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1.
Mol Reprod Dev ; 91(4): e23742, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38644727

RESUMO

Preeclampsia (PE) is a common pregnancy complication with a high mortality rate. Abnormally activated endoplasmic reticulum stress (ERS) is believed to be responsible for the destruction of key placental cells-trophoblasts. Phenylbutyric acid (4-PBA), an ERS inhibitor, is involved in regulating the development of ERS-related diseases. At present, how 4-PBA affects trophoblasts and its mechanisms is still unclear. In this study, PE cell models were established by stimulating HTR-8/SVneo cells with hypoxia. To verify the underlying mechanisms of 4-PBA on PE, CCT020312, an activator of PERK, was also used. The results showed that 4-PBA restored hypoxia-induced trophoblast viability, inhibited HIF-1α protein expression, inflammation, and PERK/ATF-4/CHOP pathway. Hoechst 33342 staining and flow cytometry results confirmed that 4-PBA decreased hypoxia-induced apoptosis in trophoblasts. The results of the JC-1 analysis and apoptosis initiation enzyme activity assay also demonstrated that 4-PBA inhibited apoptosis related to the mitochondrial pathway. Furthermore, by detecting autophagy in trophoblasts, an increased number of autophagic vesicles, damaged mitochondria, enhanced dansylcadaverine fluorescence, enhanced levels of autophagy proteins Beclin-1, LC3II, and decreased p62 were seen in hypoxia-stimulated cells. These changes were reversed by 4-PBA. Furthermore, it was observed that CCT020312 reversed the effects of 4-PBA on the viability, apoptosis, and autophagosome number of hypoxia-induced trophoblasts. In summary, 4-PBA reduces autophagy and apoptosis via the PERK/ATF-4/CHOP pathway and mitochondrial pathway, thereby restoring the viability of hypoxic trophoblasts. These findings provide a solid evidence base for the use of 4-PBA in PE treatment and guide a new direction for improving the outcomes of patients with PE.


Assuntos
Fator 4 Ativador da Transcrição , Apoptose , Autofagia , Hipóxia Celular , Fenilbutiratos , Pré-Eclâmpsia , Fator de Transcrição CHOP , Trofoblastos , eIF-2 Quinase , Trofoblastos/efeitos dos fármacos , Trofoblastos/metabolismo , Trofoblastos/patologia , Feminino , Humanos , Pré-Eclâmpsia/metabolismo , Pré-Eclâmpsia/tratamento farmacológico , Pré-Eclâmpsia/patologia , Autofagia/efeitos dos fármacos , Fator de Transcrição CHOP/metabolismo , Apoptose/efeitos dos fármacos , Gravidez , Fenilbutiratos/farmacologia , eIF-2 Quinase/metabolismo , Fator 4 Ativador da Transcrição/metabolismo , Hipóxia Celular/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Linhagem Celular
2.
Cell Death Dis ; 15(4): 276, 2024 Apr 18.
Artigo em Inglês | MEDLINE | ID: mdl-38637497

RESUMO

The Unfolded Protein Response (UPR) is an essential cellular process activated by the accumulation of unfolded proteins within the Endoplasmic Reticulum (ER), a condition referred to as ER stress. Three ER anchored receptors, IRE1, PERK and ATF6 act as ER stress sensors monitoring the health of the ER. Upon detection of ER stress, IRE1, PERK and ATF6 initiate downstream signaling pathways collectively referred to as the UPR. The overarching aim of the UPR is to restore ER homeostasis by reducing ER stress, however if that is not possible, the UPR transitions from a pro-survival to a pro-death response. While our understanding of the key signaling pathways central to the UPR is well defined, the same is not true of the subtle signaling events that help fine tune the UPR, supporting its ability to adapt to varying amplitudes or durations of ER stress. In this study, we demonstrate cross talk between the IRE1 and PERK branches of the UPR, wherein IRE1 via XBP1s signaling helps to sustain PERK expression during prolonged ER stress. Our findings suggest cross talk between UPR branches aids adaptiveness thereby helping to support the plasticity of UPR signaling responses.


Assuntos
Proteínas Serina-Treonina Quinases , eIF-2 Quinase , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , eIF-2 Quinase/genética , eIF-2 Quinase/metabolismo , Estresse do Retículo Endoplasmático/fisiologia , Transdução de Sinais , Resposta a Proteínas não Dobradas
3.
Rom J Morphol Embryol ; 65(1): 27-33, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38527981

