Inactivation of platelet-derived growth factor-BB following modification by ADP-ribosyltransferase.

1. Arginine-specific ADP-ribosyltransferase (ART1) is expressed on the surface of a number of cell types, and catalyses the transfer of ADP-ribose from NAD(+) to target proteins. We investigated whether extracellular proteins such as growth factors may serve as substrates for this enzyme, with subsequent alteration in their biological activity. Experiments were performed with rat skeletal muscle membranes and V79 Chinese hamster lung fibroblasts with doxycycline-inducible expression of human ART. 2. From a panel of growth factors, platelet-derived growth factor-BB (PDGF-BB) was found to be the best substrate for ART1, whereas the structural homologue PDGF-AA was not a substrate. Under conditions of maximum labelling 5 mol ADP-ribose was incorporated per mol of PDGF-BB. 3. Purified (ADP-ribosyl)-PDGF-BB did not stimulate a mitogenic or chemotactic response in human pulmonary smooth muscle cells, and showed a reduced capacity to bind to PDGF receptors in competition binding experiments, when compared to unmodified PDGF-BB. 4. PDGF-dependent [(3)H-methyl]-thymidine incorporation was measured in the ART1-transfected fibroblast cell line at physiological concentrations of PDGF-BB, and without addition of extracellular NAD(+). Fibroblasts expressing human ART1 at the cell surface showed reduced mitogenic responses to PDGF-BB, but not to PDGF-AA. This loss of mitogenic response in cells expressing ART1 activity was reversed by the addition of agmatine (an ART1 substrate). 5. In conclusion, we propose that PDGF-BB-dependent signalling may be regulated by post-translational modification of the growth factor by ART1 at the cell surface. This has been demonstrated in membranes of rat skeletal muscle, and the reaction confirmed in ART1-transfected fibroblasts.

Polymorphic forms of human ADP-ribosyltransferase-1 differences in their catalytic activities revealed by labeling of membrane-associated substrates.

Full length cDNA encoding ADP-ribosyltransferase-1 (ART1) was generated from human skeletal muscle. A single base variation from the published sequence was observed (C770-->T), and was established as a polymorphism by the screening of a population of 50 Caucasians. The base variation predicted a nonconservative substitution of Leu for Pro at codon 257. Cell lines with stable and doxycycline-inducible expression of the two polymorphic forms of ART1 were generated from Chinese hamster V79 cells, and exploited in studies to compare the activities of ART1-Pro257 and ART1-Leu257. The results revealed no differences in the capacity of phosphoinositide-specific phospholipase C to cleave the two ART1 isoforms from the plasma membrane. Furthermore, the capacities of ART1-Pro257 and ART1-Leu257 to ADP-ribosylate agmatine or fibroblast growth factor-2 were similar. Differences in the catalytic activities of ART1-Pro257 and ART1-Leu257 were however, identified when measurements were made of their capacities to ADP-ribosylate membrane-associated proteins on the surface of V79 cells. Protein(s) of molecular mass 80-110 kDa were more extensively ADP-ribosylated by ART1-Pro257 than ART1-Leu257, in accordance with the Vmax (59.5 +/- 5.5 and 37.0 +/- 3.0) and Km values (12.5 +/- 4.5 and 5.0 +/- 1. 9) for ART1-Pro257 and ART1-Leu257, respectively.

Inhibition of chemotaxis in A7r5 rat smooth muscle cells by a novel panel of inhibitors.

1. Arginine-specific ADP-ribosyltransferase (ART) activity has been implicated in white cell chemotaxis. In this study, we examined the capacity of a panel of structurally unrelated inhibitors and pseudosubstrates of ART to inhibit chemotaxis of A7r5 rat vascular smooth muscle cells in response to PDGF-BB. 2. The IC50 values for nicotinamide (12 mM) and novobiocin (165 microM) were similar to those observed for inhibition of chemotaxis by human polymorphonuclear neutrophil leucocytes (PMN), whereas vitamins K3 (IC50=22 microM) and K1 (IC50=95 microM) were less potent than previously described in PMNs. The pseudo-substrates for the enzyme (DEA-BAG, agmatine and arginine-methylester) also inhibited A7r5 chemotaxis, and in addition inhibited cell adhesion at similar concentrations. Vitamin K3 was unique among the inhibitors of ART, in that it also inhibited cell adhesion. 3. A rat ART1 transcript was amplified by rtPCR from rat skeletal muscle, and was noted to share 94% homology with the mouse ART1 cDNA sequence. No such transcript could be detected in A7r5 cells by Northern blot analysis or rtPCR. 4. Evidence for ART activity on the surface of A7r5 cells was investigated using 32P-NAD+ as substrate, and labelled membrane proteins were observed with MWt values of 116, 100, 90 and 70 kDa. Exposure of the labelled proteins to phosphodiesterase yielded 32P-AMP, and hydrolysis with NaOH yielded 32P-NAD+. These results indicated that the labelled proteins were adducts with NAD+, and not the products of ART activity. The absence of ART catalytic activity in A7r5 cells was confirmed in protocols designed to show ADP-ribosylation of agmatine. 5. We conclude that the chemotactic activity of A7r5 cells is independent of ART activity, and the mechanism whereby the novel panel of inhibitors reduced cell migration remains undefined.

