RESUMO
Charge transport (CT) through the DNA base pairs provides a means to promote redox reactions at a remote site and potentially to effect signaling between molecules bound to DNA. Here we describe the oxidation of a cell-cycle regulatory protein, p53, from a distance through DNA-mediated CT. A consensus p53 binding site as well as three DNA promoters regulated by p53 were synthesized containing a tethered DNA photooxidant, anthraquinone. Photoinduced oxidation of the protein occurs from a distance; introduction of an intervening CA mismatch, which inhibits DNA-mediated CT, prevents oxidation of p53. DNA-mediated oxidation is shown to promote dissociation of p53 from only some promoters, and this sequence-selectivity in oxidative dissociation correlates with the biological regulation of p53. Under severe oxidative stress, effected here through oxidation at long range, p53 dissociates from a promoter that activates DNA repair as well as the promoter for the negative regulator of p53, Mdm2, but not from a promoter activating cell-cycle arrest. Mass spectrometry results are consistent with disulfide bond formation in p53 upon DNA-mediated oxidation. Furthermore, DNA-bound p53 oxidation is shown in vivo by up-regulation of p53 and subsequent irradiation in the presence of a rhodium photooxidant to give a new p53 adduct that can be reversed with thiol treatment. This DNA-mediated oxidation of p53 parallels that seen by treating cells with hydrogen peroxide. These results indicate a unique mechanism using DNA-mediated CT chemistry by which p53 activity on different promoters may be controlled globally under conditions of oxidative stress.
Assuntos
DNA/metabolismo , Transporte de Elétrons/fisiologia , Proteína Supressora de Tumor p53/metabolismo , Antraquinonas/efeitos da radiação , Ciclo Celular/genética , Proteínas de Ciclo Celular/genética , Sequência Consenso , Inibidor de Quinase Dependente de Ciclina p21/genética , Cistina/química , DNA/química , Reparo do DNA/genética , Ensaio de Desvio de Mobilidade Eletroforética , Substâncias Intercalantes/efeitos da radiação , Proteínas Nucleares/genética , Compostos Organometálicos/efeitos da radiação , Oxidantes Fotoquímicos/efeitos da radiação , Oxirredução , Estresse Oxidativo , Fotoquímica , Regiões Promotoras Genéticas , Ligação Proteica/efeitos da radiação , Conformação Proteica , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteína Supressora de Tumor p53/químicaRESUMO
DNA-mediated charge transport (CT) is exquisitely sensitive to the integrity of the bridging pi-stack and is characterized by a shallow distance dependence. These properties are obscured by poor coupling between the donor/acceptor pair and the DNA bridge, or by convolution with other processes. Previously, we found a surprising periodic length dependence for the rate of DNA-mediated CT across adenine tracts monitored by 2-aminopurine fluorescence. Here we report a similar periodicity by monitoring N 2-cyclopropylguanosine decomposition by rhodium and anthraquinone photooxidants. Furthermore, we find that this periodicity is attenuated by consequent back-electron transfer (BET), as observed by direct comparison between sequences that allow and suppress BET. Thus, the periodicity can be controlled by engineering the extent of BET across the bridge. The periodic length dependence is not consistent with a periodicity predicted by molecular wire theory but is consistent with a model where multiples of four to five base pairs form an ideal CT-active length of a bridging adenine domain.
Assuntos
Adenina/química , DNA/química , 2-Aminopurina/química , Antraquinonas/química , Sequência de Bases , Elétrons , Modelos Moleculares , Oligonucleotídeos/síntese química , Oligonucleotídeos/química , Oxirredução , Teoria Quântica , Ródio/químicaRESUMO
Inhibition of caspase-6 is a potential therapeutic strategy for some neurodegenerative diseases, but it has been difficult to develop selective inhibitors against caspases. We report the discovery and characterization of a potent inhibitor of caspase-6 that acts by an uncompetitive binding mode that is an unprecedented mechanism of inhibition against this target class. Biochemical assays demonstrate that, while exquisitely selective for caspase-6 over caspase-3 and -7, the compound's inhibitory activity is also dependent on the amino acid sequence and P1' character of the peptide substrate. The crystal structure of the ternary complex of caspase-6, substrate-mimetic and an 11 nM inhibitor reveals the molecular basis of inhibition. The general strategy to develop uncompetitive inhibitors together with the unique mechanism described herein provides a rationale for engineering caspase selectivity.
Assuntos
Caspase 6/metabolismo , Inibidores de Caspase/química , Inibidores de Caspase/farmacologia , Sequência de Aminoácidos , Caspase 6/química , Inibidores de Caspase/análise , Cristalografia por Raios X , Avaliação Pré-Clínica de Medicamentos , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/metabolismo , Ligação Proteica/efeitos dos fármacos , Reprodutibilidade dos Testes , Especificidade por Substrato/efeitos dos fármacos , Ressonância de Plasmônio de SuperfícieRESUMO
A novel tris heteroleptic dipyridophenazine complex of ruthenium(II), [{Ru(phen)(dppz)(bpy'-his)}{Ru(NH3)5}]5+, containing a covalently tethered ruthenium pentammine quencher coordinated through a bridging histidine has been synthesized and characterized spectroscopically and biochemically in a DNA environment and in organic solvent. Steady-state and time-resolved luminescence measurements indicate that the tethered Ru complex is quenched relative to the parent complexes [Ru(phen)(dppz)(bpy')]2+ and [Ru(phen)(dppz)(bpy'-his)]2+ in DNA and acetonitrile, consistent with intramolecular photoinduced electron transfer. Intercalated into guanine-containing DNA, [{Ru(phen)(dppz)(bpy'-his)}{Ru(NH3)5}]5+, upon excitation and intramolecular quenching, is capable of injecting charge into the duplex based upon the EPR detection of guanine radicals. DNA-mediated charge transport is also indicated using a kinetically fast cyclopropylamine-substituted base as an electron hole trap. Guanine damage is not observed, however, in measurements using the guanine radical as the kinetically slower hole trap, indicating that back electron-transfer reactions are competitive with guanine oxidation. Moreover, transient absorption measurements reveal a novel photophysical reaction pathway for [{Ru(phen)(dppz)(bpy'-his)}{Ru(NH3)5}]5+ in the presence of DNA that is competitive with the intramolecular flash-quench process. These results illustrate the remarkably rich redox chemistry that can occur within a bimolecular ruthenium complex intercalated in duplex DNA.
Assuntos
DNA/química , Rutênio/química , Espectroscopia de Ressonância de Spin Eletrônica , Cinética , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
The fluorescent adenosine analogue 4-amino-8-(2-deoxy-beta-d-ribofuranosyl)-5'-O-dimethoxytrityl-6-methyl-7(8H)-pteridone (6MAP) has been used to perform residue specific analyses of DNA A-tracts during the premelting transition. DNA A-tracts, which exhibit sequence-induced curvature, adopt a B-DNA conformation as a function of increasing temperature. Fluorescence melting curves indicate that 6MAP is a more sensitive reporter of the premelting transition than UV absorption spectroscopy. Further, residue specific fluorescence analyses of A-tract and control duplexes reveal that some of the conformational changes associated with the premelting transition occur within A-tract regions. Analyses of the energetics of the premelting transition indicate that ApA steps make a larger enthalpic contribution to the premelting transition than ApT steps. To explore the effect of cations on the premelting transition, fluorescence melts were performed in the presence of NH(4)(+), Mg(2+), and low (0.05 M) and high (0.5 M) concentrations of Na(+). These studies show that the fluorescence intensity changes associated with the premelting transition are sensitive to cation type and concentration and are larger and more pronounced in the presence of 0.5 M Na(+), NH(4)(+), and Mg(2+). Incorporation of 6MAP into longer duplexes containing phased A-tracts shows that the local environment of adenosines in phased A-tracts is similar to that of individual A-tracts. Fluorescence quenching results indicate that ApA and ApT steps within A-tracts are less solvent exposed than their counterparts in control sequence isomers, possibly because of the narrowed minor groove of A-tract sequences.