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1.
J Exp Clin Cancer Res ; 42(1): 10, 2023 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-36609396

RESUMO

BACKGROUND: Posttranscriptional modification of tumor-associated factors plays a pivotal role in breast cancer progression. However, the underlying mechanism remains unknown. M6A modifications in cancer cells are dynamic and reversible and have been found to impact tumor initiation and progression through various mechanisms. In this study, we explored the regulatory mechanism of breast cancer cell proliferation and metabolism through m6A methylation in the Hippo pathway.  METHODS: A combination of MeRIP-seq, RNA-seq and metabolomics-seq was utilized to reveal a map of m6A modifications in breast cancer tissues and cells. We conducted RNA pull-down assays, RIP-qPCR, MeRIP-qPCR, and RNA stability analysis to identify the relationship between m6A proteins and LATS1 in m6A regulation in breast cancer cells. The expression and biological functions of m6A proteins were confirmed in breast cancer cells in vitro and in vivo. Furthermore, we investigated the phosphorylation levels and localization of YAP/TAZ to reveal that the activity of the Hippo pathway was affected by m6A regulation of LATS1 in breast cancer cells.  RESULTS: We demonstrated that m6A regulation plays an important role in proliferation and glycolytic metabolism in breast cancer through the Hippo pathway factor, LATS1. METTL3 was identified as the m6A writer, with YTHDF2 as the reader protein of LATS1 mRNA, which plays a positive role in promoting both tumorigenesis and glycolysis in breast cancer. High levels of m6A modification were induced by METTL3 in LATS1 mRNA. YTHDF2 identified m6A sites in LATS1 mRNA and reduced its stability. Knockout of the protein expression of METTL3 or YTHDF2 increased the expression of LATS1 mRNA and suppressed breast cancer tumorigenesis by activating YAP/TAZ in the Hippo pathway. CONCLUSIONS: In summary, we discovered that the METTL3-LATS1-YTHDF2 pathway plays an important role in the progression of breast cancer by activating YAP/TAZ in the Hippo pathway.


Assuntos
Neoplasias da Mama , Humanos , Feminino , Metilação , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Transformação Celular Neoplásica/genética , Carcinogênese/genética , Fatores de Transcrição/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Metiltransferases/genética , Metiltransferases/metabolismo
2.
Int J Mol Sci ; 24(1)2023 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-36614187

RESUMO

Ladderane lipids (found in the membranes of anaerobic ammonium-oxidizing [anammox] bacteria) have unique ladder-like hydrophobic groups, and their highly strained exotic structure has attracted the attention of scientists. Although enzymes encoded in type II fatty acid biosynthesis (FASII) gene clusters in anammox bacteria, such as S-adenosyl-l-methionine (SAM)-dependent enzymes, have been proposed to construct a ladder-like structure using a substrate connected to acyl carrier protein from anammox bacteria (AmxACP), no experimental evidence to support this hypothesis was reported to date. Here, we report the crystal structure of a SAM-dependent methyltransferase from anammox bacteria (AmxMT1) that has a substrate and active site pocket between a class I SAM methyltransferase-like core domain and an additional α-helix inserted into the core domain. Structural comparisons with homologous SAM-dependent C-methyltransferases in polyketide synthase, AmxACP pull-down assays, AmxACP/AmxMT1 complex structure predictions by AlphaFold, and a substrate docking simulation suggested that a small compound connected to AmxACP could be inserted into the pocket of AmxMT1, and then the enzyme transfers a methyl group from SAM to the substrate to produce branched lipids. Although the enzymes responsible for constructing the ladder-like structure remain unknown, our study, for the first time, supports the hypothesis that biosynthetic intermediates connected to AmxACP are processed by SAM-dependent enzymes, which are not typically involved in the FASII system, to produce the ladder-like structure of ladderane lipids in anammox bacteria.


