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1.
Nat Commun ; 12(1): 1248, 2021 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-33623001

RESUMO

Mutations in human equilibrative nucleoside transporter 3 (ENT3) encoded by SLC29A3 results in anemia and erythroid hypoplasia, suggesting that ENT3 may regulate erythropoiesis. Here, we demonstrate that lysosomal ENT3 transport of taurine-conjugated bile acids (TBA) facilitates TBA chemical chaperone function and alleviates endoplasmic reticulum (ER) stress in expanding mouse hematopoietic stem and progenitor cells (HSPCs). Slc29a3-/- HSPCs accumulate less TBA despite elevated levels of TBA in Slc29a3-/- mouse plasma and have elevated basal ER stress, reactive oxygen species (ROS), and radiation-induced apoptosis. Reintroduction of ENT3 allows for increased accumulation of TBA into HSPCs, which results in TBA-mediated alleviation of ER stress and erythroid apoptosis. Transplanting TBA-preconditioned HSPCs expressing ENT3 into Slc29a3-/- mice increase bone marrow repopulation capacity and erythroid pool size and prevent early mortalities. Together, these findings suggest a putative role for a facilitative lysosomal transporter in the bile acid regulation of ER stress in mouse HSPCs which may have implications in erythroid biology, the treatment of anemia observed in ENT3-mutated human genetic disorders, and nucleoside analog drug therapy.


Assuntos
Ácidos e Sais Biliares/metabolismo , Estresse do Retículo Endoplasmático , Células-Tronco Hematopoéticas/metabolismo , Lisossomos/metabolismo , Animais , Apoptose/efeitos dos fármacos , Ácidos e Sais Biliares/sangue , Transporte Biológico/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Diferenciação Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Células Eritroides/efeitos dos fármacos , Células Eritroides/metabolismo , Transplante de Células-Tronco Hematopoéticas , Concentração de Íons de Hidrogênio , Lisossomos/efeitos dos fármacos , Metabolômica , Camundongos , Proteínas de Transporte de Nucleosídeos/metabolismo , Taurina/metabolismo , Ácido Tauroquenodesoxicólico/farmacologia
2.
Nat Commun ; 12(1): 61, 2021 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-33397928

RESUMO

Coat protein complex I (COP-I) mediates the retrograde transport from the Golgi apparatus to the endoplasmic reticulum (ER). Mutation of the COPA gene, encoding one of the COP-I subunits (α-COP), causes an immune dysregulatory disease known as COPA syndrome. The molecular mechanism by which the impaired retrograde transport results in autoinflammation remains poorly understood. Here we report that STING, an innate immunity protein, is a cargo of the retrograde membrane transport. In the presence of the disease-causative α-COP variants, STING cannot be retrieved back to the ER from the Golgi. The forced Golgi residency of STING results in the cGAS-independent and palmitoylation-dependent activation of the STING downstream signaling pathway. Surf4, a protein that circulates between the ER/ ER-Golgi intermediate compartment/ Golgi, binds STING and α-COP, and mediates the retrograde transport of STING to the ER. The STING/Surf4/α-COP complex is disrupted in the presence of the disease-causative α-COP variant. We also find that the STING ligand cGAMP impairs the formation of the STING/Surf4/α-COP complex. Our results suggest a homeostatic regulation of STING at the resting state by retrograde membrane traffic and provide insights into the pathogenesis of COPA syndrome.


Assuntos
Retículo Endoplasmático/metabolismo , Homeostase , Proteínas de Membrana/metabolismo , Animais , Brefeldina A/farmacologia , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/efeitos dos fármacos , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/ultraestrutura , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/ultraestrutura , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/ultraestrutura , Complexo de Golgi/efeitos dos fármacos , Complexo de Golgi/metabolismo , Complexo de Golgi/ultraestrutura , Células HEK293 , Humanos , Lipoilação , Luciferases/metabolismo , Camundongos , Nucleotidiltransferases/metabolismo , Ligação Proteica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos
3.
Carbohydr Polym ; 255: 117477, 2021 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-33436240

RESUMO

The cell surface and extracellular matrix polysaccharide, heparan sulfate (HS) conveys chemical information to control crucial biological processes. HS chains are synthesized in a non-template driven process mainly in the Golgi apparatus, involving a large number of enzymes capable of subtly modifying its substitution pattern, hence, its interactions and biological effects. Changes in the localization of HS-modifying enzymes throughout the Golgi were found to correlate with changes in the structure of HS, rather than protein expression levels. Following BFA treatment, the HS-modifying enzymes localized preferentially in COPII vesicles and at the trans-Golgi. Shortly after heparin treatment, the HS-modifying enzyme moved from cis to trans-Golgi, which coincided with increased HS sulfation. Finally, it was shown that COPI subunits and Sec24 gene expression changed. Collectively, these findings demonstrate that knowledge of the ER-Golgi dynamics of HS-modifying enzymes via vesicular trafficking is a critical prerequisite for the complete delineation of HS biosynthesis.


