Selective augmentation of histone H1 phosphorylation sites by interaction of poly(ADP-ribose) polymerase and cdc2-kinase: comparison with protein kinase C.

The molecular interactions between PARP I, cdc2-kinase, PKC and histone H1 were determined with the aid of the common phosphate acceptor function of histone H1 to both kinases. PKC phosphorylates both histone H1 and PARP I and PARP I augments the acceptor function of histone H1. When both acceptors (PARP I and histone H1) are present an apparent distributive phosphorylation of both acceptors takes place. In contrast, cdc2-kinase only phosphorylates histone H1, and the activation of this reaction by PARP I does not involve PARP I-cdc2-kinase binding only PARP I-histone H1 association. Since the phosphorylation of histone H1 by PKC is a model reaction with no apparent physiologic consequences, the PARP I activated phosphorylation of histone H1 by cdc2-kinase, by contrast, reflects a physiologically meaningful regulation of the linker histone by a cyclin dependent kinase (cdc2-kinase). The increased phosphorylation of histone H1 by cdc2-kinase following PARP I-histone H1 binding results in the appearance of new phosphorylated histone H1 polypeptides as measured by proteolytic digestion and re-electrophoresis of cdc2-kinase phosphorylated polypeptides, indicating a probable conformational change in histone H1, following PARP I binding. The cell biologic significance of this reaction in PARP I ligand-induced enzyme induction is briefly analysed.

The somatostatin analogue TT-232 induces apoptosis in A431 cells: sustained activation of stress-activated kinases and inhibition of signalling to extracellular signal-regulated kinases.

TT-232 is a somatostatin analogue containing a five-residue ring structure. The present report describes TT-232-induced signalling events in A431 cells, where a 4-h preincubation with the peptide irreversibly induced a cell death program, which involves DNA-laddering and the appearance of shrunken nuclei, but is unrelated to somatostatin signalling. Early intracellular signals of TT-232 include a transient two-fold activation of the extracellular signal-regulated kinase (ERK2) and a strong and sustained activation of the stress-activated protein kinases c-Jun NH(2)-terminal kinase (JNK)/SAPK and p38MAPK. Blocking the signalling to ERK or p38MAPK activation had no effect on the TT-232-induced cell killing. At the commitment time for inducing cell death, TT-232 decreased EGFR-tyrosine phosphorylation and prevented epidermial growth factor (EGF)-induced events like cRaf-1 and ERK2 activation. Signalling to ERK activation by FCS, phorbol 12-myristate 13-acetate (PMA) and platelet-derived growth factor (PDGF) was similarly blocked. Our data suggest that TT-232 triggers an apoptotic type of cell death, concomitant with a strong activation of JNK and a blockade of cellular ERK2 activation pathways.

Molecular interactions between poly(ADP-ribose) polymerase (PARP I) and topoisomerase I (Topo I): identification of topology of binding.

The molecular interactions of poly(ADP-ribose) polymerase I (PARP I) and topoisomerase I (Topo I) have been determined by the analysis of physical binding of the two proteins and some of their polypeptide components and by the effect of PARP I on the enzymatic catalysis of Topo I. Direct association of Topo I and PARP I as well as the binding of two Topo I polypeptides to PARP I are demonstrated. The effect of PARP I on the 'global' Topo I reaction (scission and religation), and the activation of Topo I by the 36 kDa polypeptide of PARP I and catalytic modifications by poly(ADP-ribosyl)ation are also shown. The covalent binding of Topo I to circular DNA is activated by PARP I similar to the degree of activation of the 'global' Topo I reaction, whereas the religation of DNA is unaffected by PARP I. The geometry of PARP I-Topo I interaction compared to automodified PARP I was reconstructed from direct binding assays between glutathione S-transferase fusion polypeptides of Topo I and PARP I demonstrating highly selective binding, which was correlated with amino acid sequences and with the 'C clamp' model derived from X-ray crystallography.

Binding of topo I to PARP I - antibody immunocomplex.

