RESUMEN
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.
Asunto(s)
Citrinina , Proteínas Fúngicas , Regulación Fúngica de la Expresión Génica , Monascus , Metabolismo Secundario , Monascus/genética , Monascus/metabolismo , Monascus/crecimiento & desarrollo , Monascus/enzimología , Metabolismo Secundario/genética , Citrinina/biosíntesis , Citrinina/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Pigmentos Biológicos/biosíntesis , Pigmentos Biológicos/metabolismo , Esporas Fúngicas/crecimiento & desarrollo , Esporas Fúngicas/genética , Técnicas de Inactivación de Genes , Familia de Multigenes , Enzimas AlkB/genética , Enzimas AlkB/metabolismo , Metilación de ADNRESUMEN
BACKGROUND: The oncogenic role of circPUM1 has been revealed in multiple cancers. Nevertheless, the specific role and molecular mechanism of circPUM1 in neuroblastoma (NB) have never been reported. METHODS: The expression of genes was detected using RT-qPCR and Western Blot assay. The proliferation, migration, and invasion of NB cells were evaluated by CCK-8 and Transwell assays. Besides, mouse model was established to evaluate the effect of circPUM1 on the progression of NB. The interaction among genes was verified through RIP, MeRIP, or Luciferase reporter assay. RESULTS: Through our investigation, it was discovered that circPUM1 expression was abnormally elevated in NB tissues and the abundance of circPUM1 was correlated with unfavorable clinical outcomes in NB patients. Besides, the viability and mobility of NB cells as well as NB tumor growth were suppressed by silencing circPUM1. Moreover, bioinformatics prediction and experimental verification demonstrated that circPUM1 was a sponge for miR-423-5p which further targeted proliferation-associated protein 2G4 (PA2G4). The oncogenic effect of circPUM1 on NB was exerted through suppressing miR-423-5p to elevate PA2G4 expression. Finally, we investigated the transcriptional factor causing the upregulation of circPUM1 in NB. The result was that ALKB homolog 5 (ALKBH5), an m6A demethylase, suppressed the m6A modification of circPUM1 and caused the elevation of circPUM1 expression in NB. CONCLUSION: ALKBH5 induced the upregulation of circPUM1 to accelerate the development of NB through regulating miR-423-5p/PA2G4 axis.
Asunto(s)
MicroARNs , Neuroblastoma , Animales , Ratones , MicroARNs/genética , MicroARNs/metabolismo , Regulación hacia Arriba , Proliferación Celular/genética , Neuroblastoma/metabolismo , Enzimas AlkB/genética , Enzimas AlkB/metabolismo , Regulación Neoplásica de la Expresión Génica/genética , Línea Celular TumoralRESUMEN
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.
Asunto(s)
Neoplasias de Cabeza y Cuello , Vía de Señalización Wnt , Humanos , Enzimas AlkB/genética , Enzimas AlkB/metabolismo , beta Catenina/genética , beta Catenina/metabolismo , Línea Celular Tumoral , Proliferación Celular , Familia , Regulación Neoplásica de la Expresión Génica , Neoplasias de Cabeza y Cuello/genética , Cinesinas/genética , Cinesinas/metabolismo , ARN , Carcinoma de Células Escamosas de Cabeza y Cuello/genética , Vía de Señalización Wnt/genéticaRESUMEN
Background: RNA-binding proteins (RBPs) have important roles in orchestrating posttranscriptional regulation and modulating many tumorigenesis events. SERBP1 has been recognized as an important regulator in multiple cancers, while it remains unclear whether SERBP1-regulated gene expression at the transcriptome-wide level is significantly correlated with tumorigenesis. Methods: We overexpressed SERBP1 in HeLa cells and explored whether SERBP1 overexpression (SERBP1-OE) affects the proliferation and apoptosis of HeLa cells. We analyzed the transcriptome-wide gene expression changes and alternative splicing changes mediated by SERBP1-OE using the transcriptome sequencing method (RNA-seq). RT-qPCR was conducted to assay SERBP1-regulated alternative splicing. Results: SERBP1-OE induced the apoptosis of HeLa cells. The downregulated genes were strongly enriched in the cell proliferation and apoptosis pathways according to the GO analysis, including FOS, FOSB, PAK6 and RAB26. The genes undergoing at least one SERBP1-regulated alternative splicing event were enriched in transcriptional regulation, suggesting a mechanism of the regulation of gene expression, and in pyruvate and fatty acid metabolic processes critical for tumorigenesis events. The SERBP1-regulated alternative splicing of ME3, LPIN3, CROT, PDP1, SLC27A1 and ALKBH7 was validated by RT-qPCR analysis. Conclusions: We for the first time demonstrated the cellular function and molecular targets of SERBP1 in HeLa cells at transcriptional and post-transcriptional levels. The SERBP1-regulated gene expression and alternative splicing networks revealed by this study provide important information for exploring the functional roles and regulatory mechanisms of SERBP1 in cancer development and progression.
