Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 11.977
Filtrar
1.
J Agric Food Chem ; 67(41): 11428-11435, 2019 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-31589037

RESUMO

Diosgenin and diosgenyl saponins as the major bioactive compounds isolated from dietary fenugreek seeds, yam roots, etc. possessed strong antitumor effects. To understand their detailed antitumor mechanisms, a fluorophore-appended derivative of diosgenin [Glc/CNHphth-diosgenin (GND)] was synthesized, starting from diosgenin and glucosamine hydrochloride in overall yields of 7-12% over 7-10 steps. Co-localization of GND with organelle-specific stains, transmission electron microscopy, and relative protein analyses demonstrated that GND crossed the plasma membrane through organic anion-transporting polypeptide 1B1 and distributed in the endoplasmic reticulum (ER), lysosome, and mitochondria. In this process, GND induced ER swelling, mitochondrial damage, and autophagosome and upregulating IRE-1α to induce autophagy and apoptosis. Furthermore, autophagy inhibitor chloroquine delayed the appearance of cleaved poly(ADP-ribose) polymerase and inhibited cleaved caspase 8, which indicated that GND induced autophagy to activate caspase-8-dependent apoptosis. These observations suggested that diosgenyl saponin was a potent anticancer agent that elicited ER stress and mitochondria-mediated apoptotic pathways in liver cancer.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Autofagia/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Neoplasias Hepáticas/fisiopatologia , Extratos Vegetais/farmacologia , Saponinas/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Lisossomos/efeitos dos fármacos , Lisossomos/genética , Lisossomos/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Transportadores de Ânions Orgânicos/genética , Transportadores de Ânions Orgânicos/metabolismo
2.
Int J Nanomedicine ; 14: 7003-7016, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31564862

RESUMO

Background: Yttria-stabilized zirconia (Y2O3/ZrO2) nanoparticles are one of the important nanoparticles extensively used in manufacturing of plastics, textiles, catalyst, etc. Still, the cytotoxic and apoptotic effects of yttria-stabilized zirconia nanoparticles have not been well identified on human skin keratinocyte (HaCaT) cells. Therefore, in this study, we have designed to examine the cytotoxic potential of yttria-stabilized zirconia nanoparticles in HaCaT cells. Methods: Prior to treatment, the yttria-stabilized zirconia nanoparticles were characterized by using different advanced instruments viz. dynamic light scattering (DLS), scanning electron microscope (SEM) and transmission electron microscope (TEM). Cell viability of HaCaT cells was measured by using MTS and NRU assays and viability of cells was reduced in a dose- and time-dependent manner. Results: Reduction in the viability of cells was correlated with the rise of reactive oxygen species generation, increased caspase-3, mitochondria membrane potential and evidence of DNA strand breakage. These were consistent with the possibility that mitochondria damage can play a significant role in the cytotoxic response. Moreover, the activity of oxidative enzymes such as lipid peroxide (LPO) was increased and glutathione was reduced in HaCaT cells exposed with yttria-stabilized zirconia nanoparticles. It is also important to indicate that HaCaT cells appear to be more susceptible to yttria-stabilized zirconia nanoparticles exposure after 24 hrs. Conclusion: This result provides a dose- and time-dependent apoptosis and genotoxicity of yttria-stabilized zirconia nanoparticles in HaCaT cells.


Assuntos
Apoptose , Dano ao DNA , Células Epiteliais/citologia , Nanopartículas Metálicas/química , Pele/citologia , Ítrio/química , Zircônio/química , Acetilcisteína/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular , Forma Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Fragmentação do DNA/efeitos dos fármacos , Células Epiteliais/metabolismo , Glutationa/metabolismo , Humanos , Membranas Intracelulares/efeitos dos fármacos , Membranas Intracelulares/metabolismo , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Nanopartículas Metálicas/ultraestrutura , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo
3.
Chem Commun (Camb) ; 55(77): 11567-11570, 2019 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-31495830

RESUMO

A small series of fluorescent lysosome-targeting probes based on the BODIPY fluorophore and containing morpholine and nitro groups were rationally designed. These probes emitted light from green to NIR wavelengths, and provided specificity for imaging the lysosomes of hypoxic cells. The electron-withdrawing nitrophenyl group at the meso position was found to lead to highly efficient nonradiative decay of the S1 state, and hence a recovery of fluorescence when reduction of the nitro group occurred under hypoxic conditions.