It is frequently quoted in the literature that the cellular role of PARP I is its participation in the recognition of single strand breaks of genomic DNA (l.c.1). Although there is little doubt that PARP I can be made to respond powerfully as an factor in the recognition of DNA damage, it seems unlikely that this auxilliary, or telelogically defined, role of this highly abundant nuclear protein exhausts its physiologic cellular function. We have reported that Topo I is greatly activated by its association with PARP I (J Mol Med 5: 533-540, 2000). Translation of this in vitro model experiments to physiologic conditions was accomplished by the demonstration of the quantitative binding of Topo I to a PARP I - antibody complex, as reported here. This experiment demonstrates for the first time that the colligative action of PARP I can regulate a highly significant cellular process, the control of readability of genomic DNA, i.e., gene expression, without the artificiality of induced DNA damage.

Activation of topoisomerase I by poly [ADP-ribose] polymerase.

Poly(ADP-ribose) polymerase (PARP I) and Topoisomerase I (Topo I) were reisolated from calf thymus to eliminate cross contamination as tested by immunotransblots. The specific activity of Topo I was greatly increased by added PARP I, following saturation kinetics. Recombinant PARP I and isolated PARP I at final purity were indistinguishable in terms of their activation of Topo I. There was a coincidence of experimentally obtained binding constants and computer generated values based on the kinetic model, indicating that the association of PARP I and Topo I is rate limiting in the catalytic activation of Topo I by PARP I. Polypeptide domains of PARP I that are required for protein-protein binding and protein-DNA binding also activate Topo I. Fluorescence resonance energy transfer between fluorophor-labeled PARP I and Topo I was demonstrated. The binding of Topo I to circular SV40 DNA, assayed either by the formation of a) the sum of non-covalently and covalently attached Topo I to DNA or b) by the covalently bound transient intermediate in the presence of camptothecin, was augmented when PARP I protein was bound to SV40 DNA. These binding experiments provide a molecular basis for the kinetic activation of Topo I by PARP I inasmuch as the increased superhelicity of SV40 DNA induced by PARP I may facilitate the formation of a more Topo I-DNA complex that increases the rate of the DNA breakage-reunion cycle of Topo I catalysis.

Peptidyl beta-homo-aspartals (3-amino-4-carboxybutyraldehydes): new specific inhibitors of caspases.

Interleukin-1 beta (IL-1 beta)-converting enzyme (ICE, caspase-1) processes the IL-1 beta precursor to mature inflammatory cytokine IL-1 beta. ICE has been identified as a unique cysteine protease, which cleaves Asp-X bonds, shows resistance to E-64 (an inhibitor of most cysteine proteases) and has a primary structure that is homologous to CED-3, a protein required for apoptosis (programmed cell death) in the nematode Caenorhabditis elegans, and to mammalian cysteine proteases that initiate and execute apoptosis, e.g., apopain/CPP32/caspase-3. The inhibitors of the ICE/CED-3 family or caspases, as they are called recently, may constitute therapeutic agents for amelioration of inflammatory and apoptosis-associated diseases. The most efficient ICE inhibitors are peptide aldehydes and peptidyl chloro or (acyloxy)methanes. A recent study revealed that both D- and L-Asp are accepted by ICE at the P1 of such inhibitors, and the peptidyl (acyloxy)methane analogues having the beta-homo-aspartyl residue [-NH-CH(CH2COOH)-CH2CO-] are inactive. These findings we reexamined in terms of two issues. (a) ICE's resistance to E-64. Since it was thought to be caused by the enzyme's unique substrate specificity, we prepared substrate-based analogues, which were not inhibitory suggesting significant structural difference between the active centers of ICE and papain-like enzymes. (b) Tolerance for D-stereochemistry at the P1 of these inhibitors. In view of the mechanism of cysteine protease inhibition by peptidyl X-methanes, we thought that this phenomenon should be a general characteristic of cysteine proteases and the hAsp-containing analogues should behave as reversible inhibitors. Here, we analyzed the inhibition of ICE and apopain in comparison with that of papain, thrombin, and trypsin by peptide L/D-alpha-aldehydes and their L-beta-homo-aldehyde [-NH-CH(R)-CH2-CHO] analogues. The following results were found. (1) The peptidyl L-beta-homo-aspartals are potent inhibitors for caspases. (2) The L-beta-homo analogues of peptide aldehyde inhibitors designed for other proteases are not inhibitory. (3) Unlike trypsin and thrombin (serine proteases), papain (cysteine protease) shows tolerance for D-stereochemistry at the P1 site of peptide aldehydes in proportion to the lability of the alpha-hydrogen of the P1-D-residue. The complete tolerance of ICE for P1-D-Asp may arise from this residue's high tendency to epimerization. (4) Reaction of cysteine proteases with peptide aldehyde or peptidyl X-methane inhibitors containing P1-D-residues may include alpha-proton abstraction followed by asymmetric induction leading to P1-L-residue-containing products.

