Effects of chlorophyllin on acetaldehyde: lack of modulation of the rate of sister-chromatid exchanges in mouse bone marrow, and of complex formation in aqueous solution.

Acetaldehyde (Ace) is a reactive compound widely found in natural and industrialized products. On the other hand, chlorophyllin (Chl) is a chloropyll derivative which has shown DNA modulatory effects in several models. The first aim of the present study was to determine the capacity of Ace to increase the rate of sister-chromatid exchanges (SCEs) in mouse bone marrow cells in vivo, as well as to determine its capacity to modify the mitotic index (MI) and the average generation time (AGT). For this experiment we tested four dosages of Ace by the i.p. route (0.4, 4.0, 40.0 and 400 mg/kg), and found a genotoxic effect with the two highest dosages (more than double the basal level was observed with 400 mg/kg). We also found that none of the doses tested modified the MI or the AGT. A second objective was to explore the potential of Chl to modulate the genotoxicity of Ace in the same model. We evaluated whether an oral administration of Chl (2.0, 6.0 and 10.0 mg/kg), given 1 h before an i.p. administration of Ace (100 mg/kg), could modulate the SCEs produced by the mutagen. The result showed a similar SCE rate in both, the Ace-treated mice and those administered with the two chemicals, indicating that Chl was not a modulatory chemical on the genotoxicity of Ace. No modifications were observed concerning the MI or the AGT either. A third objective was to determine whether the two compounds (Ace and Chl) may form a molecular complex in aqueous solution. In agreement with the lack of modulatory effect by Chl, a reversed HPLC and a spectrophotometric analysis showed that the two compounds were unable to form a complex. This report confirms the importance of the specificity concerning the interaction mutagen/antimutagen.

The sequence-specific DNA binding of NF-kappa B is reversibly regulated by the automodification reaction of poly (ADP-ribose) polymerase 1.

Recent studies suggest that the synthesis of protein-bound ADP-ribose polymers catalyzed by poly(ADP-ribose) polymerase-1 (PARP-1) regulates eucaryotic gene expression, including the NF-kappaB-dependent pathway. Here, we report the molecular mechanism by which PARP-1 activates the sequence-specific binding of NF-kappaB to its oligodeoxynucleotide. We co-incubated pure recombinant human PARP-1 and the p50 subunit of NF-kappaB (NF-kappaB-p50) in the presence or absence of betaNAD+ in vitro. Electrophoretic mobility shift assays showed that, when PARP-1 was present, NF-kappaB-p50 DNA binding was dependent on the presence of betaNAD+. DNA binding by NF-kappaB-p50 was not efficient in the absence of betaNAD+. In fact, the binding was not efficient in the presence of 3-aminobenzamide (3-AB) either. Thus, we conclude that NF-kappaB-p50 DNA binding is protein-poly(ADP-ribosyl)ation dependent. Co-immunoprecipitation and immunoblot analysis revealed that PARP-1 physically interacts with NF-kappaB-p50 with high specificity in the absence of betaNAD+. Because NF-kB-p50 was not an efficient covalent target for poly(ADP-ribosyl)ation, our results are consistent with the conclusion that the auto-poly(ADP-ribosyl)ation reaction catalyzed by PARP-1 facilitates the binding of NF-kappaB-p50 to its DNA by inhibiting the specific protein.protein interactions between NF-kappaB-p50 and PARP-1. We also report the activation of NF-kappaB DNA binding by the automodification reaction of PARP-1 in cultured HeLa cells following exposure to H(2)O(2). In these experiments, preincubation of HeLa cells with 3-AB, prior to oxidative damage, strongly inhibited NF-kappaB activation in vivo as well.

Regulation of p53 sequence-specific DNA-binding by covalent poly(ADP-ribosyl)ation.

