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1.
Int J Mol Sci ; 24(2)2023 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-36675295

RESUMO

Cardiac fibrosis is a common pathological feature of different cardiovascular diseases, characterized by the aberrant deposition of extracellular matrix (ECM) proteins in the cardiac interstitium, myofibroblast differentiation and increased fibrillar collagen deposition stimulated by transforming growth factor (TGF)-ß activation. Biglycan (BGN), a small leucine-rich proteoglycan (SLRPG) integrated within the ECM, plays a key role in matrix assembly and the phenotypic control of cardiac fibroblasts. Moreover, BGN is critically involved in pathological cardiac remodeling through TGF-ß binding, thus causing myofibroblast differentiation and proliferation. Adenosine receptors (ARs), and in particular A2AR, may play a key role in stimulating fibrotic damage through collagen production/deposition, as a consequence of cyclic AMP (cAMP) and AKT activation. For this reason, A2AR modulation could be a useful tool to manage cardiac fibrosis in order to reduce fibrotic scar deposition in heart tissue. Therefore, the aim of the present study was to investigate the possible crosstalk between A2AR and BGN modulation in an in vitro model of TGF-ß-induced fibrosis. Immortalized human cardiac fibroblasts (IM-HCF) were stimulated with TGF-ß at the concentration of 10 ng/mL for 24 h to induce a fibrotic phenotype. After applying the TGF-ß stimulus, cells were treated with two different A2AR antagonists, Istradefylline and ZM241385, for an additional 24 h, at the concentration of 10 µM and 1 µM, respectively. Both A2AR antagonists were able to regulate the oxidative stress induced by TGF-ß through intracellular reactive oxygen species (ROS) reduction in IM-HCFs. Moreover, collagen1a1, MMPs 3/9, BGN, caspase-1 and IL-1ß gene expression was markedly decreased following A2AR antagonist treatment in TGF-ß-challenged human fibroblasts. The results obtained for collagen1a1, SMAD3, α-SMA and BGN were also confirmed when protein expression was evaluated; phospho-Akt protein levels were also reduced following Istradefylline and ZM241385 use, thus suggesting that collagen production involves AKT recruited by the A2AR. These results suggest that A2AR modulation might be an effective therapeutic option to reduce the fibrotic processes involved in heart pathological remodeling.


Assuntos
Fibroblastos , Proteínas Proto-Oncogênicas c-akt , Humanos , Biglicano/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fibroblastos/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Colágeno/metabolismo , Fibrose , Adenosina/farmacologia , Adenosina/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Células Cultivadas
2.
Autoimmunity ; 56(1): 2167983, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36708146

RESUMO

Skin diseases are global health issues caused by multiple pathogenic factors, in which epigenetics plays an invaluable role. Post-transcriptional RNA modifications are important epigenetic mechanism that regulate gene expression at the genome-wide level. N6-methyladenosine (m6A) is the most prevalent modification that occurs in the messenger RNAs (mRNA) of most eukaryotes, which is installed by methyltransferases called "writers", removed by demethylases called "erasers", and recognised by RNA-binding proteins called "readers". To date, m6A is emerging to play essential part in both physiological processes and pathological progression, including skin diseases. However, a systematic summary of m6A in skin disease has not yet been reported. This review starts by illustrating each m6A-related modifier specifically and their roles in RNA processing, and then focus on the existing research advances of m6A in immune homeostasis and skin diseases.


Assuntos
Metiltransferases , Dermatopatias , Humanos , Metilação , Metiltransferases/genética , Metiltransferases/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Adenosina/genética , Adenosina/metabolismo , Dermatopatias/genética , RNA/metabolismo
3.
Int J Mol Sci ; 24(1)2023 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-36614216

RESUMO

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


Assuntos
MicroRNAs , Animais , MicroRNAs/genética , MicroRNAs/metabolismo , Adenosina/metabolismo , Metilação , Metiltransferases/metabolismo , Epigênese Genética , Proteínas de Transporte/metabolismo , Fatores de Transcrição/metabolismo , Mamíferos/metabolismo
5.
J Am Chem Soc ; 145(2): 929-943, 2023 Jan 18.
Artigo em Inglês | MEDLINE | ID: mdl-36608272

