RESUMEN
DNA cross-links severely challenge replication and transcription in cells, promoting senescence and cell death. In this paper, we report a novel type of DNA interstrand cross-link (ICL) produced as a side product during the attempted repair of 1,N6-ethenoadenine (εA) by human α-ketoglutarate/Fe(II)-dependent enzyme ALKBH2. This stable/nonreversible ICL was characterized by denaturing polyacrylamide gel electrophoresis analysis and quantified by high-resolution LC-MS in well-matched and mismatched DNA duplexes, yielding 5.7% as the highest level for cross-link formation. The binary lesion is proposed to be generated through covalent bond formation between the epoxide intermediate of εA repair and the exocyclic N6-amino group of adenine or the N4-amino group of cytosine residues in the complementary strand under physiological conditions. The cross-links occur in diverse sequence contexts, and molecular dynamics simulations rationalize the context specificity of cross-link formation. In addition, the cross-link generated from attempted εA repair was detected in cells by highly sensitive LC-MS techniques, giving biological relevance to the cross-link adducts. Overall, a combination of biochemical, computational, and mass spectrometric methods was used to discover and characterize this new type of stable cross-link both in vitro and in human cells, thereby uniquely demonstrating the existence of a potentially harmful ICL during DNA repair by human ALKBH2.
Asunto(s)
Adenina/análogos & derivados , Dioxigenasas , Ácidos Cetoglutáricos , Humanos , Dioxigenasas/metabolismo , ADN/química , Reparación del ADN , Compuestos Ferrosos , Aductos de ADN , Dioxigenasa Dependiente de Alfa-Cetoglutarato, Homólogo 2 de AlkB/metabolismoRESUMEN
The human DNA repair enzyme AlkB homologue-2 (ALKBH2) repairs methyl adducts from genomic DNA. Overexpression of ALKBH2 has been implicated in both tumorigenesis and chemotherapy resistance in some cancers, including glioblastoma and renal cancer rendering it a potential therapeutic target and a diagnostic marker. However, no inhibitor is available against these important DNA repair proteins. Intending to repurpose a drug as an inhibitor of ALKBH2, we performed in silico evaluation of HIV protease inhibitors and identified Ritonavir as an ALKBH2-interacting molecule. Using molecular dynamics simulation, we elucidated the molecular details of Ritonavir-ALKBH2 interaction. The present work highlights that Ritonavir might be used to target the ALKBH2-mediated DNA alkylation repair.
Asunto(s)
Inhibidores de la Proteasa del VIH , Ritonavir , Humanos , Ritonavir/farmacología , Inhibidores de la Proteasa del VIH/farmacología , Simulación de Dinámica Molecular , Enzimas Reparadoras del ADN/genética , Enzimas Reparadoras del ADN/metabolismo , Reparación del ADN , Dioxigenasa Dependiente de Alfa-Cetoglutarato, Homólogo 2 de AlkB/genética , Dioxigenasa Dependiente de Alfa-Cetoglutarato, Homólogo 2 de AlkB/metabolismoRESUMEN
Elucidation of physicochemical mechanisms of enzymatic processes is one of the main tasks of modern biology. High efficiency and selectivity of enzymatic catalysis are mostly ensured by conformational dynamics of enzymes and substrates. Here, we applied a stopped-flow kinetic analysis based on fluorescent spectroscopy to investigate mechanisms of conformational transformations during the removal of alkylated bases from DNA by ALKBH2, a human homolog of Escherichia coli AlkB dioxygenase. This enzyme protects genomic DNA against various alkyl lesions through a sophisticated catalytic mechanism supported by a cofactor (Fe(II)), a cosubstrate (2-oxoglutarate), and O2. We present here a comparative study of conformational dynamics in complexes of the ALKBH2 protein with double-stranded DNA substrates containing N1-methyladenine, N3-methylcytosine, or 1,N6-ethenoadenine. By means of fluorescent labels of different types, simultaneous detection of conformational transitions in the protein globule and DNA substrate molecule was performed. Fitting of the kinetic curves by a nonlinear-regression method yielded a molecular mechanism and rate constants of its individual steps. The results shed light on overall conformational dynamics of ALKBH2 and damaged DNA during the catalytic cycle.
