ABSTRACT
We used a [(32)P] p53 sequence-specific oligodeoxynucleotide and Electrophoretic-Mobility-Shift-Assays to monitor p53 DNA sequence-specific binding with p53-R267W, a nonbinding point mutant; and p53-Δ30, a deletion-mutant which lacks the carboxy-terminus that recognizes DNA-strand-breaks. Recombinant p53 and poly(ADP-ribose)polymerase-1 (PARP-1) were incubated with labeled ßNAD(+) with/without DNA. The poly(ADP-ribosyl)ation of each protein increased with incubation-time and ßNAD(+) and p53 concentration(s). Since p53-Δ30 was efficiently labeled, poly(ADP-ribosyl)ation target site(s) of wt-p53 must reside outside its carboxy-terminal-domain. The poly(ADP-ribosyl)ation of p53-Δ30 did not diminish its DNA binding; Instead, it enhanced DNA-sequence-specific-binding. Therefore, we conclude that DNA-sequence-specific-binding and DNA-nick-sensing of mutant-p53 are differentially regulated by poly(ADP-ribosyl)ation.
Subject(s)
DNA Breaks, Single-Stranded , DNA/metabolism , Poly Adenosine Diphosphate Ribose/metabolism , Transcriptional Activation , Tumor Suppressor Protein p53/metabolism , Binding Sites , Electrophoretic Mobility Shift Assay , Humans , NAD/metabolism , Poly (ADP-Ribose) Polymerase-1 , Poly(ADP-ribose) Polymerases/metabolism , Protein Processing, Post-Translational , Recombinant Proteins/metabolism , Sequence Deletion , Time Factors , Tumor Suppressor Protein p53/genetics , Up-RegulationABSTRACT
The integrity of the genome in higher eukaryotes, as well as the modulation of its complex structure and functions, is exquisitely regulated. This genomic regulation occurs as a function of time in a very sophisticated and elaborate biological process called cell cycle progression, resulting in cell division, and is also controlled by a highly coordinated and intricate network of molecular signaling pathways, which in turn orchestrate very specific macromolecular interactions among nuclear proteins and DNA at the biochemical level. Among the latter, a prominent enzymatic cycle that is involved in maintaining the integrity of mammalian chromosomes is covalent protein-poly[adenosine diphosphate (ADP)-ribosyl]ation. The importance of this posttranslational modification is illustrated by the close cooperation between two "guardian angels" of the genome, one constitutive and one inducible protein, namely poly(ADP-ribose) polymerase-1 (PARP-1) and p53, and the integration of these pivotal signaling processes with genomic maintenance.
Subject(s)
Cell Nucleus/metabolism , Eukaryotic Cells/metabolism , Genomic Instability/physiology , Poly Adenosine Diphosphate Ribose/metabolism , Protein Processing, Post-Translational/physiology , Tumor Suppressor Protein p53/metabolism , Animals , HumansABSTRACT
The 40 kDa carboxy-terminal catalytic domain (CD) of avian poly(ADP-ribose) polymerase (PARP-1) was cloned, expressed in a baculovirus expression system, and purified to homogeneity by affinity chromatography. The purified polypeptide synthesized covalent CD-poly(ADP-ribose) conjugates in the absence of DNA. Electrophoretic analysis of the ADP-ribose chain length distribution generated indicated that recombinant CD was able to catalyze the initiation, elongation, and branching reactions of poly(ADP-ribose) synthesis, although at a 500-fold lower efficiency than wild-type PARP-1. Kinetic evaluation of poly(ADP-ribose) synthesis showed that the enzymatic activities of CD increased for up to 60 minutes in a time-dependent manner. Moreover, the rates of CD auto-poly(ADP-ribosyl)ation increased with second-order kinetics as a function of the protein concentration with either betaNAD(+) or 3'-deoxyNAD(+) as a substrate. Furthermore, the formation of catalytically competent CD-[PARP-1] heterodimers was also observed in specific ultrafiltration experiments. Thus, we conclude that the 40 kDa carboxy terminus of PARP-1 forms a competent catalytic dimer in the absence of DNA, and that its automodification reaction is intermolecular.