Arginine-specific ADP-ribosyltransferases in leukocytes.

The posttranslational modification of proteins by the addition of an ADP-ribose group is mediated by ADP-ribosyltransferases, which are expressed widely in many eukaryotic tissues, including leukocytes. DNA encoding arginine-specific ADP-ribosyltransferases has been cloned from both polymorphonuclear neutrophil leukocytes and lymphocytes, and their primary structures are widely conserved, particularly in those domains of the enzyme implicated in NAD+ binding and catalysis. In most cases the enzymes are tethered to the outer aspect of the cell surface or are released directly from the cell surface. The protein substrates of some of the ADP-ribosyltransferases have been identified and the catalytic activity of these enzymes has been implicated in several immune responses as well as white cell chemotaxis. This review describes recent significant advances in this field, and it seems likely that additional leukocyte functions, most particularly those linked to the activity of surface integrins, will be assigned to this class of enzymes.

An ADP-ribosyltransferase from bovine erythrocytes apparently specific for cysteine residues.

An NAD+:cysteine glycohydrolase purified from bovine erythrocytes had a specific activity of 1900 (nmol nicotinamide released).min-1.mg-1, a K(m) for cysteine of 4.0 mM, and an M, of 45,000. The enzyme also catalysed the dose-dependent ADP-ribosylation of several bovine erythrocyte proteins, including a doublet of high M(r) and proteins of M(r) 60,000, 55,000, and 29,000. ADP-ribosylation of the M(r) 55,000 protein was blocked by pre-treatment of the erythrocyte membranes with N-ethylmaleimide, and ADP-ribose was released by treatment with mercuric ions, but not with hydroxylamine. The enzyme therefore appears to be a cysteine-specific ADP-ribosyltransferase.

ATP-citrate lyase as a target for hypolipidemic intervention. Design and synthesis of 2-substituted butanedioic acids as novel, potent inhibitors of the enzyme.

ATP-citrate lyase is the primary enzyme responsible for the synthesis of cytosolic acetyl-CoA in many tissues. Inhibitors of the enzyme represent a potentially novel class of hypolipidemic agent, which are anticipated to have combined hypocholesterolemic and hypotriglyceridemic properties. A series of 2-substituted butanedioic acids have been designed and synthesized as inhibitors of the enzyme. The best compounds, 58, 68, 71, 74 have reversible Ki's in the 1-3 microM range against the isolated rat enzyme. As representative of this compound class, 58, has been shown to exert its inhibitory action through a mainly competitive mechanism with respect to citrate and a noncompetitive one with respect to CoA. None of the inhibitors were able to inhibit cholesterol and/or fatty acid synthesis in HepG2 cells. This has been attributed to the adverse physicochemical properties of the molecules leading to a lack of cell penetration. Despite this, a lead structural class of compound has been identified with the potential for modification into potent, cell-penetrant, and efficacious inhibitors of ATP-citrate lyase.

Expression of protamine P2 in the testis of the common marmoset and man visualized using non-radioactive in-situ hybridization.

Information on the organization of the spermatogenic cycle of the common marmoset (Callíthrix jacchus), a small New World primate, is limited to a single histological report on the differentiation of spermatids. In the present study we have used non-radioactive in-situ hybridization with a cRNA probe directed against marmoset protamine 2, on fixed sections of marmoset and human testis to elucidate the organization of mature germ cells within the seminiferous epithelium. Specificity of the probe was checked on Northern blots; mP2 hybridized exclusively to mRNA in samples extracted from marmoset and human testis. In sections from human and marmoset testis, positive staining for mRNA was confined to round and elongating spermatids and in the human was reduced in samples from patients with incomplete spermatogenesis. In the human, P2 mRNA was present in groups of cells consistent with the presence of more than one stage of the spermatogenic cycle in transverse sections of individual tubules. In the marmoset, P2-positive cells were detected as a continuous ring of staining in the majority of sections of tubules whilst in others only a group(s) of cells was positive. We conclude that the arrangement of the spermatogenic wave in this New World primate may be intermediate between that seen in rodents (segmental) and in the human (helical).