Assuntos
Compostos de Boro/química , Hipóxia Celular , Corantes Fluorescentes/química , Lisossomos/metabolismo , Teoria da Densidade Funcional , Células Hep G2 , Humanos , Luz , Morfolinas/química , Espectrometria de Fluorescência/métodos
4.
Nat Cell Biol ; 21(10): 1206-1218, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31548609

RESUMO

Cholesterol activates the master growth regulator, mTORC1 kinase, by promoting its recruitment to the surface of lysosomes by the Rag guanosine triphosphatases (GTPases). The mechanisms that regulate lysosomal cholesterol content to enable mTORC1 signalling are unknown. Here, we show that oxysterol binding protein (OSBP) and its anchors at the endoplasmic reticulum (ER), VAPA and VAPB, deliver cholesterol across ER-lysosome contacts to activate mTORC1. In cells lacking OSBP, but not other VAP-interacting cholesterol carriers, the recruitment of mTORC1 by the Rag GTPases is inhibited owing to impaired transport of cholesterol to lysosomes. By contrast, OSBP-mediated cholesterol trafficking drives constitutive mTORC1 activation in a disease model caused by the loss of the lysosomal cholesterol transporter, Niemann-Pick C1 (NPC1). Chemical and genetic inactivation of OSBP suppresses aberrant mTORC1 signalling and restores autophagic function in cellular models of Niemann-Pick type C (NPC). Thus, ER-lysosome contacts are signalling hubs that enable cholesterol sensing by mTORC1, and targeting the sterol-transfer activity of these signalling hubs could be beneficial in patients with NPC.


Assuntos
Colesterol/metabolismo , Retículo Endoplasmático/metabolismo , Lisossomos/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Doenças de Niemann-Pick/metabolismo , Receptores de Esteroides/metabolismo , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Células HEK293 , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Receptores de Esteroides/genética , Transdução de Sinais , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo
5.
Aquat Toxicol ; 215: 105266, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31401474

RESUMO

The persistent pollutants polybrominated diphenyl ethers (PBDEs) have been demonstrated to produce several negative effects on marine organisms. Although Mytilus galloprovincialis was extensively studied as model system, the effects of PBDEs on the innate immune system of mussels remains unclear. In this study, except for the control treatment, specimens of M. galloprovincialis were fed with microalgae treated with increasing concentrations of PBDEs (maximum level 100 ng L-1 of BDE-47 per day). BDE-47 treatment was maintained for 15 days and then the animals were fed with the same control diet, without contaminants, for 15 days. Samples of haemolymph (HL) were obtained at T0, T15 and T30 days of the experiment to evaluate different parameters related to immunity, such as neutral red retention time, and peroxidase, protease, antiprotease, lysozyme and bactericidal activities. BDE-47 exposure for 15 days affected both the stability of haemocytes and humoral parameters. In addition, the obtained results indicated that, at 30 days, after 15 days of culture without contaminant, the immune parameters were still affected, as some of them did not return to the basal levels, and others remained stimulated. Overall the results indicate that BDE-47 exposures at environmentally realistic levels may affect various aspects of immune function in M. galloprovincialis, acting as stressor that can compromise the general welfare.


Assuntos
Exposição Ambiental , Éteres Difenil Halogenados/toxicidade , Mytilus/imunologia , Animais , Antibacterianos/farmacologia , Comportamento Alimentar/efeitos dos fármacos , Hemócitos/efeitos dos fármacos , Hemócitos/metabolismo , Hemolinfa/efeitos dos fármacos , Hemolinfa/metabolismo , Hemolinfa/microbiologia , Membranas Intracelulares/efeitos dos fármacos , Membranas Intracelulares/metabolismo , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Microalgas/fisiologia , Mytilus/efeitos dos fármacos , Mytilus/microbiologia , Peptídeo Hidrolases/metabolismo , Análise de Sobrevida , Poluentes Químicos da Água/toxicidade
6.
Chem Commun (Camb) ; 55(73): 10916-10919, 2019 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-31441466
7.
Chem Commun (Camb) ; 55(70): 10440-10443, 2019 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-31410422

RESUMO

In this work, a unique dual-site controlled fluorescent probe was presented for the sensitive and concurrent detection of pH in the cytoplasm and lysosomes. With the probe, the simultaneous down-regulation of pH in the lysosomes and cytoplasm during autophagy has been successfully revealed for the first time.


