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1.
Proc Natl Acad Sci U S A ; 117(31): 18591-18599, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32690681

RESUMO

Repeat associated non-AUG (RAN) translation is found in a growing number of microsatellite expansion diseases, but the mechanisms remain unclear. We show that RAN translation is highly regulated by the double-stranded RNA-dependent protein kinase (PKR). In cells, structured CAG, CCUG, CAGG, and G4C2 expansion RNAs activate PKR, which leads to increased levels of multiple RAN proteins. Blocking PKR using PKR-K296R, the TAR RNA binding protein or PKR-KO cells, reduces RAN protein levels. p-PKR is elevated in C9orf72 ALS/FTD human and mouse brains, and inhibiting PKR in C9orf72 BAC transgenic mice using AAV-PKR-K296R or the Food and Drug Administration (FDA)-approved drug metformin, decreases RAN proteins, and improves behavior and pathology. In summary, targeting PKR, including by use of metformin, is a promising therapeutic approach for C9orf72 ALS/FTD and other expansion diseases.


Assuntos
Esclerose Amiotrófica Lateral/metabolismo , Proteína C9orf72 , Metformina/farmacologia , Biossíntese de Proteínas/efeitos dos fármacos , eIF-2 Quinase , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Proteína C9orf72/genética , Proteína C9orf72/metabolismo , Modelos Animais de Doenças , Demência Frontotemporal/metabolismo , Humanos , Camundongos , Camundongos Transgênicos , Repetições de Microssatélites/genética , eIF-2 Quinase/genética , eIF-2 Quinase/metabolismo
2.
Environ Toxicol ; 35(10): 1100-1113, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32506763

RESUMO

Chronic exposure to arsenic remains a worldwide environmental health issue, affecting hundreds of millions of people. Although, arsenic-induced oxidative stress and apoptosis have been determined, the underlying apoptosis mechanism has not been fully elucidated yet. Oxidative stress integrated-ER stress plays an important role in Life-and-Death decision of cells. The current study was to investigate whether NaAsO2 utilizes oxidative stress integrated-ER stress signaling to exert pro-apoptotic activity in L-02 cells. Results showed that death receptor 5 (DR5) was a mediator of NaAsO2 -induced apoptosis by enhancing construction of the death-inducing signaling complex (DISC). NaAsO2 -sensitized DR5 elevation required maintainable transcription and its transcription factor C/EBP homologous protein (CHOP). Further results showed that NaAsO2 increased expression in biomarker of endoplasmic reticulum (ER) stress and activated the protein kinase R-like ER kinase (PERK)-eukaryotic translation initiation 2α (eIF2α)-activating transcription factor 4 (ATF4) pathway. PERK inhibitor and ATF4 siRNA significantly attenuated NaAsO2 -induced CHOP and DR5 expressions. In addition, the antioxidant N-acetyl-l-cysteine (NAC) treatment led to amelioration of NaAsO2 -induced production of reactive oxygen species (ROS) and some ER stress- and apoptosis- related protein levels and cell viability. Taken together, the results indicate that ROS-mediated PERK-eIF2α-ATF4 pathway activated by NaAsO2 is the critical upstream event for subsequent apoptosis induction via regulating CHOP-DR5 signaling in L-02 cells when chronic exposure to arsenic, and support that antioxidants might be potential therapeutic agents for preventing or delaying the onset and progress of arsenic-induced hepatotoxicity.


Assuntos
Fator 4 Ativador da Transcrição/metabolismo , Apoptose/efeitos dos fármacos , Arsenitos/toxicidade , Poluentes Ambientais/toxicidade , Espécies Reativas de Oxigênio/metabolismo , Fator de Transcrição CHOP/metabolismo , eIF-2 Quinase/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Humanos , Transdução de Sinais
3.
PLoS One ; 15(6): e0234147, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32479530

RESUMO

Conversion of cellular prion protein (PrPC) into the pathogenic isoform of prion protein (PrPSc) in neurons is one of the key pathophysiological events in prion diseases. However, the molecular mechanism of neurodegeneration in prion diseases has yet to be fully elucidated because of a lack of suitable experimental models for analyzing neuron-autonomous responses to prion infection. In the present study, we used neuron-enriched primary cultures of cortical and thalamic mouse neurons to analyze autonomous neuronal responses to prion infection. PrPSc levels in neurons increased over the time after prion infection; however, no obvious neuronal losses or neurite alterations were observed. Interestingly, a finer analysis of individual neurons co-stained with PrPSc and phosphorylated protein kinase RNA-activated-like endoplasmic reticulum (ER) kinase (p-PERK), the early cellular response of the PERK-eukaryotic initiation factor 2 (eIF2α) pathway, demonstrated a positive correlation between the number of PrPSc granular stains and p-PERK granular stains, in cortical neurons at 21 dpi. Although the phosphorylation of PERK was enhanced in prion-infected cortical neurons, there was no sign of subsequent translational repression of synaptic protein synthesis or activations of downstream unfolded protein response (UPR) in the PERK-eIF2α pathway. These results suggest that PrPSc production in neurons induces ER stress in a neuron-autonomous manner; however, it does not fully activate UPR in prion-infected neurons. Our findings provide insights into the autonomous neuronal responses to prion propagation and the involvement of neuron-non-autonomous factor(s) in the mechanisms of neurodegeneration in prion diseases.


