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1.
Environ Pollut ; 292(Pt A): 118279, 2022 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-34619179

RESUMO

Arsenic (As) contamination in groundwater is responsible for numerous adverse health outcomes among millions of people. Epigenetic alterations are among the most widely studied mechanisms of As toxicity. To understand how As exposure alters gene expression through epigenetic modifications, a systematic genome-wide study was designed to address the impact of multiple important single nucleotide polymorphisms (SNPs) related to As exposure on the methylome of drinking water As-exposed rural subjects from Pakistan. Urinary As levels were used to stratify subjects into low, medium and high exposure groups. Genome-wide DNA methylation was investigated using MeDIP in combination with NimbleGen 2.1 M Deluxe Promotor arrays. Transcriptome levels were measured using Agilent 8 × 60 K expression arrays. Genotyping of selected SNPs (As3MT, DNMT1a, ERCC2, EGFR and MTHFR) was measured and an integrated genetic risk factor for each respondent was calculated by assigning a specific value to the measured genotypes based on known risk allele numbers. To select a representative model related to As exposure we compared 9 linear mixed models comprising of model 1 (including the genetic risk factor), model 2 (without the genetic risk factor) and models with individual SNPs incorporated into the methylome data. Pathway analysis was performed using ConsensusPathDB. Model 1 comprising the integrated genetic risk factor disclosed biochemical pathways including muscle contraction, cardio-vascular diseases, ATR signaling, GPCR signaling, methionine metabolism and chromatin modification in association with hypo- and hyper-methylated gene targets. A unique pathway (direct P53 effector) was found associated with the individual DNMT1a polymorphism due to hyper-methylation of CSE1L and TRRAP. Most importantly, we provide here the first evidence of As-associated DNA methylation in relation with gene expression of ATR, ATF7IP, TPM3, UBE2J2. We report the first evidence that integrating SNPs data with methylome data generates a more representative epigenome profile and discloses a better insight in disease risks of As-exposed individuals.


Assuntos
Arsênio , Metilação de DNA , Epigenômica , Estudo de Associação Genômica Ampla , Humanos , Metiltransferases/genética , Polimorfismo de Nucleotídeo Único , Fatores de Risco , Enzimas de Conjugação de Ubiquitina , Proteína Grupo D do Xeroderma Pigmentoso
2.
Theriogenology ; 177: 94-102, 2022 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-34687941

RESUMO

Seminal plasma plays an important role in sperm physiology. Seminal plasma proteins vehiculated in microvesicles, carry RNAs and proteins with a potential role in early embryo development. Additionally, proteins present in seminal plasma participate in redox regulation and energy metabolism. In view of these facts, we hypothesized that differences in protein composition of the seminal plasma among stallions may help to explain differences in freeze-ability seen among them. Three independent ejaculates from 10 different stallions of varying breeds were frozen using standard protocols in our laboratory. Aliquots of the ejaculate were separated and stored at -80 °C until further proteomic analysis. Semen analysis was performed using computer assisted sperm analysis and flow cytometry. Significant differences in proteome composition of seminal plasma were observed in the group of stallions showing better motility post thaw. 3116 proteins were identified, and of these, 34 were differentially expressed in stallions with better motility post thaw, 4 of them were also differentially expressed in stallions with different percentages of linearly motile sperm post thaw and 1 protein, Midasin, was expressed in stallions showing high circular velocity post thaw. Seminal plasma proteins may play a major role in sperm functionality; being vehiculated through extracellular vesicles and participating in sperm physiology. Bioinformatic analysis identifies discriminant proteins able to predict the outcome of cryopreservation, identifying potential new biomarkers to assess ejaculate quality.


Assuntos
Preservação do Sêmen , Adenina , Animais , Arginina , Criopreservação/veterinária , Cavalos , Masculino , Metiltransferases , Proteômica , Sêmen , Preservação do Sêmen/veterinária , Proteínas de Plasma Seminal , Motilidade Espermática , Espermatozoides
3.
Theriogenology ; 177: 140-150, 2022 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-34700071