Asunto(s)
Dioxigenasa Dependiente de Alfa-Cetoglutarato, Homólogo 2 de AlkB , Reparación del ADN , Proteínas de Escherichia coli , Dioxigenasa Dependiente de Alfa-Cetoglutarato, Homólogo 2 de AlkB/genética , Dioxigenasa Dependiente de Alfa-Cetoglutarato, Homólogo 2 de AlkB/metabolismo , ADN/química , Reparación del ADN/fisiología , Dioxigenasas/genética , Dioxigenasas/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Humanos , Cinética , Conformación Proteica , Espectrometría de FluorescenciaRESUMEN
E. coli AlkB and human ALKBH2 belong to the AlkB family enzymes, which contain several α-ketoglutarate (α-KG)/Fe(II)-dependent dioxygenases that repair alkylated DNA. Specifically, the AlkB enzymes catalyze decarboxylation of α-KG to generate a high-valent Fe(IV)-oxo species that oxidizes alkyl groups on DNA adducts. AlkB and ALKBH2 have been reported to differentially repair select etheno adducts, with preferences for 1,N6-ethenoadenine (1,N6-εA) and 3,N4-ethenocytosine (3,N4-εC) over 1,N2-ethenoguanine (1,N2-εG). However, N2,3-ethenoguanine (N2,3-εG), the most common etheno adduct, is not repaired by the AlkB enzymes. Unfortunately, a structural understanding of the differential activity of E. coli AlkB and human ALKBH2 is lacking due to challenges acquiring atomistic details for a range of substrates using experiments. This study uses both molecular dynamics (MD) simulations and ONIOM(QM:MM) calculations to determine how the active site changes upon binding each etheno adduct and characterizes the corresponding catalytic impacts. Our data reveal that the preferred etheno substrates (1,N6-εA and 3,N4-εC) form favorable interactions with catalytic residues that situate the lesion near the Fe(IV)-oxo species and permit efficient oxidation. In contrast, although the damage remains correctly aligned with respect to the Fe(IV)-oxo moiety, repair of 1,N2-εG is mitigated by increased solvation of the active site and a larger distance between Fe(IV)-oxo and the aberrant carbons. Binding of non-substrate N2,3-εG in the active site disrupts key DNA-enzyme interactions, and positions the aberrant carbon atoms even further from the Fe(IV)-oxo species, leading to prohibitively high barriers for oxidative catalysis. Overall, our calculations provide the first structural insight required to rationalize the experimentally-reported substrate specificities of AlkB and ALKBH2 and thereby highlight the roles of several active site residues in the repair of etheno adducts that directly correlates with available experimental data. These proposed catalytic strategies can likely be generalized to other α-KG/Fe(II)-dependent dioxygenases that play similar critical biological roles, including epigenetic and post-translational regulation.
