RESUMO
ABSTRACT: N(6)-methyladenosine (m6A) methylation modification is involved in the progression of myocardial infarction (MI). In this study, we investigated the effects of demethylase alkylation repair homolog 5 (ALKBH5) on cell apoptosis and oxidative stress in MI. The ischemia/reperfusion (I/R) injury mouse model and hypoxia/reoxygenation (H/R) cell model were established. The levels of ALKBH5 and mitsugumin 53 (MG53) were measured by quantitative real-time polymerase chain reaction, immunohistochemical, and immunofluorescence analysis. Apoptosis was evaluated by TUNEL assay, flow cytometry, and western blot. Oxidative stress was assessed by antioxidant index kits. Methylation was analyzed by RNA binding protein immunoprecipitation (RIP), MeRIP, and dual-luciferase reporter assay. We observed that ALKBH5 and MG53 were highly expressed in MI. Overexpression of ALKBH5 inhibited H/R-induced cardiomyocyte apoptosis and oxidative stress in vitro, and it inhibited I/R-induced collagen deposition, cardiac function, and apoptosis in vivo. ALKBH5 could bind to MG53, inhibit m6A methylation of MG53, and increase its mRNA stability. Silencing of MG53 counteracted the inhibition of apoptosis and oxidative stress induced by ALKBH5. In conclusion, ALKBH5 suppressed m6A methylation of MG53 and inhibited MG53 degradation to inhibit apoptosis and oxidative stress of cardiomyocytes, thereby attenuating MI. The results provided a theoretical basis that ALKBH5 is a potential target for MI treatment.
Assuntos
Adenosina , Enzimas AlkB , Homólogo AlkB 5 da RNA Desmetilase , Infarto do Miocárdio , Estresse Oxidativo , Animais , Camundongos , Adenina/análogos & derivados , Adenosina/análogos & derivados , Enzimas AlkB/metabolismo , Homólogo AlkB 5 da RNA Desmetilase/metabolismo , Apoptose , Proteínas de Membrana , Metilação , Infarto do Miocárdio/metabolismoRESUMO
Class-II fumarases (fumarate hydratase, FH) are dual-targeted enzymes occurring in the mitochondria and cytosol of all eukaryotes. They are essential components in the DNA damage response (DDR) and, more specifically, protect cells from DNA double-strand breaks. Similarly, the gram-positive bacterium Bacillus subtilis class-II fumarase, in addition to its role in the tricarboxylic acid cycle, participates in the DDR. Escherichia coli harbors three fumarase genes: class-I fumA and fumB and class-II fumC Notably, class-I fumarases show no sequence similarity to class-II fumarases and are of different evolutionary origin. Strikingly, here we show that E. coli fumarase functions are distributed between class-I fumarases, which participate in the DDR, and the class-II fumarase, which participates in respiration. In E. coli, we discover that the signaling molecule, alpha-ketoglutarate (α-KG), has a function, complementing DNA damage sensitivity of fum-null mutants. Excitingly, we identify the E. coli α-KG-dependent DNA repair enzyme AlkB as the target of this interplay of metabolite signaling. In addition to α-KG, fumarate (fumaric acid) is shown to affect DNA damage repair on two different levels, first by directly inhibiting the DNA damage repair enzyme AlkB demethylase activity, both in vitro and in vivo (countering α-KG). The second is a more global effect on transcription, because fum-null mutants exhibit a decrease in transcription of key DNA damage repair genes. Together, these results show evolutionary adaptable metabolic signaling of the DDR, in which fumarases and different metabolites are recruited regardless of the evolutionary enzyme class performing the function.
