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1.
Gene ; 724: 144157, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31629820

RESUMO

Cellular microRNAs are known to modulate the life-cycle of different viruses. Surprisingly, very little data exists on AAV-induced changes to the cellular microRNAome in general and in hepatic and retinal cells, in particular. We reasoned that inducible microRNA in response to recombinant AAV infection may regulate immediate and long-lived cellular responses necessary for the cell's own survival as well as its ability to control several aspects of viral life-cycle. To study this, we performed a global small RNA sequencing analysis in Adeno-associated virus (AAV) serotypes 2 and 3 infected hepatic and retinal cell models. This screen identified multiple differentially expressed microRNAs, in AAV infected Huh-7 and ARPE-19 cells. Among these, one microRNA (miR-4488) was found to be significantly down regulated (-2.24 fold for AAV2 and -3.32 fold for ARPE-19) in AAV infected cells. An enrichment and pathway analysis of miR-4488 predicted its possible effects on gene targets involved in multiple biological processes including cell-cycle regulation, endoplasmic reticulum stress response and lipid-signalling pathways. Moreover, validation studies in miR-4488 mimic or sponge transfected cells revealed modulation of these target pathways in a cell-specific manner. Further studies demonstrated that overexpression of miR-4488, modestly increased gene expression (126-128%) from AAV2 and AAV3 vectors in Huh-7 cells whereas miR-4488 inhibition in ARPE-19 cells had a similar increase (142-158%) on AAV2 or AAV3 transduction. Our results highlight that recombinant AAV mediated microRNA expression is cell-type and serotype-specific and can target specific host cellular biological pathways.


Assuntos
Dependovirus/genética , MicroRNAs/genética , Infecções por Parvoviridae/genética , Epitélio Pigmentado da Retina/virologia , Transdução Genética/métodos , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/virologia , Ciclo Celular/genética , Linhagem Celular , Linhagem Celular Tumoral , Estresse do Retículo Endoplasmático/genética , Perfilação da Expressão Gênica , Humanos , Metabolismo dos Lipídeos/genética , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/virologia , Parvovirinae/genética , Reprodutibilidade dos Testes , Epitélio Pigmentado da Retina/citologia , Transgenes
2.
Chemosphere ; 235: 1134-1145, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31561304

RESUMO

Particulate matter (PM) from layer house has adverse effect on people and chicken respiratory health, which can further influence animal performance and reduce production efficiency. However, little study focus on the respiratory inflammation induced by PM2.5 from layer house and the underlying mechanism also unclear. In this study, human adenocarcinoma alveolar basal epithelial cells (A549 cell) was subjected to the PM2.5 from layer house to evaluate the inflammation reaction caused by PM2.5 and explore the role of Nrf2 and autophagy in regulating the inflammation. Results showed that the viability of A549 cell decreased in a time - and concentration - dependent manner after PM2.5 treatment. TNFα, IL6, and IL8 increased significantly treated with PM2.5 at 12 h. RNA sequencing indicated differentially expressed genes were enriched in immune system process, oxidative stress (OS), endoplasmic reticulum stress (ERS), and autophagy. Further studies showed TLR4 - NFκB p65 signal pathway involved in the inflammation reaction caused by PM2.5. The overexpression of Nrf2 decreased the level of TNFα, IL6, IL8 markedly as well as the level of NFκB p65 and NFκB pp65. OS and ERS were also limited under overactivation of Nrf2 in PM2.5 treated cells. Autophagy induced by PM2.5 promoted the inflammation through increasing the level of NFκB p65 and NFκB pp65. Autophagy deficient strengthened the expression of Nrf2. Collectively, our study revealed Nrf2 prevents inflammation caused by layer house PM2.5 stimulation, however, autophagy exerts a promotive role in TLR4 - NFκB p65 mediating inflammation in A549 cell.


Assuntos
Autofagia/fisiologia , Inflamação/etiologia , Fator 2 Relacionado a NF-E2/fisiologia , Material Particulado/efeitos adversos , Receptor 4 Toll-Like/metabolismo , Fator de Transcrição RelA/metabolismo , Células A549 , Animais , Autofagia/efeitos dos fármacos , Estresse do Retículo Endoplasmático/genética , Humanos , Inflamação/induzido quimicamente , Inflamação/prevenção & controle , Fator 2 Relacionado a NF-E2/farmacologia , Estresse Oxidativo/genética , Transdução de Sinais
3.
DNA Cell Biol ; 38(9): 969-981, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31355672

RESUMO

Analysis of gene expression can be challenging, especially if it involves genetically diverse populations that exhibit high variation in their individual expression profile. Despite this variation, it is conceivable that in the same individuals a high degree of coordination is maintained between transcripts that belong to the same signaling modules and are associated with related biological functions. To explore this further, we calculated the correlation in the expression levels between each of ATF4, CHOP (DDIT3), GRP94, DNAJB9 (ERdj4), DNAJ3C (P58IPK), and HSPA5 (BiP/GRP78) with the whole transcriptome in primary fibroblasts from deer mice following induction of endoplasmic reticulum (ER) stress. Since these genes are associated with different transducers of the unfolded protein response (UPR), we postulated that their profile, in terms of correlation of transcripts, reflects distinct UPR branches engaged, and therefore different biological processes. Standard gene ontology analysis was able to predict major functions associated with the corresponding transcript, and of the UPR arm related to that, namely regulation of the apoptotic response by ATF4 (PERK arm) and the ER stress-associated degradation for GRP94 (IRE1). BiP, being a global regulator of the UPR, was associated with activation of ER stress in a rather global manner. Pairwise comparison in the correlation coefficients for these genes' associated transcriptome showed the relevance of selected genes in terms of expression profiles. Conventional assessment of differential gene expression was incapable of providing meaningful information and pointed only to a generic association with stress. Collectively, this approach suggests that by evaluating the degree of coordination in gene expression, in genetically diverse biological specimens, may be useful in assigning genes in transcriptome networks, and more importantly in linking signaling nodules to specific biological functions and processes.