Subject(s)
DNA/metabolism , Poly(ADP-ribose) Polymerases/chemistry , Poly(ADP-ribose) Polymerases/metabolism , Protein Conformation , Animals , Catalytic Domain , Chickens , Dimerization , Humans , NAD/chemistry , NAD/metabolism , Peptide Fragments/chemistry , Peptide Fragments/metabolism , Poly(ADP-ribose) Polymerases/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolismABSTRACT
We have evaluated the influence of protein poly(ADP-ribosyl)ation in the relaxation of chromatin by exposing a rat liver polynucleosomal extract to micrococcal nuclease (MNase) digestion. The kinetic susceptibility of polynucleosomes to endonuclease digestion was determined as a function of the time of incubation as well as endonuclease concentration. To validate our assay, we also ran control experiments with protein-free calf thymus DNA as the opposite of polynucleosomal DNA. Rat liver chromatin was also incubated in the absence or presence of exogenously added 200 microM betaNAD(+), the poly(ADP-ribosyl)ation substrate, before MNase digestion. For incubations in the presence of betaNAD(+), the synthesis of polynucleosomal poly(ADP-ribose) was stopped with 1 mM benzamide. After addition of MNase, endonuclease digestion was blocked with EDTA to chelate the Mg(2+) ions needed for enzymatic activation, and the samples were subjected to electrophoresis through 1.5% agarose gels. As expected, a faster degradation of chromatin into oligonucleosomal DNA ladders was observed upon protein poly(ADP-ribosyl)ation when the chromatin extract was preincubated with 200 microM betaNAD(+). Thus, our results are consistent with the conclusion that the covalent poly(ADP-ribosyl)ation of polynucleosomal proteins favors a more "relaxed" or "open" structure, which renders chromatin more susceptible to MNase digestion.
Subject(s)
Chromatin/metabolism , Micrococcal Nuclease/metabolism , Nucleosomes/metabolism , Poly Adenosine Diphosphate Ribose/biosynthesis , Animals , Cattle , Chromatin/chemistry , Hydrolysis , Kinetics , Protein DenaturationABSTRACT
We have characterized the biochemical association of two DNA damage-dependent enzymes, poly(ADP-ribose) polymerase-1 (PARP-1) [EC 2.4.2.30] and DNA polymerase beta (pol beta) [2.7.7.7]. We reproducibly observed that pol beta is an efficient covalent target for ADP-ribose polymers under standard conditions of enzymatically catalyzed ADP-ribosylation of betaNAD+ as a substrate. The efficiency of poly(ADP-ribosyl)ation increased as a function of the pol beta and betaNAD+ concentrations. To further characterize the molecular interactions between these two unique polymerases, we also subjected human recombinant PARP-1 to peptide-specific enzymatic degradation with either caspase-3 or caspase-7 in vitro. This proteolytic treatment, commonly referred to as 'a hallmark of apoptosis', generated the two physiologically relevant peptide fragments of PARP-1, e.g., a 24-kDa amino-terminus and an 89-kDa carboxy-terminal domain. Interestingly, co-incubation of the two peptide fragments of PARP-1 with full-length pol beta resulted in their domain-specific molecular association as determined by co-immunoprecipitation and reciprocal immunoblotting. Therefore, our data strongly suggest that, once PARP-1 is proteolyzed by either caspase-3 or caspase-7 during cell death, the specific association of its apoptotic fragments with DNA repair enzymes, such as pol beta, may serve a regulatory molecular role in the execution phase of apoptosis.
