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1.
Life Sci ; 249: 117499, 2020 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-32142763

RESUMO

AIMS: Since several factors are involved in the tumorigenesis process, targeting only one factor most probably cannot overwhelm cancer progression. Therefore, it seems that combination therapy through targeting more than one cancer-related factor may lead to cancer control. The expression and function of p68 (DDX5; DEAD-Box Helicase 5) are dysregulated in various cancers. P68 is also a co-activator of many oncogenic transcription factors such as the signal transducer and activator of transcription-3 (STAT3), which contributes to cancer progression. This close connection between p68 and STAT3 plays an important role in the growth and development of cancer. MATERIALS AND METHODS: We decided to suppress the p68/STAT3 axis in various cancer cells by using Polyethylene glycol-trimethyl Chitosan-Hyaluronic acid (PEG-TMC-HA) nanoparticles (NPs) loaded with siRNA molecules. We assessed the impact of this combination therapy on apoptosis, proliferation, angiogenesis, and tumor growth, both in vitro and in vivo. KEY FINDINGS: The results showed that siRNA-loaded NPs notably suppressed the expression of p68/STAT3 axis in cancer cells, which was associated with blockade of tumor growth, colony formation, angiogenesis, and cancer cell migration. In addition to apoptosis induction, this combined therapy also reduced the expression of several tumor-promoting factors including Fibroblast growth factors (FGF), vascular endothelial growth factor (VEGF), transforming growth factor-ß (TGF-ß), matrix metallopeptidases-2 (MMP-2), MMP-9, hypoxia-inducible factor-(HIF-1α), interleukin-6 (IL-6), IL-33, Bcl-x, vimentin, and snail. SIGNIFICANCE: These findings indicate the potential of this nano-based anti-cancer therapeutic strategy for efficient cancer therapy which should be further investigated in future studies.


Assuntos
Inativação Gênica , Neoplasias/patologia , Fator de Transcrição STAT3/genética , eIF-2 Quinase/genética , Apoptose , Linhagem Celular Tumoral , Progressão da Doença , Humanos , Neoplasias/metabolismo
2.
BMC Med Genet ; 21(1): 61, 2020 03 27.
Artigo em Inglês | MEDLINE | ID: mdl-32216767

RESUMO

BACKGROUND: Wolcott-Rallison Syndrome (WRS) is a rare autosomal recessive disease that is the most common cause of neonatal diabetes in consanguineous families. WRS is caused by various genetic alterations of the Eukaryotic Translation Initiation Factor 2-Alpha Kinase 3 (EIF2AK3) gene. METHODS: Genetic analysis of a consanguineous family where two children were diagnosed with WRS was performed by Sanger sequencing. The altered protein was investigated by in vitro cloning, expression and immunohistochemistry. RESULTS: The first cases in Hungary, - two patients in one family, where the parents were fourth-degree cousins - showed the typical clinical features of WRS: early onset diabetes mellitus with hyperglycemia, growth retardation, infection-induced multiple organ failure. The genetic background of the disease was a novel alteration in the EIF2AK3 gene involving the splice site of exon 11- intron 11-12 boundary: g.53051_53062delinsTG. According to cDNA sequencing this created a new splice site and resulted in a frameshift and the development of an early termination codon at amino acid position 633 (p.Pro627AspfsTer7). Based on in vitro cloning and expression studies, the truncated protein was functionally inactive. Immunohistochemistry revealed that the intact protein was absent in the islets of pancreas, furthermore insulin expressing cells were also dramatically diminished. Elevated GRP78 and reduced CHOP protein expression were observed in the liver. CONCLUSIONS: The novel genetic alteration causing the absence of the EIF2AK3 protein resulted in insufficient handling of severe endoplasmic reticulum stress, leading to liver failure and demise of the patients.


Assuntos
Diabetes Mellitus Tipo 1/genética , Epífises/anormalidades , Mutação INDEL , Osteocondrodisplasias/genética , Sítios de Splice de RNA/genética , eIF-2 Quinase/genética , Pré-Escolar , Consanguinidade , Diabetes Mellitus Tipo 1/diagnóstico , Diabetes Mellitus Tipo 1/patologia , Estresse do Retículo Endoplasmático/genética , Epífises/patologia , Evolução Fatal , Feminino , Mutação da Fase de Leitura , Humanos , Hungria , Lactente , Falência Hepática/complicações , Falência Hepática/genética , Falência Hepática/patologia , Osteocondrodisplasias/diagnóstico , Osteocondrodisplasias/patologia , Linhagem , Irmãos , Viroses/complicações , Viroses/patologia
3.
Am J Hum Genet ; 106(4): 570-583, 2020 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-32197074

RESUMO

EIF2AK1 and EIF2AK2 encode members of the eukaryotic translation initiation factor 2 alpha kinase (EIF2AK) family that inhibits protein synthesis in response to physiologic stress conditions. EIF2AK2 is also involved in innate immune response and the regulation of signal transduction, apoptosis, cell proliferation, and differentiation. Despite these findings, human disorders associated with deleterious variants in EIF2AK1 and EIF2AK2 have not been reported. Here, we describe the identification of nine unrelated individuals with heterozygous de novo missense variants in EIF2AK1 (1/9) or EIF2AK2 (8/9). Features seen in these nine individuals include white matter alterations (9/9), developmental delay (9/9), impaired language (9/9), cognitive impairment (8/9), ataxia (6/9), dysarthria in probands with verbal ability (6/9), hypotonia (7/9), hypertonia (6/9), and involuntary movements (3/9). Individuals with EIF2AK2 variants also exhibit neurological regression in the setting of febrile illness or infection. We use mammalian cell lines and proband-derived fibroblasts to further confirm the pathogenicity of variants in these genes and found reduced kinase activity. EIF2AKs phosphorylate eukaryotic translation initiation factor 2 subunit 1 (EIF2S1, also known as EIF2α), which then inhibits EIF2B activity. Deleterious variants in genes encoding EIF2B proteins cause childhood ataxia with central nervous system hypomyelination/vanishing white matter (CACH/VWM), a leukodystrophy characterized by neurologic regression in the setting of febrile illness and other stressors. Our findings indicate that EIF2AK2 missense variants cause a neurodevelopmental syndrome that may share phenotypic and pathogenic mechanisms with CACH/VWM.


Assuntos
Deficiências do Desenvolvimento/genética , Variação Genética/genética , Leucoencefalopatias/genética , Malformações do Sistema Nervoso/genética , eIF-2 Quinase/genética , Adolescente , Ataxia/genética , Criança , Pré-Escolar , Feminino , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/genética , Humanos , Lactente , Masculino , Substância Branca/patologia
4.
PLoS One ; 15(3): e0229948, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32155190

RESUMO

The integrated stress response (ISR) is one of the most important cytoprotective mechanisms and is integrated by phosphorylation of the α subunit of eukaryotic translation initiation factor 2 (eIF2α). Four eIF2α kinases, heme-regulated inhibitor (HRI), double-stranded RNA-dependent protein kinase (PKR), PKR-like endoplasmic reticulum kinase (PERK), and general control nonderepressible 2 (GCN2), are activated in response to several stress conditions. We previously reported that nanosecond pulsed electric fields (nsPEFs) are a potential therapeutic tool for ISR activation. In this study, we examined which eIF2α kinase is activated by nsPEF treatment. To assess the responsible eIF2α kinase, we used previously established eIF2α kinase quadruple knockout (4KO) and single eIF2α kinase-rescued 4KO mouse embryonic fibroblast (MEF) cells. nsPEFs 70 ns in duration with 30 kV/cm electric fields caused eIF2α phosphorylation in wild-type (WT) MEF cells. On the other hand, nsPEF-induced eIF2α phosphorylation was completely abolished in 4KO MEF cells and was recovered by HRI overexpression. CM-H2DCFDA staining showed that nsPEFs generated reactive oxygen species (ROS), which activated HRI. nsPEF-induced eIF2α phosphorylation was blocked by treatment with the ROS scavenger N-acetyl-L-cysteine (NAC). Our results indicate that the eIF2α kinase HRI is responsible for nsPEF-induced ISR activation and is activated by nsPEF-generated ROS.


Assuntos
Eletricidade/efeitos adversos , Fibroblastos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Estresse Fisiológico/fisiologia , Acetilcisteína/farmacologia , Animais , Linhagem Celular , Técnicas de Inativação de Genes , Camundongos , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Espécies Reativas de Oxigênio/antagonistas & inibidores , Estresse Fisiológico/efeitos dos fármacos , eIF-2 Quinase/genética
5.
Parasite Immunol ; 42(1): e12678, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31610026

RESUMO

Dominant-negative mutation of LdeK1 gene, an eIF2α kinase from Leishmania donovani, revealed its role in translation regulation in response to nutrient starvation earlier. However, whether the kinase influences the infectivity of the parasites which naturally encounters nutrient deprivation during its life cycle was interesting to investigate. Both in vitro and in vivo experiments resulted in decrease of the parasite burden in peritoneal macrophages and in splenic/ hepatic load, respectively. An insight into the immune response of mice infected with mutant parasite showed enhanced pro-inflammatory cytokines and nitric oxide levels but reduced TH 2 and Treg population. The significantly reduced loss of infectivity of the parasites lacking a functional LdeK1 by modulating the immune response towards host protection makes it a potential vaccine candidate against Leishmaniasis.


Assuntos
Leishmania donovani/genética , Leishmania donovani/patogenicidade , Leishmaniose Visceral/imunologia , Leishmaniose Visceral/parasitologia , eIF-2 Quinase/genética , Animais , Citocinas/imunologia , Feminino , Imunidade Celular , Leishmania donovani/imunologia , Fígado/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Óxido Nítrico/metabolismo , Carga Parasitária , Baço/imunologia , Baço/parasitologia , Linfócitos T/imunologia , Virulência
6.
Mol Cells ; 42(11): 783-793, 2019 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-31707777

RESUMO

When endoplasmic reticulum (ER) functions are perturbed, the ER induces several signaling pathways called unfolded protein response to reestablish ER homeostasis through three ER transmembrane proteins: inositol-requiring enzyme 1 (IRE1), PKR-like ER kinase (PERK), and activating transcription factor 6 (ATF6). Although it is important to measure the activity of ATF6 that can indicate the status of the ER, no specific cell-based reporter assay is currently available. Here, we report a new cell-based method for monitoring ER stress based on the cleavage of ATF6α by sequential actions of proteases at the Golgi apparatus during ER stress. A new expressing vector was constructed by using fusion gene of GAL4 DNA binding domain (GAL4DBD) and activation domain derived from herpes simplex virus VP16 protein (VP16AD) followed by a human ATF6α N-terminal deletion variant. During ER stress, the GAL4DBD-VP16AD(GV)-hATF6α deletion variant was cleaved to liberate active transcription activator encompassing GV-hATF6α fragment which could translocate into the nucleus. The translocated GV-hATF6α fragment strongly induced the expression of firefly luciferase in HeLa Luciferase Reporter cell line containing a stably integrated 5X GAL4 site-luciferase gene. The established double stable reporter cell line HLR-GV-hATF6α(333) represents an innovative tool to investigate regulated intramembrane proteolysis of ATF6α. It can substitute active pATF6(N) binding motif-based reporter cell lines.


Assuntos
Fator 6 Ativador da Transcrição/metabolismo , Retículo Endoplasmático/metabolismo , Luciferases/metabolismo , Proteínas de Membrana/metabolismo , Resposta a Proteínas não Dobradas , eIF-2 Quinase/metabolismo , Fator 6 Ativador da Transcrição/genética , Linhagem Celular Tumoral , Ditiotreitol/farmacologia , Retículo Endoplasmático/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Endorribonucleases/genética , Endorribonucleases/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Genes Reporter/genética , Células HeLa , Humanos , Luciferases/genética , Proteínas de Membrana/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteólise , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , eIF-2 Quinase/genética
7.
Science ; 366(6467): 843-849, 2019 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-31727829

RESUMO

Down syndrome (DS) is the most common genetic cause of intellectual disability. Protein homeostasis is essential for normal brain function, but little is known about its role in DS pathophysiology. In this study, we found that the integrated stress response (ISR)-a signaling network that maintains proteostasis-was activated in the brains of DS mice and individuals with DS, reprogramming translation. Genetic and pharmacological suppression of the ISR, by inhibiting the ISR-inducing double-stranded RNA-activated protein kinase or boosting the function of the eukaryotic translation initiation factor eIF2-eIF2B complex, reversed the changes in translation and inhibitory synaptic transmission and rescued the synaptic plasticity and long-term memory deficits in DS mice. Thus, the ISR plays a crucial role in DS, which suggests that tuning of the ISR may provide a promising therapeutic intervention.


Assuntos
Síndrome de Down/fisiopatologia , Síndrome de Down/psicologia , Plasticidade Neuronal , Proteostase/fisiologia , Estresse Fisiológico/fisiologia , Transmissão Sináptica , Animais , Encéfalo/fisiopatologia , Fator de Iniciação 2 em Eucariotos/metabolismo , Memória de Longo Prazo , Camundongos , Camundongos Mutantes , Biossíntese de Proteínas , Estresse Fisiológico/efeitos dos fármacos , Estresse Fisiológico/genética , eIF-2 Quinase/antagonistas & inibidores , eIF-2 Quinase/genética , eIF-2 Quinase/metabolismo
8.
PLoS Pathog ; 15(11): e1008169, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31738790

RESUMO

The unfolded protein response (UPR) in the endoplasmic reticulum (ER) constitutes a critical component of host innate immunity against microbial infections. In this report, we show that porcine reproductive and respiratory syndrome virus (PRRSV) utilizes the UPR machinery for its own benefit. We provide evidence that the virus targets the UPR central regulator GRP78 for proteasomal degradation via a mechanism that requires viral glycoprotein GP2a, while both IRE1-XBP1s and PERK-eIF2α-ATF4 signaling branches of the UPR are turned on at early stage of infection. The activated effector XBP1s was found to enter the nucleus, but ATF4 was unexpectedly diverted to cytoplasmic viral replication complexes by means of nonstructural proteins nsp2/3 to promote viral RNA synthesis. RNAi knockdown of either ATF4 or XBP1s dramatically attenuated virus titers, while overexpression caused increases. These observations reveal attractive host targets (e.g., ATF4 and XBP1s) for antiviral drugs and have implications in vaccine development.


Assuntos
Retículo Endoplasmático/virologia , Síndrome Respiratória e Reprodutiva Suína/virologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/fisiologia , Resposta a Proteínas não Dobradas , Replicação Viral , eIF-2 Quinase/metabolismo , Fator 4 Ativador da Transcrição/genética , Fator 4 Ativador da Transcrição/metabolismo , Animais , Retículo Endoplasmático/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Síndrome Respiratória e Reprodutiva Suína/genética , Síndrome Respiratória e Reprodutiva Suína/metabolismo , Transdução de Sinais , Suínos , Proteína 1 de Ligação a X-Box/genética , Proteína 1 de Ligação a X-Box/metabolismo , eIF-2 Quinase/genética
9.
Mol Cell ; 75(6): 1203-1217.e5, 2019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31494035

RESUMO

In response to foreign and endogenous double-stranded RNA (dsRNA), protein kinase R (PKR) and ribonuclease L (RNase L) reprogram translation in mammalian cells. PKR inhibits translation initiation through eIF2α phosphorylation, which triggers stress granule (SG) formation and promotes translation of stress responsive mRNAs. The mechanisms of RNase L-driven translation repression, its contribution to SG assembly, and its regulation of dsRNA stress-induced mRNAs are unknown. We demonstrate that RNase L drives translational shut-off in response to dsRNA by promoting widespread turnover of mRNAs. This alters stress granule assembly and reprograms translation by allowing translation of mRNAs resistant to RNase L degradation, including numerous antiviral mRNAs such as interferon (IFN)-ß. Individual cells differentially activate dsRNA responses revealing variation that can affect cellular outcomes. This identifies bulk mRNA degradation and the resistance of antiviral mRNAs as the mechanism by which RNase L reprograms translation in response to dsRNA.


Assuntos
Reprogramação Celular , Endorribonucleases/metabolismo , Interferon beta/biossíntese , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , eIF-2 Quinase/metabolismo , Células A549 , Endorribonucleases/genética , Células HEK293 , Humanos , Interferon beta/genética , Estabilidade de RNA , RNA de Cadeia Dupla/genética , RNA de Cadeia Dupla/metabolismo , RNA Mensageiro/genética , eIF-2 Quinase/genética
10.
Molecules ; 24(17)2019 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-31461933

RESUMO

In response to cellular stresses, activating transcriptional factor 4 (ATF4) regulates the expression of both stress-relieving genes and apoptosis-inducing genes, eliciting cell fate determination. Since pharmacological activation of ATF4 exerts potent anti-tumor effects, modulators of ATF4 activation may have potential in cancer therapy. We herein attempted to identify small molecules that activate ATF4. A cell-based screening to monitor TRB3 promoter activation was performed using crude drugs used in traditional Japanese Kampo medicine. We found that an extract from Sophora flavescens roots exhibited potent TRB3 promoter activation. The activity-guided fractionation revealed that kurarinone was identified as the active ingredient. Intriguingly, ATF4 activation in response to kurarinone required PKR-like endoplasmic reticulum kinase (PERK). Moreover, kurarinone induced the cyclin-dependent kinase inhibitor p21 as well as cytostasis in cancer cells. Importantly, the cytostatic effect of kurarinone was reduced by pharmacological inhibition of PERK. These results indicate that kurarinone triggers ATF4 activation through PERK and exerts cytostatic effects on cancer cells. Taken together, our results suggest that modulation of the PERK-ATF4 pathway with kurarinone has potential as a cancer treatment.


Assuntos
Fator 4 Ativador da Transcrição/metabolismo , Proteínas de Ciclo Celular/genética , Flavonoides/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Repressoras/genética , Sophora/química , eIF-2 Quinase/metabolismo , Fator 4 Ativador da Transcrição/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Células HeLa , Humanos , Fosforilação , Regiões Promotoras Genéticas/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/genética , eIF-2 Quinase/genética
11.
Oncogene ; 38(36): 6382-6398, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31312024

RESUMO

The major impediment to effective cancer therapy has been the development of drug resistance. The tumour suppressive transcription factor FOXO3 promotes cell cycle arrest, senescence and cell death, and mediates the cytotoxic and cytostatic functions of cancer therapeutics. In consequence, FOXO3 is often downregulated as an adaptive response in cancer and particularly in chemotherapeutic drug-resistant cells. Consistently, we find that FOXO3 expression is attenuated in the drug-resistant MCF-7-EpiR and MCF-7-TaxR compared to the parental MCF-7 breast cancer cells. Using ChIP, short-interfering RNA (siRNA) knockdown, and overexpression assays as well as Foxo1/3/4-/- MEFs, we establish the endoplasmic reticulum (ER)-stress defence modulator PERK (eIF2AK3) as a direct downstream transcriptional target of FOXO3. In agreement, there is also a positive correlation between FOXO3 and PERK expression at the protein and RNA levels in breast cancer patient samples. We uncover that PERK expression is downregulated but its activity constitutively elevated in the drug-resistant cells. With this in mind, we exploit this adaptive response of low FOXO3 and PERK expression, and high PERK activity in drug-resistant breast cancer cells and show that these drug-resistant cells are specifically sensitive to PERK inhibition. In support of this finding, we show that ectopic overexpression of FOXO3 can reduce the sensitivity of the resistant cells to the PERK inhibitor GSK2606414, while the Foxo1/3/4-/- MEFs expressing lower levels of PERK are more sensitive to PERK inhibition compared to wild-type MEFs. PERK inhibitor-titration and -time course experiments showed that the drug-resistant cells, which express lower expression and higher activity levels of PERK, are more sensitive to the increasing concentrations of PERK inhibitor compared to parental MCF-7 cells. Our present work thus reveals a chemotherapeutic drug-resistant cancer cell vulnerability in PERK and suggests PERK as a potential target for cancer therapy, specifically in the context of drug-resistant cancers.


Assuntos
Resistencia a Medicamentos Antineoplásicos/genética , Proteína Forkhead Box O3/fisiologia , eIF-2 Quinase/genética , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Células Cultivadas , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Estresse do Retículo Endoplasmático/genética , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Células MCF-7 , Camundongos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Transdução de Sinais/efeitos dos fármacos
12.
Biomed Res Int ; 2019: 5320747, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31341901

RESUMO

Autosomal dominant polycystic kidney disease (ADPKD) is mainly caused by mutations in the PKD1 (~85%) or PKD2 (~15%) gene which, respectively, encode polycystin-1 (PC1) and polycystin-2 (PC2). How PC1 regulates cell proliferation and apoptosis has been studied for decades but the underlying mechanisms remain controversial. Protein kinase RNA-activated (PKR) is activated by interferons or double-stranded RNAs, inhibits protein translation, and induces cell apoptosis. In a previous study, we found that PC1 reduces apoptosis through suppressing the PKR/eIF2α signaling. Whether and how PKR is involved in PC1-inhibited proliferation and protein synthesis remains unknown. Here we found that knockdown of PKR abolishes PC1-inhibited proliferation and translation. Because suppressed PKR-eIF2α signaling/activity by PC1 would stimulate, rather than inhibit, the proliferation and translation, we examined the effect of dominant negative PKR mutant K296R that has no kinase activity and found that it enhances the inhibition of proliferation and translation by PC1. Thus, our study showed that inhibition of cell proliferation and protein synthesis by PC1 is mediated by the total expression but not the kinase activity of PKR, possibly through physical association.


Assuntos
Proliferação de Células , Biossíntese de Proteínas/genética , Canais de Cátion TRPP/metabolismo , eIF-2 Quinase/metabolismo , Proliferação de Células/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Células HEK293 , Células HeLa , Humanos , Serina-Treonina Quinases TOR/metabolismo , Canais de Cátion TRPP/genética , eIF-2 Quinase/genética
13.
Nat Commun ; 10(1): 2871, 2019 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-31253805

RESUMO

Adenovirus Virus-Associated (VA) RNAs are the first discovered viral noncoding RNAs. By mimicking double-stranded RNAs (dsRNAs), the exceptionally abundant, multifunctional VA RNAs sabotage host machineries that sense, transport, process, or edit dsRNAs. How VA-I suppresses PKR activation despite its strong dsRNA character, and inhibits the crucial antiviral kinase to promote viral translation, remains largely unknown. Here, we report a 2.7 Å crystal structure of VA-I RNA. The acutely bent VA-I features an unusually structured apical loop, a wobble-enriched, coaxially stacked apical and tetra-stems necessary and sufficient for PKR inhibition, and a central domain pseudoknot that resembles codon-anticodon interactions and prevents PKR activation by VA-I. These global and local structural features collectively define VA-I as an archetypal PKR inhibitor made of RNA. The study provides molecular insights into how viruses circumnavigate cellular rules of self vs non-self RNAs to not only escape, but further compromise host innate immunity.


Assuntos
Conformação de Ácido Nucleico , RNA de Cadeia Dupla/química , RNA Viral/química , Adenovírus Humanos/genética , Sequência de Bases , Cristalização , Luz , RNA de Cadeia Dupla/genética , RNA Viral/genética , eIF-2 Quinase/genética , eIF-2 Quinase/metabolismo
14.
Curr Med Sci ; 39(3): 356-362, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31209803

RESUMO

Poly(ADP-ribose) polymerase 1 (PARP1) plays important roles in the regulation of transcription factors. Mounting evidence has shown that inhibition of PARP1 influences the expression of genes associated with inflammatory response. Interferon regulatory factor 1 (IRF1) is a critical transcription factor for the development of both the innate and adaptive immune responses against infections. However, the molecular mechanism through which PARP1 mediates the effects has not been clearly demonstrated. Jurkat cells were exposed to dexamethasone (Dex) or PARP1 inhibitor PJ34. The expression levels of IL-12, LMP2, OAS1 and PKR were detected using real-time RT-PCR. The interactions between PARP1 and IRF1 were examined by co-immunoprecipitation (co-IP) assays. We further explored the mechanism of PARP1 suppressing IRF1 by assessing the activities of interferon stimulated response element (ISRE). The mRNA expression of IL-12, LMP2, OAS1 and PKR was obviously suppressed by Dex in Jurkat cells, which could be rescued by PJ34 treatment. Luciferase study revealed that poly(ADP-ribosyl)- ation suppressed IRF1-mediated transcription through preventing the binding of IRF1 to ISREs. PARP1 inhibited IRF1-mediated transcription in Jurkat cells by preventing IRF1 binding to ISREs in the promoters of target genes. It is suggested that PARP1 is a crucial regulator of IRF1-mediated immune response. This study provides experimental evidence for the possible application of PARP1 inhibitors in the treatment of IRF1-related immune anergy.


Assuntos
Dexametasona/farmacologia , Regulação Leucêmica da Expressão Gênica , Fator Regulador 1 de Interferon/genética , Poli(ADP-Ribose) Polimerase-1/genética , Transcrição Genética/efeitos dos fármacos , 2',5'-Oligoadenilato Sintetase/antagonistas & inibidores , 2',5'-Oligoadenilato Sintetase/genética , 2',5'-Oligoadenilato Sintetase/imunologia , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/imunologia , Humanos , Fator Regulador 1 de Interferon/imunologia , Interleucina-12/antagonistas & inibidores , Interleucina-12/genética , Interleucina-12/imunologia , Células Jurkat , Fenantrenos/farmacologia , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Poli(ADP-Ribose) Polimerase-1/imunologia , Poli Adenosina Difosfato Ribose/imunologia , Poli Adenosina Difosfato Ribose/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica/efeitos dos fármacos , RNA Mensageiro/antagonistas & inibidores , RNA Mensageiro/genética , RNA Mensageiro/imunologia , Elementos de Resposta , Transdução de Sinais , eIF-2 Quinase/antagonistas & inibidores , eIF-2 Quinase/genética , eIF-2 Quinase/imunologia
15.
Redox Biol ; 26: 101232, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31181458

RESUMO

There is more skeletal muscle tissue in the body than any other tissue and, as it is the organ of the majority of metabolic activity, muscle defect can affect the health of the entire body. Endoplasmic reticulum (ER) stress due to defects in protein folding/degradation balance, altered calcium and lipid levels and alterations in ER-mitochondria contacts has recently been recognised as the pathogenic cause of many different myopathies. In addition, a maladaptive ER stress response triggered by ER stress and mediated by three ER stress sensors (PERK, IRE1 and ATF6) is involved in a failure to relieve muscle tissue from this stress. Targeting ER stress and the ER stress response pathway offers a broad range of opportunities for treating myopathies but, as the inhibition of the three ER stress sensors may not be safe because it could lead to unexpected effects; it therefore calls for careful analysis of the changes in downstream signal transduction in the different myopathies so these sub-pathways can be pharmacologically targeted. This review summarises the known inhibitors of the ER stress response and the successful results obtained using some of them in mouse models of muscle diseases caused by ER stress/ER stress response.


Assuntos
Retículo Endoplasmático/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Doenças Musculares/tratamento farmacológico , Fenilbutiratos/farmacologia , Sulfonamidas/farmacologia , Tiofenos/farmacologia , Fator 6 Ativador da Transcrição/antagonistas & inibidores , Fator 6 Ativador da Transcrição/genética , Fator 6 Ativador da Transcrição/metabolismo , Adenina/análogos & derivados , Adenina/farmacologia , Aldeídos/farmacologia , Animais , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/patologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Endorribonucleases/antagonistas & inibidores , Endorribonucleases/genética , Endorribonucleases/metabolismo , Humanos , Indóis/farmacologia , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Terapia de Alvo Molecular/métodos , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patologia , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Doenças Musculares/genética , Doenças Musculares/metabolismo , Doenças Musculares/patologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , eIF-2 Quinase/antagonistas & inibidores , eIF-2 Quinase/genética , eIF-2 Quinase/metabolismo
16.
RNA ; 25(9): 1192-1201, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31239298

RESUMO

Protein kinase RNA-activated (PKR) is an interferon-inducible kinase that is potently activated by long double-stranded RNA (dsRNA). In a previous study, we found that snoRNAs exhibit increased association with PKR in response to metabolic stress. While it was unclear if snoRNAs also activated PKR in cells, activation in vitro was observed. snoRNAs do not exhibit the double-stranded character typically required for activation of PKR, but some studies suggest such RNAs can activate PKR if triphosphorylated at the 5' terminus, or if they are able to form intermolecular dimers. To interrogate the mechanism of PKR activation by snoRNAs in vitro we focused on SNORD113. Using multiple methods for defining the 5'-phosphorylation state, we find that activation of PKR by SNORD113 does not require a 5'-triphosphate. Gel purification from a native gel followed by analysis using analytical ultracentrifugation showed that dimerization was also not responsible for activation. We isolated distinct conformers of SNORD113 from a native polyacrylamide gel and tracked the activating species to dsRNA formed from antisense RNA synthesized during in vitro transcription with T7 RNA polymerase. Similar studies with additional snoRNAs and small RNAs showed the generality of our results. Our studies suggest that a 5' triphosphate is not an activating ligand for PKR, and emphasize the insidious nature of antisense contamination.


Assuntos
Ativação Enzimática/genética , Polifosfatos/metabolismo , eIF-2 Quinase/genética , eIF-2 Quinase/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Dimerização , Humanos , Ligantes , Fosforilação/genética , Ligação Proteica/genética , RNA de Cadeia Dupla/genética , RNA Nucleolar Pequeno/genética , Transcrição Genética/genética , Ultracentrifugação/métodos , Proteínas Virais/metabolismo
17.
J Pediatr Endocrinol Metab ; 32(6): 607-613, 2019 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-31141482

RESUMO

Background Wolcott-Rallison syndrome is a rare autosomal recessive disorder characterized by neonatal/early-onset non-autoimmune insulin-dependent diabetes, multiple epiphyseal dysphasia and growth retardation. It is caused by mutations in the gene encoding eukaryotic translation initiation factor 2α kinase 3 (EIF2AK3). We aimed to study the clinical characteristics and frequency of the disease in the Iranian population. Methods We recruited 42 patients who referred to the endocrine and metabolism clinic at Mashhad Imam Reza Hospital with neonatal diabetes. Molecular screening of KCNJ11, INS, ABCC8 and EIF2AK3 was performed at the Exeter Molecular Genetics Laboratory, UK. We calculated the frequency of the disease in 124 patients referred from Iran to the Exeter Molecular Genetics Laboratory for genetic screening and compared it to other countries worldwide. Results We identified seven patients as having Wolcott-Rallison syndrome. Genetic testing confirmed the clinical diagnosis and indicated five novel mutations. Only two patients developed clinical features of the syndrome by 6 months of age. Of all 124 cases of Iranian neonatal diabetes referred to the Exeter Molecular Genetics Laboratory for genetic screening, 28 patients (22.58%) had a recessive mutation in EIF2AK3. Conclusions The results of this study raises awareness of the condition and provides further accurate data on the genetic and clinical presentation of Wolcott-Rallison syndrome in the Iranian population. Our study highlights the importance of genetic testing in patients from consanguineous families with diabetes diagnosed within the first 6 months of life.


Assuntos
Biomarcadores/análise , Diabetes Mellitus Tipo 1/complicações , Diabetes Mellitus/etiologia , Epífises/anormalidades , Doenças do Recém-Nascido/etiologia , Osteocondrodisplasias/complicações , eIF-2 Quinase/genética , Criança , Pré-Escolar , Consanguinidade , Diabetes Mellitus/diagnóstico , Diabetes Mellitus/epidemiologia , Feminino , Humanos , Recém-Nascido , Doenças do Recém-Nascido/diagnóstico , Doenças do Recém-Nascido/epidemiologia , Irã (Geográfico)/epidemiologia , Masculino , Mutação , Prognóstico
18.
Int J Mol Sci ; 20(7)2019 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-30939763

RESUMO

The demyelinating canine distemper virus (CDV)-leukoencephalitis represents a translational animal model for multiple sclerosis. The present study investigated the expression of type I interferon (IFN-I) pathway members in CDV-induced cerebellar lesions to gain an insight into their role in lesion development. Gene expression of 110 manually selected genes in acute, subacute and chronic lesions was analyzed using pre-existing microarray data. Interferon regulatory factor (IRF) 3, IRF7, signal transducer and activator of transcription (STAT) 1, STAT2, MX protein, protein kinase R (PKR), 2'-5'-oligoadenylate synthetase (OAS) 1 and interferon-stimulated gene (ISG) 15 expression were also evaluated using immunohistochemistry. Cellular origin of STAT1, STAT2, MX and PKR were determined using immunofluorescence. CDV infection caused an increased expression of the antiviral effector proteins MX, PKR, OAS1 and ISG15, which probably contributed to a restricted viral replication, particularly in neurons and oligodendrocytes. This increase might be partly mediated by IRF-dependent pathways due to the lack of changes in IFN-I levels and absence of STAT2 in astrocytes. Nevertheless, activated microglia/macrophages showed a strong expression of STAT1, STAT2 and MX proteins in later stages of the disease, indicating a strong activation of the IFN-I signaling cascade, which might be involved in the aggravation of bystander demyelination.


Assuntos
Cinomose/genética , Imunidade Inata/genética , Interferons/farmacologia , 2',5'-Oligoadenilato Sintetase/genética , 2',5'-Oligoadenilato Sintetase/metabolismo , Animais , Cerebelo/efeitos dos fármacos , Cerebelo/metabolismo , Cinomose/imunologia , Cães , Fator Regulador 7 de Interferon/genética , Fator Regulador 7 de Interferon/metabolismo , Proteínas de Resistência a Myxovirus/genética , Proteínas de Resistência a Myxovirus/metabolismo , Fatores de Transcrição STAT/genética , Fatores de Transcrição STAT/metabolismo , Ubiquitinas/genética , Ubiquitinas/metabolismo , eIF-2 Quinase/genética , eIF-2 Quinase/metabolismo
19.
Cell Death Dis ; 10(4): 300, 2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30931942

RESUMO

IRE1, PERK, and ATF6 are the three transducers of the mammalian canonical unfolded protein response (UPR). GSK2606414 is a potent inhibitor of PERK, while KIRA6 inhibits the kinase activity of IRE1. Both molecules are frequently used to probe the biological roles of the UPR in mammalian cells. In a direct binding assay, GSK2606414 bound to the cytoplasmic domain of KIT with dissociation constants (Kd) value of 664 ± 294 nM whereas KIRA6 showed a Kd value of 10.8 ± 2.9 µM. In silico docking studies confirmed a compact interaction of GSK2606414 and KIRA6 with KIT ATP binding pocket. In cultured cells, GSK2606414 inhibited KIT tyrosine kinase activity at nanomolar concentrations and in a PERK-independent manner. Moreover, in contrast to other KIT inhibitors, GSK2606414 enhanced KIT endocytosis and its lysosomal degradation. Although KIRA6 also inhibited KIT at nanomolar concentrations, it did not prompt KIT degradation, and rescued KIT from GSK2606414-mediated degradation. Consistent with KIT inhibition, nanomolar concentrations of GSK2606414 and KIRA6 were sufficient to induce cell death in a KIT signaling-dependent mast cell leukemia cell line. Our data show for the first time that KIT is a shared target for two seemingly unrelated UPR inhibitors at concentrations that overlap with PERK and IRE1 inhibition. Furthermore, these data underscore discrepancies between in vitro binding measurements of kinase inhibitors and inhibition of the tyrosine kinase receptors in living cells.


Assuntos
Adenina/análogos & derivados , Indóis/farmacologia , Proteínas Proto-Oncogênicas c-kit/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-kit/metabolismo , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Adenina/química , Adenina/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Endocitose/efeitos dos fármacos , Células HEK293 , Células Hep G2 , Humanos , Indóis/química , Cinética , Lisossomos/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-kit/genética , eIF-2 Quinase/antagonistas & inibidores , eIF-2 Quinase/genética , eIF-2 Quinase/metabolismo
20.
Int J Mol Sci ; 20(7)2019 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-30970629

RESUMO

Proline-rich extensin-like receptor kinases (PERKs) are an important class of receptor kinases in plants. Receptor kinases comprise large gene families in many plant species, including the 15 PERK genes in Arabidopsis. At present, there is no comprehensive published study of PERK genes in G. hirsutum. Our study identified 33 PERK genes in G. hirsutum. Phylogenetic analysis of conserved PERK protein sequences from 15 plant species grouped them into four well defined clades. The GhPERK gene family is an evolutionarily advanced gene family that lost its introns over time. Several cis-elements were identified in the promoter regions of the GhPERK genes that are important in regulating growth, development, light responses and the response to several stresses. In addition, we found evidence for gene loss or addition through segmental or whole genome duplication in cotton. Gene duplication and synteny analysis identified 149 orthologous/paralogous gene pairs. Ka/Ks values show that most GhPERK genes experienced strong purifying selection during the rapid evolution of the gene family. GhPERK genes showed high expression levels in leaves and during ovule development. Furthermore, the expression of GhPERK genes can be regulated by abiotic stresses and phytohormone treatments. Additionally, PERK genes could be involved in several molecular, biological and physiological processes that might be the result of functional divergence.


Assuntos
Duplicação Gênica , Gossypium/genética , Folhas de Planta/genética , eIF-2 Quinase/genética , Sequência de Aminoácidos , Simulação por Computador , Evolução Molecular , Regulação da Expressão Gênica de Plantas , Genoma de Planta , Família Multigênica , Filogenia , Proteínas de Plantas/genética
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