Assuntos
Neurônios/metabolismo , Proteínas PrPSc/metabolismo , eIF-2 Quinase/metabolismo , Animais , Células Cultivadas , Estresse do Retículo Endoplasmático , Camundongos , Camundongos Endogâmicos ICR , Crescimento Neuronal , Neurônios/citologia , Fosforilação , Proteínas PrPSc/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Resposta a Proteínas não Dobradas
4.
Life Sci ; 255: 117842, 2020 Aug 15.
Artigo em Inglês | MEDLINE | ID: covidwho-403331

RESUMO

The outbreak of COVID-19 caused by 2019-nCov/SARS-CoV-2 has become a pandemic with an urgent need for understanding the mechanisms and identifying a treatment. Viral infections including SARS-CoV are associated with increased levels of reactive oxygen species, disturbances of Ca++ caused by unfolded protein response (UPR) mediated by endoplasmic reticulum (ER) stress and is due to the exploitation of virus's own protein i.e., viroporins into the host cells. Several clinical trials are on-going including testing Remdesivir (anti-viral), Chloroquine and Hydroxychloroquine derivatives (anti-malarial drugs) etc. Unfortunately, each drug has specific limitations. Herein, we review the viral protein involvement to activate ER stress transducers (IRE-1, PERK, ATF-6) and their downstream signals; and evaluate combination therapies for COVID-19 mediated ER stress alterations. Melatonin is an immunoregulator, anti-pyretic, antioxidant, anti-inflammatory and ER stress modulator during viral infections. It enhances protective mechanisms for respiratory tract disorders. Andrographolide, isolated from Andrographis paniculata, has versatile biological activities including immunomodulation and determining SARS-CoV-2 binding site. Considering the properties of both compounds in terms of anti-inflammatory, antioxidant, anti-pyrogenic, anti-viral and ER stress modulation and computational approaches revealing andrographolide docks with the SARS-CoV2 binding site, we predict that this combination therapy may have potential utility against COVID-19.


Assuntos
Betacoronavirus/metabolismo , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/virologia , Diterpenos/farmacologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Melatonina/farmacologia , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/virologia , Fator 6 Ativador da Transcrição/metabolismo , Antivirais/farmacologia , Sinergismo Farmacológico , Estresse do Retículo Endoplasmático/fisiologia , Endorribonucleases/metabolismo , Humanos , Terapia de Alvo Molecular , Pandemias , Proteínas Serina-Treonina Quinases/metabolismo , Resposta a Proteínas não Dobradas/efeitos dos fármacos , eIF-2 Quinase/metabolismo
5.
Proc Natl Acad Sci U S A ; 117(20): 10935-10945, 2020 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-32355000

RESUMO

The circadian clock in eukaryotes controls transcriptional and posttranscriptional events, including regulation of the levels and phosphorylation state of translation factors. However, the mechanisms underlying clock control of translation initiation, and the impact of this potential regulation on rhythmic protein synthesis, were not known. We show that inhibitory phosphorylation of eIF2α (P-eIF2α), a conserved translation initiation factor, is clock controlled in Neurospora crassa, peaking during the subjective day. Cycling P-eIF2α levels required rhythmic activation of the eIF2α kinase CPC-3 (the homolog of yeast and mammalian GCN2), and rhythmic activation of CPC-3 was abolished under conditions in which the levels of charged tRNAs were altered. Clock-controlled accumulation of P-eIF2α led to reduced translation during the day in vitro and was necessary for the rhythmic synthesis of select proteins in vivo. Finally, loss of rhythmic P-eIF2α levels led to reduced linear growth rates, supporting the idea that partitioning translation to specific times of day provides a growth advantage to the organism. Together, these results reveal a fundamental mechanism by which the clock regulates rhythmic protein production, and provide key insights into how rhythmic translation, cellular energy, stress, and nutrient metabolism are linked through the levels of charged versus uncharged tRNAs.


Assuntos
Relógios Circadianos/fisiologia , Fator de Iniciação 2 em Eucariotos/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Animais , Ritmo Circadiano , Fator de Iniciação 2 em Eucariotos/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Neurospora crassa/crescimento & desenvolvimento , Neurospora crassa/metabolismo , Fosforilação , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , eIF-2 Quinase/metabolismo
6.
Clin Sci (Lond) ; 134(10): 1127-1141, 2020 05 29.
Artigo em Inglês | MEDLINE | ID: mdl-32400851

RESUMO

Vitiligo is a depigmentation disorder that develops as a result of the progressive disappearance of epidermal melanocytes. The elevated level of amino acid metabolite homocysteine (Hcy) has been identified as circulating marker of oxidative stress and known as a risk factor for vitiligo. However, the mechanism underlying Hcy-regulated melanocytic destruction is currently unknown. The present study aims to elucidate the effect of Hcy on melanocytic destruction and its involvement in the pathogenesis of vitiligo. Our results showed that Hcy level was significantly elevated in the serum of progressive vitiligo patients. Notably, Hcy induced cell apoptosis in melanocytes via activating reactive oxygen species (ROS) and endoplasmic reticulum (ER) stress protein kinase RNA-like ER kinase (PERK)-eukaryotic translation initiation factor 2α (eIF2α)-C/EBP homologous protein (CHOP) pathway. More importantly, folic acid, functioning in the transformation of Hcy, could lower the intracellular Hcy level and further reverse the apoptotic effect of Hcy on melanocytes. Additionally, Hcy disrupted melanogenesis whereas folic acid supplementation could reverse the melanogenesis defect induced by Hcy in melanocytes. Taken together, Hcy is highly increased in vitiligo patients at progressive stage, and our in vitro studies revealed that folic acid could protect melanocytes from Hcy-induced apoptosis and melanin synthesis inhibition, indicating folic acid as a potential benefit agent for patients with progressive vitiligo.


Assuntos
Apoptose , Fator de Iniciação 2 em Eucariotos/metabolismo , Homocisteína/metabolismo , Melanócitos/metabolismo , Melanócitos/patologia , Fator de Transcrição CHOP/metabolismo , Vitiligo/metabolismo , eIF-2 Quinase/metabolismo , Adulto , Apoptose/efeitos dos fármacos , Estudos de Casos e Controles , Proliferação de Células/efeitos dos fármacos , Progressão da Doença , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Feminino , Ácido Fólico/farmacologia , Homocisteína/sangue , Humanos , Masculino , Melaninas/biossíntese , Melanócitos/efeitos dos fármacos , Modelos Biológicos , Transdução de Sinais/efeitos dos fármacos , Vitiligo/sangue
7.
Life Sci ; 255: 117842, 2020 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-32454157

RESUMO

The outbreak of COVID-19 caused by 2019-nCov/SARS-CoV-2 has become a pandemic with an urgent need for understanding the mechanisms and identifying a treatment. Viral infections including SARS-CoV are associated with increased levels of reactive oxygen species, disturbances of Ca++ caused by unfolded protein response (UPR) mediated by endoplasmic reticulum (ER) stress and is due to the exploitation of virus's own protein i.e., viroporins into the host cells. Several clinical trials are on-going including testing Remdesivir (anti-viral), Chloroquine and Hydroxychloroquine derivatives (anti-malarial drugs) etc. Unfortunately, each drug has specific limitations. Herein, we review the viral protein involvement to activate ER stress transducers (IRE-1, PERK, ATF-6) and their downstream signals; and evaluate combination therapies for COVID-19 mediated ER stress alterations. Melatonin is an immunoregulator, anti-pyretic, antioxidant, anti-inflammatory and ER stress modulator during viral infections. It enhances protective mechanisms for respiratory tract disorders. Andrographolide, isolated from Andrographis paniculata, has versatile biological activities including immunomodulation and determining SARS-CoV-2 binding site. Considering the properties of both compounds in terms of anti-inflammatory, antioxidant, anti-pyrogenic, anti-viral and ER stress modulation and computational approaches revealing andrographolide docks with the SARS-CoV2 binding site, we predict that this combination therapy may have potential utility against COVID-19.


Assuntos
Betacoronavirus/metabolismo , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/virologia , Diterpenos/farmacologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Melatonina/farmacologia , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/virologia , Fator 6 Ativador da Transcrição/metabolismo , Antivirais/farmacologia , Sinergismo Farmacológico , Estresse do Retículo Endoplasmático/fisiologia , Endorribonucleases/metabolismo , Humanos , Terapia de Alvo Molecular , Pandemias , Proteínas Serina-Treonina Quinases/metabolismo , Resposta a Proteínas não Dobradas/efeitos dos fármacos , eIF-2 Quinase/metabolismo
8.
PLoS One ; 15(5): e0231894, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32365111

RESUMO

Stress granules (SG) are cytoplasmic RNA granules that form during various types of stress known to inhibit general translation, including oxidative stress, hypoxia, endoplasmic reticulum stress (ER), ionizing radiations or viral infection. Induction of these SG promotes cell survival in part through sequestration of proapoptotic molecules, resulting in the inactivation of cell death pathways. SG also form in cancer cells, but studies investigating their formation upon treatment with chemotherapeutics are very limited. Here we identified Lapatinib (Tykerb / Tyverb®), a tyrosine kinase inhibitor used for the treatment of breast cancers as a new inducer of SG in breast cancer cells. Lapatinib-induced SG formation correlates with the inhibition of general translation initiation which involves the phosphorylation of the translation initiation factor eIF2α through the kinase PERK. Disrupting PERK-SG formation by PERK depletion experiments sensitizes resistant breast cancer cells to Lapatinib. This study further supports the assumption that treatment with anticancer drugs activates the SG pathway, which may constitute an intrinsic stress response used by cancer cells to resist treatment.


Assuntos
Grânulos Citoplasmáticos/efeitos dos fármacos , Lapatinib/uso terapêutico , Neoplasias/tratamento farmacológico , Biossíntese de Proteínas/efeitos dos fármacos , Linhagem Celular Tumoral , Grânulos Citoplasmáticos/metabolismo , Grânulos Citoplasmáticos/patologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Estresse do Retículo Endoplasmático/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Humanos , Lapatinib/farmacologia , Células MCF-7 , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Fosforilação/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genética , eIF-2 Quinase/metabolismo
9.
Life Sci ; 252: 117654, 2020 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-32277979