RESUMO

Adenosine tripolyphosphate (ATP) is a small polyvalent anion that has recently been shown to interact with proteins and have a major impact on assembly processes involved in biomolecular condensate formation and protein aggregation. However, the nature of non-specific protein-ATP interactions and their effects on protein solubility are largely unknown. Here, the binding of ATP to the globular model protein is characterized in detail using X-ray crystallography and nuclear magnetic resonance (NMR). Using NMR, we identified six ATP binding sites on the lysozyme surface, with one known high-affinity nucleic acid binding site and five non-specific previously unknown sites with millimolar affinities that also bind tripolyphosphate (TPP). ATP binding occurs primarily through the polyphosphate moiety, which was confirmed by the X-ray structure of the lysozyme-ATP complex. Importantly, ATP binds preferentially to arginine over lysine in non-specific binding sites. ATP and TPP have similar effects on solution-phase protein-protein interactions. At low salt concentrations, ion binding to lysozyme causes precipitation, while at higher salt concentrations, redissolution occurs. The addition of an equimolar concentration of magnesium to ATP does not alter ATP binding affinities but prevents lysozyme precipitation. These findings have important implications for both protein crystallization and cell biology. Crystallization occurs readily in ATP solutions outside the well-established crystallization window. In the context of cell biology, the findings suggest that ATP binds non-specifically to folded proteins in physiological conditions. Based on the nature of the binding sites identified by NMR, we propose several mechanisms for how ATP binding can prevent the aggregation of natively folded proteins.


Assuntos
Adenosina , Muramidase , Adenosina/metabolismo , Muramidase/química , Sítios de Ligação , Polifosfatos , Trifosfato de Adenosina/metabolismo , Ligação Proteica
6.
Biomolecules ; 13(1)2023 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-36671491

RESUMO

Adenosine receptors mainly control synaptic function, and excessive activation of adenosine receptors may worsen the onset of many neurological disorders. Accordingly, the regular intake of moderate doses of caffeine antagonizes adenosine receptors and affords robust neuroprotection. Although caffeine intake alters brain functional connectivity and multi-omics analyses indicate that caffeine intake modifies synaptic and metabolic processes, it is unclear how caffeine intake affects behavior, synaptic plasticity and its modulation by adenosine. We now report that male mice drinking caffeinated water (0.3 g/L) for 2 weeks were behaviorally indistinguishable (locomotion, mood, memory) from control mice (drinking water) and displayed superimposable synaptic plasticity (long-term potentiation) in different brain areas (hippocampus, prefrontal cortex, amygdala). Moreover, there was a general preservation of the efficiency of adenosine A1 and A2A receptors to control synaptic transmission and plasticity, although there was a tendency for lower levels of endogenous adenosine ensuring A1 receptor-mediated inhibition. In spite of similar behavioral and neurophysiological function, caffeine intake increased the energy charge and redox state of cortical synaptosomes. This increased metabolic competence likely involved a putative increase in the glycolytic rate in synapses and a prospective greater astrocyte-synapse lactate shuttling. It was concluded that caffeine intake does not trigger evident alterations of behavior or of synaptic plasticity but increases the metabolic competence of synapses, which might be related with the previously described better ability of animals consuming caffeine to cope with deleterious stimuli triggering brain dysfunction.


Assuntos
Adenosina , Cafeína , Masculino , Camundongos , Animais , Cafeína/farmacologia , Adenosina/farmacologia , Adenosina/metabolismo , Estudos Prospectivos , Receptores Purinérgicos P1/metabolismo , Hipocampo/metabolismo
7.
Nat Commun ; 14(1): 489, 2023 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-36717587

RESUMO

Vascular repair is considered a key restorative measure to improve long-term outcomes after ischemic stroke. N6-methyladenosine (m6A), the most prevalent internal modification in eukaryotic mRNAs, functionally mediates vascular repair. However, whether circular RNA SCMH1 (circSCMH1) promotes vascular repair by m6A methylation after stroke remains to be elucidated. Here, we identify the role of circSCMH1 in promoting vascular repair in peri-infarct cortex of male mice and male monkeys after photothrombotic (PT) stroke, and attenuating the ischemia-induced m6A methylation in peri-infarct cortex of male mice after PT stroke. Mechanically, circSCMH1 increased the translocation of ubiquitination-modified fat mass and obesity-associated protein (FTO) into nucleus of endothelial cells (ECs), leading to m6A demethylation of phospholipid phosphatase 3 (Plpp3) mRNA and subsequently the increase of Plpp3 expression in ECs. Our data demonstrate that circSCMH1 enhances vascular repair via FTO-regulated m6A methylation after stroke, providing insights into the mechanism of circSCMH1 in promoting stroke recovery.


