RESUMO
The human DNA repair enzyme AlkB homologue-2 (ALKBH2) repairs methyl adducts from genomic DNA and is overexpressed in several cancers. However, there are no known inhibitors available for this crucial DNA repair enzyme. The aim of this study was to examine whether the first-generation HIV protease inhibitors having strong anti-cancer activity can be repurposed as inhibitors of ALKBH2. We selected four such inhibitors and performed in vitro binding analysis against ALKBH2 based on alterations of its intrinsic tryptophan fluorescence and differential scanning fluorimetry. The effect of these HIV protease inhibitors on the DNA repair activity of ALKBH2 was also evaluated. Interestingly, we observed that one of the inhibitors, ritonavir, could inhibit ALKBH2-mediated DNA repair significantly via competitive inhibition and sensitized cancer cells to alkylating agent methylmethane sulfonate (MMS). This work may provide new insights into the possibilities of utilizing HIV protease inhibitor ritonavir as a DNA repair antagonist.
Assuntos
Homólogo AlkB 2 da Dioxigenase Dependente de alfa-Cetoglutarato , Reparo do DNA , Inibidores da Protease de HIV , Metanossulfonato de Metila , Ritonavir , Humanos , Homólogo AlkB 2 da Dioxigenase Dependente de alfa-Cetoglutarato/metabolismo , Ritonavir/farmacologia , Inibidores da Protease de HIV/farmacologia , Metanossulfonato de Metila/farmacologia , Dano ao DNA , Alquilação , Linhagem Celular TumoralRESUMO
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.
Assuntos
Adenina/análogos & derivados , Dioxigenases , Ácidos Cetoglutáricos , Humanos , Dioxigenases/metabolismo , DNA/química , Reparo do DNA , Compostos Ferrosos , Adutos de DNA , Homólogo AlkB 2 da Dioxigenase Dependente de alfa-Cetoglutarato/metabolismoRESUMO
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.
Assuntos
Inibidores da Protease de HIV , Ritonavir , Humanos , Ritonavir/farmacologia , Inibidores da Protease de HIV/farmacologia , Simulação de Dinâmica Molecular , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Reparo do DNA , Homólogo AlkB 2 da Dioxigenase Dependente de alfa-Cetoglutarato/genética , Homólogo AlkB 2 da Dioxigenase Dependente de alfa-Cetoglutarato/metabolismoRESUMO
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.
Assuntos
Homólogo AlkB 2 da Dioxigenase Dependente de alfa-Cetoglutarato , Reparo do DNA , Proteínas de Escherichia coli , Homólogo AlkB 2 da Dioxigenase Dependente de alfa-Cetoglutarato/genética , Homólogo AlkB 2 da Dioxigenase Dependente de alfa-Cetoglutarato/metabolismo , DNA/química , Reparo do DNA/fisiologia , Dioxigenases/genética , Dioxigenases/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Humanos , Cinética , Conformação Proteica , Espectrometria de FluorescênciaRESUMO
BACKGROUND: The alkB homolog 2, alpha-ketoglutarate-dependent dioxygenase (ALKBH2) gene is involved in DNA repair and is expressed in different types of malignancies. However, the role of ALKBH2 in colorectal carcinoma (CRC) remains unclear. This study aimed to explore the potential mechanism of ALKBH2 and its function in CRC. METHODS: The expression levels of ALKBH2 in CRC tissues and cells were determined by qRT-PCR. Following that, the role of ALKBH2 in cell proliferation, invasion, and epithelial-mesenchymal transition (EMT) in CRC cells (Caco-2 and LOVO) were assessed by Cell Counting Kit-8 (CCK-8), transwell assays, and Western blotting, respectively. The effect of ALKBH2 on B cell-specific Moloney murine leukemia virus integration site 1 (BMI1) and downstream NF-κB pathway was determined by Western blotting and luciferase reporter assay. RESULTS: The expression of ALKBH2 was significantly upregulated both in CRC tissues and cells. Further experiments demonstrated that reduction of ALKBH2 suppressed Caco-2 and LOVO cell proliferation and invasion. Moreover, ALKBH2 knockdown also suppressed EMT, which increased E-cadherin expression and reduced N-cadherin expression. Besides, ALKBH2 silencing inhibited BMI1 expression and reduced nuclear accumulation of the NF-κB p65 protein, as well as the luciferase activity of NF-κB p65. Upregulation of BMI1 reversed the effect of ALKBH2 knockdown on the proliferation and invasion in CRC cells. CONCLUSIONS: Our findings suggest that suppression of ALKBH2 alleviates malignancy in CRC by regulating BMI1-mediated activation of NF-κB pathway. ALKBH2 may serve as a potential treatment target for human CRC.
