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Introduction: Recent advances in induced pluripotent stem (iPS) technology and regenerative medicine require effective cryopreservation of iPSC-derived differentiated cells and three-dimensional cell aggregates (eg. Spheroids and organoids). Moreover, innovative freezing technologies for keeping food fresh over the long-term rapidly developed in the food industry. Therefore, we examined whether one of such freezing technologies, called "Dynamic Effect Powerful Antioxidation Keeping (DEPAK)," could be effective for the cryopreservation of biological materials. Methods: We evaluated the efficiency of cryopreservation using DEPAK and Proton freezers, both of which are used in the food industry, compared with conventional slow-freezing methods using a programmable freezer and a cell-freezing vessel. As they are highly susceptible cells to freeze-thaw damage, we selected two suspension cell lines (KHYG-1 derived from human natural killer cell leukemia and THP-1 derived from human acute monocyte leukemia) and two adherent cell lines (OVMANA derived from human ovarian tumors and HuH-7 derived from human hepatocarcinoma). We used two human iPS cell lines, 201B7-Ff and 1231A3, which were either undifferentiated or differentiated into neurospheres. After freezing using the above methods, the frozen cells and neurospheres were immediately transferred to liquid nitrogen. After thawing, we assessed the cryopreservation efficiency of cell viability, proliferation, neurosphere formation, and neurite outgrowth after thawing. Results: Among the four cryopreservation methods, DEPAK freezing resulted in the highest cell proliferation in suspension and adherent cell lines. Similar results were obtained for the cryopreservation of undifferentiated human iPS cells. In addition, we demonstrated that the DEPAK freezing method sustained the neurosphere formation capacity of differentiated iPS cells to the same extent as unfrozen controls. In addition, we observed that DEPAK-frozen neurospheres exhibited higher viability after thawing and underwent neural differentiation more efficiently than slow-freezing methods. Conclusions: Our results suggest that diversifying food-freezing technologies can overcome the difficulties associated with the cryopreservation of various biological materials, including three-dimensional cell aggregates.
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BACKGROUND: SOX4 is a transcription factor belonging to the SOX (Sry-related High Mobility Group [HMG] box) family and plays a pivotal role in various biological processes at various stages of life. SOX4 is also expressed in the skin in adults and has been reported to be involved in wound healing, tumor formation, and metastasis. METHODS AND RESULTS: In this study, we investigated the role of SOX4 in keratinocyte phenotypic changes. We generated a SOX4-overexpressing keratinocyte cell line that expresses SOX4 in a doxycycline (DOX)-inducible manner. DOX treatment induced a change from a paving stone-like morphology to a spindle-like morphology under microscopic observation. Comprehensive gene analysis by RNA sequencing revealed increased expression of genes related to anatomical morphogenesis and cell differentiation as well as decreased expression of genes related to epithelial formation and keratinization, suggesting that SOX4 induced EMT-like phenotype in keratinocytes. Differentially expressed genes (DEGs) obtained by RNA-seq were confirmed using qRT-PCR. DOX-treated TY-1 SOX4 showed a decrease in the epithelial markers (KRT15, KRT13, KRT5, and CLDN1) and an increase in the mesenchymal marker FN1. Protein expression changes by Western blotting also showed a decrease in the epithelial marker proteins keratin 15, keratin 13, and claudin 1, and an increase in the mesenchymal marker fibronectin. Removal of DOX from DOX-treated cells also restored the epithelial and mesenchymal markers altered by SOX4. CONCLUSION: Our results indicate that SOX4 reversibly induces an EMT-like phenotype in human keratinocytes via suppression of epithelial marker genes.