Assuntos
Dano ao DNA , Escherichia coli/genética , Fumarato Hidratase/metabolismo , Fumaratos/metabolismo , Ácidos Cetoglutáricos/metabolismo , Enzimas AlkB , Bacillus subtilis/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Ciclo do Ácido Cítrico , Quebras de DNA de Cadeia Dupla , DNA Bacteriano/genética , Fumarato Hidratase/química , Genes BacterianosRESUMO
Nucleic acid demethylases of α-ketoglutarate-dependent dioxygenase (AlkB) family can reversibly erase methyl adducts from nucleobases, thus dynamically regulating the methylation status of DNA/RNA and playing critical roles in multiple cellular processes. But little is known about AlkB demethylases in filamentous fungi so far. The present study reports that Monascus purpureus genomes contain a total of five MpAlkB genes. The MpAlkB1 gene was disrupted and complemented through homologous recombination strategy to analyze its biological functions in M. purpureus. MpAlkB1 knockout significantly accelerated the growth of strain, increased biomass, promoted sporulation and cleistothecia development, reduced the content of Monascus pigments (Mps), and strongly inhibited citrinin biosynthesis. The downregulated expression of the global regulator gene LaeA, and genes of Mps biosynthesis gene cluster (BGC) or citrinin BGC in MpAlkB1 disruption strain supported the pleiotropic trait changes caused by MpAlkB1 deletion. These results indicate that MpAlkB1-mediated demethylation of nucleic acid plays important roles in regulating the growth and development, and secondary metabolism in Monascus spp.
Assuntos
Citrinina , Proteínas Fúngicas , Regulação Fúngica da Expressão Gênica , Monascus , Metabolismo Secundário , Monascus/genética , Monascus/metabolismo , Monascus/crescimento & desenvolvimento , Monascus/enzimologia , Metabolismo Secundário/genética , Citrinina/biossíntese , Citrinina/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Pigmentos Biológicos/biossíntese , Pigmentos Biológicos/metabolismo , Esporos Fúngicos/crescimento & desenvolvimento , Esporos Fúngicos/genética , Técnicas de Inativação de Genes , Família Multigênica , Enzimas AlkB/genética , Enzimas AlkB/metabolismo , Metilação de DNARESUMO
Human AlkB homolog 6, ALKBH6, plays key roles in nucleic acid damage repair and tumor therapy. However, no precise structural and functional information are available for this protein. In this study, we determined atomic resolution crystal structures of human holo-ALKBH6 and its complex with ligands. AlkB members bind nucleic acids by NRLs (nucleotide recognition lids, also called Flips), which can recognize DNA/RNA and flip methylated lesions. We found that ALKBH6 has unusual Flip1 and Flip2 domains, distinct from other AlkB family members both in sequence and conformation. Moreover, we show that its unique Flip3 domain has multiple unreported functions, such as discriminating against double-stranded nucleic acids, blocking the active center, binding other proteins, and in suppressing tumor growth. Structural analyses and substrate screening reveal how ALKBH6 discriminates between different types of nucleic acids and may also function as a nucleic acid demethylase. Structure-based interacting partner screening not only uncovered an unidentified interaction of transcription repressor ZMYND11 and ALKBH6 in tumor suppression but also revealed cross talk between histone modification and nucleic acid modification in epigenetic regulation. Taken together, these results shed light on the molecular mechanism underlying ALKBH6-associated nucleic acid damage repair and tumor therapy.