Subject(s)
Apoptosis/physiology , DNA Polymerase beta/metabolism , Peptide Fragments/metabolism , Poly(ADP-ribose) Polymerases/metabolism , Animals , DNA Polymerase beta/chemistry , Humans , Peptide Fragments/chemistry , Poly (ADP-Ribose) Polymerase-1 , Poly(ADP-ribose) Polymerases/chemistry , Protein Binding/physiology , RatsABSTRACT
BACKGROUND/AIM: Nucleic acid metabolism is biochemically compartmentalized to the nucleus. Thus, it is necessary to define the proteome of the various macromolecular structures within this organelle. MATERIALS AND METHODS: We isolated the nuclear matrix (NM) fraction from rat liver by sequential centrifugation steps at 13,000 rpm, staggered between endogenous nuclease treatment for 2 h at 37°C, followed by high-salt (H.S.; 2.0 M NaCl) and non-ionic detergent extractions (0.1%- or 1.0% Triton X-100) to eliminate the bulk of chromosomal DNA/RNA, histone proteins and the nuclear envelope (NE). RESULTS: Integrity of the NM and NE structures was confirmed by electron microscopy. Next, we analyzed the NM proteome on a 20% polyacrylamide gel using the PhastSystem. We observed the absence of histone proteins and the characteristic presence of the lamins by Coomassie blue staining. By contrast, upon silver staining, following electrophoretic separation with a Tris-Borate-EDTA buffer, we observed the NM-associated nucleic RNA and protein-free ADP-ribose polymers. While polymers are found in much lower concentration than RNA in NM, they were purified by affinity chromatography on boronate resin prior to electrophoresis. We observed the electrophoretic resolution of free ADP-ribose chains (5-25 units) by silver staining. CONCLUSION: The significance of our observations to cancer studies and carcinogenesis is discussed.
Subject(s)
Neoplasms/chemistry , Nuclear Envelope/chemistry , Nuclear Matrix-Associated Proteins/chemistry , Nuclear Matrix/chemistry , Poly(ADP-ribose) Polymerases/chemistry , Proteome/chemistry , Animals , Cell Nucleolus/chemistry , Cell Nucleolus/metabolism , Electrophoresis/methods , Neoplasms/metabolism , Nuclear Envelope/metabolism , Nuclear Matrix/metabolism , Nuclear Matrix-Associated Proteins/metabolism , Poly(ADP-ribose) Polymerases/metabolism , Proteome/metabolism , Rats , Rats, Sprague-DawleyABSTRACT
The structural integrity of eukaryotic genomes, to a great extent, depends on highly regulated and -coordinated enzymatic chromosomal poly(ADP-ribosyl)ation cycles that target chromatin proteins for specific covalent epigenetic poly(ADP-ribose) modification. As a result, the accurate determination of poly(ADP-ribosyl)ation amino acid specificity, as well as, a detailed characterization of the structural -complexity of the protein-bound ADP-ribose polymers generated, e.g., linear versus branched ADP-ribose chains, need to be carefully sorted out. In this chapter, we describe well-established and reproducible laboratory methods and protocols typically used to determine: (1) the ADP-ribose chain length(s) and (2) the molecular stoichiometry of the protein-poly(ADP-ribosyl)ation reaction, e.g., number of ADP-ribose chains/polypeptide unit. While the methodology described here is exclusively for in vitro purified systems that can be used with high reliability, the reader is advised that application of these protocols to whole cell extracts and tissue systems must take into consideration the rapid turnover rate of protein-bound ADP-ribose polymers in vivo. Indeed, these extremely low-abundance chromatin-bound polymeric molecules are notoriously characterized for displaying a short half-life, typically from a few seconds to a few minutes. We also discuss potential methodological pitfalls, such as: (1) the chemical stability of protein-(ADP-ribose)n adducts and (2) the requirement for polymeric radiolabeling.
