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1.
FASEB J ; 35(11): e21935, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34591327

RESUMO

Inosine monophosphate (IMP) is the intracellular precursor for both adenosine monophosphate and guanosine monophosphate and thus plays a central role in intracellular purine metabolism. IMP can also serve as an extracellular signaling molecule, and can regulate diverse processes such as taste sensation, neutrophil function, and ischemia-reperfusion injury. How IMP regulates inflammation induced by bacterial products or bacteria is unknown. In this study, we demonstrate that IMP suppressed tumor necrosis factor (TNF)-α production and augmented IL-10 production in endotoxemic mice. IMP exerted its effects through metabolism to inosine, as IMP only suppressed TNF-α following its CD73-mediated degradation to inosine in lipopolysaccharide-activated macrophages. Studies with gene targeted mice and pharmacological antagonism indicated that A2A , A2B, and A3 adenosine receptors are not required for the inosine suppression of TNF-α production. The inosine suppression of TNF-α production did not require its metabolism to hypoxanthine through purine nucleoside phosphorylase or its uptake into cells through concentrative nucleoside transporters indicating a role for alternative metabolic/uptake pathways. Inosine augmented IL-ß production by macrophages in which inflammasome was activated by lipopolysaccharide and ATP. In contrast to its effects in endotoxemia, IMP failed to affect the inflammatory response to abdominal sepsis and pneumonia. We conclude that extracellular IMP and inosine differentially regulate the inflammatory response.


Assuntos
Endotoxemia/metabolismo , Inosina Monofosfato/metabolismo , Inosina/metabolismo , Pneumonia Pneumocócica/metabolismo , Streptococcus pneumoniae , Antagonistas do Receptor A2 de Adenosina/farmacologia , Antagonistas do Receptor A3 de Adenosina/farmacologia , Animais , Modelos Animais de Doenças , Interleucina-10/biossíntese , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pneumonia Pneumocócica/microbiologia , Quinazolinas/farmacologia , Receptor A2A de Adenosina/metabolismo , Receptor A2B de Adenosina/metabolismo , Receptor A3 de Adenosina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Triazóis/farmacologia , Fator de Necrose Tumoral alfa/biossíntese
2.
Immunity ; 54(9): 1961-1975.e5, 2021 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-34525337

RESUMO

Nucleic acids are powerful triggers of innate immunity and can adopt the Z-conformation, an unusual left-handed double helix. Here, we studied the biological function(s) of Z-RNA recognition by the adenosine deaminase ADAR1, mutations in which cause Aicardi-Goutières syndrome. Adar1mZα/mZα mice, bearing two point mutations in the Z-nucleic acid binding (Zα) domain that abolish Z-RNA binding, displayed spontaneous induction of type I interferons (IFNs) in multiple organs, including in the lung, where both stromal and hematopoietic cells showed IFN-stimulated gene (ISG) induction. Lung neutrophils expressed ISGs induced by the transcription factor IRF3, indicating an initiating role for neutrophils in this IFN response. The IFN response in Adar1mZα/mZα mice required the adaptor MAVS, implicating cytosolic RNA sensing. Adenosine-to-inosine changes were enriched in transposable elements and revealed a specific requirement of ADAR1's Zα domain in editing of a subset of RNAs. Thus, endogenous RNAs in Z-conformation have immunostimulatory potential curtailed by ADAR1, with relevance to autoinflammatory disease in humans.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/imunologia , Adenosina Desaminase/genética , Interferon Tipo I/imunologia , RNA de Cadeia Dupla/genética , Adenosina/genética , Adenosina/metabolismo , Animais , Doenças Autoimunes do Sistema Nervoso/genética , Doenças Autoimunes do Sistema Nervoso/imunologia , Inosina/genética , Inosina/metabolismo , Interferon Tipo I/genética , Camundongos , Mutação , Malformações do Sistema Nervoso/genética , Malformações do Sistema Nervoso/imunologia , Edição de RNA/genética , RNA de Cadeia Dupla/metabolismo
3.
Nucleic Acids Res ; 49(12): 7011-7034, 2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34125917