Assuntos
Neoplasias Colorretais , NF-kappa B , Homólogo AlkB 2 da Dioxigenase Dependente de alfa-Cetoglutarato , Células CACO-2 , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Neoplasias Colorretais/genética , Transição Epitelial-Mesenquimal , Regulação Neoplásica da Expressão Gênica , Humanos , NF-kappa B/genética , NF-kappa B/metabolismo , Complexo Repressor Polycomb 1/genética , Prognóstico , Proteínas Proto-Oncogênicas/genéticaRESUMO
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.
Assuntos
Homólogo AlkB 2 da Dioxigenase Dependente de alfa-Cetoglutarato/metabolismo , Domínio Catalítico , Adutos de DNA/metabolismo , Reparo do DNA , Proteínas de Escherichia coli/metabolismo , Oxigenases de Função Mista/metabolismo , Simulação de Dinâmica Molecular , Adenina/análogos & derivados , Adenina/metabolismo , Homólogo AlkB 2 da Dioxigenase Dependente de alfa-Cetoglutarato/química , Biologia Computacional , Citosina/análogos & derivados , Citosina/metabolismo , Adutos de DNA/química , Escherichia coli/enzimologia , Escherichia coli/genética , Proteínas de Escherichia coli/química , Guanina/análogos & derivados , Guanina/metabolismo , Humanos , Oxigenases de Função Mista/química , Modelos Moleculares , Conformação Proteica , Especificidade por SubstratoRESUMO
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.
Assuntos
Inibidores da Monoaminoxidase/química , Monoaminoxidase/metabolismo , Doenças Neurodegenerativas/tratamento farmacológico , Fármacos Neuroprotetores/química , Acetilcolinesterase/metabolismo , Homólogo AlkB 2 da Dioxigenase Dependente de alfa-Cetoglutarato/metabolismo , Desenho de Fármacos , Humanos , Hidrazinas/química , Simulação de Acoplamento Molecular , Inibidores da Monoaminoxidase/farmacologia , Fármacos Neuroprotetores/farmacologia , Relação Estrutura-AtividadeRESUMO
AlkB is a bacterial Fe(II)- and 2-oxoglutarate-dependent dioxygenase that repairs a wide range of alkylated nucleobases in DNA and RNA as part of the adaptive response to exogenous nucleic acid-alkylating agents. Although there has been longstanding interest in the structure and specificity of Escherichia coli AlkB and its homologs, difficulties in assaying their repair activities have limited our understanding of their substrate specificities and kinetic mechanisms. Here, we used quantitative kinetic approaches to determine the transient kinetics of recognition and repair of alkylated DNA by AlkB. These experiments revealed that AlkB is a much faster alkylation repair enzyme than previously reported and that it is significantly faster than DNA repair glycosylases that recognize and excise some of the same base lesions. We observed that whereas 1,N6-ethenoadenine can be repaired by AlkB with similar efficiencies in both single- and double-stranded DNA, 1-methyladenine is preferentially repaired in single-stranded DNA. Our results lay the groundwork for future studies of AlkB and its human homologs ALKBH2 and ALKBH3.