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Queratinócitos , Fatores de Transcrição SOXC , Pele , Humanos , Western Blotting , Doxiciclina , Expressão Gênica , Fenótipo , Fatores de Transcrição SOXC/genéticaRESUMO
The endoplasmic reticulum (ER)-embedded transcription factors, sterol regulatory element-binding proteins (SREBPs), master regulators of lipid biosynthesis, are transported to the Golgi for proteolytic activation to tune cellular cholesterol levels and regulate lipogenesis. However, mechanisms by which the cell responds to the levels of saturated or unsaturated fatty acids remain underexplored. Here, we show that RHBDL4/RHBDD1, a rhomboid family protease, directly cleaves SREBP-1c at the ER. The p97/VCP, AAA-ATPase complex then acts as an auxiliary segregase to extract the remaining ER-embedded fragment of SREBP-1c. Importantly, the enzymatic activity of RHBDL4 is enhanced by saturated fatty acids (SFAs) but inhibited by polyunsaturated fatty acids (PUFAs). Genetic deletion of RHBDL4 in mice fed on a Western diet enriched in SFAs and cholesterol prevented SREBP-1c from inducing genes for lipogenesis, particularly for synthesis and incorporation of PUFAs, and secretion of lipoproteins. The RHBDL4-SREBP-1c pathway reveals a regulatory system for monitoring fatty acid composition and maintaining cellular lipid homeostasis.
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Histone posttranslational modifications play critical roles in a variety of eukaryotic cellular processes. In particular, methylation at lysine and arginine residues is an epigenetic mark that determines the chromatin state. In addition, histone "histidine" methylation was initially reported over 50 years ago; however, further studies in this area were not conducted, leaving a gap in our understanding. Here, we aimed to investigate the occurrence of histidine methylation in histone proteins using highly sensitive mass spectrometry. We found that acid hydrolysates of whole histone fraction from calf thymus contained Nτ-methylhistidine, but not Nπ-methylhistidine. Both core and linker histones carried a Nτ-methylhistidine modification, and methylation levels were relatively high in histone H3. Furthermore, through MALDI-TOF MS, we identified two histidine methylation sites at His-82 in the structured globular domain of histone H2A and His-39 in the N-terminal tail of histones H3. Importantly, these histidine methylation signals were also detected in histones purified from a human cell line HEK293T. Moreover, we revealed the overall methylation status of histone H3, suggesting that methylation is enriched primarily at lysine residues and to a lesser extent at arginine and histidine residues. Thus, our findings established histidine Nτ-methylation as a new histone modification, which may serve as a chemical flag that mediates the epigenetic mark of adjacent residues of the N-terminal tail and the conformational properties of the globular domain.
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Protein methylation is mainly observed in lysine, arginine and histidine residues. Histidine methylation occurs at one of two different nitrogen atoms of the imidazole ring, producing Nτ-methylhistidine and Nπ-methylhistidine, and it has recently attracted attention with the identification of SETD3, METTL18 and METTL9 as catalytic enzymes in mammals. Although accumulating evidence had suggested the presence of more than 100 proteins containing methylated histidine residues in cells, much less information has been known regarding histidine-methylated proteins than lysine- and arginine-methylated ones, because no method has been developed to identify substrates for histidine methylation. Here, we established a method to screen novel target proteins for histidine methylation, using biochemical protein fractionation combined with the quantification of methylhistidine by LC-MS/MS. Interestingly, the differential distribution pattern of Nτ-methylated proteins was found between the brain and skeletal muscle, and identified γ-enolase where the His-190 at the Nτ position is methylated in mouse brain. Finally, in silico structural prediction and biochemical analysis showed that the His-190 in γ-enolase is involved in the intermolecular homodimeric formation and enzymatic activity. In the present study, we provide a new methodology to find histidine-methylated proteins in vivo and suggest an insight into the importance of histidine methylation.