RESUMO

The modification of adenosine to inosine at the wobble position (I34) of tRNA anticodons is an abundant and essential feature of eukaryotic tRNAs. The expansion of inosine-containing tRNAs in eukaryotes followed the transformation of the homodimeric bacterial enzyme TadA, which generates I34 in tRNAArg and tRNALeu, into the heterodimeric eukaryotic enzyme ADAT, which modifies up to eight different tRNAs. The emergence of ADAT and its larger set of substrates, strongly influenced the tRNA composition and codon usage of eukaryotic genomes. However, the selective advantages that drove the expansion of I34-tRNAs remain unknown. Here we investigate the functional relevance of I34-tRNAs in human cells and show that a full complement of these tRNAs is necessary for the translation of low-complexity protein domains enriched in amino acids cognate for I34-tRNAs. The coding sequences for these domains require codons translated by I34-tRNAs, in detriment of synonymous codons that use other tRNAs. I34-tRNA-dependent low-complexity proteins are enriched in functional categories related to cell adhesion, and depletion in I34-tRNAs leads to cellular phenotypes consistent with these roles. We show that the distribution of these low-complexity proteins mirrors the distribution of I34-tRNAs in the phylogenetic tree.


Assuntos
Inosina/metabolismo , Biossíntese de Proteínas , RNA de Transferência/metabolismo , Adenosina Desaminase/genética , Adesão Celular , Processos de Crescimento Celular , Linhagem Celular , Códon , Eucariotos/genética , Feminino , Células HEK293 , Humanos , Domínios Proteicos/genética , Inibidores da Síntese de Proteínas/farmacologia , RNA Mensageiro/metabolismo , RNA de Transferência/química , Ribossomos/metabolismo
4.
Nutr Res ; 91: 44-56, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34134040

RESUMO

Severe acute malnutrition (SAM), due to poor energy and/or protein intake, is associated with poor growth, depressed immune function, and long-term impacts on metabolic function. As the liver is a major metabolic organ and malnutrition poses metabolic stress, we hypothesize that SAM will be associated with alterations in the hepatic metabolome reflective of oxidative stress, gluconeogenesis, and ketogenesis. Thus, the purpose of this secondary analysis was to understand how SAM alters hepatic metabolism using a piglet model. Weanling piglets were feed either a reference (REF) or protein-energy deficient diet (MAL) for 5 weeks. After dietary treatment MAL piglets were severely underweight (weight-for-age Z-score of -3.29, Welch's t test, P = .0007), moderately wasted (weight-for-length Z-score of-2.49, Welch's t test, P = .003), and tended toward higher hepatic triglyceride content (Welch's t test, P = .07). Hematologic and blood biochemical measurements were assessed at baseline and after dietary treatment. The hepatic metabolome was investigated using 1H-NMR spectroscopy. Hepatic concentrations of betaine, cysteine, and glutathione tended to be lower in MAL (Welch's t test with FDR correction, P < .1), while inosine, lactate, and methionine sulfoxide concentrations were higher in MAL (inosine: P = .0448, lactate: P = .0258, methionine sulfoxide: P = .0337). These changes suggest that SAM is associated with elevated hepatic oxidative stress, increased gluconeogenesis, and alterations in 1-carbon metabolism.


Assuntos
Fígado/metabolismo , Metaboloma , Estresse Oxidativo , Desnutrição Aguda Grave/metabolismo , Animais , Betaína/metabolismo , Cisteína/metabolismo , Dieta , Gluconeogênese , Glutationa/metabolismo , Inosina/metabolismo , Ácido Láctico/metabolismo , Masculino , Metabolômica/métodos , Metionina/análogos & derivados , Metionina/metabolismo , Safrol/análogos & derivados , Safrol/metabolismo , Desnutrição Aguda Grave/complicações , Suínos , Magreza , Triglicerídeos
5.
Nucleic Acids Res ; 49(11): 6529-6548, 2021 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-34057470

RESUMO

Post-transcriptional modification of tRNA wobble adenosine into inosine is crucial for decoding multiple mRNA codons by a single tRNA. The eukaryotic wobble adenosine-to-inosine modification is catalysed by the ADAT (ADAT2/ADAT3) complex that modifies up to eight tRNAs, requiring a full tRNA for activity. Yet, ADAT catalytic mechanism and its implication in neurodevelopmental disorders remain poorly understood. Here, we have characterized mouse ADAT and provide the molecular basis for tRNAs deamination by ADAT2 as well as ADAT3 inactivation by loss of catalytic and tRNA-binding determinants. We show that tRNA binding and deamination can vary depending on the cognate tRNA but absolutely rely on the eukaryote-specific ADAT3 N-terminal domain. This domain can rotate with respect to the ADAT catalytic domain to present and position the tRNA anticodon-stem-loop correctly in ADAT2 active site. A founder mutation in the ADAT3 N-terminal domain, which causes intellectual disability, does not affect tRNA binding despite the structural changes it induces but most likely hinders optimal presentation of the tRNA anticodon-stem-loop to ADAT2.


