RESUMO
PURPOSE: Vault (vt) RNAs are noncoding (nc) RNAs transcribed by RNA polymerase III (RNA Pol III) with 5'-triphosphate (5'-PPP) termini that play significant roles and are recognized by innate immune sensors, including retinoic acid-inducible protein 1 (RIG-I). In addition, vtRNAs adopt secondary structures that can be targets of interferon-inducible protein kinase R (PKR) and the oligoadenylate synthetase (OAS)/RNase L system, both of which are important for activating antiviral defenses. However, changes in the expression of vtRNAs have been associated with pathological processes that activate proinflammatory pathways, which influence cellular events such as differentiation, aging, autophagy, apoptosis, and drug resistance in cancer cells. RESULTS: In this review, we summarized the biology of vtRNAs and focused on their interactions with the innate immune system. These findings provide insights into the diverse roles of vtRNAs and their correlation with various cellular processes to improve our understanding of their biological functions.
Assuntos
Imunidade Inata , Interferons , Imunidade Inata/genética , Apoptose , AutofagiaRESUMO
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 protozoan parasite Leishmania (L.) amazonensis infects and replicates inside host macrophages due to subversion of the innate host cell response. In the present study, we demonstrate that TLR3 is required for the intracellular growth of L. (L.) amazonensis. We observed restricted intracellular infection of TLR3-/- mouse macrophages, reduced levels of IFN1ß and IL-10, and increased levels of IL-12 upon L. (L.) amazonensis infection, compared with their wild-type counterparts. Accordingly, in vivo infection of TLR3-/- mice with L. (L.) amazonensis displayed a significant reduction in lesion size. Leishmania (L.) amazonensis infection induced TLR3 proteolytic cleavage, which is a process required for TLR3 signaling. The chemical inhibition of TLR3 cleavage or infection by CPB-deficient mutant L. (L.) mexicana resulted in reduced parasite load and restricted the expression of IFN1ß and IL-10. Furthermore, we show that the dsRNA sensor molecule PKR (dsRNA-activated protein kinase) cooperates with TLR3 signaling to potentiate the expression of IL-10 and IFN1ß and parasite survival. Altogether, our results show that TLR3 signaling is engaged during L. (L.) amazonensis infection and this component of innate immunity modulates the host cell response.
Assuntos
Leishmania mexicana , Leishmaniose , Parasitos , Receptor 3 Toll-Like , Animais , Interleucina-10/metabolismo , Interleucina-12/metabolismo , Leishmania mexicana/metabolismo , Leishmaniose/metabolismo , Leishmaniose/parasitologia , Camundongos , Parasitos/metabolismo , Proteínas Quinases/metabolismo , Receptor 3 Toll-Like/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
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
GM2-gangliosidosis, a subgroup of lysosomal storage disorders, is caused by deficiency of hexosaminidase activity, and comprises the closely related Tay-Sachs and Sandhoff diseases. The enzyme deficiency prevents normal metabolization of ganglioside GM2, usually resulting in progressive neurodegenerative disease. The molecular mechanisms whereby GM2 accumulation in neurons triggers neurodegeneration remain unclear. In vitro experiments, using microsomes from Sandhoff mouse model brain, showed that increase of GM2 content negatively modulates sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) (Pelled et al., 2003). Furthermore, Ca2+ depletion in endoplasmic reticulum (ER) triggers Unfolded Protein Response (UPR), which tends to restore homeostasis in the ER; however, if cellular damage persists, an apoptotic response is initiated. We found that ER GM2 accumulation in cultured neurons induces luminal Ca2+ depletion, which in turn activates PERK (protein kinase RNA [PKR]-like ER kinase), one of three UPR sensors. PERK signaling displayed biphasic activation; i.e., early upregulation of cytoprotective calcineurin (CN) and, under prolonged ER stress, enhanced expression of pro-apoptotic transcription factor C/EBP homologous protein (CHOP). Moreover, GM2 accumulation in neuronal cells induced neurite atrophy and apoptosis. Both processes were effectively modulated by treatment with the selective PERK inhibitor GSK2606414, by CN knockdown, and by CHOP knockdown. Overall, our findings demonstrate the essential role of PERK signaling pathway contributing to neurodegeneration in a model of GM2-gangliosidosis.
