RESUMEN
Poly(ADP-ribose)polymerases (PARP-1 and -2) are activated by DNA strand breaks to synthesize protein-bound ADP-ribose polymers from NAD+. The two enzymes are overexpressed in rat spermatocytes and are likely to play a role in meiosis. Indeed parp-2-/- mice, but not parp-1 knockouts, show hypofertility. Aside, PARP-1 and PARP-2 are both involved in DNA damage repair and signalling, but their relative contributions to such processes remain as yet unknown, largely because of the lack of PARP isoform-specific inhibitors that has precluded in vivo studies. Here, we used permeabilized rat primary spermatocytes or isolated spermatocyte nuclei and radiolabelled NAD+ to investigate potential isoform-specific effects on basic features of the poly(ADP-ribosyl)ation reaction, including size of ADP-ribose polymers at different NAD+ concentrations, extent of auto- versus etheromodification, and modulation of such reactions by the PARP inhibitor, PJ34. We found that PARP-1 automodification prevailed over PARP-2 modification. In addition, over 50% of cellular poly(ADP-ribose) was covalently bound to histones H1 and H2. The inhibitory effect of PJ34 appeared to be targeted mainly to the elongation step of the reaction. We propose that a different propensity of PARP-1 and PARP-2 to undergo automodification and/or catalyze etheromodification, both in terms of number of enzyme molecules being involved and amount of bound poly(ADP-ribose), may underlie distinct roles in the regulation of spermatocyte functions.
Asunto(s)
Poli Adenosina Difosfato Ribosa/metabolismo , Poli(ADP-Ribosa) Polimerasas/metabolismo , Espermatocitos/metabolismo , Animales , Núcleo Celular/metabolismo , Electroforesis en Gel Bidimensional , Electroforesis en Gel de Poliacrilamida , Histonas/metabolismo , Masculino , Ratas , Ratas WistarRESUMEN
Using a poly(ADP-ribose) binding assay on protein blots we examined the ability of rat testis histone H1 variants to establish non-covalent interactions with the polymer. All the H1 variants bound ADP-ribose polymers; the binding was salt resistant and highly specific, occurring even in the presence of a large excess of competitor DNA. A comparison among the H1 variants showed that H1t has the highest affinity for poly(ADP-ribose). Long and branched poly(ADP-ribose) molecules were found to be preferentially involved in the interaction with the histone variants. The results further corroborate the concept that non-covalent interactions of poly(ADP-ribose) with target proteins may constitute an important mechanism to modulate chromatin structure.
Asunto(s)
Histonas/química , Poli Adenosina Difosfato Ribosa/química , ADN/química , Electroforesis en Gel de Poliacrilamida , Endopeptidasa K , Histonas/clasificación , Poli Adenosina Difosfato Ribosa/síntesis químicaRESUMEN
ADP-ribosylation of a specific basic protein has been investigated in isolated intact bull testis nuclei incubated with NAD+. The electrophoretic mobility, molecular weight and amino-acid composition of the purified bull testis specific protein are similar to those of rat testis protein. About 1-5% of the total radioactivity incorporated in the 20% acid-insoluble fraction was associated with testis protein and was identified as ADP-ribose.
Asunto(s)
Adenosina Difosfato Ribosa/metabolismo , Proteínas/metabolismo , Testículo/análisis , Aminoácidos/análisis , Animales , Bovinos , Núcleo Celular/análisis , Masculino , Procesamiento Proteico-PostraduccionalRESUMEN
Poly(ADPribosylation) of nuclear proteins has been investigated in the nuclei from growing oviducts of intact and estrogen-treated spayed females of the lizard Podarcis s. sicula Raf. Isolated nuclei were incubated with [14C] NAD and nuclear proteins extracted in 0.2 m H2SO4. Labeled acid-soluble proteins were analysed by reverse-phase high-performance liquid chromatography (HPLC) and acetic acid-urea gel electrophoresis. The results reported here indicate that the ADPribosylation reaction is involved in modifying besides histone H2b, tissue specific proteins (SNPs and LMG-O). Moreover, comparable results have been obtained from nuclei prepared from the fully active oviduct of intact animals and spayed lizards stimulated with 17 beta-estradiol. It is concluded that the poly-(ADPribosylation) of hormone-induced proteins might play a role in the differentiation of the lizard oviduct.