Assuntos
Adenosina Desaminase/química , Adenosina/metabolismo , Adenosina Desaminase/genética , Adenosina Desaminase/metabolismo , Animais , Domínio Catalítico , Linhagem Celular Tumoral , Movimento Celular , Cristalografia por Raios X , Ferredoxinas/química , Inosina/metabolismo , Camundongos , Modelos Moleculares , Mutação , Neurônios/fisiologia , Domínios Proteicos , RNA de Transferência/química , RNA de Transferência/metabolismo
6.
Genes (Basel) ; 12(4)2021 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-33921764

RESUMO

The nucleoside inosine plays an important role in purine biosynthesis, gene translation, and modulation of the fate of RNAs. The editing of adenosine to inosine is a widespread post-transcriptional modification in transfer RNAs (tRNAs) and messenger RNAs (mRNAs). At the wobble position of tRNA anticodons, inosine profoundly modifies codon recognition, while in mRNA, inosines can modify the sequence of the translated polypeptide or modulate the stability, localization, and splicing of transcripts. Inosine is also found in non-coding and exogenous RNAs, where it plays key structural and functional roles. In addition, molecular inosine is an important secondary metabolite in purine metabolism that also acts as a molecular messenger in cell signaling pathways. Here, we review the functional roles of inosine in biology and their connections to human health.


Assuntos
Códon , Doença/genética , Inosina/genética , Biossíntese de Proteínas , RNA Mensageiro/genética , RNA de Transferência/genética , Animais , Humanos , Inosina/metabolismo , RNA Mensageiro/metabolismo , RNA de Transferência/metabolismo
7.
Biomolecules ; 11(4)2021 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-33917025

RESUMO

During the preparative synthesis of 2-fluorocordycepin from 2-fluoroadenosine and 3'-deoxyinosine catalyzed by E. coli purine nucleoside phosphorylase, a slowdown of the reaction and decrease of yield down to 5% were encountered. An unknown nucleoside was found in the reaction mixture and its structure was established. This nucleoside is formed from the admixture of 2',3'-anhydroinosine, a byproduct in the preparation of 3-'deoxyinosine. Moreover, 2',3'-anhydroinosine forms during radical dehalogenation of 9-(2',5'-di-O-acetyl-3'-bromo- -3'-deoxyxylofuranosyl)hypoxanthine, a precursor of 3'-deoxyinosine in chemical synthesis. The products of 2',3'-anhydroinosine hydrolysis inhibit the formation of 1-phospho-3-deoxyribose during the synthesis of 2-fluorocordycepin. The progress of 2',3'-anhydroinosine hydrolysis was investigated. The reactions were performed in D2O instead of H2O; this allowed accumulating intermediate substances in sufficient quantities. Two intermediates were isolated and their structures were confirmed by mass and NMR spectroscopy. A mechanism of 2',3'-anhydroinosine hydrolysis in D2O is fully determined for the first time.


Assuntos
Desoxiadenosinas/biossíntese , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Purina-Núcleosídeo Fosforilase/metabolismo , Adenosina/análogos & derivados , Adenosina/química , Adenosina/metabolismo , Biocatálise , Desoxiadenosinas/química , Óxido de Deutério/química , Hidrólise , Inosina/análogos & derivados , Inosina/química , Inosina/metabolismo , Especificidade por Substrato
8.
Mol Cell ; 81(11): 2374-2387.e3, 2021 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-33905683

RESUMO

Adenosine-to-inosine editing is catalyzed by ADAR1 at thousands of sites transcriptome-wide. Despite intense interest in ADAR1 from physiological, bioengineering, and therapeutic perspectives, the rules of ADAR1 substrate selection are poorly understood. Here, we used large-scale systematic probing of ∼2,000 synthetic constructs to explore the structure and sequence context determining editability. We uncover two structural layers determining the formation and propagation of A-to-I editing, independent of sequence. First, editing is robustly induced at fixed intervals of 35 bp upstream and 30 bp downstream of structural disruptions. Second, editing is symmetrically introduced on opposite sites on a double-stranded structure. Our findings suggest a recursive model for RNA editing, whereby the structural alteration induced by the editing at one site iteratively gives rise to the formation of an additional editing site at a fixed periodicity, serving as a basis for the propagation of editing along and across both strands of double-stranded RNA structures.


Assuntos
Adenosina Desaminase/genética , Adenosina/metabolismo , Inosina/metabolismo , Edição de RNA , RNA de Cadeia Dupla/genética , Proteínas de Ligação a RNA/genética , Células A549 , Adenosina/genética , Adenosina Desaminase/metabolismo , Animais , Pareamento de Bases , Células HEK293 , Humanos , Inosina/genética , Células MCF-7 , Camundongos , Células NIH 3T3 , Conformação de Ácido Nucleico , RNA de Cadeia Dupla/química , RNA de Cadeia Dupla/metabolismo , Proteínas de Ligação a RNA/metabolismo
9.
Nat Commun ; 12(1): 2287, 2021 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-33863894

RESUMO

Both adenine base editors (ABEs) and cytosine base editors (CBEs) have been recently revealed to induce transcriptome-wide RNA off-target editing in a guide RNA-independent manner. Here we construct a reporter system containing E.coli Hokb gene with a tRNA-like motif for robust detection of RNA editing activities as the optimized ABE, ABEmax, induces highly efficient A-to-I (inosine) editing within an E.coli tRNA-like structure. Then, we design mutations to disrupt the potential interaction between TadA and tRNAs in structure-guided principles and find that Arginine 153 (R153) within TadA is essential for deaminating RNAs with core tRNA-like structures. Two ABEmax or mini ABEmax variants (TadA* fused with Cas9n) with deletion of R153 within TadA and/or TadA* (named as del153/del153* and mini del153) are successfully engineered, showing minimized RNA off-targeting, but comparable DNA on-targeting activities. Moreover, R153 deletion in recently reported ABE8e or ABE8s can also largely reduce their RNA off-targeting activities. Taken together, we develop a strategy to generate engineered ABEs (eABEs) with minimized RNA off-targeting activities.


Assuntos
Adenosina Desaminase/genética , Proteína 9 Associada à CRISPR/genética , DNA/genética , Proteínas de Escherichia coli/genética , Edição de Genes/métodos , Adenina/metabolismo , Adenosina Desaminase/metabolismo , Toxinas Bacterianas/genética , Proteína 9 Associada à CRISPR/metabolismo , Linhagem Celular Tumoral , Citosina/metabolismo , DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Genes Reporter , Células HEK293 , Humanos , Inosina/genética , Inosina/metabolismo , Engenharia de Proteínas , Edição de RNA/genética , RNA de Transferência/genética , RNA de Transferência/metabolismo , RNA-Seq , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo
10.
J Immunol ; 206(8): 1691-1696, 2021 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-33782089

RESUMO

Severe COVID-19 disease is associated with elevated inflammatory responses. One form of Aicardi-Goutières syndrome caused by inactivating mutations in ADAR results in reduced adenosine-to-inosine (A-to-I) editing of endogenous dsRNAs, induction of IFNs, IFN-stimulated genes, other inflammatory mediators, morbidity, and mortality. Alu elements, ∼10% of the human genome, are the most common A-to-I-editing sites. Using leukocyte whole-genome RNA-sequencing data, we found reduced A-to-I editing of Alu dsRNAs in patients with severe COVID-19 disease. Dendritic cells infected with COVID-19 also exhibit reduced A-to-I editing of Alu dsRNAs. Unedited Alu dsRNAs, but not edited Alu dsRNAs, are potent inducers of IRF and NF-κB transcriptional responses, IL6, IL8, and IFN-stimulated genes. Thus, decreased A-to-I editing that may lead to accumulation of unedited Alu dsRNAs and increased inflammatory responses is associated with severe COVID-19 disease.


Assuntos
Adenosina/genética , Elementos Alu/genética , COVID-19/genética , Inosina/genética , Edição de RNA/genética , RNA de Cadeia Dupla/genética , SARS-CoV-2 , Índice de Gravidade de Doença , Adenosina/metabolismo , COVID-19/patologia , Células Dendríticas/metabolismo , Células Dendríticas/virologia , Genoma Humano , Humanos , Inosina/metabolismo , Fatores Reguladores de Interferon/metabolismo , NF-kappa B/metabolismo , RNA-Seq , Transdução de Sinais/genética
11.
Curr Gene Ther ; 21(3): 258-269, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33573552