Assuntos
Autofagia , Citoplasma/metabolismo , Regulação para Baixo , Concentração de Íons de Hidrogênio , Lisossomos/metabolismo , Sondas Moleculares/química , Sobrevivência Celular , Células Hep G2 , Humanos , Espectrometria de Fluorescência
8.
Cell Physiol Biochem ; 53(2): 400-412, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31403270

RESUMO

BACKGROUND/AIMS: Mutations in ABCA4 cause Stargardt macular degeneration, which invariably ends in legal blindness. We studied two common mutants, A1038V (in NBD1) and G1961E (in NBD2), with the purpose of exploring how they interact with the cell's quality control mechanism. The study was designed to determine how these mutants can be rescued. METHODS: We expressed wt and mutant ABCA4 in HEK293 cells and studied the effect of the mutations on trafficking and processing and the ability of correctors to rescue them. We used a combination of western blotting, confocal microscopy and surface biotinylation coupled with pulldown of plasma membrane proteins. RESULTS: G1961E is sensitive to inhibitors of the aggresome, tubacin and the lysosome, bafilomycin A. Both mutants cause a reduction in heat shock protein, Hsp27. Incubation of HEK293 cells expressing the mutants with VX-809, an FDA approved drug for the treatment of cystic fibrosis, increased the levels of A1038V and G1961E by 2- to 3-fold. Importantly, VX-809 increased the levels of both mutants at the plasma membrane suggesting that trafficking had been restored. Transfecting additional Hsp27 to the cells also increased the steady state levels of both mutants. However, in combination with VX-809 the addition of Hsp27 caused a dramatic increase in the protein expression particularly in the G1961 mutant which increased approximately 5-fold. CONCLUSION: Our results provide a new mechanism for the rescue of ABCA4 trafficking mutants based on the restoration of Hsp27. Our results provide a pathway for the treatment of Stargardt disease.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Aminopiridinas/farmacologia , Benzodioxóis/farmacologia , Transportadores de Cassetes de Ligação de ATP/genética , Aminopiridinas/uso terapêutico , Anilidas/farmacologia , Benzodioxóis/uso terapêutico , Membrana Celular/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Proteínas de Choque Térmico HSP27/metabolismo , Humanos , Ácidos Hidroxâmicos/farmacologia , Leupeptinas/farmacologia , Lisossomos/metabolismo , Degeneração Macular/congênito , Degeneração Macular/tratamento farmacológico , Degeneração Macular/metabolismo , Degeneração Macular/patologia , Mutação , Transporte Proteico/efeitos dos fármacos
9.
Nat Cell Biol ; 21(9): 1093-1101, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31451768

RESUMO

Mechanistic target of rapamycin (mTOR) kinase functions in two multiprotein complexes: lysosomal mTOR complex 1 (mTORC1) and mTORC2 at the plasma membrane. mTORC1 modulates the cell response to growth factors and nutrients by increasing protein synthesis and cell growth, and repressing the autophagy-lysosomal pathway1-4; however, dysfunction in mTORC1 is implicated in various diseases3,5,6. mTORC1 activity is regulated by phosphoinositide lipids7-10. Class I phosphatidylinositol-3-kinase (PI3K)-mediated production of phosphatidylinositol-3,4,5-trisphosphate6,11 at the plasma membrane stimulates mTORC1 signalling, while local synthesis of phosphatidylinositol-3,4-bisphosphate by starvation-induced recruitment of class II PI3K-ß (PI3KC2-ß) to lysosomes represses mTORC1 activity12. How the localization and activity of PI3KC2-ß are regulated by mitogens is unknown. We demonstrate that protein kinase N (PKN) facilitates mTORC1 signalling by repressing PI3KC2-ß-mediated phosphatidylinositol-3,4-bisphosphate synthesis downstream of mTORC2. Active PKN2 phosphorylates PI3KC2-ß to trigger PI3KC2-ß complex formation with inhibitory 14-3-3 proteins. Conversely, loss of PKN2 or inactivation of its target phosphorylation site in PI3KC2-ß represses nutrient signalling via mTORC1. These results uncover a mechanism that couples mTORC2-dependent activation of PKN2 to the regulation of mTORC1-mediated nutrient signalling by local lipid signals.


Assuntos
Lipídeos , Lisossomos/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Transdução de Sinais/fisiologia , Proliferação de Células/fisiologia , Fibroblastos/metabolismo , Humanos , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Complexos Multiproteicos/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina-Treonina Quinases TOR/metabolismo
10.
Ecotoxicol Environ Saf ; 183: 109512, 2019 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-31398584

RESUMO

Azadirachtin, a botanical insecticide with high potential, has been widely used in pest control. Azadirachtin has shown strong biological activity against Bactrocera dorsalis in toxicological reports, but its mechanism remains unclear. This study finds that azadirachtin A inhibits the growth and development of Bactrocera dorsalis larvae. The larval weights and body sizes of the azadirachtin-treated group were significantly less than those of the control group in a concentration-dependent manner. Further, pathological sections revealed that azadirachtin destroyed the midgut cell structure and intestinal walls, while TUNEL staining showed that azadirachtin could induce apoptosis of midgut cells, and Western blot analysis indicated that Bcl-XL expression was inhibited and cytochrome c (CytC) released into the cytoplasm. The results also imply azadirachtin-induced structural alterations in the Bactrocera dorsalis larvae midgut by activation of apoptosis. RNA-seq analysis of midgut cells found that 482 and 708 unique genes were upregulated and downregulated, respectively. These differentially expressed genes (DEGs) were enriched in apoptotic and lysosomal signaling pathways and included 26 genes of the cathepsin family. qRT-PCR verified the expression patterns of some DEGs, indicating that Cathepsin F was upregulated by 278.47-fold and that Cathepsin L and Cathepsin D were upregulated by 28.06- and 8.97-fold, respectively. Finally, association analysis between DEGs and DEMs (differentially expressed metabolites) revealed that azadirachtin significantly reduced the digestion and absorption of carbohydrates, proteins, fats, vitamins and minerals in the midgut. In conclusion, azadirachtin induces the release of cathepsin from lysosomes, causing apoptosis in the midgut. Ultimately, this leads to reduced digestion and absorption of nutrient metabolites in the midgut and inhibition of the growth and development of Bactrocera dorsalis larvae.


Assuntos
Catepsinas/metabolismo , Inseticidas/toxicidade , Intestinos/efeitos dos fármacos , Larva/efeitos dos fármacos , Limoninas/toxicidade , Tephritidae/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Intestinos/patologia , Larva/crescimento & desenvolvimento , Larva/metabolismo , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Lisossomos/patologia , Transdução de Sinais , Tephritidae/metabolismo
11.
Ecotoxicol Environ Saf ; 183: 109492, 2019 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-31421534

RESUMO

Bisphenol A (BPA) is an artificial xenoestrogen widely used in consumer products containing polycarbonate plastics and epoxy resins. Exposure to BPA occurs through various channels, including ingestion of contaminated food and water. Autophagy is an important catabolic pathway that plays an important role in liver lipid metabolism. Evidence suggests that BPA exposure causes abnormal lipid droplet accumulation in liver, but the mechanism remains unknown. Here, we investigate the function of BPA in lipid metabolism and autophagy. BPA exposure increases lipid droplet and ROS accumulation which is accompanied by a defect in the fusion of the autophagosome to the lysosome. BPA exposure decreases the translocation of Stx17 to lysosome resulting in the autophagogome-lysosome fusion defect. There is no defect in the formation of the autophagosome indicated by increased LC3-II, p62 level, GFP/mRFP-LC3 ratios and decreased colocalization between LAMP2 with LC3. Mechanistically, BPA exposure reduces autophagy SNARE complex formation. Promoting autophagy by autophagy inducer (Torin2) partially reverses lipid droplet accumulation caused by BPA exposure. In summary, our results demonstrate BPA exposure inhibits autophagy resulting in decreased lipid droplet degradation and increased ROS levels. These results also provide a novel implication between autophagosome-lysosome fusion.