Assuntos
Dioxigenase FTO Dependente de alfa-Cetoglutarato , Fosfatidato Fosfatase , RNA Circular , Acidente Vascular Cerebral , Animais , Masculino , Camundongos , Adenosina/metabolismo , Dioxigenase FTO Dependente de alfa-Cetoglutarato/genética , Dioxigenase FTO Dependente de alfa-Cetoglutarato/metabolismo , Células Endoteliais/metabolismo , Infarto , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Acidente Vascular Cerebral/genética , Acidente Vascular Cerebral/terapia , Fosfatidato Fosfatase/genética , Fosfatidato Fosfatase/metabolismo
8.
Eur J Med Chem ; 247: 115052, 2023 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-36599229

RESUMO

The purinergic signaling has drawn attention from academia and more recently from pharmaceutical industries as a potential therapeutic route for cancer treatment, since ATP may act as chemotactic agent and possess in vitro antineoplastic activity. On the other way, adenosine, produced in extracellular medium by ecto-5'-NT, acts as immunosuppressor and is related to neoangiogenesis, vasculogenesis and evasion to the immune system. Consequently, inhibitors of ecto-5'-NT may prevent tumor progression, reducing adenosine concentrations, preventing escape from the host's immune system and slowing cancer's growth. This review aims to highlight important biochemical and structural features of ecto-5'NT, highlight its expression profile in normal and cancer cell lines detailing compounds which may act as expression regulators and to review the several classes of ecto-5'NT inhibitors developed in the past 12 years, in order to build a general structure-activity relationship model to guide further compound design.


Assuntos
5'-Nucleotidase , Antineoplásicos , Adenosina/farmacologia , Adenosina/metabolismo , Monofosfato de Adenosina , Antineoplásicos/farmacologia , Linhagem Celular
9.
Metabolism ; 138: 155339, 2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-36302453

RESUMO

N6-methyladenosine (m6A) methyltransferase writer proteins (METTL3/METTL14) have been shown to regulate ß-cell function and diabetes. However, whether and which m6A reader proteins regulate ß-cell function and the pathogenesis of diabetes are largely unknown. In this study, we showed that YTHDC1 (YTH domain-containing protein 1), a key m6A nuclear reader protein, plays an essential role in maintaining ß-cell function. YTHDC1 is downregulated in islet ß cells in type 2 diabetes, which is due to lipotoxicity and chronic inflammation. ß-Cell specific deletion of Ythdc1 results in ß-cell failure and diabetes, which is likely due to the decreased expression of ß-cell specific transcription factors and insulin secretion-related genes. Taken together, YTHDC1 is required for maintaining ß-cell function, and the downregulation of YTHDC1 leads to ß-cell failure and diabetes.


Assuntos
Diabetes Mellitus Tipo 2 , Humanos , Fatores de Processamento de RNA/genética , Fatores de Processamento de RNA/metabolismo , Regulação para Baixo , Proteínas do Tecido Nervoso/genética , Adenosina/metabolismo , Proteínas Nucleares/metabolismo , Metiltransferases/genética
10.
Neuropharmacology ; 224: 109370, 2023 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-36493858

RESUMO

Purine-based molecules play ancient, fundamental, and evolutionarily-conserved roles across life on Earth, ranging from DNA and RNA, to the universal energy currency, ATP. In mammals, the two primary routes for the synthesis of the adenine nucleotides ATP, ADP and AMP, and, as a consequence, the major bioactive metabolite adenosine, are the de novo purine biosynthesis (DNPB) pathway, and the purine salvage pathway (PSP). Of the two, the PSP dominates in both the mammalian brain and heart. This is because the PSP utilizes the breakdown products of ATP, occasioned by the high energy demands of these organs, to rapidly regenerate adenine nucleotides. This resynthesis route, while efficient and energetically favourable, leaves these organs vulnerable to loss of salvageable metabolites, with the potential for protracted depletion of the means to synthesize ATP, and the ability to deploy neuro- and cardioprotective adenosine. Having previously shown that hippocampal cellular ATP and adenosine release can be increased by supplying substrates for the PSP (d-ribose and adenine), we now explore the expression of DNPB and PSP enzymes in hippocampal neurons and astrocytes based on available transcriptomic data. We find that key enzymes of the PSP are expressed at higher levels than those in the DNPB pathway, and that PSP enzymes are expressed at higher levels in neurons than in astrocytes. These data reflect the importance of the PSP in the mammalian brain and imply that pharmacological targeting of the PSP may be particularly beneficial to neurons at times of metabolic stress. This article is part of the Special Issue on 'Purinergic Signaling: 50 years'.