RESUMO

BACKGROUND: Recent studies have revealed thousands of A-to-I RNA editing events in primates. These events are closely related to the occurrence and development of multiple cancers, but the origination and general functions of these events in ovarian cancer remain incompletely understood. OBJECTIVE: To further the determination of molecular mechanisms of ovarian cancer from the perspective of RNA editing. METHODS: Here, we used the SNP-free RNA editing Identification Toolkit (SPRINT) to detect RNA editing sites. These editing sites were then annotated, and related functional analysis was performed. RESULTS: In this study, about 1.7 million RES were detected in each sample, and 98% of these sites were due to A-to-G editing and were mainly distributed in non-coding regions. More than 1,000 A-- to-G RES were detected in CDS regions, and nearly 700 could lead to amino acid changes. Our results also showed that editing in the 3'UTR regions could influence miRNA-target binding. We predicted the network of changed miRNA-mRNA interaction caused by the A-to-I RNA editing sites. We also screened the differential RNA editing sites between ovarian cancer and adjacent normal tissues. We then performed GO and KEGG pathway enrichment analysis on the genes that contained these differential RNA editing sites. Finally, we identified the potential dysregulated RNA editing events in ovarian cancer samples. CONCLUSION: This study systematically identified and analyzed RNA editing events in ovarian cancer and laid a foundation to explore the regulatory mechanism of RNA editing and its function in ovarian cancer.


Assuntos
Cistadenoma Seroso/genética , Cistadenoma Seroso/metabolismo , MicroRNAs/metabolismo , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Edição de RNA , RNA Mensageiro/metabolismo , Adenosina/metabolismo , Feminino , Estudo de Associação Genômica Ampla , Humanos , Inosina/metabolismo , RNA-Seq
12.
Curr Opin Struct Biol ; 69: 1-10, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-33445115

RESUMO

RNA undergoes extensive biochemical modification following transcription. In addition to RNA splicing, transcripts are processed by a suite of enzymes that alter the chemical structure of different nucleobases. Broadly termed as 'RNA editing,' these modifications impart significant functional changes to translation, localization, and stability of individual transcripts within the cell. These changes are dynamic and required for a number of critical cellular processes, and dysregulation of these pathways is responsible for several disease states. Accurately detecting, measuring, and mapping different RNA modifications across the transcriptome is vital to understanding their broader functions as well as leveraging these events as diagnostic biomarkers. Here, we review recent advances in profiling several types of RNA modifications, with particular emphasis on adenosine-to-inosine (A-to-I) and N6-methyladenosine (m6A) RNA editing. We especially highlight approaches that utilize proteins to detect or enrich modified RNA transcripts before sequencing, and we summarize recent insights yielded from these techniques.


Assuntos
Inosina , Edição de RNA , Adenosina/metabolismo , Inosina/metabolismo , RNA/genética , RNA/metabolismo , Transcriptoma
13.
Brief Bioinform ; 22(5)2021 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-33401309

RESUMO

A-to-I RNA editing, contributing to nearly 90% of all editing events in human, has been reported to involve in the pathogenesis of Alzheimer's disease (AD) due to its roles in brain development and immune regulation, such as the deficient editing of GluA2 Q/R related to cell death and memory loss. Currently, there are urgent needs for the systematic annotations of A-to-I RNA editing events in AD. Here, we built ADeditome, the annotation database of A-to-I RNA editing in AD available at https://ccsm.uth.edu/ADeditome, aiming to provide a resource and reference for functional annotation of A-to-I RNA editing in AD to identify therapeutically targetable genes in an individual. We detected 1676 363 editing sites in 1524 samples across nine brain regions from ROSMAP, MayoRNAseq and MSBB. For these editing events, we performed multiple functional annotations including identification of specific and disease stage associated editing events and the influence of editing events on gene expression, protein recoding, alternative splicing and miRNA regulation for all the genes, especially for AD-related genes in order to explore the pathology of AD. Combing all the analysis results, we found 108 010 and 26 168 editing events which may promote or inhibit AD progression, respectively. We also found 5582 brain region-specific editing events with potentially dual roles in AD across different brain regions. ADeditome will be a unique resource for AD and drug research communities to identify therapeutically targetable editing events. Significance: ADeditome is the first comprehensive resource of the functional genomics of individual A-to-I RNA editing events in AD, which will be useful for many researchers in the fields of AD pathology, precision medicine, and therapeutic researches.