Assuntos
Enzimas AlkB , Proteínas de Ciclo Celular , Proteínas Correpressoras , Proteínas de Ligação a DNA , Enzimas AlkB/genética , Enzimas AlkB/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Correpressoras/metabolismo , DNA/genética , DNA/metabolismo , Reparo do DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Epigênese Genética , Proteínas de Escherichia coli/metabolismo , Humanos , Proteínas/metabolismo , RNA/metabolismoRESUMO
Kinesin family member C1 (KIFC1) is a kinesin-14 motor protein, and its abnormal upregulation promotes the malignant behavior of cancer cells. N6-methyladenosine (m6A) RNA methylation is a common modification of eukaryotic messenger RNA and affects RNA expression. In this study, we explored how KIFC1 regulated head and neck squamous cell carcinoma (HNSCC) tumorigenesis and how m6A modification affected KIFC1 expression. A bioinformatics analysis was performed to screen for genes of interest, and in vitro and in vivo studies were carried out to investigate the function and mechanism of KIFC1 in HNSCC tissues. We observed that the expression of KIFC1 in HNSCC tissues was significantly higher than that in normal or adjacent normal tissues. Patients with cancer with higher KIFC1 expression have a lower tumor differentiation status. Demethylase alkB homolog 5, a cancer-promoting factor in HNSCC tissues, could interact with KIFC1 messenger RNA and posttranscriptionally activate KIFC1 through m6A modification. KIFC1 downregulation suppressed HNSCC cell growth and metastasis in vivo and in vitro. However, overexpression of KIFC1 promoted these malignant behaviors. We demonstrated that KIFC1 overexpression activated the oncogenic Wnt/ß-catenin pathway. KIFC1 interacted with the small GTPase Ras-related C3 botulinum toxin substrate 1 (Rac1) at the protein level and increased its activity. The Rho GTPase Rac1 was indicated to be an upstream activator of the Wnt/ß-catenin signaling pathway, and its Rac1 inhibitor, NSC-23766, treatment reversed the effects caused by KIFC1 overexpression. Those observations demonstrate that abnormal expression of KIFC1 may be regulated by demethylase alkB homolog 5 in an m6A-dependent manner and promote HNSCC progression via the Rac1/Wnt/ß-catenin pathway.
Assuntos
Neoplasias de Cabeça e Pescoço , Via de Sinalização Wnt , Humanos , Enzimas AlkB/genética , Enzimas AlkB/metabolismo , beta Catenina/genética , beta Catenina/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Família , Regulação Neoplásica da Expressão Gênica , Neoplasias de Cabeça e Pescoço/genética , Cinesinas/genética , Cinesinas/metabolismo , RNA , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Via de Sinalização Wnt/genéticaRESUMO
DNA repair is essential to prevent the cytotoxic or mutagenic effects of various types of DNA lesions, which are sensed by distinct pathways to recruit repair factors specific to the damage type. Although biochemical mechanisms for repairing several forms of genomic insults are well understood, the upstream signalling pathways that trigger repair are established for only certain types of damage, such as double-stranded breaks and interstrand crosslinks. Understanding the upstream signalling events that mediate recognition and repair of DNA alkylation damage is particularly important, since alkylation chemotherapy is one of the most widely used systemic modalities for cancer treatment and because environmental chemicals may trigger DNA alkylation. Here we demonstrate that human cells have a previously unrecognized signalling mechanism for sensing damage induced by alkylation. We find that the alkylation repair complex ASCC (activating signal cointegrator complex) relocalizes to distinct nuclear foci specifically upon exposure of cells to alkylating agents. These foci associate with alkylated nucleotides, and coincide spatially with elongating RNA polymerase II and splicing components. Proper recruitment of the repair complex requires recognition of K63-linked polyubiquitin by the CUE (coupling of ubiquitin conjugation to ER degradation) domain of the subunit ASCC2. Loss of this subunit impedes alkylation adduct repair kinetics and increases sensitivity to alkylating agents, but not other forms of DNA damage. We identify RING finger protein 113A (RNF113A) as the E3 ligase responsible for upstream ubiquitin signalling in the ASCC pathway. Cells from patients with X-linked trichothiodystrophy, which harbour a mutation in RNF113A, are defective in ASCC foci formation and are hypersensitive to alkylating agents. Together, our work reveals a previously unrecognized ubiquitin-dependent pathway induced specifically to repair alkylation damage, shedding light on the molecular mechanism of X-linked trichothiodystrophy.