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Histidina , Metilistidinas , Camundongos , Animais , Metilistidinas/análise , Histidina/metabolismo , Lisina/metabolismo , Isoenzimas , Cromatografia Líquida , Espectrometria de Massas em Tandem , Proteínas , Fosfopiruvato Hidratase , Arginina , MamíferosRESUMO
Protein methylation is one of the most common post-translational modifications observed in basic amino acid residues, including lysine, arginine, and histidine. Histidine methylation occurs on the distal or proximal nitrogen atom of its imidazole ring, producing two isomers: Nτ-methylhistidine or Nπ-methylhistidine. However, the biological significance of protein histidine methylation remains largely unclear owing in part to the very limited knowledge about its contributing enzymes. Here, we identified mammalian seven-ß-strand methyltransferase METTL9 as a histidine Nπ-methyltransferase by siRNA screening coupled with methylhistidine analysis using LC-tandem MS. We demonstrated that METTL9 catalyzes Nπ-methylhistidine formation in the proinflammatory protein S100A9, but not that of myosin light chain kinase MYLK2, in vivo and in vitro. METTL9 does not affect the heterodimer formation of S100A9 and S100A8, although Nπ-methylation of S100A9 at His-107 overlaps with a zinc-binding site, attenuating its affinity for zinc. Given that S100A9 exerts an antimicrobial activity, probably by chelation of zinc essential for the growth of bacteria and fungi, METTL9-mediated S100A9 methylation might be involved in the innate immune response to bacterial and fungal infection. Thus, our findings suggest a functional consequence for protein histidine Nπ-methylation and may add a new layer of complexity to the regulatory mechanisms of post-translational methylation.
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Calgranulina B , Metiltransferases , Processamento de Proteína Pós-Traducional , RNA Interferente Pequeno , Animais , Calgranulina B/genética , Calgranulina B/metabolismo , Células HEK293 , Células HeLa , Humanos , Inflamação/genética , Inflamação/metabolismo , Metilação , Metilistidinas/metabolismo , Metiltransferases/genética , Metiltransferases/metabolismo , Camundongos , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismoRESUMO
The nucleolus is a membrane-less nuclear structure that disassembles when cells undergo mitosis. During mitosis, nucleolar factors are thus released from the nucleolus and dynamically change their subcellular localization; however, their functions remain largely uncharacterised. Here, we found that a nucleolar factor called nucleolar protein 11 (NOL11) forms a protein complex with two tryptophan-aspartic acid (WD) repeat proteins named WD-repeat protein 43 (WDR43) and Cirhin in mitotic cells. This complex, referred to here as the NWC (NOL11-WDR43-Cirhin) complex, exists in nucleoli during interphase and translocates to the periphery of mitotic chromosomes, i.e., perichromosomal regions. During mitotic progression, both the congression of chromosomes to the metaphase plate and sister chromatid cohesion are impaired in the absence of the NWC complex, as it is required for the centromeric enrichment of Aurora B and the associating phosphorylation of histone H3 at threonine 3. These results reveal the characteristics of a novel protein complex consisting of nucleolar proteins, which is required for regulating kinetochores and centromeres to ensure faithful chromosome segregation.
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Aurora Quinase B/metabolismo , Segregação de Cromossomos , Mitose , Proteínas Nucleares/metabolismo , Células HeLa , Humanos , Proteínas Nucleares/genética , Ligação ProteicaRESUMO
The tricarboxylic acid (TCA) cycle (or citric acid cycle) is responsible for the complete oxidation of acetyl-CoA and formation of intermediates required for ATP production and other anabolic pathways, such as amino acid synthesis. Here, we uncovered an additional mechanism that may help explain the essential role of the TCA cycle in the early embryogenesis of Caenorhabditis elegans. We found that knockdown of citrate synthase (cts-1), the initial and rate-limiting enzyme of the TCA cycle, results in early embryonic arrest, but that this phenotype is not because of ATP and amino acid depletions. As a possible alternative mechanism explaining this developmental deficiency, we observed that cts-1 RNAi embryos had elevated levels of intracellular acetyl-CoA, the starting metabolite of the TCA cycle. Of note, we further discovered that these embryos exhibit hyperacetylation of mitochondrial proteins. We found that supplementation with acetylase-inhibiting polyamines, including spermidine and putrescine, counteracted the protein hyperacetylation and developmental arrest in the cts-1 RNAi embryos. Contrary to the hypothesis that spermidine acts as an acetyl sink for elevated acetyl-CoA, the levels of three forms of acetylspermidine, N1-acetylspermidine, N8-acetylspermidine, and N1,N8-diacetylspermidine, were not significantly increased in embryos treated with exogenous spermidine. Instead, we demonstrated that the mitochondrial deacetylase sirtuin 4 (encoded by the sir-2.2 gene) is required for spermidine's suppression of protein hyperacetylation and developmental arrest in the cts-1 RNAi embryos. Taken together, these results suggest the possibility that during early embryogenesis, acetyl-CoA consumption by the TCA cycle in C. elegans prevents protein hyperacetylation and thereby protects mitochondrial function.