Assuntos
Gangliosidoses GM2/metabolismo , Neuritos/fisiologia , eIF-2 Quinase/metabolismo , Adenina/análogos & derivados , Adenina/farmacologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Atrofia/metabolismo , Linhagem Celular Tumoral , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático/fisiologia , Gangliosídeo G(M2)/metabolismo , Gangliosídeo G(M2)/fisiologia , Gangliosidoses GM2/genética , Indóis/farmacologia , Camundongos , Neuritos/metabolismo , Doenças Neurodegenerativas/metabolismo , Neurônios/metabolismo , Transdução de Sinais/genética , Fator de Transcrição CHOP/metabolismo , Resposta a Proteínas não Dobradas/fisiologia , eIF-2 Quinase/fisiologiaRESUMO
Activation of an appropriate innate immune response to bacterial infection is critical to limit microbial spread and generate cytokines and chemokines to instruct appropriate adaptive immune responses. Recognition of bacteria or bacterial products by pattern recognition molecules is crucial to initiate this response. However, it is increasingly clear that the context in which this recognition occurs can dictate the quality of the response and determine the outcome of an infection. The cross talk established between host and pathogen results in profound alterations on cellular homeostasis triggering specific cellular stress responses. In particular, the highly conserved integrated stress response (ISR) has been shown to shape the host response to bacterial pathogens by sensing cellular insults resulting from infection and modulating transcription of key genes, translation of new proteins and cell autonomous antimicrobial mechanisms such as autophagy. Here, we review the growing body of evidence demonstrating a role for the ISR as an integral part of the innate immune response to bacterial pathogens.
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Excessive exposure to ultraviolet (UV) rays can cause damage of the skin and may induce cancer, immunosuppression, photoaging, and inflammation. The long non-coding RNA (lncRNA) HOX antisense intergenic RNA (HOTAIR) is involved in multiple human biological processes. However, its role in UVB-induced keratinocyte injury is unclear. This study was performed to investigate the effects of HOTAIR in UVB-induced apoptosis and inflammatory injury in human keratinocytes (HaCaT cells). Quantitative real-time polymerase chain reaction was performed to analyze the expression levels of HOTAIR, PKR, TNF-α, and IL-6. Cell viability was measured using trypan blue exclusion method and cell apoptosis using flow cytometry and western blot. ELISA was used to measure the concentrations of TNF-α and IL-6. Western blot was used to measure the expression of PKR, apoptosis-related proteins, and PI3K/AKT and NF-κB pathway proteins. UVB induced HaCaT cell injury by inhibiting cell viability and promoting cell apoptosis and expressions of IL-6 and TNF-α. UVB also promoted the expression of HOTAIR. HOTAIR suppression increased cell viability and decreased apoptosis and expression of inflammatory factors in UVB-treated cells. HOTAIR also promoted the expression of PKR. Overexpression of HOTAIR decreased cell viability and increased cell apoptosis and expression of inflammatory factors in UVB-treated cells by upregulating PKR. Overexpression of PKR decreased cell viability and promoted cell apoptosis in UVB-treated cells. Overexpression of PKR activated PI3K/AKT and NF-κB pathways. Our findings identified an essential role of HOTAIR in promoting UVB-induced apoptosis and inflammatory injury by up-regulating PKR in keratinocytes.
Assuntos
Humanos , Queratinócitos/metabolismo , Apoptose/fisiologia , eIF-2 Quinase/metabolismo , Fator de Indução de Apoptose/metabolismo , RNA Longo não Codificante/metabolismo , Raios Ultravioleta/efeitos adversos , Expressão Gênica , Queratinócitos/efeitos da radiação , Regulação para Cima , Sobrevivência Celular/fisiologia , NF-kappa B/efeitos dos fármacos , NF-kappa B/metabolismo , Interleucina-6/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Apoptose/efeitos da radiação , Fosfatidilinositol 3-Quinases/metabolismo , Inflamação/etiologiaRESUMO
Leishmania parasites infect macrophages, causing a wide spectrum of human diseases, from cutaneous to visceral forms. In search of novel therapeutic targets, we performed comprehensive in vitro and ex vivo mapping of the signaling pathways upstream and downstream of antioxidant transcription factor [nuclear factor erythroid 2-related factor 2 (Nrf2)] in cutaneous leishmaniasis (CL), by combining functional assays in human and murine macrophages with a systems biology analysis of in situ (skin biopsies) CL patient samples. First, we show the PKR pathway controls the expression and activation of Nrf2 in Leishmania amazonensis infection in vitro. Nrf2 activation also required PI3K/Akt signaling and autophagy mechanisms. Nrf2- or PKR/Akt-deficient macrophages exhibited increased levels of ROS/RNS and reduced expression of Sod1 Nrf2-dependent gene and reduced parasite load. L. amazonensis counteracted the Nrf2 inhibitor Keap1 through the upregulation of p62 via PKR. This Nrf2/Keap1 observation was confirmed in situ in skin biopsies from Leishmania-infected patients. Next, we explored the ex vivo transcriptome in CL patients, as compared to healthy controls. We found the antioxidant response element/Nrf2 signaling pathway was significantly upregulated in CL, including downstream target p62. In silico enrichment analysis confirmed upstream signaling by interferon and PI3K/Akt, and validated our in vitro findings. Our integrated in vitro, ex vivo, and in silico approach establish Nrf2 as a central player in human cutaneous leishmaniasis and reveal Nrf2/PKR crosstalk and PI3K/Akt pathways as potential therapeutic targets.