Assuntos
Enzimas AlkB/química , Reparo do DNA , DNA Bacteriano/química , Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , Enzimas AlkB/genética , Homólogo AlkB 2 da Dioxigenase Dependente de alfa-Cetoglutarato/química , Homólogo AlkB 2 da Dioxigenase Dependente de alfa-Cetoglutarato/genética , Homólogo AlkB 3 da Dioxigenase Dependente de alfa-Cetoglutarato/química , Homólogo AlkB 3 da Dioxigenase Dependente de alfa-Cetoglutarato/genética , DNA/química , DNA/genética , DNA Bacteriano/genética , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , HumanosRESUMO
1,N6-ethenoadenine (εA) is a mutagenic lesion and biomarker observed in numerous cancerous tissues. Two pathways are responsible for its repair: base excision repair (BER) and direct reversal repair (DRR). Alkyladenine DNA glycosylase (AAG) is the primary enzyme that excises εA in BER, generating stable intermediates that are processed by downstream enzymes. For DRR, the Fe(II)/α-ketoglutarate-dependent ALKBH2 enzyme repairs εA by direct conversion of εA to A. While the molecular mechanism of each enzyme is well understood on unpackaged duplex DNA, less is known about their actions on packaged DNA. The nucleosome core particle (NCP) forms the minimal packaging unit of DNA in eukaryotic organisms and is composed of 145-147 base pairs wrapped around a core of eight histone proteins. In this work, we investigated the activity of AAG and ALKBH2 on εA lesions globally distributed at positions throughout a strongly positioned NCP. Overall, we examined the repair of εA at 23 unique locations in packaged DNA. We observed a strong correlation between rotational positioning of εA and AAG activity but not ALKBH2 activity. ALKBH2 was more effective than AAG at repairing occluded εA lesions, but only AAG was capable of full repair of any εA in the NCP. However, notable exceptions to these trends were observed, highlighting the complexity of the NCP as a substrate for DNA repair. Modeling of binding of the repair enzymes to NCPs revealed that some of these observations can be explained by steric interference caused by DNA packaging. Specifically, interactions between ALKBH2 and the histone proteins obstruct binding to DNA, which leads to diminished activity. Taken together, these results support in vivo observations of alkylation damage profiles and contribute to our understanding of mutational hotspots.
Assuntos
Adenina/análogos & derivados , Reparo do DNA , Homólogo AlkB 2 da Dioxigenase Dependente de alfa-Cetoglutarato/química , DNA/química , DNA Glicosilases/química , Modelos Moleculares , NucleossomosRESUMO
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.
Assuntos
Homólogo AlkB 2 da Dioxigenase Dependente de alfa-Cetoglutarato/metabolismo , Homólogo AlkB 3 da Dioxigenase Dependente de alfa-Cetoglutarato/metabolismo , Dano ao DNA , DNA de Cadeia Simples/genética , Reparo do DNA , Replicação do DNA , DNA de Cadeia Simples/efeitos dos fármacos , Humanos , Recombinação Genética , Proteína de Replicação A/metabolismoRESUMO
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.
Assuntos
5-Metilcitosina/análogos & derivados , 5-Metilcitosina/metabolismo , Enzimas AlkB/metabolismo , Homólogo AlkB 2 da Dioxigenase Dependente de alfa-Cetoglutarato/metabolismo , Homólogo AlkB 3 da Dioxigenase Dependente de alfa-Cetoglutarato/metabolismo , Citosina/análogos & derivados , Enzimas AlkB/genética , Homólogo AlkB 2 da Dioxigenase Dependente de alfa-Cetoglutarato/genética , Homólogo AlkB 3 da Dioxigenase Dependente de alfa-Cetoglutarato/genética , Animais , Biologia Computacional , Ilhas de CpG , Citosina/metabolismo , DNA/genética , Metilação de DNA , Epigênese Genética , Humanos , Estrutura Molecular , OxirreduçãoRESUMO
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.
Assuntos
Homólogo AlkB 2 da Dioxigenase Dependente de alfa-Cetoglutarato/antagonistas & inibidores , Reparo do DNA , Inibidores Enzimáticos/farmacologia , Taninos Hidrolisáveis/farmacologia , Quelantes de Ferro/farmacologia , Homólogo AlkB 2 da Dioxigenase Dependente de alfa-Cetoglutarato/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/química , Humanos , Taninos Hidrolisáveis/química , Quelantes de Ferro/química , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
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.
Assuntos
Homólogo AlkB 2 da Dioxigenase Dependente de alfa-Cetoglutarato/química , Reparo do DNA , Resistencia a Medicamentos Antineoplásicos , Corantes Fluorescentes/química , Homólogo AlkB 2 da Dioxigenase Dependente de alfa-Cetoglutarato/genética , Homólogo AlkB 2 da Dioxigenase Dependente de alfa-Cetoglutarato/metabolismo , Alquilação , Antineoplásicos Alquilantes/uso terapêutico , Corantes Fluorescentes/metabolismo , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Cinética , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Espectrometria de Fluorescência , Temozolomida/uso terapêuticoRESUMO
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.