RESUMO

Cyclophosphamide (CP) is an alkylating chemotherapeutic agent commonly used in cancer treatments. In this study, we aimed to investigate the effects of 4-Hydroperoxy cyclophosphamide (4-HC), which is active form of CP, on glucose-regulated protein 78 (GRP78), activating transcription factor 6 (ATF6), phospho-protein kinase R (PKR)-like endoplasmic reticulum (ER) kinase (p-PERK), phospho-inositol-requiring enzyme 1 alpha (p-IRE1α), eukaryotic translation initiation factor 2 alpha (eIF2α), and caspase-3 messenger ribonucleic acids (mRNAs) and proteins that play roles in the ER stress pathway and apoptosis in U87 and T98 human glioblastoma cell lines. U87 and T98 human glioblastoma cell lines were divided into control and 4-HC-treated groups. Cell viability assay was used to detect the half maximal inhibitory concentration (IC50) for 24 hours of 4-HC. Immunocytochemistry and quantitative polymerase chain reaction (qPCR) methods were used to evaluate the levels of proteins and their mRNAs. The IC50 values of U87 and T98 cells were calculated as 15.67±0.58 µM and 19.92±1 µM, respectively. The levels of GRP78, ATF6, p-PERK, p-IRE1α, eIF2α, and caspase-3 protein expressions in the 4-HC-treated group compared to that in the control group. These increased protein expressions also were correlated with the mRNA levels. The ER stress signal pathway could be active in 4-HC-induced cell death. Further studies of ER-related stress mechanisms in anticancer treatment would be important for effective therapeutic strategies.


Assuntos
Glioblastoma , Proteínas Serina-Treonina Quinases , Humanos , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/farmacologia , Endorribonucleases/farmacologia , eIF-2 Quinase/genética , eIF-2 Quinase/metabolismo , eIF-2 Quinase/farmacologia , Caspase 3/farmacologia , Chaperona BiP do Retículo Endoplasmático , Estresse do Retículo Endoplasmático , Linhagem Celular , Apoptose , Ciclofosfamida/farmacologia
4.
Ecotoxicol Environ Saf ; 274: 116193, 2024 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-38460407

RESUMO

Chlorocholine chloride (CCC) is a plant growth regulator used worldwide that is detectable in cereals, fruits and animal products. The health effects of CCC exposure have raised public concern. Our previous research showed that CCC exposure decreased testosterone synthesis in pubertal rats. However, little is known about whether and how pubertal CCC exposure impacts spermatogenesis. In this study, we used BALB/c mice and spermatogonia-derived GC-1 cells to examine CCC-induced spermatogenic dysfunction. In vivo, pubertal CCC exposure led to decreased testicular weight, decreased testicular germ cells and poor sperm quality. This effect worsened after cessation of CCC exposure for the next 30 days. RNA-seq and western blot analysis revealed that CCC induced aryl hydrocarbon receptor (AhR) signaling, endoplasmic reticulum stress (ERS) and ferritinophagy. Increased iron content and lipid peroxidation levels were also observed in CCC-treated testes. In vitro, it was identified that iron overload mediated by enhanced ferritinophagy occurred in CCC-treated GC-1 cells, which might be attributed to the PERK pathway in ERS. Further, for the first time, our study elucidated the involvement of AhR in CCC-induced iron overload, which aggravated testicular oxidative damage via lipid peroxidation. Considering the adverse impact of CCC exposure on rodents, supportive evidence from GC-1 cells, and the critical importance of spermatogenesis on male development, the effects of CCC on the male reproduction warrant increased attention.


Assuntos
Acetatos , Clormequat , Sobrecarga de Ferro , Fenóis , Espermatogênese , Animais , Masculino , Camundongos , Ratos , Clormequat/metabolismo , Clormequat/toxicidade , Sobrecarga de Ferro/metabolismo , Receptores de Hidrocarboneto Arílico/genética , Receptores de Hidrocarboneto Arílico/metabolismo , Sementes , Espermatogênese/efeitos dos fármacos , Testículo , eIF-2 Quinase/efeitos dos fármacos , eIF-2 Quinase/metabolismo
5.
J Med Chem ; 67(7): 5259-5271, 2024 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-38530741

RESUMO

A series of activators of GCN2 (general control nonderepressible 2) kinase have been developed, leading to HC-7366, which has entered the clinic as an antitumor therapy. Optimization resulted in improved permeability compared to that of the original indazole hinge binding scaffold, while maintaining potency at GCN2 and selectivity over PERK (protein kinase RNA-like endoplasmic reticulum kinase). The improved ADME properties of this series led to robust in vivo compound exposure in both rats and mice, allowing HC-7366 to be dosed in xenograft models, demonstrating that activation of the GCN2 pathway by this compound leads to tumor growth inhibition.


Assuntos
Proteínas Serina-Treonina Quinases , eIF-2 Quinase , Humanos , Camundongos , Ratos , Animais , Proteínas Serina-Treonina Quinases/metabolismo , eIF-2 Quinase/metabolismo , Camundongos Endogâmicos C57BL , RNA , Retículo Endoplasmático/metabolismo
6.
Biochem Biophys Res Commun ; 706: 149728, 2024 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-38479246

RESUMO

Influenza A virus is the cause of a widespread human disease with high morbidity and mortality rates. The influenza virus encodes non-structural protein 1 (NS1), an exceedingly multifunctional virulence component. NS1 plays essential roles in viral replication and evasion of the cellular innate immune system. Protein kinase RNA-activated also known as protein kinase R (PKR) phosphorylates translation initiation factor eIF-2α on serine 51 to inhibit protein synthesis in virus-infected mammalian cells. Consequently, PKR activation inhibits mRNA translation, which results in the assert of both viral protein synthesis and cellular and possibly apoptosis in response to virus infection. Host signaling pathways are important in the replication of influenza virus, but the mechanisms involved remain to be characterized. Herein, the structure of NS1 and PKR complex was determined using Cryo-EM. We found the N91, E94, and G95 residues of PKR bind directly with N188, D125, and K126, respectively, of NS1. Furthermore, the study shows that PKR peptide offers a potential treatment for Influenza A virus infections.