Assuntos
Enzimas AlkB/metabolismo , Adutos de DNA/metabolismo , Reparo do DNA , Complexos Multiproteicos/metabolismo , Transdução de Sinais , Síndromes de Tricotiodistrofia/genética , Ubiquitina/metabolismo , Homólogo AlkB 3 da Dioxigenase Dependente de alfa-Cetoglutarato/metabolismo , Alquilantes/farmacologia , Alquilação , Sequência de Aminoácidos , Adutos de DNA/química , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Retículo Endoplasmático/metabolismo , Genes Ligados ao Cromossomo X , Humanos , Cinética , Modelos Moleculares , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Poliubiquitina/metabolismo , RNA Polimerase II/metabolismo , Splicing de RNA , Síndromes de Tricotiodistrofia/metabolismo , Síndromes de Tricotiodistrofia/patologia , UbiquitinaçãoRESUMO
ALKBH proteins, the homologs of Escherichia coli AlkB dioxygenase, constitute a single-protein repair system that safeguards cellular DNA and RNA against the harmful effects of alkylating agents. ALKBH10B, the first discovered N6-methyladenosine (m6A) demethylase in Arabidopsis (Arabidopsis thaliana), has been shown to regulate plant growth, development, and stress responses. However, until now, the functional role of the plant ALKBH10B has solely been reported in arabidopsis, cotton, and poplar, leaving its functional implications in other plant species shrouded in mystery. In this study, we identified the AlkB homolog SlALKBH10B in tomato (Solanum lycopersicum) through phylogenetic and gene expression analyses. SlALKBH10B exhibited a wide range of expression patterns and was induced by exogenous abscisic acid (ABA) and abiotic stresses. By employing CRISPR/Cas9 gene editing techniques to knock out SlALKBH10B, we observed an increased sensitivity of mutants to ABA treatment and upregulation of gene expression related to ABA synthesis and response. Furthermore, the Slalkbh10b mutants displayed an enhanced tolerance to drought and salt stress, characterized by higher water retention, accumulation of photosynthetic products, proline accumulation, and lower levels of reactive oxygen species and cellular damage. Collectively, these findings provide insights into the negative impact of SlALKBH10B on drought and salt tolerance in tomato plant, expanding our understanding of the biological functionality of SlALKBH10B.
Assuntos
Arabidopsis , Proteínas de Escherichia coli , Solanum lycopersicum , Tolerância ao Sal/genética , Secas , Filogenia , Solanum lycopersicum/genética , Ácido Abscísico , Escherichia coli , Enzimas AlkB , Oxigenases de Função MistaRESUMO
RNA N6-methyladenosine (m6A) is essential for many bioprocesses in many species, but its role in goat testis development remains elusive, especially alkB homolog 5 (ALKBH5), one of the m6A demethylases. To this end, nine healthy Haimen goats of different ages were chosen randomly to provide testes. The results showed that the expression level of ALKBH5 was increased significantly (P < 0.05) in the 9-month group compared with the 0-day and 3-month groups, and ALKBH5 was located in goat spermatocytes with the highest expression level compared with Leydig cells and Sertoli cells. Thus, pcDNA3.1-ALKBH5 was constructed to explore the influences of the ALKBH5 increase in goat spermatogonial stem cells (SSC) in vitro. The results showed that the expression level of ALKBH5 in SSC transfected with pcDNA3.1-ALKBH5 (OE_ALKBH5) was significantly increased (P < 0.001) compared with that in SSC transfected with pcDNA3.1-EGFP (EGFP). With ALKBH5 overexpression in SSC, flow cytometry analysis showed that cells at G1 phase were significantly reduced (P < 0.01), while cells at S phase significantly increased (P < 0.01), and cell apoptosis was inhibited. Accordingly, the mRNA degradation of CCND1, CCNE1, and BCL2 was suppressed with ALKBH5 overexpression in SSC after treatment with actinomycin D. Furthermore, the mRNA levels of pluripotency maintenance- and cell differentiation-associated genes were changed between the two groups. Overall, the results indicated the crucial role of ALKBH5 during Haimen goat testis development. The results of this study provide a theoretical basis and technical means for RNA methylation participating in goat testis development.