RESUMO

It has been reported that N6-methyladenosine (m6A) methyltransferase-like 3 (METTL3) plays an important role in zygote genome activation during embryonic development, but the effects of METTL3 under oxidative stress in the early development of goat embryos remain largely unknown. In this study, zygotes were monitored at 72 and 168 h after fertilization, and they developed to the 8-cell stage and blastocyst stage under hypoxic conditions and normoxic conditions. Single-cell transcriptome sequencing was performed at the 8-cell stage and the blastocyst stage in the goat embryos, the differentially expressed METTL3 was screened from the sequencing results. We found that microinjection of small interfering RNA (siRNA) against METTL3 caused developmental arrest, both 8-cell rates (37.45 ± 2.21% vs. 47.09 ± 1.38%; P < 0.01) and blastocyst rates of Si-METTL3 (12.17% ± 2.84 vs. 20.83 ± 3.61%; P < 0.01) in Si-METTL3 group were significantly decreased compared with that of control under hypoxic conditions, significant changes were found in the m6A-related genes and the expression levels of critical transcription factors, such as, NANOG, GATA3, CDX2 and SOX17, were decreased. This study revealed the key role of METTL3 in the regulation of embryonic development under oxidative stress, and laid the foundation for further study of the crucial mechanism of oxidative stress during the early embryonic development of goats.


Assuntos
Cabras , Metiltransferases , Adenosina , Animais , Desenvolvimento Embrionário , Metiltransferases/genética , RNA Mensageiro
4.
World J Gastroenterol ; 27(38): 6348-6356, 2021 Oct 14.
Artigo em Inglês | MEDLINE | ID: mdl-34720526

RESUMO

Thiopurines are immunomodulators used in the treatment of acute lymphoblastic leukemia and inflammatory bowel diseases. Adverse reactions to these agents are one of the main causes of treatment discontinuation or interruption. Myelosuppression is the most frequent adverse effect; however, approximately 5%-20% of patients develop gastrointestinal toxicity. The identification of biomarkers able to prevent and/or monitor these adverse reactions would be useful for clinicians for the proactive management of long-term thiopurine therapy. In this editorial, we discuss evidence supporting the use of PACSIN2, RAC1, and ITPA genes, in addition to TPMT and NUDT15, as possible biomarkers for thiopurine-related gastrointestinal toxicity.


Assuntos
Mercaptopurina , Pirofosfatases , Azatioprina/efeitos adversos , Biomarcadores , Humanos , Fatores Imunológicos , Mercaptopurina/efeitos adversos , Metiltransferases/genética
5.
Anal Chim Acta ; 1184: 339018, 2021 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-34625260

RESUMO

DNA methylation is an epigenetic modification that plays a vital role in X chromosome inactivation, genome imprinting, and gene expression. DNA methyltransferase establishes and maintains a stable methylation state in genomic DNA. Efficient and specific DNA methyltransferase testing is essential for the early diagnosis and treatment of cancer. In this study, we designed an ultra-sensitive fluorescent biosensor, based on a 3D tetrahedral fluorescent scaffold assisted by symmetrical double-ring dumbbells, for the detection of DNA-[N 6-adenine]-methyltransferase (Dam MTase). Double-stranded DNA was methylated by Dam MTase and then digested by DpnI to form two identical dumbbell rings. The 3D tetrahedral fluorescent scaffold was synthesized from four oligonucleotide chains containing hairpins. When the sheared dumbbells reacted with the 3D tetrahedral fluorescent scaffold, the hairpins opened and a fluorescence signal could be detected. The strategy was successful over a wide detection range, from 0.002 to 100 U mL-1 Dam MTase, and the lowest detection limit was 0.00036 U mL-1. Control experiments with M.SssI methyltransferase and HpaII methylation restriction endonuclease confirmed the specificity of the method. Experiments with spiked human serum and the 5-fluorouracil inhibitor proved the suitability of the method for early cancer diagnosis.


Assuntos
Metilação de DNA , DNA Metiltransferases Sítio Específica (Adenina-Específica) , Adenina , DNA/genética , DNA/metabolismo , Humanos , Metiltransferases , DNA Metiltransferases Sítio Específica (Adenina-Específica)/metabolismo
6.
Sci Rep ; 11(1): 19752, 2021 10 05.
Artigo em Inglês | MEDLINE | ID: mdl-34611227

RESUMO

Although metabolic syndrome (MetS) is linked to an elevated risk of cardiovascular disease (CVD), the cardiac-specific risk mechanism is unknown. Obesity, hypertension, and diabetes (all MetS components) are the most common form of CVD and represent risk factors for worse COVID-19 outcomes compared to their non MetS peers. Here, we use obese Yorkshire pigs as a highly relevant animal model of human MetS, where pigs develop the hallmarks of human MetS and reproducibly mimics the myocardial pathophysiology in patients. Myocardium-specific mass spectroscopy-derived metabolomics, proteomics, and transcriptomics enabled the identity and quality of proteins and metabolites to be investigated in the myocardium to greater depth. Myocardium-specific deregulation of pro-inflammatory markers, propensity for arterial thrombosis, and platelet aggregation was revealed by computational analysis of differentially enriched pathways between MetS and control animals. While key components of the complement pathway and the immune response to viruses are under expressed, key N6-methyladenosin RNA methylation enzymes are largely overexpressed in MetS. Blood tests do not capture the entirety of metabolic changes that the myocardium undergoes, making this analysis of greater value than blood component analysis alone. Our findings create data associations to further characterize the MetS myocardium and disease vulnerability, emphasize the need for a multimodal therapeutic approach, and suggests a mechanism for observed worse outcomes in MetS patients with COVID-19 comorbidity.


Assuntos
COVID-19/patologia , Suscetibilidade a Doenças , Síndrome Metabólica/patologia , Animais , Fatores de Coagulação Sanguínea/genética , Fatores de Coagulação Sanguínea/metabolismo , COVID-19/complicações , COVID-19/virologia , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Dieta Hiperlipídica/veterinária , Modelos Animais de Doenças , Humanos , Imunidade Inata/genética , Síndrome Metabólica/complicações , Síndrome Metabólica/metabolismo , Metiltransferases/genética , Metiltransferases/metabolismo , Miocárdio/metabolismo , Estresse Oxidativo/genética , Agregação Plaquetária , Receptores Purinérgicos P2Y1/genética , Receptores Purinérgicos P2Y1/metabolismo , Sistema Renina-Angiotensina , Fatores de Risco , SARS-CoV-2/isolamento & purificação , Suínos , Ativador de Plasminogênio Tipo Uroquinase/genética , Ativador de Plasminogênio Tipo Uroquinase/metabolismo
7.
PLoS One ; 16(10): e0258726, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34648604

RESUMO

Fusarium head blight (FHB) is an economically important disease of wheat that results in yield loss and grain contaminated with fungal mycotoxins that are harmful to human and animal health. Herein we characterised two wheat genes involved in the FHB response in wheat: a wheat mitochondrial phosphate transporter (TaMPT) and a methyltransferase (TaSAM). Wheat has three sub-genomes (A, B, and D) and gene expression studies demonstrated that TaMPT and TaSAM homoeologs were differentially expressed in response to FHB infection and the mycotoxigenic Fusarium virulence factor deoxynivalenol (DON) in FHB resistant wheat cv. CM82036 and susceptible cv. Remus. Virus-induced gene silencing (VIGS) of either TaMPT or TaSAM enhanced the susceptibility of cv. CM82036 to FHB disease, reducing disease spread (Type II disease resistance). VIGS of TaMPT and TaSAM significantly reduced grain number and grain weight. This indicates TaSAM and TaMPT genes also contribute to grain development in wheat and adds to the increasing body of evidence linking FHB resistance genes to grain development. Hence, Fusarium responsive genes TaSAM and TaMPT warrant further study to determine their potential to enhance both disease resistance and grain development in wheat.


Assuntos
Resistência à Doença , Fusarium/patogenicidade , Proteínas de Plantas/genética , Triticum/crescimento & desenvolvimento , Produtos Agrícolas/crescimento & desenvolvimento , Produtos Agrícolas/microbiologia , Fusarium/metabolismo , Metiltransferases/genética , Proteínas de Transporte de Fosfato/genética , Tricotecenos/toxicidade , Triticum/efeitos dos fármacos , Triticum/microbiologia
8.
Nat Commun ; 12(1): 5959, 2021 10 13.
Artigo em Inglês | MEDLINE | ID: mdl-34645844

RESUMO

The directed evolution of antibodies has yielded important research tools and human therapeutics. The dependence of many antibodies on disulfide bonds for stability has limited the application of continuous evolution technologies to antibodies and other disulfide-containing proteins. Here we describe periplasmic phage-assisted continuous evolution (pPACE), a system for continuous evolution of protein-protein interactions in the disulfide-compatible environment of the E. coli periplasm. We first apply pPACE to rapidly evolve novel noncovalent and covalent interactions between subunits of homodimeric YibK protein and to correct a binding-defective mutant of the anti-GCN4 Ω-graft antibody. We develop an intein-mediated system to select for soluble periplasmic expression in pPACE, leading to an eight-fold increase in soluble expression of the Ω-graft antibody. Finally, we evolve disulfide-containing trastuzumab antibody variants with improved binding to a Her2-like peptide and improved soluble expression. Together, these results demonstrate that pPACE can rapidly optimize proteins containing disulfide bonds, broadening the applicability of continuous evolution.


