Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 19 de 19
Filtrar
Mais filtros

Base de dados
País/Região como assunto
Tipo de documento
Intervalo de ano de publicação
1.
Proc Natl Acad Sci U S A ; 117(28): 16616-16625, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32601203

RESUMO

Enhanced inflammation is believed to contribute to overnutrition-induced metabolic disturbance. Nutrient flux has also been shown to be essential for immune cell activation. Here, we report an unexpected role of nutrient-sensing O-linked ß-N-acetylglucosamine (O-GlcNAc) signaling in suppressing macrophage proinflammatory activation and preventing diet-induced metabolic dysfunction. Overnutrition stimulates an increase in O-GlcNAc signaling in macrophages. O-GlcNAc signaling is down-regulated during macrophage proinflammatory activation. Suppressing O-GlcNAc signaling by O-GlcNAc transferase (OGT) knockout enhances macrophage proinflammatory polarization, promotes adipose tissue inflammation and lipolysis, increases lipid accumulation in peripheral tissues, and exacerbates tissue-specific and whole-body insulin resistance in high-fat-diet-induced obese mice. OGT inhibits macrophage proinflammatory activation by catalyzing ribosomal protein S6 kinase beta-1 (S6K1) O-GlcNAcylation and suppressing S6K1 phosphorylation and mTORC1 signaling. These findings thus identify macrophage O-GlcNAc signaling as a homeostatic mechanism maintaining whole-body metabolism under overnutrition.


Assuntos
Macrófagos/imunologia , N-Acetilglucosaminiltransferases/imunologia , Obesidade/imunologia , Proteínas Quinases S6 Ribossômicas 90-kDa/imunologia , Acetilglucosamina/imunologia , Tecido Adiposo/imunologia , Animais , Humanos , Ativação de Macrófagos , Macrófagos/enzimologia , Camundongos , Camundongos Knockout , N-Acetilglucosaminiltransferases/genética , Obesidade/enzimologia , Obesidade/genética , Obesidade/metabolismo , Fosforilação , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Transdução de Sinais
2.
Int J Mol Sci ; 24(11)2023 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-37298123

RESUMO

Through a comprehensive analysis of the gene expression and dependency in HCC patients and cell lines, LAT1 was identified as the top amino acid transporter candidate supporting HCC tumorigenesis. To assess the suitability of LAT1 as a HCC therapeutic target, we used CRISPR/Cas9 to knockout (KO) LAT1 in the epithelial HCC cell line, Huh7. Knockout of LAT1 diminished its branched chain amino acid (BCAA) transport activity and significantly reduced cell proliferation in Huh7. Consistent with in vitro studies, LAT1 ablation led to suppression of tumor growth in a xenograft model. To elucidate the mechanism underlying the observed inhibition of cell proliferation upon LAT1 KO, we performed RNA-sequencing analysis and investigated the changes in the mTORC1 signaling pathway. LAT1 ablation resulted in a notable reduction in phosphorylation of p70S6K, a downstream target of mTORC1, as well as its substrate S6RP. This reduced cell proliferation and mTORC1 activity were rescued when LAT1 was overexpressed. These findings imply an essential role of LAT1 for maintenance of tumor cell growth and additional therapeutic angles against liver cancer.


Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , Transdução de Sinais , Linhagem Celular , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo
3.
Int J Mol Sci ; 23(16)2022 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-36012379

RESUMO

Ultraviolet (UV) germicidal tools have recently gained attention as a disinfection strategy against the COVID-19 pandemic, but the safety profile arising from their exposure has been controversial and impeded larger-scale implementation. We compare the emerging 222-nanometer far UVC and 277-nanometer UVC LED disinfection modules with the traditional UVC mercury lamp emitting at 254 nm to understand their effects on human retinal cell line ARPE-19 and HEK-A keratinocytes. Cells illuminated with 222-nanometer far UVC survived, while those treated with 254-nanometer and 277-nanometer wavelengths underwent apoptosis via the JNK/ATF2 pathway. However, cells exposed to 222-nanometer far UVC presented the highest degree of DNA damage as evidenced by yH2AX staining. Globally, these cells displayed transcriptional changes in cell-cycle and senescence pathways. Thus, the introduction of 222-nanometer far UVC lamps for disinfection purposes should be carefully considered and designed with the inherent dangers involved.