Asunto(s)
Estrógenos/farmacología , Proteínas Nucleares/metabolismo , Oviductos/metabolismo , Poli Adenosina Difosfato Ribosa/metabolismo , Ribonucleoproteínas/metabolismo , Aminoácidos/análisis , Animales , Cromatografía Líquida de Alta Presión , Electroforesis , Técnicas In Vitro , Lagartos , Ribonucleoproteínas Nucleares PequeñasRESUMEN
Nanoparticles made of metal-organic frameworks (nanoMOFs) attract a growing interest in gas storage, separation, catalysis, sensing and more recently, biomedicine. Achieving stable, versatile coatings on highly porous nanoMOFs without altering their ability to adsorb molecules of interest represents today a major challenge. Here we bring the proof of concept that the outer surface of porous nanoMOFs can be specifically functionalized in a rapid, biofriendly and non-covalent manner, leading to stable and versatile coatings. Cyclodextrin molecules bearing strong iron complexing groups (phosphates) were firmly anchored to the nanoMOFs' surface, within only a few minutes, simply by incubation with aqueous nanoMOF suspensions. The coating procedure did not affect the nanoMOF porosity, crystallinity, adsorption and release abilities. The stable cyclodextrin-based coating was further functionalized with: i) targeting moieties to increase the nanoMOF interaction with specific receptors and ii) poly(ethylene glycol) chains to escape the immune system. These results pave the way towards the design of surface-engineered nanoMOFs of interest for applications in the field of targeted drug delivery, catalysis, separation and sensing.
Asunto(s)
Materiales Biocompatibles Revestidos/química , Ensayo de Materiales , Nanopartículas/química , Animales , Línea Celular , Ratones , PorosidadRESUMEN
Down regulation of poly(ADP-ribose)polymerase (ADPRP) activity was observed in mouse LW-cells after treatment with 2'-5'oligoadenylates or with fibroblast interferon and poly(rI) poly(rC). The poly(rI) poly(rC)-induced inhibition of the enzymatic activity correlates with the observed increase of endogenous 2',5'-oligoadenylate cores which were reported to be potent inhibitors of ADPRP in vitro.
Asunto(s)
Nucleótidos de Adenina/farmacología , Células L/enzimología , Oligorribonucleótidos/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Nucleótidos de Adenina/análisis , Animales , Células Cultivadas , Cromatografía Líquida de Alta Presión , Interferones/farmacología , Ratones , Oligorribonucleótidos/análisisRESUMEN
In the present paper, we investigated the relationship between the growth inhibitory effects of recombinant interferon-alpha2b (rIFN-alpha2b) and poly (ADPR) polymerase-1 (PARP-1) activity in the human squamous KB cancer cell line. Growth inhibition of the KB cells mediated by 1000 IU/ml of rIFN-alpha2b was accompanied by a transient rise in PARP-1 specific activity 24 h after rIFN-alpha2b treatment, confirmed by both the increase of intracellular poly (ADP-ribose) content and the PARP-1 auto-modification level. At longer times of incubation, the onset of apoptosis accompanied KB cell growth inhibition, as demonstrated by both flow cytometry and western-blotting analysis showing an 89 kDa apoptotic fragment of PARP-1. Moreover, pretreatment of the cells with the PARP-1 inhibitor, 3-aminobenzamide (3-ABA), at non-cytotoxic concentrations (1 mM), reduced the cell-growth inhibition, cell-cycle perturbation and apoptosis caused by rIFN-alpha2b. Taken together, these results strongly suggest that PARP-1 may be directly involved in the effects of rIFN-alpha2b in the KB cancer cell line.