BCNU is a caspase-mediated inhibitor of drug-induced apoptosis.

BCNU [1,3-bis(2-chloroethyl)-1-nitrosourea], a bifunctional (alkylating/carbamoylating) anticancer agent, in noncytotoxic doses (12-50 microM) inhibited drug-induced apoptosis in HT58 human lymphoma cells exposed to etoposide (ETO; 50 microM) as well as in mouse thymocytes exposed to dexamethasone (5 microg/ml) in vitro in 4-h cultures. The cytoplasmic extracts of ETO-treated HT58 cells cleaved both purified poly(ADP-ribose)polymerase and Ac-Asp-Glu-Val-Asp-7-amino-4-methylcoumarin fluorogenic caspase substrate, indicating the presence of active caspases, and these effects were inhibited by BCNU concentration dependently. The carbamoylating decomposite, 2-chloroethyl-isocyanate (6-25 microM), also decreased ETO-induced apoptosis in HT58 cells in vitro and their caspase 3-like activity ex vivo, whereas N-(2-chloroethyl)-N-nitrosocarbamoyl-valinamide, an alkylating and mainly intramolecularly carbamoylating nitrosourea derivative (400 microM), did not influence these phenomena. Furthermore, the activity of recombinant caspase 3 was also strongly inhibited by BCNU and 2-chloroethyl-isocyanate. These results indicate that BCNU, via its carbamoylating capacity, can inactivate cysteine protease(s) essential for ETO-induced apoptosis. This apoptosis-modulating property of BCNU, in turn, may influence the efficacy of chemotherapeutic protocols in the treatment of cancer.

Isolation and identification of a proteinase from calf thymus that cleaves poly(ADP-ribose) polymerase and histone H1.

A proteinase was isolated from calf thymus that degraded pADPRT, histone H1 and alpha-casein in a Ca(2+)-dependent manner. In a five-step procedure, a homogenous proteinase was obtained with a subunit structure of 80 and 30 kDa. The amino-acid homology of an internal sequence as well as kinetic and inhibitor assays identified the proteinase as calpain I. It is suggested that even though the general substrate alpha-casein is widely used for the assaying of calpains, more appropriately physiological cellular components (pADPRT and histone H1) specify the thymus proteinase.

Apoptotic cell death induced by inhibitors of energy conservation--Bcl-2 inhibits apoptosis downstream of a fall of ATP level.

Energy charge controls intermediary metabolism and cellular regulation. Here we show that inhibition of energy conservation at the level of glucose uptake, glycolysis, citric acid cycle, and oxidative phosphorylation induces cell death, leading to fragmentation of DNA into an oligonucleosomal ladder and morphological changes typical for apoptosis. Bcl-2, the prototype of oncogenes that suppress cell death, efficiently inhibits apoptosis induced by metabolic inhibitors. Bcl-2 does not antagonize the inhibitory potential of mitochondrial inhibitors, and cannot prevent or delay the decrease of the cellular ATP level subsequent to metabolic inhibition. Thus, we propose that Bcl-2 blocks apoptosis at a point downstream of the collapse of the cellular-energy homeostasis.

Identification of domains of poly(ADP-ribose) polymerase for protein binding and self-association.

Cellular proteins extracted from normal and cancer cells bind polymerizing ADP-ribose transferase (pADPRT) on nitrocellulose membrane transblots. Histones at 1 mg/ml concentration completely prevent the binding of pADPRT to cellular proteins, indicating that the binding of histones to pADPRT sites competitively blocks the association of pADPRT to proteins other than histones. The direct binding of pADPRT to histones is shown by cross-linking with glutaraldehyde. The COOH-terminal basic histone H1 tail binds to the basic polypeptide domain of pADPRT. The basic domain present in the NH2-terminal part of core histones is the probable common structural feature of all core histones that accounts for their binding to pADPRT. Two polypeptide domains of pADPRT were identified, by way of CNBr fragments, to bind histones. These two domains are located within the 64-kDa fragment of pADPRT and are contiguous with the polypeptide domains that were shown to participate in self-association of pADPRT, ending at the 606th amino acid residue. The polypeptide domains of pADPRT which participate in DNA binding are thus shown to associate also with other proteins. Intact pADPRT binds to both the zinc-free or zinc-reconstituted basic polypeptide fragments of pADPRT. Histones activate auto-poly(ADP)-ribosylation of pADPRT by increasing the number of short oligomers on pADPRT. This reaction is also dependent in a biphasic manner on the concentration of pADPRT. Histones in solution are only marginally poly(ADP)-ribosylated but are good polymer acceptors when incorporated into artificial nucleosome structures.