Asunto(s)
Empalme Alternativo , Transcriptoma , Humanos , Empalme Alternativo/genética , Células HeLa , Proliferación Celular/genética , Carcinogénesis , Enzimas AlkB/genética , Proteínas Mitocondriales/genéticaRESUMEN
Breast cancer is the most common lethal carcinoma worldwide and better targeted therapies are still worthy of exploration, having had some great successes already. Abnormal expression of ALKBH members were found in various cancers, and the roles played by it were the focus of attention. The ALKBH gene family encodes nine homologous enzymes (ALKBH1-8 and FTO) to repair DNA or RNA depending on Fe2+ and α-ketoglutarate (α-KG), which is related to carcinogenesis. In this study, we applied several databases to explore the roles of ALKBHs in breast cancer. We found that ALKBH members were abnormal expression in breast cancer and associated with tumor stage and subclasses. Higher alteration rates of ALKBH family were found in breast cancer. Function enrichment revealed that several cancer-associated signal pathways were related to ALKBH family such as PI3K-Akt signaling pathway and axon guidance. Infiltration of immune cells (Eosinophiles, NK CD56bright cells, mast cells, T helper cells and so on) were strongly related to ALKBHs. Moreover, we further found that there was strong correlation between ALKBH7 and higher age, later T stage, ER/PR positive and post-menopause of breast cancer patients, and patients with higher ALKBH7 expression had shorter overall survival (OS) and post progression survival (PPS). In conclusion, our findings may provide novel insights into ALKBH-targeted therapy for breast cancer patients, and ALKBH7 may be a potential prognostic biomarker.
Asunto(s)
Neoplasias de la Mama , Carcinoma , Enzimas AlkB/genética , Histona H2a Dioxigenasa, Homólogo 1 de AlkB , Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato/genética , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Femenino , Humanos , Proteínas Mitocondriales , Fosfatidilinositol 3-Quinasas , PronósticoRESUMEN
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.
Asunto(s)
Enzimas AlkB , Proteínas de Ciclo Celular , Proteínas Co-Represoras , Proteínas de Unión al ADN , Enzimas AlkB/genética , Enzimas AlkB/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Co-Represoras/metabolismo , ADN/genética , ADN/metabolismo , Reparación del ADN , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Epigénesis Genética , Proteínas de Escherichia coli/metabolismo , Humanos , Proteínas/metabolismo , ARN/metabolismoRESUMEN
Members of the mammalian AlkB family are known to mediate nucleic acid demethylation1,2. ALKBH7, a mammalian AlkB homologue, localizes in mitochondria and affects metabolism3, but its function and mechanism of action are unknown. Here we report an approach to site-specifically detect N1-methyladenosine (m1A), N3-methylcytidine (m3C), N1-methylguanosine (m1G) and N2,N2-dimethylguanosine (m22G) modifications simultaneously within all cellular RNAs, and discovered that human ALKBH7 demethylates m22G and m1A within mitochondrial Ile and Leu1 pre-tRNA regions, respectively, in nascent polycistronic mitochondrial RNA4-6. We further show that ALKBH7 regulates the processing and structural dynamics of polycistronic mitochondrial RNAs. Depletion of ALKBH7 leads to increased polycistronic mitochondrial RNA processing, reduced steady-state mitochondria-encoded tRNA levels and protein translation, and notably decreased mitochondrial activity. Thus, we identify ALKBH7 as an RNA demethylase that controls nascent mitochondrial RNA processing and mitochondrial activity.