Asunto(s)
Dioxigenasa Dependiente de Alfa-Cetoglutarato, Homólogo 2 de AlkB/metabolismo , Dominio Catalítico , Aductos de ADN/metabolismo , Reparación del ADN , Proteínas de Escherichia coli/metabolismo , Oxigenasas de Función Mixta/metabolismo , Simulación de Dinámica Molecular , Adenina/análogos & derivados , Adenina/metabolismo , Dioxigenasa Dependiente de Alfa-Cetoglutarato, Homólogo 2 de AlkB/química , Biología Computacional , Citosina/análogos & derivados , Citosina/metabolismo , Aductos de ADN/química , Escherichia coli/enzimología , Escherichia coli/genética , Proteínas de Escherichia coli/química , Guanina/análogos & derivados , Guanina/metabolismo , Humanos , Oxigenasas de Función Mixta/química , Modelos Moleculares , Conformación Proteica , Especificidad por SustratoRESUMEN
Fourteen (hetero-)(arylidene)arylhydrazide derivatives (ABH1-ABH14) were synthesized, and their inhibitory activities against monoamine oxidases (MAOs) and acetylcholinesterase (AChE) were evaluated. Compound ABH5 most potently inhibited MAO-B with an IC50 value of 0.025 ± 0.0019 µM; ABH2 and ABH3 exhibited high IC50 values as well. Most of the compounds weakly inhibited MAO-A, except ABH5 (IC50 = 3.31 ± 0.41 µM). Among the active compounds, ABH2 showed the highest selectivity index (SI) of 174 for MAO-B, followed by ABH5 (SI = 132). ABH3 and ABH5 effectively inhibited AChE with IC50 values of 15.7 ± 6.52 and 16.5 ± 7.29 µM, respectively, whereas the other compounds were weak inhibitors of AChE. ABH5 was shown to be a reversible competitive inhibitor for MAO-A and MAO-B with Ki values of 0.96 ± 0.19 and 0.024 ± 0.0077 µM, respectively, suggesting that this molecule can be considered as an interesting candidate for further development as a multitarget inhibitor relating to neurodegenerative disorders.
Asunto(s)
Inhibidores de la Monoaminooxidasa/química , Monoaminooxidasa/metabolismo , Enfermedades Neurodegenerativas/tratamiento farmacológico , Fármacos Neuroprotectores/química , Acetilcolinesterasa/metabolismo , Dioxigenasa Dependiente de Alfa-Cetoglutarato, Homólogo 2 de AlkB/metabolismo , Diseño de Fármacos , Humanos , Hidrazinas/química , Simulación del Acoplamiento Molecular , Inhibidores de la Monoaminooxidasa/farmacología , Fármacos Neuroprotectores/farmacología , Relación Estructura-ActividadRESUMEN
Cellular processes, such as DNA replication, recombination and transcription, require DNA strands separation and single-stranded DNA is formation. The single-stranded DNA is promptly wrapped by human single-stranded DNA binding proteins, replication protein A (RPA) complex. RPA binding not only prevent nuclease degradation and annealing, but it also coordinates cell-cycle checkpoint activation and DNA repair. However, RPA binding offers little protection against the chemical modification of DNA bases. This review focuses on the type of DNA base damage that occurs in single-stranded DNA and how the damage is rectified in human cells. The discovery of DNA repair proteins, such as ALKBH3, AGT, UNG2, NEIL3, being able to repair the damaged base in the single-stranded DNA, renewed the interest to study single-stranded DNA repair. These mechanistically different proteins work independently from each other with the overarching goal of increasing fidelity of recombination and promoting error-free replication.
Asunto(s)
Dioxigenasa Dependiente de Alfa-Cetoglutarato, Homólogo 2 de AlkB/metabolismo , Dioxigenasa Dependiente de Alfa-Cetoglutarato, Homólogo 3 de AlkB/metabolismo , Daño del ADN , ADN de Cadena Simple/genética , Reparación del ADN , Replicación del ADN , ADN de Cadena Simple/efectos de los fármacos , Humanos , Recombinación Genética , Proteína de Replicación A/metabolismoRESUMEN
5-Methylcytosine (5mC) in DNA CpG islands is an important epigenetic biomarker for mammalian gene regulation. It is oxidized to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) by the ten-eleven translocation (TET) family enzymes, which are α-ketoglutarate (α-KG)/Fe(II)-dependent dioxygenases. In this work, we demonstrate that the epigenetic marker 5mC is modified to 5hmC, 5fC, and 5caC in vitro by another class of α-KG/Fe(II)-dependent proteins-the DNA repair enzymes in the AlkB family, which include ALKBH2, ALKBH3 in huamn and AlkB in Escherichia coli. Theoretical calculations indicate that these enzymes may bind 5mC in the syn-conformation, placing the methyl group comparable to 3-methylcytosine, the prototypic substrate of AlkB. This is the first demonstration of the AlkB proteins to oxidize a methyl group attached to carbon, instead of nitrogen, on a DNA base. These observations suggest a broader role in epigenetics for these DNA repair proteins.