Reversion of malignant phenotype by 5-iodo-6-amino-1,2-benzopyrone a non-covalently binding ligand of poly(ADP-ribose) polymerase.

A non-covalently binding inhibitory ligand of poly(ADP-ribose) polymerase, 5-iodo-6-amino-1,2-benzopyrone, when incubated at 5-600 microM external concentration with an E-ras-transformed tumorigenic cell line or with human prostatic carcinoma cells for 40 to 60 days converts both cancer cells to a non-tumorigenic phenotype that is characterized by drastic changes in cell morphology, absence of tumorigenicity in nude mice, and a high rate of aerobic glycolysis.

Phosphorylation of poly(ADP-ribose)polymerase protein in human peripheral lymphocytes stimulated with phytohemagglutinin.

Intracellular phosphorylation of poly(ADP-ribose)polymerase was assayed in streptolysin-O-permeabilized human lymphocytes. Whereas 32P incorporation from [gamma-32P]ATP into immunoprecipitated enzyme protein was undetectable in resting cells, significant phosphorylation of this enzyme was observed in lymphocytes treated with phytohemagglutinin. The phosphorylation of poly(ADP-ribose)polymerase in permeabilized cells was not stimulated by phorbol ester, while phorbol-induced phosphorylation of other proteins and of a specific oligopeptide substrate of protein kinase C was observed. However, the specific inhibitory pseudosubstrate peptide of protein kinase C blocked the phosphorylation of poly(ADP-ribose)polymerase induced by phytohemagglutinin. Therefore, a potential role of a member of the protein kinase C family in the phytohemagglutinin stimulated intracellular phosphorylation of poly(ADP-ribose)polymerase is conceivable.

Inhibition of DNA binding by the phosphorylation of poly ADP-ribose polymerase protein catalysed by protein kinase C.

Purified type II (beta) and type III (alpha) protein kinase C phosphorylates highly purified polyADP-ribose polymerase in vitro whereby 2 mols of phosphate are transferred from ATP to serine and threonine residues present in the 36 and 56 kDa polypeptide domains of the polymerase protein. Calf thymus DNA was a non-competitive inhibitor of the protein kinase C catalyzed phosphorylation of polyADP-ribose polymerase. Coincidental with the phosphorylation of the protein the polymerase activity and DNA binding capacity of polyADP-ribose polymerase were inhibited. These in vitro findings may have possible cell biological significance in cellular signal transduction.

Inhibitory binding of adenosine diphosphoribosyl transferase to the DNA primer site of reverse transcriptase templates.

Purified adenosine diphosphoribose transferase protein binds to RNA-DNA hybrid templates of reverse transcriptase at the DNA primer site and inhibits RT activity of HIV and MMu RTs. This action is prevented by auto-poly-ADP-ribosylation of the transferase but is reinduced by inhibitory ligands of the enzyme.

Inhibition of HIV-1 IIIb replication in AA-2 and MT-2 cells in culture by two ligands of poly (ADP-ribose) polymerase: 6-amino-1,2-benzopyrone and 5-iodo-6-amino-1,2-benzopyrone.

