RESUMO
IMPORTANCE: One of the fundamental features that make viruses intracellular parasites is the necessity to use cellular translational machinery. Hence, this is a crucial checkpoint for controlling infections. Here, we show that dengue and Zika viruses, responsible for nearly 400 million infections every year worldwide, explore such control for optimal replication. Using immunocompetent cells, we demonstrate that arrest of protein translations happens after sensing of dsRNA and that the information required to avoid this blocking is contained in viral 5'-UTR. Our work, therefore, suggests that the non-canonical translation described for these viruses is engaged when the intracellular stress response is activated.
Assuntos
Vírus da Dengue , Estresse Fisiológico , Replicação Viral , Zika virus , eIF-2 Quinase , Animais , Humanos , Células A549 , Chlorocebus aethiops , Dengue/imunologia , Dengue/virologia , Vírus da Dengue/fisiologia , eIF-2 Quinase/genética , eIF-2 Quinase/metabolismo , Fator de Iniciação 2 em Eucariotos/metabolismo , Deleção de Genes , Biossíntese de Proteínas/genética , Biossíntese de Proteínas/imunologia , Estresse Fisiológico/genética , Estresse Fisiológico/imunologia , Células Vero , Replicação Viral/genética , Replicação Viral/imunologia , Zika virus/fisiologia , Infecção por Zika virus/imunologia , Infecção por Zika virus/virologia , RNA de Cadeia Dupla/metabolismoRESUMO
The TcK2 protein kinase of Trypanosoma cruzi, the causative agent of Chagas disease, is structurally similar to the human kinase PERK, which phosphorylates the initiation factor eIF2α and, in turn, inhibits translation initiation. We have previously shown that absence of TcK2 kinase impairs parasite proliferation within mammalian cells, positioning it as a potential target for treatment of Chagas disease. To better understand its role in the parasite, here we initially confirmed the importance of TcK2 in parasite proliferation by generating CRISPR/Cas9 TcK2-null cells, albeit they more efficiently differentiate into infective forms. Proteomics indicates that the TcK2 knockout of proliferative forms expresses proteins including trans-sialidases, normally restricted to infective and nonproliferative trypomastigotes explaining decreased proliferation and better differentiation. TcK2 knockout cells lost phosphorylation of eukaryotic initiation factor 3 and cyclic AMP responsive-like element, recognized to promote growth, likely explaining both decreased proliferation and augmented differentiation. To identify specific inhibitors, a library of 379 kinase inhibitors was screened by differential scanning fluorimetry using a recombinant TcK2 encompassing the kinase domain and selected molecules were tested for kinase inhibition. Only Dasatinib and PF-477736, inhibitors of Src/Abl and ChK1 kinases, showed inhibitory activity with IC50 of 0.2 ± 0.02 mM and 0.8 ± 0.1, respectively. In infected cells Dasatinib inhibited growth of parental amastigotes (IC50 = 0.6 ± 0.2 mM) but not TcK2 of depleted parasites (IC50 > 34 mM) identifying Dasatinib as a potential lead for development of therapeutics for Chagas disease targeting TcK2.
Assuntos
Doença de Chagas , Parasitos , Trypanosoma cruzi , Animais , Humanos , Trypanosoma cruzi/genética , eIF-2 Quinase/genética , eIF-2 Quinase/metabolismo , Dasatinibe , Doença de Chagas/tratamento farmacológico , Doença de Chagas/parasitologia , Proliferação de Células , Mamíferos/metabolismoRESUMO
The accumulation of unfolded proteins within the Endoplasmic Reticulum (ER) activates a signal transduction pathway termed the unfolded protein response (UPR), which attempts to restore ER homoeostasis. If this cannot be done, UPR signalling ultimately induces apoptosis. Ca2+ depletion in the ER is a potent inducer of ER stress. Despite the ubiquity of Ca2+ as an intracellular messenger, the precise mechanism(s) by which Ca2+ release affects the UPR remains unknown. Tethering a genetically encoded Ca2+ indicator (GCamP6) to the ER membrane revealed novel Ca2+ signalling events initiated by Ca2+ microdomains in human astrocytes under ER stress, induced by tunicamycin (Tm), an N-glycosylation inhibitor, as well as in a cell model deficient in all three inositol triphosphate receptor isoforms. Pharmacological and molecular studies indicate that these local events are mediated by translocons and that the Ca2+ microdomains impact (PKR)-like-ER kinase (PERK), an UPR sensor, activation. These findings reveal the existence of a Ca2+ signal mechanism by which stressor-mediated Ca2+ release regulates ER stress.