Assuntos
Metionina , S-Adenosilmetionina , S-Adenosilmetionina/metabolismo , Metionina/metabolismo , Proteína de Transporte de Acila/metabolismo , Metiltransferases/metabolismo , Oxidação Anaeróbia da Amônia , Bactérias/metabolismo , Racemetionina/metabolismo , Lipídeos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo
3.
Int J Mol Sci ; 24(1)2023 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-36614216

RESUMO

N6-metyladenosine (m6A), one of the most common RNA methylation modifications in mammals, has attracted extensive attentions owing to its regulatory roles in a variety of physiological and pathological processes. As a reversible epigenetic modification on RNAs, m6A is dynamically mediated by the functional interplay among the regulatory proteins of methyltransferases, demethylases and methyl-binding proteins. In recent years, it has become increasingly clear that m6A modification is associated with the production and function of microRNAs (miRNAs). In this review, we summarize the specific kinds of m6A modification methyltransferases, demethylases and methyl-binding proteins. In particular, we focus on describing the roles of m6A modification and its regulatory proteins in the production and function of miRNAs in a variety of pathological and physiological processes. More importantly, we further discuss the mediating mechanisms of miRNAs in m6A modification and its regulatory proteins during the occurrence and development of various diseases.


Assuntos
MicroRNAs , Animais , MicroRNAs/genética , MicroRNAs/metabolismo , Adenosina/metabolismo , Metilação , Metiltransferases/metabolismo , Epigênese Genética , Proteínas de Transporte/metabolismo , Fatores de Transcrição/metabolismo , Mamíferos/metabolismo
4.
Proc Natl Acad Sci U S A ; 120(4): e2208941120, 2023 Jan 24.
Artigo em Inglês | MEDLINE | ID: mdl-36656859

RESUMO

p97 is an essential AAA+ ATPase that extracts and unfolds substrate proteins from membranes and protein complexes. Through its mode of action, p97 contributes to various cellular processes, such as membrane fusion, ER-associated protein degradation, DNA repair, and many others. Diverse p97 functions and protein interactions are regulated by a large number of adaptor proteins. Alveolar soft part sarcoma locus (ASPL) is a unique adaptor protein that regulates p97 by disassembling functional p97 hexamers to smaller entities. An alternative mechanism to regulate the activity and interactions of p97 is by posttranslational modifications (PTMs). Although more than 140 PTMs have been identified in p97, only a handful of those have been described in detail. Here we present structural and biochemical data to explain how the p97-remodeling adaptor protein ASPL enables the metastasis promoting methyltransferase METTL21D to bind and trimethylate p97 at a single lysine side chain, which is deeply buried inside functional p97 hexamers. The crystal structure of a heterotrimeric p97:ASPL:METTL21D complex in the presence of cofactors ATP and S-adenosyl homocysteine reveals how structural remodeling by ASPL exposes the crucial lysine residue of p97 to facilitate its trimethylation by METTL21D. The structure also uncovers a role of the second region of homology (SRH) present in the first ATPase domain of p97 in binding of a modifying enzyme to the AAA+ ATPase. Investigation of this interaction in the human, fish, and plant reveals fine details on the mechanism and significance of p97 trimethylation by METTL21D across different organisms.


Assuntos
ATPases Associadas a Diversas Atividades Celulares , Adenosina Trifosfatases , Metiltransferases , Animais , Humanos , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adenosina Trifosfatases/metabolismo , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Lisina/metabolismo , Metilação , Ligação Proteica , Processamento de Proteína Pós-Traducional , Fatores de Transcrição/metabolismo , Proteína com Valosina/metabolismo , Metiltransferases/metabolismo
5.
Molecules ; 28(2)2023 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-36677825

RESUMO

SARS-CoV-2 nsp14 guanine-N7-methyltransferase plays an important role in the viral RNA translation process by catalyzing the transfer of a methyl group from S-adenosyl-methionine (SAM) to viral mRNA cap. We report a structure-guided design and synthesis of 3-(adenosylthio)benzoic acid derivatives as nsp14 methyltransferase inhibitors resulting in compound 5p with subnanomolar inhibitory activity and improved cell membrane permeability in comparison with the parent inhibitor. Compound 5p acts as a bisubstrate inhibitor targeting both SAM and mRNA-binding pockets of nsp14. While the selectivity of 3-(adenosylthio)benzoic acid derivatives against human glycine N-methyltransferase was not improved, the discovery of phenyl-substituted analogs 5p,t may contribute to further development of SARS-CoV-2 nsp14 bisubstrate inhibitors.