The effects of two adenosine diphosphoribose transferase (ADPRT) enzyme inhibitory ligands, 6-amino-1,2-benzopyrone and its 5-iodo-derivative, were determined in AA-2 and MT-2 cell cultures on the replication of HIV-1 IIIb, assayed by an immunochemical test for the HIV protein p24, and syncytium formation, characteristic of HIV-infected cells. Intracellular concentrations of both drugs were sufficient to inhibit poly(ADP-ribose) polymerase activity within the intact cell. Both drugs inhibited HIV replication parallel to their inhibitory potency on ADPRT, but distinct differences were ascertained between the two cell lines. In AA-2 cells both p24 and syncytium formation were depressed simultaneously, whereas in MT-2 cells only syncytium formation was inhibited by the drugs, and the p24 production, which remained unchanged during viral growth, was unaffected. Both drugs only moderately depressed the growth rate of the AA-2 and MT-2 cells and there was no detectable cellular toxicity. Results suggest the feasibility of the development of a new line of ADPRT ligand anti-HIV drugs that fundamentally differ in their mode of action from currently used chemotherapeutics.

Destabilization of Zn2+ coordination in ADP-ribose transferase (polymerizing) by 6-nitroso-1,2-benzopyrone coincidental with inactivation of the polymerase but not the DNA binding function.

6-Nitroso-1,2-benzopyrone, an oxidation product of 6-amino-1,2-benzopyrone, binds to the DNA-recognizing domain of the ADP-ribose transferase protein and preferentially destabilizes Zn2+ from one of the two zinc finger polypeptide complexes present in the intact enzyme, as determined by the loss of 50% of 65Zn2+ from the 65Zn(2+)-isolated protein molecule, coincidental with the loss of 99% of enzymatic activity. The 50% zinc-deficient enzyme still binds to a DNA template, consisting of a 17-mer DNA primer annealed to M13 positive strand, resulting in the blocking of DNA synthesis by the Klenow fragment of Pol I. Auto-poly-ADP-ribosylated ADP-ribose transferase, which is the probable physiological state of this protein in intact cells, does not bind to primer-template DNA and does not block DNA synthesis by the Klenow fragment. On the basis of this in vitro model it is proposed that molecules which inhibit or inactivate ADP-ribose transferase in intact cells can induce significant alteration in DNA structure and replication.

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.

Apparent role of adenosine diphosphoribosyl transferase in the development of Mytilus edulis and the inhibition of differentiation by ligands of the enzyme protein.

The poly(ADP-ribose) polymerase or transferase (ADPRT) activity of developing embryos of Mytilus edulis increases with the progression of larval growth. ADPRT protein was partially purified from 2-hr-old embryos and identified by gel electrophoresis and immunotransblot, demonstrating cross-reactivity with anti-ADPRT IgG produced against the calf thymus enzyme. Two inhibitors of ADPRT, benzamide, competing with NAD at the nicotinamide binding site, and 6-amino-1,2-benzopyrone, which competes with DNA at the DNA binding site(s), both selectively arrest differentiation at the prodissoconch stage. The DNA site-oriented inhibitor, 6-amino-1,2-benzopyrone, has a much larger differentiation arresting effect than benzamide. The arrest of differentiation by 6-amino-1,2-benzopyrone is reversible. A probable ecotoxicity of ADPRT ligands on mussel differentiation is proposed.

Suppression of dexamethasone-stimulated DNA synthesis in an oncogene construct containing rat cell line by a DNA site-oriented ligand of poly-ADP-ribose polymerase: 6-amino-1,2-benzopyrone.

The cellular inhibitory effects of 6-amino-1,2-benzopyrone (6-ABP), a DNA site-specific ligand of adenosine diphosphoribosyl transferase (ADPRT), were determined in a dexamethasone-sensitive EJ-ras gene construct containing cell line (14C cells). Dexamethasone in vitro transforms these cells to a tumorigenic phenotype and also stimulates cell replication. At a non-toxic concentrations (0.2 mM) 6-ABP treatment of intact cells for 4 days inhibits the dexamethasone-stimulated increment of cellular DNA content, depresses replicative DNA synthesis as assayed by thymidine incorporation to the level of cells that were not exposed to dexamethasone, and in permeabilized cells reduces the dexamethasone-stimulated increase of deoxyribonucleotide incorporation into DNA to the level of untreated cells. In situ pulse labeling of cells pretreated with 6-ABP indicated an inhibition of DNA synthesis at a stage prior to the formation of the 10-kb intermediate species. The drug had no direct effect on cellular DNA polymerases as tested in vitro, and the inhibition of DNA synthesis in permeabilized cells following drug treatment for 4 days was abolished by externally added DNA templates. Neither dexamethasone nor the drug influenced the cellular quantity of ADPRT molecules, tested immunochemically.