Asunto(s)
Enzimas AlkB/metabolismo , Mitocondrias/enzimología , Proteínas Mitocondriales/metabolismo , Procesamiento Postranscripcional del ARN , ARN Mitocondrial/metabolismo , Adenosina/análogos & derivados , Adenosina/metabolismo , Enzimas AlkB/genética , Citidina/análogos & derivados , Citidina/metabolismo , Guanosina/análogos & derivados , Guanosina/metabolismo , Células HEK293 , Células HeLa , Células Hep G2 , Humanos , Mitocondrias/genética , Proteínas Mitocondriales/genética , Biosíntesis de Proteínas , ARN Mitocondrial/genética , ARN de Transferencia/genética , ARN de Transferencia/metabolismoRESUMEN
AlkB family of Fe (II) and α-ketoglutarate-dependent dioxygenases plays essential roles in development of ovarian serous carcinoma (OV). However, the molecular profiles of AlkB family in OV have not been clarified. The results indicated that the expression of ALKBH1/3/5/8 and FTO was lower in OV patients while ALKBH2/4/6/7 expression was higher. There was a strong correlation between ALKBH5/7 and pathological stage of OV patients. Kaplan-Meier plotter revealed that OV patients with high ALKBH4 level showed longer overall survival (OS). However, patients with high levels of ALKBH5/6 and FTO showed shorter OS and progression-free survival (PFS). Genetic alterations using cBioPortal revealed that the alteration rates of FTO were the highest. We also found that the functions of AlkB family were linked to several cancer-associated signaling pathways, including chemokine receptor signaling. TIMER database indicated that the AlkB family had a strong relationship with the infiltration of six types of immune cells (macrophages, neutrophils, CD8+ T-cells, B-cells, CD4+ T-cells and dendritic cells). Next, DiseaseMeth databases revealed that the global methylation levels of ALKBH1/2/3/4/5/6/7/8 and FTO were all lower in OV patients. Thus, our findings will enhance the understanding of AlkB family in OV pathology, and provide novel insights into AlkB-targeted therapy for OV patients.
Asunto(s)
Enzimas AlkB/metabolismo , Cistadenocarcinoma Seroso/metabolismo , Regulación Neoplásica de la Expresión Génica , Neoplasias Ováricas/metabolismo , Enzimas AlkB/genética , Cistadenocarcinoma Seroso/genética , Cistadenocarcinoma Seroso/patología , Metilación de ADN , Bases de Datos Genéticas , Femenino , Perfilación de la Expresión Génica , Humanos , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Pronóstico , Tasa de SupervivenciaRESUMEN
Petroleum-contaminated soil is considered among the most important potential anthropogenic atmospheric methane sources. Additionally, various rhizoremediation factors can affect methane emissions by altering soil ecosystem carbon cycles. Nonetheless, greenhouse gas emissions from soil have not been given due importance as a potentially relevant parameter in rhizoremediation techniques. Therefore, in this study we sought to investigate the effects of different plant and soil amendments on both remediation efficiencies and methane emission characteristics in dieselcontaminated soil. An indoor pot experiment consisting of three plant treatments (control, maize, tall fescue) and two soil amendments (chemical nutrient, compost) was performed for 95 days. Total petroleum hydrocarbon (TPH) removal efficiency, dehydrogenase activity, and alkB (i.e., an alkane compound-degrading enzyme) gene abundance were the highest in the tall fescue and maize soil system amended with compost. Compost addition enhanced both the overall remediation efficiencies, as well as pmoA (i.e., a methane-oxidizing enzyme) gene abundance in soils. Moreover, the potential methane emission of diesel-contaminated soil was relatively low when maize was introduced to the soil system. After microbial community analysis, various TPH-degrading microorganisms (Nocardioides, Marinobacter, Immitisolibacter, Acinetobacter, Kocuria, Mycobacterium, Pseudomonas, Alcanivorax) and methane-oxidizing microorganisms (Methylocapsa, Methylosarcina) were observed in the rhizosphere soil. The effects of major rhizoremediation factors on soil remediation efficiency and greenhouse gas emissions discussed herein are expected to contribute to the development of sustainable biological remediation technologies in response to global climate change.