We have characterized the covalent poly(ADP-ribosyl)ation of p53 using an in vitro reconstituted system. We used recombinant wild type p53, recombinant poly(ADP-ribose) polymerase-1 (PARP-1) (EC ), and betaNAD(+). Our results show that the covalent poly(ADP-ribosyl)ation of p53 is a time-dependent protein-poly(ADP-ribosyl)ation reaction and that the addition of this tumor suppressor protein to a PARP-1 automodification mixture stimulates total protein-poly(ADP-ribosyl)ation 3- to 4-fold. Electrophoretic analysis of the products synthesized indicated that short oligomers predominate early during hetero-poly(ADP-ribosyl)ation, whereas longer ADP-ribose chains are synthesized at later times of incubation. A more drastic effect in the complexity of the ADP-ribose chains generated was observed when the betaNAD(+) concentration was varied. As expected, increasing the betaNAD(+) concentration from low nanomolar to high micromolar levels resulted in the slower electrophoretic migration of the p53-(ADP-ribose)(n) adducts. Increasing the concentration of p53 protein from low nanomolar (40 nm) to low micromolar (1.0 microm) yielded higher amounts of poly(ADP-ribosyl)ated p53 as well. Thus, the reaction was acceptor protein concentration-dependent. The hetero-poly(ADP-ribosyl)ation of p53 also showed that high concentrations of p53 specifically stimulated the automodification reaction of PARP-1. The covalent modification of p53 resulted in the inhibition of the binding ability of this transcription factor to its DNA consensus sequence as judged by electrophoretic mobility shift assays. In fact, controls carried out with calf thymus DNA, betaNAD(+), PARP-1, and automodified PARP-1 confirmed our conclusion that the covalent poly(ADP-ribosyl)ation of p53 results in the transcriptional inactivation of this tumor suppressor protein.

Comparative characterisation of poly(ADP-ribose) polymerase-1 from two mammalian species with different life span.

DNA damage induced in higher eukaryotes by alkylating agents, oxidants or ionising radiation triggers the synthesis of protein-conjugated poly(ADP-ribose) catalysed by poly(ADP-ribose) polymerase-1 (PARP-1). Previously, cellular poly(ADP-ribosyl)ation capacity has been shown to correlate positively with the life span of mammalian species [Proc. Natl. Acad. Sci. USA 89 (1992) 11,759-11,763]. Here, we have tested whether this correlation results from differences in kinetic parameters of the enzymatic activity of PARP-1. We therefore compared recombinant enzymes, expressed in a baculovirus system, from rat and man as two mammalian species with extremely divergent life span. In standard activity assays performed in the presence of histones as poly(ADP-ribose) acceptors both enzymes showed saturation kinetics with [NAD(+)]. The kinetic parameters (k(cat), k(m) and k(cat)/k(m)) of the two enzymes were not significantly different. However, in assays assessing the auto-poly(ADP-ribosyl)ation reaction, both enzymes displayed second-order kinetics with respect to [PARP-1], and up to two-fold higher specific activity was observed for human versus rat PARP-1. We conclude that the correlation of poly(ADP-ribosyl)ation capacity with life span is not reflected in the kinetic parameters, but that subtle differences in primary structure of PARP-1 from mammalian species of different longevity may control the extent of the automodification reaction.

Expression of c-jun and c-fos in apoptotic cells after DNA damage.

Apoptosis was induced in HeLa cells by exposure to 50 microM N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) for various time intervals (up to 120 min). Apoptotic death was confirmed by the microscopic observation of cell blebbing, cell granulation, and cell aggregation. Cells also showed loss of phospholipid symmetry as judged by immunofluorescent microscopy with fluorescently labeled phosphatidyl serine-specific annexin V. In addition, staining of cells with ethidium bromide showed the presence of genomic DNA apoptotic bodies. The protein expression levels of c-jun and c-fos increased in DNA-damaged HeLa cells after MNNG treatment in a time-dependent fashion. Although the levels of c-fos increased rapidly during the first 30 min and remained high for 2 hr, the increase in c-jun expression was more gradual and slower (60-120 min) after MNNG treatment. These results are consistent with the conclusion that c-fos is important in the initial stages (commitment phase) of apoptosis and c-jun is involved in the late stages (execution phase) of apoptosis induced with alkylating agents.

Metabolic changes in the poly(ADP-ribosyl)ation pathway of differentiating rat germinal cells.