The purification of a cysteine-dependent NAD+ glycohydrolase activity from bovine erythrocytes and evidence that it exhibits a novel ADP-ribosyltransferase activity.

An NAD+:cysteine ADP-ribosyltransferase activity was purified from bovine erythrocytes on the assumption that, like pertussis toxin, the enzyme would exhibit a cysteine-dependent NAD+ glycohydrolase activity. A three-step purification procedure was developed involving (1) precipitation with 40% (NH4)2SO4, (2) binding to a cysteine-Sepharose affinity column, and (3) binding to an NAD+ affinity column. PAGE showed a single band of M(r) 45,000. The enzyme had been purified 47,000-fold and had a specific activity of 1900 nmol nicotinamide released/min per mg. A study of the kinetic properties of this enzyme showed saturation kinetics for cysteine (Km = 4.0 mM). The ability of this enzyme to ADP-ribosylate protein was investigated using re-sealed inverted bovine erythrocyte ghosts. Incubation of the purified enzyme with erythrocyte ghosts and [adenylate-32P]NAD+ led to the enhanced dose-dependent labelling of several proteins, a doublet of high M(r) and proteins of M(r) 60,000, 55,000 and 29,000, identified by autoradiography of separated proteins on SDS/PAGE. The enzyme-catalysed labelling of the major component at M(r) 55,000 was blocked by pre-treatment of the erythrocyte ghosts with N-ethymaleimide, a sulphydryl alkylating agent, and the label was released by mercuric ion, but not by hydroxylamine. These experiments suggested that a cysteine residue on the target protein had been mono-ADP-ribosylated. This supposition was further supported by identification of the mercf1p4ion-released radiolabelled product as ADP-ribose by HPLC, and the observation that free ADP-ribose was unable to modify the membrane target protein directly.

Synthesis of novel thiol-containing citric acid analogues. Kinetic evaluation of these and other potential active-site-directed and mechanism-based inhibitors of ATP citrate lyase.

ATP citrate lyase is an enzyme involved in mammalian lipogenesis and cholesterogenesis. Inhibitors of the enzyme represent a potentially novel class of hypolipidemic agents. Citric acid analogues 5-16 bearing electrophilic and latent electrophilic substituents were synthesized and evaluated as irreversible inhibitors of the enzyme. The design of these agents was based on the classical enzymatic mechanism where an active-site nucleophile (thiol) was believed to be critically involved in catalysis. Reversible inhibition (Ki's ranging from ca. 20 to 500 microM) was observed for compounds 5, 10, and 12-16. Compounds 6-9 and 11 had no appreciable affinity for enzyme (Ki > 1 mM). Time-dependent inactivation of the enzyme by 5-16 was not detected following long incubation times (> 1 h, 37 degrees C) at 2 mM inhibitor concentrations.

ATP-citrate lyase as a target for hypolipidemic intervention. Sulfoximine and 3-hydroxy-beta-lactam containing analogues of citric acid as potential tight-binding inhibitors.

Citric acid analogues (+/-)-12a,b and (+/-)-17a,b, where one of the primary carboxylates has been replaced by a sulfoximinoyl and a 3-(3-hydroxy-beta-lactamyl) moiety, respectively, have been synthesized and evaluated as inhibitors of ATP-citrate lyase. The design of these inhibitors was based on methionine sulfoximine and tabtoxinine beta-lactam, potent, tight-binding inhibitors of glutamine synthetase. Both ATP-citrate lyase and glutamine synthetase employ phosphate-carboxylate anhydrides as a method for carboxylate activation during catalysis. Only one diastereomer, (+/-)-12a, displayed weak, reversible inhibition, while the remaining citrate analogues (+/-)-12b and (+/-)-17a,b were inactive against the lyase. No time-dependent inactivation of the enzyme was observed.

Cloning and expression of a human ATP-citrate lyase cDNA.