Asunto(s)
Restauración y Remediación Ambiental , Metano/metabolismo , Petróleo , Fenómenos Fisiológicos de las Plantas , Contaminantes del Suelo , Suelo/química , Enzimas AlkB/genética , Biodegradación Ambiental , Compostaje , Hidrocarburos , Microbiota , Plantas , Pseudomonas , Rizosfera , Microbiología del SueloRESUMEN
Iron-dependent dioxygenases of the AlkB protein family found in most organisms throughout the tree of life play a major role in oxidative dealkylation processes. Many of these enzymes have attracted the attention of researchers across different fields and have been subjected to thorough biochemical characterization because of their link to human health and disease. For example, several mammalian AlkB homologues are involved in the direct reversal of alkylation damage in DNA, while others have been shown to play a regulatory role in epigenetic or epitranscriptomic nucleic acid methylation or in post-translational modifications such as acetylation of actin filaments. These studies show that that divergence in amino acid sequence and structure leads to different characteristics and substrate specificities. In this review, we aim to summarize current insights in the structural features involved in the substrate selection of AlkB homologues, with focus on nucleic acid interactions.
Asunto(s)
Enzimas AlkB/metabolismo , Enzimas AlkB/química , Enzimas AlkB/genética , Animales , Bacterias/enzimología , Bacterias/genética , ADN/metabolismo , Reparación del ADN , Epigénesis Genética , Proteínas de Escherichia coli , Eucariontes/enzimología , Eucariontes/genética , Humanos , Oxigenasas de Función Mixta , Modelos Moleculares , Conformación Proteica , Especificidad por SustratoRESUMEN
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.
Asunto(s)
Enzimas AlkB/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Arabidopsis/fisiología , ARN/metabolismo , Estrés Fisiológico/fisiología , Ácido Abscísico/metabolismo , Enzimas AlkB/genética , Arabidopsis/genética , Regulación hacia Abajo/genética , Sequías , Regulación de la Expresión Génica de las Plantas/genética , Germinación/genética , Germinación/fisiología , Mutación/genética , Reguladores del Crecimiento de las Plantas/genética , Reguladores del Crecimiento de las Plantas/metabolismo , Raíces de Plantas/genética , Raíces de Plantas/metabolismo , Raíces de Plantas/fisiología , ARN/genética , Plantones/genética , Plantones/metabolismo , Plantones/fisiología , Semillas/genética , Semillas/metabolismo , Semillas/fisiología , Transducción de Señal/genética , Cloruro de Sodio/metabolismo , Estrés Fisiológico/genéticaRESUMEN
Alkb homolog 7 (ALKBH7) is a mitochondrial α-ketoglutarate dioxygenase required for DNA alkylation-induced necrosis, but its function and substrates remain unclear. Herein, we show ALKBH7 regulates dialdehyde metabolism, which impacts the cardiac response to ischemia-reperfusion (IR) injury. Using a multi-omics approach, we find no evidence ALKBH7 functions as a prolyl-hydroxylase, but we do find Alkbh7-/- mice have elevated glyoxalase I (GLO-1), a dialdehyde detoxifying enzyme. Metabolic pathways related to the glycolytic by-product methylglyoxal (MGO) are rewired in Alkbh7-/- mice, along with elevated levels of MGO protein adducts. Despite greater glycative stress, hearts from Alkbh7-/- mice are protected against IR injury, in a manner blocked by GLO-1 inhibition. Integrating these observations, we propose ALKBH7 regulates glyoxal metabolism, and that protection against necrosis and cardiac IR injury bought on by ALKBH7 deficiency originates from the signaling response to elevated MGO stress.
Asunto(s)
Enzimas AlkB/genética , Glioxal/metabolismo , Redes y Vías Metabólicas , Necrosis/genética , Daño por Reperfusión/metabolismo , Enzimas AlkB/metabolismo , Animales , Femenino , Masculino , RatonesRESUMEN
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.