RESUMO

BACKGROUND: Septic encephalopathy, the most frequent complication of sepsis, is orchestrated by a complex interplay of signals that leads to high mortality rates among intensive care unit patients. However, the role of the vascular endothelial growth factor receptor-2 (VEGFR2) in endoplasmic reticulum stress response (ERSR), during septic encephalopathy, is still elusive. AIM: This study was aimed to examine the effect of an in-house designed/synthesized VEGFR2 antagonist, named WAG4S, on septic encephalopathy using cecal ligation and perforation (CLP). MAIN METHODS: Rats were intraperitoneally injected with WAG-4S (1 mg/kg/d) for 7 days post-CLP. KEY FINDINGS: In septic animals, VEGFR2 antagonism declined the expression of cortical p-VEGFR2 and p-mammalian target of rapamycin complex-1 (p-mTORC1). It also worsened the behavioral and histopathological alterations beyond CLP. However, and contrary to CLP, WAG-4S decreased the p-protein kinase R-like ER kinase (p-PERK) and eukaryotic initiation factor-2α (p-eIF2α) expression. Moreover, VEGFR2 blockade upregulated the mRNA expression of activating transcription factor-4 (ATF4), binding immunoglobulin protein/glucose-regulated protein-78 (Bip/GRP78), growth arrest and DNA damage-34 (GADD34) and spliced X-box binding protein-1 (XBP1s) above CLP. Similarly, it boosted inositol requiring enzyme-1α (IRE1α) activation and redox imbalance. In the same context, WAG-4S augmented the protein levels of CLP-induced ERSR apoptotic markers, namely C/EBP homologous protein (CHOP/GADD153), c-jun N-terminal kinase (JNK) and caspase-3. SIGNIFICANCE: In conclusion, the PERK/eIF2α axis inhibition, during septic encephalopathy, is VEGFR2-independent, whereas the activated IRE1α/XBP1s/CHOP/JNK/caspase-3 cue promotes the ERSR execution module through VEGFR2 inhibition. This has turned VEGFR2 into a potential therapeutic target for ameliorating such an ailment.


Assuntos
Encefalopatias/fisiopatologia , Estresse do Retículo Endoplasmático/fisiologia , Sepse/complicações , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator 4 Ativador da Transcrição/genética , Animais , Encefalopatias/etiologia , Encefalopatias/prevenção & controle , Modelos Animais de Doenças , Masculino , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Sepse/tratamento farmacológico , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , eIF-2 Quinase/metabolismo
10.
Res Vet Sci ; 130: 237-239, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32224353

RESUMO

Monocrotaline (MCT) belongs to the category of Pyrrolizdine Alkaloids (PAs), which is one of important hepatotoxic alkaloid in Crotalaria Lin. Apoptosis is one mechanism of toxic responses induced by MCT. However, the underlying mechanism of liver apoptosis caused by MCT through Endoplasmic reticulum (ER) stress continues to be incompletely understood. In this study, we describe the role of ER stress in MCT induced hepatotoxicity in rats. 24 male rats were randomly divided into 3 groups: normal saline group, 45 mg/kg MCT group and 90 mg/kg MCT group. After 48 h of saline/MCT administration, the livers were collected for analysis of ER stress-related proteins by Western blotting. The expression of GRP78, p-IRE1α, ATF6 and caspase-12 showed a dose-dependent increase. PERK/eIF2α/ATF4/CHOP pathway is one of the major ER stress pathways which is required for cell survival. Therefore, through analyzing the effects of MCT on this pathway, we found the protein levels of p-PERK, p-eIF2α, ATF4 and CHOP were increase obviously. All these results indicate that MCT induces ER stress in rat liver. The PERK/eIF2α/ATF4/CHOP pathway is involved in the regulation of MCT-induced ER stress in the liver of rat.


Assuntos
Estresse do Retículo Endoplasmático/efeitos dos fármacos , Fígado/efeitos dos fármacos , Monocrotalina/efeitos adversos , Fator 4 Ativador da Transcrição/metabolismo , Animais , Fator de Iniciação 2 em Eucariotos/metabolismo , Fígado/fisiopatologia , Masculino , Ratos , Ratos Sprague-Dawley , Fator de Transcrição CHOP/metabolismo , eIF-2 Quinase/metabolismo
11.
Clin Sci (Lond) ; 134(7): 907-920, 2020 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-32236445

RESUMO

BACKGROUND: Increased keratinocyte proliferation occurs in the skin of psoriatic patients and is supposed to play a role in the pathogenesis of this disorder. Compounds interfering with keratinocyte proliferation could be useful in the management of psoriatic patients. AIM: To investigate whether albendazole, an anti-helmintic drug that regulates epithelial cell function in various systems, inhibits keratinocyte proliferation in models of psoriasis. METHODS: Aldara-treated mice received daily topical application of albendazole. Keratinocyte proliferation and keratin (K) 6 and K16 expression were evaluated by immunohistochemistry and Western blotting and inflammatory cells/mediators were analysed by immunohistochemistry and real-time PCR. In human keratinocytes (HEKa and HaCaT) treated with albendazole, cell cycle and proliferation, keratins and cell cycle-associated factors were evaluated by flow cytometry, colorimetric assay and Western blotting respectively. RESULTS: Aldara-treated mice given albendazole exhibited reduced epidermal thickness, decreased number of proliferating keratinocytes and K6/K16 expression. Reduction of CD3- and Ly6G-positive cells in the skin of albendazole-treated mice associated with inhibition of IL-6, TNF-α, IL-1ß, IL-17A, IL-36, CCL17, CXCL1, CXCL2 and CXCL5 expression. Treatment of keratinocytes with albendazole reduced K6/K16 expression and reversibly inhibited cell growth by promoting accumulation of cells in S-phase. This phenomenon was accompanied by down-regulation of CDC25A, a phosphatase regulating progression of cell cycle through S-phase, and PKR-dependent hyper-phosphorylation of eIF2α, an inhibitor of CDC25 translation. In Aldara-treated mice, albendazole activated PKR, enhanced eIF2α phosphorylation and reduced CDC25A expression. CONCLUSIONS: Data show that albendazole inhibits keratinocyte proliferation and exerts therapeutic effect in a murine model of psoriasis.