Assuntos
Estresse do Retículo Endoplasmático/genética , Animais , Células Cultivadas , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Peromyscus , Transcriptoma , Tunicamicina/farmacologia
4.
Nat Cell Biol ; 21(7): 889-899, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31263264

RESUMO

The c-Myc oncogene drives malignant progression and induces robust anabolic and proliferative programmes leading to intrinsic stress. The mechanisms enabling adaptation to MYC-induced stress are not fully understood. Here we reveal an essential role for activating transcription factor 4 (ATF4) in survival following MYC activation. MYC upregulates ATF4 by activating general control nonderepressible 2 (GCN2) kinase through uncharged transfer RNAs. Subsequently, ATF4 co-occupies promoter regions of over 30 MYC-target genes, primarily those regulating amino acid and protein synthesis, including eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1), a negative regulator of translation. 4E-BP1 relieves MYC-induced proteotoxic stress and is essential to balance protein synthesis. 4E-BP1 activity is negatively regulated by mammalian target of rapamycin complex 1 (mTORC1)-dependent phosphorylation and inhibition of mTORC1 signalling rescues ATF4-deficient cells from MYC-induced endoplasmic reticulum stress. Acute deletion of ATF4 significantly delays MYC-driven tumour progression and increases survival in mouse models. Our results establish ATF4 as a cellular rheostat of MYC activity, which ensures that enhanced translation rates are compatible with survival and tumour progression.


Assuntos
Fator 4 Ativador da Transcrição/genética , Genes myc/genética , Ativação Transcricional/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Estresse do Retículo Endoplasmático/genética , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos Transgênicos , Fosfoproteínas/genética , Fosforilação , Biossíntese de Proteínas/fisiologia , Serina-Treonina Quinases TOR/metabolismo
5.
Genes Dev ; 33(15-16): 1083-1094, 2019 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-31296559

RESUMO

The orphan nuclear receptor SHP (small heterodimer partner) is a well-known transcriptional corepressor of bile acid and lipid metabolism in the liver; however, its function in other tissues is poorly understood. Here, we report an unexpected role for SHP in the exocrine pancreas as a modulator of the endoplasmic reticulum (ER) stress response. SHP expression is induced in acinar cells in response to ER stress and regulates the protein stability of the spliced form of X-box-binding protein 1 (XBP1s), a key mediator of ER stress response. Loss of SHP reduces XBP1s protein level and transcriptional activity, which in turn attenuates the ER stress response during the fasting-feeding cycle. Consequently, SHP-deficient mice also are more susceptible to cerulein-induced pancreatitis. Mechanistically, we show that SHP physically interacts with the transactivation domain of XBP1s, thereby inhibiting the polyubiquitination and degradation of XBP1s by the Cullin3-SPOP (speckle-type POZ protein) E3 ligase complex. Together, our data implicate SHP in governing ER homeostasis and identify a novel posttranslational regulatory mechanism for the key ER stress response effector XBP1.


Assuntos
Estresse do Retículo Endoplasmático/genética , Proteólise , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteína 1 de Ligação a X-Box/metabolismo , Células Acinares/metabolismo , Animais , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/genética , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Pâncreas Exócrino/metabolismo , Pancreatite/genética , Processamento de Proteína , Estabilidade Proteica , Receptores Citoplasmáticos e Nucleares/deficiência , Receptores Citoplasmáticos e Nucleares/genética , Ubiquitinação/genética
6.
J Ovarian Res ; 12(1): 53, 2019 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-31176373

RESUMO

OBJECT: To explore the mechanisms of ovarian aging, we performed overall analysis on the age-related alterations of gene expression profiles in mouse germinal vesicle (GV) stage oocytes by means of single-cell RNA-sequencing method (scRNA-seq). METHODS: Two age groups (5-week-old and 32-week-old) female KM mice were used as young and old models. Subsequently, GV oocytes were collected for scRNA-seq. The bioinformatics was performed to analyze and compare the differences of gene expression profile between GV oocytes of young and old mice. RESULTS: The analysis of scRNA-seq data showed that there were 624 differential expressed genes (DEGs) between two age groups of mouse GV stage oocytes. Four hundred forty-nine DEGs were up-regulated while 175 DEGs were down-regulated in the GV oocytes of the old group. KEGG pathway analysis revealed that the genes involved in mitochondrial function including oxidative phosphorylation and ATP production pathway were significantly down-regulated in GV oocytes of 32-week-old mice, especially the mitochondrial encoded NADH dehydrogenase (mt-Nd), including mt-Nd2, mt-Nd3, mt-Nd4, mt-Nd4L and mt-Nd5. Analysis of DEGs revealed that endoplasmic reticulum stress-related genes including AdipoR2, IRAK-1, RCAN1 and MsrB1 were significantly down-regulated in GV oocytes of 32-week-old mice. Also, analysis of DEGs demonstrated that anti-oxidation-related genes including Erbb3、Rcan1、Gsto2 and Msrb1 were significantly down-regulated in GV oocytes of old group. CONCLUSION: The disorder of mitochondrial function, endoplasmic reticulum stress and the reduced antioxidant capability might be involved in the progression of oocyte aging. Especially, the down regulation of mitochondrial encoded subunits of respiratory chain complexes might play critical roles in the relevant mechanisms.