Assuntos
Vírus da Influenza A , eIF-2 Quinase , Animais , Humanos , eIF-2 Quinase/metabolismo , Proteínas não Estruturais Virais/química , Vírus da Influenza A/genética , Microscopia Crioeletrônica , Linhagem Celular , Antivirais/metabolismo , Replicação Viral , Mamíferos/metabolismo
7.
Curr Biol ; 34(7): 1390-1402.e4, 2024 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-38428416

RESUMO

Collective cell migration is integral to many developmental and disease processes. Previously, we discovered that protein phosphatase 1 (Pp1) promotes border cell collective migration in the Drosophila ovary. We now report that the Pp1 phosphatase regulatory subunit dPPP1R15 is a critical regulator of border cell migration. dPPP1R15 is an ortholog of mammalian PPP1R15 proteins that attenuate the endoplasmic reticulum (ER) stress response. We show that, in collectively migrating border cells, dPPP1R15 phosphatase restrains an active physiological protein kinase R-like ER kinase- (PERK)-eIF2α-activating transcription factor 4 (ATF4) stress pathway. RNAi knockdown of dPPP1R15 blocks border cell delamination from the epithelium and subsequent migration, increases eIF2α phosphorylation, reduces translation, and drives expression of the stress response transcription factor ATF4. We observe similar defects upon overexpression of ATF4 or the eIF2α kinase PERK. Furthermore, we show that normal border cells express markers of the PERK-dependent ER stress response and require PERK and ATF4 for efficient migration. In many other cell types, unresolved ER stress induces initiation of apoptosis. In contrast, border cells with chronic RNAi knockdown of dPPP1R15 survive. Together, our results demonstrate that the PERK-eIF2α-ATF4 pathway, regulated by dPPP1R15 activity, counteracts the physiological ER stress that occurs during collective border cell migration. We propose that in vivo collective cell migration is intrinsically "stressful," requiring tight homeostatic control of the ER stress response for collective cell cohesion, dynamics, and movement.


Assuntos
Transdução de Sinais , eIF-2 Quinase , Animais , eIF-2 Quinase/genética , eIF-2 Quinase/metabolismo , Estresse do Retículo Endoplasmático/fisiologia , Apoptose , Movimento Celular , Monoéster Fosfórico Hidrolases/metabolismo , Fator de Iniciação 2 em Eucariotos/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Mamíferos
8.
Int J Mol Sci ; 25(5)2024 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-38474017

RESUMO

Ubiquitin-specific protease 7 inhibitors (USP7i) are considered a novel class of anticancer drugs. Cancer cells occasionally become insensitive to anticancer drugs, known as chemoresistance, by acquiring multidrug resistance, resulting in poor clinical outcomes in patients with cancer. However, the chemoresistance of cancer cells to USP7i (P22077 and P5091) and mechanisms to overcome it have not yet been investigated. In the present study, we generated human cancer cells with acquired resistance to USP7i-induced cell death. Gene expression profiling showed that heat stress response (HSR)- and unfolded protein response (UPR)-related genes were largely upregulated in USP7i-resistant cancer cells. Biochemical studies showed that USP7i induced the phosphorylation and activation of heat shock transcription factor 1 (HSF1), mediated by the endoplasmic reticulum (ER) stress protein kinase R-like ER kinase (PERK) signaling pathway. Inhibition of HSF1 and PERK significantly sensitized cancer cells to USP7i-induced cytotoxicity. Our study demonstrated that the ER stress-PERK axis is responsible for chemoresistance to USP7i, and inhibiting PERK is a potential strategy for improving the anticancer efficacy of USP7i.