Asunto(s)
5-Metilcitosina/análogos & derivados , 5-Metilcitosina/metabolismo , Enzimas AlkB/metabolismo , Dioxigenasa Dependiente de Alfa-Cetoglutarato, Homólogo 2 de AlkB/metabolismo , Dioxigenasa Dependiente de Alfa-Cetoglutarato, Homólogo 3 de AlkB/metabolismo , Citosina/análogos & derivados , Enzimas AlkB/genética , Dioxigenasa Dependiente de Alfa-Cetoglutarato, Homólogo 2 de AlkB/genética , Dioxigenasa Dependiente de Alfa-Cetoglutarato, Homólogo 3 de AlkB/genética , Animales , Biología Computacional , Islas de CpG , Citosina/metabolismo , ADN/genética , Metilación de ADN , Epigénesis Genética , Humanos , Estructura Molecular , Oxidación-ReducciónRESUMEN
Hydrolyzable tannins are a class of polyphenolic compounds commonly found in natural products. In this work, we studied the in vitro inhibitory mechanism of six molecules in this class on ALKBH2, an Fe(II)/α-ketoglutarate-dependent DNA repair enzyme in the AlkB family. We determined the IC50 values of these compounds on the repair of 3-methylcytosine and 1-methyladenine, the prototypical substrates of ALKBH2. A structure-activity relationship was also observed between the strength of inhibition and the number of galloyl moieties in a molecule. In addition, we found that the inhibition by this class of polyphenolic compounds on ALKBH2 is through an iron-chelating mechanism.
Asunto(s)
Dioxigenasa Dependiente de Alfa-Cetoglutarato, Homólogo 2 de AlkB/antagonistas & inhibidores , Reparación del ADN , Inhibidores Enzimáticos/farmacología , Taninos Hidrolizables/farmacología , Quelantes del Hierro/farmacología , Dioxigenasa Dependiente de Alfa-Cetoglutarato, Homólogo 2 de AlkB/metabolismo , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/química , Humanos , Taninos Hidrolizables/química , Quelantes del Hierro/química , Estructura Molecular , Relación Estructura-ActividadRESUMEN
The DNA repair enzyme ALKBH2 is implicated in both tumorigenesis as well as resistance to chemotherapy in certain cancers. It is currently under study as a potential diagnostic marker and has been proposed as a therapeutic target. To date, however, there exist no direct methods for measuring the repair activity of ALKBH2 inâ vitro or in biological samples. Herein, we report a highly specific, fluorogenic probe design based on an oligonucleotide scaffold that reports directly on ALKBH2 activity both inâ vitro and in cell lysates. Importantly, the probe enables the monitoring of cellular regulation of ALKBH2 activity in response to treatment with the chemotherapy drug temozolomide through a simple fluorescence assay, which has only previously been observed through indirect means such as qPCR and western blots. Furthermore, the probe provides a viable high-throughput assay for drug discovery.