Assuntos
Lesões Encefálicas , Purinas , Animais , Purinas/metabolismo , Adenosina/metabolismo , Mamíferos/metabolismo , Trifosfato de Adenosina/metabolismo , Metabolismo Energético , Nucleotídeos de Purina/metabolismo
11.
Chem Biol Interact ; 369: 110288, 2023 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-36509115

RESUMO

Huntington's disease (HD) is an inherited neurodegenerative disease characterized by progressive motor, behavioral, and cognitive impairments. Intrastriatal injection of 3- nitropropionic acid (3NP) was used to induce HD-like symptoms by inhibiting succinate dehydrogenase enzyme (SDH) in the mitochondrial complex II. The adenosine A1 receptor has long been known to have a crucial role in neuroprotection, mainly by blocking Ca2+ influx, which causes inhibition of glutamate (Glu) and a decline in its excitatory effects at the postsynaptic level. To this end, this study investigated the possible involvement of TrKB/PI3K/Akt/CREB/BDNF pathway in mediating protection afforded by the central N6-cyclohexyladenosine (CHA), an adenosine A1 receptor agonist. A single intrastriatal CHA injection (6.25 nM/1 µL); 45min after 3-NP injection, attenuated neuronal death, and improved cognitive and motor deficits caused by 3-NP neurotoxin. This effect was shown to parallel an enhanced activation of PI3K/Akt/CREB/BDNF axis as well as boosting pERK1/2 levels. Moreover, CHA attenuated neuroinflammatory and oxidative stress status via reducing NFκB p65, TNFα and iNOS contents and increasing SOD. Furthermore, immunohistochemical data showed a reduction in the glial fibrillary acidic protein (GFAP) immunoreactivity to a marker for astrocyte and microglia activation following CHA treatment. The results of this study suggest that CHA may have protective effect against HD via modulating oxidative stress, excitotoxic and inflammatory pathways.


Assuntos
Doença de Huntington , Doenças Neurodegenerativas , Fármacos Neuroprotetores , Ratos , Animais , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Doença de Huntington/induzido quimicamente , Doença de Huntington/tratamento farmacológico , Doença de Huntington/metabolismo , Doenças Neurodegenerativas/tratamento farmacológico , Corpo Estriado , Adenosina/metabolismo , Nitrocompostos/toxicidade , Fármacos Neuroprotetores/uso terapêutico
12.
Methods Mol Biol ; 2584: 347-370, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36495460

RESUMO

RNA editing is a widespread molecular phenomenon occurring in a variety of organisms. In humans, it mainly involves the deamination of adenosine to inosine (A-to-I) in double-stranded RNAs by ADAR enzymes. A-to-I RNA editing has been investigated in different tissues as well as in diverse experimental and pathological conditions. By contrast, its biological role in single cells has not been explored in depth. Recent methodologies for cell sorting in combination with deep sequencing technologies have enabled the study of eukaryotic transcriptomes at single cell resolution, paving the way to the profiling of their epitranscriptomic dynamics.Here we describe a step-by-step protocol to detect and characterize A-to-I events occurring in publicly available single-cell RNAseq experiments from human alpha and beta pancreatic cells.


Assuntos
Adenosina Desaminase , Edição de RNA , Humanos , Adenosina Desaminase/genética , Inosina/genética , Inosina/metabolismo , Adenosina/genética , Adenosina/metabolismo , RNA de Cadeia Dupla , RNA/genética
13.
Am J Physiol Cell Physiol ; 324(2): C327-C338, 2023 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-36503240