Assuntos
Adenosina/metabolismo , Doença de Alzheimer/genética , Amnésia/genética , Inosina/metabolismo , Proteínas do Tecido Nervoso/genética , Edição de RNA , Transcriptoma , Adenosina/genética , Processamento Alternativo , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Amnésia/metabolismo , Amnésia/patologia , Encéfalo/metabolismo , Encéfalo/patologia , Mapeamento Encefálico , Bases de Dados Genéticas , Ontologia Genética , Humanos , Inosina/genética , MicroRNAs/classificação , MicroRNAs/genética , MicroRNAs/metabolismo , Anotação de Sequência Molecular , Proteínas do Tecido Nervoso/classificação , Proteínas do Tecido Nervoso/metabolismo , Receptores de AMPA/genética , Receptores de AMPA/metabolismo
14.
Crit Rev Biochem Mol Biol ; 56(1): 54-87, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33356612

RESUMO

Adenosine deaminases that act on RNA (ADARs) are present in all animals and function to both bind double-stranded RNA (dsRNA) and catalyze the deamination of adenosine (A) to inosine (I). As inosine is a biological mimic of guanosine, deamination by ADARs changes the genetic information in the RNA sequence and is commonly referred to as RNA editing. Millions of A-to-I editing events have been reported for metazoan transcriptomes, indicating that RNA editing is a widespread mechanism used to generate molecular and phenotypic diversity. Loss of ADARs results in lethality in mice and behavioral phenotypes in worm and fly model systems. Furthermore, alterations in RNA editing occur in over 35 human pathologies, including several neurological disorders, metabolic diseases, and cancers. In this review, a basic introduction to ADAR structure and target recognition will be provided before summarizing how ADARs affect the fate of cellular RNAs and how researchers are using this knowledge to engineer ADARs for personalized medicine. In addition, we will highlight the important roles of ADARs and RNA editing in innate immunity and cancer biology.


Assuntos
Adenosina Desaminase/metabolismo , Carcinogênese/metabolismo , Imunidade Inata , Neoplasias/metabolismo , RNA de Cadeia Dupla/metabolismo , Proteínas de Ligação a RNA/metabolismo , Adenosina/metabolismo , Adenosina Desaminase/química , Adenosina Desaminase/genética , Animais , Desaminação , Humanos , Inosina/metabolismo , Edição de RNA , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética
15.
J Sep Sci ; 44(5): 954-962, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33348445

RESUMO

Creatinine and purines are gout-related metabolites commonly quantified by liquid chromatography coupled with ultraviolet and mass spectrometry. However, the high cost of liquid chromatography coupled with mass spectrometry hindered its extensive use in ordinary hospitals and clinical laboratories. Using the traditional liquid chromatography method, the full separation of these metabolites in complex biological samples is still not achieved. In this study, an improved ultra-high-performance liquid chromatography with ultraviolet spectroscopy method was reported for quantitative determination of five gout-related metabolites (i.e., creatinine, uric acid, hypoxanthine, xanthine, and inosine) in human serum within 10 min. A UHPLC system equipped with a hydrophilic C18 column was used to improve separation, shorten analysis time, and increase analysis throughput. The performance of the method was validated by evaluating linearity (squared correlation coefficient > 0.9991), recovery (92.8-100.0%, with relative standard deviation < 4.7%), accuracy (relative errors < 14.6%), precision (0.2-4.1% for intraday and 2.1-7.3% for interday) and stability (-14.1 to 8.3% in autosampler for 12 h and -13.3 to 2.2% for freeze-thaw cycles). This method was successfully applied to quantify gout-related metabolites in serum samples of healthy controls and gout patients, which was expected to be used in the clinical investigation of gout at different stages.


Assuntos
Creatinina/sangue , Gota/sangue , Hipoxantina/sangue , Inosina/sangue , Ácido Úrico/sangue , Xantina/sangue , Cromatografia Líquida de Alta Pressão , Creatinina/metabolismo , Gota/metabolismo , Humanos , Hipoxantina/metabolismo , Inosina/metabolismo , Ácido Úrico/metabolismo , Xantina/metabolismo
16.
Int J Mol Sci ; 21(19)2020 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-33023260