Assuntos
Autofagossomos/efeitos dos fármacos , Compostos Benzidrílicos/toxicidade , Poluentes Ambientais/toxicidade , Gotículas Lipídicas/efeitos dos fármacos , Lisossomos/efeitos dos fármacos , Fenóis/toxicidade , Animais , Autofagossomos/metabolismo , Autofagia/efeitos dos fármacos , Linhagem Celular , Células HEK293 , Células HeLa , Humanos , Gotículas Lipídicas/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Lisossomos/metabolismo , Camundongos , Naftiridinas/farmacologia , Proteínas Qa-SNARE/metabolismo , Espécies Reativas de Oxigênio/metabolismo
12.
Nat Commun ; 10(1): 2943, 2019 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-31270333

RESUMO

Mutations exclusively in equilibrative nucleoside transporter 3 (ENT3), the only intracellular nucleoside transporter within the solute carrier 29 (SLC29) gene family, cause an expanding spectrum of human genetic disorders (e.g., H syndrome, PHID syndrome, and SHML/RDD syndrome). Here, we identify adult stem cell deficits that drive ENT3-related abnormalities in mice. ENT3 deficiency alters hematopoietic and mesenchymal stem cell fates; the former leads to stem cell exhaustion, and the latter leads to breaches of mesodermal tissue integrity. The molecular pathogenesis stems from the loss of lysosomal adenosine transport, which impedes autophagy-regulated stem cell differentiation programs via misregulation of the AMPK-mTOR-ULK axis. Furthermore, mass spectrometry-based metabolomics and bioenergetics studies identify defects in fatty acid utilization, and alterations in mitochondrial bioenergetics can additionally propel stem cell deficits. Genetic, pharmacologic and stem cell interventions ameliorate ENT3-disease pathologies and extend the lifespan of ENT3-deficient mice. These findings delineate a primary pathogenic basis for the development of ENT3 spectrum disorders and offer critical mechanistic insights into treating human ENT3-related disorders.


Assuntos
Células-Tronco Adultas/metabolismo , Proteínas de Transporte de Nucleosídeos/metabolismo , Adenosina/metabolismo , Adenilato Quinase/metabolismo , Células-Tronco Adultas/ultraestrutura , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacologia , Animais , Autofagia , Transporte Biológico , Diferenciação Celular , Autorrenovação Celular , Metabolismo Energético , Ácidos Graxos/metabolismo , Células HEK293 , Humanos , Metabolismo dos Lipídeos , Lisossomos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Biológicos , Fenótipo , Ribonucleotídeos/farmacologia , Transdução de Sinais , Análise de Sobrevida , Serina-Treonina Quinases TOR/metabolismo
13.
Int J Nanomedicine ; 14: 4867-4880, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31308663

RESUMO

Background: The demand for an effective vaccine delivery system that drives a suitable immune response is increasing. The oxidized carbon nanosphere (OCN), a negatively charged carbon nanoparticle, has the potential to fulfill this requirement because it can efficiently deliver macromolecules into cells and allows endosomal leakage. However, fundamental insights into how OCNs are taken up by antigen-presenting cells, and the intracellular behavior of delivered molecules is lacking. Furthermore, how immune responses are stimulated by OCN-mediated delivery has not been investigated. Purpose: In this study, the model protein antigen ovalbumin (OVA) was used to investigate the uptake mechanism and intracellular fate of OCN-mediated delivery of protein in macrophages. Moreover, the immune response triggered by OVA delivered by OCNs was characterized. Methods: Bone-marrow-derived macrophages (BMDMs) from mice were used to study antigen uptake and intracellular trafficking. Mice were immunized using OCN-OVA combined with known adjuvants, and the specific immune response was measured. Results: OCNs showed no cytotoxicity against BMDMs. OCN-mediated delivery of OVA into BMDMs was partially temperature independent process. Using specific inhibitors, it was revealed that intracellular delivery of OCN-OVA does not rely on phagocytosis or the clathrin- and lipid raft/caveolae-mediated pathways. Delivered OVA was found to colocalize with compartments containing MHC class I, but not with early endosomes, lysosomes, and autophagosomes. Immunization of OVA using OCNs in combination with the known adjuvant monophosphoryl lipid A specifically enhanced interferon gamma (IFNγ)- and granzyme B-producing cytotoxic T cells (CTLs). Conclusion: OCNs effectively delivered protein antigens into macrophages that localized with compartments containing MHC class I partially by the temperature independent, but not clathrin- and lipid raft/caveolae-mediated pathways. Increased CD8+ T-cell activity was induced by OCN-delivered antigens, suggesting antigen processing toward antigen presentation for CTLs. Taken together, OCNs are a potential protein antigen delivery system that stimulates the cell-mediated immune response.