Assuntos
Células-Tronco Germinativas Adultas/metabolismo , Enzimas AlkB/metabolismo , Espermatogônias/metabolismo , Testículo/fisiologia , Animais , Diferenciação Celular , Cabras , Humanos , Masculino , TransfecçãoRESUMO
AlkB homologs (ALKBH) are a family of specific demethylases that depend on Fe2+ and α-ketoglutarate to catalyze demethylation on different substrates, including ssDNA, dsDNA, mRNA, tRNA, and proteins. Previous studies have made great progress in determining the sequence, structure, and molecular mechanism of the ALKBH family. Here, we first review the multi-substrate selectivity of the ALKBH demethylase family from the perspective of sequence and structural evolution. The construction of the phylogenetic tree and the comparison of key loops and non-homologous domains indicate that the paralogs with close evolutionary relationship have similar domain compositions. The structures show that the lack and variations of four key loops change the shape of clefts to cause the differences in substrate affinity, and non-homologous domains may be related to the compatibility of multiple substrates. We anticipate that the new insights into selectivity determinants of the ALKBH family are useful for understanding the demethylation mechanisms.
Assuntos
Enzimas AlkB/metabolismo , Enzimas AlkB/química , Enzimas AlkB/classificação , Animais , DNA/metabolismo , Reparo do DNA , Humanos , Filogenia , Domínios Proteicos , RNA Mensageiro/metabolismo , RNA de Transferência/metabolismo , Especificidade por SubstratoRESUMO
Emerging evidence shows that m6A, one of the most abundant RNA modifications in mammals, is involved in the entire process of spermatogenesis, including mitosis, meiosis, and spermiogenesis. "Writers" catalyze m6A formation on stage-specific transcripts during male germline development, while "erasers" remove m6A modification to maintain a balance between methylation and demethylation. The different functions of RNA-m6A transcripts depend on their recognition by "readers". m6A modification mediates RNA metabolism, including mRNA splicing, translation, and degradation, as well as the maturity and biosynthesis of non-coding RNAs. Sperm RNA profiles are easily affected by environmental exposure and can even be inherited for several generations, similar to epigenetic inheritance. Here, we review and summarize the critical role of m6A in different developmental stages of male germ cells, to understand of the mechanisms and epigenetic regulation of m6A modifications. In addition, we also outline and discuss the important role of non-coding RNAs in spermatogenesis and RNA modifications in epigenetic inheritance.
Assuntos
Epigênese Genética , RNA/metabolismo , Espermatozoides/metabolismo , Enzimas AlkB/metabolismo , Animais , Humanos , Masculino , Metiltransferases/metabolismo , Fatores de Processamento de Serina-Arginina/metabolismo , EspermatogêneseRESUMO
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
The Fe(II) and 2-oxoglutarate-dependent oxygenase Alkb homologue 1 (Alkbh1) has been shown to act on a wide range of substrates, like DNA, tRNA and histones. Thereby different enzymatic activities have been identified including, among others, demethylation of N3-methylcytosine (m3C) in RNA- and single-stranded DNA oligonucleotides, demethylation of N1-methyladenosine (m1A) in tRNA or formation of 5-formyl cytosine (f5C) in tRNA. In accordance with the different substrates, Alkbh1 has also been proposed to reside in distinct cellular compartments in human and mouse cells, including the nucleus, cytoplasm and mitochondria. Here, we describe further evidence for a role of human Alkbh1 in regulation of mitochondrial protein biogenesis, including visualizing localization of Alkbh1 into mitochondrial RNA granules with super-resolution 3D SIM microscopy. Electron microscopy and high-resolution respirometry analyses revealed an impact of Alkbh1 level on mitochondrial respiration, but not on mitochondrial structure. Downregulation of Alkbh1 impacts cell growth in HeLa cells and delays development in Caenorhabditis elegans, where the mitochondrial role of Alkbh1 seems to be conserved. Alkbh1 knockdown, but not Alkbh7 knockdown, triggers the mitochondrial unfolded protein response (UPRmt) in C. elegans.