Asunto(s)
Antineoplásicos/uso terapéutico , Carcinoma de Células Escamosas/tratamiento farmacológico , Interferón-alfa/uso terapéutico , Poli(ADP-Ribosa) Polimerasas/metabolismo , Western Blotting , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patología , Ciclo Celular , División Celular , Activación Enzimática , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Humanos , Interferón alfa-2 , Células KB , Proteínas Recombinantes , Células Tumorales CultivadasRESUMEN
The molecular mechanisms whereby poly(ADP-ribosyl)ation primes chromatin proteins for an active role in DNA excision repair are not understood. The prevalent view is that the covalent linkage of ADP-ribose polymers is essential for the modification of target protein function. By contrast, we have focused on the possibility that ADP-ribose polymers interact non-covalently with nuclear proteins and thereby modulate their function. The results show that ADP-ribose polymers engage in highly specific and strong non-covalent interactions with a small number of nuclear proteins, predominantly histones, and among these only with specific polypeptide domains. The binding affinities were largely determined by two factors, ie the polymer sizes and the presence of branches. This provides an explanation for the target specificity of the histone shuttle mechanism that was previously reported by our laboratory. Interestingly, the polymer molecules being most effective in protein targeting in vitro, are strictly regulated in mammalian cells during DNA repair in vivo.
Asunto(s)
Proteínas Nucleares/química , Poli Adenosina Difosfato Ribosa/química , Animales , Línea Celular , Reparación del ADN , Histonas/química , Humanos , SolucionesRESUMEN
In mammalian cells, the incision step of DNA excision repair triggers a dramatic metabolic response in chromatin. The reaction starts with the binding of a zinc-finger protein, i.e. poly(ADP-ribose)polymerase to DNA nicks, activation of four resident catalytic activities leading to poly(ADP-ribose) synthesis, conversion of the polymerase into a protein modified with up to 28 variably sized ADP-ribose polymers, and rapid degradation of polymerase-bound polymers by poly(ADP-ribose)glycohydrolase. This automodification cycle catalyzes a transient and reversible dissociation of histones from DNA. Shuttling of histones on the DNA allows selected other proteins, such as DNA helicase A and topoisomerase I, to gain access to DNA. Histone shuttling in vitro mimics nucleosomal unfolding/refolding in vivo that accompanies the postincisional steps of DNA excision repair. Suppression of the automodification cycle in mammalian cells prevents nucleosomal unfolding and nucleotide excision repair.
Asunto(s)
Histonas/metabolismo , Poli(ADP-Ribosa) Polimerasas/metabolismo , Animales , ADN/metabolismo , Reparación del ADN/fisiología , Glicósido Hidrolasas/metabolismo , Humanos , Modelos GenéticosRESUMEN
PURPOSE: To characterize the response to X-irradiation of the poly ADP-ribosylation system in two closely related murine lymphoma sublines, L5178Y-R (LY-R) and L5178Y-S (LY-S), with differential sensitivity to various DNA damaging agents (UV-C and ionizing radiation, hydrogen peroxide). MATERIALS AND METHODS: LY cells were X-irradiated (2 Gy). NAD+ was determined in cell extracts by high-pressure liquid chromatography. ADP-ribose polymers were purified and analysed by densitometry after polyacrylamide gel electrophoresis. Nuclear matrix proteins were separated by SDS-polyacrylamide gel electrophoresis and processed for ADP-ribose polymer blots to estimate their ability to bind poly(ADP-ribose). RESULTS: In the radiosensitive LY-S cells, the constitutive levels of ADP-ribose polymers were twofold higher than in radioresistant LY-R cells, but unresponsive to a challenge with 2 Gy X-rays. The concentrations of NAD+ - the substrate for poly(ADP-ribose) synthesis - were identical in the two cell lines. X-rays (2 Gy) depleted NAD+ only in LY-S cells. These cells also produced shorter poly(ADP-ribose) molecules as compared with LY-R cells. Nuclear matrix preparations of LY-S cells exhibited lower poly(ADP-ribose)-binding capacity than those of LY-R cells. CONCLUSION: The results demonstrate disturbances in the poly ADP-ribosylation response of the radiosensitive LY-S cells and reduced poly(ADP-ribose)-binding affinity of the nuclear matrix of these cells.