Endogenous levels of poly(ADP-ribose) and betaNAD+ have been determined in rat male germinal cells at different stages of differentiation. The levels of both metabolites decreased progressively from primary spermatocytes to secondary spermatocytes and especially in spermatids. We have also determined the size and complexity of the ADP-ribose polymers synthesized in permeabilized germ cells. Polymers of different chain length and complexity were observed in cells incubated with different concentrations of [32P]betaNAD+; short polymers characterized primary spermatocytes incubated with low betaNAD+ concentration. In all cell fractions, polymers of over 20 residues in size were observed at high betaNAD+ levels. Long polymers were associated with the sulfuric acid-insoluble proteins (nonhistone proteins such as PARP itself). By contrast, oligomers of 20 ADP-ribose units or less were found in the sulfuric acid-soluble proteins (histone proteins). We have also identified the main ADP-ribose protein acceptors formed in each cell type. In all cells examined, PARP appears to be extensively automodified. However, by far, the H1t variant of histone H1 appeared to be the preferred ADP-ribose target among the acid-soluble proteins separated by reverse-phase HPLC. Therefore, we conclude that an active protein-poly(ADP-ribosyl)ation system is concentrated in primary spermatocytes, based on a high level of PARP automodification accompanied by the preferential heteromodification of the histone H1 variant specifically expressed in the cells undergoing the pachytene phase of the meiotic division.

Chain length analysis of ADP-ribose polymers generated by poly(ADP-ribose) polymerase (PARP) as a function of beta-NAD+ and enzyme concentrations.

Bireactant autopoly(ADP-ribosyl)ation of poly(ADP-ribose) polymerase (PARP) (EC 2.4.2.30) was carried out by using either increasing concentrations of beta-NAD+ (donor substrate) at a fixed protein concentration or increasing concentrations of PARP (acceptor substrate) at a fixed beta-NAD+ concentration. The [32P]ADP-ribose polymers synthesized were chemically detached from PARP by alkaline hydrolysis of the monoester bond between the carboxylate moiety of Glu and the polymer. Nucleic acid-like polymers were then analyzed by high-resolution polyacrylamide gel electrophoresis and autoradiography. The ADP-ribose chain lengths observed displayed substrate concentration-dependent elongation from 0.2 microM to 2 mM beta-NAD+. Similar results were observed at fixed concentrations of 4.5, 9, 18, 27, and 36 nM PARP. Therefore, we conclude that the concentration of the ADP-ribose donor substrate determines the average chain length of the polymer synthesized. In contrast, the polymer size was unaltered when the concentration of PARP was varied from 4.5 to 18 nM at a fixed beta-NAD+ concentration. However, when PARP concentrations > 18 nM were used, the total amount of monomeric ADP-ribose produced was noticeably less. Therefore, we conclude that high concentrations of PARP lead to acceptor substrate inhibition at the level of the ADP-ribose chain initiation reaction.

Selective loss of poly(ADP-ribose) and the 85-kDa fragment of poly(ADP-ribose) polymerase in nucleoli during alkylation-induced apoptosis of HeLa cells.

Alkylation treatment of HeLa cells results in the rapid induction of apoptosis as revealed by DNA laddering and cleavage of poly(ADP-ribose) polymerase (PARP) into the 29-and 85-kDa fragments (Kumari S. R., Mendoza-Alvarez, H. & Alvarez-Gonzalez, R. (1998) Cancer Res. 58, 5075-5078). Here, we performed a time-course analysis of (i) poly(ADP-ribose) synthesis and degradation as well as (ii) the subnuclear localization of PARP and its fragments by using confocal laser scanning immunofluorescence microscopy. PARP was activated within 15 min post-treatment, as revealed by nuclear immunostaining with antibody 10H (recognizing poly(ADP-ribose)). This was followed by a late, time-dependent, progressive decline of 10H signals that coincide with the time of PARP cleavage. Strikingly, nucleolar immunostaining with antibodies 10H and C-II-10 (recognizing the 85-kDa PARP fragment) was lost by 15 min post-treatment, whereas F-I-23 signals (recognizing the 29-kDa fragment) persisted. We hypothesize that the 85-kDa PARP fragment is translocated, along with covalently bound poly(ADP-ribose), from nucleoli to the nucleoplasm, whereas the 29-kDa fragment is retained, because it binds to DNA strand breaks. Our data (i) provide a link between the known time-dependent bifunctional role of PARP in apoptosis and the subcellular localization of PARP fragments and also (ii) add to the evidence for early proteolytic changes in nucleoli during apoptosis.

Measurement of poly(ADP-ribose) glycohydrolase activity by high resolution polyacrylamide gel electrophoresis: specific inhibition by histones and nuclear matrix proteins.

We have developed a novel enzyme assay that allows the simultaneous determination of noncovalent interactions of poly(ADP-ribose) with nuclear proteins as well as poly(ADP-ribose) glycohydrolase (PARG) activity by high resolution polyacrylamide gel electrophoresis. ADP-ribose chains between 2 and 70 residues in size were enzymatically synthesized with pure poly(ADP-ribose) polymerase (PARP) and were purified by affinity chromatography on a boronate resin following alkaline release from protein. This preparation of polymers of ADP-ribose was used as the enzyme substrate for purified PARG. We also obtained the nuclear matrix fraction from rat liver nuclei and measured the enzyme activity of purified PARG in the presence or absence of either histone proteins or nuclear matrix proteins. Both resulted in a marked inhibition of PARG activity as determined by the decrease in the formation of monomeric ADP-ribose. The inhibition of PARG was presumably due to the non-covalent interactions of these proteins with free ADP-ribose polymers. Thus, the presence of histone and nuclear matrix proteins should be taken into consideration when measuring PARG activity.

Regulatory mechanisms of poly(ADP-ribose) polymerase.

Here, we describe the latest developments on the mechanistic characterization of poly(ADP-ribose) polymerase (PARP) [EC 2.4.2.30], a DNA-dependent enzyme that catalyzes the synthesis of protein-bound ADP-ribose polymers in eucaryotic chromatin. A detailed kinetic analysis of the automodification reaction of PARP in the presence of nicked dsDNA indicates that protein-poly(ADP-ribosyl)ation probably occurs via a sequential mechanism since enzyme-bound ADP-ribose chains are not reaction intermediates. The multiple enzymatic activities catalyzed by PARP (initiation, elongation, branching and self-modification) are the subject of a very complex regulatory mechanism that may involve allosterism. For instance, while the NAD+ concentration determines the average ADP-ribose polymer size (polymerization reaction), the frequency of DNA strand breaks determines the total number of ADP-ribose chains synthesized (initiation reaction). A general discussion of some of the mechanisms that regulate these multiple catalytic activities of PARP is presented below.

Biochemical characterization of mono(ADP-ribosyl)ated poly(ADP-ribose) polymerase.

Here, we report the biochemical characterization of mono(ADP-ribosyl)ated poly(ADP-ribose) polymerase (PARP) (EC 2.4.2. 30). PARP was effectively mono(ADP-ribosyl)ated both in solution and via an activity gel assay following SDS-PAGE with 20 microM or lower concentrations of [32P]-3'-dNAD+ as the ADP-ribosylation substrate. We observed the exclusive formation of [32P]-3'-dAMP and no polymeric ADP-ribose molecules following chemical release of enzyme-bound ADP-ribose units and high-resolution polyacrylamide gel electrophoresis. The reaction in solution (i) was time-dependent, (ii) was activated by nicked dsDNA, and (iii) increased with the square of the enzyme concentration. Stoichiometric analysis of the reaction indicated that up to four amino acid residues per mole of enzyme were covalently modified with single units of 3'-dADP-ribose. Peptide mapping of mono(3'-dADP-ribosyl)ated-PARP following limited proteolysis with either papain or alpha-chymotrypsin indicated that the amino acid acceptor sites for chain initiation with 3'-dNAD+ as a substrate are localized within an internal 22 kDa automodification domain. Neither the amino-terminal DNA-binding domain nor the carboxy-terminal catalytic fragment became ADP-ribosylated with [32P]-3'-dNAD+ as a substrate. Finally, the apparent rate constant of mono(ADP-ribosyl)ation in solution indicates that the initiation reaction catalyzed by PARP proceeds 232-fold more slowly than ADP-ribose polymerization.

Functional interactions of p53 with poly(ADP-ribose) polymerase (PARP) during apoptosis following DNA damage: covalent poly(ADP-ribosyl)ation of p53 by exogenous PARP and noncovalent binding of p53 to the M(r) 85,000 proteolytic fragment.

We have examined the domain-specific interactions between p53 and poly(ADP-ribose)polymerase (PARP) (E.C. 2.4.2.30) in apoptotic HeLa cells. Apoptosis was induced by exposing cells to 50 microM N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) for increasing lengths of time and was confirmed by: (a) oligonucleosomal fragmentation of chromatin; (b) increase in p53 levels; and (c) degradation of PARP into the characteristic M(r) 85,000 (COOH-terminal catalytic domain) and M(r) 29,000 (DNA-binding domain) peptide fragments. We also immunodetected p53 in immunoprecipitates obtained with a PARP-specific antibody. However, intact PARP coimmunoprecipitated with a p53-specific antibody during the initial 30 min of MNNG treatment. After 60 min, only the COOH-terminal fragment coimmunoprecipitated with p53, indicating that PARP noncovalently binds p53 via its M(r) 85,000 catalytic domain. Therefore, we next examined p53 as a covalent target for poly(ADP-ribosyl)ation. Although p53 was not endogenously poly (ADP-ribosyl)ated in situ, incubation of cell extracts with full-length PARP from calf thymus and [32P]beta NAD+ resulted in its time-dependent poly(ADP-ribosyl)ation. In summary, our results are consistent with the conclusion that PARP and p53 are activated with nonoverlapping kinetics during apoptosis.

TFIIF, a basal eukaryotic transcription factor, is a substrate for poly(ADP-ribosyl)ation.

We have examined the susceptibility of some of the basal eukaryotic transcription factors as covalent targets for poly(ADP-ribosyl)ation. Human recombinant TATA-binding protein, transcription factor (TF)IIB and TFIIF (made up of the 30 and 74 kDa RNA polymerase II-associated proteins RAP30 and RAP74) were incubated with calf thymus poly(ADP-ribose) polymerase and [32P]NAD+ at 37 degrees C. On lithium dodecyl sulphate/PAGE and autoradiography, two bands of radioactivity, coincident with RAP30 and RAP74, were observed. No radioactivity co-migrated with TATA-binding protein or TFIIB. The phenomenon was dependent on the presence of nicked DNA, which is essential for poly(ADP-ribose) polymerase activity. Covalent modification of TFIIF increased with time of incubation, with increasing TFIIF concentration and with increasing NAD+ concentration. High-resolution PAGE confirmed that the radioactive species associated with RAP30 and RAP74 were ADP-ribose polymers. From these observations, we conclude that both TFIIF subunits are highly specific substrates for covalent poly(ADP-ribosyl)ation.

Purification and partial characterization of a chromate reductase from Bacillus.

A soluble NADH-dependent enzyme capable of reducing hexavalent chromium [Cr(VI)] to the trivalent form [Cr(III)] was purified from chromate-resistant Bacillus QC1-2. An enriched single protein band of 24 kDa was observed by SDS-PAGE following HPLC ion-exchange and size-exclusion procedures. In the latter step, the chromate reductase showed a molecular mass of 44 kDa, which suggested that the enzyme consists of two subunits of about 24 kDa. Purified chromate reductase displayed optimal activity at a temperature and pH of 37 degrees C and 7.0, respectively. The enzyme showed a Km of 0.35 mM for chromate and a Vmax of 50 nmol Cr(VI) reduced per minute per mg protein.

Dissection of ADP-ribose polymer synthesis into individual steps of initiation, elongation, and branching.

We have studied the automodification reaction of poly(ADP-ribose)polymerase (PARP) (EC 2.4.2.30). The individual reactions of initiation, elongation, and branching catalyzed by this enzyme have been dissected out by manipulating the concentration of beta NAD, the ADP-ribosylation substrate. While PARP-mono(ADP-ribose) conjugates were the predominant products of automodification at 200 nM NAD (initiation), highly branched and complex polymers were synthesized at 200 microM NAD (polymerization). Initial rates of automodification increased with second order kinetics as a function of the enzyme concentration at both 200 nM and 200 microM NAD. These results are consistent with the conclusion that two molecules of PARP are required for ADP-ribose polymer synthesis during enzyme automodification. Thus, the auto-poly(ADP-ribosyl)ation reaction of PARP is intermolecular. In agreement with this notion, we observed that initial rates of the initiation reaction with 3'-deoxyNAD as a substrate also increased with the square of the enzyme concentration. In addition, the auto-poly(ADP-ribosyl)ation reaction of PARP increased with second order kinetics as a function of the NAD concentration at nanomolar levels (0.2-106 microM). Therefore, the dimeric structure of PARP also requires two molecules of bound NAD for efficient ADP-ribose polymerization.