Subject(s)
Poly Adenosine Diphosphate Ribose/chemistry , Boronic Acids/chemistry , Chromatography, Affinity , Chromatography, Gel , Chromatography, High Pressure Liquid , Electrophoresis, Polyacrylamide Gel , Molecular Structure , Poly Adenosine Diphosphate Ribose/isolation & purificationABSTRACT
El trauma quirúrgico produce sensibilización nociceptiva; ésta, a su vez, se traduce en la amplificación y prolongación del dolor posoperatorio. Existen diferentes formas de bloquearlo, por ejemplo la analgesia preventiva asociada a la utilización de más de un analgésico. Este procedimiento se conoce como sinergia farmacológica multimodal. El objetivo del presente estudio fue el de comprobar que la anestesia regional tipo peridural con fentanilo y lidocaína asociada a la administración de ketorolaco e.v. más la infiltración preincisional de bupivacaína tiene mayores beneficios que la anestesia general con infiltración preincisional de bupivacaína solamente. Material y métodos: Estudio prospectivo de 20 pacientes divididos en dos grupos: I) Anestesia regional tipo peridural con lidocaína y fentanilo asociada a la administración de ketorolaco e.v. más la infiltración preincisional de bupivacaína. II) Anestesia general con infiltración antes de la incisión quirúrgica de bupivacaína. La evaluación del dolor se realizó mediante la escala analógica del dolor; ambos grupos se compararon con el método estadístico de regresión lineal o mínimos cuadrados considerando significativa una r: > 0.88. Resultados: En el grupo I el promedio de dolor referido mediante la escala analógica del dolor en las primeras 12 horas fue de 0.43, mientras que en el grupo II fue de 3.273 (r > 0.88). En la sala de recuperación el valor fue de 0 y 4.35, respectivamente; en el segundo grupo se administró una dosis mayor y más temprana de analgésico. Conclusión: El bloqueo peridural como método de analgesia preventiva demostró ser superior a la anestesia general en la prevención del dolor posoperatorio, disminuyendo los requerimientos de analgésicos en el posoperatorio.
Subject(s)
Humans , Male , Female , Adult , Analgesia , Anesthesia , Anesthesia, Epidural/statistics & numerical data , Pain, Postoperative/prevention & control , Analgesics/administration & dosage , Anesthesia, GeneralABSTRACT
El consumo de oxígeno (VO2) puede medirse de forma no invasiva durante la anestesia mediante un analizador de gases. Diversos estudios experimentales han demostrado que los anestésicos influyen en el consumo de oxígeno de todo el organismo: la cirugía realizada durante la anestesia general produce un incremento del consumo de oxígeno. Los objetivos de este estudio fueron: observar el comportamiento del consumo e oxígeno evaluado en forma no invasiva durante la anestesia total endovenosa y determinar cómo pueden detectarse de manera inmediata los cambios en el consumo de oxígeno, estableciendo la relación que existe entre el consumo de oxígeno y otros parámetros de profundidad anestésica. Fueron incluidos 40 pacientes en estado I-II de acuerdo a la clasificación de la American Society of Anesthesiology (ASA I-II), sometidos a anestesia total endovenosa con propofol y fentanil, ambos en régimen de infusión y cisatracurio por vía intravenosa, con una FIO2 de 68 por ciento; se comparó el consumo de oxígeno medido en forma no invasiva, la presión arterial y la frecuencia cardiaca, durante diferentes procedimientos quirúrgicos. Las alteraciones en el intercambio de gases y en las variables hemodinámicas fueron valoradas mediante análisis de varianza (ANOVA) para repetidas mediciones, considerando significativa una p < 0.05. El intercambio de gases respiratorios fue evaluado por medio del analizador de gases (DATEX-OHMEDA AS/3) durante 60 minutos. En el análisis estadístico se utilizó primero estadística descriptiva (univariada) y posteriormente estos valores fueron sometidos a estadística comparativa (bivariada) con el coeficiente de Pearson de correlación lineal. Se observó una correlación importante en las variaciones del consumo de oxígeno, de la presión arterial y de la frecuencia cardiaca que resultó estadísticamente significativa, p = 0.01 con una R2 = 0.3865. La disminución del consumo de oxígeno obtenida coincidió con la notificada en otros estudios. La monitorización no invasiva del intercambio de gases puede proveer un método adicional de vigilancia durante la anestesia y de evaluación del grado de profundidad de la anestesia.