Assuntos
Homólogo AlkB 2 da Dioxigenase Dependente de alfa-Cetoglutarato/metabolismo , Homólogo AlkB 3 da Dioxigenase Dependente de alfa-Cetoglutarato/metabolismo , Glutaratos/metabolismo , Homólogo AlkB 2 da Dioxigenase Dependente de alfa-Cetoglutarato/antagonistas & inibidores , Homólogo AlkB 2 da Dioxigenase Dependente de alfa-Cetoglutarato/genética , Homólogo AlkB 3 da Dioxigenase Dependente de alfa-Cetoglutarato/antagonistas & inibidores , Homólogo AlkB 3 da Dioxigenase Dependente de alfa-Cetoglutarato/genética , Sequência de Bases , Cromatografia Líquida de Alta Pressão , Reparo do DNA , Ensaios Enzimáticos , Glutaratos/análise , Glutaratos/química , Humanos , Concentração Inibidora 50 , Ácidos Cetoglutáricos/análise , Ácidos Cetoglutáricos/química , Ácidos Cetoglutáricos/metabolismo , Cinética , Ligação Proteica , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , EstereoisomerismoRESUMO
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.
Assuntos
Homólogo AlkB 2 da Dioxigenase Dependente de alfa-Cetoglutarato/metabolismo , Homólogo AlkB 3 da Dioxigenase Dependente de alfa-Cetoglutarato/metabolismo , Adutos de DNA/metabolismo , DNA Glicosilases/metabolismo , Alquilação , Animais , Linhagem Celular , Adutos de DNA/química , Adutos de DNA/genética , Reparo do DNA , Humanos , Camundongos , Timidina/análogos & derivados , Timidina/química , Timidina/genética , Timidina/metabolismoRESUMO
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.
Assuntos
Antraquinonas/farmacologia , Enzimas Reparadoras do DNA/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Proteínas de Escherichia coli/antagonistas & inibidores , Oxigenases de Função Mista/antagonistas & inibidores , Homólogo AlkB 2 da Dioxigenase Dependente de alfa-Cetoglutarato/antagonistas & inibidores , Homólogo AlkB 2 da Dioxigenase Dependente de alfa-Cetoglutarato/genética , Homólogo AlkB 2 da Dioxigenase Dependente de alfa-Cetoglutarato/metabolismo , Homólogo AlkB 3 da Dioxigenase Dependente de alfa-Cetoglutarato/antagonistas & inibidores , Homólogo AlkB 3 da Dioxigenase Dependente de alfa-Cetoglutarato/genética , Homólogo AlkB 3 da Dioxigenase Dependente de alfa-Cetoglutarato/metabolismo , Antraquinonas/química , Domínio Catalítico , Linhagem Celular , Cristalografia por Raios X , Dano ao DNA , Metilação de DNA , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Inibidores Enzimáticos/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Humanos , Metanossulfonato de Metila/farmacologia , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Modelos Moleculares , Interferência de RNA , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
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.
Assuntos
Homólogo AlkB 2 da Dioxigenase Dependente de alfa-Cetoglutarato/metabolismo , Homólogo AlkB 3 da Dioxigenase Dependente de alfa-Cetoglutarato/metabolismo , Adutos de DNA/metabolismo , DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Guanina/análogos & derivados , Oxigenases de Função Mista/metabolismo , Timina/análogos & derivados , DNA/química , Adutos de DNA/química , Reparo do DNA , Escherichia coli/química , Guanina/química , Guanina/metabolismo , Humanos , Especificidade por Substrato , Timina/química , Timina/metabolismoRESUMO
Chemotherapy of a combination of DNA alkylating agents, procarbazine and lomustine (CCNU), and a microtubule poison, vincristine, offers a significant benefit to a subset of glioma patients. The benefit of this regimen, known as PCV, was recently linked to IDH mutation that occurs frequently in glioma and produces D-2-hydroxyglutarate (D-2-HG), a competitive inhibitor of α-ketoglutarate (α-KG). We report here that D-2-HG inhibits the α-KG-dependent alkB homolog (ALKBH) DNA repair enzymes. Cells expressing mutant IDH display reduced repair kinetics, accumulate more DNA damages, and are sensitized to alkylating agents. The observed sensitization to alkylating agents requires the catalytic activity of mutant IDH to produce D-2-HG and can be reversed by the deletion of mutant IDH allele or overexpression of ALKBH2 or AKLBH3. Our results suggest that impairment of DNA repair may contribute to tumorigenesis driven by IDH mutations and that alkylating agents may merit exploration for treating IDH-mutated cancer patients.