Asunto(s)
Leucemia L5178/metabolismo , Leucemia L5178/radioterapia , Poli Adenosina Difosfato Ribosa/metabolismo , Poli Adenosina Difosfato Ribosa/efectos de la radiación , Animales , Daño del ADN , Reparación del ADN , Peróxido de Hidrógeno/farmacología , Ratones , NAD/metabolismo , NAD/efectos de la radiación , Matriz Nuclear/efectos de los fármacos , Matriz Nuclear/metabolismo , Matriz Nuclear/efectos de la radiación , Tolerancia a Radiación , Transducción de Señal/efectos de la radiación , Células Tumorales Cultivadas , Rayos UltravioletaRESUMEN
Poly(ADP-ribose) (PAR) has been identified as a DNA damage-inducible cell death signal upstream of apoptosis-inducing factor (AIF). PAR causes the translocation of AIF from mitochondria to the nucleus and triggers cell death. In living cells, PAR molecules are subject to dynamic changes pending on internal and external stress factors. Using RNA interference (RNAi), we determined the roles of poly(ADP-ribose) polymerases-1 and -2 (PARP-1, PARP-2) and poly(ADP-ribose) glycohydrolase (PARG), the key enzymes configuring PAR molecules, in cell death induced by an alkylating agent. We found that PARP-1, but not PARP-2 and PARG, contributed to alkylation-induced cell death. Likewise, AIF translocation was only affected by PARP-1. PARP-1 seems to play a major role configuring PAR as a death signal involving AIF translocation regardless of the death pathway involved.
Asunto(s)
Muerte Celular/fisiología , Poli Adenosina Difosfato Ribosa/metabolismo , Poli(ADP-Ribosa) Polimerasas/metabolismo , Alquilación , Animales , Apoptosis/fisiología , Daño del ADN , Fibroblastos/enzimología , Células HeLa , Humanos , Cinética , Ratones , Poli(ADP-Ribosa) Polimerasas/genética , Reacción en Cadena de la Polimerasa , ARN Interferente Pequeño/genéticaRESUMEN
Protein modification by ADP-ribose polymers is a common regulatory mechanism in eukaryotic cells and is involved in several aspects of brain physiology and physiopathology, including neurotransmission, memory formation, neurotoxicity, ageing and age-associated diseases. Here we show age-related misregulation of poly(ADP-ribose) synthesis in rat cerebellum as revealed by: (i) reduced poly(ADP-ribose) polymerase-1 (PARP-1) activation in response to enzymatic DNA cleavage, (ii) altered protein poly(ADP-ribosyl)ation profiles in isolated nuclei, and (iii) cell type-specific loss of poly(ADP-ribosyl)ation capacity in granule cell layer and Purkinje cells in vivo. In particular, although PARP-1 could be detected in virtually all granule cells, only a fraction of them appeared to be actively engaged in poly(ADP-ribose) synthesis and this fraction was reduced in old rat cerebellum. NAD(+), quantified in tissue homogenates, was essentially the same in the cerebellum of young and old rats suggesting that in vivo factors other than PARP-1 content and/or NAD(+) levels may be responsible for the age-associated lowering of poly(ADP-ribose) synthesis. Moreover, PARP-1 expression was substantially down-regulated in Purkinje cells of senescent rats.
Asunto(s)
Envejecimiento/metabolismo , Encéfalo/enzimología , Poli Adenosina Difosfato Ribosa/metabolismo , Poli(ADP-Ribosa) Polimerasas/metabolismo , Animales , Western Blotting/métodos , Encéfalo/citología , Encéfalo/crecimiento & desarrollo , Química Encefálica/fisiología , Núcleo Celular/enzimología , Inmunohistoquímica/métodos , Masculino , NAD/metabolismo , Ratas , Ratas WistarRESUMEN
Poly(ADP-ribose) is a nuclear polynucleotide involved in the regulation of chromatin functions via covalent and/or noncovalent modification of nuclear proteins. Using a binding assay on protein blots, we searched for poly(ADP-ribose) binding proteins in nuclear matrices from testes of differently aged rats as well as from various adult rat tissues (brain, liver, spleen). We found that nuclear matrix proteins represent a significant subset of the nuclear proteins that can establish noncovalent interactions with poly(ADP-ribose). The profiles of poly(ADP-ribose) binding nuclear matrix proteins appeared to be tissue-specific and changed during postnatal development in the testis. The isolation and analysis of endogenous poly-(ADP-ribose) from rat testes showed that the ADP-ribose polymers that bind nuclear matrix proteins in vitro are also present under physiologic conditions in vivo. These results further substantiate the possibility that poly(ADP-ribose) may affect chromatin functions through noncovalent interaction with specific protein targets, including nuclear matrix components.