Assuntos
Albendazol/farmacologia , Proliferação de Células/efeitos dos fármacos , Fármacos Dermatológicos/farmacologia , Queratinócitos/efeitos dos fármacos , Psoríase/tratamento farmacológico , Pele/efeitos dos fármacos , Animais , Linhagem Celular , Citocinas/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Fator de Iniciação 2 em Eucariotos/metabolismo , Humanos , Imiquimode , Mediadores da Inflamação/metabolismo , Queratinócitos/metabolismo , Queratinócitos/patologia , Queratinas/genética , Queratinas/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Fosforilação , Psoríase/induzido quimicamente , Psoríase/metabolismo , Psoríase/patologia , Pontos de Checagem da Fase S do Ciclo Celular/efeitos dos fármacos , Transdução de Sinais , Pele/metabolismo , Pele/patologia , Fosfatases cdc25/metabolismo , eIF-2 Quinase/metabolismo
12.
Biochemistry ; 59(12): 1252-1260, 2020 03 31.
Artigo em Inglês | MEDLINE | ID: mdl-32176479

RESUMO

Protein kinase R (PKR) is a key antiviral component of the innate immune pathway and is activated by viral double-stranded RNAs (dsRNAs). Adenovirus-associated RNA 1 (VAI) is an abundant, noncoding viral RNA that functions as a decoy by binding PKR but not inducing activation, thereby inhibiting the antiviral response. In VAI, coaxial stacking produces an extended helix that mediates high-affinity PKR binding but is too short to result in activation. Like adenovirus, Epstein-Barr virus produces high concentrations of a noncoding RNA, EBER1. Here, we compare interactions of PKR with VAI and EBER1 and present a structural model of EBER1. Both RNAs function as inhibitors of dsRNA-mediated PKR activation. However, EBER1 weakly activates PKR whereas VAI does not. PKR binds EBER1 more weakly than VAI. Assays at physiological ion concentrations indicate that both RNAs can accommodate two PKR monomers and induce PKR dimerization. A structural model of EBER1 was obtained using constraints derived from chemical structure probing and small-angle X-ray scattering experiments. The central stem of EBER1 coaxially stacks with stem loop 4 and stem loop 1 to form an extended RNA duplex of ∼32 bp that binds PKR and promotes activation. Our observations that EBER1 binds PKR much more weakly than VAI and exhibits weak PKR activation suggest that EBER1 is less well suited to function as an RNA decoy.


Assuntos
Herpesvirus Humano 4/genética , Interações entre Hospedeiro e Microrganismos/genética , RNA Viral/metabolismo , eIF-2 Quinase/genética , Infecções por Vírus Epstein-Barr/imunologia , Infecções por Vírus Epstein-Barr/virologia , Herpesvirus Humano 4/imunologia , Humanos , Imunidade Inata/genética , Modelos Moleculares , Conformação de Ácido Nucleico , Multimerização Proteica/genética , Multimerização Proteica/imunologia , Estabilidade de RNA , RNA Viral/química , Espalhamento a Baixo Ângulo , Difração de Raios X , eIF-2 Quinase/química , eIF-2 Quinase/imunologia , eIF-2 Quinase/metabolismo
13.
Nature ; 579(7799): 433-437, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32132706

RESUMO

Mitochondrial fidelity is tightly linked to overall cellular homeostasis and is compromised in ageing and various pathologies1-3. Mitochondrial malfunction needs to be relayed to the cytosol, where an integrated stress response is triggered by the phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) in mammalian cells4,5. eIF2α phosphorylation is mediated by the four eIF2α kinases GCN2, HRI, PERK and PKR, which are activated by diverse types of cellular stress6. However, the machinery that communicates mitochondrial perturbation to the cytosol to trigger the integrated stress response remains unknown1,2,7. Here we combine genome engineering and haploid genetics to unbiasedly identify genes that affect the induction of C/EBP homologous protein (CHOP), a key factor in the integrated stress response. We show that the mitochondrial protease OMA1 and the poorly characterized protein DELE1, together with HRI, constitute the missing pathway that is triggered by mitochondrial stress. Mechanistically, stress-induced activation of OMA1 causes DELE1 to be cleaved into a short form that accumulates in the cytosol, where it binds to and activates HRI via its C-terminal portion. Obstruction of this pathway can be beneficial or adverse depending on the type of mitochondrial perturbation. In addition to the core pathway components, our comparative genetic screening strategy identifies a suite of additional regulators. Together, these findings could be used to inform future strategies to modulate the cellular response to mitochondrial dysfunction in the context of human disease.