Assuntos
Senescência Celular/genética , Estresse do Retículo Endoplasmático/genética , Mitocôndrias/genética , Oócitos/metabolismo , Envelhecimento , Animais , Feminino , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Camundongos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Oócitos/citologia , Oócitos/crescimento & desenvolvimento , Estresse Oxidativo/genética , Análise de Célula Única
7.
BMC Plant Biol ; 19(1): 193, 2019 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-31072347

RESUMO

BACKGROUND: Wheat production is largely restricted by adverse environmental stresses. Under many undesirable conditions, endoplasmic reticulum (ER) stress can be induced. However, the physiological and molecular responses of wheat to ER stress remain poorly understood. We used dithiothreitol (DTT) and tauroursodeoxycholic acid (TUDCA) to induce or suppress ER stress in wheat cells, respectively, with the aim to reveal the molecular background of ER stress responses using a combined approach of transcriptional profiling and morpho-physiological characterization. METHODS: To understand the mechanism of wheat response to ER stress, three wheat cultivars were used in our pre-experiments. Among them, the cultivar with a moderate stress tolerance, Yunong211 was used in the following experiments. We used DTT (7.5 mM) to induce ER stress and TUDCA (25 µg·mL- 1) to suppress the stress. Under three treatment groups (Control, DTT and DTT + TUDCA), we firstly monitored the morphological, physiological and cytological changes of wheat seedlings. Then we collected leaf samples from each group for RNA extraction, library construction and RNA sequencing on an Illumina Hiseq platform. The sequencing data was then validated by qRT-PCR. RESULTS: Morpho-physiological results showed DTT significantly reduced plant height and biomass, decreased contents of chlorophyll and water, increased electrolyte leakage rate and antioxidant enzymes activity, and accelerated the cell death ratio, whereas these changes were all remarkably alleviated after TUDCA co-treatment. Therefore, RNA sequencing was performed to determine the genes involved in regulating wheat response to stress. Transcriptomic analysis revealed that 8204 genes were differentially expressed in three treatment groups. Among these genes, 158 photosynthesis-related genes, 42 antioxidant enzyme genes, 318 plant hormone-related genes and 457 transcription factors (TFs) may play vital roles in regulating wheat response to ER stress. Based on the comprehensive analysis, we propose a hypothetical model to elucidate possible mechanisms of how plants adapt to environmental stresses. CONCLUSIONS: We identified several important genes that may play vital roles in wheat responding to ER stress. This work should lay the foundations of future studies in plant response to environmental stresses.


Assuntos
Estresse do Retículo Endoplasmático/genética , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Transcriptoma/genética , Triticum/genética , Triticum/fisiologia , Ditiotreitol/farmacologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Ontologia Genética , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/genética , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/genética , Reprodutibilidade dos Testes , Análise de Sequência de RNA , Ácido Tauroquenodesoxicólico/farmacologia , Fatores de Transcrição/metabolismo , Transcriptoma/efeitos dos fármacos , Triticum/anatomia & histologia
8.
Nat Immunol ; 20(7): 865-878, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31086333

RESUMO

Natural killer (NK) cells are critical mediators of host immunity to pathogens. Here, we demonstrate that the endoplasmic reticulum stress sensor inositol-requiring enzyme 1 (IRE1α) and its substrate transcription factor X-box-binding protein 1 (XBP1) drive NK cell responses against viral infection and tumors in vivo. IRE1α-XBP1 were essential for expansion of activated mouse and human NK cells and are situated downstream of the mammalian target of rapamycin signaling pathway. Transcriptome and chromatin immunoprecipitation analysis revealed c-Myc as a new and direct downstream target of XBP1 for regulation of NK cell proliferation. Genetic ablation or pharmaceutical blockade of IRE1α downregulated c-Myc, and NK cells with c-Myc haploinsufficency phenocopied IRE1α-XBP1 deficiency. c-Myc overexpression largely rescued the proliferation defect in IRE1α-/- NK cells. Like c-Myc, IRE1α-XBP1 also promotes oxidative phosphorylation in NK cells. Overall, our study identifies a IRE1α-XBP1-cMyc axis in NK cell immunity, providing insight into host protection against infection and cancer.