Assuntos
Antígenos/administração & dosagem , Carbono/química , Sistemas de Liberação de Medicamentos , Imunidade Celular , Nanopartículas/química , Adjuvantes Imunológicos/farmacologia , Animais , Antígenos/imunologia , Autofagossomos/efeitos dos fármacos , Autofagossomos/metabolismo , Linhagem Celular , Endocitose/efeitos dos fármacos , Endossomos/efeitos dos fármacos , Endossomos/metabolismo , Feminino , Imunidade Celular/efeitos dos fármacos , Cinética , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos Endogâmicos BALB C , Nanopartículas/toxicidade , Oxirredução , Linfócitos T Citotóxicos/efeitos dos fármacos , Linfócitos T Citotóxicos/imunologia
15.
J Biochem ; 166(2): 175-185, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31329883

RESUMO

TMEM55B is first identified as phosphatidylinositol-4,5-P24-phosphatases (PtdIns-4,5-P24-phosphatases) that catalyse dephosphorylation of PtdIns-4,5-P2 to PtdIns-5-P. We demonstrate for the first time that TMEM55B is phosphorylated by Erk/MAPK and that this mechanism participates in regulation of lysosomal clustering. Exposure of RAW264.7 macrophages to various stimuli induces phosphorylation of TMEM55B on Ser76 and Ser169, sites corresponding to consensus sequences (PX(S/T)P) for phosphorylation by MAPK. Of these stimuli, Toll-like receptor ligands most strongly induce TMEM55B phosphorylation, and this is blocked by the MEK1/2 inhibitor U0126. However, phosphorylation does not impact intrinsic phosphatase activity of TMEM55B. TMEM55B has recently been implicated in starvation induced lysosomal translocation. Amino acid starvation induces perinuclear lamp1 clustering in RAW264.7 macrophages, which was attenuated by shRNA-mediated knock-down or CRISPR/Cas9-mediated knock-out of TMEM55B. Cells exposed to U0126 also exhibit attenuated lamp1 clustering. Overexpression of TMEM55B but not TMEM55A notably enhances lamp1 clustering, with TMEM55B mutants (lacking phosphorylation sites or mimicking the phosphorylated state) exhibiting lower and higher efficacies (respectively) than wild-type TMEM55B. Collectively, results suggest that phosphorylation of TMEM55B by Erk/MAPK impacts lysosomal dynamics.


Assuntos
Lisossomos/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosfatases de Fosfoinositídeos/química , Fosfatases de Fosfoinositídeos/metabolismo , Animais , Camundongos , Fosforilação , Células RAW 264.7
16.
Talanta ; 202: 34-41, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31171192

RESUMO

Lysosomes are organelles containing many hydrolytic enzymes responsible for degrading macromolecules. Abnormal lysosomal pH changes are known to associate with dysfunction of cells linking to various diseases such as cancer and neurodegenerative disorders. Thus, it is of paramount importance to monitor lysosomal pH changes in order to investigate the pathological conditions. We report herein two novel, highly sensitive and fast responsive bis-chromophoric ratiometric two-photon fluorescent probes with different emission wavelengths, namely VP and VL for acidic pH sensing in live cells. Importantly, by adopting bis-chromophoric approach, the VP and VL probes bearing pyridyl and quinolyl as acid sensing sites exhibit pKa values of 4.62 and 5.26, respectively, which are ideal for quantitative analysis of lysosomal pH changes in live cells. These two biocompatible probes are not only highly lysosomal targeting, sensitive towards pH change with distinct emission color shifting but also highly two-photon active in cells with excellent photostability and reversibility. These probes were successfully applied to ratiometrically track and image pH fluctuation in lysosomes of HeLa cells by one- and two-photon excited fluorescence microscopy. For the first time, we have demonstrated here that the bis-chromophoric strategy is a useful tool to effectively modify and tune the pKa of a fluorescent probe.