Assuntos
Homólogo AlkB 1 da Histona H2a Dioxigenase/metabolismo , Mitocôndrias/metabolismo , RNA Mitocondrial/metabolismo , Células A549 , Enzimas AlkB/genética , Enzimas AlkB/metabolismo , Homólogo AlkB 1 da Histona H2a Dioxigenase/genética , Animais , Caenorhabditis elegans , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Eletroforese em Gel de Poliacrilamida , Células HEK293 , Células HT29 , Células HeLa , Humanos , Camundongos , Microscopia Eletrônica , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Consumo de Oxigênio/fisiologia , Fator Tu de Elongação de Peptídeos/genética , Fator Tu de Elongação de Peptídeos/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Resposta a Proteínas não Dobradas/genética , Resposta a Proteínas não Dobradas/fisiologiaRESUMO
Esophageal squamous cell carcinoma (ESCC) is one of the most fatal types of malignant tumors worldwide. Epitranscriptome, such as N6 -methyladenosine (m6 A) of mRNA, is an abundant post-transcriptional mRNA modification and has been recently implicated to play roles in several cancers, whereas the significance of m6 A modifications is virtually unknown in ESCC. Analysis of tissue microarray of the tumors in 177 ESCC patients showed that higher expression of m6 A demethylase ALKBH5 correlated with poor prognosis and that ALKBH5 was an independent prognostic factor of the survival of patients. There was no correlation between the other demethylase FTO and prognosis. siRNA knockdown of ALKBH5 but not FTO significantly suppressed proliferation and migration of human ESCC cells. ALKBH5 knockdown delayed progression of cell cycle and accumulated the cells to G0/G1 phase. Mechanistically, expression of CDKN1A (p21) was significantly up-regulated in ALKBH5-depleted cells, and m6 A modification and stability of CDKN1A mRNA were increased by ALKBH5 knockdown. Furthermore, depletion of ALKBH5 substantially suppressed tumor growth of ESCC cells subcutaneously transplanted in BALB/c nude mice. Collectively, we identify ALKBH5 as the first m6 A demethylase that accelerates cell cycle progression and promotes cell proliferation of ESCC cells, which is associated with poor prognosis of ESCC patients.
Assuntos
Homólogo AlkB 5 da RNA Desmetilase/metabolismo , Carcinoma de Células Escamosas do Esôfago/genética , Adenosina/análogos & derivados , Adenosina/genética , Adenosina/metabolismo , Adulto , Idoso , Enzimas AlkB/metabolismo , Homólogo AlkB 5 da RNA Desmetilase/genética , Dioxigenase FTO Dependente de alfa-Cetoglutarato/metabolismo , Animais , Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Progressão da Doença , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patologia , Carcinoma de Células Escamosas do Esôfago/metabolismo , Carcinoma de Células Escamosas do Esôfago/patologia , Feminino , Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Prognóstico , Estabilidade de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
DNA alkylation is used as the key epigenetic mark in eukaryotes, however, most alkylation in DNA can result in deleterious effects. Therefore, this process needs to be tightly regulated. The enzymes of the AlkB and Ten-Eleven Translocation (TET) families are members of the Fe and alpha-ketoglutarate-dependent superfamily of enzymes that are tasked with dealkylating DNA and RNA in cells. Members of these families span all species and are an integral part of transcriptional regulation. While both families catalyze oxidative dealkylation of various bases, each has specific preference for alkylated base type as well as distinct catalytic mechanisms. This perspective aims to provide an overview of computational work carried out to investigate several members of these enzyme families including AlkB, ALKB Homolog 2, ALKB Homolog 3 and Ten-Eleven Translocate 2. Insights into structural details, mutagenesis studies, reaction path analysis, electronic structure features in the active site, and substrate preferences are presented and discussed.
Assuntos
Enzimas AlkB/metabolismo , Proteínas de Escherichia coli/metabolismo , Ferro/metabolismo , Ácidos Cetoglutáricos/metabolismo , Simulação de Dinâmica Molecular , Enzimas AlkB/química , Escherichia coli/enzimologia , Proteínas de Escherichia coli/química , Ferro/química , Ácidos Cetoglutáricos/químicaRESUMO
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
Programmed necrosis has emerged as a crucial modulator of cell death in response to several forms of cellular stress. In one form of programmed necrotic cell death, induced by cytotoxic alkylating agents, hyperactivation of poly-ADP-ribose polymerase (PARP) leads to cellular NAD and ATP depletion, mitochondrial dysfunction, reactive oxygen species formation, and ensuing cell death. Here, we show that the protein encoded by the human AlkB homolog 7 (ALKBH7) gene plays a pivotal role in DNA-damaging agent-induced programmed necrosis by triggering the collapse of mitochondrial membrane potential and large-scale loss of mitochondrial function that lead to energy depletion and cellular demise. Depletion of ALKBH7 suppresses necrotic cell death induced by numerous alkylating and oxidizing agents while having no effect on apoptotic cell death. Like wild-type cells, ALKBH7-depleted cells undergo PARP hyperactivation and NAD depletion after severe DNA damage but, unlike wild-type cells, exhibit rapid recovery of intracellular NAD and ATP levels. Consistent with the recovery of cellular bioenergetics, ALKBH7-depleted cells maintain their mitochondrial membrane potential, plasma membrane integrity, and viability. Our results uncover a novel role for a mammalian AlkB homolog in programmed necrosis, presenting a new target for therapeutic intervention in cancer cells that are resistant to apoptotic cell death.
Assuntos
Apoptose , Dano ao DNA , Proteínas Mitocondriais/metabolismo , Proteínas Nucleares/metabolismo , Estresse Oxidativo , Trifosfato de Adenosina/metabolismo , Enzimas AlkB , Alquilação , Apoptose/efeitos dos fármacos , Linhagem Celular , Dano ao DNA/efeitos dos fármacos , Resistência a Medicamentos , Metabolismo Energético , Ativação Enzimática , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Proteínas Mitocondriais/deficiência , NAD/metabolismo , Necrose/metabolismo , Necrose/patologia , Proteínas Nucleares/deficiência , Oxirredução , Poli(ADP-Ribose) Polimerases/metabolismo , Transporte ProteicoRESUMO
Site-specific DNA methylation plays an important role in epigenetic regulation of gene expression. Chemical methylation of DNA, including the formation of various methylated nitrogenous bases, leads to the formation of genotoxic modifications that impair DNA functions. Despite the fact that different pathways give rise to methyl groups in DNA, the main pathway for their removal is oxidative demethylation, which is catalyzed by nonheme Fe(II)/α-ketoglutarate-dependent DNA dioxygenases. DNA dioxygenases share a common catalytic mechanism of the oxidation of the alkyl groups on nitrogenous bases in nucleic acids. This review presents generalized data on the catalytic mechanism of action of DNA dioxygenases and on the participation of typical representatives of this superfamily, such as prokaryotic enzyme AlkB and eukaryotic enzymes ALKBH1-8 and TET1-3, in both processes of direct repair of alkylated DNA adducts and in the removal of an epigenetic mark (5-methylcytosine).
Assuntos
Enzimas AlkB , Metilação de DNA , Reparo do DNA , Epigênese Genética , Enzimas AlkB/química , Enzimas AlkB/metabolismo , Animais , HumanosRESUMO
The α-ketoglutarate-dependent (AlkB) superfamily of FeII/2-oxoglutarate (2-OG)-dependent dioxygenases consists of a unique class of nucleic acid repair enzymes that reversibly remove alkyl substituents from nucleobases through oxidative dealkylation. Recent studies have verified the involvement of AlkB dioxygenases in a variety of human diseases. However, the development of small organic molecules that can function as enzyme inhibitors to block the action of oxidative dealkylation is still in its infancy. These purposeful chemical motifs, if capable of influencing the dealkylation activity, would have a potential clinical value by controlling genetic information expression. In this Perspective, we will summarize some of the most updated inhibitors of AlkB family demethylases and hope to provide a thought for the follow-up screening optimization.
