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1.
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
2.
Planta ; 254(5): 88, 2021 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-34586497

RESUMO

MAIN CONCLUSION: An Arabidopsis S-adenosyl-L-methionine-dependent methyltransferase belonging to the SABATH family catalyzes the specific carboxymethylation of (11R)-carlactonoic acid. Methyl carlactonoate (MeCLA), found in Arabidopsis (Arabidopsis thaliana) as a non-canonical strigolactone (SL), may be a biosynthetic intermediate of various non-canonical SLs and biologically active as a plant hormone. MeCLA is formed from carlactonoic acid (CLA), but the methyltransferases (MTs) converting CLA to MeCLA remain unclear. Previous studies have demonstrated that the carboxymethylation of acidic plant hormones is catalyzed by the same protein family, the SABATH family (Wang et al. in Evol Bioinform 15:117693431986086. https://doi.org/10.1177/1176934319860864 , 2019). In the present study, we focused on the At4g36470 gene, an Arabidopsis SABATH MT gene co-expressed with the MAX1 gene responsible for CLA formation for biochemical characterization. The recombinant At4g36470 protein expressed in Escherichia coli exhibited exclusive activity against naturally occurring (11R)-CLA among the substrates, including CLA enantiomers and a variety of acidic plant hormones. The apparent Km value for (11R)-CLA was 1.46 µM, which was relatively smaller than that of the other Arabidopsis SABATH MTs responsible for the carboxymethylation of acidic plant hormones. The strict substrate specificity and high affinity of At4g36470 suggested it is an (11R)-CLA MT. We also confirmed the function of the identified gene by reconstructing MeCLA biosynthesis using transient expression in Nicotiana benthamiana. Phylogenetic analysis demonstrated that At4g36470 and its orthologs in non-canonical SL-producing plants cluster together in an exclusive clade, suggesting that the SABATH MTs of this clade may be involved in the carboxymethylation of CLA and the biosynthesis of non-canonical SLs.


Assuntos
Arabidopsis , Arabidopsis/genética , Arabidopsis/metabolismo , Metilação , Metiltransferases/genética , Metiltransferases/metabolismo , Filogenia , Reguladores de Crescimento de Plantas
3.
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
4.
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
5.
Nat Commun ; 12(1): 5355, 2021 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-34504067

RESUMO

Peptide backbone α-N-methylations change the physicochemical properties of amide bonds to provide structural constraints and other favorable characteristics including biological membrane permeability to peptides. Borosin natural product pathways are the only known ribosomally encoded and posttranslationally modified peptides (RiPPs) pathways to incorporate backbone α-N-methylations on translated peptides. Here we report the discovery of type IV borosin natural product pathways (termed 'split borosins'), featuring an iteratively acting α-N-methyltransferase and separate precursor peptide substrate from the metal-respiring bacterium Shewanella oneidensis. A series of enzyme-precursor complexes reveal multiple conformational states for both α-N-methyltransferase and substrate. Along with mutational and kinetic analyses, our results give rare context into potential strategies for iterative maturation of RiPPs.


Assuntos
Proteínas de Bactérias/metabolismo , Produtos Biológicos/metabolismo , Metiltransferases/metabolismo , Peptídeos/metabolismo , Processamento de Proteína Pós-Traducional , Algoritmos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sítios de Ligação/genética , Cristalografia por Raios X , Cinética , Metilação , Metiltransferases/química , Metiltransferases/genética , Mutação , Peptídeos/química , Peptídeos/genética , Conformação Proteica , Multimerização Proteica , Ribossomos/genética , Ribossomos/metabolismo , Shewanella/enzimologia , Shewanella/genética , Especificidade por Substrato
6.
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
7.
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
8.
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
9.
Nat Methods ; 18(9): 1060-1067, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34480159

RESUMO

N6-methyladenosine (m6A) is the most prevalent modification of messenger RNA in mammals. To interrogate its functions and dynamics, there is a critical need to quantify m6A at three levels: site, gene and sample. Current approaches address these needs in a limited manner. Here we develop m6A-seq2, relying on multiplexed m6A-immunoprecipitation of barcoded and pooled samples. m6A-seq2 allows a big increase in throughput while reducing technical variability, requirements of input material and cost. m6A-seq2 is furthermore uniquely capable of providing sample-level relative quantitations of m6A, serving as an orthogonal alternative to mass spectrometry-based approaches. Finally, we develop a computational approach for gene-level quantitation of m6A. We demonstrate that using this metric, roughly 30% of the variability in RNA half life in mouse embryonic stem cells can be explained, establishing m6A as a main driver of RNA stability. m6A-seq2 thus provides an experimental and analytic framework for dissecting m6A-mediated regulation at three different levels.