Asunto(s)
Dioxigenasa Dependiente de Alfa-Cetoglutarato, Homólogo 2 de AlkB/química , Reparación del ADN , Resistencia a Antineoplásicos , Colorantes Fluorescentes/química , Dioxigenasa Dependiente de Alfa-Cetoglutarato, Homólogo 2 de AlkB/genética , Dioxigenasa Dependiente de Alfa-Cetoglutarato, Homólogo 2 de AlkB/metabolismo , Alquilación , Antineoplásicos Alquilantes/uso terapéutico , Colorantes Fluorescentes/metabolismo , Técnicas de Inactivación de Genes , Células HEK293 , Humanos , Cinética , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Espectrometría de Fluorescencia , Temozolomida/uso terapéuticoRESUMEN
Cancer-associated mutations often lead to perturbed cellular energy metabolism and accumulation of potentially harmful oncometabolites. One example is the chiral molecule 2-hydroxyglutarate (2HG); its two stereoisomers (d- and l-2HG) have been found at abnormally high concentrations in tumors featuring anomalous metabolic pathways. 2HG has been demonstrated to competitively inhibit several α-ketoglutarate (αKG)- and non-heme iron-dependent dioxygenases, including some of the AlkB family DNA repair enzymes, such as ALKBH2 and ALKBH3. However, previous studies have only provided the IC50 values of d-2HG on the enzymes, and the results have not been correlated to physiologically relevant concentrations of 2HG and αKG in cancer cells. In this work, we performed detailed kinetic analyses of DNA repair reactions catalyzed by ALKBH2, ALKBH3, and the bacterial AlkB in the presence of d- and l-2HG in both double- and single-stranded DNA contexts. We determined the kinetic parameters of inhibition, including kcat, KM, and Ki. We also correlated the relative concentrations of 2HG and αKG previously measured in tumor cells with the inhibitory effect of 2HG on the AlkB family enzymes. Both d- and l-2HG significantly inhibited the human DNA repair enzymes ALKBH2 and ALKBH3 at pathologically relevant concentrations (73-88% for d-2HG and 31-58% for l-2HG inhibition). This work provides a new perspective that the elevation of the d- or l-2HG concentration in cancer cells may contribute to an increased mutation rate by inhibiting the DNA repair performed by the AlkB family enzymes and thus exacerbate the genesis and progression of tumors.
Asunto(s)
Dioxigenasa Dependiente de Alfa-Cetoglutarato, Homólogo 2 de AlkB/metabolismo , Dioxigenasa Dependiente de Alfa-Cetoglutarato, Homólogo 3 de AlkB/metabolismo , Glutaratos/metabolismo , Dioxigenasa Dependiente de Alfa-Cetoglutarato, Homólogo 2 de AlkB/antagonistas & inhibidores , Dioxigenasa Dependiente de Alfa-Cetoglutarato, Homólogo 2 de AlkB/genética , Dioxigenasa Dependiente de Alfa-Cetoglutarato, Homólogo 3 de AlkB/antagonistas & inhibidores , Dioxigenasa Dependiente de Alfa-Cetoglutarato, Homólogo 3 de AlkB/genética , Secuencia de Bases , Cromatografía Líquida de Alta Presión , Reparación del ADN , Pruebas de Enzimas , Glutaratos/análisis , Glutaratos/química , Humanos , Concentración 50 Inhibidora , Ácidos Cetoglutáricos/análisis , Ácidos Cetoglutáricos/química , Ácidos Cetoglutáricos/metabolismo , Cinética , Unión Proteica , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , EstereoisomerismoRESUMEN
Environmental and endogenous genotoxic agents can result in a variety of alkylated and carboxymethylated DNA lesions, including N3-ethylthymidine (N3-EtdT), O(2)-EtdT, and O(4)-EtdT as well as N3-carboxymethylthymidine (N3-CMdT) and O(4)-CMdT. By using nonreplicative double-stranded vectors harboring a site-specifically incorporated DNA lesion, we assessed the potential roles of alkyladenine DNA glycosylase (Aag); alkylation repair protein B homologue 2 (Alkbh2); or Alkbh3 in modulating the effects of N3-EtdT, O(2)-EtdT, O(4)-EtdT, N3-CMdT, or O(4)-CMdT on DNA transcription in mammalian cells. We found that the depletion of Aag did not significantly change the transcriptional inhibitory or mutagenic properties of all five examined lesions, suggesting a negligible role of Aag in the repair of these DNA adducts in mammalian cells. In addition, our results revealed that N3-EtdT, but not other lesions, could be repaired by Alkbh2 and Alkbh3 in mammalian cells. Furthermore, we demonstrated the direct reversal of N3-EtdT by purified human Alkbh2 protein in vitro. These findings provided important new insights into the repair of the carboxymethylated and alkylated thymidine lesions in mammalian cells.