Assuntos
Citosol/metabolismo , Citosol/patologia , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Proteínas Mitocondriais/metabolismo , Ativação Enzimática , Fator de Iniciação 2 em Eucariotos/metabolismo , Genoma Humano/genética , Humanos , Metaloendopeptidases/metabolismo , Mitocôndrias/enzimologia , Fosforilação , Ligação Proteica , Estresse Fisiológico , Fator de Transcrição CHOP/metabolismo , eIF-2 Quinase/metabolismo
14.
J Pathol ; 251(2): 123-134, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32166747

RESUMO

Spontaneously regressing infantile haemangiomas and aggressive angiosarcomas are vascular tumours with excessive angiogenesis. When analysing haemangiomas and angiosarcomas immunohistochemically with respect to their chaperone profiles we found that angiosarcomas have significantly elevated protein levels of binding immunoglobulin protein (BIP) and PERK with concomitant attenuated IRE1α levels, whereas haemangioma tissue exhibits the same pattern as embryonal skin tissue. We show that BiP is essential for the maintenance of VEGFR2 protein, which is expressed in the endothelium of both tumour types. When studying the effects of BiP, the IRE1α/Xbp1 -, and PERK/ATF4-signalling pathways on the migration and tube-forming potential of endothelial cells, we show that downregulation of BiP, as well as inhibition of the kinase activity of IRE1α, inhibit in vitro angiogenesis. Downregulation of PERK (PKR-like kinase; PKR = protein kinase R) levels promotes Xbp1 splicing in endoplasmic reticulum (ER)-stressed cells, indicating that in angiosarcoma the elevated PERK levels might result in high levels of unspliced Xbp1, which have been reported to promote cell proliferation and increase tumour malignancy. The data presented in this study revealed that in addition to BiP or PERK, the kinase domains of IRE1α and Xbp1 could be potential targets for the development of novel therapeutic approaches for treating angiosarcomas and to control tumour angiogenesis. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.


Assuntos
Endorribonucleases/metabolismo , Células Endoteliais/enzimologia , Proteínas de Choque Térmico/metabolismo , Hemangioma/enzimologia , Hemangiossarcoma/enzimologia , Neovascularização Patológica , Proteínas Serina-Treonina Quinases/metabolismo , eIF-2 Quinase/metabolismo , Movimento Celular , Proliferação de Células , Células Cultivadas , Endorribonucleases/genética , Células Endoteliais/patologia , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Proteínas de Choque Térmico/genética , Hemangioma/genética , Hemangioma/patologia , Hemangiossarcoma/genética , Hemangiossarcoma/patologia , Células Endoteliais da Veia Umbilical Humana/enzimologia , Humanos , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Proteína 1 de Ligação a X-Box/genética , Proteína 1 de Ligação a X-Box/metabolismo , eIF-2 Quinase/genética
15.
Nature ; 579(7799): 427-432, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32132707

RESUMO

In mammalian cells, mitochondrial dysfunction triggers the integrated stress response, in which the phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) results in the induction of the transcription factor ATF41-3. However, how mitochondrial stress is relayed to ATF4 is unknown. Here we show that HRI is the eIF2α kinase that is necessary and sufficient for this relay. In a genome-wide CRISPR interference screen, we identified factors upstream of HRI: OMA1, a mitochondrial stress-activated protease; and DELE1, a little-characterized protein that we found was associated with the inner mitochondrial membrane. Mitochondrial stress stimulates OMA1-dependent cleavage of DELE1 and leads to the accumulation of DELE1 in the cytosol, where it interacts with HRI and activates the eIF2α kinase activity of HRI. In addition, DELE1 is required for ATF4 translation downstream of eIF2α phosphorylation. Blockade of the OMA1-DELE1-HRI pathway triggers an alternative response in which specific molecular chaperones are induced. The OMA1-DELE1-HRI pathway therefore represents a potential therapeutic target that could enable fine-tuning of the integrated stress response for beneficial outcomes in diseases that involve mitochondrial dysfunction.


Assuntos
Citosol/metabolismo , Metaloendopeptidases/metabolismo , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Proteínas Mitocondriais/metabolismo , Estresse Fisiológico , eIF-2 Quinase/metabolismo , Fator 4 Ativador da Transcrição/biossíntese , Fator 4 Ativador da Transcrição/metabolismo , Sistemas CRISPR-Cas , Linhagem Celular , Citosol/enzimologia , Ativação Enzimática , Fator de Iniciação 2 em Eucariotos/metabolismo , Humanos , Masculino , Proteínas Mitocondriais/química , Chaperonas Moleculares/metabolismo , Fosforilação , Ligação Proteica
16.
Phytomedicine ; 69: 153183, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32113150