Assuntos
COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/metabolismo , Metilação , Proteínas não Estruturais Virais/metabolismo , Metiltransferases/metabolismo , RNA Mensageiro/metabolismo , S-Adenosilmetionina/química , RNA Viral/genética
6.
Free Radic Biol Med ; 195: 343-358, 2023 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-36587923

RESUMO

BACKGROUND: Alzheimer's disease (AD) is a severe neurodegenerative disorder that progressively destroys cognitive skills. Exploring the mechanism underlying autophagic clearance of phosphorylated tau (p-Tau) contributes to developing novel therapeutic strategies for AD. METHODS: SH-SY5Y and HT22 cells were treated with Aß1-42 to establish an in vitro model of AD. Cell viability was examined using CCK-8. TUNEL staining was applied to evaluate cell apoptosis. LC3 puncta was examined by IF staining. m6A modification level was evaluated through MeRIP. RNA pull-down and RIP assays were used for analyzing the interaction between IGF2BP1 and STUB1 transcripts. The binding of KDM1A to the promoter of METTL3 was confirmed by ChIP assays. APP/PS1 transgenic mice were used as an in vivo model of AD. Cognitive skills of mice were evaluated with the Morris water maze. Hippocampal damage and Aß deposition were detected through H&E and IHC staining. RESULTS: Dysregulated levels of autophagy, p-Tau and m6A was observed in an in vitro model of AD. Overexpression of METTL3 or STUB1 enhanced autophagy but reduced p-Tau level in Aß1-42-treated cells. METTL3 stabilized STUB1 mRNA through the m6A-IGF2BP1-dependent mechanism and naturally promoted STUB1 expression, thereby enhancing autophagic p-Tau clearance in Aß1-42-treated cells. Overexpression of KDM1A enhanced autophagy, m6A modification and autophagic p-Tau clearance in Aß1-42-treated cells. KDM1A-mediated upregulation of METTL3 promoted autophagic p-Tau clearance and ameliorated Alzheimer's disease both in vitro and in vivo. CONCLUSION: KDM1A-mediated upregulation of METTL3 enhances autophagic clearance of p-Tau through m6A-dependent regulation of STUB1, thus ameliorating Alzheimer's disease. Our study provides novel mechanistic insights into AD pathogenesis and potential drug targets for AD.


Assuntos
Doença de Alzheimer , Neuroblastoma , Humanos , Camundongos , Animais , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/genética , Peptídeos beta-Amiloides/metabolismo , Regulação para Cima , Camundongos Transgênicos , Autofagia/genética , Ubiquitina-Proteína Ligases/genética , Metiltransferases/genética , Metiltransferases/metabolismo , Metiltransferases/uso terapêutico , Histona Desmetilases/genética
7.
Sci Rep ; 13(1): 350, 2023 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-36611052

RESUMO

In recent years, the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), as the cause of the coronavirus disease (COVID-19) global pandemic, and its variants, especially those with higher transmissibility and substantial immune evasion, have highlighted the imperative for developing novel therapeutics as sustainable solutions other than vaccination to combat coronaviruses (CoVs). Beside receptor recognition and virus entry, members of the SARS-CoV-2 replication/transcription complex are promising targets for designing antivirals. Here, the interacting residues that mediate protein-protein interactions (PPIs) of nsp10 with nsp16 and nsp14 were comprehensively analyzed, and the key residues' interaction maps, interaction energies, structural networks, and dynamics were investigated. Nsp10 stimulates both nsp14's exoribonuclease (ExoN) and nsp16's 2'O-methyltransferase (2'O-MTase). Nsp14 ExoN is an RNA proofreading enzyme that supports replication fidelity. Nsp16 2'O-MTase is responsible for the completion of RNA capping to ensure efficient replication and translation and escape from the host cell's innate immune system. The results of the PPIs analysis proposed crucial information with implications for designing SARS-CoV-2 antiviral drugs. Based on the predicted shared protein-protein interfaces of the nsp16-nsp10 and nsp14-nsp10 interactions, a set of dual-target peptide inhibitors was designed. The designed peptides were evaluated by molecular docking, peptide-protein interaction analysis, and free energy calculations, and then further optimized by in silico saturation mutagenesis. Based on the predicted evolutionary conservation of the interacted target residues among CoVs, the designed peptides have the potential to be developed as dual target pan-coronavirus inhibitors.


Assuntos
COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/genética , Simulação de Acoplamento Molecular , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/química , Replicação Viral/genética , Metiltransferases/genética , Peptídeos/farmacologia , Antivirais/farmacologia , RNA/farmacologia , Exorribonucleases/genética , Exorribonucleases/química
8.
Microb Cell Fact ; 22(1): 15, 2023 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-36658647

RESUMO

BACKGROUND: Spinosad is a macrolide insecticide with the tetracyclic lactone backbone to which forosamine and tri-O-methylrhamnose are attached. Both the sugar moieties are essential for its insecticidal activity. In biosynthesis of spinosad, the amino group of forosamine is dimethylated by SpnS and then transferred onto the lactone backbone by SpnP. Because the spinosad native producer is difficult to genetically manipulate, we previously changed promoters, ribosome binding sites and start codons of 23 spinosad biosynthetic genes to construct an artificial gene cluster which resulted in a 328-fold yield improvement in the heterologous host Streptomyces albus J1074 compared with the native gene cluster. However, in fermentation of J1074 with the artificial gene cluster, the N-monodesmethyl spinosad with lower insecticidal activity was always produced with the same titer as spinosad. RESULTS: By tuning expression of SpnS with an inducible promotor, we found that the undesired less active byproduct N-monodesmethyl spinosad was produced when SpnS was expressed at low level. Although N-monodesmethyl spinosad can be almost fully eliminated with high SpnS expression level, the titer of desired product spinosad was only increased by less than 38%. When the forosaminyl transferase SpnP was further overexpressed together with SpnS, the titer of spinosad was improved by 5.3 folds and the content of N-desmethyl derivatives was decreased by ~ 90%. CONCLUSION: N-monodesmethyl spinosad was produced due to unbalanced expression of spnS and upstream biosynthetic genes in the refactored artificial gene cluster. The accumulated N-desmethyl forosamine was transferred onto the lactone backbone by SpnP. This study suggested that balanced expression of biosynthetic genes should be considered in the refactoring strategy to avoid accumulation of undesired intermediates or analogues which may affect optimal production of desired compounds.


Assuntos
Streptomyces griseus , Transferases , Transferases/genética , Metiltransferases/genética , Streptomyces griseus/metabolismo , Macrolídeos/metabolismo , Família Multigênica
9.
Cell Stem Cell ; 30(1): 52-68.e13, 2023 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-36608679

RESUMO

N6-methyladenosine (m6A), the most prevalent internal modification in mammalian mRNAs, is involved in many pathological processes. METTL16 is a recently identified m6A methyltransferase. However, its role in leukemia has yet to be investigated. Here, we show that METTL16 is a highly essential gene for the survival of acute myeloid leukemia (AML) cells via CRISPR-Cas9 screening and experimental validation. METTL16 is aberrantly overexpressed in human AML cells, especially in leukemia stem cells (LSCs) and leukemia-initiating cells (LICs). Genetic depletion of METTL16 dramatically suppresses AML initiation/development and maintenance and significantly attenuates LSC/LIC self-renewal, while moderately influencing normal hematopoiesis in mice. Mechanistically, METTL16 exerts its oncogenic role by promoting expression of branched-chain amino acid (BCAA) transaminase 1 (BCAT1) and BCAT2 in an m6A-dependent manner and reprogramming BCAA metabolism in AML. Collectively, our results characterize the METTL16/m6A/BCAT1-2/BCAA axis in leukemogenesis and highlight the essential role of METTL16-mediated m6A epitranscriptome and BCAA metabolism reprograming in leukemogenesis and LSC/LIC maintenance.


Assuntos
Autorrenovação Celular , Leucemia Mieloide Aguda , Camundongos , Humanos , Animais , Leucemia Mieloide Aguda/patologia , Carcinogênese/patologia , RNA Mensageiro/metabolismo , Aminoácidos de Cadeia Ramificada/genética , Aminoácidos de Cadeia Ramificada/metabolismo , Células-Tronco Neoplásicas/patologia , Mamíferos/metabolismo , Transaminases/genética , Transaminases/metabolismo , Metiltransferases/genética , Metiltransferases/metabolismo
10.
Nature ; 613(7943): 383-390, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36599982

RESUMO

Specific, regulated modification of RNAs is important for proper gene expression1,2. tRNAs are rich with various chemical modifications that affect their stability and function3,4. 7-Methylguanosine (m7G) at tRNA position 46 is a conserved modification that modulates steady-state tRNA levels to affect cell growth5,6. The METTL1-WDR4 complex generates m7G46 in humans, and dysregulation of METTL1-WDR4 has been linked to brain malformation and multiple cancers7-22. Here we show how METTL1 and WDR4 cooperate to recognize RNA substrates and catalyse methylation. A crystal structure of METTL1-WDR4 and cryo-electron microscopy structures of METTL1-WDR4-tRNA show that the composite protein surface recognizes the tRNA elbow through shape complementarity. The cryo-electron microscopy structures of METTL1-WDR4-tRNA with S-adenosylmethionine or S-adenosylhomocysteine along with METTL1 crystal structures provide additional insights into the catalytic mechanism by revealing the active site in multiple states. The METTL1 N terminus couples cofactor binding with conformational changes in the tRNA, the catalytic loop and the WDR4 C terminus, acting as the switch to activate m7G methylation. Thus, our structural models explain how post-translational modifications of the METTL1 N terminus can regulate methylation. Together, our work elucidates the core and regulatory mechanisms underlying m7G modification by METTL1, providing the framework to understand its contribution to biology and disease.


Assuntos
Microscopia Crioeletrônica , Proteínas de Ligação ao GTP , Metilação , Metiltransferases , Processamento Pós-Transcricional do RNA , RNA de Transferência , Humanos , Domínio Catalítico , Cristalografia por Raios X , Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/metabolismo , Proteínas de Ligação ao GTP/ultraestrutura , Metiltransferases/química , Metiltransferases/metabolismo , Metiltransferases/ultraestrutura , RNA de Transferência/química , RNA de Transferência/metabolismo , RNA de Transferência/ultraestrutura , S-Adenosil-Homocisteína/metabolismo , S-Adenosilmetionina/metabolismo , Especificidade por Substrato , Biocatálise
11.
Nature ; 613(7943): 391-397, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36599985

RESUMO

Chemical modifications of RNA have key roles in many biological processes1-3. N7-methylguanosine (m7G) is required for integrity and stability of a large subset of tRNAs4-7. The methyltransferase 1-WD repeat-containing protein 4 (METTL1-WDR4) complex is the methyltransferase that modifies G46 in the variable loop of certain tRNAs, and its dysregulation drives tumorigenesis in numerous cancer types8-14. Mutations in WDR4 cause human developmental phenotypes including microcephaly15-17. How METTL1-WDR4 modifies tRNA substrates and is regulated remains elusive18. Here we show,  through structural, biochemical and cellular studies of human METTL1-WDR4, that WDR4 serves as a scaffold for METTL1 and the tRNA T-arm. Upon tRNA binding, the αC region of METTL1 transforms into a helix, which together with the α6 helix secures both ends of the tRNA variable loop. Unexpectedly, we find that the predicted disordered N-terminal region of METTL1 is part of the catalytic pocket and essential for methyltransferase activity. Furthermore, we reveal that S27 phosphorylation in the METTL1 N-terminal region inhibits methyltransferase activity by locally disrupting the catalytic centre. Our results provide a molecular understanding of tRNA substrate recognition and phosphorylation-mediated regulation of METTL1-WDR4, and reveal the presumed disordered N-terminal region of METTL1 as a nexus of methyltransferase activity.


Assuntos
Proteínas de Ligação ao GTP , Metiltransferases , Processamento Pós-Transcricional do RNA , RNA de Transferência , Humanos , Biocatálise , Domínio Catalítico , Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/metabolismo , Metiltransferases/antagonistas & inibidores , Metiltransferases/química , Metiltransferases/metabolismo , Fosforilação , RNA de Transferência/química , RNA de Transferência/metabolismo , Especificidade por Substrato
12.
J Cell Sci ; 136(2)2023 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-36647772

RESUMO

N-terminal methylation of the α-amine group (Nα-methylation) is a post-translational modification (PTM) that was discovered over 40 years ago. Although it is not the most abundant of the Nα-PTMs, there are more than 300 predicted substrates of the three known mammalian Nα-methyltransferases, METTL11A and METTL11B (also known as NTMT1 and NTMT2, respectively) and METTL13. Of these ∼300 targets, the bulk are acted upon by METTL11A. Only one substrate is known to be Nα-methylated by METTL13, and METTL11B has no proven in vivo targets or predicted targets that are not also methylated by METTL11A. Given that METTL11A could clearly handle the entire substrate burden of Nα-methylation, it is unclear why three distinct Nα-methyltransferases have evolved. However, recent evidence suggests that many methyltransferases perform important biological functions outside of their catalytic activity, and the Nα-methyltransferases might be part of this emerging group. Here, we describe the distinct expression, localization and physiological roles of each Nα-methyltransferase, and compare these characteristics to other methyltransferases with non-catalytic functions, as well as to methyltransferases with both catalytic and non-catalytic functions, to give a better understanding of the global roles of these proteins. Based on these comparisons, we hypothesize that these three enzymes do not just have one common function but are actually performing three unique jobs in the cell.


Assuntos
Mamíferos , Metiltransferases , Animais , Metiltransferases/genética , Metiltransferases/metabolismo , Metilação , Catálise , Mamíferos/metabolismo
13.
Cell Stem Cell ; 30(1): 3-4, 2023 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-36608677

RESUMO

Chemical modifications of RNA are regulated by a series of readers, writers, and erasers that dictate gene expression. Two new studies in Cell Stem Cell1,2identify roles for the N6-methyladenosine (m6A) methyltransferase METTL16 and the m6A reader IGF2BP2 in leukemia-initiating cells, illuminating exciting new therapeutic targets for leukemia.


Assuntos
Leucemia , RNA , Humanos , RNA/genética , RNA/metabolismo , Metilação , Metiltransferases/genética , Leucemia/genética , Células-Tronco/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo
14.
Int J Biol Sci ; 19(2): 705-720, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36632456

RESUMO

Autophagy is an evolutionarily conserved cellular degradation and recycling process. It is important for maintaining vital cellular function and metabolism. Abnormal autophagy activity can cause the development of various diseases. N6-methyladenosine (m6A) methylation is the most prevalent and abundant internal modification in eukaryotes, affecting almost all aspects of RNA metabolism. The process of m6A modification is dynamic and adjustable. Its regulation depends on the regulation of m6A methyltransferases, m6A demethylases, and m6A binding proteins. m6A methylation and autophagy are two crucial and independent cellular events. Recent studies have shown that m6A modification mediates the transcriptional and post-transcriptional regulation of autophagy-related genes, affecting autophagy regulatory networks in multiple diseases. However, the regulatory effects of m6A regulators on autophagy in human diseases are not adequately acknowledged. In the present review, we summarized the latest knowledge of m6A modification in autophagy and elucidated the molecular regulatory mechanisms underlying m6A modification in autophagy regulatory networks. Moreover, we discuss the potentiality of m6A regulators serving as promising predictive biomarkers for human disease diagnosis and targets for therapy. This review will increase our understanding of the relationship between m6A methylation and autophagy, and provide novel insights to specifically target m6A modification in autophagy-associated therapeutic strategies.


Assuntos
Regulação da Expressão Gênica , Metiltransferases , Humanos , Metilação , Metiltransferases/genética , Metiltransferases/metabolismo , Autofagia/genética
15.
Viruses ; 15(1)2023 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-36680231

RESUMO

Since late 2016, a yellow fever virus (YFV) variant carrying a set of nine amino acid variations has circulated in South America. Three of them were mapped on the methyltransferase (MTase) domain of viral NS5 protein. To assess whether these changes affected viral infectivity, we synthesized YFV carrying the MTase of circulating lineage as well as its isoform with the residues of the previous strains (NS5 K101R, NS5 V138I, and NS5 G173S). We observed a slight difference in viral growth properties and plaque phenotype between the two synthetic YFVs. However, the MTase polymorphisms associated with the Brazilian strain of YFV (2016-2019) confer more susceptibility to the IFN-I. In addition, in vitro MTase assay revealed that the interaction between the YFV MTase and the methyl donor molecule (SAM) is altered in the Brazilian MTase variant. Altogether, the results reported here describe that the MTase carrying the molecular signature of the Brazilian YFV circulating since 2016 might display a slight decrease in its catalytic activity but virtually no effect on viral fitness in the parameters comprised in this study. The most marked influence of these residues stands in the immune escape against the antiviral response mediated by IFN-I.