Asunto(s)
Proteínas Nucleares/metabolismo , Poli Adenosina Difosfato Ribosa/metabolismo , Animales , Antígenos Nucleares , Núcleo Celular/metabolismo , Masculino , Matriz Nuclear/metabolismo , Polímeros , Ratas , Ratas Wistar , Testículo/metabolismoRESUMEN
We have found that human cells respond to treatment with an alkylating carcinogen by producing a specific, highly conserved array of poly(ADP-ribose) molecules. Using in situ radiolabeling and boronate affinity chromatography, we have been able to isolate ADP-ribose polymers from living cells and to quantify individual molecular size classes on high resolution polyacrylamide gels. Despite carcinogen-induced changes in poly(ADP-ribose) turnover, the relative frequency of linear polymers was strictly conserved. However, the abundance of branched polymers with high affinity for histones increased 2.6-fold. These polymers were degraded more slowly than linear polymers. Our results indicate that the poly(ADP-ribose) molecules involved in the shuttling of histones on DNA in vitro also play a role in the repair of DNA damage in vivo.
Asunto(s)
Reparación del ADN , Poli Adenosina Difosfato Ribosa/biosíntesis , Alquilación , Línea Celular , Cromatografía de Afinidad , Daño del ADN , Electroforesis en Gel de Poliacrilamida , Humanos , Queratinocitos/metabolismo , Poli Adenosina Difosfato Ribosa/aislamiento & purificaciónRESUMEN
Recent evidence suggests that poly(ADP-ribose) may take part in DNA strand break signalling due to its ability to interact with and affect the function of specific target proteins. Using a poly(ADP-ribose) blot assay, we have found that several nuclear matrix proteins from human and murine cells bind ADP-ribose polymers with high affinity. The binding was observed regardless of the procedure used to isolate nuclear matrices, and it proved resistant to high salt concentrations. In murine lymphoma LY-cell cultures, the spontaneous appearance of radiosensitive LY-S sublines was associated with a loss of poly(ADP-ribose)-binding of several nuclear matrix proteins. Because of the importance of the nuclear matrix in DNA processing reactions, the targeting of matrix proteins could be an important aspect of DNA damage signalling via the poly ADP-ribosylation system.
Asunto(s)
Proteínas Nucleares/metabolismo , Poli Adenosina Difosfato Ribosa/metabolismo , Animales , Antígenos Nucleares , Línea Celular , Núcleo Celular/metabolismo , Humanos , Queratinocitos/citología , Queratinocitos/metabolismo , Ratones , Unión Proteica , Fracciones Subcelulares/metabolismo , Células Tumorales CultivadasRESUMEN
Poly(ADPR)polymerase activity and poly(ADP-ribosyl)ation of nuclear proteins have been investigated in ventral prostate nuclei of different aged rats (14, 28, 60, 180, 360 day old animals), by reverse-phase HPLC and acetic acid-urea polyacrylamide gel electrophoresis. The major ADP-ribose acceptor proteins were identified as histone H1 and H2b. It is concluded that concomitant with major changes to chromatin organization, poly(ADP-ribosyl)ation reaction is progressively inhibited during aging of rat ventral prostate. These results support the hypothesis that prostatic dysfunction in senescent animals is related to a failure of DNA repair mechanisms and deregulated template activity.
Asunto(s)
Envejecimiento , Proteínas Nucleares/metabolismo , Poli(ADP-Ribosa) Polimerasas/metabolismo , Próstata/enzimología , Aminoácidos/análisis , Animales , Núcleo Celular/enzimología , Cromatina/análisis , Cromatografía Líquida de Alta Presión , Reparación del ADN , Masculino , Poli(ADP-Ribosa) Polimerasas/genética , Próstata/fisiología , Ratas , Ratas EndogámicasRESUMEN
Culture and differentiation parameters of a human thyroid transformed cell line (HuT) were analyzed. Treatment with high concentrations of chemical agents namely dimethyl sulphoxide and retinoic acid, exerted a dramatic cytotoxic effect. The exposure of these cells to the lowest doses of retinoic acid as well as to 8 mM-16 mM 3-aminobenzamide a potent inhibitor of poly(ADPribose)polymerase, resulted in a delay of cell proliferation. Poly(ADPribose)polymerase activity was differently affected by retinoic acid (stimulation) and 3-aminobenzamide (inhibition).