Assuntos
Estresse do Retículo Endoplasmático/genética , Endorribonucleases/genética , Regulação da Expressão Gênica , Genes myc , Imunidade/genética , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Proteínas Serina-Treonina Quinases/genética , Animais , Biomarcadores , Sobrevivência Celular/genética , Sobrevivência Celular/imunologia , Citotoxicidade Imunológica , Interações Hospedeiro-Patógeno/imunologia , Humanos , Ativação Linfocitária/imunologia , Melanoma Experimental , Camundongos , Camundongos Knockout , Mitocôndrias/metabolismo , Fosforilação Oxidativa , Transdução de Sinais , Proteína 1 de Ligação a X-Box/metabolismo
9.
Mol Carcinog ; 58(9): 1623-1630, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31041814

RESUMO

Cancer is associated with a number of conditions such as hypoxia, nutrient deprivation, cellular redox, and pH changes that result in accumulation of unfolded or misfolded proteins in the endoplasmic reticulum (ER) and trigger a stress response known as the unfolded protein response (UPR). The UPR is a conserved cellular survival mechanism mediated by the ER transmembrane proteins activating transcription factor 6, protein kinase-like endoplasmic reticulum kinase, and inositol-requiring enzyme 1α (IRE1α) that act to resolve ER stress and promote cell survival. IRE1α is a kinase/endoribonuclease (RNase) with multiple activities including unconventional splicing of the messenger RNA (mRNA) for the transcription factor X-Box Binding Protein 1 (XBP1), degradation of other mRNAs in a process called regulated IRE1α-dependent decay (RIDD) and activation of a pathway leading to c-Jun N-terminal kinase phosphorylation. Each of these outputs plays a role in the adaptive and cell death responses to ER stress. Many studies indicate an important role for XBP1 and RIDD functions in cancer and new studies suggest that these two functions of the IRE1α RNase can have opposing functions in the early and later stages of cancer pathogenesis. Finally, as more is learned about the context-dependent role of IRE1α in cancer development, specific small molecule inhibitors and activators of IRE1α could play an important role in counteracting the protective shield provided by ER stress signaling in cancer cells.


Assuntos
Endorribonucleases/genética , Regulação da Expressão Gênica/genética , Neoplasias/genética , Resposta a Proteínas não Dobradas/genética , Animais , Retículo Endoplasmático/genética , Estresse do Retículo Endoplasmático/genética , Humanos , RNA Mensageiro/genética , Transdução de Sinais/genética
10.
Br J Anaesth ; 123(1): 51-59, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31084986

RESUMO

BACKGROUND: Macrophage phagocytosis constitutes an essential part of the host defence against microbes and the resolution of inflammation. Hyperglycaemia during sepsis is reported to reduce macrophage function, and thus, potentiate inflammatory deterioration. We investigated whether high-glucose concentrations augment lipopolysaccharide-induced reduction in macrophage phagocytosis via the endoplasmic stress-C/EBP homologous protein (CHOP) pathway using animal and laboratory investigations. METHODS: Peritoneal macrophages of artificially ventilated male Wistar rats, divided into four groups based on target blood glucose concentrations achieved by glucose administration with or without lipopolysaccharide, were obtained after 24 h. Human macrophages were also cultured in normal or high glucose with or without lipopolysaccharide exposure for 72 h. Changes in the phagocytic activity, intranuclear CHOP expression, and intracellular Akt phosphorylation status of macrophages were evaluated. These changes were also evaluated in human macrophages after genetic knock-down of CHOP by specific siRNA transfection or resolvin D2 treatment. RESULTS: Lipopolysaccharide impaired phagocytosis, increased intranuclear expression of CHOP, and inhibited Akt phosphorylation in both rat peritoneal and human macrophages. Hyperglycaemic glucose concentrations augmented these changes. Genetic knock-down of CHOP restored phagocytic ability and Akt phosphorylation in human macrophages. Furthermore, resolvin D2 co-incubation restored the inhibited phagocytosis and Akt phosphorylation along with the inhibition of intranuclear CHOP expression in human macrophages. CONCLUSIONS: These findings imply that controlling endoplasmic reticulum stress might provide new strategies for restoring reduced macrophage phagocytosis in sepsis-induced hyperglycaemia.


Assuntos
Hiperglicemia/metabolismo , Lipopolissacarídeos/metabolismo , Macrófagos/metabolismo , Fagocitose/fisiologia , Fator de Transcrição CHOP/metabolismo , Adulto , Animais , Células Cultivadas , Modelos Animais de Doenças , Estresse do Retículo Endoplasmático/genética , Humanos , Masculino , Ratos , Ratos Wistar , Transdução de Sinais , Fator de Transcrição CHOP/genética
11.
Mol Med Rep ; 20(1): 350-358, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31115545

RESUMO

Endoplasmic reticulum (ER) stress and autophagy are involved in myocardial ischemia­reperfusion (I/R) injury; however, their roles in this type of injury remain unclear. The present study investigated the roles of ER stress and autophagy, and their underlying mechanisms, in H9c2 cells during hypoxia/reoxygenation (H/R) injury. Cell viability was detected by CCK­8 assay. The autophagy flux was monitored with mCherry­GFP­LC3­adenovirus transfection. The expression levels of autophagy­related proteins and ER stress­related proteins were measured by western blotting. Apoptosis was detected by flow cytometry and western blotting. The results indicated that autophagy was induced, ER stress was activated and apoptosis was promoted in H9c2 cells during H/R injury. The inhibition of ER stress by 4­phenylbutyrate or C/EBP homologous protein (CHOP)­targeting small interfering RNA (siRNA) decreased autophagy and ameliorated cell apoptosis during H/R injury. Activation of autophagy by rapamycin attenuated ER stress and ameliorated cell apoptosis. Inhibition of autophagy by 3­methyladenine or Beclin1­targeting siRNA aggravated ER stress and exacerbated cell apoptosis, and activation of ER stress by thapsigargin decreased autophagy and induced cell apoptosis. Collectively, the findings of the present study demonstrated that H/R induced apoptosis and autophagy via ER stress in H9c2 cells, and that CHOP may serve an important role in ER stress­induced autophagy and apoptosis. Autophagy, as an adaptive response, was activated by ER stress and alleviated ER stress­induced cell apoptosis during H/R injury.


Assuntos
Autofagia/genética , Estresse do Retículo Endoplasmático/genética , Traumatismo por Reperfusão Miocárdica/genética , Miócitos Cardíacos/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Proteínas Relacionadas à Autofagia/genética , Proteína Beclina-1/genética , Hipóxia Celular/genética , Linhagem Celular , Regulação da Expressão Gênica/genética , Humanos , Traumatismo por Reperfusão Miocárdica/patologia , Traumatismo por Reperfusão Miocárdica/terapia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Fenilbutiratos/farmacologia , RNA Interferente Pequeno/genética , Ratos , Transdução de Sinais/efeitos dos fármacos , Fator de Transcrição CHOP/genética
12.
Int J Mol Med ; 44(1): 125-134, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31059078

RESUMO

Mutations in myelin protein zero (MPZ) cause inherited peripheral neuropathies, including Charcot­Marie­Tooth disease (CMT) and Dejerine­Sottas neuropathy. Mutant MPZ proteins have previously been reported to cause CMT via enhanced endoplasmic reticulum (ER) stress and Schwann cell (SC) death, although the pathological mechanisms have not yet been elucidated. In this study, we generated an in vitro model of rat SCs expressing mutant MPZ (MPZ V169fs or R98C) proteins and validated the increase in cell death and ER stress induced by the overexpression of the MPZ mutants. Using this model, we examined the efficacy of 3 different aminosalicylic acids (ASAs; 4­ASA, sodium 4­ASA and 5­ASA) in alleviating pathological phenotypes. FACS analysis indicated that the number of apoptotic rat SCs, RT4 cells, induced by mutant MPZ overexpression was significantly reduced following treatment with each ASA. In particular, treatment with 4­ASA reduced the levels of ER stress markers in RT4 cells induced by V169fs MPZ mutant overexpression and relieved the retention of V169fs mutant proteins in the ER. Additionally, the level of an apoptotic signal mediator (p­JNK) was only decreased in the RT4 cells expressing R98C MPZ mutant protein following treatment with 4­ASA. Although 4­ASA is known as a free radical scavenger, treatment with 4­ASA in the in vitro model did not moderate the level of reactive oxygen species, which was elevated by the expression of mutant MPZ proteins. On the whole, the findings of this study indicate that treatment with 4­ASA reduced the ER stress and SC death caused by 2 different MPZ mutants and suggest that ASA may be a potential therapeutic agent for CMT.


Assuntos
Ácido Aminossalicílico/farmacologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Mutação de Sentido Incorreto , Proteína P0 da Mielina/metabolismo , Células de Schwann/metabolismo , Substituição de Aminoácidos , Animais , Morte Celular/efeitos dos fármacos , Morte Celular/genética , Linhagem Celular , Doença de Charcot-Marie-Tooth/tratamento farmacológico , Doença de Charcot-Marie-Tooth/genética , Doença de Charcot-Marie-Tooth/metabolismo , Doença de Charcot-Marie-Tooth/patologia , Estresse do Retículo Endoplasmático/genética , Humanos , Proteína P0 da Mielina/genética , Ratos , Células de Schwann/patologia
13.
Chemosphere ; 227: 541-550, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31004821

RESUMO

Isoniazid (INH) is a first-line anti-tuberculosis drug. INH has been detected in surface waters which may create a risk to aquatic organisms. In this study, the hepatotoxicity of INH was elucidated using zebrafish. The liver morphology, transaminase level, redox-related enzyme activity, reactive oxygen species (ROS) content and mRNA levels of liver injury-related genes were measured. The results showed that INH (4, 6 mM) significantly caused liver atrophy and increased levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in zebrafish. INH (6 mM) led to decreased catalase (CAT) activity, glutathione peroxidase (GPx) activity and glutathione (GSH) content but increased ROS and malondialdehyde (MDA) levels. Moreover, INH (6 mM) decreased expression levels of miR-122 and pparα but increased mRNA levels of ap-1 and c-jun. Furthermore, mRNA levels of factors related to endoplasmic reticulum stress (ERS) (grp78, atf6, perk, ire1, xbp1s and chop), apoptosis (bax, cyt, caspase-3, caspase-8 and caspase-9) and the Nrf2 signalling pathway (nrf2, ho-1, nqo1, gclm and gclc) were significantly upregulated. INH may act on hepatotoxicity in zebrafish by increasing ROS content, which weakens the antioxidant capacity, leading to ERS, cell apoptosis and liver injury. In addition, the Nrf2 signalling pathway is activated as a stress compensation mechanism during INH-induced liver injury, but it is not sufficient to counteract INH-induced hepatotoxicity.


Assuntos
Doença Hepática Induzida por Substâncias e Drogas/etiologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Isoniazida/toxicidade , Larva/metabolismo , Espécies Reativas de Oxigênio , Peixe-Zebra/metabolismo , Animais , Antioxidantes/metabolismo , Antituberculosos/toxicidade , Doença Hepática Induzida por Substâncias e Drogas/genética , Doença Hepática Induzida por Substâncias e Drogas/patologia , Estresse do Retículo Endoplasmático/genética , Regulação da Expressão Gênica , Larva/efeitos dos fármacos , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo/fisiologia , Espécies Reativas de Oxigênio/efeitos adversos , Transdução de Sinais , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
14.
Mol Cell ; 74(5): 909-921.e6, 2019 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-31006538

RESUMO

Certain proteins and organelles can be selectively degraded by autophagy. Typical substrates and receptors of selective autophagy have LC3-interacting regions (LIRs) that bind to autophagosomal LC3 and GABARAP family proteins. Here, we performed a differential interactome screen using wild-type LC3B and a LIR recognition-deficient mutant and identified TEX264 as a receptor for autophagic degradation of the endoplasmic reticulum (ER-phagy). TEX264 is an ER protein with a single transmembrane domain and a LIR motif. TEX264 interacts with LC3 and GABARAP family proteins more efficiently and is expressed more ubiquitously than previously known ER-phagy receptors. ER-phagy is profoundly blocked by deletion of TEX264 alone and almost completely by additional deletion of FAM134B and CCPG1. A long intrinsically disordered region of TEX264 is required for its ER-phagy receptor function to bridge the gap between the ER and autophagosomal membranes independently of its amino acid sequence. These results suggest that TEX264 is a major ER-phagy receptor.


Assuntos
Proteínas Relacionadas à Autofagia/genética , Autofagia/genética , Retículo Endoplasmático/genética , Proteínas Intrinsicamente Desordenadas/genética , Sequência de Aminoácidos/genética , Proteínas Relacionadas à Autofagia/química , Proteínas de Ciclo Celular/genética , Retículo Endoplasmático/química , Estresse do Retículo Endoplasmático/genética , Humanos , Proteínas Intrinsicamente Desordenadas/química , Proteínas Associadas aos Microtúbulos/genética , Proteínas de Neoplasias/genética , Proteólise
15.
Immunity ; 50(5): 1218-1231.e5, 2019 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-30952607

RESUMO

Patients with the neurological disorder HSAN-I suffer frequent infections, attributed to a lack of pain sensation and failure to seek care for minor injuries. Whether protective CD8+ T cells are affected in HSAN-I patients remains unknown. Here, we report that HSAN-I-associated mutations in serine palmitoyltransferase subunit SPTLC2 dampened human T cell responses. Antigen stimulation and inflammation induced SPTLC2 expression, and murine T-cell-specific ablation of Sptlc2 impaired antiviral-T-cell expansion and effector function. Sptlc2 deficiency reduced sphingolipid biosynthetic flux and led to prolonged activation of the mechanistic target of rapamycin complex 1 (mTORC1), endoplasmic reticulum (ER) stress, and CD8+ T cell death. Protective CD8+ T cell responses in HSAN-I patient PBMCs and Sptlc2-deficient mice were restored by supplementing with sphingolipids and pharmacologically inhibiting ER stress-induced cell death. Therefore, SPTLC2 underpins protective immunity by translating extracellular stimuli into intracellular anabolic signals and antagonizes ER stress to promote T cell metabolic fitness.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Neuropatias Hereditárias Sensoriais e Autônomas/genética , Coriomeningite Linfocítica/imunologia , Vírus da Coriomeningite Linfocítica/imunologia , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Serina C-Palmitoiltransferase/genética , Animais , Proliferação de Células , Células Cultivadas , Citocinas/biossíntese , Estresse do Retículo Endoplasmático/genética , Estresse do Retículo Endoplasmático/imunologia , Feminino , Humanos , Coriomeningite Linfocítica/virologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Transdução de Sinais/imunologia , Esfingolipídeos/biossíntese
16.
Mol Cell ; 74(5): 891-908.e10, 2019 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-31006537

RESUMO

Cells respond to nutrient stress by trafficking cytosolic contents to lysosomes for degradation via macroautophagy. The endoplasmic reticulum (ER) serves as an initiation site for autophagosomes and is also remodeled in response to nutrient stress through ER-phagy, a form of selective autophagy. Quantitative proteome analysis during nutrient stress identified an unstudied single-pass transmembrane ER protein, TEX264, as an ER-phagy receptor. TEX264 uses an LC3-interacting region (LIR) to traffic into ATG8-positive puncta that often initiate from three-way ER tubule junctions and subsequently fuse with lysosomes. Interaction and proximity biotinylation proteomics identified a cohort of autophagy regulatory proteins and cargo adaptors located near TEX264 in an LIR-dependent manner. Global proteomics and ER-phagy flux analysis revealed the stabilization of a cohort of ER proteins in TEX264-/- cells during nutrient stress. This work reveals TEX264 as an unrecognized ER-phagy receptor that acts independently of other candidate ER-phagy receptors to remodel the ER during nutrient stress.


Assuntos
Família da Proteína 8 Relacionada à Autofagia/genética , Proteínas Relacionadas à Autofagia/genética , Autofagia/genética , Retículo Endoplasmático/genética , Animais , Autofagossomos/metabolismo , Proteínas Relacionadas à Autofagia/metabolismo , Células COS , Cercopithecus aethiops , Citosol/metabolismo , Estresse do Retículo Endoplasmático/genética , Células HCT116 , Células HEK293 , Humanos , Lisossomos/genética , Lisossomos/metabolismo , Nutrientes/metabolismo , Transporte Proteico/genética , Proteoma/genética
17.
J Exp Clin Cancer Res ; 38(1): 160, 2019 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-30987650

RESUMO

BACKGROUND: Glucose-6-phospate dehydrogenase (G6PD) is the limiting enzyme of the pentose phosphate pathway (PPP) correlated to cancer progression and drug resistance. We previously showed that G6PD inhibition leads to Endoplasmic Reticulum (ER) stress often associated to autophagy deregulation. The latter can be induced by target-based agents such as Lapatinib, an anti-HER2 tyrosine kinase inhibitor (TKI) largely used in breast cancer treatment. METHODS: Here we investigate whether G6PD inhibition causes autophagy alteration, which can potentiate Lapatinib effect on cancer cells. Immunofluorescence and flow cytometry for LC3B and lysosomes tracker were used to study autophagy in cells treated with lapatinib and/or G6PD inhibitors (polydatin). Immunoblots for LC3B and p62 were performed to confirm autophagy flux analyses together with puncta and colocalization studies. We generated a cell line overexpressing G6PD and performed synergism studies on cell growth inhibition induced by Lapatinib and Polydatin using the median effect by Chou-Talay. Synergism studies were additionally validated with apoptosis analysis by annexin V/PI staining in the presence or absence of autophagy blockers. RESULTS: We found that the inhibition of G6PD induced endoplasmic reticulum stress, which was responsible for the deregulation of autophagy flux. Indeed, G6PD blockade caused a consistent increase of autophagosomes formation independently from mTOR status. Cells engineered to overexpress G6PD became resilient to autophagy and resistant to lapatinib. On the other hand, G6PD inhibition synergistically increased lapatinib-induced cytotoxic effect on cancer cells, while autophagy blockade abolished this effect. Finally, in silico studies showed a significant correlation between G6PD expression and tumour relapse/resistance in patients. CONCLUSIONS: These results point out that autophagy and PPP are crucial players in TKI resistance, and highlight a peculiar vulnerability of breast cancer cells, where impairment of metabolic pathways and autophagy could be used to reinforce TKI efficacy in cancer treatment.


Assuntos
Antineoplásicos/farmacologia , Autofagia/efeitos dos fármacos , Neoplasias da Mama/metabolismo , Glucosefosfato Desidrogenase/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Apoptose/efeitos dos fármacos , Autofagia/genética , Neoplasias da Mama/genética , Neoplasias da Mama/mortalidade , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Sinergismo Farmacológico , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Estresse do Retículo Endoplasmático/genética , Feminino , Expressão Gênica , Glucosefosfato Desidrogenase/genética , Glucosefosfato Desidrogenase/metabolismo , Humanos , Lapatinib/farmacologia , Prognóstico , Recidiva
18.
Mol Cell ; 74(5): 877-890.e6, 2019 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-31023583

RESUMO

Endoplasmic reticulum (ER) stress and unfolded protein response are energetically challenging under nutrient stress conditions. However, the regulatory mechanisms that control the energetic demand under nutrient and ER stress are largely unknown. Here we show that ER stress and glucose deprivation stimulate mitochondrial bioenergetics and formation of respiratory supercomplexes (SCs) through protein kinase R-like ER kinase (PERK). Genetic ablation or pharmacological inhibition of PERK suppresses nutrient and ER stress-mediated increases in SC levels and reduces oxidative phosphorylation-dependent ATP production. Conversely, PERK activation augments respiratory SCs. The PERK-eIF2α-ATF4 axis increases supercomplex assembly factor 1 (SCAF1 or COX7A2L), promoting SCs and enhanced mitochondrial respiration. PERK activation is sufficient to rescue bioenergetic defects caused by complex I missense mutations derived from mitochondrial disease patients. These studies have identified an energetic communication between ER and mitochondria, with implications in cell survival and diseases associated with mitochondrial failures.


Assuntos
Fator 4 Ativador da Transcrição/genética , Metabolismo Energético/genética , Fator de Iniciação 2 em Eucariotos/genética , Mitocôndrias/genética , eIF-2 Quinase/genética , Trifosfato de Adenosina/metabolismo , Animais , Apoptose , Linhagem Celular , Sobrevivência Celular/genética , Complexo I de Transporte de Elétrons/genética , Complexo I de Transporte de Elétrons/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/genética , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático/genética , Glucose/metabolismo , Humanos , Camundongos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Doenças Mitocondriais/genética , Doenças Mitocondriais/metabolismo , Doenças Mitocondriais/patologia , Mutação de Sentido Incorreto/genética , Nutrientes/metabolismo , Fosforilação , Fatores de Processamento de Serina-Arginina/genética , Transdução de Sinais
19.
Arch Virol ; 164(5): 1323-1334, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30877450

RESUMO

Porcine circovirus type 2 (PCV2) is the essential infectious agent causing porcine circovirus-associated disease (PCVD) in pigs and one of the important viruses that severely jeopardize the swine husbandry industry. PCV2 elicits the unfolded protein response (UPR) via activation of the PERK pathway, and its capsid protein (Cap) has also been found to induce UPR with subsequent activation of apoptosis. The open reading frame 5 (ORF5) protein is a recently discovered non-structural protein, and its function in PCV2 pathogenesis remains unknown. The aim of this study was to determine whether the PCV2 ORF5 protein could induce endoplasmic reticulum stress (ERS) and UPR in porcine alveolar macrophages (PAMs). pEGFP-tagged ORF5 protein was transiently overexpressed in PAMs. Transmission electron microscopy (TEM) was employed to examine changes in ER morphology, and quantitative real-time PCR and western blotting analysis were used to measure UPR-related cell signaling alterations. We found that the ORF5 protein triggers swelling and degranulation of the ER and upregulates the expression of ERS markers. Further experiments demonstrated that the PCV2 ORF5 protein induces ERS and UPR via the PERK (RNA-activated protein kinase-like endoplasmic reticulum kinase), ATF6 (activating transcription factor 6) and IRE1 (inositol requiring enzyme 1) signaling pathways. Together with previous studies, we provide new information on the ERS-UPR induced by the PCV2 ORF5 protein.


Assuntos
Circovirus/genética , Estresse do Retículo Endoplasmático/genética , Retículo Endoplasmático/ultraestrutura , Macrófagos Alveolares/patologia , Resposta a Proteínas não Dobradas/genética , Proteínas do Envelope Viral/genética , Proteínas não Estruturais Virais/genética , Fator 6 Ativador da Transcrição/metabolismo , Animais , Linhagem Celular , Infecções por Circoviridae/patologia , Infecções por Circoviridae/veterinária , Retículo Endoplasmático/virologia , Endorribonucleases/metabolismo , Macrófagos Alveolares/virologia , Microscopia Eletrônica de Transmissão , Suínos , Doenças dos Suínos , Proteínas do Envelope Viral/metabolismo , eIF-2 Quinase/metabolismo
20.
J Exp Clin Cancer Res ; 38(1): 116, 2019 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-30845964

RESUMO

BACKGROUND: Angiotensin II (ANGII) and its receptor (AGTR1) have been proposed as significant contributors to metastasis in multiple cancers. Further, high AGTR1 levels are associated with poor epithelial ovarian cancer (EOC) outcomes. However, the mechanistic basis for these effects is unknown. Recent studies have suggested that ovarian cancer metastasis is highly dependent on the formation of multicellular spheroids (MCS). To understand the associations between the ANGII/AGTR1 pathway and cancer outcomes, we evaluated the effects of ANGII on MCS formation by ovarian cancer cells and used a proteomic approach to analyze the mechanistic basis. METHODS: We used the data from the GENT database and immunohistochemistry staining to assess the AGTR1 expression in epithelial ovarian cancer (EOC) patients and to assess its role in cancer progression. Colony formation assay, 3D culture assay, and transwell assays were used to analyze the effect of ANGII on the MCS formation and cell migration. The signaling pathways of AGTR1 and transactivation of epidermal growth factor receptor (EGFR) transactivation were investigated by the western blotting analysis. Xenograft models were used to determine the role of AGTR1 in ovarian cancer metastasis. ANGII release from ovarian cancer cells and ANGII levels in the EOC ascites fluid were measured by immunoassay. A shotgun proteomic approach was used to explore the detail molecular mechanism. Modulation of lipid desaturation and endoplasmic reticulum stress were verified by the in vitro and in vivo functional assays. RESULTS: AGTR1 expression was negatively correlated with EOC prognosis. AGTR1activation significantly enhanced the MCS formation and cell migration. ANGII triggered both of the classical AGTR1 pathway and the EGFR transactivation. ANGII administration increased peritoneal metastasis. In addition, ovarian cancer cells secreted ANGII and enhanced cancer metastasis in a positive feedback manner. Based on the proteomic data, lipid desaturation was activated by induction of stearoyl-CoA desaturase-1 (SCD1), which suggests that inhibition of SCD1 may significantly reduce MCS formation by increasing endoplasmic reticulum stress. CONCLUSIONS: ANGII promotes MCS formation and peritoneal metastasis of EOC cells. AGTR1 activation increases the lipid desaturation via SCD1 upregulation, which ultimately reduces endoplasmic reticulum stress in MCS. This mechanism explained the association between high levels of AGTR1 and poor clinical outcomes in EOC patients.


Assuntos
Carcinoma Epitelial do Ovário/genética , Neoplasias Peritoneais/genética , Receptor Tipo 1 de Angiotensina/genética , Estearoil-CoA Dessaturase/genética , Angiotensina II/genética , Angiotensina II/metabolismo , Animais , Carcinoma Epitelial do Ovário/patologia , Movimento Celular/genética , Estresse do Retículo Endoplasmático/genética , Receptores ErbB/genética , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Metabolismo dos Lipídeos/genética , Lipídeos/genética , Camundongos , Metástase Neoplásica , Neoplasias Peritoneais/patologia , Neoplasias Peritoneais/secundário , Prognóstico , Proteômica , Esferoides Celulares/metabolismo , Esferoides Celulares/patologia , Ensaios Antitumorais Modelo de Xenoenxerto
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