Assuntos
COVID-19 , Raios Ultravioleta , Animais , Dano ao DNA , Desinfecção/métodos , Humanos , Mamíferos , Pandemias , Raios Ultravioleta/efeitos adversos
4.
J Gastroenterol Hepatol ; 36(12): 3477-3486, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34547820

RESUMO

BACKGROUND AND AIM: Protein O-GlcNAcylation is a critical post-translational modification regulating gene expression and fundamental cell functions. O-GlcNAc transferase (OGT) emerged as a key regulator of liver pathophysiology and disease. In this study, we aimed to evaluate the role of OGT in hepatic stellate cells (HSCs) and its consequent role in liver fibrosis. METHODS: Primary HSCs were isolated from C57/B6 mice. Cell morphology and α-SMA immunofluorescence staining were observed under scanning confocal microscope. Transcriptomic profile was evaluated by RNAseq (Illumina). Promoter activity was examined by luciferase and ß--Galactosidase reporter assays. Liver fibrosis mouse models were induced either by intraperitoneal injection of CCl4 at 3 times/week for 4 weeks or by feeding with methionine and choline deficient (MCD) diet for 4 weeks. RESULTS: OGT protein expression and protein O-GlcNAcylation were significantly decreased in CCl4 - or MCD diet-induced liver fibrosis as compared with normal liver in mice. OGT expression and protein O-GlcNAcylation were also decreased in primary HSCs isolated from liver with CCl4 -induced fibrosis compared with those from normal liver. RNA-seq showed that OGT knockdown in HSCs modulated key signaling pathways involved in HSC activation. Promoter sequence analysis of the differentially expressed genes predicted serum response factor (SRF) as a key transcription factor regulated by OGT. Luciferase reporter assay confirmed that OGT repressed activity of SRF to induce α-SMA transcription. Mutations of specific O-GlcNAcylation sites on SRF increased its transcriptional activity, validating negative regulation of SRF by OGT-mediated O-GlcNAcylation. CONCLUSIONS: Our results suggest that OGT functions as a negative regulator of HSC activation by promoting SRF O-GlcNAcylation to protect against liver fibrosis.


Assuntos
Células Estreladas do Fígado , N-Acetilglucosaminiltransferases , Processamento de Proteína Pós-Traducional , Animais , Células Estreladas do Fígado/fisiologia , Cirrose Hepática/prevenção & controle , Camundongos , N-Acetilglucosaminiltransferases/metabolismo
5.
Cancer Sci ; 111(11): 4288-4302, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32945042

RESUMO

Non-alcoholic fatty liver disease (NAFLD) is one of the most common causes of hepatocellular carcinoma (HCC), but the underlying mechanisms behind the correlation of NAFLD with HCC are unclear. We aimed to uncover the genes and potential mechanisms that drive this progression. This study uncovered the genes and potential mechanisms through a multiple 'omics integration approach. Quantitative proteomics combined with phenotype-association analysis was performed. To investigate the potential mechanisms, a comprehensive transcriptome/lipidome/phenome-wide association analysis was performed in genetic reference panel BXD mice strains. The quantitative proteomics combined with phenotype-association results showed that VDAC1 was significantly increased in tumor tissues and correlated with NAFLD-related traits. Gene co-expression network analysis indicated that VDAC1 is involved in mitochondria dysfunction in the tumorigenic/tumor progression. The association between VDAC1 and mitochondria dysfunction can be explained by the fact that VDAC1 was associated with mitochondria membrane lipids cardiolipin (CL) composition shift. VDAC1 was correlated with the suppression of mature specie CL(LLLL) and elevation level of nascent CL species. Such profiling shift was supported by the significant positive correlation between VDAC1 and PTPMT1, as well as negative correlation with CL remodeling enzyme Tafazzin (TAZ). This study confirmed that the expression of VADC1 was dysregulated in NAFLD-driven HCC and associated with NAFLD progression. The VDAC1-driven mitochondria dysfunction is associated with cardiolipin composition shift, which causes alteration of mitochondria membrane properties.