Assuntos
Evolução Molecular Direcionada/métodos , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Metiltransferases/genética , Periplasma/genética , Isomerases de Dissulfetos de Proteínas/genética , Trastuzumab/genética , Sítios de Ligação , Clonagem Molecular , Colífagos/genética , Colífagos/metabolismo , Dissulfetos/química , Dissulfetos/metabolismo , Escherichia coli/metabolismo , Escherichia coli/virologia , Proteínas de Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Inteínas/genética , Metiltransferases/metabolismo , Modelos Moleculares , Periplasma/metabolismo , Periplasma/virologia , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Isomerases de Dissulfetos de Proteínas/metabolismo , Domínios e Motivos de Interação entre Proteínas , Processamento de Proteína , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Trastuzumab/química , Trastuzumab/metabolismo
9.
Int J Clin Oncol ; 26(12): 2229-2236, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34689291

RESUMO

OBJECTIVES: This study aimed to investigate the role of the tRNA aspartic acid methyltransferase 1 (TRDMT1) protein in the development and progression of gastric cancer (GC). METHODS: The 90 GC tissues and 35 paracancerous tissues (gastric mucosa) were collected from patients (31 males and 59 females; average age 66), who were pathologically diagnosed as GC. The expression of TRDMT1 in three GC cell lines (MKN28, BGC823, and MGC803) and tissues from GC patients were detected by western blotting and immunological staining, respectively. The relationship between TRDMT1 expression and clinicopathological parameters in GC patients was explored. TRDMT1 was knocked down by RNAi lentivirus in GC cells. GC cell migration and invasion were analyzed using scratch and transwell assays. RESULTS: TRDMT1 expression in the GC cell lines was higher than that in the normal gastric mucosal epithelial cell line (P < 0.05). Positive TRDMT1 protein expression in the GC tissue was higher than that in the adjacent tissue. The expression of TRDMT1 was positively associated with tumor size, histological grade, invasion depth, lymph node metastasis, and tumor node metastasis (TNM) stage (P < 0.05). High TRDMT1 expression predicted poor OS of GC patients. Tumor size, differentiation degree, invasion depth, lymph node metastasis, TNM stage, and TRDMT1 expression were independent predictors of the OS of GC patients. Knockdown of TRDMT1 inhibited the migration and invasion of MKN28 cells. CONCLUSION: TRDMT1 was highly expressed in GC cell lines and tissues. TRDMT1 expression was independent predictor of the OS of GC patients. TRDMT1 knockdown reduced GC cell migration and invasion. All these results suggested that TRDMT1 has the potential to be used as a target for the diagnosis and treatment of GC.


Assuntos
Ácido Aspártico , Neoplasias Gástricas , Idoso , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Humanos , Masculino , Metiltransferases , Invasividade Neoplásica , RNA de Transferência , Neoplasias Gástricas/genética
10.
Methods Enzymol ; 658: 161-190, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34517946

RESUMO

The RNA methyltransferase (MTase) complex METTL3-METTL14 transfers methyl groups from S-adenosyl-l-methionine (AdoMet) to the N6-position of adenosines within its consensus sequence, the DRACH motif (D=A, G, U; R=A, G; H=A, C, U). Interestingly, this MTase complex shows remarkable promiscuity regarding the cosubstrate. This can be exploited to install nonnatural modifications, like clickable or photocaging groups. Clickable groups are widely used for subsequent functionalization and open a broad range of possibilities for downstream applications. Here, we elaborate on click chemistry for coupling of RNA to biotin to enrich MTase targets via streptavidin-coated magnetic beads. Importantly, after clicking and coupling to beads the modification becomes sterically demanding and stalls reverse transcriptases, leading to termination adjacent to the MTase target site. Using radioactively labeled primers in the reverse transcription, the modified position can be precisely identified on a sequencing gel via phosphor imaging.


Assuntos
Metiltransferases , RNA , Adenosina , Metionina , Metiltransferases/genética , S-Adenosilmetionina
11.
Methods Enzymol ; 658: 251-275, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34517950

RESUMO

The application of in vitro kinetic tools has the potential to provide important insight into the molecular mechanisms of RNA modification enzymes. Utilizing quantitative biochemical approaches can reveal information about enzyme preferences for specific substrates that are relevant for understanding modification reactions in their biological contexts. Moreover, kinetic tools have been powerfully applied to identify and characterize roles for specific amino acid residues in catalysis, which can be essential information for understanding the molecular basis for human disease, as well as for targeting these enzymes for potential therapeutic interventions. RNA methyltransferases are a particularly interesting group of RNA modification enzymes because of the diversity in structure and mechanism that has been revealed among members of this group, even including some examples of enzymes that use entirely distinct reaction mechanisms to form identical methylated nucleotides in RNA. Yet, many questions remain unanswered about how these distinct catalytic strategies are facilitated by the relevant enzyme families. We have applied in vitro kinetic analysis to specifically focus on catalytically relevant ionizations in the context of tRNA methyltransferase reactions, by measuring rates under conditions of varied pH. This analysis can be applied broadly to RNA methyltransferases to expand our understanding of these important enzymes.


Assuntos
Metiltransferases , tRNA Metiltransferases , Catálise , Humanos , Cinética , Metiltransferases/metabolismo , RNA , RNA de Transferência , Especificidade por Substrato , tRNA Metiltransferases/metabolismo
12.
Methods Enzymol ; 658: 49-72, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34517959

RESUMO

RNAs from various cells and tissues are modified in nearly 200 chemically distinct ways. These modifications can be deposited either on the 5' or 3' ends, or internally on the nucleobases or sugar backbone. 5'-end modifications are crucial for protecting RNAs from untimely degradation/processing, regulating their cellular functions, or discriminating endogenous RNAs from pathogenic RNAs. 5'-end phospho-methylation is a remarkable RNA modification that is enzymatically deposited either on the γ-phosphate of nascent triphosphorylated RNAs by human BCDIN3/MePCE, or on the α-phosphate of processed monophosphorylated RNAs by human BCDIN3D. These 5'-phospho-methyltransferases are part of the BIN3 family of O-methyltransferases conserved from S. pombe to humans and play important cellular and biological roles, many of which await further elucidation. Here, we quickly recapitulate historical methods for the detection of 5'-end phospho-methyl modifications, and focus more specifically on a method that can be used to detect and quantify α-monophosphate methylation from as low as 10-100ng of total RNA from cells or tissues. This method is important for deciphering the roles of BCDIN3D and its homologs across species, as well as serves as starting point for the development of new methods for detection of 5'-end modifications.


Assuntos
Metiltransferases , RNA , Humanos , Metilação , Metiltransferases/metabolismo , Processamento Pós-Transcricional do RNA
13.
Enzyme Microb Technol ; 150: 109862, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34489021

RESUMO

Glycosylation and methylation of flavonoids are the main types of structural modifications and can endow flavonoids with greater stability, bioactivity, and bioavailability. In this study, five types of O-methyltransferases were screened for producing O-methylated luteolin, and the biosynthesis strategy of 3'-O-methylisoorientin from luteolin was determined. To improve the production of 3'-O-methylluteolin, the S-adenosyl-l-methionine synthesis pathway was reconstructed in the recombinant strain by introducing S-adenosyl-l-methionine synthetase genes. After optimizing the conversion conditions, maximal 3'-O-methylluteolin production reached 641 ± 25 mg/L with a corresponding molar conversion of 76.5 %, which was the highest titer of methylated flavonoids reported to date in Escherichia coli. 3'-O-Methylluteolin (127 mg) was prepared from 250 mL of the broth by silica gel column chromatography and preparative HPLC with a yield of 79.4 %. Subsequently, we used the biocatalytic cascade of Gentiana triflora C-glycosyltransferase (Gt6CGT) and Glycine max sucrose synthase (GmSUS) to biosynthesize 3'-O-methylisoorientin from 3'-O-methylluteolin in vitro. By optimizing the coupled reaction conditions and using the fed-batch operation, maximal 3'-O-methylisoorientin production reached 226 ± 8 mg/L with a corresponding molar conversion of 98 %. Therefore, this study provides an efficient method for the production of novel 3'-O-methylisoorientin and the biosynthesis strategy for methylated C-glycosylation flavonoids by selective O-methylation/C-glycosylation motif on flavonoids.


Assuntos
Flavonoides , Luteolina , Glicosilação , Metilação , Metiltransferases/metabolismo
14.
Math Biosci Eng ; 18(5): 6608-6619, 2021 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-34517547

RESUMO

Lung adenocarcinoma (LUAD) is a frequently diagnosed malignant tumor that is highly invasive and lethal. The prognosis of patients with LUAD still needs to be improved, as conventional treatment is remarkably well tolerated. In this study, the expression profile of LUAD in the TCGA database was used for differential expression analysis, and differential expression genes were determined to construct a weighted gene co-expression network analysis (WGCNA) for dividing and finding the gene modules with the highest correlation with tumor stage. Here, METTL5, DDX23, GPSM2, CEP95, WDCP, and METL17 were identified as hub genes. According to the relation degree, METTL5 was determined as the candidate gene in this study. Difference analysis and receiver operating characteristic (ROC) curve were applied to identify the predictive performance of METTL5 in LUAD, and Kaplan-Meier (KM) analysis showed that the prognosis of LUAD patients with high METTL5 expression was poor. Further GSEA analysis showed that high-expressed METTL5 was related to epithelial-mesenchymal transition and other pathways. Therefore, METTL5 may be involved in the occurrence and malignant progression of LUAD. The current findings provide an effective molecular target for early diagnosis of LUAD, helping monitor the malignant progression of LUAD and improve the prognosis of LUAD patients.


Assuntos
Adenocarcinoma de Pulmão , Neoplasias Pulmonares , Metiltransferases/genética , Adenocarcinoma de Pulmão/genética , Biomarcadores Tumorais/genética , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Neoplasias Pulmonares/genética
15.
Nat Commun ; 12(1): 5522, 2021 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-34535671

RESUMO

Natural killer (NK) cells exert critical roles in anti-tumor immunity but how their functions are regulated by epitranscriptional modification (e.g., N6-methyladenosine (m6A) methylation) is unclear. Here we report decreased expression of the m6A "writer" METTL3 in tumor-infiltrating NK cells, and a positive correlation between protein expression levels of METTL3 and effector molecules in NK cells. Deletion of Mettl3 in NK cells alters the homeostasis of NK cells and inhibits NK cell infiltration and function in the tumor microenvironment, leading to accelerated tumor development and shortened survival in mice. The gene encoding SHP-2 is m6A modified, and its protein expression is decreased in METTL3-deficient NK cells. Reduced SHP-2 activity renders NK cells hyporesponsive to IL-15, which is associated with suppressed activation of the AKT and MAPK signaling pathway in METTL3-deficient NK cells. These findings show that m6A methylation safeguards the homeostasis and tumor immunosurveillance function of NK cells.


Assuntos
Adenosina/análogos & derivados , Células Matadoras Naturais/imunologia , Metiltransferases/metabolismo , Neoplasias/imunologia , Neoplasias/metabolismo , RNA/metabolismo , Adenosina/metabolismo , Animais , Carcinogênese/patologia , Linhagem Celular Tumoral , Deleção de Genes , Homeostase , Interleucina-15/metabolismo , Linfócitos do Interstício Tumoral/imunologia , Metilação , Metiltransferases/deficiência , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Microambiente Tumoral
16.
Nat Struct Mol Biol ; 28(9): 713-723, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34489609

RESUMO

Human mitochondrial transcripts contain messenger and ribosomal RNAs flanked by transfer RNAs (tRNAs), which are excised by mitochondrial RNase (mtRNase) P and Z to liberate all RNA species. In contrast to nuclear or bacterial RNase P, mtRNase P is not a ribozyme but comprises three protein subunits that carry out RNA cleavage and methylation by unknown mechanisms. Here, we present the cryo-EM structure of human mtRNase P bound to precursor tRNA, which reveals a unique mechanism of substrate recognition and processing. Subunits TRMT10C and SDR5C1 form a subcomplex that binds conserved mitochondrial tRNA elements, including the anticodon loop, and positions the tRNA for methylation. The endonuclease PRORP is recruited and activated through interactions with its PPR and nuclease domains to ensure precise pre-tRNA cleavage. The structure provides the molecular basis for the first step of RNA processing in human mitochondria.


Assuntos
3-Hidroxiacil-CoA Desidrogenases/química , Metiltransferases/química , Precursores de RNA/metabolismo , Processamento Pós-Transcricional do RNA , Ribonuclease P/química , 3-Hidroxiacil-CoA Desidrogenases/metabolismo , Anticódon/química , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Proteínas Arqueais/química , Proteínas Arqueais/metabolismo , Microscopia Crioeletrônica , Humanos , Metilação , Metiltransferases/genética , Metiltransferases/metabolismo , Mitocôndrias/enzimologia , Modelos Moleculares , Mutação de Sentido Incorreto , Conformação de Ácido Nucleico , Ligação Proteica , Conformação Proteica , Mapeamento de Interação de Proteínas , RNA Fúngico/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Ribonuclease P/metabolismo , Especificidade da Espécie , Relação Estrutura-Atividade , Especificidade por Substrato
17.
Viruses ; 13(9)2021 08 30.
Artigo em Inglês | MEDLINE | ID: mdl-34578302

RESUMO

The ongoing COVID-19 pandemic exemplifies the general need to better understand viral infections. The positive single-strand RNA genome of its causative agent, the SARS coronavirus 2 (SARS-CoV-2), encodes all viral enzymes. In this work, we focused on one particular methyltransferase (MTase), nsp16, which, in complex with nsp10, is capable of methylating the first nucleotide of a capped RNA strand at the 2'-O position. This process is part of a viral capping system and is crucial for viral evasion of the innate immune reaction. In light of recently discovered non-canonical RNA caps, we tested various dinucleoside polyphosphate-capped RNAs as substrates for nsp10-nsp16 MTase. We developed an LC-MS-based method and discovered four types of capped RNA (m7Gp3A(G)- and Gp3A(G)-RNA) that are substrates of the nsp10-nsp16 MTase. Our technique is an alternative to the classical isotope labelling approach for the measurement of 2'-O-MTase activity. Further, we determined the IC50 value of sinefungin to illustrate the use of our approach for inhibitor screening. In the future, this approach may be an alternative technique to the radioactive labelling method for screening inhibitors of any type of 2'-O-MTase.


Assuntos
COVID-19/virologia , Metiltransferases/metabolismo , SARS-CoV-2/enzimologia , Proteínas não Estruturais Virais/metabolismo , Proteínas Virais Reguladoras e Acessórias/metabolismo , Cromatografia Líquida , Regulação Viral da Expressão Gênica , Humanos , Espectrometria de Massas , Metilação , Metiltransferases/genética , Capuzes de RNA , RNA Viral/genética , SARS-CoV-2/genética , Especificidade por Substrato , Proteínas não Estruturais Virais/genética , Proteínas Virais Reguladoras e Acessórias/genética
18.
J Biol Chem ; 297(4): 101205, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34543624

RESUMO

The histone chaperone Spt6 is involved in promoting elongation of RNA polymerase II (RNAPII), maintaining chromatin structure, regulating cotranscriptional histone modifications, and controlling mRNA processing. These diverse functions of Spt6 are partly mediated through its interactions with RNAPII and other factors in the transcription elongation complex. In this study, we used mass spectrometry to characterize the differences in RNAPII-interacting factors between wildtype cells and those depleted for Spt6, leading to the identification of proteins that depend on Spt6 for their interaction with RNAPII. The altered association of some of these factors could be attributed to changes in steady-state protein levels. However, Abd1, the mRNA cap methyltransferase, had decreased association with RNAPII after Spt6 depletion despite unchanged Abd1 protein levels, showing a requirement for Spt6 in mediating the Abd1-RNAPII interaction. Genome-wide studies showed that Spt6 is required for maintaining the level of Abd1 over transcribed regions, as well as the level of Spt5, another protein known to recruit Abd1 to chromatin. Abd1 levels were particularly decreased at the 5' ends of genes after Spt6 depletion, suggesting a greater need for Spt6 in Abd1 recruitment over these regions. Together, our results show that Spt6 is important in regulating the composition of the transcription elongation complex and reveal a previously unknown function for Spt6 in the recruitment of Abd1.


Assuntos
Chaperonas de Histonas/metabolismo , Metiltransferases/metabolismo , Elementos de Resposta , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Transcrição Genética , Fatores de Elongação da Transcrição/metabolismo , Cromatina/genética , Cromatina/metabolismo , Chaperonas de Histonas/genética , Espectrometria de Massas , Metiltransferases/genética , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Fatores de Elongação da Transcrição/genética
19.
Molecules ; 26(18)2021 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-34577183

RESUMO

Despite many efforts, malaria remains among the most problematic infectious diseases worldwide, mainly due to the development of drug resistance by P. falciparum. Over the past decade, new essential pathways have been emerged to fight against malaria. Among them, epigenetic processes and mitochondrial metabolism appear to be important targets. This review will focus on recent evolutions concerning worldwide efforts to conceive, synthesize and evaluate new drug candidates interfering selectively and efficiently with these two targets and pathways. The focus will be on compounds/scaffolds that possess biological/pharmacophoric properties on DNA methyltransferases and HDAC's for epigenetics, and on cytochrome bc1 and dihydroorotate dehydrogenase for mitochondrion.


Assuntos
Antimaláricos/química , Malária Falciparum/tratamento farmacológico , Mitocôndrias/metabolismo , Plasmodium falciparum/efeitos dos fármacos , Animais , Antimaláricos/farmacologia , DNA/química , Descoberta de Drogas , Resistência a Medicamentos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Epigênese Genética , Histona Desacetilases/metabolismo , Humanos , Metiltransferases/antagonistas & inibidores , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/antagonistas & inibidores , Quinazolinas/química , Quinazolinas/farmacologia , Transdução de Sinais , Relação Estrutura-Atividade
20.
Microbiol Spectr ; 9(2): e0131221, 2021 10 31.
Artigo em Inglês | MEDLINE | ID: mdl-34585949

RESUMO

The large (L) polymerase proteins of most nonsegmented, negative-stranded (NNS) RNA viruses have conserved methyltransferase motifs, (G)-G-G-D and K-D-K-E, which are important for the stabilization and translation of mRNA. However, the function of the (G)-G-G-D and K-D-K-E motifs in the NNS RNA virus Newcastle disease virus (NDV) remains unclear. We observed G-G-D and K-D-K-E motifs in all NDV genotypes. By using the infection cloning system of NDV rSG10 strain, recombinant NDVs with a single amino acid mutated to alanine in one motif (G-G-D or K-D-K-E) were rescued. The intracerebral pathogenicity index and mean death time assay results revealed that the G-G-D motif and K-D-K-E motif attenuate the virulence of NDV to various degrees. The replication, transcription, and translation levels of the K-D-K-E motif-mutant strains were significantly higher than those of wild-type virus owing to their altered regulation of the affinity between nucleocapsid protein and eukaryotic translation initiation factor 4E. When the infection dose was changed from a multiplicity of infection (MOI) of 10 to an MOI of 0.01, the cell-to-cell spread abilities of G-G-D- and K-D-K-E-mutant strains were reduced, according to plaque assay and dynamic indirect immunofluorescence assay results. Finally, we found that NDV strains with G-G-D or K-D-K-E motif mutations had less pathogenicity in 3-week-old specific-pathogen-free chickens than wild-type NDV. Therefore, these methyltransferase motifs can affect virulence by regulating the translation and cell-to-cell spread abilities of NDV. This work provides a feasible approach for generating vaccine candidates for viruses with methyltransferase motifs. IMPORTANCE Newcastle disease virus (NDV) is an important pathogen that is widespread globally. Research on its pathogenic mechanism is an important means of improving prevention and control efforts. Our study found that a deficiency in its methyltransferase motifs (G-G-D and K-D-K-E motifs) can attenuate NDV and revealed the molecular mechanism by which these motifs affect pathogenicity, which provides a new direction for the development of NDV vaccines. In addition to the (G)-G-G-D and K-D-K-E motifs of many nonsegmented, negative-stranded RNA viruses, similar motifs have been found in dengue virus, Zika virus, Japanese encephalitis virus (JEV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This suggests that such motifs may be present in more viruses. Our finding also provides a molecular basis for the discovery and functional study of (G)-G-G-D and K-D-K-E motifs of other viruses.


Assuntos
Motivos de Aminoácidos/genética , Metiltransferases/genética , Doença de Newcastle/transmissão , Vírus da Doença de Newcastle/crescimento & desenvolvimento , Vírus da Doença de Newcastle/genética , Proteínas Virais/genética , Animais , Linhagem Celular , Galinhas , Chlorocebus aethiops , Cricetinae , Genoma Viral/genética , Vírus da Doença de Newcastle/patogenicidade , Doenças das Aves Domésticas/transmissão , Doenças das Aves Domésticas/virologia , RNA Viral/biossíntese , RNA Viral/genética , Células Vero , Virulência/genética , Replicação Viral/genética
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