Assuntos
Interferon Tipo I , Vírus da Febre Amarela , Vírus da Febre Amarela/fisiologia , Interferon Tipo I/genética , Aminoácidos , Evasão da Resposta Imune , Brasil , Metiltransferases/metabolismo , Proteínas não Estruturais Virais/genética
16.
Commun Biol ; 6(1): 54, 2023 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-36646841

RESUMO

The 22nd genetically encoded amino acid, pyrrolysine, plays a unique role in the key step in the growth of methanogens on mono-, di-, and tri-methylamines by activating the methyl group of these substrates for transfer to a corrinoid cofactor. Previous crystal structures of the Methanosarcina barkeri monomethylamine methyltransferase elucidated the structure of pyrrolysine and provide insight into its role in monomethylamine activation. Herein, we report the second structure of a pyrrolysine-containing protein, the M. barkeri trimethylamine methyltransferase MttB, and its structure bound to sulfite, a substrate analog of trimethylamine. We also report the structure of MttB in complex with its cognate corrinoid protein MttC, which specifically receives the methyl group from the pyrrolysine-activated trimethylamine substrate during methanogenesis. Together these structures provide key insights into the role of pyrrolysine in methyl group transfer from trimethylamine to the corrinoid cofactor in MttC.


Assuntos
Corrinoides , Metiltransferases , Metiltransferases/metabolismo , Metilaminas/metabolismo , Corrinoides/metabolismo
17.
Comput Biol Med ; 153: 106492, 2023 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-36621191

RESUMO

BACKGROUND: The O6-methylguanine-DNA methyltransferase (MGMT) is a deoxyribonucleic acid (DNA) repairing enzyme that has been established as an essential clinical brain tumor biomarker for Glioblastoma Multiforme (GBM). Knowing the status of MGMT methylation biomarkers using multi-parametric MRI (mp-MRI) helps neuro-oncologists to analyze GBM and its treatment plan. METHOD: The hand-crafted radiomics feature extraction of GBM's subregions, such as edema(ED), tumor core (TC), and enhancing tumor (ET) in the machine learning (ML) framework, was investigated using support vector machine(SVM), K-Nearest Neighbours (KNN), random forest (RF), LightGBM, and extreme gradient boosting (XGB). For tissue-level analysis of the promotor genes in GBM, we used the deep residual neural network (ResNet-18) with 3D architecture, followed by EfficientNet-based investigation for variants as B0 and B1. Lastly, we analyzed the fused deep learning (FDL) framework that combines ML and DL frameworks. RESULT: Structural mp-MRI consisting of T1, T2, FLAIR, and T1GD having a size of 400 and 185 patients, respectively, for discovery and replication cohorts. Using the CV protocol in the ResNet-3D framework, MGMT methylation status prediction in mp-MRI gave the AUC of 0.753 (p < 0.0001) and 0.72 (p < 0.0001) for the discovery and replication cohort, respectively. We presented that the FDL is ∼7% superior to solo DL and ∼15% to solo ML. CONCLUSION: The proposed study aims to provide solutions for building an efficient predictive model of MGMT for GBM patients using deep radiomics features obtained from mp-MRI with the end-to-end ResNet-18 3D and FDL imaging signatures.


Assuntos
Neoplasias Encefálicas , Aprendizado Profundo , Glioblastoma , Humanos , Glioblastoma/diagnóstico por imagem , Glioblastoma/genética , Glioblastoma/tratamento farmacológico , Metiltransferases/genética , Metiltransferases/uso terapêutico , Metilação de DNA/genética , Metilases de Modificação do DNA/genética , Metilases de Modificação do DNA/metabolismo , Metilases de Modificação do DNA/uso terapêutico , Imageamento por Ressonância Magnética/métodos , Neoplasias Encefálicas/diagnóstico por imagem , Neoplasias Encefálicas/genética , DNA , Biomarcadores
18.
Int J Mol Sci ; 24(2)2023 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-36674842

RESUMO

The PRDM family of methyltransferases has been implicated in cellular proliferation and differentiation and is deregulated in human diseases, most notably in cancer. PRDMs are related to the SET domain family of methyltransferases; however, from the 19 PRDMs only a few PRDMs with defined enzymatic activities are known. PRDM15 is an uncharacterized transcriptional regulator, with significant structural disorder and lack of defined small-molecule binding pockets. Many aspects of PRDM15 are yet unknown, including its structure, substrates, reaction mechanism, and its methylation profile. Here, we employ a series of computational approaches for an exploratory investigation of its potential substrates and reaction mechanism. Using the knowledge of PRDM9 and current knowledge of PRDM15 as basis, we tried to identify genuine substrates of PRDM15. We start from histone-based peptides and learn that the native substrates of PRDM15 may be non-histone proteins. In the future, a combination of sequence-based approaches and signature motif analysis may provide new leads. In summary, our results provide new information about the uncharacterized methyltransferase, PRDM15.