DeoxyNAD and deoxyADP-ribosylation of proteins.

Recently, two deoxyribose analogs of beta NAD+ (2'-deoxy and 3'-deoxyNAD+) have been synthesized and purified in this laboratory. Whereas 2'-deoxyNAD+ was an efficient substrate for arg-specific mon(ADP-ribosyl) transferases, it was not a substrate for poly(ADP-ribose) polymerase (PARP). Instead, it was a non-competitive inhibitor of beta NAD+ in the ADP-ribose polymerization reaction catalyzed by PARP. Thus, 2'-deoxyNAD+ has been utilized to distinguish between mono(ADP-ribose) and poly(ADP-ribose) acceptor proteins. 2'-deoxyNAD+ has also been used to characterize the arg-specific mono(2'-deoxyADP-ribosyl)ation reaction of PARP with cholera toxin or avian mono(ADP-ribosyl)transferase. By contrast, 3'-deoxyNAD+ can effectively be utilized as a substrate by PARP. However, while the estimated Km and Kcat of polymerization with 3'-deoxyNAD+ were 20 microM and 0.11 moles/sec, the Km and Kcat with beta NAD+ as a substrate were 59 microM and 1.29 moles/sec, respectively. Determination of the average size of 3'-deoxyADP-ribose polymers indicated that chains no larger than four residues are synthesized with this substrate. Thus, the utilization of 3'-deoxyNAD+ has facilitated the electrophoretic identification of poly(ADP-ribose) acceptor proteins in mammalian chromatin.

Enzymology of ADP-ribose polymer synthesis.

In this minireview, we summarize recent advances on the enzymology of ADP-ribose polymer synthesis. First, a short discussion of the primary structure and cloning of poly(ADP-ribose) polymerase (PARP) [EC 2.4.2.30], the enzyme that catalyzes the synthesis of poly(ADP-ribose), is presented. A catalytic distinction between the multiple enzymatic activities of PARP is established. The direction of ADP-ribose chain growth as well as the molecular mechanism of the automodification reaction catalyzed by PARP are described. Current approaches to dissect ADP-ribose polymer synthesis into individual reactions of initiation, elongation and branching, as well as a partial mechanistic characterization of the ADP-ribose elongation reaction at the chemical level are also presented. Finally, recent developments in the catalytic characterization of PARP by site-directed mutagenesis are also briefly summarized.

Poly(ADP-ribose) polymerase is a catalytic dimer and the automodification reaction is intermolecular.

We have determined the molecular mechanism of the automodification reaction of poly(ADP-ribose) polymerase (PARP) (EC 2.4.2.30). While PARP-mono(ADP-ribose) conjugates were the predominant products of automodification at 200 nM NAD, enzyme-bound branched polymers were preferentially synthesized at 200 microM NAD. Thus, the initiation, elongation, and branching reactions catalyzed by PARP appear to be [NAD]-dependent. Initial rates of automodification increased with second order kinetics as a function of [PARP] at both 200 nM and 200 microM NAD. Therefore, 2 molecules of PARP, i.e. a catalytic dimer, are required for the auto-mono(ADP-ribosyl)ation and the auto-poly(ADP-ribosyl)ation reactions. Initial rates of automodification also increased with second order kinetics at low NAD concentrations. Therefore, the catalytic dimer also requires 2 molecules of NAD. These results are consistent with the conclusion that the automodification reaction of PARP is intermolecular and that the 2 monomeric units of PARP may simultaneously function as catalyst and acceptor molecules in the automodification reaction. Confirmatory evidence for the catalytic role of protein-protein interactions in the automodification reaction was manifested by a marked inhibition of auto-poly(ADP-ribosyl)ation at 40 nM or higher [PARP].