Assuntos
Estresse do Retículo Endoplasmático , eIF-2 Quinase , Apoptose , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático/fisiologia , Humanos , Transdução de Sinais , Resposta a Proteínas não Dobradas , eIF-2 Quinase/genética , eIF-2 Quinase/metabolismoRESUMO
Leishmania donovani is a protozoan parasite that causes visceral leishmaniasis, provoking liver and spleen tissue destruction that is lethal unless treated. The parasite replicates in macrophages and modulates host microbicidal responses. We have previously reported that neutrophil elastase (NE) is required to sustain L. donovani intracellular growth in macrophages through the induction of interferon beta (IFN-ß). Here, we show that the gene expression of IFN-ß by infected macrophages was reduced by half when TLR4 was blocked by pre-treatment with neutralizing antibodies or in macrophages from tlr2-/- mice, while the levels in macrophages from myd88-/- mice were comparable to those from wild-type C57BL/6 mice. The neutralization of TLR4 in tlr2-/- macrophages completely abolished induction of IFN-ß gene expression upon parasite infection, indicating an additive role for both TLRs. Induction of type I interferon (IFN-I), OASL2, SOD1, and IL10 gene expression by L. donovani was completely abolished in macrophages from NE knock-out mice (ela2-/-) or from protein kinase R (PKR) knock-out mice (pkr-/-), and in C57BL/6 macrophages infected with transgenic L. donovani expressing the inhibitor of serine peptidase 2 (ISP2). Parasite intracellular growth was impaired in pkr-/- macrophages but was fully restored by the addition of exogenous IFN-ß, and parasite burdens were reduced in the spleen of pkr-/- mice at 7 days, as compared to the 129Sv/Ev background mice. Furthermore, parasites were unable to grow in macrophages lacking TLR3, which correlated with lack of IFN-I gene expression. Thus, L. donovani engages innate responses in infected macrophages via TLR2, TLR4, and TLR3, via downstream PKR, to induce the expression of pro-survival genes in the host cell, and guarantee parasite intracellular development.
Assuntos
Interferon-alfa/metabolismo , Interferon beta/metabolismo , Leishmania donovani/imunologia , Leishmaniose Visceral/imunologia , Macrófagos Peritoneais/imunologia , Transdução de Sinais/genética , Receptor 2 Toll-Like/metabolismo , Receptor 3 Toll-Like/metabolismo , Receptor 4 Toll-Like/metabolismo , eIF-2 Quinase/metabolismo , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/farmacologia , Feminino , Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/imunologia , Técnicas de Inativação de Genes , Interferon-alfa/genética , Interferon beta/genética , Leishmaniose Visceral/parasitologia , Elastase de Leucócito/antagonistas & inibidores , Elastase de Leucócito/genética , Elastase de Leucócito/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Sulfonamidas/farmacologia , Receptor 2 Toll-Like/genética , Receptor 3 Toll-Like/genética , Receptor 4 Toll-Like/antagonistas & inibidores , Receptor 4 Toll-Like/imunologia , eIF-2 Quinase/genéticaRESUMO
Ulcerative colitis (UC) is characterized by a chronic overproduction of proinflammatory cytokines. During an acute phase, the endoplasmic reticulum (ER) is overloaded and the protein folding process is impaired, a condition named ER stress. This state induces a response (unfolded protein response (UPR)), initiated by the activation of IRE1/Xbp-1, PERK/eIF2α, and ATF6 pathways, which has previously been linked to intestinal inflammation in experimental models. ER stress and UPR activation trigger the activation of proinflammatory, autophagy, and apoptosis genes, in addition to promoting protein degradation. Therefore, the goal of this study was to evaluate the activation of ER stress and UPR in colonic mucosa of UC patients. Patient and Methods. Transcriptional analysis of ER stress- and UPR-related genes was performed by qPCR from intestinal mucosa of patients with UC. We also performed in situ hybridization (ISH) and immunohistochemistry (IHQ) of PERK/eIF2α and IRE1/Xbp-1 pathways and UPR-related chaperones. Results. We first evaluated inflammatory genes via qPCR, and we observed that all analyzed proinflammatory transcripts were upregulated in UC patients. ISH and IHQ images showed that ER stress is activated via PERK/eIF2α and IRE1/Xbp-1 pathways not only in intestinal epithelial cells but also in cells of the lamina propria of UC colonic mucosa. Transcriptional analysis confirmed that EIF2AK3 was upregulated in UC patients. UPR-related genes, such as ATF3, STC2, and DDIT3, along with the chaperones and cochaperones DNAJC3, CALR, HSP90B1, and HSPA5, were also upregulated in UC patients. In addition, we observed that proapoptotic and autophagy genes (Bax and ATG6L1, respectively) were also upregulated. Conclusion. Our results suggest that ER stress and UPR are indeed activated in UC patients and this may contribute to the chronic inflammatory process seen in UC. The increased apoptosis and autophagy markers further support the activation of these findings once they are activated to counterbalance tissue damage. These findings provide new insights into the molecular mechanisms that maintain UC activity and open new possibilities to attenuate intestinal inflammation.
Assuntos
Colite Ulcerativa , Estresse do Retículo Endoplasmático , Endorribonucleases , Proteínas Serina-Treonina Quinases , eIF-2 Quinase , Colite Ulcerativa/metabolismo , Estresse do Retículo Endoplasmático/genética , Endorribonucleases/genética , Endorribonucleases/metabolismo , Humanos , Mucosa Intestinal/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Resposta a Proteínas não Dobradas , eIF-2 Quinase/genética , eIF-2 Quinase/metabolismoRESUMO
HIV-1-associated neurocognitive disorders (HAND) are a major concern in HIV-infected individuals despite the currently available antiretroviral therapy regime. Impaired M1 pro-inflammatory microglial activation is considered one of the hallmark features of HAND neuropathogenesis, and it has been suggested that circulant HIV-1 transactivator protein (Tat) can play a critical role in this process. At the same time, endoplasmatic reticulum (ER) stress has also been implicated in neurodegenerative conditions resulting from the accumulation of misfolded proteins and subsequent unfolded protein response (UPR) deflagration. Here, we demonstrate that pharmacological inhibition of UPR-related protein kinase R-like endoplasmic reticulum kinase (PERK) can attenuate HIV-1 Tat-induced M1 inflammatory state in microglia in vitro. Our initial experiments demonstrate that the bystander stimulus of recombinant Tat on BV-2 microglial cells result in the coupled overexpression of central UPR markers and pro-inflammatory mediators such as iNOS, surface CD16/32 and secreted tumour necrosis factor-α (TNF-α), IL-6, monocyte chemoattractant protein (MCP)-1 and NO. We show that blocking PERK-eIF2-α-ATF4 signalling using the PERK inhibitor GSK2606414 leads to reduced inflammatory response in M1-like BV-2 cells activated by recombinant Tat. Taken together, these findings suggest that PERK targeting may provide a therapeutic intervention to mitigate against lasting neuroinflammation and neuronal loss in of HAND.
Assuntos
Microglia , Resposta a Proteínas não Dobradas , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático , Microglia/metabolismo , Fenótipo , eIF-2 Quinase/genética , eIF-2 Quinase/metabolismoRESUMO
Retinal neuron apoptosis is a key component of diabetic retinopathy (DR), one of the most common complications of diabetes. Stress due to persistent hyperglycaemia and corresponding glucotoxicity represents one of the primary pathogenic mechanisms of diabetes and its complications. Apoptosis of retinal neurons serves a critical role in the pathogenesis of DR observed in patients with diabetes and streptozotocin (STZ)induced diabetic rats. Retinal neuron apoptosis occurs one month after STZ injection, which is considered the early stage of DR. The molecular mechanism involved in the suppression of retinal neuron apoptosis during the early stage of DR remains unclear. RNAdependent protein kinase (PKR) is a stresssensitive proapoptotic kinase. Our previous study indicated that PKRassociated protein X, a stresssensitive activator of PKR, is upregulated in the early stage of STZinduced diabetes. In order to assess the role of PKR in DR prior to apoptosis of retinal neurons, immunofluorescence and western blotting were performed to investigate the cellular localization and expression of PKR in the retina in the early stage of STZinduced diabetes in rats. PKR activity was indirectly assessed by expression levels of phosphorylated eukaryotic translation initiation factor 2α (peIF2α) and the presence of apoptotic cells in the retina was investigated by TUNEL assay. The findings revealed that PKR was localized in the nucleus of retinal ganglion and inner nuclear layer cells from normal and diabetic rats. To the best of our knowledge, the present study is the first to demonstrate nuclear localization of PKR in retinal neurons. Immunofluorescence analysis demonstrated that PKR was expressed in the nuclei of retinal neurons at 3 and 6 days and its expression was decreased at 15 days after STZ treatment. In addition, peIF2α expression and cellular localization followed the trend of PKR, suggesting that this proapoptotic kinase was active in the nuclei of retinal neurons. These findings are consistent with the hypothesis that nuclear translocation of PKR may be a mechanism to sequester active PKR, thus preventing upregulation of cytosolic signalling pathways that induce apoptosis in retinal neurons. Apoptotic cells were not detected in the retina in the early stage of DR. A model was proposed to explain the mechanism by which apoptosis of retinal neurons by PKR is suppressed in the early stage of DR. The possible role of mitochondrial RNA (mtRNA) and Alu RNA in this phenomenon is also discussed since it was demonstrated that the cellular stress due to prolonged hyperglycaemia induces the release of mtRNA and transcription of Alu RNA. Moreover, it mtRNA activates PKR, whereas Alu RNA inhibits the activation of this protein kinase.
Assuntos
Diabetes Mellitus Experimental/metabolismo , Retinopatia Diabética/metabolismo , Neurônios Retinianos/metabolismo , eIF-2 Quinase/metabolismo , Animais , Apoptose/genética , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/genética , Retinopatia Diabética/etiologia , Retinopatia Diabética/genética , Retinopatia Diabética/patologia , Regulação para Baixo , Fator de Iniciação 2 em Eucariotos/metabolismo , Masculino , Ratos Wistar , Estreptozocina , eIF-2 Quinase/genéticaRESUMO
In eukaryotes, phosphorylation of the α-subunit of eIF2 is a mechanism to adjust cellular gene expression profiles in response to specific signals. The eIF2α kinases are a group of serine-threonine kinases that perform important functions in response to infection, proteotoxicity, and nutrient scavenging. The conserved nature of eIF2α kinases among fungi makes them potential evolutionary markers, which may contribute to deeper understanding of taxonomy and evolution. To date, only few studies are available of eIF2α kinases in black yeasts, which are members of Chaetothyriales containing potential agents of a gamut of major human diseases, such as chromoblastomycosis, phaeohyphomycosis and mycetoma. To establish the phylogenetic validity of sequences of eIF2α kinases hypothetical genes, we compared these genes between members of different classes of fungi, including black yeasts and allies, aiming at evaluation of the phylogeny of this group using an alternative molecular marker, compared to standard ribosomal genes. Trees generated with eIF2α kinase sequences of fungi were compared with those generated by ribosomal internal transcribed spacers (ITS rDNA) sequences from the same species. Sequences used were obtained from the protein Non-redundant database of NCBI, were aligned using CLUSTALX v1.8 and alignments were analyzed with RAxML v8.2.9 on the CIPRES Science Gateway portal. The trees generated had similar topologies, demonstrating that eIF2α kinases hypothetical gene sequences present a coherent reflection of evolution among fungi, compared to trees reconstructed by the use of ribosomal sequences. Our preliminary findings with a limited dataset strongly suggest that the evolution of kinases among black yeasts follows a similar path as revealed by ribosomal data, which underlines the validity of current taxonomy of black yeasts and relatives.
Assuntos
Ascomicetos , Genes Fúngicos , Filogenia , eIF-2 Quinase/genética , Ascomicetos/enzimologia , Ascomicetos/genética , DNA Ribossômico/genéticaRESUMO
The current standard of care for locally advanced rectal cancer (RC) is neoadjuvant radio-chemotherapy (NRC) with 5-fluorouracil (5Fu) as the main drug, followed by surgery and adjuvant chemotherapy. While a group of patients will achieve a pathological complete response, a significant percentage will not respond to the treatment. The Unfolding Protein Response (UPR) pathway is generally activated in tumors and results in resistance to radio-chemotherapy. We previously showed that RHBDD2 gene is overexpressed in the advanced stages of colorectal cancer (CRC) and that it could modulate the UPR pathway. Moreover, RHBDD2 expression is induced by 5Fu. In this study, we demonstrate that the overexpression of RHBDD2 in CACO2 cell line confers resistance to 5Fu, favors cell migration, adhesion and proliferation and has a profound impact on the expression of both, the UPR genes BiP, PERK and CHOP, and on the cell adhesion genes FAK and PXN. We also determined that RHBDD2 binds to BiP protein, the master UPR regulator. Finally, we confirmed that a high expression of RHBDD2 in RC tumors after NRC treatment is associated with the development of local or distant metastases. The collected evidence positions RHBDD2 as a promising prognostic biomarker to predict the response to neoadjuvant therapy in patients with RC.
Assuntos
Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica , Proteínas de Membrana/genética , Neoplasias Retais/terapia , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Antimetabólitos Antineoplásicos/farmacologia , Células CACO-2 , Adesão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Chaperona BiP do Retículo Endoplasmático , Fluoruracila/farmacologia , Quinase 1 de Adesão Focal/genética , Quinase 1 de Adesão Focal/metabolismo , Adesões Focais/efeitos dos fármacos , Células HCT116 , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Metástase Linfática , Proteínas de Membrana/metabolismo , Terapia Neoadjuvante/métodos , Paxilina/genética , Paxilina/metabolismo , Ligação Proteica , Neoplasias Retais/genética , Neoplasias Retais/metabolismo , Neoplasias Retais/patologia , Transdução de Sinais , Fator de Transcrição CHOP/genética , Fator de Transcrição CHOP/metabolismo , eIF-2 Quinase/genética , eIF-2 Quinase/metabolismoRESUMO
Reactivation of fetal hemoglobin remains a critical goal in the treatment of patients with sickle cell disease and ß-thalassemia. Previously, we discovered that silencing of the fetal γ-globin gene requires the erythroid-specific eIF2α kinase heme-regulated inhibitor (HRI), suggesting that HRI might present a pharmacologic target for raising fetal hemoglobin levels. Here, via a CRISPR-Cas9-guided loss-of-function screen in human erythroblasts, we identify transcription factor ATF4, a known HRI-regulated protein, as a novel γ-globin regulator. ATF4 directly stimulates transcription of BCL11A, a repressor of γ-globin transcription, by binding to its enhancer and fostering enhancer-promoter contacts. Notably, HRI-deficient mice display normal Bcl11a levels, suggesting species-selective regulation, which we explain here by demonstrating that the analogous ATF4 motif at the murine Bcl11a enhancer is largely dispensable. Our studies uncover a linear signaling pathway from HRI to ATF4 to BCL11A to γ-globin and illustrate potential limits of murine models of globin gene regulation.