Asunto(s)
Enzimas AlkB/química , Reparación del ADN , ADN Bacteriano/química , Proteínas de Escherichia coli/química , Escherichia coli/enzimología , Enzimas AlkB/genética , Dioxigenasa Dependiente de Alfa-Cetoglutarato, Homólogo 2 de AlkB/química , Dioxigenasa Dependiente de Alfa-Cetoglutarato, Homólogo 2 de AlkB/genética , Dioxigenasa Dependiente de Alfa-Cetoglutarato, Homólogo 3 de AlkB/química , Dioxigenasa Dependiente de Alfa-Cetoglutarato, Homólogo 3 de AlkB/genética , ADN/química , ADN/genética , ADN Bacteriano/genética , ADN de Cadena Simple/química , ADN de Cadena Simple/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , HumanosRESUMEN
The complete genome sequence of Rhodococcus sp. WAY2 (WAY2) consists of a circular chromosome, three linear replicons and a small circular plasmid. The linear replicons contain typical actinobacterial invertron-type telomeres with the central CGTXCGC motif. Comparative phylogenetic analysis of the 16S rRNA gene along with phylogenomic analysis based on the genome-to-genome blast distance phylogeny (GBDP) algorithm and digital DNA-DNA hybridization (dDDH) with other Rhodococcus type strains resulted in a clear differentiation of WAY2, which is likely a new species. The genome of WAY2 contains five distinct clusters of bph, etb and nah genes, putatively involved in the degradation of several aromatic compounds. These clusters are distributed throughout the linear plasmids. The high sequence homology of the ring-hydroxylating subunits of these systems with other known enzymes has allowed us to model the range of aromatic substrates they could degrade. Further functional characterization revealed that WAY2 was able to grow with biphenyl, naphthalene and xylene as sole carbon and energy sources, and could oxidize multiple aromatic compounds, including ethylbenzene, phenanthrene, dibenzofuran and toluene. In addition, WAY2 was able to co-metabolize 23 polychlorinated biphenyl congeners, consistent with the five different ring-hydroxylating systems encoded by its genome. WAY2 could also use n-alkanes of various chain-lengths as a sole carbon source, probably due to the presence of alkB and ladA gene copies, which are only found in its chromosome. These results show that WAY2 has a potential to be used for the biodegradation of multiple organic compounds.
Asunto(s)
Bifenilos Policlorados/química , Rhodococcus/clasificación , Rhodococcus/crecimiento & desarrollo , Secuenciación Completa del Genoma/métodos , Enzimas AlkB/genética , Enzimas AlkB/metabolismo , Biodegradación Ambiental , Análisis por Conglomerados , Secuenciación de Nucleótidos de Alto Rendimiento , Naftalenos/metabolismo , Filogenia , ARN Ribosómico 16S/genética , Rhodococcus/genética , Xilenos/metabolismoRESUMEN
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.
Asunto(s)
Enzimas AlkB/genética , Inhibidores Enzimáticos/farmacología , Complejo Cetoglutarato Deshidrogenasa/genética , Enzimas AlkB/antagonistas & inhibidores , Enzimas AlkB/química , Daño del ADN/efectos de los fármacos , Metilación de ADN/efectos de los fármacos , Reparación del ADN/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Complejo Cetoglutarato Deshidrogenasa/antagonistas & inhibidores , Ácidos Cetoglutáricos/antagonistas & inhibidoresRESUMEN
AlkB family proteins are enzymes that repair alkylated DNA and RNA by oxidative demethylation. Nine homologs have been identified and characterized in mammals. ALKBH1 is conserved among metazoans including Drosophila. Although the ALKBH1 mouse homolog, Alkbh1 functions in neurogenesis, it currently remains unclear whether ALKBH1 plays a role in neuronal disorders induced by ultraviolet-induced DNA damage. We herein demonstrated that the Drosophila ALKBH1 homolog, AlkB contributed to recovery from neuronal disorders induced by ultraviolet damage. The knockdown of AlkB resulted in not only learning defects but also altered crawling behavior in Drosophila larvae after ultraviolet irradiation. A molecular analysis revealed that AlkB contributed to the repair of ultraviolet-induced DNA damage in the central nervous system of larvae. Therefore, we propose that ALKBH1 plays a role in the repair of ultraviolet-induced DNA damage in central nervous system. Ultraviolet-induced DNA damage is involved in the pathogenesis of xeroderma pigmentosum, and has recently been implicated in Parkinson's disease. The present results will contribute to our understanding of neuronal diseases induced by ultraviolet-induced DNA damage.
Asunto(s)
Enzimas AlkB/fisiología , Neuronas/patología , Neuronas/efectos de la radiación , Rayos Ultravioleta , Enzimas AlkB/genética , Animales , Sistema Nervioso Central/patología , Sistema Nervioso Central/efectos de la radiación , Daño del ADN/efectos de la radiación , Drosophila , Anomalías del Ojo/genética , Técnicas de Silenciamiento del Gen , Inmunohistoquímica , Larva , Discapacidades para el Aprendizaje/genética , Discapacidades para el Aprendizaje/psicología , Locomoción/efectos de la radiaciónRESUMEN
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.