Assuntos
Alquilantes/toxicidade , Dano ao DNA/efeitos dos fármacos , Enzimas Reparadoras do DNA/metabolismo , Glutaratos/farmacologia , Isocitrato Desidrogenase/metabolismo , Homólogo AlkB 2 da Dioxigenase Dependente de alfa-Cetoglutarato , Homólogo AlkB 3 da Dioxigenase Dependente de alfa-Cetoglutarato , Bussulfano/toxicidade , Linhagem Celular Tumoral , Reparo do DNA/efeitos dos fármacos , Enzimas Reparadoras do DNA/antagonistas & inibidores , Enzimas Reparadoras do DNA/genética , Dioxigenases/antagonistas & inibidores , Dioxigenases/genética , Dioxigenases/metabolismo , Glutaratos/metabolismo , Humanos , Isocitrato Desidrogenase/genética , Ácidos Cetoglutáricos/metabolismo , Mutação , Ligação Proteica , Interferência de RNA , RNA Interferente Pequeno/metabolismoRESUMO
Human AlkB homolog 2 (ALKBH2) is a DNA repair enzyme that catalyzes the direct reversal of DNA methylation damage through oxidative demethylation. While ALKBH2 colocalizes with proliferating cell nuclear antigen (PCNA) in DNA replication foci, it remains unknown whether these two proteins alone form a complex or require additional components for interaction. Here, we demonstrate that ALKBH2 can directly interact with PCNA independent from other cellular factors, and we identify the hydrophobic pocket of PCNA as the key domain mediating this interaction. Moreover, we find that PCNA association with ALKBH2 increases significantly during DNA replication, suggesting that ALKBH2 forms a cell-cycle dependent complex with PCNA. Intriguingly, we show that an ALKBH2 germline variant, as well as a variant found in cancer, display altered interaction with PCNA. Our studies reveal the ALKBH2 binding interface of PCNA and indicate that both germline and somatic ALKBH2 variants could have cellular effects on ALKBH2 function in DNA repair.
Assuntos
Enzimas Reparadoras do DNA/química , Reparo do DNA , Dioxigenases/química , Antígeno Nuclear de Célula em Proliferação/química , Homólogo AlkB 2 da Dioxigenase Dependente de alfa-Cetoglutarato , Substituição de Aminoácidos , Enzimas Reparadoras do DNA/genética , Replicação do DNA , Dioxigenases/genética , Mutação em Linhagem Germinativa , Células HEK293 , Humanos , Interações Hidrofóbicas e Hidrofílicas , Neoplasias/genética , Neoplasias/metabolismo , Antígeno Nuclear de Célula em Proliferação/genética , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Estrutura Terciária de Proteína , Fase SRESUMO
Repair of DNA alkylation damage is critical for genomic stability and involves multiple conserved enzymatic pathways. Alkylation damage resistance, which is critical in cancer chemotherapy, depends on the overexpression of alkylation repair proteins. However, the mechanisms responsible for this upregulation are unknown. Here, we show that an OTU domain deubiquitinase, OTUD4, is a positive regulator of ALKBH2 and ALKBH3, two DNA demethylases critical for alkylation repair. Remarkably, we find that OTUD4 catalytic activity is completely dispensable for this function. Rather, OTUD4 is a scaffold for USP7 and USP9X, two deubiquitinases that act directly on the AlkB proteins. Moreover, we show that loss of OTUD4, USP7, or USP9X in tumor cells makes them significantly more sensitive to alkylating agents. Taken together, this work reveals a novel, noncanonical mechanism by which an OTU family deubiquitinase regulates its substrates, and provides multiple new targets for alkylation chemotherapy sensitization of tumors.