Assuntos
Metiltransferases , Neoplasias , Humanos , Metiltransferases/metabolismo , Metilação , Histonas/genética , Histonas/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo
19.
Int J Mol Sci ; 24(2)2023 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-36674903

RESUMO

Methylation of histone 3 at lysine 79 (H3K79) and its catalyst, a disrupter of telomeric silencing (DOT1l), have been coupled to multiple forms of stress, such as bioenergetic and ER challenges. However, studies on H3K79 methylation and Dot1l in the (aging) brain and neurons are limited. This, together with the increasing evidence of a dynamic neuroepigenome, made us wonder if H3K79 methylation and its activator Dot1l could play important roles in brain aging and associated disorders. In aged humans, we found strong and consistent global hypermethylation of H3K79 in neurons. Specific in dopaminergic neurons, we found a strong increase in H3K79 methylation in lipofucsin positive neurons, which are linked to pathology. In animals, where we conditionally removed Dot1l, we found a rapid loss of H3K79 methylation. As a consequence, we found some decrease in specific dopaminergic genes, and surprisingly, a clear up-regulation of almost all genes belonging to the family of the respiratory chain. These data, in relation to the observed increase in global H3K79 methylation, suggest that there is an inverse relationship between H3K79 methylation and the capacity of energy metabolism in neuronal systems.


Assuntos
Genes Mitocondriais , Metiltransferases , Animais , Humanos , Idoso , Metiltransferases/metabolismo , Histonas/genética , Histonas/metabolismo , Metilação de DNA , Neurônios/metabolismo , Encéfalo/metabolismo , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo
20.
Int J Mol Sci ; 24(2)2023 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-36674918

RESUMO

Excessive differentiation of osteoclasts contributes to the disruption of bone homeostasis in inflammatory bone diseases. Methyltransferase-like 3 (METTL3), the core methyltransferase that installs an N6-methyladenosine (m6A) modification on RNA, has been reported to participate in bone pathophysiology. However, whether METTL3-mediated m6A affects osteoclast differentiation in inflammatory conditions remains unelucidated. In this study, we observed that the total m6A content and METTL3 expression decreased during LPS-induced osteoclastogenesis. After knocking down METTL3, we found reduced levels of the number of osteoclasts, osteoclast-related gene expression and bone resorption area. A METTL3 deficiency increased osteoclast apoptosis and pro-apoptotic protein expression. RNA sequencing analysis showed that differentially expressed genes in METTL3-deficient cells were mainly associated with the mitochondrial function. The expression of the mitochondrial function-related genes, ATP production and mitochondrial membrane potential decreased after METTL3 knockdown. Moreover, the most obviously upregulated gene in RNA-Seq was Nos2, which encoded the iNOS protein to induce nitric oxide (NO) synthesis. METTL3 knockdown increased the levels of Nos2 mRNA, iNOS protein and NO content. NOS inhibitor L-NAME rescued the inhibited mitochondrial function and osteoclast formation while suppressing osteoclast apoptosis in METTL3-silenced cells. Mechanistically, a METTL3 deficiency promoted the stability and expression of Nos2 mRNA, and similar results were observed after m6A-binding protein YTHDF1 knockdown. Further in vivo evidence revealed that METTL3 knockdown attenuated the inflammatory osteolysis of the murine calvaria and suppressed osteoclast formation. In conclusion, these data suggested that METTL3 knockdown exacerbated iNOS/NO-mediated mitochondrial dysfunction by promoting a Nos2 mRNA stability in a YTHDF1-dependent manner and further inhibited osteoclast differentiation and increased osteoclast apoptosis in inflammatory conditions.


Assuntos
Reabsorção Óssea , Osteoclastos , Camundongos , Animais , Osteoclastos/metabolismo , Óxido Nítrico/metabolismo , Reabsorção Óssea/metabolismo , Metiltransferases/genética , Metiltransferases/metabolismo , RNA Mensageiro/genética
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