Asunto(s)
Histona H2a Dioxigenasa, Homólogo 1 de AlkB/metabolismo , Mitocondrias/metabolismo , ARN Mitocondrial/metabolismo , Células A549 , Enzimas AlkB/genética , Enzimas AlkB/metabolismo , Histona H2a Dioxigenasa, Homólogo 1 de AlkB/genética , Animales , Caenorhabditis elegans , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Electroforesis en Gel de Poliacrilamida , Células HEK293 , Células HT29 , Células HeLa , Humanos , Ratones , Microscopía Electrónica , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Consumo de Oxígeno/fisiología , Factor Tu de Elongación Peptídica/genética , Factor Tu de Elongación Peptídica/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Respuesta de Proteína Desplegada/genética , Respuesta de Proteína Desplegada/fisiologíaRESUMEN
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
Alkylation B (AlkB) proteins are ubiquitous among diverse cellular organisms, where they act to reverse the damage in DNA and RNA due to methylation, such as 1-methyladenine and 3-methylcytosine. This process is found in virtually all forms of life, with the notable exception of archaea and yeast. This protein family is so significant to all forms of life that it was recently discovered that an AlkB domain is encoded as part of the replicase (poly)protein in a small subset of single-stranded, positive-sense RNA viruses, mainly belonging to the families Alphaflexiviridae, Betaflexiviridae and Closteroviridae. Interestingly, these AlkB-containing viruses are mostly important pathogens of woody perennials such as fruit crops, and are responsible for significant economic losses. As a newly identified protein domain in RNA viruses, the origin and molecular boundary of the viral AlkB domain, as well as its function in viral replication, virus-host interactions and infection are unknown. This is due to the limited sequence conservation of viral AlkB domains, especially at the N-terminal region corresponding to the nucleotide recognition lid. Here we apply several independent analytical approaches (homology modelling, principal component analysis and the Shannon diversity index) for the first time, to better understand this viral domain. We conclude that a functional AlkB domain in these viruses comprises approximately 150-170 amino acids. Although the exact function of the viral AlkB domain remains unknown, we hypothesize that it counteracts a host defence mechanism that is unique in these perennial plants and was acquired to enhance the long-term survival of these RNA viruses that infect perennial plants. Interestingly, a majority of these viruses have a tissue tropism for the phloem. Furthermore, we identified several additional amino acid residues that are uniquely conserved among viral AlkBs. This work helps to provide a foundation for further investigation of the function of viral AlkBs and critical residues involved in AlkB function.
Asunto(s)
Enzimas AlkB/genética , Alquilación/genética , Dominios Proteicos/genética , Virus ARN/genética , Proteínas Virales/genética , Secuencia de Aminoácidos , Aminoácidos/genética , Análisis de Componente Principal/métodos , Homología de Secuencia de AminoácidoRESUMEN
Plants and bacteria individually as well as in synergism with each other hold a great potential to degrade a wide range of environmental pollutants. Floating treatment wetlands (FTWs) is an efficient and low-cost technology that uses the synergistic interaction between plant roots and microbes for in situ remediation of wastewater. The present study aims to assess the feasibility of FTW-based remediation of oil field-produced wastewater using an interaction between two plant species, Typha domingensis and Leptochloa fusca, in partnership with a consortium of crude oil-degrading bacterial species, Bacillus subtilis LORI66, Klebsiella sp. LCRI87, Acinetobacter Junii TYRH47, and Acinetobacter sp. BRSI56. All the treatments reduced contaminant levels, but T. domingensis, in combination with bacterial inoculation, exhibited the highest reduction in hydrocarbon (95%), COD (90%), and BOD content (93%) as compared to L. fusca. This combination maximally promoted increases in fresh biomass (31%), dry biomass (52%), and length (25%) of plants as well. This effect was further signified by the persistence of bacteria (40%) and considerable abundance (27%) and expression (28.5%) of the alkB gene in the rhizoplane of T. domingensis in comparison to that of L. fusca. The study, therefore, suggests that T. domingensis, in combination with bacterial consortium, has significant potential for treatment of oil field-produced water and can be exploited on large scale in FTWs.