Assuntos
Vesículas Revestidas pelo Complexo de Proteína do Envoltório/enzimologia , Retículo Endoplasmático/enzimologia , Complexo de Golgi/enzimologia , Heparitina Sulfato/biossíntese , Transporte Biológico/efeitos dos fármacos , Brefeldina A/farmacologia , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/genética , Membrana Celular/química , Membrana Celular/efeitos dos fármacos , Membrana Celular/enzimologia , Retículo Endoplasmático/química , Retículo Endoplasmático/efeitos dos fármacos , Regulação da Expressão Gênica , Complexo de Golgi/química , Complexo de Golgi/efeitos dos fármacos , Heparina/farmacologia , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/enzimologia , Humanos , Plasmídeos/química , Plasmídeos/metabolismo , Cultura Primária de Células , Transfecção , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo
4.
Chemosphere ; 262: 127878, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33182097

RESUMO

Reactive oxygen species (ROS)-mediated endoplasmic reticulum (ER) stress and mitochondrial dysfunction are known to affect the structural and functional damage in the neural system. Cadmium (Cd) is an environmental contaminant that is widely found in numerous environmental matrices and exhibits potential neurotoxic risk. However, it remains unclear how mitochondrial redox status induces, and whether Cd destabilizes, the ER-mitochondria crosstalk to have a toxic effect on the nervous system. Herein, in our present study, bioinformatics analysis revealed an important role of protein interaction and mitochondrial machinery in brain samples from Alzheimer's disease (AD) patients. Furthermore, we established a neurotoxicity model in vivo and in vitro induced by cadmium chloride (CdCl2). We demonstrated that CdCl2 exposure disrupts the balance in mitochondrial redox represented by enhanced mitochondrial ROS (mitoROS) levels, which enhance mitofusin 2 (Mfn2) S-glutathionylation and interrupt the mitochondria-associated ER membranes (MAMs) for crosstalk between the ER and mitochondria to induce neuronal necroptosis. Mechanistically, it was shown that CdCl2 exposure significantly enhances the mitochondria-associated degradation (MAD) of Mfn2 via S-glutathionylation, which inhibits Mfn2 localization to the MAMs and subsequently leads to the formation of the RIPK1-RIPK3-p-MLKL complex (a key component of the necrosome) at MAMs, to promote neuronal necroptosis. Furthermore, the glutaredoxin 1 (Grx1) catalyzed and Mfn2 overexpression restored S-glu-Mfn2, MAMs perturbation, necrosome formation, and necroptosis in neurons induced by CdCl2 exposure in vitro. Moreover, the intervention with antioxidants to reduce mitochondrial redox, such as N-acetyl-l-cysteine (NAC) and mitochondria-targeted antioxidant Mito-TEMPO, reduced the S-glutathionylation of Mfn2 involved in the antagonism of CdCl2-induced necroptosis and neurotoxicity in vivo and in vitro. Taken together, our results are the first time to demonstrate that S-glutathionylation of Mfn2 promotes neuronal necroptosis via disruption of ER-mitochondria crosstalk in CdCl2-induced neurotoxicity, providing the novel mechanistic insight into how hazardous chemical-induced adverse effects in various organs and tissues could be interpreted by intraorganellar pathways under the control of MAMs components in neurons.


Assuntos
Cádmio/toxicidade , Poluentes Ambientais/toxicidade , Necroptose , Animais , Cádmio/metabolismo , Cloreto de Cádmio/farmacologia , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Humanos , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Neurônios/metabolismo , Oxirredução , Espécies Reativas de Oxigênio/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores
5.
Aquat Toxicol ; 231: 105721, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33373863

RESUMO

Previous studies illustrated that gibel carp F strain displays better lipid mobilization and antioxidant ability and compared to the A strain. We therefore hypothesized that the F strain would exhibit superior defense to cadmium exposure. Comparative studies were conducted between A and F strains using plasma stress biomarkers, histological observations, and analysis of hepatic molecular events to examine exposure to waterborne Cd (11.9 mg L-1) for 48 h and 96 h. Waterborne Cd exposure stimulated stress response and hepatic metallothionein mRNA induction in both gibel carp strains confirming exposure. Antioxidant responses were stimulated to counteract Cd toxicity, suggested by the upregulation of mRNA levels of genes associated with nuclear factor erythroid 2-related factor 2 (nrf2) signaling. Cd exposure induced endoplasmic reticulum (ER) stress, meanwhile, branches of genes in unfolded protein response (UPR) were activated. Slight time-dependent effects were implied by greater ER stress, UPR, and apoptosis signals with the duration of Cd exposure. Genotype-specific effects were identified, revealing that the F strain showed greater stress at 96 h exposure and higher antioxidant response compared to the A strain, as indicated by the mRNA levels of genes in nrf2 signaling. ER stress and UPR were also stronger in the F strain after Cd exposure. In contrast, the A strain showed higher autophagy and apoptosis response compared to the F strain. Collectively, combined autophagy and apoptosis were triggered under ER stress, which might serve as defense strategies in both gibel carp strains. The F strain showed greater antioxidant detoxification response and UPR to mitigate Cd toxicity, whereas excessive ER stress contributed to higher autophagy and apoptosis in the A strain. The present study uncovered the differential regulation and defense strategies in fish strains exposed to metal exposure.