RESUMO

BACKGROUND: Osteosarcoma (OS) is a significant threat to the lives of children and young adults. Although neoadjuvant chemotherapy is the first choice of treatment for OS, it is limited by serious side-effects and cancer metastasis. ß-Elemonic acid (ß-EA), an active component extracted from Boswellia carterii Birdw., has been reported to exhibit potential anti-inflammatory and anticancer activities. However, the anti-tumor effects and underlying mechanisms on OS as well as pharmacokinetic characteristics of ß-EA remain unknown. PURPOSE: This study was aimed to investigating the anti-tumor effects of ß-EA on human OS, the underlying mechanisms, and the pharmacokinetic and tissue distribution characteristics. STUDY DESIGN AND METHODS: Cell viability and colony formation assays were performed to determine the effect of ß-EA cell on cell proliferation. Apoptosis rates, mitochondrial membrane potential and cell cycle features were analyzed by flow cytometry. qRT-PCR, Western blot, immunofluorescence and immunohistochemical assays were conducted to evaluate the expression levels of genes or proteins related to the pathways affected by ß-EA in vitro and in vivo. Cell migration and invasion were evaluated in wound healing and Transwell chamber assays. The effects and pharmacokinetic characteristics of ß-EA in vivo were evaluated by analyzing tumor suppression, pharmacokinetics and tissue distribution. RESULTS: Explorations indicated that endoplasmic reticulum (ER) stress conditions provoked by ß-EA activated the PERK/eIF2α/ATF4 branch of the unfolded protein reaction (UPR), stimulating C/EBP homologous protein (CHOP)-regulated apoptosis and inducing Ca2+ leakage leading to caspase-dependent apoptosis. Furthermore, ß-EA induced G0/G1 cell cycle arrest and inhibited metastasis of HOS and 143B cells by attenuating Wnt/ß-catenin signaling effects, which included decreased levels of p-Akt(Ser473), p-Gsk3ß (Ser9), Wnt/ß-catenin target genes (c-Myc and CyclinD1) along with a decline in nuclear ß-catenin accumulation. The fast absorption, short elimination half-life, and linear pharmacokinetic characteristics of ß-EA were also revealed. The distribution of ß-EA was detected in the tumor and bone tissues. CONCLUSIONS: Overall, both in vitro and in vivo investigations showed the potential of ß-EA for the treatment of human OS. The pharmacokinetic profile and considerable distribution in the tumor and bone tissues warrant further preclinical or even clinical studies.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Neoplasias Ósseas/tratamento farmacológico , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Osteossarcoma/tratamento farmacológico , Fenantrenos/farmacologia , Fator 4 Ativador da Transcrição/metabolismo , Animais , Antineoplásicos Fitogênicos/farmacocinética , Apoptose/efeitos dos fármacos , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Estresse do Retículo Endoplasmático/fisiologia , Fator de Iniciação 2 em Eucariotos/metabolismo , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Humanos , Masculino , Camundongos Endogâmicos BALB C , Osteossarcoma/metabolismo , Osteossarcoma/patologia , Distribuição Tecidual , Fator de Transcrição CHOP/genética , Fator de Transcrição CHOP/metabolismo , Via de Sinalização Wnt/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto , beta Catenina/metabolismo , eIF-2 Quinase/metabolismo
17.
Nat Commun ; 11(1): 1304, 2020 03 11.
Artigo em Inglês | MEDLINE | ID: mdl-32161259

RESUMO

The integrated stress response (ISR) converges on eIF2α phosphorylation to regulate protein synthesis. ISR is activated by several stress conditions, including endoplasmic reticulum (ER) stress, executed by protein kinase R-like endoplasmic reticulum kinase (PERK). We report that ER stress combined with ISR inhibition causes an impaired maturation of several tyrosine kinase receptors (RTKs), consistent with a partial block of their trafficking from the ER to the Golgi. Other proteins mature or are secreted normally, indicating selective retention in the ER (sERr). sERr is relieved upon protein synthesis attenuation and is accompanied by the generation of large mixed disulfide bonded complexes, including ERp44. sERr was pharmacologically recapitulated by combining the HIV-protease inhibitor nelfinavir with ISRIB, an experimental drug that inhibits ISR. Nelfinavir/ISRIB combination is highly effective to inhibit the growth of RTK-addicted cell lines and hepatocellular (HCC) cells in vitro and in vivo. Thus, pharmacological sERr can be utilized as a modality for cancer treatment.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Carcinoma Hepatocelular/tratamento farmacológico , Retículo Endoplasmático/efeitos dos fármacos , Neoplasias Hepáticas/tratamento farmacológico , eIF-2 Quinase/metabolismo , Acetamidas/farmacologia , Acetamidas/uso terapêutico , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Sistemas CRISPR-Cas/genética , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Cicloexilaminas/farmacologia , Cicloexilaminas/uso terapêutico , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Técnicas de Inativação de Genes , Complexo de Golgi/metabolismo , Humanos , Neoplasias Hepáticas/patologia , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Nelfinavir/farmacologia , Nelfinavir/uso terapêutico , Ensaios Antitumorais Modelo de Xenoenxerto , eIF-2 Quinase/genética
18.
Life Sci ; 246: 117415, 2020 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-32035932

RESUMO

High-mobility group box 1 protein (HMGB1) is a crucial proinflammatory cytokine that contributes to acute lung injury (ALI). Macrophages are known to express the primary receptors (Toll-like receptor [TLR] 2, and TLR4) of HMGB1 for transmitting intracellular signals. Studies have revealed that double-stranded RNA activated protein kinase R (PKR), which is expressed in macrophages, participates in ALI by regulating macrophage polarization and proinflammatory cytokine release, and that PKR is normally activated by a subset of TLRs. The present study investigated whether HMGB1 engages in ALI by activating PKR in macrophages and inducing classically activated macrophage (M1) polarization via TLR2- and TLR4-mediated nuclear factor (NF)-κB signaling pathways. In an vivo mouse model of lipopolysaccharide (LPS)-induced ALI, anti-HMGB1, rHMGB1, LPS-RS (TLR2 and TLR4 antagonist), or C16 (PKR inhibitor) was administered to mice 2 h after LPS challenge or 1 h before LPS challenge. In vitro, bone marrow-derived macrophages from mice primed with LPS were stimulated with or without anti-HMGB1, rHMGB1, LPS-RS, or C16. Our studies revealed that rHMGB1 stimulation induced M1 polarization in ALI, and that anti-HMGB1 and C16 treatments had the opposite effect. Anti-HMGB1 and LPS-RS significantly inhibited LPS-induced PKR expression in macrophages; however, rHMGB1 administration increased PKR expression. These results indicate that HMGB1 participates in the pathogenesis of ALI by activating PKR in macrophages and inducing M1 polarization through TLR2- and TLR4-mediated NF-κB signaling pathways.


Assuntos
Lesão Pulmonar Aguda/metabolismo , Proteína HMGB1/fisiologia , Ativação de Macrófagos , eIF-2 Quinase/metabolismo , Lesão Pulmonar Aguda/etiologia , Animais , Western Blotting , Modelos Animais de Doenças , Ativação Enzimática , Ensaio de Imunoadsorção Enzimática , Proteína HMGB1/metabolismo , Interleucinas/metabolismo , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase em Tempo Real , Proteínas da Matriz Viral
19.
Respir Med ; 161: 105852, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-32056726

RESUMO

OBJECTIVE: The aim of the study was to investigate the mechanism and effect of FBXL10 in myocardial ischemia reperfusion injury in vivo and in vitro. METHODS: The myocardial ischemia reperfusion (I/R) model was established by 30 min of coronary occlusion followed by 2 h of reperfusion in rats. Western blot and TUNEL assay were used to measure the apoptosis during I/R. The expression levels of endoplasmic reticulum related proteins in myocardial tissues and H9c2 cells were detected by immunohistochemistry staining and immunofluorescence staining. Flow cytometry and CCK-8 were used to detect the apoptosis and viability of H9c2 cells. RESULTS: The results revealed that FBXL10 significantly reduced myocardial infarction, improved the pathological morphology of myocardium, markedly reduced inflammatory response in the myocardial ischemia reperfusion rats. Moreover the expressions of endoplasmic reticulum stress key proteins were caused by I/R were suppressed significantly by FBXL10 treatment, including CHOP, GRP78, ATF4 and p-PERK. Additionally FBXL10 inhibited the expression of endoplasmic reticulum stress key proteins in H/R H9c2 cells. Furthermore, FBXL10 reduced the levels of apoptotic cells and inflammatory response compared with I/R and H/R group. CONCLUSION: Taken together, we found that FBXL10 could attenuate I/R injury through inhibiting endoplasmic reticulum stress (ERs).


Assuntos
Estresse do Retículo Endoplasmático/genética , Proteínas F-Box/fisiologia , Histona Desmetilases com o Domínio Jumonji/fisiologia , Traumatismo por Reperfusão Miocárdica/genética , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Fator 4 Ativador da Transcrição/genética , Fator 4 Ativador da Transcrição/metabolismo , Animais , Apoptose/genética , Células Cultivadas , Expressão Gênica/genética , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Masculino , Traumatismo por Reperfusão Miocárdica/patologia , Ratos , Ratos Sprague-Dawley , Fator de Transcrição CHOP/genética , Fator de Transcrição CHOP/metabolismo , eIF-2 Quinase/genética , eIF-2 Quinase/metabolismo
20.
Artigo em Inglês | MEDLINE | ID: mdl-31940218

RESUMO

Airway inflammation is a key aspect of diseases such as asthma. Proinflammatory cytokines such as TNFα mediate the inflammatory response. In various diseases, inflammation leads to endoplasmic reticulum (ER) stress, the accumulation of unfolded proteins, which triggers homeostatic responses to restore normal cellular function. We hypothesized that TNFα triggers ER stress through an increase in reactive oxygen species generation in human airway smooth muscle (hASM) with a downstream effect on mitofusin 2 (Mfn2). In hASM cells isolated from lung specimens incidental to patient surgery, dose- and time-dependent effects of TNFα exposure were assessed. Exposure of hASM to tunicamycin was used as a positive control. Tempol (500 µM) was used as superoxide scavenger. Activation of three ER stress pathways were evaluated by Western blotting: 1) autophosphorylation of inositol-requiring enzyme1 (IRE1α) leading to splicing of X-box binding protein 1 (XBP1); 2) autophosphorylation of protein kinase RNA-like endoplasmic reticulum kinase (PERK) leading to phosphorylation of eukaryotic initiation factor 2α; and 3) translocation and cleavage of activating transcription factor 6 (ATF6). We found that exposure of hASM cells to tunicamycin activated all three ER stress pathways. In contrast, TNFα selectively activated the IRE1α/XBP1 pathway in a dose- and time-dependent fashion. Our results indicate that TNFα does not activate the PERK and ATF6 pathways. Exposure of hASM cells to TNFα also decreased Mfn2 protein expression. Concurrent exposure to TNFα and tempol reversed the effect of TNFα on IRE1α phosphorylation and Mfn2 protein expression. Selective activation of the IRE1α/XBP1 pathway in hASM cells after exposure to TNFα may reflect a unique homeostatic role of this pathway in the inflammatory response of hASM cells.


Assuntos
Estresse do Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/patologia , Endorribonucleases/metabolismo , Músculo Liso Vascular/patologia , Proteínas Serina-Treonina Quinases/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Proteína 1 de Ligação a X-Box/metabolismo , Células Cultivadas , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Endorribonucleases/genética , Fator de Iniciação 2 em Eucariotos/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Humanos , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Proteína 1 de Ligação a X-Box/genética , eIF-2 Quinase/genética , eIF-2 Quinase/metabolismo
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