RESUMO

Arterial calcification due to deficiency of CD73 (ACDC) is a rare genetic disease caused by a loss-of-function mutation in the NT5E gene encoding the ecto-5'-nucleotidase (cluster of differentiation 73, CD73) enzyme. Patients with ACDC develop vessel arteriomegaly, tortuosity, and vascular calcification in their lower extremity arteries. Histological analysis shows that patients with ACDC vessels exhibit fragmented elastin fibers similar to that seen in aneurysmal-like pathologies. It is known that alterations in transforming growth factor ß (TGFß) pathway signaling contribute to this elastin phenotype in several connective tissue diseases, as TGFß regulates extracellular matrix (ECM) remodeling. Our study investigates whether CD73-derived adenosine modifies TGFß signaling in vascular smooth muscle cells (SMCs). We show that Nt5e-/- SMCs have elevated contractile markers and elastin gene expression compared with Nt5e+/+ SMCs. Ecto-5'-nucleotidase (Nt5e)-deficient SMCs exhibit increased TGFß-2 and activation of small mothers against decapentaplegic (SMAD) signaling, elevated elastin transcript and protein, and potentiate SMC contraction. These effects were diminished when the A2b adenosine receptor was activated. Our results identify a novel link between adenosine and TGFß signaling, where adenosine signaling via the A2b adenosine receptor attenuates TGFß signaling to regulate SMC homeostasis. We discuss how disruption in adenosine signaling is implicated in ACDC vessel tortuosity and could potentially contribute to other aneurysmal pathogenesis.


Assuntos
5'-Nucleotidase , Adenosina , Adenosina/metabolismo , Elastina/genética , Transdução de Sinais , Fator de Crescimento Transformador beta
14.
J Med Chem ; 66(1): 934-950, 2023 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-36581322

RESUMO

Antivirulence agents targeting endospore-transmitted Clostridioides difficile infections are urgently needed. C. difficile-specific DNA adenine methyltransferase (CamA) is required for efficient sporulation and affects persistence in the colon. The active site of CamA is conserved and closely resembles those of hundreds of related S-adenosyl-l-methionine (SAM)-dependent methyltransferases, which makes the design of selective inhibitors more challenging. We explored the solvent-exposed edge of the SAM adenosine moiety and systematically designed 42 analogs of adenosine carrying substituents at the C6-amino group (N6) of adenosine. We compare the inhibitory properties and binding affinity of these diverse compounds and present the crystal structures of CamA in complex with 14 of them in the presence of substrate DNA. The most potent of these inhibitors, compound 39 (IC50 ∼ 0.4 µM and KD ∼ 0.2 µM), is selective for CamA against closely related bacterial and mammalian DNA and RNA adenine methyltransferases, protein lysine and arginine methyltransferases, and human adenosine receptors.


Assuntos
Clostridioides difficile , Metiltransferases , Animais , Humanos , Metiltransferases/química , Adenosina/metabolismo , Adenina/farmacologia , Adenina/metabolismo , S-Adenosilmetionina/metabolismo , DNA/metabolismo , Proteína-Arginina N-Metiltransferases , Mamíferos/metabolismo
15.
Mol Ther ; 30(2): 932-946, 2022 02 02.
Artigo em Inglês | MEDLINE | ID: mdl-34547464

RESUMO

N6-methyladenosine (m6A) mRNA modification plays critical roles in various biological events and is involved in multiple complex diseases. However, the role of m6A modification in autophagy in nonalcoholic fatty liver disease (NAFLD) remains largely unknown. Here, we report that m6A modification was increased in livers of NAFLD mouse models and in free fatty acid (FFA)-treated hepatocytes, and the abnormal m6A modification was attributed to the upregulation of methyltransferase like 3 (METTL3) induced by lipotoxicity. Knockdown of METTL3 promoted hepatic autophagic flux and clearance of lipid droplets (LDs), while overexpression of METTL3 inhibited these processes. Mechanistically, METTL3 directly bound to Rubicon mRNA and mediated the m6A modification, while YTH N6-methyladenosine RNA binding protein 1 (YTHDF1), as a partner of METTL3, interacted with the m6A-marked Rubicon mRNA and promoted its stability. Subsequently, RUBICON inhibited autophagosome-lysosome fusion and further blocked clearance of LDs. Taken together, our results showed a critical role of METTL3 and YTHDF1 in regulating lipid metabolism via the autophagy pathway and provided a novel insight into m6A mRNA methylation in NAFLD.


Assuntos
Hepatopatia Gordurosa não Alcoólica , Adenosina/metabolismo , Animais , Autofagia/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Metilação , Metiltransferases/genética , Metiltransferases/metabolismo , Camundongos , Hepatopatia Gordurosa não Alcoólica/genética , Proteínas de Ligação a RNA
16.
Mol Cancer ; 21(1): 220, 2022 12 14.
Artigo em Inglês | MEDLINE | ID: mdl-36517820

RESUMO

Cancer drug resistance represents the main obstacle in cancer treatment. Drug-resistant cancers exhibit complex molecular mechanisms to hit back therapy under pharmacological pressure. As a reversible epigenetic modification, N6-methyladenosine (m6A) RNA modification was regarded to be the most common epigenetic RNA modification. RNA methyltransferases (writers), demethylases (erasers), and m6A-binding proteins (readers) are frequently disordered in several tumors, thus regulating the expression of oncoproteins, enhancing tumorigenesis, cancer proliferation, development, and metastasis. The review elucidated the underlying role of m6A in therapy resistance. Alteration of the m6A modification affected drug efficacy by restructuring multidrug efflux transporters, drug-metabolizing enzymes, and anticancer drug targets. Furthermore, the variation resulted in resistance by regulating DNA damage repair, downstream adaptive response (apoptosis, autophagy, and oncogenic bypass signaling), cell stemness, tumor immune microenvironment, and exosomal non-coding RNA. It is highlighted that several small molecules targeting m6A regulators have shown significant potential for overcoming drug resistance in different cancer categories. Further inhibitors and activators of RNA m6A-modified proteins are expected to provide novel anticancer drugs, delivering the therapeutic potential for addressing the challenge of resistance in clinical resistance.


Assuntos
Adenosina , Neoplasias , Humanos , Adenosina/metabolismo , Metiltransferases/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/patologia , RNA/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Microambiente Tumoral
17.
Nucleic Acids Res ; 50(22): 12979-12996, 2022 12 09.
Artigo em Inglês | MEDLINE | ID: mdl-36533443

RESUMO

Aggregation of the microtubule-associated protein tau characterizes tauopathies, including Alzheimer's disease and frontotemporal lobar degeneration (FTLD-Tau). Gene expression regulation of tau is complex and incompletely understood. Here we report that the human tau gene (MAPT) generates two circular RNAs (circRNAs) through backsplicing of exon 12 to either exon 7 (12→7 circRNA) or exon 10 (12→10 circRNA). Both circRNAs lack stop codons. The 12→7 circRNA contains one start codon and is translated in a rolling circle, generating a protein consisting of multimers of the microtubule-binding repeats R1-R4. For the 12→10 circRNA, a start codon can be introduced by two FTLD-Tau mutations, generating a protein consisting of multimers of the microtubule-binding repeats R2-R4, suggesting that mutations causing FTLD may act in part through tau circRNAs. Adenosine to inosine RNA editing dramatically increases translation of circRNAs and, in the 12→10 circRNA, RNA editing generates a translational start codon by changing AUA to AUI. Circular tau proteins self-aggregate and promote aggregation of linear tau proteins. Our data indicate that adenosine to inosine RNA editing initiates translation of human circular tau RNAs, which may contribute to tauopathies.


Assuntos
Tauopatias , Proteínas tau , Humanos , Adenosina/metabolismo , Códon de Iniciação , Inosina/metabolismo , RNA/genética , RNA/metabolismo , Edição de RNA , RNA Circular/metabolismo , Proteínas tau/genética , Proteínas tau/metabolismo , Tauopatias/genética , Tauopatias/metabolismo
18.
Biochemistry (Mosc) ; 87(11): 1301-1309, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36509721

RESUMO

RNA editing by adenosine deaminases of the ADAR family can lead to protein recoding, since inosine formed from adenosine in mRNA is complementary to cytosine; the resulting codon editing might introduce amino acid substitutions into translated proteins. Proteome recoding can have functional consequences which have been described in many animals including humans. Using protein recoding database derived from publicly available transcriptome data, we identified for the first time the recoding sites in the zebrafish shotgun proteomes. Out of more than a hundred predicted recoding events, ten substitutions were found in six used datasets. Seven of them were in the AMPA glutamate receptor subunits, whose recoding has been well described, and are conserved among vertebrates. Three sites were specific for zebrafish proteins and were found in the transmembrane receptors astrotactin 1 and neuregulin 3b (proteins involved in the neuronal adhesion and signaling) and in the rims2b gene product (presynaptic membrane protein participating in the neurotransmitter release), respectively. Further studies are needed to elucidate the role of recoding of the said three proteins in the zebrafish.


Assuntos
Proteômica , Peixe-Zebra , Animais , Humanos , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteômica/métodos , Proteínas de Peixe-Zebra/genética , Adenosina Desaminase/genética , Adenosina Desaminase/metabolismo , Proteoma/metabolismo , Adenosina/metabolismo , RNA Mensageiro/genética
19.
BMC Genomics ; 23(1): 829, 2022 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-36517742

RESUMO

BACKGROUND: Heat stress has a substantial negative economic impact on the dairy industry. N6-methyladenosine (m6A) is the most common internal RNA modification in eukaryotes and plays a key role in regulating heat stress response in animals. In dairy cows, however, this modification remains largely unexplored. Therefore, we examined the effects of heat stress on the m6A modification and gene expression in bovine mammary epithelial cells to elucidate the mechanism of heat stress response. In this study, Mammary alveolar cells-large T antigen (MAC-T) cells were incubated at 37 °C (non-heat stress group, NH) and 40 °C (heat stress group, H) for 2 hours, respectively. HSP70, HSF1, BAX and CASP3 were up regulated in H group compared with those in the NH group. RESULTS: Methylated RNA immunoprecipitation sequencing (MeRIP-seq) and RNA sequencing (RNA-seq) were conducted to identify m6A peaks and to produce gene expression data of MAC-T cells in the H and NH groups. In total, we identified 17,927 m6A peaks within 9355 genes in the H group, and 18,974 peaks within 9660 genes in the NH groups using MeRIP-seq. Compared with the NH group, 3005 significantly differentially enriched m6A peaks were identified, among which 1131 were up-regulated and 1874 were down-regulated. In addition, 1502 significantly differentially expressed genes were identified using RNA-seq, among which 796 were up-regulated and 706 were down-regulated in the H group compared to the NH group. Furthermore, 199 differentially expressed and synchronously differentially methylated genes were identified by conjoint analysis of the MeRIP-seq and RNA-seq data, which were subsequently divided into four groups: 47 hyper-up, 53 hyper-down, 59 hypo-up and 40 hypo-down genes. In addition, GO enrichment and KEGG analyses were used to analyzed the potential functions of the genes in each section. CONCLUSION: The comparisons of m6A modification patterns and conjoint analyses of m6A modification and gene expression profiles suggest that m6A modification plays a critical role in the heat stress response by regulating gene expression.


Assuntos
Perfilação da Expressão Gênica , Transcriptoma , Feminino , Bovinos , Animais , Adenosina/metabolismo , Resposta ao Choque Térmico/genética , Células Epiteliais/metabolismo , RNA/metabolismo
20.
J Transl Med ; 20(1): 605, 2022 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-36527141

RESUMO

BACKGROUND: N6-methyladenosine (m6A) is the most prevalent epigenetic modification in eukaryotic messenger RNAs and plays a critical role in cell fate transition. However, it remains to be elucidated how m6A marks functionally impact the transcriptional cascades that orchestrate stem cell differentiation. The present study focuses on the biological function and mechanism of m6A methylation in dental pulp stem cell (DPSC) differentiation. METHODS: m6A RNA immunoprecipitation sequencing was utilized to assess the m6A-mRNA landscape during DPSC differentiation. Ectopic transplantation of DPSCs in immunodeficient mice was conducted to verify the in vitro findings. RNA sequencing and m6A RNA immunoprecipitation sequencing were combined to identify the candidate targets. RNA immunoprecipitation and RNA/protein stability of Noggin (NOG) were evaluated. The alteration in poly(A) tail was measured by 3'-RACE and poly(A) tail length assays. RESULTS: We characterized a dynamic m6A-mRNA landscape during DPSC mineralization with increasing enrichment in the 3' untranslated region (UTR). Methyltransferase-like 3 (METTL3) was identified as the key m6A player, and METTL3 knockdown disrupted functional DPSC differentiation. Moreover, METTL3 overexpression enhanced DPSC mineralization. Increasing m6A deposition in the 3' UTR restricted NOG expression, which is required for DPSC mineralization. This stage-specific m6A methylation and destabilization of NOG was suppressed by METTL3 knockdown only in differentiated DPSCs. Furthermore, METTL3 promotes the degradation of m6A-tagged NOG by shortening the poly(A) tail length in the differentiated stage. CONCLUSIONS: Our results address an essential role of dynamic m6A signaling in the temporal control of DPSC differentiation and provide new insight into epitranscriptomic mechanisms in stem cell-based therapy.


Assuntos
Adenosina , Metiltransferases , Camundongos , Animais , Metiltransferases/genética , Metiltransferases/metabolismo , Adenosina/metabolismo , Polpa Dentária , Diferenciação Celular , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
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