Asunto(s)
Benzamidas/farmacología , Ciclo Celular/efectos de los fármacos , Dimetilsulfóxido/farmacología , Poli(ADP-Ribosa) Polimerasas/metabolismo , Tretinoina/farmacología , División Celular , Línea Celular Transformada , Supervivencia Celular/efectos de los fármacos , ADN/análisis , Femenino , Citometría de Flujo , Humanos , Microscopía Electrónica , Persona de Mediana Edad , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Virus 40 de los Simios/fisiología , Glándula Tiroides/citologíaRESUMEN
ADP-ribose polymers were isolated from living mammalian cells, separated by polyacrylamide gel electrophoresis, visualized in the gel with a novel silver staining agent, and quantified by computer-aided scanning densitometry. This method detects as little as approximately 40 fmol of ADP-ribose polymers of a particular size class and reduces the gel exposure times required for conventionally radiolabeled polymers from 2 months to about 1 h. The method also detects polymers with slow turnover which may be underestimated by techniques requiring metabolic radiolabeling of poly(ADP-ribose).
Asunto(s)
Adenosina Difosfato Ribosa/metabolismo , Tinción con Nitrato de Plata/métodos , Biopolímeros , Línea Celular , Glicósido Hidrolasas , Humanos , Microquímica , Estándares de Referencia , Sensibilidad y EspecificidadRESUMEN
DNA strand breaks are potential interaction sites for the nuclear enzyme poly(ADP-ribose) polymerase (PARP; E.C. 2.4.2.30) and the tumor suppressor protein p53. Both proteins bind and respond to DNA breaks and both play a role in DNA damage signaling. A temporary colocalization and complex formation between these proteins has been demonstrated in mammalian cells. Here we show that free and poly(ADP-ribose) polymerase-bound ADP-ribose polymers target three domains in p53 protein for strong noncovalent interactions. The polymer binding sites could be mapped to two amino acid sequences in the sequence-specific core DNA binding domain of p53 (amino acid positions 153-178 and 231-253) and another one in the oligomerization domain (amino acids 326-348). In mobility shift experiments, poly(ADP-ribose) effectively prevented and reversed p53 binding to the palindromic p53 consensus sequence. Additionally, poly(ADP-ribose) also interfered with the DNA single strand end binding of p53. The results suggest that ADP-ribose polymers could play a role in regulating the DNA binding properties of p53.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Poli Adenosina Difosfato Ribosa/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Animales , Sitios de Unión , Unión Competitiva , Secuencia de Consenso , ADN de Cadena Simple/metabolismo , Humanos , Ratones , Poli(ADP-Ribosa) Polimerasa-1 , Poli(ADP-Ribosa) Polimerasas , Unión Proteica , Proteínas/metabolismo , Relación Estructura-ActividadRESUMEN
Poly(ADPR)polymerase (PARP) is a chromatin-associated enzyme with a presumptive role in DNA repair during replication and recovery from strand breaks caused by genotoxic agents. It catalyses the attachment and elongation of ADPribose polymers (pADPR) to a variety of acceptor proteins (including PARP itself, and histones) and is particularly active in the testis where its expression varies according to the stage of germ cell differentiation. PARP degradation is also one of the classic indicators of apoptosis. In this investigation we have examined the effects of heat stress on the adult rat testis with respect to the concentration and activity of PARP, the nature of the pADRP nuclear acceptor proteins, the length of ADPR polymers and the activity of the ADPR depolymerizing enzyme, poly(ADPR)glycohydrolase (PARG). Our results show a significant reduction in the concentration and activity of PARP 4 and 8 days after artificial cryptorchidism, but no significant changes were observed in PARG activity or in pADPR length. Unexpectedly, the apoptotic degradation of PARP was not detected following heat stress. These results confirm that PARP gene expression is developmentally regulated during spermatogenesis and indicate that it is suppressed coincidentally with the loss of meiotic spermatocytes during artificial cryptorchidism.