Assuntos
Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Cádmio/toxicidade , Carpas/metabolismo , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Poluentes Químicos da Água/toxicidade , Animais , Antioxidantes/metabolismo , Apoptose/genética , Autofagia/genética , Biomarcadores/sangue , Carpas/genética , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Estresse do Retículo Endoplasmático/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Metalotioneína/genética , Metalotioneína/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacos , Estresse Fisiológico/efeitos dos fármacos , Resposta a Proteínas não Dobradas/efeitos dos fármacos
6.
Arch Biochem Biophys ; 698: 108723, 2021 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-33321111

RESUMO

PURPOSE: Indirubin-3'-monoxime (I3M) induces cell death in many cancer cells; however, whether I3M regulates paraptosis is unclear. The present study aimed to investigate I3M-induced paraptosis. METHODS: We treated various cancer cells with I3M, and measured vacuole formation (a paraptosis marker) and the regulating signaling pathway such as endoplasmic reticulum (ER) stress, reactive oxygen species, and proteasomal dysfunction. RESULTS: We found that I3M induced small vacuole formation in MDA-MB-231 breast cancer cells and transient knockdown of eIF2α and CHOP significantly downregulated vacuolation in the ER and mitochondria, as well as cell death in response to I3M, indicating that I3M-meditaed paraptosis was upregulated by ER stress. Moreover, I3M accumulated ubiquitinylated proteins via proteasome dysfunction, which stimulated ER stress-mediated Ca2+ release. A Ca2+ chelator significantly downregulated vacuolation in the ER and mitochondria as well as cell death, suggesting that Ca2+ was a key regulator in I3M-induced paraptosis. Our results also revealed that Ca2+ finally transited in mitochondria through mitochondrial Ca2+ uniporter (MCU), causing I3M-mediated paraptosis; however, the paraptosis was completely inhibited by, ruthenium red, an MCU inhibitor. CONCLUSION: I3M induced proteasomal dysfunction-mediated ER stress and subsequently promoted Ca2+ release, which was accumulated in the mitochondria via MCU, thus causing paraptosis in MDA-MB-231 breast cancer cells.


Assuntos
Antineoplásicos/farmacologia , Cálcio/metabolismo , Retículo Endoplasmático/efeitos dos fármacos , Indóis/farmacologia , Mitocôndrias/efeitos dos fármacos , Oximas/farmacologia , Morte Celular Regulada/efeitos dos fármacos , Linhagem Celular Tumoral , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Humanos , Complexo de Endopeptidases do Proteassoma/metabolismo , Fator de Transcrição CHOP/metabolismo , Vacúolos/metabolismo
7.
Chem Biol Interact ; 334: 109353, 2021 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-33309543

RESUMO

Perhexiline is a coronary vasodilator for angina treatment that was first developed in the 1960s. Perhexiline enjoyed worldwide success before reports of severe side effects, such as hepatotoxicity and neurotoxicity, caused its withdrawal from most of the markets. The underlying mechanism of the cytotoxicity of perhexiline, however, is not yet well understood. Here we demonstrated that perhexiline induced cellular damage in primary human hepatocytes, HepaRG cells and HepG2 cells. Analysis of gene and protein expression levels of endoplasmic reticulum (ER) stress markers showed that perhexiline caused ER stress in primary human hepatocytes and HepG2 cells. The splicing of XBP1 mRNA, a hallmark of ER stress, was observed upon perhexiline treatment. Using Gluc-Fluc-HepG2 cell line, we demonstrated that protein secretion was impaired upon perhexiline treatment, suggesting functional deficits in ER. Inhibition of ER stress using ER inhibitor 4-PBA or salubrinal attenuated the cytotoxicity of perhexiline. Directly knocking down ATF4 using siRNA also partially rescued HepG2 cells upon perhexiline exposure. In addition, inhibition of ER stress using either inhibitors or siRNA transfection attenuated perhexiline-induced increase in caspase 3/7 activity, indicating that ER stress contributed to perhexiline-induced apoptosis. Moreover, perhexiline treatment resulted in activation of p38 and JNK signaling pathways, two branches of MAPK cascade. Pre-treating HepG2 cells with p38 inhibitor SB239063 attenuated perhexiline-induced apoptosis and cell death. The inhibitor also prevented the activation of CHOP and ATF4. Overall, our study demonstrated that ER stress is one important mechanism underlying the hepatotoxicity of perhexiline, and p38 signaling pathway contributes to this process. Our finding shed light on the role of both ER stress and p38 signaling pathway in drug-induced liver injury.


Assuntos
Estresse do Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Perexilina/farmacologia , Transdução de Sinais/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Fator 4 Ativador da Transcrição/metabolismo , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Doença Hepática Induzida por Substâncias e Drogas/tratamento farmacológico , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Células Hep G2 , Hepatócitos/metabolismo , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Fator de Transcrição CHOP/metabolismo
8.
Nat Commun ; 11(1): 5807, 2020 11 16.
Artigo em Inglês | MEDLINE | ID: mdl-33199780

RESUMO

Chronic nonalcoholic steatohepatitis (NASH) is a metabolic disorder that often leads to liver fibrosis, a condition with limited therapy options. Adiponectin is an adipocytokine that regulates glucose and lipid metabolism via binding to its receptors AdipoR1 and AdipoR2, and AdipoRs signaling is reported to enhance fatty acid oxidation and glucose uptake. Here, we synthesize and report an adiponectin-based agonist JT003, which potently improves insulin resistance in high fat diet induced NASH mice and suppresses hepatic stellate cells (HSCs) activation in CCl4 induced liver fibrosis. Mechanistic studies indicate that JT003 simultaneously stimulates AdipoR1- and AdipoR2- mediated signaling pathways as well as the PI3K-Akt pathway. Moreover, JT003 treatment significantly improves ER-mitochondrial axis function, which contributes to the reduced HSCs activation. Thus, the AdipoR1/AdipoR2 dual agonist improves both NASH and fibrosis in mice models, which provides the pharmacological and biological foundation for developing AdipoRs-based therapeutic agents on liver fibrosis.


Assuntos
Retículo Endoplasmático/metabolismo , Mitocôndrias/metabolismo , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Hepatopatia Gordurosa não Alcoólica/patologia , Receptores de Adiponectina/agonistas , Adenilato Quinase/metabolismo , Alanina Transaminase/sangue , Animais , Tetracloreto de Carbono , Dieta Hiperlipídica , Modelos Animais de Doenças , Retículo Endoplasmático/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Fibrose , Células Hep G2 , Células Estreladas do Fígado/metabolismo , Células Estreladas do Fígado/patologia , Humanos , Resistência à Insulina , Cirrose Hepática/sangue , Cirrose Hepática/induzido quimicamente , Cirrose Hepática/tratamento farmacológico , Cirrose Hepática/patologia , Camundongos Endogâmicos C57BL , Mitocôndrias/efeitos dos fármacos , Hepatopatia Gordurosa não Alcoólica/sangue , Obesidade/sangue , Obesidade/complicações , Obesidade/tratamento farmacológico , PPAR alfa/metabolismo , Peptídeos/química , Peptídeos/farmacologia , Peptídeos/uso terapêutico , Fosfatidilinositol 3-Quinases/metabolismo , Domínios Proteicos , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Adiponectina/metabolismo , Transdução de Sinais , Ganho de Peso/efeitos dos fármacos
9.
Mol Pharmacol ; 98(5): 586-597, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32938721

RESUMO

This study investigated the roles of transient receptor potential (TRP) ankyrin-1 (TRPA1) and TRP vanilloid-3 (TRPV3) in regulating endoplasmic reticulum stress (ERS) and cytotoxicity in human bronchial epithelial cells (HBECs) treated with pneumotoxic wood smoke particulate matter (WSPM) and chemical agonists of each channel. Functions of TRPA1 and TRPV3 in pulmonary epithelial cells remain largely undefined. This study shows that TRPA1 activity localizes to the plasma membrane and endoplasmic reticulum (ER) of cells, whereas TRPV3 resides primarily in the ER. Additionally, treatment of cells using moderately cytotoxic concentrations of pine WSPM, carvacrol, and other TRPA1 agonists caused ERS as a function of both TRPA1 and TRPV3 activities. Specifically, ERS and cytotoxicity were attenuated by TRPA1 inhibition, whereas inhibiting TRPV3 exacerbated ERS and cytotoxicity. Interestingly, after treatment with pine WSPM, TRPA1 transcription was suppressed, whereas TRPV3 was increased. TRPV3 overexpression in HBECs conferred resistance to ERS and an attenuation of ERS-associated cell cycle arrest caused by WSPM and multiple prototypical ERS-inducing agents. Alternatively, short hairpin RNA-mediated knockdown of TRPV3, like the TRPV3 antagonist, exacerbated ERS. This study reveals previously undocumented roles for TRPA1 in promoting pathologic ERS and cytotoxicity elicited by pneumotoxic WSPM and TRPA1 agonists, and a unique role for TRPV3 in fettering pathologic facets of the integrated ERS response. SIGNIFICANCE STATEMENT: These findings provide new insights into how wood smoke particulate matter and other transient receptor potential ankyrin-1 (TRPA1) and transient receptor potential vanilloid-3 (TRPV3) agonists can affect human bronchial epithelial cells and highlight novel physiological and pathophysiological roles for TRPA1 and TRPV3 in these cells.


Assuntos
Estresse do Retículo Endoplasmático/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Pulmão/efeitos dos fármacos , Material Particulado/administração & dosagem , Fumaça/efeitos adversos , Canal de Cátion TRPA1/metabolismo , Canais de Cátion TRPV/metabolismo , Linhagem Celular , Cimenos/efeitos adversos , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Células Epiteliais/metabolismo , Células HEK293 , Humanos , Pulmão/metabolismo , Pinus/efeitos adversos , Canais de Receptores Transientes de Potencial/metabolismo , Madeira/efeitos adversos
10.
Aquat Toxicol ; 228: 105628, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32971353

RESUMO

Cadmium (Cd) is a non-essential metal with no known biological function and a broad range of toxic effects in biological systems. We used whole-transcriptome sequencing (RNA-seq) to study the effects of Cd2+ toxicity in zebrafish liver cells, ZFL. The results of an RNA-Seq analysis of ZFL cells exposed to 5, 10 or 20 µM Cd2+ for 4- or 24-h. The differentially expressed genes affected by Cd2+ were analyzed by using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis to study the regulated pathways. Cd2+ regulated the expression of genes associated with cellular Cu, Zn, and Fe homeostasis, DNA replication leading to cell cycle arrest and apoptosis, and glutathione metabolism. Cd2+ boosted up the amino acid synthesis, possibly to support the glutathione metabolism for tackling the oxidative stress generated from Cd2+. Cd2+ stimulation was similar to heat or xenobiotics, based on the responses from ZFL such as endoplasmic reticulum stress and protein folding. We linked also those finding of gene activations relating to carcinogenesis of Cd. This paper provides a comprehensive analysis of the expression profiles induced by Cd2+ exposure in ZFL cells, as well as useful insights into the specific toxic effects.


Assuntos
Cádmio/toxicidade , Exposição Ambiental/análise , Fígado/citologia , RNA-Seq , Transcriptoma/genética , Peixe-Zebra/genética , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Ontologia Genética , Hepatócitos/efeitos dos fármacos , Íons , Fígado/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Transcriptoma/efeitos dos fármacos , Poluentes Químicos da Água/toxicidade , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
11.
Life Sci ; 261: 118364, 2020 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-32866516

RESUMO

AIMS: Prenatal hypoxia (PH) could affect peripheral vascular tone of the offspring, thus increasing the risk of cardiovascular diseases in adult. However, it's still unknown whether functions of coronary arteries (COA) in adult offspring would be influenced by PH. The present study aimed at effects of PH on vascular tone of COA and its related mechanisms. METHODS: Coronary arteries of adult offspring exposed to hypoxic or normoxic circumstances during gestational day 5 to 21 were collected. Wire myograph system, whole-cell patch clamp technique, IonOptix MyoCam system, PCR, and western blot were used to detect vascular function of adult offspring COA. KEY FINDINGS: PH significantly attenuated serotonin- and phorbol 12, 13-dibutyrate (PDBu)-induced constriction. Iberiotoxin potentiated PDBu-induced constriction and the effect was augmented by PH, however, no significant differences were found in whole-cell BKCa channel currents and its protein expression. Nifedipine inhibited PDBu-mediated constriction and the inhibitory effect was reduced in PH group, and whole-cell calcium channel current was decreased in offspring COA. Besides, PH reduced the capability of calcium release from the endoplasmic reticulum in COA. The phosphorylated PKCß protein expression at Ser660 site, not Thr641 site, was significantly decreased in PH offspring. Chronic hypoxia during pregnancy attenuated PDBu-mediated constriction in offspring COA, presumably through decreased phosphorylated PKCß at serine660 sites and decreased intracellular calcium-related weaker PKC activation. SIGNIFICANCE: The findings provided new information on the influence of prenatal hypoxia on COA, and suggested potential use of PKCß-serine660 for early prevention of coronary heart diseases in developmental origins.


Assuntos
Cálcio/metabolismo , Vasos Coronários/fisiopatologia , Hipóxia/complicações , Espaço Intracelular/metabolismo , Fosfosserina/metabolismo , Efeitos Tardios da Exposição Pré-Natal/fisiopatologia , Proteína Quinase C beta/metabolismo , Vasoconstrição , Animais , Peso Corporal/efeitos dos fármacos , Cafeína/farmacologia , Canais de Cálcio/metabolismo , Vasos Coronários/efeitos dos fármacos , Vasos Coronários/patologia , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Feminino , Miocárdio/patologia , Tamanho do Órgão/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Gravidez , Ratos Sprague-Dawley , Serotonina/metabolismo , Acetato de Tetradecanoilforbol
12.
Phys Rev Lett ; 125(5): 058101, 2020 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-32794890

RESUMO

Diffusion of tracer particles in the cytoplasm of mammalian cells is often anomalous with a marked heterogeneity even within individual particle trajectories. Despite considerable efforts, the mechanisms behind these observations have remained largely elusive. To tackle this problem, we performed extensive single-particle tracking experiments on quantum dots in the cytoplasm of living mammalian cells at varying conditions. Analyses of the trajectories reveal a strong, microtubule-dependent subdiffusion with antipersistent increments and a substantial heterogeneity. Furthermore, particles stochastically switch between different mobility states, most likely due to transient associations with the cytoskeleton-shaken endoplasmic reticulum network. Comparison to simulations highlight that all experimental observations can be fully described by an intermittent fractional Brownian motion, alternating between two states of different mobility.


Assuntos
Citoplasma/metabolismo , Modelos Biológicos , Citoesqueleto de Actina/metabolismo , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Simulação por Computador , Citocalasina D/farmacologia , Citoplasma/efeitos dos fármacos , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Difusão , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Células HeLa , Humanos , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Nocodazol/farmacologia , Pontos Quânticos , Processos Estocásticos , Tiazolidinas/farmacologia
13.
Nat Commun ; 11(1): 2936, 2020 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-32522993

RESUMO

Stress response pathways are critical for cellular homeostasis, promoting survival through adaptive changes in gene expression and metabolism. They play key roles in numerous diseases and are implicated in cancer progression and chemoresistance. However, the underlying mechanisms are only poorly understood. We have employed a multi-omics approach to monitor changes to gene expression after induction of a stress response pathway, the unfolded protein response (UPR), probing in parallel the transcriptome, the proteome, and changes to translation. Stringent filtering reveals the induction of 267 genes, many of which have not previously been implicated in stress response pathways. We experimentally demonstrate that UPR-mediated translational control induces the expression of enzymes involved in a pathway that diverts intermediate metabolites from glycolysis to fuel mitochondrial one-carbon metabolism. Concomitantly, the cells become resistant to the folate-based antimetabolites Methotrexate and Pemetrexed, establishing a direct link between UPR-driven changes to gene expression and resistance to pharmacological treatment.


Assuntos
Antimetabólitos/farmacologia , Ácido Fólico/farmacologia , Regulon/genética , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Resposta a Proteínas não Dobradas/genética , Animais , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Humanos , Metotrexato/farmacologia , Pemetrexede/farmacologia , Proteoma/efeitos dos fármacos , Proteoma/genética , Regulon/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Transcriptoma/efeitos dos fármacos , Transcriptoma/genética
14.
Eur J Pharmacol ; 882: 173288, 2020 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-32561291

RESUMO

In December 2019, many pneumonia cases with unidentified sources appeared in Wuhan, Hubei, China, with clinical symptoms like viral pneumonia. Deep sequencing analysis of samples from lower respiratory tract revealed a novel coronavirus, called 2019 novel coronavirus (2019-nCoV). Currently there is a rapid global spread. World Health Organization declare the disease a pandemic condition. The pathologic source of this disease was a new RNA virus from Coronaviridae family, which was named COVID-19. SARS-CoV-2 entry starts with the binding of the spike glycoprotein expressed on the viral envelope to ACE2 on the alveolar surface followed by clathrin-dependent endocytosis of the SARS-CoV-2 and ACE2 complex. SARS-CoV-2 enters the cells through endocytosis process, which is possibly facilitated, via a pH dependent endosomal cysteine protease cathepsins. Once inside the cells, SARS-CoV-2 exploits the endogenous transcriptional machinery of alveolar cells to replicate and spread through the entire lung. Endosomal acidic pH for SARS-CoV-2 processing and internalization is critical. After entering the cells, it possibly activates or hijack many intracellular pathways in favor of its replication. In the current opinion article, we will explain the possible involvement of unfolded protein response as a cellular stress response to the SARS-CoV-2 infection.


Assuntos
Células Epiteliais Alveolares/efeitos dos fármacos , Infecções por Coronavirus/tratamento farmacológico , Retículo Endoplasmático/efeitos dos fármacos , Ionóforos/farmacologia , Pneumonia Viral/tratamento farmacológico , Células Epiteliais Alveolares/citologia , Células Epiteliais Alveolares/metabolismo , Células Epiteliais Alveolares/virologia , Betacoronavirus/metabolismo , Vesículas Revestidas por Clatrina/efeitos dos fármacos , Vesículas Revestidas por Clatrina/metabolismo , Infecções por Coronavirus/virologia , Endocitose/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Endossomos/efeitos dos fármacos , Endossomos/metabolismo , Humanos , Ionóforos/uso terapêutico , Pandemias , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/virologia , Resposta a Proteínas não Dobradas/efeitos dos fármacos
15.
Chemosphere ; 251: 126466, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32443253

RESUMO

Arsenic is a naturally occurring environmental toxicant. Chronic exposure to arsenic is linked with neurological damage. Although the mechanisms remain to be elucidated, it is currently believed that neural cell apoptosis is one of the underlying mechanisms of arsenic-induced neurotoxicity. Calreticulin (CRT) is a quality control chaperone located in the lumen of the endoplasmic reticulum (ER), which participates in many signaling pathways including apoptosis. However, the role of CRT in apoptosis is controversial. Whether CRT plays a role in arsenite-induced apoptosis and the relationship between CRT and ER stress-mediated apoptosis have not been mentioned before. In this study, we found that CRT expression as well as the cell apoptosis levels increased in a dose dependent manner upon arsenite exposure in HT-22 cells, a mouse hippocampal neural cell line. In addition, arsenite exposure resulted in the up-regulation of ER stress indicator GRP78 and ER stress-related proteins including p-PERK, ATF4, CHOP, calpain2 and cleaved caspases-12, accompanied by the down-regulation of Bcl-2 and up-regulation of Bax and cleaved caspase-3. Silence of CRT remarkably alleviated arsenite-induced apoptosis and reversed the expression of the proteins above. Our findings confirmed the role of CRT in the induction of apoptosis upon arsenite exposure and suggested that CRT mediated the intrinsic apoptotic cell death including both mitochondria-dependent (PERK/ATF4/CHOP/Bcl-2) and independent (calpain2/caspases-12) pathways initiated by ER stress, which we believed to be a previously undocumented property of arsenite-induced apoptosis.


Assuntos
Apoptose/efeitos dos fármacos , Arsenitos/toxicidade , Calreticulina/metabolismo , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Animais , Caspase 3/metabolismo , Linhagem Celular , Regulação para Baixo , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Camundongos , Mitocôndrias/metabolismo , Neurônios/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Transdução de Sinais , Regulação para Cima
16.
Nat Commun ; 11(1): 2444, 2020 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-32415068

RESUMO

The essential role of ORAI1 channels in receptor-evoked Ca2+ signaling is well understood, yet little is known about the physiological activation of the ORAI channel trio natively expressed in all cells. The roles of ORAI2 and ORAI3 have remained obscure. We show that ORAI2 and ORAI3 channels play a critical role in mediating the regenerative Ca2+ oscillations induced by physiological receptor activation, yet ORAI1 is dispensable in generation of oscillations. We reveal that ORAI2 and ORAI3 channels multimerize with ORAI1 to expand the range of sensitivity of receptor-activated Ca2+ signals, reflecting their enhanced basal STIM1-binding and heightened Ca2+-dependent inactivation. This broadened bandwidth of Ca2+ influx is translated by cells into differential activation of NFAT1 and NFAT4 isoforms. Our results uncover a long-sought role for ORAI2 and ORAI3, revealing an intricate control mechanism whereby heteromerization of ORAI channels mediates graded Ca2+ signals that extend the agonist-sensitivity to fine-tune transcriptional control.


Assuntos
Canais de Cálcio Ativados pela Liberação de Cálcio/metabolismo , Sinalização do Cálcio , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Carbacol/farmacologia , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Células HEK293 , Humanos , Modelos Biológicos , Fatores de Transcrição NFATC/metabolismo , Proteína ORAI1/metabolismo , Ligação Proteica/efeitos dos fármacos , Isoformas de Proteínas/metabolismo , Multimerização Proteica/efeitos dos fármacos , Molécula 1 de Interação Estromal/metabolismo , Imagem com Lapso de Tempo
17.
Environ Pollut ; 260: 113873, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32369892

RESUMO

Cadmium (Cd), a heavy metal contaminant, exists in humans and animals throughout life and closely associate with severe hepatotoxicity. Selenium (Se) has been recognized as an effective chemo-protectant of Cd, but the underlying mechanisms remain unclear. The objective of the present study is to illustrate the antagonistic effect of Se against Cd-induced hepatotoxicity. Primary hepatocytes were cultured in the presence of 5 µM Cd, 1 µM Se and the mixture of 1 µM Se and 5 µM Cd for 24 h. Cell viability and morphology, antioxidant status, endoplasmic reticulum (ER) stress response and selenotranscriptome were assessed. It was observed that Se treatment dramatically alleviated Cd-induced hepatocytes death and morphological change. Simultaneously, Se mitigated Cd-induced oxidative stress by reducing ROS production, increasing reduced glutathione (GSH) level and increasing selenoenzyme (glutathione peroxidase, GPX) activity. Cd induced hepatotoxicity via disordering ER-resident selenoproteins transcription and triggering ER stress and unfolded protein response. Supplementary Se evidently relieved hepatocytes injury via modulating ER-resident selenoproteins transcription to inhibit ER stress. Collectively, our findings showed a potential protection of Se against Cd-induced hepatotoxicity via suppressing ER stress response.


Assuntos
Cádmio/toxicidade , Retículo Endoplasmático/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Selênio/farmacologia , Selenoproteínas/biossíntese , Animais , Doença Hepática Induzida por Substâncias e Drogas , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático
18.
Biochim Biophys Acta Proteins Proteom ; 1868(7): 140426, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32272193

RESUMO

Lipotoxicity, an important factor in the pathogenesis of diabetes, leads to defective ß-cell proliferation and increased apoptosis. Glucagon-like peptide-1 (GLP-1) analogs, which are used to treat type 2 diabetes, reduce endoplasmic reticulum stress and inflammation in pancreatic ß-cells and improve their survival. However, their effects on the heat shock response (HSR) have not been elucidated yet. We investigated whether the GLP-1 analog exendin-4 exerts its protective effect by modulating the HSR and mitogen-activated protein kinases (MAPKs) in BTC-6 mouse pancreatic cells under palmitic acid (PA) stress. Expression patterns were analyzed using mass spectrometry, Western blotting, and qRT-PCR in the presence of 250 or 400 µM PA and 100 nM exendin-4. Additionally, we measured MAPK expression and phosphorylation using qRT-PCR and Western blotting, respectively. Upregulation of heat shock protein (HSP), notably HSP72, in the presence of PA, was attenuated by exendin-4. Despite the absence of global effects on the HSR system, exendin-4 attenuated the expression of other non-classical HSPs (GRP94, DNAJA1, and DNAJB6) in the presence of PA. Regarding MAPKs, only extracellular signal-regulated kinase (ERK) phosphorylation was highly increased by exendin-4 in both the presence and absence of PA. Furthermore, exendin-4 significantly alleviated PA-induced cell death, which was further confirmed with proteomics analysis where key cellular functions, including cellular growth, assembly, and organization, were improved by exendin-4 treatment. Thus, our results expand the protective role of GLP-1 analogs to include other cellular mechanisms involved in restoring normal ß-cell homeostasis.


Assuntos
Exenatida/metabolismo , Peptídeo 1 Semelhante ao Glucagon/análogos & derivados , Proteínas de Choque Térmico HSP72/metabolismo , Células Secretoras de Insulina/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Diabetes Mellitus Tipo 2/metabolismo , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Exenatida/farmacologia , Proteínas de Choque Térmico HSP40 , Glicoproteínas de Membrana , Camundongos , Chaperonas Moleculares , Fosforilação , Substâncias Protetoras/farmacologia , Mapas de Interação de Proteínas , Regulação para Cima
19.
Nat Commun ; 11(1): 1780, 2020 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-32286267

RESUMO

A promising new compound class for treating human malaria is the imidazolopiperazines (IZP) class. IZP compounds KAF156 (Ganaplacide) and GNF179 are effective against Plasmodium symptomatic asexual blood-stage infections, and are able to prevent transmission and block infection in animal models. But despite the identification of resistance mechanisms in P. falciparum, the mode of action of IZPs remains unknown. To investigate, we here combine in vitro evolution and genome analysis in Saccharomyces cerevisiae with molecular, metabolomic, and chemogenomic methods in P. falciparum. Our findings reveal that IZP-resistant S. cerevisiae clones carry mutations in genes involved in Endoplasmic Reticulum (ER)-based lipid homeostasis and autophagy. In Plasmodium, IZPs inhibit protein trafficking, block the establishment of new permeation pathways, and cause ER expansion. Our data highlight a mechanism for blocking parasite development that is distinct from those of standard compounds used to treat malaria, and demonstrate the potential of IZPs for studying ER-dependent protein processing.


Assuntos
Antimaláricos/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Concentração Inibidora 50 , Espectrometria de Massas , Proteínas de Protozoários/metabolismo , Pirazóis/farmacologia , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Via Secretória/efeitos dos fármacos
20.
J Med Chem ; 63(8): 4205-4214, 2020 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-32227946

RESUMO

Influenza and dengue viruses present a growing global threat to public health. Both viruses depend on the host endoplasmic reticulum (ER) glycoprotein folding pathway. In 2014, Sadat et al. reported two siblings with a rare genetic defect in ER α-glucosidase I (ER Glu I) who showed resistance to viral infections, identifying ER Glu I as a key antiviral target. Here, we show that a single dose of UV-4B (the hydrochloride salt form of N-(9'-methoxynonyl)-1-deoxynojirimycin; MON-DNJ) capable of inhibiting Glu I in vivo is sufficient to prevent death in mice infected with lethal viral doses, even when treatment is started as late as 48 h post infection. The first crystal structure of mammalian ER Glu I will constitute the basis for the development of potent and selective inhibitors. Targeting ER Glu I with UV-4B-derived compounds may alter treatment paradigms for acute viral disease through development of a single-dose therapeutic regime.


Assuntos
Dengue/prevenção & controle , Retículo Endoplasmático/efeitos dos fármacos , Inibidores de Glicosídeo Hidrolases/administração & dosagem , Influenza Humana/prevenção & controle , alfa-Glucosidases , Animais , Dengue/tratamento farmacológico , Dengue/enzimologia , Vírus da Dengue/efeitos dos fármacos , Vírus da Dengue/enzimologia , Relação Dose-Resposta a Droga , Retículo Endoplasmático/enzimologia , Humanos , Influenza Humana/tratamento farmacológico , Influenza Humana/enzimologia , Camundongos da Linhagem 129 , Camundongos Endogâmicos BALB C , Estrutura Secundária de Proteína , alfa-Glucosidases/metabolismo
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