Assuntos
Carcinoma Hepatocelular/etiologia , Neoplasias Hepáticas/etiologia , Mitocôndrias/genética , Mitocôndrias/metabolismo , Hepatopatia Gordurosa não Alcoólica/complicações , Hepatopatia Gordurosa não Alcoólica/genética , Canais de Ânion Dependentes de Voltagem/genética , Idoso , Animais , Carcinoma Hepatocelular/diagnóstico , Modelos Animais de Doenças , Suscetibilidade a Doenças , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Variação Genética , Humanos , Neoplasias Hepáticas/diagnóstico , Masculino , Camundongos , Pessoa de Meia-Idade , Gradação de Tumores , Estadiamento de Neoplasias , Hepatopatia Gordurosa não Alcoólica/metabolismo , Hepatopatia Gordurosa não Alcoólica/patologia , Proteoma , Proteômica/métodos , Locos de Características Quantitativas , Transdução de Sinais , Canais de Ânion Dependentes de Voltagem/metabolismo
6.
Nano Lett ; 19(10): 6955-6963, 2019 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-31552740

RESUMO

Magnetogenetics is a new field that leverages genetically encoded proteins and protein assemblies that are sensitive to magnetic fields to study and manipulate cell behavior. Theoretical studies show that many proposed magnetogenetic proteins do not contain enough iron to generate substantial magnetic forces. Here, we have engineered a genetically encoded ferritin-containing protein crystal that grows inside mammalian cells. Each of these crystals contains more than 10 million ferritin subunits and is capable of mineralizing substantial amounts of iron. When isolated from cells and loaded with iron in vitro, these crystals generate magnetic forces that are 9 orders of magnitude larger than the forces from the single ferritin cages used in previous studies. These protein crystals are attracted to an applied magnetic field and move toward magnets even when internalized into cells. While additional studies are needed to realize the full potential of magnetogenetics, these results demonstrate the feasibility of engineering protein assemblies for magnetic sensing.


Assuntos
Ferritinas/química , Imãs/química , Animais , Cristalização , Ferritinas/genética , Células HEK293 , Humanos , Ferro/química , Campos Magnéticos , Camundongos , Engenharia de Proteínas , Células RAW 264.7
7.
Heliyon ; 8(10): e11132, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-36276725

RESUMO

To interrupt SARS-CoV-2 transmission chains, Ultraviolet-C (UVC) irradiation has emerged as a potential disinfection tool to aid in blocking the spread of coronaviruses. While conventional 254-nm UVC mercury lamps have been used for disinfection purposes, other UVC wavelengths have emerged as attractive alternatives but a direct comparison of these tools is lacking with the inherent mechanistic properties unclear. Our results using human coronaviruses, hCoV-229E and hCoV-OC43, have indicated that 277-nm UVC LED is most effective in viral inactivation, followed by 222-nm far UVC and 254-nm UVC mercury lamp. While UVC mercury lamp is more effective in degrading viral genomic content compared to 277-nm UVC LED, the latter results in a pronounced photo-degradation of spike proteins which potentially contributed to the higher efficacy of coronavirus inactivation. Hence, inactivation of coronaviruses by 277-nm UVC LED irradiation constitutes a more promising method for disinfection.

8.
Sci Rep ; 11(1): 22612, 2021 11 19.
Artigo em Inglês | MEDLINE | ID: mdl-34799671

RESUMO

This paper proposes an investigating SARS-CoV-2 inactivation on surfaces with UV-C LED irradiation using our in-house-developed ray-tracing simulator. The results are benchmarked with experiments and Zemax OpticStudio commercial software simulation to demonstrate our simulator's easy accessibility and high reliability. The tool can input the radiant profile of the flexible LED source and accurately yield the irradiance distribution emitted from an LED-based system in 3D environments. The UV-C operating space can be divided into the safe, buffer, and germicidal zones for setting up a UV-C LED system. Based on the published measurement data, the level of SARS-CoV-2 inactivation has been defined as a function of UV-C irradiation. A realistic case of public space, i.e., a food court in Singapore, has been numerically investigated to demonstrate the relative impact of environmental UV-C attenuation on the SARS-CoV-2 inactivation. We optimise a specific UV-C LED germicidal system and its corresponding exposure time according to the simulation results. These ray-tracing-based simulations provide a useful guideline for safe deployment and efficient design for germicidal UV-C LED technology.


Assuntos
SARS-CoV-2/efeitos da radiação , Raios Ultravioleta , Inativação de Vírus/efeitos da radiação , Simulação por Computador , Desinfecção/instrumentação , Imageamento Tridimensional , Singapura , Esterilização/instrumentação
9.
Nat Commun ; 11(1): 181, 2020 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-31924761

RESUMO

Excessive visceral fat accumulation is a primary risk factor for metabolically unhealthy obesity and related diseases. The visceral fat is highly susceptible to the availability of external nutrients. Nutrient flux into the hexosamine biosynthetic pathway leads to protein posttranslational modification by O-linked ß-N-acetylglucosamine (O-GlcNAc) moieties. O-GlcNAc transferase (OGT) is responsible for the addition of GlcNAc moieties to target proteins. Here, we report that inducible deletion of adipose OGT causes a rapid visceral fat loss by specifically promoting lipolysis in visceral fat. Mechanistically, visceral fat maintains a high level of O-GlcNAcylation during fasting. Loss of OGT decreases O-GlcNAcylation of lipid droplet-associated perilipin 1 (PLIN1), which leads to elevated PLIN1 phosphorylation and enhanced lipolysis. Moreover, adipose OGT overexpression inhibits lipolysis and promotes diet-induced obesity. These findings establish an essential role for OGT in adipose tissue homeostasis and indicate a unique potential for targeting O-GlcNAc signaling in the treatment of obesity.


Assuntos
Dieta/efeitos adversos , Gordura Intra-Abdominal/efeitos dos fármacos , Lipólise/efeitos dos fármacos , N-Acetilglucosaminiltransferases/antagonistas & inibidores , Obesidade/metabolismo , Acetilglucosamina/metabolismo , Animais , Linhagem Celular Tumoral , Jejum , Deleção de Genes , Células HEK293 , Células HeLa , Homeostase , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Knockout , N-Acetilglucosaminiltransferases/genética , Perilipina-1/metabolismo , Fosforilação , Processamento de Proteína Pós-Traducional , Transdução de Sinais
10.
Oncogene ; 39(3): 560-573, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31501520

RESUMO

Cancer cells are known to adopt aerobic glycolysis in order to fuel tumor growth, but the molecular basis of this metabolic shift remains largely undefined. O-GlcNAcase (OGA) is an enzyme harboring O-linked ß-N-acetylglucosamine (O-GlcNAc) hydrolase and cryptic lysine acetyltransferase activities. Here, we report that OGA is upregulated in a wide range of human cancers and drives aerobic glycolysis and tumor growth by inhibiting pyruvate kinase M2 (PKM2). PKM2 is dynamically O-GlcNAcylated in response to changes in glucose availability. Under high glucose conditions, PKM2 is a target of OGA-associated acetyltransferase activity, which facilitates O-GlcNAcylation of PKM2 by O-GlcNAc transferase (OGT). O-GlcNAcylation inhibits PKM2 catalytic activity and thereby promotes aerobic glycolysis and tumor growth. These studies define a causative role for OGA in tumor progression and reveal PKM2 O-GlcNAcylation as a metabolic rheostat that mediates exquisite control of aerobic glycolysis.


Assuntos
Antígenos de Neoplasias/metabolismo , Proteínas de Transporte/metabolismo , Histona Acetiltransferases/metabolismo , Hialuronoglucosaminidase/metabolismo , Proteínas de Membrana/metabolismo , N-Acetilglucosaminiltransferases/metabolismo , Neoplasias/patologia , Hormônios Tireóideos/metabolismo , Acetilação , Acetilglucosamina/metabolismo , Animais , Linhagem Celular Tumoral , Conjuntos de Dados como Assunto , Progressão da Doença , Feminino , Perfilação da Expressão Gênica , Glicólise , Células HEK293 , Humanos , Masculino , Camundongos , Gradação de Tumores , Estadiamento de Neoplasias , Neoplasias/metabolismo , Processamento de Proteína Pós-Traducional , Análise Serial de Tecidos , Regulação para Cima , Ensaios Antitumorais Modelo de Xenoenxerto , Proteínas de Ligação a Hormônio da Tireoide
11.
Artigo em Inglês | MEDLINE | ID: mdl-30464755

RESUMO

O-GlcNAcylation is an important posttranslational modification governed by a single pair of enzymes-O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). These two enzymes mediate the dynamic cycling of O-GlcNAcylation on a wide variety of cytosolic, nuclear and mitochondrial proteins in a nutrient- and stress-responsive fashion. While cellular functions of O-GlcNAcylation have been emerging, little is known regarding the precise mechanisms how the enzyme pair senses the environmental cues to elicit molecular and physiological changes. In this review, we discuss how the OGT/OGA pair acts as a metabolic sensor that integrates signaling pathways, given their capability of receiving signaling inputs from various partners, targeting multiple substrates with spatiotemporal specificity and translocating to different parts of the cell. We also discuss how the pair maintains homeostatic signaling within the cell and its physiological relevance. A better understanding of the mechanisms of OGT/OGA action would enable us to derive therapeutic benefits of resetting cellular O-GlcNAc levels within an optimal range.

12.
ACS Synth Biol ; 7(7): 1685-1693, 2018 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-29975841

RESUMO

Nerve growth factor/tropomyosin receptor kinase A (NGF/TrkA) signaling plays a key role in neuronal development, function, survival, and growth. The pathway is implicated in neurodegenerative disorders including Alzheimer's disease, chronic pain, inflammation, and cancer. NGF binds the extracellular domain of TrkA, leading to the activation of the receptor's intracellular kinase domain. As TrkA signaling is highly dynamic, mechanistic studies would benefit from a tool with high spatial and temporal resolution. Here we present the design and evaluation of four strategies for light-inducible activation of TrkA in the absence of NGF. Our strategies involve the light-sensitive protein Arabidopsis cryptochrome 2 and its binding partner CIB1. We demonstrate successful recapitulation of native NGF/TrkA functions by optical induction of plasma membrane recruitment and homo-interaction of the intracellular domain of TrkA. This approach activates PI3K/AKT and Raf/ERK signaling pathways, promotes neurite growth in PC12 cells, and supports survival of dorsal root ganglion neurons in the absence of NGF. This ability to activate TrkA using light bestows high spatial and temporal resolution for investigating NGF/TrkA signaling.


Assuntos
Receptor trkA/metabolismo , Animais , Membrana Celular/metabolismo , Sobrevivência Celular/genética , Sobrevivência Celular/fisiologia , Gânglios Espinais/metabolismo , Fator de Crescimento Neural/metabolismo , Células PC12 , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação/genética , Fosforilação/fisiologia , Ratos , Receptor trkA/genética , Transdução de Sinais
13.
Nat Commun ; 8(1): 547, 2017 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-28916751

RESUMO

Arabidopsis cryptochrome 2 (CRY2) can simultaneously undergo light-dependent CRY2-CRY2 homo-oligomerization and CRY2-CIB1 hetero-dimerization, both of which have been widely used to optically control intracellular processes. Applications using CRY2-CIB1 interaction desire minimal CRY2 homo-oligomerization to avoid unintended complications, while those utilizing CRY2-CRY2 interaction prefer robust homo-oligomerization. However, selecting the type of CRY2 interaction has not been possible as the molecular mechanisms underlying CRY2 interactions are unknown. Here we report CRY2-CIB1 and CRY2-CRY2 interactions are governed by well-separated protein interfaces at the two termini of CRY2. N-terminal charges are critical for CRY2-CIB1 interaction. Moreover, two C-terminal charges impact CRY2 homo-oligomerization, with positive charges facilitating oligomerization and negative charges inhibiting it. By engineering C-terminal charges, we develop CRY2high and CRY2low with elevated or suppressed oligomerization respectively, which we use to tune the levels of Raf/MEK/ERK signaling. These results contribute to our understanding of the mechanisms underlying light-induced CRY2 interactions and enhance the controllability of CRY2-based optogenetic systems.Cryptochrome 2 (CRY2) can form light-regulated CRY2-CRY2 homo-oligomers or CRY2-CIB1 hetero-dimers, but modulating these interactions is difficult owing to the lack of interaction mechanism. Here the authors identify the interactions facilitating homo-oligomers and introduce mutations to create low and high oligomerization versions.


Assuntos
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Arabidopsis/efeitos da radiação , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Criptocromos/química , Criptocromos/metabolismo , Motivos de Aminoácidos , Arabidopsis/química , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Criptocromos/genética , Dimerização , Luz , Optogenética , Ligação Proteica , Transdução de Sinais
14.
PLoS One ; 11(4): e0153487, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27082641

RESUMO

Acute brain injuries such as ischemic stroke or traumatic brain injury often cause massive neural death and irreversible brain damage with grave consequences. Previous studies have established that a key participant in the events leading to neural death is the excessive production of reactive oxygen species. Protecting neuronal cells by activating their endogenous defense mechanisms is an attractive treatment strategy for acute brain injuries. In this work, we investigate how the precise timing of the Raf/ERK and the AKT pathway activation affects their protective effects against oxidative stress. For this purpose, we employed optogenetic systems that use light to precisely and reversibly activate either the Raf/ERK or the AKT pathway. We find that preconditioning activation of the Raf/ERK or the AKT pathway immediately before oxidant exposure provides significant protection to cells. Notably, a 15-minute transient activation of the Raf/ERK pathway is able to protect PC12 cells against oxidant strike that is applied 12 hours later, while the transient activation of the AKT pathway fails to protect PC12 cells in such a scenario. On the other hand, if the pathways are activated after the oxidative insult, i.e. postconditioning, the AKT pathway conveys greater protective effect than the Raf/ERK pathway. We find that postconditioning AKT activation has an optimal delay period of 2 hours. When the AKT pathway is activated 30min after the oxidative insult, it exhibits very little protective effect. Therefore, the precise timing of the pathway activation is crucial in determining its protective effect against oxidative injury. The optogenetic platform, with its precise temporal control and its ability to activate specific pathways, is ideal for the mechanistic dissection of intracellular pathways in protection against oxidative stress.


Assuntos
Citoproteção , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Estresse Oxidativo/fisiologia , Quinases raf/metabolismo , Animais , Citoproteção/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Peróxido de Hidrogênio/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Células NIH 3T3 , Estresse Oxidativo/efeitos dos fármacos , Células PC12 , Ratos , Fatores de Tempo
15.
ACS Chem Neurosci ; 6(1): 130-7, 2015 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-25544156

RESUMO

U0126 is a potent and selective inhibitor of MEK1 and MEK2 kinases. It has been widely used as an inhibitor for the Ras/Raf/MEK/ERK signaling pathway with over 5000 references on the NCBI PubMed database. In particular, U0126 has been used in a number of studies to show that inhibition of the Raf/MEK/ERK pathway protects neuronal cells against oxidative stress. Here, we report that U0126 can function as an antioxidant that protects PC12 cells against a number of different oxidative-stress inducers. This protective effect of U0126 is independent of its function as a MEK inhibitor, as several other MEK inhibitors failed to show similar protective effects. U0126 reduces reactive oxygen species (ROS) in cells. We further demonstrate that U0126 is a direct ROS scavenger in vitro, and the oxidation products of U0126 exhibit fluorescence. Our finding that U0126 is a strong antioxidant signals caution for its future usage as a MEK inhibitor and for interpreting some previous results.


Assuntos
Butadienos/farmacologia , Inibidores Enzimáticos/farmacologia , MAP Quinase Quinase Quinases/antagonistas & inibidores , Nitrilas/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Animais , Morte Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Peróxido de Hidrogênio/toxicidade , Imageamento por Ressonância Magnética , Células PC12 , Fosforilação/efeitos dos fármacos , Ratos , Espécies Reativas de Oxigênio/metabolismo , Fatores de Tempo
16.
Chem Biol ; 22(5): 671-82, 2015 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-25963241

RESUMO

Intracellular transport and distribution of organelles play important roles in diverse cellular functions, including cell polarization, intracellular signaling, cell survival, and apoptosis. Here, we report an optogenetic strategy to control the transport and distribution of organelles by light. This is achieved by optically recruiting molecular motors onto organelles through the heterodimerization of Arabidopsis thaliana cryptochrome 2 (CRY2) and its interacting partner CIB1. CRY2 and CIB1 dimerize within subseconds upon exposure to blue light, which requires no exogenous ligands and low intensity of light. We demonstrate that mitochondria, peroxisomes, and lysosomes can be driven toward the cell periphery upon light-induced recruitment of kinesin, or toward the cell nucleus upon recruitment of dynein. Light-induced motor recruitment and organelle movements are repeatable, reversible, and can be achieved at subcellular regions. This light-controlled organelle redistribution provides a new strategy for studying the causal roles of organelle transport and distribution in cellular functions in living cells.


Assuntos
Proteínas de Arabidopsis/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Criptocromos/metabolismo , Optogenética , Animais , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Transporte Biológico/efeitos da radiação , Células COS , Núcleo Celular/metabolismo , Chlorocebus aethiops , Criptocromos/genética , Cinesinas/metabolismo , Cinética , Luz , Lisossomos/metabolismo , Lisossomos/efeitos da radiação , Microscopia de Fluorescência , Mitocôndrias/metabolismo , Mitocôndrias/efeitos da radiação , Ligação Proteica , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética
17.
PLoS One ; 9(3): e92917, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24667437

RESUMO

It has been proposed that differential activation kinetics allows cells to use a common set of signaling pathways to specify distinct cellular outcomes. For example, nerve growth factor (NGF) and epidermal growth factor (EGF) induce different activation kinetics of the Raf/MEK/ERK signaling pathway and result in differentiation and proliferation, respectively. However, a direct and quantitative linkage between the temporal profile of Raf/MEK/ERK activation and the cellular outputs has not been established due to a lack of means to precisely perturb its signaling kinetics. Here, we construct a light-gated protein-protein interaction system to regulate the activation pattern of the Raf/MEK/ERK signaling pathway. Light-induced activation of the Raf/MEK/ERK cascade leads to significant neurite outgrowth in rat PC12 pheochromocytoma cell lines in the absence of growth factors. Compared with NGF stimulation, light stimulation induces longer but fewer neurites. Intermittent on/off illumination reveals that cells achieve maximum neurite outgrowth if the off-time duration per cycle is shorter than 45 min. Overall, light-mediated kinetic control enables precise dissection of the temporal dimension within the intracellular signal transduction network.


Assuntos
MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Luz , Sistema de Sinalização das MAP Quinases/efeitos da radiação , Neuritos/metabolismo , Neuritos/efeitos da radiação , Quinases raf/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/efeitos da radiação , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Membrana Celular/efeitos da radiação , Criptocromos/química , Criptocromos/metabolismo , Relação Dose-Resposta à Radiação , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/efeitos da radiação , Cinética , MAP Quinase Quinase Quinases/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Células NIH 3T3 , Fator de Crescimento Neural/farmacologia , Neuritos/efeitos dos fármacos , Células PC12 , Estrutura Terciária de Proteína , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/efeitos da radiação , Proteínas Proto-Oncogênicas c-raf , Ratos
19.
Org Lett ; 14(22): 5676-9, 2012 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-23101562

RESUMO

3,3-Difluoro-2-oxindoles can be obtained directly from indoles in moderate yields via electrophilic fluorination using N-fluorobenzenesulfonimide as a mild fluorinating reagent. The presence of tert-butyl hydroperoxide during the reaction, together with additional heating after quenching the reaction with triethylamine, is beneficial to the formation of the desired product.

SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA