatment, were only approximately 50% in magnitude of those observed in response to morphine. Unlike chronic morphine, chronic clonidine produced no change in G-protein ADP-ribosylation levels in the LC. Chronic administration of a number of other drugs, namely diazepam, chloral hydrate, and dextromethorphan, which produce electrophysiological actions distinct from those of clonidine and morphine in the LC, failed to alter adenylate cyclase and cyclic AMP-dependent protein kinase in this brain region. The results indicate that increased levels of adenylate cyclase and cyclic AMP-dependent protein kinase represent common adaptations by LC neurons to chronic clonidine and morphine, and raise the possibility that such changes contribute to the development of clonidine and morphine tolerance and dependence in these neurons.     

An endogenous inhibitor of the ADP-ribosylation of GTP-binding proteins by pertussis toxin is present in bovine brain

The ADP-ribosylation of GTP-binding proteins (G-proteins) catalyzed by pertussis toxin was inhibited by endogenous inhibitor activity in the membrane extract of bovine brain. Most of the activity appeared in the fractions eluted from a DEAE-Sephacel column by 0.5 M NaCl. The activity was heat-stable and sensitive to pronase K. The results suggest the presence of an endogenous inhibitor of pertussis toxin in bovine brain.