RESUMO

Adenosine is a neuromodulator that has been involved in aging and neurodegenerative diseases as Alzheimer's disease (AD). In the present work, we analyzed the possible modulation of purine metabolites, 5'nucleotidase (5'NT) and adenosine deaminase (ADA) activities, and adenosine monophosphate (AMP)-activated protein kinase (AMPK) and its phosphorylated form during aging in the cerebral cortex. Three murine models were used: senescence-accelerated mouse-resistant 1 (SAMR1, normal senescence), senescence-accelerated mouse-prone 8 (SAMP8, a model of AD), and the wild-type C57BL/6J (model of aging) mice strains. Glutamate and excitatory amino acid transporter 2 (EAAT2) levels were also measured in these animals. HPLC, Western blotting, and enzymatic activity evaluation were performed to this aim. 5'-Nucleotidase (5'NT) activity was decreased at six months and recovered at 12 months in SAMP8 while opposite effects were observed in SAMR1 at the same age, and no changes in C57BL/6J mice. ADA activity significantly decreased from 3 to 12 months in the SAMR1 mice strain, while a significant decrease from 6 to 12 months was observed in the SAMP8 mice strain. Regarding purine metabolites, xanthine and guanosine levels were increased at six months in SAMR1 without significant differences in SAMP8 mice. In C57BL/6J mice, inosine and xanthine were increased, while adenosine decreased, from 4 to 24 months. The AMPK level was decreased at six months in SAMP8 without significant changes nor in SAMR1 or C57BL/6J strains. Glutamate and EAAT2 levels were also modulated during aging. Our data show a different modulation of adenosine metabolism participants in the cerebral cortex of these animal models. Interestingly, the main differences between SAMR1 and SAMP8 mice were found at six months of age, SAMP8 being the most affected strain. As SAMP8 is an AD model, results suggest that adenosinergic metabolism is involved in the neurodegeneration of AD.


Assuntos
Adenosina/metabolismo , Envelhecimento/metabolismo , Doença de Alzheimer/genética , Córtex Cerebral/metabolismo , Envelhecimento/genética , Envelhecimento/patologia , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Animais , Senescência Celular/genética , Córtex Cerebral/patologia , Modelos Animais de Doenças , Hipocampo/metabolismo , Hipocampo/patologia , Humanos , Inosina/metabolismo , Camundongos , Fosforilação/genética , Xantina/metabolismo
17.
J Proteome Res ; 19(10): 4046-4060, 2020 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-32866021

RESUMO

Adenosine-to-inosine RNA editing is an enzymatic post-transcriptional modification which modulates immunity and neural transmission in multicellular organisms. In particular, it involves editing of mRNA codons with the resulting amino acid substitutions. We identified such sites for developmental proteomes of Drosophila melanogaster at the protein level using available data for 15 stages of fruit fly development from egg to imago and 14 time points of embryogenesis. In total, 40 sites were obtained, each belonging to a unique protein, including four sites related to embryogenesis. The interactome analysis has revealed that the majority of the editing-recoded proteins were associated with synaptic vesicle trafficking and actomyosin organization. Quantitation data analysis suggested the existence of a phase-specific RNA editing regulation with yet unknown mechanisms. These findings supported the transcriptome analysis results, which showed that a burst in the RNA editing occurs during insect metamorphosis from pupa to imago. Finally, targeted proteomic analysis was performed to quantify editing-recoded and genomically encoded versions of five proteins in brains of larvae, pupae, and imago insects, which showed a clear tendency toward an increase in the editing rate for each of them. These results will allow a better understanding of the protein role in physiological effects of RNA editing.


Assuntos
Proteínas de Drosophila , Edição de RNA , Adenosina Desaminase/genética , Adenosina Desaminase/metabolismo , Animais , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Inosina/metabolismo , Proteoma/genética , Proteoma/metabolismo , Proteômica , RNA Mensageiro/genética
18.
Science ; 369(6510): 1481-1489, 2020 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-32792462

RESUMO

Several species of intestinal bacteria have been associated with enhanced efficacy of checkpoint blockade immunotherapy, but the underlying mechanisms by which the microbiome enhances antitumor immunity are unclear. In this study, we isolated three bacterial species-Bifidobacterium pseudolongum, Lactobacillus johnsonii, and Olsenella species-that significantly enhanced efficacy of immune checkpoint inhibitors in four mouse models of cancer. We found that intestinal B. pseudolongum modulated enhanced immunotherapy response through production of the metabolite inosine. Decreased gut barrier function induced by immunotherapy increased systemic translocation of inosine and activated antitumor T cells. The effect of inosine was dependent on T cell expression of the adenosine A2A receptor and required costimulation. Collectively, our study identifies a previously unknown microbial metabolite immune pathway activated by immunotherapy that may be exploited to develop microbial-based adjuvant therapies.