Assuntos
Corantes Fluorescentes/química , Lisossomos/química , Imagem Óptica , Corantes Fluorescentes/síntese química , Células HeLa , Humanos , Concentração de Íons de Hidrogênio , Lisossomos/metabolismo , Microscopia de Fluorescência , Estrutura Molecular , Fótons , Células Tumorais Cultivadas
17.
Toxicol Lett ; 313: 60-65, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31226396

RESUMO

The use of waterpipes in the United States is increasing in a largely unregulated market. The shisha smoked in a waterpipe is a complex matrix of tobacco, flavorings, and humectant with smoke generated by an external heat source. This study explored the relationship between shisha components and the particulate matter size distributions and toxicity of smoke generated with heating. Standard waterpipe puff topography of charcoal- or electronic- heated whole shisha and shisha components generated smoke particulate matter that was characterized using a TSI Engine Exhaust Particle Sizer. Relative toxicity of the whole smoke was determined via measurement of lysosomal integrity and measures of membrane integrity following acute exposure of type II alveolar cells at the air-liquid interface. All waterpipe aerosols exhibited a unimodal particle size distribution, the peak and concentration of which varied depending upon the shisha components present. Acute exposure to charcoal-heated whole shisha, flavoring syrup, or humectant smoke, or electronic-heated whole shisha smoke caused significant alveolar cell damage and death, indicating neither tobacco nor charcoal are needed for these cytotoxic effects to occur.


Assuntos
Células Epiteliais Alveolares/efeitos dos fármacos , Carvão Vegetal/toxicidade , Aromatizantes/análise , Higroscópicos/toxicidade , Fumaça/efeitos adversos , Tabaco para Cachimbos de Água/toxicidade , Fumar Cachimbo de Água/efeitos adversos , Aerossóis , Células Epiteliais Alveolares/metabolismo , Células Epiteliais Alveolares/patologia , Animais , Morte Celular/efeitos dos fármacos , Linhagem Celular , Carvão Vegetal/análise , Aromatizantes/efeitos adversos , Higroscópicos/análise , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Lisossomos/patologia , Tamanho da Partícula , Ratos , Medição de Risco , Fumaça/análise , Tabaco para Cachimbos de Água/análise
18.
J Microbiol Biotechnol ; 29(6): 989-998, 2019 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-31154748

RESUMO

Autophagy is crucial for immune defense against Mycobacterium tuberculosis (Mtb) infection. Mtb can evade host immune attack and survival within macrophages by manipulating the autophagic process. MicroRNAs (miRNAs) are small, non-coding RNAs that are involved in regulating vital genes during Mtb infection. The precise role of miRNAs in autophagy with the exits of Mtb remains largely unknown. In this study, we found miR-1958, a new miRNA that could regulate autophagy by interacting with 3'UTR of autophagy-related gene 5 (Atg5). In addition, Mtb infection triggered miR-1958 expression in RAW264.7 cells. What's more, miR- 1958 overexpression blocked autophagic flux by impairing the fusion of autophagosomes and lysosomes. Overexpression of miR-1958 reduced Atg5 expression and LC3 puncta while inhibition of miR-1958 brought an increase of Atg5 and LC3 puncta; the opposite results were observed in detection of p62. The survival of Mtb in RAW264.7 cells transfected with mimic of miR-1958 was enhanced. Taken together, our research demonstrated that a novel miR-1958 could inhibit autophagy by interacting with Atg5 and favored intracellular Mtb survival in RAW264.7 cells.


Assuntos
Proteína 5 Relacionada à Autofagia/genética , Autofagia/fisiologia , MicroRNAs/metabolismo , Viabilidade Microbiana/genética , Mycobacterium tuberculosis/fisiologia , Regiões 3' não Traduzidas , Animais , Autofagossomos/metabolismo , Expressão Gênica , Evasão da Resposta Imune , Lisossomos/metabolismo , Macrófagos/metabolismo , Macrófagos/microbiologia , Macrófagos/patologia , Camundongos , MicroRNAs/genética , Mycobacterium tuberculosis/imunologia , Células RAW 264.7
19.
Cell Mol Biol Lett ; 24: 33, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31160892