Assuntos
Enzimas AlkB/genética , Inibidores Enzimáticos/farmacologia , Complexo Cetoglutarato Desidrogenase/genética , Enzimas AlkB/antagonistas & inibidores , Enzimas AlkB/química , Dano ao DNA/efeitos dos fármacos , Metilação de DNA/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Complexo Cetoglutarato Desidrogenase/antagonistas & inibidores , Ácidos Cetoglutáricos/antagonistas & inibidoresRESUMO
Driven by oncogenic signaling, glutamine addiction exhibited by cancer cells often leads to severe glutamine depletion in solid tumors. Despite this nutritional environment that tumor cells often experience, the effect of glutamine deficiency on cellular responses to DNA damage and chemotherapeutic treatment remains unclear. Here, we show that glutamine deficiency, through the reduction of alpha-ketoglutarate, inhibits the AlkB homolog (ALKBH) enzymes activity and induces DNA alkylation damage. As a result, glutamine deprivation or glutaminase inhibitor treatment triggers DNA damage accumulation independent of cell death. In addition, low glutamine-induced DNA damage is abolished in ALKBH deficient cells. Importantly, we show that glutaminase inhibitors, 6-Diazo-5-oxo-L-norleucine (DON) or CB-839, hypersensitize cancer cells to alkylating agents both in vitro and in vivo. Together, the crosstalk between glutamine metabolism and the DNA repair pathway identified in this study highlights a potential role of metabolic stress in genomic instability and therapeutic response in cancer.
Assuntos
Enzimas AlkB/antagonistas & inibidores , Antineoplásicos Alquilantes/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Inibidores Enzimáticos/uso terapêutico , Glutaminase/antagonistas & inibidores , Proteínas de Neoplasias/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Enzimas AlkB/genética , Enzimas AlkB/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 , Alquilação/efeitos dos fármacos , Animais , Antineoplásicos Alquilantes/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Dano ao DNA , Inibidores Enzimáticos/farmacologia , Glutaminase/metabolismo , Humanos , Masculino , Camundongos , Camundongos Nus , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Interferência de RNA , Distribuição Aleatória , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
RNA methylation and demethylation, which is mediated by RNA methyltransferases (referred to as "writers") and demethylases (referred to as "erasers"), respectively, are emerging as a key regulatory process in plant development and stress responses. Although several studies have shown that AlkB homolog (ALKBH) proteins are potential RNA demethylases, the function of most ALKBHs is yet to be determined. The Arabidopsis thaliana genome contains thirteen genes encoding ALKBH proteins, the functions of which are largely unknown. In this study, we characterized the function of a potential eraser protein, ALKBH6 (At4g20350), during seed germination and seedling growth in Arabidopsis under abiotic stresses. The seeds of T-DNA insertion alkbh6 knockdown mutants germinated faster than the wild-type seeds under cold, salt, or abscisic acid (ABA) treatment conditions but not under dehydration stress conditions. Although no differences in seedling and root growth were observed between the alkbh6 mutant and wild-type under normal conditions, the alkbh6 mutant showed a much lower survival rate than the wild-type under salt, drought, or heat stress. Cotyledon greening of the alkbh6 mutants was much higher than that of the wild-type upon ABA application. Moreover, the transcript levels of ABA signaling-related genes, including ABI3 and ABI4, were down-regulated in the alkbh6 mutant compared to wild-type plants. Importantly, the ALKBH6 protein had an ability to bind to both m6A-labeled and m5C-labeled RNAs. Collectively, these results indicate that the potential eraser ALKBH6 plays important roles in seed germination, seedling growth, and survival of Arabidopsis under abiotic stresses.