A full-length cDNA clone of 4.3 kb encoding the human ATP-citrate lyase enzyme has been isolated by screening a human cDNA library with the recently isolated rat ATP-citrate lyase cDNA clone [Elshourbagy et al. (1990) J. Biol. Chem. 265, 1430]. Nucleic-acid sequence data indicate that the cDNA contains the complete coding region for the enzyme, which is 1105 amino acids in length with a calculated molecular mass of 121,419 Da. Comparison of the human and rat ATP-citrate lyase cDNA sequences reveals 96.3% amino acid identity throughout the entire sequence. Further sequence analysis identified the His765 catalytic phosphorylation site, the ATP-binding site, as well as the CoA binding site. The human ATP-citrate lyase cDNA clone was subcloned into a mammalian expression vector for expression in African green monkey kidney cells (COS) and Chinese hamster ovary cells (CHO) cells. Transfected COS cells expressed detectable levels of an enzymatically active recombinant ATP-citrate lyase enzyme. Stable, amplified expression of ATP-citrate lyase in CHO cells as achieved by using coamplification with dihydrofolate reductase. Resistant cells expressed high levels of enzymatically active ATP-citrate lyase (3 pg/cell/d). Site-specific mutagenesis of His765----Ala diminishes the catalytic activity of the expressed ATP-citrate lyase protein. Since catalysis of ATP-citrate lyase is postulated to involve the formation of phosphohistidine, these results are consistent with the pattern of earlier observations of the significance of the histidine residue in catalysis of the human ATP-citrate lyase.

Redox control of catalysis in ATP-citrate lysate from rat liver.

In thiol redox buffers at pH 8.0, rat liver ATP-citrate lyase is in equilibrium between an oxidised inactive form and a reduced active form. The reduced enzyme is inactivated by oxidised glutathione (GSSG) at a rate of 45 min-1.M-1 and the oxidised enzyme is activated by reduced glutathione (GSH) at a rate of 3.2 min-1.M-1. At redox equilibrium, the enzyme activity depends on the ratio [GSH]2/[GSSG]. The inactivation involves formation of a protein-protein disulphide rather than a protein-glutathione complex. This reaction has Keq = 78 +/- 7 mM for the oxidative reaction. Activity can therefore be controlled by the redox state of the cell, being more active in the fed state than in the oxidatively stressed state. This redox process is also important in the in vitro enzyme assay, where ATP-citrate lyase is in redox equilibrium with oxygen and either dithiothreitol or 2-mercaptoethanol. Reduction is a two-step process, requiring high concentrations of reductant for full activation (30 mM dithiothreitol or 200 mM 2-mercaptoethanol). The enzyme inhibitor, Medica-16 raises the redox equilibrium constant to greater than 400 mM. It binds more tightly to the oxidised form of the enzyme, with Ki less than 40 microM compared to 180 microM for the reduced form.

Synthesis and evaluation of (+) and (-)-2,2-difluorocitrate as inhibitors of rat-liver ATP-citrate lyase and porcine-heart aconitase.

The enantiomers (+) and (-)-2,2-difluorocitrate have been synthesized. Both are good inhibitors of ATP-citrate lyase, showing competitive inhibition against citrate, with Kis = 0.7 microM for (+)-2,2-difluorocitrate and 3.2 microM for (-)-2,2-difluorocitrate. The inhibition patterns with either ATP or CoA as the varied substrate were uncompetitive and mixed, respectively, but with much weaker inhibition constants. Neither isomer undergoes carbon-carbon bond cleavage as a substrate and there is no evidence of irreversible time-dependent inactivation. When ATP-citrate lyase is incubated with CoA and difluorocitrate, the maximal intrinsic ATPase rate is 10% of the citrate-induced rate for the (+)-enantiomer and 2% for the (-)-enantiomer. 19F-NMR studies confirm that only the (+)-enantiomer is chemically processed. The effects of the difluorocitrate enantiomers on the reaction catalysed by aconitase were examined. (-)-2,2-Difluorocitrate is a competitive inhibitor against citrate (Kis = 1.5 microM), whereas the (+)-enantiomer is a relatively poor mixed inhibitor (Ki greater than 300 microM). The (-)-enantiomer irreversibly inactivates aconitase at 1.1 min-1.mM-1 at 25 degrees C and pH 7.4, whereas no irreversible inhibition is seen with the (+)-enantiomer. Therefore, it would be expected that the (+)-enantiomer would slow the rate of acetyl-CoA synthesis in vivo, without inhibiting the citric acid cycle.