Assuntos
Antineoplásicos , Neoplasias , Humanos , Peptidase 7 Específica de Ubiquitina/genética , eIF-2 Quinase/metabolismo , Estresse do Retículo Endoplasmático , Resposta a Proteínas não Dobradas , Antineoplásicos/farmacologia
9.
PLoS One ; 19(2): e0292190, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38359044

RESUMO

Amyotrophic lateral sclerosis (ALS) has been linked to overactivity of the protein kinase RNA-like ER kinase (PERK) branch of the unfolded protein response (UPR) pathway, both in ALS patients and mouse models. However, attempts to pharmacologically modulate PERK for therapeutic benefit have yielded inconsistent and often conflicting results. This study sought to address these discrepancies by comprehensively evaluating three commonly used, CNS-penetrant, PERK modulators (GSK2606414, salubrinal, and Sephin1) in the same experimental models, with the goal of assessing the viability of targeting the PERK pathway as a therapeutic strategy for ALS. To achieve this goal, a tunicamycin-challenge assay was developed using wild-type mice to monitor changes in liver UPR gene expression in response to PERK pathway modulation. Subsequently, multiple dosing regimens of each PERK modulator were tested in standardized, well-powered, gender-matched, and litter-matched survival efficacy studies using the SOD1G93A mouse model of ALS. The alpha-2-adrenergic receptor agonist clonidine was also tested to elucidate the results obtained from the Sephin1, and of the previously reported guanabenz studies, by comparing the effects of presence or absence of α-2 agonism. The results revealed that targeting PERK may not be an ideal approach for ALS treatment. Inhibiting PERK with GSK2606414 or activating it with salubrinal did not confer therapeutic benefits. While Sephin1 showed some promising therapeutic effects, it appears that these outcomes were mediated through PERK-independent mechanisms. Clonidine also produced some favorable therapeutic effects, which were unexpected and not linked to the UPR. In conclusion, this study highlights the challenges of pharmacologically targeting PERK for therapeutic purposes in the SOD1G93A mouse model and suggests that exploring other targets within, and outside, the UPR may be more promising avenues for ALS treatment.


Assuntos
Adenina/análogos & derivados , Esclerose Amiotrófica Lateral , Cinamatos , Guanabenzo , Guanabenzo/análogos & derivados , Indóis , Tioureia/análogos & derivados , Camundongos , Humanos , Animais , Guanabenzo/farmacologia , Guanabenzo/uso terapêutico , eIF-2 Quinase/genética , eIF-2 Quinase/metabolismo , Esclerose Amiotrófica Lateral/tratamento farmacológico , Esclerose Amiotrófica Lateral/genética , Clonidina , Resposta a Proteínas não Dobradas , Agonistas de Receptores Adrenérgicos alfa 2
10.
Artigo em Chinês | MEDLINE | ID: mdl-38311942

RESUMO

Objective: To investigate the effects of carbon black and cadmium (Cd) combined exposure on autophagy and inflammatory response mediated by protein kinase R-like endoplasmic reticulum kinase (PERK) pathway in human bronchial epithelial (16HBE) cells. Methods: In January 2022, human bronchial epithelial (16HBE) cells were resuscitated and cultured. Carbon black nanoparticles (CBNPs) were oxidized to adsorb Cd ions to construct "CBNPs-Cd" complexes. CCK-8 assay was used to detect the effects of different concentrations and time combinations of CBNPs and Cd on the viability of 16HBE cells. The subsequent dose groups were exposed to 2 µg/ml Cd, 100 µg/ml CBNPs, 100 µg/ml CBNPs+2 µg/ml Cd for 24 h. The number of autophagosomes and autolysosomes was detected by transmission electron microscopy. Western blotting was used to detect the protein expressions of PERK, eukaryotic initiation factor 2α (eIf2α), activating transcription factor 4 (ATF4), sequestosome 1 (SQSTM1/P62), and microtubule-associated protein 1 light chain 3 (LC3). After PERK gene was silenced by siRNA technology, the changes of autophagy marker proteins P62 and LC3 were detected, and the expressions of inflammatory factors interleukin-6 (IL6) and interleukin-8 (IL8) were detected by fluorescence quantitative PCR technique. One-way ANOVA analysis was used to compare three groups or more. LSD test was used for comparison between two groups. Factorial analysis was used for multivariate component analysis. Results: There was no significant change in cell viability of 16HBE after 24 h exposure to CBNPs and Cd alone or combined (P>0.05). Compared with the control group, the expressions of P62 and LC3 in 16HBE cells were significantly increased in the CBNPs and Cd alone/combined exposure group (P<0.05), and the number of autophagosomes and autophagolysosomes in the combined exposure group was increased compared with other groups. Compared with the control group, CBNPs and Cd alone exposure group had no significant effects on p-PERK/PERK and p-eIf2α/eIf2α protein expression (P>0.05). However, the protein expressions of p-PERK/PERK and p-eIf2α/eIf2α and ATF4 were all increased in the combined exposure group (P<0.05), and the levels of IL6 and IL8 in 16HBE cells in the combined exposure group of CBNPs and Cd were significantly higher than those in the control group (P<0.05). The levels of LC3 protein, IL6 and IL8 were decreased in the CBNPs-Cd combined exposure group after knockdown of PERK gene (P<0.05). The results of factorial analysis showed that exposure to CBNPs and Cd had significant effects on the expression of P62, LC3 and IL6 (P<0.05), but the interaction between the two chemicals had no statistical significance (P>0.05) . Conclusion: CBNPs-Cd combined exposure may inhibit autophagy and increase inflammation in human bronchial epithelial cells through activation of PERK-eIf2α-ATF4 pathway.


Assuntos
Cádmio , Fuligem , Humanos , Cádmio/toxicidade , Fuligem/toxicidade , Interleucina-8 , Interleucina-6 , eIF-2 Quinase/genética , eIF-2 Quinase/metabolismo , eIF-2 Quinase/farmacologia , Autofagia , Células Epiteliais/metabolismo , Estresse do Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Inflamação
11.
Pharmacol Ther ; 255: 108604, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38360205

RESUMO

The endoplasmic reticulum (ER) is a cellular organelle that is physiologically responsible for protein folding, calcium homeostasis, and lipid biosynthesis. Pathological stimuli such as oxidative stress, ischemia, disruptions in calcium homeostasis, and increased production of normal and/or folding-defective proteins all contribute to the accumulation of misfolded proteins in the ER, causing ER stress. The adaptive response to ER stress is the activation of unfolded protein response (UPR), which affect a wide variety of cellular functions to maintain ER homeostasis or lead to apoptosis. Three different ER transmembrane sensors, including PKR-like ER kinase (PERK), activating transcription factor 6 (ATF6), and inositol-requiring enzyme-1 (IRE1), are responsible for initiating UPR. The UPR involves a variety of signal transduction pathways that reduce unfolded protein accumulation by boosting ER-resident chaperones, limiting protein translation, and accelerating unfolded protein degradation. ER is now acknowledged as a critical organelle in sensing dangers and determining cell life and death. On the other hand, UPR plays a critical role in the development and progression of several diseases such as cardiovascular diseases (CVD), metabolic disorders, chronic kidney diseases, neurological disorders, and cancer. Here, we critically analyze the most current knowledge of the master regulatory roles of ER stress particularly the PERK pathway as a conditional danger receptor, an organelle crosstalk regulator, and a regulator of protein translation. We highlighted that PERK is not only ER stress regulator by sensing UPR and ER stress but also a frontier sensor and direct senses for gut microbiota-generated metabolites. Our work also further highlighted the function of PERK as a central hub that leads to metabolic reprogramming and epigenetic modification which further enhanced inflammatory response and promoted trained immunity. Moreover, we highlighted the contribution of ER stress and PERK in the pathogenesis of several diseases such as cancer, CVD, kidney diseases, and neurodegenerative disorders. Finally, we discuss the therapeutic target of ER stress and PERK for cancer treatment and the potential novel therapeutic targets for CVD, metabolic disorders, and neurodegenerative disorders. Inhibition of ER stress, by the development of small molecules that target the PERK and UPR, represents a promising therapeutic strategy.


Assuntos
Doenças Cardiovasculares , Microbioma Gastrointestinal , Doenças Metabólicas , Neoplasias , Doenças Neurodegenerativas , Humanos , eIF-2 Quinase/genética , eIF-2 Quinase/metabolismo , Cálcio/metabolismo , Resposta a Proteínas não Dobradas , Estresse do Retículo Endoplasmático , Doenças Neurodegenerativas/tratamento farmacológico , Doença Crônica , Doenças Cardiovasculares/tratamento farmacológico , Imunidade , Alimentos Marinhos , Neoplasias/tratamento farmacológico
12.
Cell Death Dis ; 15(2): 156, 2024 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-38378666

RESUMO

Traumatic brain injury (TBI) is a common trauma with high mortality and disability rates worldwide. However, the current management of this disease is still unsatisfactory. Therefore, it is necessary to investigate the pathophysiological mechanisms of TBI in depth to improve the treatment options. In recent decades, abundant evidence has highlighted the significance of endoplasmic reticulum stress (ERS) in advancing central nervous system (CNS) disorders, including TBI. ERS following TBI leads to the accumulation of unfolded proteins, initiating the unfolded protein response (UPR). Protein kinase RNA-like ER kinase (PERK), inositol-requiring protein 1 (IRE1), and activating transcription factor 6 (ATF6) are the three major pathways of UPR initiation that determine whether a cell survives or dies. This review focuses on the dual effects of ERS on TBI and discusses the underlying mechanisms. It is suggested that ERS may crosstalk with a series of molecular cascade responses, such as mitochondrial dysfunction, oxidative stress, neuroinflammation, autophagy, and cell death, and is thus involved in the progression of secondary injury after TBI. Hence, ERS is a promising candidate for the management of TBI.


Assuntos
Lesões Encefálicas Traumáticas , eIF-2 Quinase , Humanos , eIF-2 Quinase/genética , eIF-2 Quinase/metabolismo , Estresse do Retículo Endoplasmático , Resposta a Proteínas não Dobradas , Autofagia
13.
J Virol ; 98(3): e0188323, 2024 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-38376197

RESUMO

Many viruses, including mammarenaviruses, have evolved mechanisms to counteract different components of the host cell innate immunity, which is required to facilitate robust virus multiplication. The double-stranded RNA (dsRNA) sensor protein kinase receptor (PKR) pathway plays a critical role in the cell anti-viral response. Whether PKR can restrict the multiplication of the Old World mammarenavirus lymphocytic choriomeningitis virus (LCMV) and the mechanisms by which LCMV may counteract the anti-viral functions of PKR have not yet been investigated. Here we present evidence that LCMV infection results in very limited levels of PKR activation, but LCMV multiplication is enhanced in the absence of PKR. In contrast, infection with a recombinant LCMV with a mutation affecting the 3'-5' exonuclease (ExoN) activity of the viral nucleoprotein resulted in robust PKR activation in the absence of detectable levels of dsRNA, which was associated with severely restricted virus multiplication that was alleviated in the absence of PKR. However, pharmacological inhibition of PKR activation resulted in reduced levels of LCMV multiplication. These findings uncovered a complex role of the PKR pathway in LCMV-infected cells involving both pro- and anti-viral activities.IMPORTANCEAs with many other viruses, the prototypic Old World mammarenavirus LCMV can interfere with the host cell innate immune response to infection, which includes the dsRNA sensor PKR pathway. A detailed understanding of LCMV-PKR interactions can provide novel insights about mammarenavirus-host cell interactions and facilitate the development of effective anti-viral strategies against human pathogenic mammarenaviruses. In the present work, we present evidence that LCMV multiplication is enhanced in PKR-deficient cells, but pharmacological inhibition of PKR activation unexpectedly resulted in severely restricted propagation of LCMV. Likewise, we document a robust PKR activation in LCMV-infected cells in the absence of detectable levels of dsRNA. Our findings have revealed a complex role of the PKR pathway during LCMV infection and uncovered the activation of PKR as a druggable target for the development of anti-viral drugs against human pathogenic mammarenaviruses.


Assuntos
Arenaviridae , Coriomeningite Linfocítica , Humanos , Arenaviridae/metabolismo , Linhagem Celular , Proteínas Quinases/metabolismo , Interações Hospedeiro-Patógeno , Vírus da Coriomeningite Linfocítica/metabolismo , Proteínas de Transporte , Antivirais , eIF-2 Quinase/genética , eIF-2 Quinase/metabolismo
14.
Mol Cancer Res ; 22(4): 360-372, 2024 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-38236939

RESUMO

Rapidly proliferating cancer cells require a microenvironment where essential metabolic nutrients like glucose, oxygen, and growth factors become scarce as the tumor volume surpasses the established vascular capacity of the tissue. Limits in nutrient availability typically trigger growth arrest and/or apoptosis to prevent cellular expansion. However, tumor cells frequently co-opt cellular survival pathways thereby favoring cell survival under this environmental stress. The unfolded protein response (UPR) pathway is typically engaged by tumor cells to favor adaptation to stress. PERK, an endoplasmic reticulum (ER) protein kinase and UPR effector is activated in tumor cells and contributes tumor cell adaptation by limiting protein translation and balancing redox stress. PERK also induces miRNAs that contribute to tumor adaptation. miR-211 and miR-216b were previously identified as PERK-ATF4-regulated miRNAs that regulate cell survival. We have identified another PERK-responsive miRNA, miR-217, with increased expression under prolonged ER stress. Key targets of miR-217 are identified as TRPM1, the host gene for miR-211 and EZH2. Evidence is provided that miR-217 expression is essential for the rapid loss of miR-211 in prolonged ER stress and provides a functional link for determining whether cells adapt to stress or commit to apoptosis. IMPLICATIONS: PERK-dependent induction of miR-217 limits accumulation and function of the prosurvival miRNA, miR-211, to establish cell fate and promote cell commitment to apoptosis.


Assuntos
MicroRNAs , Neoplasias , Canais de Cátion TRPM , Humanos , eIF-2 Quinase/genética , eIF-2 Quinase/metabolismo , Estresse do Retículo Endoplasmático/genética , Resposta a Proteínas não Dobradas , MicroRNAs/genética , MicroRNAs/metabolismo , Apoptose/fisiologia , Neoplasias/genética , Microambiente Tumoral , Canais de Cátion TRPM/genética
15.
Mol Pharmacol ; 105(3): 155-165, 2024 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-38164594

RESUMO

The three arms of the unfolded protein response (UPR) surveil the luminal environment of the endoplasmic reticulum (ER) and transmit information through the lipid bilayer to the cytoplasm to alert the cell of stress conditions within the ER lumen. That same lipid bilayer is the site of de novo synthesis of phospholipids and sphingolipids. Thus, it is no surprise that lipids are modulated by and are modulators of ER stress. Given that sphingolipids have both prosurvival and proapoptotic effects, they also exert opposing effects on life/death decisions in the face of prolonged ER stress detected by the UPR. In this review, we will focus on several recent studies that demonstrate how sphingolipids affect each arm of the UPR. We will also discuss the role of sphingolipids in the process of immunogenic cell death downstream of the protein kinase RNA-like endoplasmic reticulum kinase (PERK)/eukaryotic initiating factor 2α (eIF2α) arm of the UPR. Furthermore, we will discuss strategies to target the sphingolipid metabolic pathway that could potentially act synergistically with agents that induce ER stress as novel anticancer treatments. SIGNIFICANCE STATEMENT: This review provides the readers with a brief discussion of the sphingolipid metabolic pathway and the unfolded protein response. The primary focus of the review is the mechanism(s) by which sphingolipids modulate the endoplasmic reticulum (ER) stress response pathways and the critical role of sphingolipids in the process of immunogenic cell death associated with the ER stress response.