Asunto(s)
Dioxigenasa Dependiente de Alfa-Cetoglutarato, Homólogo 2 de AlkB/metabolismo , Dioxigenasa Dependiente de Alfa-Cetoglutarato, Homólogo 3 de AlkB/metabolismo , Aductos de ADN/metabolismo , ADN Glicosilasas/metabolismo , Alquilación , Animales , Línea Celular , Aductos de ADN/química , Aductos de ADN/genética , Reparación del ADN , Humanos , Ratones , Timidina/análogos & derivados , Timidina/química , Timidina/genética , Timidina/metabolismoRESUMEN
The AlkB repair enzymes, including Escherichia coli AlkB and two human homologues, ALKBH2 and ALKBH3, are iron(II)- and 2-oxoglutarate-dependent dioxygenases that efficiently repair N(1)-methyladenine and N(3)-methylcytosine methylated DNA damages. The development of small molecule inhibitors of these enzymes has seen less success. Here we have characterized a previously discovered natural product rhein and tested its ability to inhibit AlkB repair enzymes in vitro and to sensitize cells to methyl methane sulfonate that mainly produces N(1)-methyladenine and N(3)-methylcytosine lesions. Our investigation of the mechanism of rhein inhibition reveals that rhein binds to AlkB repair enzymes in vitro and promotes thermal stability in vivo In addition, we have determined a new structural complex of rhein bound to AlkB, which shows that rhein binds to a different part of the active site in AlkB than it binds to in fat mass and obesity-associated protein (FTO). With the support of these observations, we put forth the hypothesis that AlkB repair enzymes would be effective pharmacological targets for cancer treatment.
Asunto(s)
Antraquinonas/farmacología , Enzimas Reparadoras del ADN/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Proteínas de Escherichia coli/antagonistas & inhibidores , Oxigenasas de Función Mixta/antagonistas & inhibidores , Dioxigenasa Dependiente de Alfa-Cetoglutarato, Homólogo 2 de AlkB/antagonistas & inhibidores , Dioxigenasa Dependiente de Alfa-Cetoglutarato, Homólogo 2 de AlkB/genética , Dioxigenasa Dependiente de Alfa-Cetoglutarato, Homólogo 2 de AlkB/metabolismo , Dioxigenasa Dependiente de Alfa-Cetoglutarato, Homólogo 3 de AlkB/antagonistas & inhibidores , Dioxigenasa Dependiente de Alfa-Cetoglutarato, Homólogo 3 de AlkB/genética , Dioxigenasa Dependiente de Alfa-Cetoglutarato, Homólogo 3 de AlkB/metabolismo , Antraquinonas/química , Dominio Catalítico , Línea Celular , Cristalografía por Rayos X , Daño del ADN , Metilación de ADN , Enzimas Reparadoras del ADN/genética , Enzimas Reparadoras del ADN/metabolismo , Inhibidores Enzimáticos/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Humanos , Metilmetanosulfonato/farmacología , Oxigenasas de Función Mixta/genética , Oxigenasas de Función Mixta/metabolismo , Modelos Moleculares , Interferencia de ARN , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
The AlkB protein is a repair enzyme that uses an α-ketoglutarate/Fe(II)-dependent mechanism to repair alkyl DNA adducts. AlkB has been reported to repair highly susceptible substrates, such as 1-methyladenine and 3-methylcytosine, more efficiently in ss-DNA than in ds-DNA. Here, we tested the repair of weaker AlkB substrates 1-methylguanine and 3-methylthymine and found that AlkB prefers to repair them in ds-DNA. We also discovered that AlkB and its human homologues, ABH2 and ABH3, are able to repair the aforementioned adducts when the adduct is present in a mismatched base pair. These observations demonstrate the strong adaptability of AlkB toward repairing various adducts in different environments.