Assuntos
Adenosina/análogos & derivados , Estabilidade de RNA/genética , Análise de Sequência de RNA/métodos , Adenosina/análise , Adenosina/genética , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Expressão Gênica , Meia-Vida , Meiose , Metiltransferases/genética , Metiltransferases/metabolismo , Camundongos , Camundongos Knockout , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/fisiologia , Fatores de Processamento de RNA/genética , Fatores de Processamento de RNA/metabolismo , Leveduras/genética
10.
Biomed Res Int ; 2021: 1015293, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34485508

RESUMO

This article is aimed at exploring the relationship between the phosphatase 2A catalytic subunit Cα (PP2Acα, encoded by PPP2CA) and methyltransferase-like 3 (METTL3) in the malignant progression of gastric cancer (GC). Through analyzing the bioinformatics database and clinical tissue immunohistochemistry results, we found that abnormal PP2Acα and METTL3 levels were closely related to the malignant progression of GC. To explore the internal connection between PP2Acα and METTL3 in the progression of GC, we carried out cellular and molecular experiments and finally proved that PP2Acα inhibition can upregulate METTL3 levels by activating ATM activity, thereby promoting the malignant progression of GC.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Metiltransferases/metabolismo , Proteína Fosfatase 2/antagonistas & inibidores , Neoplasias Gástricas/metabolismo , Animais , Proteínas Mutadas de Ataxia Telangiectasia/genética , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Biologia Computacional/métodos , Progressão da Doença , Xenoenxertos , Humanos , Masculino , Metiltransferases/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Fosforilação , Proteína Fosfatase 2/genética , Proteína Fosfatase 2/metabolismo , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia , Taxa de Sobrevida
11.
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
12.
Exp Cell Res ; 406(1): 112761, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34339675

RESUMO

Stresses, such as neurohumoral activation, induced pathological cardiac hypertrophy is the main risk factor for heart failure. The ubiquitin-proteasome system (UPS) plays a key role in maintaining protein homeostasis and cardiac function. However, research on the role and mechanism of deubiquitinating enzymes (DUBs) in cardiac hypertrophy is limited. Here, we observe that the deubiquitinating enzyme ubiquitin-specific protease 12(USP12) is upregulated in Ang II-induced hypertrophic hearts and primary neonatal rat cardiomyocytes (NRCMs). Inhibition of USP12 ameliorate Ang II-induced myocardial hypertrophy, while overexpression of USP12 have the opposite effect. USP12 deficiency also significantly attenuate the phenotype of Ang II-induced cardiac hypertrophy in vivo. Moreover, we demonstrate that USP12 aggravate Ang II-induced cardiac hypertrophy by enhancing METTL3, a methyltransferase which catalyze N6-methyladenosine (m6A) modification on messenger RNA and acts as a harmful factor in pathological cardiac hypertrophy. Upregulation of METTL3 reverse the reduction of myocardial hypertrophy induced by USP12 silencing in NRCMs. In contrast, knockdown of METTL3 attenuate the aggravation of myocardial hypertrophy in USP12-overexpressing NRCMs. Furthermore, we discover that USP12 promote the expression of METTL3 via upregulating p300. Mechanistically, USP12 binds and stabilizes p300, thereby activating the transcription of its downstream gene METTL3. Finally, our data show that USP12 is partially dependent on the stabilization of p300 to activate METTL3 expression and promote myocardial hypertrophy. Taken together, our results demonstrate that USP12 acts as a pro-hypertrophic deubiquitinating enzyme via enhancing p300/METTL3 axis, indicating that targeting USP12 could be a potential treatment strategy for pathological cardiac hypertrophy.