Assuntos
Morte Celular Imunogênica , Neoplasias , Humanos , Bicamadas Lipídicas/metabolismo , eIF-2 Quinase/genética , eIF-2 Quinase/metabolismo , Estresse do Retículo Endoplasmático , Resposta a Proteínas não Dobradas , Retículo Endoplasmático/metabolismo , Neoplasias/metabolismo , Esfingolipídeos/metabolismo
16.
Nucleic Acids Res ; 52(4): 1830-1846, 2024 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-38281137

RESUMO

Diverse environmental insults induce the integrated stress response (ISR), which features eIF2 phosphorylation and translational control that serves to restore protein homeostasis. The eIF2 kinase GCN2 is a first responder in the ISR that is activated by amino acid depletion and other stresses not directly related to nutrients. Two mechanisms are suggested to trigger an ordered process of GCN2 activation during stress: GCN2 monitoring stress via accumulating uncharged tRNAs or by stalled and colliding ribosomes. Our results suggest that while ribosomal collisions are indeed essential for GCN2 activation in response to translational elongation inhibitors, conditions that trigger deacylation of tRNAs activate GCN2 via its direct association with affected tRNAs. Both mechanisms require the GCN2 regulatory domain related to histidyl tRNA synthetases. GCN2 activation by UV irradiation features lowered amino acids and increased uncharged tRNAs and UV-induced ribosome collisions are suggested to be dispensable. We conclude that there are multiple mechanisms that activate GCN2 during diverse stresses.


Assuntos
Proteínas Serina-Treonina Quinases , Aminoácidos/metabolismo , eIF-2 Quinase/genética , eIF-2 Quinase/metabolismo , Fator de Iniciação 2 em Eucariotos/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Ribossomos/metabolismo , RNA de Transferência/genética , RNA de Transferência/metabolismo , Humanos
17.
Toxicol Appl Pharmacol ; 483: 116800, 2024 02.
Artigo em Inglês | MEDLINE | ID: mdl-38219984

RESUMO

Nasopharyngeal carcinoma, a malignant tumor prevalent in southeast Asia and north Africa, still lacks effective treatment. Esketamine, an N-methyl-D-aspartatic acid (NMDA) receptor (NMDAR) antagonist, is widely used in clinical anesthesia. Emerging evidence suggests that esketamine plays an important role in inhibiting tumor cell activity. However, the underlying mechanisms of esketamine on nasopharyngeal carcinoma remain unknown. In this study, we found that esketamine inhibited the proliferation and migration of nasopharyngeal carcinoma cells. Mechanically, transcriptome sequencing and subsequent verification experiments revealed that esketamine promoted the apoptosis of nasopharyngeal carcinoma cells through endoplasmic reticulum stress PERK/ATF4/CHOP signaling pathway mediated by NMDAR. Additionally, when combined with esketamine, the inhibitory effect of cisplatin on the proliferation of nasopharyngeal carcinoma cells was significantly enhanced. These findings provide new insights into future anti-nasopharyngeal carcinoma clinical strategies via targeting the NMDAR/PERK/CHOP axis alone or in combination with cisplatin.


Assuntos
Ketamina , Neoplasias Nasofaríngeas , eIF-2 Quinase , Humanos , eIF-2 Quinase/metabolismo , Cisplatino/farmacologia , Carcinoma Nasofaríngeo/tratamento farmacológico , Apoptose , Neoplasias Nasofaríngeas/tratamento farmacológico , Estresse do Retículo Endoplasmático , Fator de Transcrição CHOP/genética , Fator de Transcrição CHOP/metabolismo , Fator 4 Ativador da Transcrição/metabolismo
18.
Int J Mol Sci ; 25(2)2024 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-38279321

RESUMO

Specific sequences within RNA encoded by human genes essential for survival possess the ability to activate the RNA-dependent stress kinase PKR, resulting in phosphorylation of its substrate, eukaryotic translation initiation factor-2α (eIF2α), either to curb their mRNA translation or to enhance mRNA splicing. Thus, interferon-γ (IFNG) mRNA activates PKR through a 5'-terminal 203-nucleotide pseudoknot structure, thereby strongly downregulating its own translation and preventing a harmful hyper-inflammatory response. Tumor necrosis factor-α (TNF) pre-mRNA encodes within the 3'-untranslated region (3'-UTR) a 104-nucleotide RNA pseudoknot that activates PKR to enhance its splicing by an order of magnitude while leaving mRNA translation intact, thereby promoting effective TNF protein expression. Adult and fetal globin genes encode pre-mRNA structures that strongly activate PKR, leading to eIF2α phosphorylation that greatly enhances spliceosome assembly and splicing, yet also structures that silence PKR activation upon splicing to allow for unabated globin mRNA translation essential for life. Regulatory circuits resulting in each case from PKR activation were reviewed previously. Here, we analyze mutations within these genes created to delineate the RNA structures that activate PKR and to deconvolute their folding. Given the critical role of intragenic RNA activators of PKR in gene regulation, such mutations reveal novel potential RNA targets for human disease.