Assuntos
Bifidobacterium/metabolismo , Microbioma Gastrointestinal , Imunoterapia , Inosina/metabolismo , Neoplasias Intestinais/terapia , Lactobacillus johnsonii/metabolismo , Melanoma/terapia , Neoplasias Cutâneas/terapia , Neoplasias da Bexiga Urinária/terapia , Animais , Anticorpos/uso terapêutico , Antígeno B7-H1/antagonistas & inibidores , Antígeno B7-H1/imunologia , Antígeno CTLA-4/antagonistas & inibidores , Antígeno CTLA-4/imunologia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias Experimentais/terapia , Receptor A2A de Adenosina/metabolismo , Linfócitos T/imunologia
19.
RNA ; 26(11): 1654-1666, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32763916

RESUMO

The deamination of adenosine to inosine at the wobble position of tRNA is an essential post-transcriptional RNA modification required for wobble decoding in bacteria and eukaryotes. In humans, the wobble inosine modification is catalyzed by the heterodimeric ADAT2/3 complex. Here, we describe novel pathogenic ADAT3 variants impairing adenosine deaminase activity through a distinct mechanism that can be corrected through expression of the heterodimeric ADAT2 subunit. The variants were identified in a family in which all three siblings exhibit intellectual disability linked to biallelic variants in the ADAT3 locus. The biallelic ADAT3 variants result in a missense variant converting alanine to valine at a conserved residue or the introduction of a premature stop codon in the deaminase domain. Fibroblast cells derived from two ID-affected individuals exhibit a reduction in tRNA wobble inosine levels and severely diminished adenosine tRNA deaminase activity. Notably, the ADAT3 variants exhibit impaired interaction with the ADAT2 subunit and alterations in ADAT2-dependent nuclear localization. Based upon these findings, we find that tRNA adenosine deaminase activity and wobble inosine modification can be rescued in patient cells by overexpression of the ADAT2 catalytic subunit. These results uncover a key role for the inactive ADAT3 deaminase domain in proper assembly with ADAT2 and demonstrate that ADAT2/3 nuclear import is required for maintaining proper levels of the wobble inosine modification in tRNA.


Assuntos
Adenosina Desaminase/genética , Adenosina Desaminase/metabolismo , Deficiência Intelectual/genética , Mutação de Sentido Incorreto , RNA de Transferência/química , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Transporte Ativo do Núcleo Celular , Adenosina/metabolismo , Adenosina Desaminase/química , Adolescente , Sítios de Ligação , Células Cultivadas , Criança , Pré-Escolar , Códon de Terminação , Feminino , Predisposição Genética para Doença , Humanos , Inosina/metabolismo , Deficiência Intelectual/metabolismo , Masculino , Linhagem , Domínios Proteicos , Proteínas de Ligação a RNA/química , Sequenciamento Completo do Exoma
20.
PLoS Pathog ; 16(8): e1008740, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32822429

RESUMO

Adenosine-to-inosine (A-to-I) RNA editing is an important posttranscriptional event in eukaryotes; however, many features remain largely unexplored in prokaryotes. This study focuses on a serine-to-proline recoding event (S128P) that originated in the mRNA of fliC, which encodes a flagellar filament protein; the editing event was observed in RNA-seq samples exposed to oxidative stress. Using Sanger sequencing, we show that the S128P editing event is induced by H2O2. To investigate the in vivo interaction between RNAs and TadA, which is the principal enzyme for A-to-I editing, genome-wide RNA immunoprecipitation-coupled high-throughput sequencing (iRIP-Seq) analysis was performed using HA-tagged TadA from Xanthomonas oryzae pv. oryzicola. We found that TadA can bind to the mRNA of fliC and the binding motif is identical to that previously reported by Bar-Yaacov and colleagues. This editing event increased motility and enhanced tolerance to oxidative stress due to changes in flagellar filament structure, which was modelled in 3D and measured by TEM. The change in filament structure due to the S128P mutant increased biofilm formation, which was measured by the 3D laser scanning confocal microscopy. RNA-seq revealed that a gene cluster that contributes to siderophore biosynthesis and Fe3+ uptake was upregulated in S128P compared with WT. Based on intracellular levels of reactive oxygen species and an oxidative stress survival assay, we found that this gene cluster can contribute to the reduction of the Fenton reaction and increases biofilm formation and bacterial virulence. This oxidative stress response was also confirmed in Pseudomonas putida. Overall, our work demonstrates that A-to-I RNA editing plays a role in bacterial pathogenicity and adaptation to oxidative stress.


Assuntos
Proteínas de Bactérias/genética , Edição de RNA , Xanthomonas/genética , Xanthomonas/metabolismo , Adenosina/genética , Adenosina/metabolismo , Proteínas de Bactérias/metabolismo , Peróxido de Hidrogênio/farmacologia , Inosina/genética , Inosina/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Doenças das Plantas/microbiologia , Virulência/efeitos dos fármacos , Xanthomonas/efeitos dos fármacos , Xanthomonas/patogenicidade
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