RESUMO

Background: Induction of lysosomal function and autophagy is regarded as an adaptive mechanism in response to cellular stress. The transcription factor EB (TFEB) has been identified as a master regulator of lysosomal function and autophagy. TFEB is a member of the microphthalmia family of bHLH-LZ transcription factors that includes other members such as micropthalmia-associated transcription factor (MITF), TFE3, and TFEC. TFEB controls lysosome biogenesis and autophagy by upregulation of a family of genes belonging to the Coordinated Lysosomal Expression and Regulation (CLEAR) network. Here, we investigated the expression of TFEB in cells subjected to nutrient deprivation and lysosomal stress. We studied transcriptional induction of TFEB-regulated genes in response to nutrient deprivation and lysosomal stress in retinal pigment epithelial (RPE) cells. Furthermore, we also investigated the induction of autophagy and lysosomal genes upon overexpression of constitutively active form of TFEB. Methods: Expression of TFEB and MITF protein levels were evaluated in cells subjected to prolonged periods of nutrient deprivation. mRNA levels of the CLEAR network genes was measured by quantitative real time PCR (qRT-PCR) analysis in cells deprived of nutrients, treated with ammonium chloride and upon overexpression of constitutively active TFEB. Immunostaining with LC3 antibody was used to measure autophagy flux. Labeling with lysoTracker dye was used to assess lysosomes. Results: Our results show that nutrient deprivation increases protein levels of TFEB and MITF in ARPE-19 cells. Nutrient stress induces the expression of lysosomal (LAMP1, CTSD MCOLN1, SGSH) and autophagy (BECN1) genes. Lysosomal stress also increases the expression of lysosomal (ATP6V0A1 and LAMP1) and autophagy (p62 and BECN1) genes. Our results show that overexpression of constitutively active TFEB also induces the expression of CLEAR network genes. Conclusions: Collectively, these observations suggest that nutrient stress induces the protein expression of both MITF and TFEB in ARPE-19 cells. TFEB-regulated transcriptional program plays an important role in adaptive response of cells during both nutrient and lysosomal stress.


Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Células Epiteliais/metabolismo , Lisossomos/metabolismo , Epitélio Pigmentado da Retina/patologia , Estresse Fisiológico , Adulto , Cloreto de Amônio/farmacologia , Animais , Linhagem Celular , Células Epiteliais/efeitos dos fármacos , Redes Reguladoras de Genes/efeitos dos fármacos , Humanos , Lisossomos/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Estresse Fisiológico/efeitos dos fármacos , Transcrição Genética/efeitos dos fármacos
20.
Food Chem Toxicol ; 131: 110591, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31212009

RESUMO

Kidney ischemia reperfusion injury (IRI) is an acute kidney injury associated with high number of mortality. We have examined the molecular mechanism and found that oxidative stress and hypoxia leads to induction of autophagy. In IRI induced autophagy, TFEB translocated to nucleus in response to IRI and induced a number of target genes of Coordinated Lysosomal Expression and Regulation (CLEAR) network. Real-time PCR analyses result showed IRI dependent increase in mRNA level to lysosomal hydrolases (Ctsa, Psap), lysosomal membranes (Lamp1), lysosomal acidification (Atp6ap1) non-lysosomal proteins involved in lysosomal biogenesis (M6pr, Nagpa) and autophagy (Becn1, VPS11). Overall, both lysosomal biogenesis and autophagy pathways were induced. Two key players of TFEB dependent proteins in autophagy, LAMP1 and BECN1 were verified by protein analyses. Pretreatment with urolithin A promoted autophagy and attenuated renal injury in kidney IRI and thus inverse relationship existed between TFEB-CLEAR pathway and kidney injury. Urolithin A also attenuated IRI induced pro-inflammatory cytokines TNFα, IL1ß, MIP1α and MIP2 mRNA and associated kidney injury. Overall, our results explored the understanding of autophagy and CLEAR network to kidney IRI and those insights may help to develop new therapeutic strategies to protect against IRI.


Assuntos
Lesão Renal Aguda/prevenção & controle , Autofagia/efeitos dos fármacos , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Cumarínicos/uso terapêutico , Substâncias Protetoras/uso terapêutico , Traumatismo por Reperfusão/prevenção & controle , Lesão Renal Aguda/fisiopatologia , Animais , Autofagia/fisiologia , Núcleo Celular/metabolismo , Citocinas/metabolismo , Inflamação/prevenção & controle , Rim/patologia , Rim/fisiopatologia , Lisossomos/metabolismo , Masculino , Camundongos Endogâmicos C57BL , RNA Mensageiro/genética , Traumatismo por Reperfusão/fisiopatologia
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA