RESUMO
Selective autophagy mediates the removal of harmful material from the cytoplasm. This cargo material is selected by cargo receptors, which orchestrate its sequestration within double-membrane autophagosomes and subsequent lysosomal degradation. The cargo receptor p62/SQSTM1 is present in cytoplasmic condensates, and a fraction of them are constantly delivered into lysosomes. However, the molecular composition of the p62 condensates is incompletely understood. To obtain insights into their composition, we develop a method to isolate these condensates and find that p62 condensates are enriched in components of the translation machinery. Furthermore, p62 interacts with translation initiation factors, and eukaryotic initiation factor 2α (eIF2α) and eIF4E are degraded by autophagy in a p62-dependent manner. Thus, p62-mediated autophagy may in part be linked to down-regulation of translation initiation. The p62 condensate isolation protocol developed here may facilitate the study of their contribution to cellular quality control and their roles in health and disease.
Assuntos
Condensados Biomoleculares , Fator de Iniciação 2 em Eucariotos , Fator de Iniciação 4E em Eucariotos , Proteínas de Ligação a RNA , Humanos , Células HEK293 , Proteínas de Ligação a RNA/metabolismo , Condensados Biomoleculares/efeitos dos fármacos , Condensados Biomoleculares/metabolismo , Fator de Iniciação 2 em Eucariotos/antagonistas & inibidores , Fator de Iniciação 2 em Eucariotos/metabolismo , Fator de Iniciação 4E em Eucariotos/antagonistas & inibidores , Fator de Iniciação 4E em Eucariotos/metabolismo , Autofagia/efeitos dos fármacos , Autofagia/genética , Wortmanina/farmacologiaRESUMO
The ability to modulate direct MHC class I (MHC I) Ag presentation is a desirable goal for the treatment of a variety of conditions, including autoimmune diseases, chronic viral infections, and cancers. It is therefore necessary to understand how changes in the cellular environment alter the cells' ability to present peptides to T cells. The unfolded protein response (UPR) is a signaling pathway activated by the presence of excess unfolded proteins in the endoplasmic reticulum. Previous studies have indicated that chemical induction of the UPR decreases direct MHC I Ag presentation, but the precise mechanisms are unknown. In this study, we used a variety of small molecule modulators of different UPR signaling pathways to query which UPR signaling pathways can alter Ag presentation in both murine and human cells. When signaling through the PERK pathway, and subsequent eIF2α phosphorylation, was blocked by treatment with GSK2656157, MHC I Ag presentation remain unchanged, whereas treatment with salubrinal, which has the opposite effect of GSK2656157, decreases both Ag presentation and overall cell-surface MHC I levels. Treatment with 4µ8C, an inhibitor of the IRE1α UPR activation pathway that blocks splicing of Xbp1 mRNA, also diminished MHC I Ag presentation. However, 4µ8C treatment unexpectedly led to an increase in eIF2α phosphorylation in addition to blocking IRE1α signaling. Given that salubrinal and 4µ8C lead to eIF2α phosphorylation and similar decreases in Ag presentation, we conclude that UPR signaling through PERK, leading to eIF2α phosphorylation, results in a modest decrease in direct MHC I Ag presentation.
Assuntos
Adenina/análogos & derivados , Endorribonucleases/metabolismo , Fator de Iniciação 2 em Eucariotos/metabolismo , Indóis/farmacologia , Proteínas Serina-Treonina Quinases/metabolismo , Resposta a Proteínas não Dobradas , Adenina/farmacologia , Animais , Apresentação de Antígeno/efeitos dos fármacos , Linhagem Celular , Cinamatos/farmacologia , Fator de Iniciação 2 em Eucariotos/antagonistas & inibidores , Humanos , Himecromona/análogos & derivados , Himecromona/farmacologia , Camundongos , Fosforilação , RNA Mensageiro/genética , Transdução de Sinais/efeitos dos fármacos , Tioureia/análogos & derivados , Tioureia/farmacologia , Proteína 1 de Ligação a X-Box/metabolismoRESUMO
Eukaryotic cells, when exposed to environmental or internal stress, activate the integrated stress response (ISR) to restore homeostasis and promote cell survival. Specific stress stimuli prompt dedicated stress kinases to phosphorylate eukaryotic initiation factor 2 (eIF2). Phosphorylated eIF2 (p-eIF2) in turn sequesters the eIF2-specific guanine exchange factor eIF2B to block eIF2 recycling, thereby halting translation initiation and reducing global protein synthesis. To circumvent stress-induced translational shutdown, viruses encode ISR antagonists. Those identified so far prevent or reverse eIF2 phosphorylation. We now describe two viral proteins-one from a coronavirus and the other from a picornavirus-that have independently acquired the ability to counteract the ISR at its very core by acting as a competitive inhibitor of p-eIF2-eIF2B interaction. This allows continued formation of the eIF2-GTP-Met-tRNAi ternary complex and unabated global translation at high p-eIF2 levels that would otherwise cause translational arrest. We conclude that eIF2 and p-eIF2 differ in their interaction with eIF2B to such effect that p-eIF2-eIF2B association can be selectively inhibited.
Assuntos
Fator de Iniciação 2B em Eucariotos/antagonistas & inibidores , Fator de Iniciação 2 em Eucariotos/antagonistas & inibidores , Estresse Fisiológico/fisiologia , Proteínas Virais/metabolismo , Animais , Sítios de Ligação , Chlorocebus aethiops , Células Eucarióticas/metabolismo , Fator de Iniciação 2 em Eucariotos/metabolismo , Fator de Iniciação 2B em Eucariotos/metabolismo , Técnicas de Inativação de Genes , Células HEK293 , Células HeLa , Humanos , Fosforilação , Picornaviridae/metabolismo , Ligação Proteica , Células VeroRESUMO
Eukaryotic initiation factor 2 (eIF2α) pathway is overactivated in Alzheimer disease and is probably associated with synaptic and memory deficiencies. EIF2α protein is principally in charge of the regulation of protein synthesis in eukaryotic cells. Four kinases responsible for eIF2α phosphorylation at ser-51 are: General control non-derepressible-2 kinase (GCN2), double-stranded RNA-activated protein kinase (PKR), PKR-like endoplasmic reticulum kinase (PERK), and heme-regulated inhibitor kinase (HRI) are the four kinases. They lead to reduced levels of general translation and paradoxical increase of stress-responsive mRNAs expression including the B-secretase (BACE1) and the transcriptional modulator activating transcription factor 4 (ATF4), which in turn accelerates the beta-amyloidogenesis, tau phosphorylation, proapoptotic pathway induction and autophagy elements formation leading to the main pathological hallmarks of AD. Findings suggest that genetic or pharmacological inhibition of correspondent kinases can restore memory and prevent neurodegeneration. This implies that inhibition of eIF2α phosphorylation through respondent kinases is indeed a feasible prospect of clinical application. This review discusses recent therapeutic approaches targeting eIF2α pathway and provides an overview of the links between correspondent kinases overactivation with neurodegeneration in AD.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/enzimologia , Fator de Iniciação 2 em Eucariotos/antagonistas & inibidores , Fator de Iniciação 2 em Eucariotos/metabolismo , Animais , Humanos , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/química , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , eIF-2 Quinase/antagonistas & inibidores , eIF-2 Quinase/metabolismoRESUMO
OBJECTIVES: Protein kinase R-like ER kinase (PERK)/eukaryotic initiation factor 2 alpha (eIF2α) is an important factor along the main pathways for endoplasmic reticulum (ER) stress-mediated apoptosis. In this study, we investigated the effects of eIF2α phosphorylation on hepatocyte apoptosis and the ER stress mechanisms in acute liver injury. METHODS: eIF2α phosphorylation and apoptosis under ER stress were monitored and measured in male BALB/c mice with acute liver injury and human hepatocyte line LO2 cells. RESULTS: Carbon tetrachloride (CCl4) administration triggered ER stress and hepatocyte apoptosis, as well as eIF2α phosphorylation in mice. Inhibition of eIF2α dephosphorylation, as the pretreatment with 4-phenylbutyric acid (chemical chaperone, ER stress inhibitor), mitigated CCl4-induced intrahepatic ER stress, apoptosis, and liver injury. In an ER stress model of LO2 cells induced by thapsigargin (disrupting ER calcium balance), inhibition of eIF2α dephosphorylation reduced ER stress and apoptosis, while PERK knockdown reduced eIF2α phosphorylation and exacerbated ER stress and apoptosis. CONCLUSIONS: eIF2α phosphorylation is one of the mechanisms employed by ER stress for restoring cellular homeostasis. Inhibition of eIF2α dephosphorylation mitigates hepatocyte apoptosis by alleviating ER stress in acute liver injuries.
Assuntos
Apoptose , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Estresse do Retículo Endoplasmático , Fator de Iniciação 2 em Eucariotos , Hepatócitos/metabolismo , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Tetracloreto de Carbono/efeitos adversos , Linhagem Celular , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Estresse do Retículo Endoplasmático/fisiologia , Fator de Iniciação 2 em Eucariotos/antagonistas & inibidores , Fator de Iniciação 2 em Eucariotos/metabolismo , Humanos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Fosforilação , eIF-2 Quinase/metabolismoRESUMO
Protein quality control is essential for the proper function of cells and the organisms that they make up. The resulting loss of proteostasis, the processes by which the health of the cell's proteins is monitored and maintained at homeostasis, is associated with a wide range of age-related human diseases. Here, we highlight how the integrated stress response (ISR), a central signaling network that responds to proteostasis defects by tuning protein synthesis rates, impedes the formation of long-term memory. In addition, we address how dysregulated ISR signaling contributes to the pathogenesis of complex diseases, including cognitive disorders, neurodegeneration, cancer, diabetes, and metabolic disorders. The development of tools through which the ISR can be modulated promises to uncover new avenues to diminish pathologies resulting from it for clinical benefit.
Assuntos
Fator de Iniciação 2 em Eucariotos/metabolismo , Proteostase , Estresse Fisiológico , Fatores de Complexo Ternário/metabolismo , Acetamidas/química , Acetamidas/farmacologia , Animais , Cicloexilaminas/química , Cicloexilaminas/farmacologia , Fator de Iniciação 2 em Eucariotos/antagonistas & inibidores , Humanos , Imunidade , Doenças Metabólicas/metabolismo , Camundongos , Neoplasias/metabolismo , Fosfotransferases/metabolismoRESUMO
Heme-regulated inhibitor (HRI), a eukaryotic translation initiation factor 2 alpha (eIF2α) kinase, is critically important for coupling protein synthesis to heme availability in reticulocytes and adaptation to various environmental stressors in all cells. HRI modifies the severity of several hemoglobin misfolding disorders including ß-thalassemia. Small molecule activators of HRI are essential for studying normal- and patho-biology of this kinase as well as for the treatment of various human disorders for which activation of HRI or phosphorylation of eIF2α may be beneficial. We previously reported development of 1-((1,4-trans)-4-aryloxycyclohexyl)-3-arylureas (cHAUs) as specific HRI activators and demonstrated their potential as molecular probes for studying HRI biology and as lead compounds for treatment of various human disorders. To develop more druglike cHAUs for in vivo studies and drug development and to expand the chemical space, we undertook bioassay guided structure-activity relationship studies replacing cyclohexyl ring with various 4-6-membered rings and explored further substitutions on the N-phenyl ring. We tested all analogs in the surrogate eIF2α phosphorylation and cell proliferation assays, and a subset of analogs in secondary mechanistic assays that included endogenous eIF2α phosphorylation and expression of C/EBP homologous protein (CHOP), a downstream effector. Finally, we determined specificity of these compounds for HRI by testing their anti-proliferative activity in cells transfected with siRNA targeting HRI or mock. These compounds have significantly improved cLogPs with no loss of potencies, making them excellent candidates for lead optimization for development of investigational new drugs that potently and specifically activate HRI.
Assuntos
Antineoplásicos/farmacologia , Fator de Iniciação 2 em Eucariotos/antagonistas & inibidores , Heme/antagonistas & inibidores , Ureia/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Fator de Iniciação 2 em Eucariotos/metabolismo , Heme/metabolismo , Humanos , Modelos Moleculares , Estrutura Molecular , Fosforilação/efeitos dos fármacos , Relação Estrutura-Atividade , Ureia/análogos & derivados , Ureia/químicaRESUMO
Activation of the integrated stress response (ISR) by a variety of stresses triggers phosphorylation of the α-subunit of translation initiation factor eIF2. P-eIF2α inhibits eIF2B, the guanine nucleotide exchange factor that recycles inactive eIF2â¢GDP to active eIF2â¢GTP. eIF2 phosphorylation thereby represses translation. Persistent activation of the ISR has been linked to the development of several neurological disorders, and modulation of the ISR promises new therapeutic strategies. Recently, a small-molecule ISR inhibitor (ISRIB) was identified that rescues translation in the presence of P-eIF2α by facilitating the assembly of more active eIF2B. ISRIB enhances cognitive memory processes and has therapeutic effects in brain-injured mice without displaying overt side effects. While using ISRIB to investigate the ISR in picornavirus-infected cells, we observed that ISRIB rescued translation early in infection when P-eIF2α levels were low, but not late in infection when P-eIF2α levels were high. By treating cells with varying concentrations of poly(I:C) or arsenite to induce the ISR, we provide additional proof that ISRIB is unable to inhibit the ISR when intracellular P-eIF2α concentrations exceed a critical threshold level. Together, our data demonstrate that the effects of pharmacological activation of eIF2B are tuned by P-eIF2α concentration. Thus, ISRIB can mitigate undesirable outcomes of low-level ISR activation that may manifest neurological disease but leaves the cytoprotective effects of acute ISR activation intact. The insensitivity of cells to ISRIB during acute ISR may explain why ISRIB does not cause overt toxic side effects in vivo.
Assuntos
Acetamidas/química , Acetamidas/farmacologia , Cicloexilaminas/química , Cicloexilaminas/farmacologia , Estresse Fisiológico/efeitos dos fármacos , Animais , Arsenitos/farmacologia , Linhagem Celular , Fator de Iniciação 2 em Eucariotos/antagonistas & inibidores , Fator de Iniciação 2 em Eucariotos/metabolismo , Humanos , Fosforilação , Picornaviridae , Infecções por Picornaviridae/metabolismo , Infecções por Picornaviridae/virologia , Poli I-C/farmacologiaRESUMO
The integrated stress response (ISR) pathway is essential for adaption of various stresses and is related to mitochondrion-to-nucleus communication. Mitochondrial dysfunction-induced reactive oxygen species (ROS) was demonstrated to activate general control nonderepressible 2 (GCN2)â»eukaryotic translation initiation factor 2α (eIF2α)â»activating transcription factor-4 (ATF4) pathway-mediated cisplatin resistance of human gastric cancer cells. However, whether or how ISR activation per se could enhance chemoresistance remains unclear. In this study, we used eIF2α phosphatase inhibitor salubrinal to activate the ISR pathway and found that salubrinal reduced susceptibility to cisplatin. Moreover, salubrinal up-regulated ATF4-modulated gene expression, and knockdown of ATF4 attenuated salubrinal-induced drug resistance, suggesting that ATF4-modulated genes contribute to the process. The ATF4-modulated genes, xCT (a cystine/glutamate anti-transporter), tribbles-related protein 3 (TRB3), heme oxygenase 1 (HO-1), and phosphoenolpyruvate carboxykinase 2 (PCK2), were associated with a poorer prognosis for gastric cancer patients. By silencing individual genes, we found that xCT, but not TRB3, HO-1, or PCK2, is responsible for salubrinal-induced cisplatin resistance. In addition, salubrinal increased intracellular glutathione (GSH) and decreased cisplatin-induced lipid peroxidation. Salubrinal-induced cisplatin resistance was attenuated by inhibition of xCT and GSH biosynthesis. In conclusion, our results suggest that ISR activation by salubrinal up-regulates ATF4-modulated gene expression, increases GSH synthesis, and decreases cisplatin-induced oxidative damage, which contribute to cisplatin resistance in gastric cancer cells.
Assuntos
Sistema y+ de Transporte de Aminoácidos/genética , Antineoplásicos/farmacologia , Cinamatos/farmacologia , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos , Glutationa/metabolismo , Neoplasias Gástricas/tratamento farmacológico , Tioureia/análogos & derivados , Fator 4 Ativador da Transcrição/metabolismo , Linhagem Celular Tumoral , Fator de Iniciação 2 em Eucariotos/antagonistas & inibidores , Fator de Iniciação 2 em Eucariotos/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Tioureia/farmacologia , Regulação para Cima/efeitos dos fármacosRESUMO
The identification of inhibitors of eukaryotic protein biosynthesis, which are targeting single translation factors, is highly demanded. Here we report on a small molecule inhibitor, gephyronic acid, isolated from the myxobacterium Archangium gephyra that inhibits growth of transformed mammalian cell lines in the nM range. In direct comparison, primary human fibroblasts were shown to be less sensitive to toxic effects of gephyronic acid than cancer-derived cells. Gephyronic acid is targeting the protein translation system. Experiments with IRES dual luciferase reporter assays identified it as an inhibitor of the translation initiation. DARTs approaches, co-localization studies and pull-down assays indicate that the binding partner could be the eukaryotic initiation factor 2 subunit alpha (eIF2α). Gephyronic acid seems to have a different mode of action than the structurally related polyketides tedanolide, myriaporone, and pederin and is a valuable tool for investigating the eukaryotic translation system. Because cancer derived cells were found to be especially sensitive, gephyronic acid could potentially find use as a drug candidate.
Assuntos
Fator de Iniciação 2 em Eucariotos/antagonistas & inibidores , Myxococcales/efeitos dos fármacos , Biossíntese de Proteínas/efeitos dos fármacos , Ácidos Graxos Monoinsaturados/farmacologia , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Técnicas Microbiológicas , Myxococcales/genética , Myxococcales/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacosRESUMO
The aggressive nature and inherent therapeutic resistance of glioblastoma multiforme (GBM) has rendered the median survival of afflicted patients to 14 months. Therefore, it is imperative to understand the molecular biology of GBM to provide new treatment options to overcome this disease. It has been demonstrated that the protein kinase R-like endoplasmic reticulum kinase (PERK) pathway is an important regulator of the endoplasmic reticulum (ER) stress response. PERK signaling has been observed in other model systems after radiation; however, less is known in the context of GBM, which is frequently treated with radiation-based therapies. To investigate the significance of PERK, we studied activation of the PERK-eIF2α-ATF4 pathway in GBM after ionizing radiation (IR). By inhibiting PERK, it was determined that ionizing radiation (IR)-induced PERK activity led to eIF2α phosphorylation. IR enhanced the prodeath component of PERK signaling in cells treated with Sal003, an inhibitor of phospho-eIF2α phosphatase. Mechanistically, ATF4 mediated the prosurvival activity during the radiation response. The data support the notion that induction of ER stress signaling by radiation contributes to adaptive survival mechanisms during radiotherapy. The data also support a potential role for the PERK/eIF2α/ATF4 axis in modulating cell viability in irradiated GBM.Implications: The dual function of PERK as a mediator of survival and death may be exploited to enhance the efficacy of radiation therapy.Visual Overview: http://mcr.aacrjournals.org/content/16/10/1447/F1.large.jpg Mol Cancer Res; 16(10); 1447-53. ©2018 AACR.
Assuntos
Fator 4 Ativador da Transcrição/genética , Fator de Iniciação 2 em Eucariotos/genética , Glioblastoma/radioterapia , Tolerância a Radiação/genética , eIF-2 Quinase/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos da radiação , Retículo Endoplasmático/efeitos da radiação , Estresse do Retículo Endoplasmático/genética , Fator de Iniciação 2 em Eucariotos/antagonistas & inibidores , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Glioblastoma/genética , Glioblastoma/patologia , Humanos , Lentivirus/genética , Fosforilação/efeitos da radiação , Radiação Ionizante , Transdução de Sinais/efeitos da radiação , TransfecçãoRESUMO
Cholangiocarcinoma (CCA) is a highly malignant and aggressive tumor of the bile duct that arises from epithelial cells. Chemotherapy is an important treatment strategy for CCA patients, but its efficacy remains limited due to drug resistance. Salubrinal, an inhibitor of eukaryotic translation initiation factor 2 alpha (eIF2α), has been reported to affect antitumor activities in cancer chemotherapy. In this study, the authors investigated the effect of salubrinal on the chemosensitivity of doxorubicin in CCA cells. They showed that doxorubicin induces CCA cell death in a dose- and time-dependent manner. Doxorubicin triggers reactive oxygen species (ROS) generation and induces DNA damage in CCA cells. In addition, ROS inhibitor N-acetylcysteine (NAC) pretreatment inhibits doxorubicin-induced CCA cell death. Importantly, these data demonstrate a synergistic death induction effect contributed by the combination of salubrinal and doxorubicin in CCA cells. It is notable that salubrinal promotes doxorubicin-induced ROS production and DNA damage in CCA cells. Taken together, these data suggest that salubrinal enhances the sensitivity of doxorubicin in CCA cells through promoting ROS-mediated DNA damage.
Assuntos
Antineoplásicos/farmacologia , Neoplasias dos Ductos Biliares/tratamento farmacológico , Colangiocarcinoma/tratamento farmacológico , Cinamatos/farmacologia , Dano ao DNA/efeitos dos fármacos , Tioureia/análogos & derivados , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Neoplasias dos Ductos Biliares/genética , Neoplasias dos Ductos Biliares/patologia , Ductos Biliares Intra-Hepáticos/patologia , Linhagem Celular Tumoral , Colangiocarcinoma/genética , Colangiocarcinoma/patologia , Cinamatos/uso terapêutico , Doxorrubicina/farmacologia , Doxorrubicina/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Sinergismo Farmacológico , Fator de Iniciação 2 em Eucariotos/antagonistas & inibidores , Humanos , Espécies Reativas de Oxigênio/metabolismo , Tioureia/farmacologia , Tioureia/uso terapêuticoRESUMO
Paraquat (PQ), as one of the most widely used herbicides in the world, can cause severe lung damage in humans and animals. This study investigated the underlying molecular mechanism of PQ-induced lung cell damage and the protective role of salubrinal. Human lung epithelial-like A549 cells were treated with PQ for 24h and were pre-incubated with salubrinal for 2h, followed by 500µM of PQ treatment. Silencing eIF2α gene of the A549 cells with siRNA interference method was conducted. Cell morphology, cell viability, apoptosis and caspase-3 activity were assessed by different assays accordingly thereafter. The expression of PERK, p-PERK, ATF6, c-ATF6, IRE1α, p-IRE1α, CHOP, GRP78, p-eIF2α and ß-actin was assayed by western blot. The data showed that PQ significantly reduced A549 cell viability, changed cell morphology, induced cell apoptosis and significantly upregulated the levels of GRP78, CHOP, p-PERK, c-ATF6 and p-IRE1α. However, 30µM salubrinal could attenuate the effects of PQ on damages to A549 cells through upregulating p-eIF2α. In contrast, knocking down eIF2α gene inhabited the effects of salubrinal. These results suggest that PQ-induced A549 cell apoptosis involved endoplasmic reticulum (ER) stress, specially the PERK-eIF2α pathway. Salubrinal attenuated A549 cells from PQ-induced damages through regulation of the PERK-eIF2α signaling.
Assuntos
Cinamatos/farmacologia , Células Epiteliais/efeitos dos fármacos , Fator de Iniciação 2 em Eucariotos/antagonistas & inibidores , Pulmão/citologia , Paraquat/toxicidade , Tioureia/análogos & derivados , eIF-2 Quinase/metabolismo , Células A549 , Chaperona BiP do Retículo Endoplasmático , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Transdução de Sinais/efeitos dos fármacos , Tioureia/farmacologiaRESUMO
Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in PKD1 or PKD2 which encodes polycystin-1 (PC1) and polycystin-2, respectively. PC1 was previously shown to slow cell proliferation and inhibit apoptosis but the underlying mechanisms remain elusive or controversial. Here we showed in cultured mammalian cells and Pkd1 knockout mouse kidney epithelial cells that PC1 and its truncation mutant comprising the last five transmembrane segments and the intracellular C-terminus (PC1-5TMC) down-regulate the phosphorylation of protein kinase R (PKR) and its substrate eukaryotic translation initiation factor 2 alpha (eIF2α). PKR is known to be activated by interferons and dsRNAs, inhibits protein synthesis and induces apoptosis. By co-immunoprecipitation experiments we found that PC1 truncation mutants associate with PKR, or with PKR and its activator PACT. Further experiments showed that PC1 and PC1-5TMC reduce phosphorylation of eIF2α through inhibiting PKR phosphorylation. Our TUNEL experiments using tunicamycin, an apoptosis inducer, and GADD34, an inhibitor of eIF2α phosphorylation, demonstrated that PC1-5TMC inhibits apoptosis of HEK293T cells in a PKR-eIF2α-dependent manner, with concurrent up- and down-regulation of Bcl-2 and Bax, respectively, revealed by Western blotting. Involvement of PC1-regulated eIF2α phosphorylation and a PKR-eIF2α pathway in cell apoptosis may be an important part of the mechanism underlying ADPKD pathogenesis.
Assuntos
Apoptose , Fator de Iniciação 2 em Eucariotos/metabolismo , Transdução de Sinais , Canais de Cátion TRPP/metabolismo , eIF-2 Quinase/metabolismo , Animais , Apoptose/genética , Fator de Iniciação 2 em Eucariotos/antagonistas & inibidores , Células HEK293 , Células HeLa , Humanos , Rim/metabolismo , Camundongos , Fosforilação , Rim Policístico Autossômico Dominante/genética , Rim Policístico Autossômico Dominante/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , eIF-2 Quinase/genéticaRESUMO
Recently we described a novel phenomenon observed during eukaryotic translation in a cell-free system: the coupling of initiation and termination on different mRNA molecules. Here we show that the phenomenon does not depend on a special mode of initiation. The mRNAs with certain leader sequences known to require different determinants for successful initiation were examined. Even in a case of using the intergenic internal ribosome entry site (IRES) of cricket paralysis virus RNA as the leader sequence, while no initiation factors are required, the effect of coupling is well expressed, including trials in the presence of hippuristanol as an inhibitor of eIF4A. Thus, the effect persists in the absence of scanning and does not depend on initiator tRNA and eIF2. The results suggest that the initiation factors are not involved in the coupling mechanism.
Assuntos
RNA Mensageiro/metabolismo , Regiões 5' não Traduzidas , Sistema Livre de Células/metabolismo , Fator de Iniciação 2 em Eucariotos/antagonistas & inibidores , Fator de Iniciação 2 em Eucariotos/metabolismo , Fator de Iniciação 4A em Eucariotos/antagonistas & inibidores , Fator de Iniciação 4A em Eucariotos/metabolismo , Genes Reporter , Sítios Internos de Entrada Ribossomal/genética , Iniciação Traducional da Cadeia Peptídica , Terminação Traducional da Cadeia Peptídica , Plasmídeos/metabolismo , Esteróis/química , Esteróis/metabolismoRESUMO
No therapies exist to prevent neuronal deficits in multiple sclerosis (MS), because the molecular mechanism responsible for the progressive neurodegeneration is unknown. We previously showed that axon injury-induced neuronal endoplasmic reticulum (ER) stress plays an important role in retinal ganglion cell (RGC) death and optic nerve degeneration in traumatic and glaucomatous optic neuropathies. Optic neuritis, one of the most common clinical manifestations of MS, is readily modeled by experimental autoimmune encephalomyelitis (EAE) in mouse. Using this in vivo model, we now show that ER stress is induced early in EAE and that modulation of ER stress by inhibition of eIF2α-CHOP and activation of XBP-1 in RGC specifically, protects RGC somata and axons and preserves visual function. This finding adds to the evidence that ER stress is a general upstream mechanism for neurodegeneration and suggests that targeting ER stress molecules is a promising therapeutic strategy for neuroprotection in MS.
Assuntos
Encefalomielite Autoimune Experimental/prevenção & controle , Fator de Iniciação 2 em Eucariotos/metabolismo , Esclerose Múltipla/prevenção & controle , Neuroproteção , Neurite Óptica/prevenção & controle , Fator de Transcrição CHOP/metabolismo , Proteína 1 de Ligação a X-Box/metabolismo , Animais , Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/metabolismo , Fator de Iniciação 2 em Eucariotos/antagonistas & inibidores , Fator de Iniciação 2 em Eucariotos/genética , Camundongos , Camundongos Knockout , Esclerose Múltipla/genética , Esclerose Múltipla/metabolismo , Neurite Óptica/genética , Neurite Óptica/metabolismo , Fator de Transcrição CHOP/antagonistas & inibidores , Fator de Transcrição CHOP/genética , Proteína 1 de Ligação a X-Box/genéticaRESUMO
Heme-regulated inhibitor (HRI), an eukaryotic translation initiation factor 2 alpha (eIF2α) kinase, plays critical roles in cell proliferation, differentiation, adaptation to stress, and hemoglobin disorders. HRI phosphorylates eIF2α, which couples cellular signals, including endoplasmic reticulum (ER) stress, to translation. We previously identified 1,3-diarylureas and 1-((1,4-trans)-4-aryloxycyclohexyl)-3-arylureas (cHAUs) as specific activators of HRI that trigger the eIF2α phosphorylation arm of ER stress response as molecular probes for studying HRI biology and its potential as a druggable target. To develop drug-like cHAUs needed for in vivo studies, we undertook bioassay-guided structure-activity relationship studies and tested them in the surrogate eIF2α phosphorylation and cell proliferation assays. We further evaluated some of these cHAUs in endogenous eIF2α phosphorylation and in the expression of the transcription factor C/EBP homologous protein (CHOP) and its mRNA, demonstrating significantly improved solubility and/or potencies. These cHAUs are excellent candidates for lead optimization for development of investigational new drugs that potently and specifically activate HRI.
Assuntos
Antineoplásicos/farmacologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Fator de Iniciação 2 em Eucariotos/antagonistas & inibidores , Fosforilação/efeitos dos fármacos , Neoplasias Cutâneas/tratamento farmacológico , Ureia/farmacologia , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Fator de Iniciação 2 em Eucariotos/metabolismo , Humanos , Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/patologia , Camundongos , Estrutura Molecular , Neoplasias Cutâneas/patologia , Relação Estrutura-Atividade , Ureia/análise , Ureia/químicaRESUMO
Steroidal alkaloids are a class of natural products that occur in several species of the Solanaceae family. In the case of the tomato plant (Lycopersicon esculentum Mill.), tomatine and its aglycone, tomatidine, are the most representative molecules. These steroidal alkaloids have already shown several potentially useful biological activities, from anticancer to anti-inflammatory or antibacterial. In this work, the toxicity of these molecules in neuronal cells, namely in the neuroblastoma cell line SH-SY5Y, was assessed, emphasis being given to the cellular mechanisms underlying the effects observed. The results show that tomatine/tomatidine-induced cell death is caspase- and RIP1 kinase-independent, as cell death is not prevented by the pan-caspase inhibitor Z-VAD.fmk or by RIP1 inhibitor necrostatin-1. Analysis of Ca2+ levels using the fluorescent probe Fura-2/AM indicates that both tomatine and tomatidine have a marked effect upon Ca2+ homeostasis by increasing cytosolic Ca2+, an event that might be associated with their effect upon the endoplasmic reticulum. We show that the toxicity of these molecules require the PERK/eIF2α branch of the unfolded protein response, but not the IRE1α branch. Given the role of the endoplasmic reticulum in proteostasis, the ability of these molecules to inhibit the proteasome was also evaluated. Tomatine was able to inhibit the chymotrypsin-like catalytic core of purified human 20S proteasome, as shown by its ability to prevent degradation of the fluorogenic substrate Suc-Leu-Leu-Val-Tyr-AMC, thus suggesting that interference with proteostasis can be responsible for the toxicity of these steroidal alkaloids. This study is relevant as it sheds a light regarding the toxicity of molecules present in one of the most consumed plants worldwide.
Assuntos
Retículo Endoplasmático/efeitos dos fármacos , Fator de Iniciação 2 em Eucariotos/antagonistas & inibidores , Neurônios/efeitos dos fármacos , Inibidores de Proteassoma/toxicidade , Tomatina/análogos & derivados , Tomatina/toxicidade , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Sinalização do Cálcio/efeitos dos fármacos , Inibidores de Caspase/farmacologia , Caspases/química , Caspases/metabolismo , Linhagem Celular Tumoral , Nucléolo Celular/efeitos dos fármacos , Nucléolo Celular/metabolismo , Tamanho do Núcleo Celular/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Retículo Endoplasmático/enzimologia , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Fator de Iniciação 2 em Eucariotos/metabolismo , Humanos , Solanum lycopersicum/química , Neurônios/citologia , Neurônios/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteína Serina-Treonina Quinases de Interação com Receptores/antagonistas & inibidores , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Tomatina/antagonistas & inibidoresRESUMO
Less is known about the roles of eukaryotic initiation factor alpha (eIF2α) in cholangiocarcinoma (CCA). Here, we report that eIF2α inhibitor salubrinal inhibits the proliferation of human CCA cells. Clinical application of mammalian target of rapamycin (mTOR) inhibitors only has moderate antitumor efficacy. Therefore, combination approaches may be required for effective clinical use of mTOR inhibitors. Here, we investigated the efficacy of the combination of salubrinal and rapamycin in the treatment of CCA. Our data demonstrate a synergistic antitumor effect of the combination of salubrinal and rapamycin against CCA cells. Rapamycin significantly inhibits the proliferation of CCA cells. However, rapamycin initiates a negative feedback activation of Akt. Inhibition of Akt by salubrinal potentiates the efficacy of rapamycin both in vitro and in vivo. Additionally, rapamycin treatment results in the up-regulation of Bcl-xL in a xenograft mouse model. It is notable that salubrinal inhibits rapamycin-induced Bcl-xL up-regulation in vivo. Taken together, our data suggest that salubrinal and rapamycin combination might be a new and effective strategy for the treatment of CCA.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias dos Ductos Biliares/tratamento farmacológico , Colangiocarcinoma/tratamento farmacológico , Cinamatos/farmacologia , Sirolimo/farmacologia , Tioureia/análogos & derivados , Animais , Neoplasias dos Ductos Biliares/metabolismo , Neoplasias dos Ductos Biliares/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Colangiocarcinoma/metabolismo , Colangiocarcinoma/patologia , Sinergismo Farmacológico , Fator de Iniciação 2 em Eucariotos/antagonistas & inibidores , Fator de Iniciação 2 em Eucariotos/metabolismo , Humanos , Masculino , Camundongos Nus , Fosforilação , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/metabolismo , Tioureia/farmacologia , Fatores de Tempo , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto , Proteína bcl-X/metabolismoRESUMO
In mammals, haem degradation to biliverdin (BV) through the action of haem oxygenase (HO) is a critical step in haem metabolism. The malaria parasite converts haem into the chemically inert haemozoin to avoid toxicity. We discovered that the knock-out of HO in P. berghei is lethal; therefore, we investigated the function of biliverdin (BV) and haem in the parasite. Addition of external BV and haem to P. falciparum-infected red blood cell (RBC) cultures delays the progression of parasite development. The search for a BV molecular target within the parasites identified P. falciparum enolase (Pf enolase) as the strongest candidate. Isothermal titration calorimetry using recombinant full-length Plasmodium enolase suggested one binding site for BV. Kinetic assays revealed that BV is a non-competitive inhibitor. We employed molecular modelling studies to predict the new binding site as well as the binding mode of BV to P. falciparum enolase. Furthermore, addition of BV and haem targets the phosphorylation of Plasmodium falciparum eIF2α factor, an eukaryotic initiation factor phosphorylated by eIF2α kinases under stress conditions. We propose that BV targets enolase to reduce parasite glycolysis rates and changes the eIF2α phosphorylation pattern as a molecular mechanism for its action.