Assuntos
Precursores de RNA , RNA , Humanos , RNA/metabolismo , Precursores de RNA/genética , Precursores de RNA/metabolismo , Biossíntese de Proteínas , eIF-2 Quinase/genética , eIF-2 Quinase/metabolismo , RNA Mensageiro/genética , Fosforilação , Fator de Necrose Tumoral alfa/metabolismo , Nucleotídeos/metabolismo , Globinas/genética , Fator de Iniciação 2 em Eucariotos/genética , Fator de Iniciação 2 em Eucariotos/metabolismo
19.
Acta Pharmacol Sin ; 45(3): 502-516, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-37880338

RESUMO

Olanzapine (OLZ) is a widely prescribed antipsychotic drug with a relatively ideal effect in the treatment of schizophrenia (SCZ). However, its severe metabolic side effects often deteriorate clinical therapeutic compliance and mental rehabilitation. The peripheral mechanism of OLZ-induced metabolic disorders remains abstruse for its muti-target activities. Endoplasmic reticulum (ER) stress is implicated in cellular energy metabolism and the progression of psychiatric disorders. In this study, we investigated the role of ER stress in the development of OLZ-induced dyslipidemia. A cohort of 146 SCZ patients receiving OLZ monotherapy was recruited, and blood samples and clinical data were collected at baseline, and in the 4th week, 12th week, and 24th week of the treatment. This case-control study revealed that OLZ treatment significantly elevated serum levels of endoplasmic reticulum (ER) stress markers GRP78, ATF4, and CHOP in SCZ patients with dyslipidemia. In HepG2 cells, treatment with OLZ (25, 50 µM) dose-dependently enhanced hepatic de novo lipogenesis accompanied by SREBPs activation, and simultaneously triggered ER stress. Inhibition of ER stress by tauroursodeoxycholate (TUDCA) and 4-phenyl butyric acid (4-PBA) attenuated OLZ-induced lipid dysregulation in vitro and in vivo. Moreover, we demonstrated that activation of PERK-CHOP signaling during ER stress was a major contributor to OLZ-triggered abnormal lipid metabolism in the liver, suggesting that PERK could be a potential target for ameliorating the development of OLZ-mediated lipid dysfunction. Taken together, ER stress inhibitors could be a potentially effective intervention against OLZ-induced dyslipidemia in SCZ.


Assuntos
Dislipidemias , Transdução de Sinais , Humanos , Olanzapina/farmacologia , Estudos de Casos e Controles , Estresse do Retículo Endoplasmático , Dislipidemias/induzido quimicamente , Lipídeos , eIF-2 Quinase/metabolismo , Apoptose
20.
J Cell Mol Med ; 28(1): e18030, 2024 01.
Artigo em Inglês | MEDLINE | ID: mdl-37929884

RESUMO

Acetylshikonin (AS) is an active component of Lithospermum erythrorhizon Sieb. et Zucc that exhibits activity against various cancers; however, the underlying mechanisms of AS against oesophageal squamous carcinoma (ESCC) need to be elusive. The research explores the anti-cancer role and potential mechanism of AS on ESCC in vitro and in vivo, providing evidences for AS treatment against ESCC. In this study, we firstly demonstrated that AS treatment effectively inhibits cell viability and proliferation of ESCC cells. In addition, AS significantly induces G1/S phage arrest and promotes apoptosis in ESCC cell lines. Further studies reveal that AS induces ER stress, as observed by dose- and time-dependently increased expression of BIP, PDI, PERK, phosphorylation of eIF2α , CHOP and splicing of XBP1. CHOP knockdown or PERK inhibition markedly rescue cell apoptosis induced by AS. Moreover, AS treatment significantly inhibits ESCC xenograft growth in nude mice. Elevated expression of BIP and CHOP is also observed in xenograft tumours. Taken together, AS inhibits proliferation and induces apoptosis through ER stress-activated PERK/eIF2α /CHOP pathway in ESCC, which indicates AS represents a promising candidate for ESCC treatment.


Assuntos
Antraquinonas , Neoplasias Esofágicas , Carcinoma de Células Escamosas do Esôfago , Camundongos , Animais , Humanos , eIF-2 Quinase/metabolismo , Fator de Iniciação 2 em Eucariotos/metabolismo , Camundongos Nus , Estresse do Retículo Endoplasmático , Apoptose , Fator de Transcrição CHOP/metabolismo
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