Assuntos
Cardiomegalia/genética , Proteína p300 Associada a E1A/genética , Metiltransferases/genética , Miócitos Cardíacos/metabolismo , Ubiquitina Tiolesterase/genética , Adenosina/análogos & derivados , Adenosina/metabolismo , Angiotensina II/administração & dosagem , Animais , Animais Recém-Nascidos , Cardiomegalia/induzido quimicamente , Cardiomegalia/metabolismo , Cardiomegalia/patologia , Proteína p300 Associada a E1A/metabolismo , Regulação da Expressão Gênica , Masculino , Metiltransferases/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miócitos Cardíacos/citologia , Cultura Primária de Células , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Ubiquitina Tiolesterase/metabolismo , Ubiquitinação
13.
J Virol ; 95(20): e0059221, 2021 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-34379509

RESUMO

The current pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to dramatic economic and health burdens. Although the worldwide SARS-CoV-2 vaccination campaign has begun, exploration of other vaccine candidates is needed due to uncertainties with the current approved vaccines, such as durability of protection, cross-protection against variant strains, and costs of long-term production and storage. In this study, we developed a methyltransferase-defective recombinant vesicular stomatitis virus (mtdVSV)-based SARS-CoV-2 vaccine candidate. We generated mtdVSVs expressing SARS-CoV-2 full-length spike (S) protein, S1, or its receptor-binding domain (RBD). All of these recombinant viruses grew to high titers in mammalian cells despite high attenuation in cell culture. The SARS-CoV-2 S protein and its truncations were highly expressed by the mtdVSV vector. These mtdVSV-based vaccine candidates were completely attenuated in both immunocompetent and immunocompromised mice. Among these constructs, mtdVSV-S induced high levels of SARS-CoV-2-specific neutralizing antibodies (NAbs) and Th1-biased T-cell immune responses in mice. In Syrian golden hamsters, the serum levels of SARS-CoV-2-specific NAbs triggered by mtdVSV-S were higher than the levels of NAbs in convalescent plasma from recovered COVID-19 patients. In addition, hamsters immunized with mtdVSV-S were completely protected against SARS-CoV-2 replication in lung and nasal turbinate tissues, cytokine storm, and lung pathology. Collectively, our data demonstrate that mtdVSV expressing SARS-CoV-2 S protein is a safe and highly efficacious vaccine candidate against SARS-CoV-2 infection. IMPORTANCE Viral mRNA cap methyltransferase (MTase) is essential for mRNA stability, protein translation, and innate immune evasion. Thus, viral mRNA cap MTase activity is an excellent target for development of live attenuated or live vectored vaccine candidates. Here, we developed a panel of MTase-defective recombinant vesicular stomatitis virus (mtdVSV)-based SARS-CoV-2 vaccine candidates expressing full-length S, S1, or several versions of the RBD. These mtdVSV-based vaccine candidates grew to high titers in cell culture and were completely attenuated in both immunocompetent and immunocompromised mice. Among these vaccine candidates, mtdVSV-S induces high levels of SARS-CoV-2-specific neutralizing antibodies (Nabs) and Th1-biased immune responses in mice. Syrian golden hamsters immunized with mtdVSV-S triggered SARS-CoV-2-specific NAbs at higher levels than those in convalescent plasma from recovered COVID-19 patients. Furthermore, hamsters immunized with mtdVSV-S were completely protected against SARS-CoV-2 challenge. Thus, mtdVSV is a safe and highly effective vector to deliver SARS-CoV-2 vaccine.


Assuntos
Vacinas contra COVID-19/imunologia , COVID-19/prevenção & controle , SARS-CoV-2/imunologia , Vírus da Estomatite Vesicular Indiana/genética , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Encéfalo/virologia , COVID-19/imunologia , Linhagem Celular , Síndrome da Liberação de Citocina/prevenção & controle , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Humanos , Imunogenicidade da Vacina , Pulmão/imunologia , Pulmão/patologia , Pulmão/virologia , Mesocricetus , Metiltransferases/genética , Metiltransferases/metabolismo , Camundongos , Domínios Proteicos , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Células Th1/imunologia , Vacinas Sintéticas/imunologia , Vírus da Estomatite Vesicular Indiana/enzimologia , Vírus da Estomatite Vesicular Indiana/fisiologia , Proteínas Virais/genética , Proteínas Virais/metabolismo , Replicação Viral
14.
Nat Commun ; 12(1): 4848, 2021 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-34381037

RESUMO

There is currently a lack of effective drugs to treat people infected with SARS-CoV-2, the cause of the global COVID-19 pandemic. The SARS-CoV-2 Non-structural protein 13 (NSP13) has been identified as a target for anti-virals due to its high sequence conservation and essential role in viral replication. Structural analysis reveals two "druggable" pockets on NSP13 that are among the most conserved sites in the entire SARS-CoV-2 proteome. Here we present crystal structures of SARS-CoV-2 NSP13 solved in the APO form and in the presence of both phosphate and a non-hydrolysable ATP analog. Comparisons of these structures reveal details of conformational changes that provide insights into the helicase mechanism and possible modes of inhibition. To identify starting points for drug development we have performed a crystallographic fragment screen against NSP13. The screen reveals 65 fragment hits across 52 datasets opening the way to structure guided development of novel antiviral agents.


Assuntos
Metiltransferases/química , RNA Helicases/química , SARS-CoV-2/química , Proteínas não Estruturais Virais/química , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Apoenzimas/química , Apoenzimas/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Desenho de Fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Metiltransferases/antagonistas & inibidores , Metiltransferases/metabolismo , Modelos Moleculares , Fosfatos/química , Fosfatos/metabolismo , Conformação Proteica , RNA Helicases/antagonistas & inibidores , RNA Helicases/metabolismo , RNA Viral/química , RNA Viral/metabolismo , SARS-CoV-2/enzimologia , Relação Estrutura-Atividade , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/metabolismo
15.
Theranostics ; 11(16): 7640-7657, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34335955

RESUMO

Background: Since primary prostate cancer (PCa) can advance to the life-threatening metastatic PCa, exploring the molecular mechanisms underlying PCa metastasis is crucial for developing the novel targeted preventive strategies for decreasing the mortality of PCa. RNA N6-methyladenosine (m6A) is an emerging regulatory mechanism for gene expression and its specific roles in PCa progression remains elusive. Methods: Western blotting, quantitative real-time PCR and immunohistochemical analyses were used to detect target gene expression in PCa cells in vitro and prostate tissues from patients. RNA immunoprecipitation was conducted to analyze the specific binding of mRNA to the target protein. Migration and invasion assays were used to assess the migratory capacities of cancer cells. The correlation between target gene expression and survival rate of PCa patients was analyzed based the TCGA database. Results: We found that total RNA N6-methyladenosine (m6A) modification levels were markedly upregulated in human PCa tissues due to increased expression of methyltransferase like 3 (METTL3). Further studies revealed that the migratory and invasive capacities of PCa cells were markedly suppressed upon METTL3 knockdown. Mechanistically, METTL3 mediates m6A modification of USP4 mRNA at A2696, and m6A reader protein YTHDF2 binds to and induces degradation of USP4 mRNA by recruiting RNA-binding protein HNRNPD to the mRNA. Decrease of USP4 fails to remove the ubiquitin group from ELAVL1 protein, resulting in a reduction of ELAVL1 protein. Lastly, downregulation of ELAVL1 in turn increases ARHGDIA expression, promoting migration and invasion of PCa cells. Conclusions: Our findings highlight the role of METTL3 in modulating invasion and metastasis of PCa cells, providing insight into promising therapeutic strategies for hindering PCa progressing to deadly metastases.


Assuntos
Metiltransferases/genética , Neoplasias da Próstata/metabolismo , Adenosina/análogos & derivados , Adenosina/genética , Adenosina/metabolismo , Movimento Celular/genética , Proliferação de Células/genética , Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/genética , Inativação Gênica/fisiologia , Humanos , Masculino , Metiltransferases/metabolismo , Invasividade Neoplásica/genética , Metástase Neoplásica/genética , Próstata/metabolismo , Próstata/patologia , Neoplasias da Próstata/genética , RNA Mensageiro/genética , Proteínas de Ligação a RNA/metabolismo , Proteases Específicas de Ubiquitina/genética
16.
Nat Commun ; 12(1): 4913, 2021 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-34389722

RESUMO

Epitranscriptomic mechanisms linking tRNA function and the brain proteome to cognition and complex behaviors are not well described. Here, we report bi-directional changes in depression-related behaviors after genetic disruption of neuronal tRNA cytosine methylation, including conditional ablation and transgene-derived overexpression of Nsun2 in the mouse prefrontal cortex (PFC). Neuronal Nsun2-deficiency was associated with a decrease in tRNA m5C levels, resulting in deficits in expression of 70% of tRNAGly isodecoders. Altogether, 1488/5820 proteins changed upon neuronal Nsun2-deficiency, in conjunction with glycine codon-specific defects in translational efficiencies. Loss of Gly-rich proteins critical for glutamatergic neurotransmission was associated with impaired synaptic signaling at PFC pyramidal neurons and defective contextual fear memory. Changes in the neuronal translatome were also associated with a 146% increase in glycine biosynthesis. These findings highlight the methylation sensitivity of glycinergic tRNAs in the adult PFC. Furthermore, they link synaptic plasticity and complex behaviors to epitranscriptomic modifications of cognate tRNAs and the proteomic homeostasis associated with specific amino acids.


Assuntos
Transtorno Depressivo/fisiopatologia , Epigênese Genética/genética , Metiltransferases/genética , Proteoma/metabolismo , RNA de Transferência/genética , Transmissão Sináptica/genética , Animais , Transtorno Depressivo/genética , Transtorno Depressivo/metabolismo , Perfilação da Expressão Gênica/métodos , Metiltransferases/deficiência , Metiltransferases/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Neurônios/metabolismo , Córtex Pré-Frontal/citologia , Córtex Pré-Frontal/metabolismo , Córtex Pré-Frontal/fisiologia , Proteômica/métodos , RNA de Transferência/metabolismo , Transdução de Sinais/genética
17.
Viruses ; 13(8)2021 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-34452352

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the coronavirus disease-19 pandemic. One of the key components of the coronavirus replication complex are the RNA methyltransferases (MTases), RNA-modifying enzymes crucial for RNA cap formation. Recently, the structure of the 2'-O MTase has become available; however, its biological characterization within the infected cells remains largely elusive. Here, we report a novel monoclonal antibody directed against the SARS-CoV-2 non-structural protein nsp10, a subunit of both the 2'-O RNA and N7 MTase protein complexes. Using this antibody, we investigated the subcellular localization of the SARS-CoV-2 MTases in cells infected with the SARS-CoV-2.


Assuntos
COVID-19/virologia , Metiltransferases/metabolismo , Capuzes de RNA/genética , RNA Viral/genética , SARS-CoV-2/enzimologia , Proteínas não Estruturais Virais/metabolismo , Proteínas Virais Reguladoras e Acessórias/metabolismo , Anticorpos Monoclonais/análise , Humanos , Metiltransferases/análise , Metiltransferases/genética , Transporte Proteico , Capuzes de RNA/metabolismo , RNA Viral/metabolismo , SARS-CoV-2/química , SARS-CoV-2/genética , Proteínas não Estruturais Virais/análise , Proteínas não Estruturais Virais/genética , Proteínas Virais Reguladoras e Acessórias/análise , Proteínas Virais Reguladoras e Acessórias/genética
18.
Cancer Sci ; 112(10): 4087-4099, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34309973

RESUMO

To explore the effect of insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2) on colorectal cancer (CRC) by recognizing the m6A modification of YAP mRNA thus activating ErbB2 expression. High expressions of IGF2BP2, YAP, and ErbB2 promoted the proliferation, migration and invasion of CRC cells and reduced their apoptosis. IGF2BP2 recognized the m6A on YAP mRNA and promoted the translation of mRNA. YAP regulated ErbB2 expression by promoting TEAD4 enrichment in ErbB2 promoter region. Therefore, IGF2BP2 promoted the expression of ErbB2 to enhance the proliferation, invasion and migration of CRC cells, to repress cell apoptosis, and to promote solid tumor formation in nude mice. IGF2BP2 activates the expression of ErbB2 by recognizing the m6A of YAP, thus affecting the cell cycle of CRC, inhibiting cell apoptosis, and promoting proliferation.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Neoplasias Colorretais/metabolismo , Proteínas de Ligação a RNA/fisiologia , Receptor ErbB-2/metabolismo , Fatores de Transcrição/metabolismo , Animais , Apoptose , Ciclo Celular , Movimento Celular , Proliferação de Células , Neoplasias Colorretais/patologia , Proteínas de Ligação a DNA/metabolismo , Progressão da Doença , Humanos , Metiltransferases/genética , Metiltransferases/metabolismo , Camundongos , Camundongos Nus , Proteínas Musculares/metabolismo , Invasividade Neoplásica , Regiões Promotoras Genéticas , Modificação Traducional de Proteínas , RNA Mensageiro/metabolismo
19.
Theor Appl Genet ; 134(10): 3411-3426, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34258645

RESUMO

KEY MESSAGE: A plant-specific Trimethylguanosine Synthase1-like homologue was identified as a candidate gene for the efl mutation in narrow-leafed lupin, which alters phenology by reducing vernalisation requirement. The vernalisation pathway is a key component of flowering time control in plants from temperate regions but is not well understood in the legume family. Here we examined vernalisation control in the temperate grain legume species, narrow-leafed lupin (Lupinus angustifolius L.), and discovered a candidate gene for an ethylene imine mutation (efl). The efl mutation changes phenology from late to mid-season flowering and additionally causes transformation from obligate to facultative vernalisation requirement. The efl locus was mapped to pseudochromosome NLL-10 in a recombinant inbred line (RIL) mapping population developed by accelerated single seed descent. Candidate genes were identified in the reference genome, and a diverse panel of narrow-leafed lupins was screened to validate mutations specific to accessions with efl. A non-synonymous SNP mutation within an S-adenosyl-L-methionine-dependent methyltransferase protein domain of a Trimethylguanosine Synthase1-like (TGS1) orthologue was identified as the candidate mutation giving rise to efl. This mutation caused substitution of an amino acid within an established motif at a position that is otherwise highly conserved in several plant families and was perfectly correlated with the efl phenotype in F2 and F6 genetic population and a panel of diverse accessions, including the original efl mutant. Expression of the TGS1 homologue did not differ between wild-type and efl genotypes, supporting altered functional activity of the gene product. This is the first time a TGS1 orthologue has been associated with vernalisation response and flowering time control in any plant species.


Assuntos
Flores/crescimento & desenvolvimento , Regulação da Expressão Gênica de Plantas , Genética Populacional , Lupinus/crescimento & desenvolvimento , Metiltransferases/metabolismo , Folhas de Planta/crescimento & desenvolvimento , Proteínas de Plantas/metabolismo , Flores/genética , Lupinus/genética , Metiltransferases/genética , Mutação , Fenótipo , Filogenia , Folhas de Planta/genética , Proteínas de Plantas/genética
20.
Appl Environ Microbiol ; 87(18): e0054321, 2021 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-34232745

RESUMO

In Serratia marcescens JNB5-1, prodigiosin was highly produced at 30°C, but it was noticeably repressed at ≥37°C. Our initial results demonstrated that both the production and the stability of the O-methyl transferase (PigF) and oxidoreductase (PigN) involved in the prodigiosin pathway in S. marcescens JNB5-1 sharply decreased at ≥37°C. Therefore, in this study, we improved mRNA stability and protein production using de novo polynucleotide fragments (PNFs) and the introduction of disulfide bonds, respectively, and observed their effects on prodigiosin production. Our results demonstrate that adding PNFs at the 3' untranslated regions of pigF and pigN significantly improved the mRNA half-lives of these genes, leading to an increase in the transcript and expression levels. Subsequently, the introduction of disulfide bonds in pigF improved the thermal stability, pH stability, and copper ion resistance of PigF. Finally, shake flask fermentation showed that the prodigiosin titer with the engineered S. marcescens was increased by 61.38% from 5.36 to 8.65 g/liter compared to the JNB5-1 strain at 30°C and, significantly, the prodigiosin yield increased 2.05-fold from 0.38 to 0.78 g/liter at 37°C. In this study, we revealed that the introduction of PNFs and disulfide bonds greatly improved the expression and stability of pigF and pigN, hence efficiently enhancing prodigiosin production with S. marcescens at 30 and 37°C. IMPORTANCE This study highlights a promising strategy to improve mRNA/enzyme stability and to increase production using de novo PNF libraries and the introduction of disulfide bonds into the protein. PNFs could increase the half-life of target gene mRNA and effectively prevent its degradation. Moreover, PNFs could increase the relative intensity of target genes without affecting the expression of other genes; as a result, it could alleviate the cellular burden compared to other regulatory elements such as promoters. In addition, we obtained a PigF variant with improved activity and stability by the introduction of disulfide bonds into PigF. Collectively, we demonstrate here a novel approach for improving mRNA/enzyme stability using PNFs, which results in enhanced prodigiosin production in S. marcescens at 30°C.


Assuntos
Proteínas de Bactérias/genética , Metiltransferases/genética , Prodigiosina/biossíntese , Serratia marcescens/genética , Serratia marcescens/metabolismo , Regiões 3' não Traduzidas , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Dissulfetos/química , Fermentação , Concentração de Íons de Hidrogênio , Metiltransferases/química , Metiltransferases/metabolismo , Simulação de Dinâmica Molecular , Polinucleotídeos/genética , Estabilidade Proteica , RNA Mensageiro/genética , Temperatura
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