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Caenorhabditis elegans/embriologia , Caenorhabditis elegans/metabolismo , Ciclo do Ácido Cítrico , Desenvolvimento Embrionário , Proteínas Mitocondriais/metabolismo , Acetilação , Trifosfato de Adenosina/metabolismo , Animais , Ácido Aspártico/metabolismo , Caenorhabditis elegans/citologia , Caenorhabditis elegans/genética , Citrato (si)-Sintase/deficiência , Citrato (si)-Sintase/genética , Ácido Cítrico/metabolismo , Ácido Glutâmico/metabolismo , Espaço Intracelular/metabolismo , Fatores de TempoRESUMO
Protein arginine methyltransferase 1 (PRMT1) catalyzes asymmetric arginine dimethylation of cellular proteins and thus modulates various biological processes, including gene regulation, RNA metabolism, cell signaling and DNA repair. Since prmt-1 null mutant completely abolishes asymmetric dimethylarginine in C. elegans, PRMT-1 is thought to play a crucial role in determining levels of asymmetric arginine dimethylation. However, the mechanism underlying the regulation of PRMT-1 activity remains largely unknown. Here, we explored for transcription factors that induce the expression of PRMT-1 by an RNAi screen using transgenic C. elegans harbouring prmt-1 promoter upstream of gfp. Of 529 clones, we identify a GATA transcription factor elt-2 as a positive regulator of Pprmt-1:: gfp expression and show that elt-2 RNAi decreases endogenous PRMT-1 expression at mRNA and protein levels. Nevertheless, surprisingly arginine methylation levels are increased when elt-2 is silenced, implying that erythroid-like transcription factor (ELT)-2 may also have ability to inhibit methyltransferase activity of PRMT-1. Supporting this idea, GST pull-down and co-immunoprecipitation assays demonstrate the interaction between ELT-2 and PRMT-1. Furthermore, we find that ELT-2 interferes with PRMT-1-induced arginine methylation in a dose-dependent manner. Collectively, our results illustrate the two modes of PRMT-1 regulation, which could determine the levels of asymmetric arginine dimethylation in C. elegans.
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Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Fatores de Transcrição GATA/metabolismo , Proteína-Arginina N-Metiltransferases/genética , Proteína-Arginina N-Metiltransferases/metabolismo , Animais , Caenorhabditis elegans/enzimologia , Células Cultivadas , Células HEK293 , HumanosRESUMO
The Nrf2 pathway is a biological defense system against oxidative stress. The pharmacological activation of the Nrf2 pathway is a promising therapy for oxidative stress-related diseases, but it has been challenging to find an Nrf2 activator with acceptable toxicity. To circumvent this problem, we focused on an already approved oral anti-arthritic drug, auranofin that has been reported to have the potential to activate Nrf2. We used a zebrafish model to investigate whether auranofin has protective action against oxidative stress in vivo. Auranofin pre-treatment considerably improved the survival of zebrafish larvae that were challenged with a lethal dose of hydrogen peroxide. This protective effect was not observed in an Nrf2 mutant zebrafish strain, suggesting that the activation of the biological defense against oxidative stress was Nrf2-dependent. Auranofin-induced protection was further tested by challenges with redox-active heavy metals. A clear protective effect was observed against arsenite, a highly redox-reactive toxicant. In addition, this effect was also demonstrated to be Nrf2-dependent based on the analysis of an Nrf2 mutant strain. These results clearly demonstrate the anti-oxidative action of auranofin and encourage the repositioning of auranofin as a drug that improves oxidative stress-related pathology.
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Antioxidantes/uso terapêutico , Antirreumáticos/uso terapêutico , Artrite/tratamento farmacológico , Auranofina/uso terapêutico , Fator 2 Relacionado a NF-E2/antagonistas & inibidores , Proteínas de Peixe-Zebra/antagonistas & inibidores , Administração Oral , Animais , Antioxidantes/farmacologia , Antirreumáticos/farmacologia , Arsenitos/toxicidade , Auranofina/farmacologia , Modelos Animais de Doenças , Reposicionamento de Medicamentos , Humanos , Peróxido de Hidrogênio/toxicidade , Larva , Metais Pesados/toxicidade , Mutação/genética , Fator 2 Relacionado a NF-E2/genética , Organismos Geneticamente Modificados , Oxirredução , Estresse Oxidativo/efeitos dos fármacos , Transdução de Sinais , Peixe-Zebra , Proteínas de Peixe-Zebra/genéticaRESUMO
Protein arginine methyltransferases (PRMTs) catalyze the transfer of a methyl group from S-adenosylmethionine to arginine residues and are classified into two types: type I producing asymmetric dimethylarginine (ADMA) and type II producing symmetric dimethylarginine (SDMA). PRMTs have been shown to regulate many cellular processes, including signal transduction, transcriptional regulation and RNA processing. Since the loss-of-function mutation of PRMT1 and PRMT5, each of which is the predominant type I and II, respectively, causes embryonic lethality in mice, their physiological significance at the whole-body level remains largely unknown. Here, we show the morphological and functional phenotypes of single or double null alleles of prmt-1 and prmt-5 in Caenorhabditis elegans. The prmt-1;prmt-5 double mutants are viable, and exhibit short body length and small brood size compared to N2 and each of the single mutants. The liquid chromatography-tandem mass spectrometry analysis demonstrated that the levels of ADMA and SDMA were abolished in the prmt-1;prmt-5 double mutants. Both prmt-1 and prmt-5 were required for resistance to heat and oxidative stresses, whereas prmt-5 is not involved in lifespan regulation even when prmt-1 is ablated. This mutant strain would be a useful model animal for investigating the role of asymmetric and symmetric arginine dimethylation in vivo.
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Arginina/metabolismo , Caenorhabditis elegans/enzimologia , Caenorhabditis elegans/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , Animais , MetilaçãoRESUMO
Protein arginine methyltransferase 1 (PRMT-1) catalyzes asymmetric arginine dimethylation on cellular proteins and modulates various aspects of biological processes, such as signal transduction, DNA repair, and transcriptional regulation. We have previously reported that the null mutant of prmt-1 in Caenorhabditis elegans exhibits a slightly shortened life span, but the physiological significance of PRMT-1 remains largely unclear. Here we explored the role of PRMT-1 in mitochondrial function as hinted by a two-dimensional Western blot-based proteomic study. Subcellular fractionation followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis showed that PRMT-1 is almost entirely responsible for asymmetric arginine dimethylation on mitochondrial proteins. Importantly, isolated mitochondria from prmt-1 mutants represent compromised ATP synthesis in vitro, and whole-worm respiration in prmt-1 mutants is decreased in vivo Transgenic rescue experiments demonstrate that PRMT-1-dependent asymmetric arginine dimethylation is required to prevent mitochondrial reactive oxygen species (ROS) production, which consequently causes the activation of the mitochondrial unfolded-protein response. Furthermore, the loss of enzymatic activity of prmt-1 induces food avoidance behavior due to mitochondrial dysfunction, but treatment with the antioxidant N-acetylcysteine significantly ameliorates this phenotype. These findings add a new layer of complexity to the posttranslational regulation of mitochondrial function and provide clues for understanding the physiological roles of PRMT-1 in multicellular organisms.
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Arginina/metabolismo , Caenorhabditis elegans/metabolismo , Metabolismo Energético , Homeostase , Mitocôndrias/metabolismo , Acetilcisteína/farmacologia , Trifosfato de Adenosina/metabolismo , Animais , Aprendizagem da Esquiva/efeitos dos fármacos , Comportamento Animal/efeitos dos fármacos , Caenorhabditis elegans/efeitos dos fármacos , Proteínas de Caenorhabditis elegans/metabolismo , Metabolismo Energético/efeitos dos fármacos , Homeostase/efeitos dos fármacos , Metilação/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Proteínas Mitocondriais/metabolismo , Mutação/genética , Fosforilação Oxidativa/efeitos dos fármacos , Consumo de Oxigênio/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Especificidade por Substrato/efeitos dos fármacos , Resposta a Proteínas não Dobradas/efeitos dos fármacosRESUMO
Forkhead box O (FOXO; DAF-16 in nematode) transcription factors activate a program of genes that control stress resistance, metabolism, and lifespan. Given the adverse impact of the stochastic DNA damage on organismal development and ageing, we examined the role of FOXO/DAF-16 in UV-induced DNA-damage response. Knockdown of FOXO1, but not FOXO3a, increases sensitivity to UV irradiation when exposed during S phase, suggesting a contribution of FOXO1 to translesion DNA synthesis (TLS), a replicative bypass of UV-induced DNA lesions. Actually, FOXO1 depletion results in a sustained activation of the ATR-Chk1 signaling and a reduction of PCNA monoubiquitination following UV irradiation. FOXO1 does not alter the expression of TLS-related genes but binds to the protein replication protein A (RPA1) that coats single-stranded DNA and acts as a scaffold for TLS. In Caenorhabditis elegans, daf-16 null mutants show UV-induced retardation in larval development and are rescued by overexpressing DAF-16 mutant lacking transactivation domain, but not substitution mutant unable to interact with RPA-1. Thus, our findings demonstrate that FOXO1/DAF-16 is a functional component in TLS independently of its transactivation activity.
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Replication stress response is a protective mechanism against defects in chromosome replication for maintaining genome integrity in eukaryotic cells. An alternative clamp loader complex termed chromosome transmission fidelity protein 18 and replication factor C (CTF18RFC) has been shown to act as a positive regulator of two types of replication stress response: Sphase checkpoint signaling and translesion DNA synthesis. However, it remains largely unknown how CTF18RFC responds to replication stress and is recruited to stalled replication forks. The present study demonstrated that endogenous CTF18 forms a physical complex with a singlestranded DNAbinding protein replication protein A (RPA) in mammalian cells. Using an in situ proximity ligation assay (PLA), it was demonstrated that the interaction between CTF18 and RPA occurs in chromatin when replication stress is elicited by treatment with hydroxyurea during S phase. Similar results were obtained after exposure to ultraviolet irradiation, which triggers translesion DNA synthesis. Furthermore, the PLA demonstrated that the kinetics of the interaction between CTF18 and RPA was positively correlated with that of checkpoint kinase 1 phosphorylation, which is an indicator of activation of the ATM and Rad3related pathway. These findings provide novel insights into the molecular mechanism underlying the participation of CTF18RFC in the regulation of replication stress response.
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Proteínas de Transporte/metabolismo , Replicação do DNA , Proteínas Nucleares/metabolismo , Proteína de Replicação A/metabolismo , Estresse Fisiológico/genética , ATPases Associadas a Diversas Atividades Celulares , Linhagem Celular , Quinase 1 do Ponto de Checagem/metabolismo , Células HEK293 , Humanos , Fosforilação , Ligação Proteica , Raios UltravioletaRESUMO
Phospholipase D (PLD) catalyzes the hydrolysis of phosphatidylcholine (PC), the most abundant phospholipids of plasma membrane, resulting in the production of choline and phosphatidic acid (PA). Choline is a precursor of the neurotransmitter acetylcholine, whereas PA functions as an intracellular lipid mediator of diverse biological functions. For assessing PLD activity in vitro, PLD-derived choline has been often analyzed with radioactive or non-radioactive methods. In this study, we have developed a new method for detecting choline and PA with MALDI-QIT-TOF/MS by using 9-aminoacridine as a matrix. The standard calibration curves showed that choline and PA could be detected with linearity over the range from 0.05 and 1 pmol, respectively. Importantly, this method enables the concomitant detection of choline and PA as a reaction product of PC hydrolysis by PLD2 proteins. Thus, our simple and direct method would be useful to characterize the enzymatic properties of PLD, thereby providing insight into mechanisms of PLD activation.
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Aminacrina/metabolismo , Biocatálise , Colina/metabolismo , Ensaios Enzimáticos/métodos , Ácidos Fosfatídicos/metabolismo , Fosfolipase D/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Células HEK293 , Humanos , Hidrólise , Limite de DetecçãoRESUMO
S-adenosyl-L-methionine (SAM) is an intermediate metabolite of methionine and serves as the methyl donor for many biological methylation reactions. The synthesis of SAM is catalyzed by SAM synthetase (SAMS), which transfers the adenosyl moiety of adenosine-5'-triphosphate to methionine. In the nematode Caenorhabditis elegans, four sams family genes, sams-1, -3, -4 and -5, are predicted to encode SAMS proteins. However, their physiological roles remain unclear. Here we show that the four predicted SAMS proteins in fact have the ability to catalyze the formation of SAM in vitro, and revealed that only sams-1 mutant animals among the family genes exhibited a significant reduction in egg-laying. Using transgenic animals carrying a transcriptional reporter for each sams gene promoter, we observed that each sams promoter confers a distinct expression pattern with respect to tissue, time of expression and expression level (i.e. promoter specificity). Promoter-swap experiments revealed that the ectopic expression of SAMS-3, -4 or -5 driven by the sams-1 promoter completely rescued egg-laying in sams-1 mutants. These data indicate that SAMS protein function is conserved throughout the entire family.
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Animais Geneticamente Modificados/fisiologia , Caenorhabditis elegans/fisiologia , Metionina Adenosiltransferase/metabolismo , Metionina/metabolismo , Oviposição/fisiologia , Animais , FemininoRESUMO
Hepatic gluconeogenesis is important for the maintenance of blood glucose homeostasis under fasting condition. Hepatocyte nuclear factor 4α (HNF4α) and FOXO1 transcription factors have implicated in this process through transcriptional regulation of glucose-6-phosphatase (G6Pase) and phosphoenolpyruvate carboxykinase (PEPCK), which are rate-limiting enzymes in gluconeogenesis. In this study, we demonstrate that glycogen synthase kinase 3ß (GSK3ß) regulates the expression of gluconeogenic genes through HNF4α and FOXO1. Silencing of GSK3ß leads to reduction in the expression of gluconeogenic genes, including G6Pase, PEPCK, and peroxisome proliferator-activated receptor γ coactivator-1α. We show that GSK3ß directly binds to both HNF4α and FOXO1. Inhibition of GSK3 by SB-216763 abolishes HNF4α-mediated activation of G6Pase promoter. We also found that overexpression of GSK3ß potentiates G6Pase promoter activation by FOXO1 in a manner dependent on its kinase activity. Treatment of SB-216763 diminishes FOXO1-mediated activation of G6Pase promoter. Taken together, these results reveal a previously unrecognized mechanism for the regulation of gluconeogenic gene expression.
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Fatores de Transcrição Forkhead/metabolismo , Regulação da Expressão Gênica , Glucose-6-Fosfatase/genética , Quinase 3 da Glicogênio Sintase/metabolismo , Proteínas de Choque Térmico/genética , Fator 4 Nuclear de Hepatócito/metabolismo , Proteínas Serina-Treonina Quinases/genética , Fatores de Transcrição/genética , Western Blotting , Células Cultivadas , Proteína Forkhead Box O1 , Fatores de Transcrição Forkhead/genética , Gluconeogênese , Glucose-6-Fosfatase/metabolismo , Quinase 3 da Glicogênio Sintase/genética , Glicogênio Sintase Quinase 3 beta , Proteínas de Choque Térmico/metabolismo , Fator 4 Nuclear de Hepatócito/genética , Humanos , Luciferases/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Fosfoenolpiruvato Carboxiquinase (ATP) , Fosforilação , Regiões Promotoras Genéticas/genética , Proteínas Serina-Treonina Quinases/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Fatores de Transcrição/metabolismoRESUMO
Arginine methylation is a widespread posttranslational modification of proteins catalyzed by a family of protein arginine methyltransferases (PRMTs). It is well established that PRMTs are implicated in various cellular processes, but their physiological roles remain unclear. Using nematodes with a loss-of-function mutation, we show that prmt-1, the major asymmetric arginine methyltransferase, is a positive regulator of longevity in C. elegans. This regulation is dependent on both its enzymatic activity and DAF-16/FoxO transcription factor, which is negatively regulated by AKT-mediated phosphorylation downstream of the DAF-2/insulin signaling. prmt-1 is also required for stress tolerance and fat storage but not dauer formation in daf-2 mutants. Biochemical analyses indicate that PRMT-1 methylates DAF-16, thereby blocking its phosphorylation by AKT. Disruption of PRMT-1 induces phosphorylation of DAF-16 with a concomitant reduction in the expression of longevity-related genes. Thus, we provide a mechanism by which asymmetric arginine dimethylation acts as an antiaging modification in C. elegans.
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Arginina/genética , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Regulação da Expressão Gênica , Longevidade/genética , Metilação , Proteína-Arginina N-Metiltransferases/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Animais , Arginina/metabolismo , Western Blotting , Caenorhabditis elegans/genética , Caenorhabditis elegans/crescimento & desenvolvimento , Proteínas de Caenorhabditis elegans/genética , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Imunoprecipitação , Insulina/genética , Insulina/metabolismo , Dados de Sequência Molecular , Mutação/genética , Fosforilação , Reação em Cadeia da Polimerase , Proteína-Arginina N-Metiltransferases/genética , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor de Insulina/genética , Receptor de Insulina/metabolismo , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Fatores de Transcrição/genéticaRESUMO
The forkhead box O transcription factors convert a variety of external stimuli, including growth factors, nutrients, and oxidative stress, into diverse biological responses through modulation of specific gene expression. Forkhead box O regulation is principally achieved by two distinct mechanisms: post-translational modifications and protein-protein interactions. Among several modifications of forkhead box O factors, we focus on reversible acetylation, describing past research and current advances. In the latter part of this review, we also provide an overview of forkhead box O-binding partners that control the transcriptional activity of forkhead box O factors. These two layers of regulation mostly overlap and thereby enable a more precise fine-tuning of forkhead box O functions involved in metabolism, longevity, and tumor suppression. This article is part of a Special Issue entitled: PI3K-AKT-FoxO axis in cancer and aging.
Assuntos
Proteínas 14-3-3/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Acetilação , Animais , Proteína Forkhead Box O1 , Humanos , Modelos Biológicos , Fosforilação , Ligação Proteica , Transporte ProteicoRESUMO
Protein arginine methylation is a common post-translational modification in eukaryotes that is catalyzed by a family of the protein arginine methyltransferases (PRMTs). PRMTs are classified into three types: type I and type II add asymmetrically and symmetrically dimethyl groups to arginine, respectively, while type III adds solely monomethyl group to arginine. However, although the enzymatic activity of type I and type II PRMTs have been reported, the substrate specificity and the methylation activity of type III PRMTs still remains unknown. Here, we report the characterization of Caenorhabditis elegans PRMT-2 and PRMT-3, both of which are highly homologous to human PRMT7. We find that these two PRMTs can bind to S-adenosyl methionine (SAM), but only PRMT-3 has methyltransferase activity for histone H2A depending on its SAM-binding domain. Importantly, thin-layer chromatographic analysis demonstrates that PRMT-3 catalyzes the formation of monomethylated, but not dimethylated arginine. Our study thus identifies the first type III PRMT in C. elegans and provides a means to elucidate the physiological significance of arginine monomethylation in multicellular organisms.