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The transforming properties of oncogenes are derived from gain-of-function mutations, shifting cell signaling from highly regulated homeostatic to an uncontrolled oncogenic state, with the contribution of the inactivating mutations in tumor suppressor genes P53 and RB, leading to tumor resistance to conventional and target-directed therapy. On the other hand, this scenario fulfills two requirements for oncolytic virus infection in tumor cells: inactivation of tumor suppressors and presence of oncoproteins, also the requirements to engage malignancy. Several of these oncogenes have a negative impact on the main interferon antiviral defense, the double-stranded RNA-activated protein kinase (PKR), which helps viruses to spontaneously target tumor cells instead of normal cells. This review is focused on the negative impact of overexpression of oncogenes on conventional and targeted therapy and their positive impact on viral oncolysis due to their ability to inhibit PKR-induced translation blockage, allowing virion release and cell death.
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The protozoan parasite Leishmania infects and replicates in macrophages, causing a spectrum of diseases in the human host, varying from cutaneous to visceral clinical forms. It is known that cytokines modulate the immunological response against Leishmania and are relevant for infection resolution. Here, we report that Interleukin (IL)-27 increases Leishmania amazonensis replication in human and murine macrophages and that the blockage of the IL-10 receptor on the surface of infected cells abolished the IL-27-mediated enhancement of Leishmania growth. IL-27 induced the activation/phosphorylation of protein kinase R (PKR) in macrophages, and PKR blockage or PKR gene deletion abrogated the enhancement of the parasite growth driven by IL-27, as well as the L. amazonensis-induced macrophage production of IL-27. We also observed that L. amazonensis-induced expression of IL-27 depends on type I interferon signaling and the engagement of Toll-like receptor 2. Treatment of Leishmania-infected mice with IL-27 increased lesion size and parasite loads in the footpad and lymph nodes of infected animals, indicating that this cytokine exerts a local and a systemic effect on parasite growth and propagation. In conclusion, we show that IL-27 is a L. amazonensis-enhancing factor and that the PKR/IFN1 axis and IL-10 are critical mediators of this IL-27 induced effect.
Assuntos
Interleucina-10/metabolismo , Interleucina-27/metabolismo , Leishmania mexicana , Leishmaniose Cutânea/metabolismo , Transdução de Sinais , eIF-2 Quinase/metabolismo , Animais , Linhagem Celular , Humanos , Interferon Tipo I/metabolismo , Interleucina-27/farmacologia , Leishmaniose Cutânea/genética , Leishmaniose Cutânea/parasitologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Macrófagos/parasitologia , Masculino , Camundongos , Camundongos Knockout , Transdução de Sinais/efeitos dos fármacos , Receptor 2 Toll-Like/metabolismo , eIF-2 Quinase/genéticaRESUMO
Hepatitis C virus (HCV) is a major cause of liver disease throughout the world. The NS5A and E2 proteins of HCV genotype 1 were reported to inhibit the double-stranded (ds) RNA-dependent protein kinase (PKR), which is involved in the cellular antiviral response induced by interferon (IFN). The response to IFN therapy is quite different between genotypes, with response rates among patients infected with types 2 and 3 that are two-three-fold higher than in patients infected with type 1. Interestingly, a significant percentage of HCV genotype 3-infected patients do not respond to treatment at all. The aim of this paper was to analyse the sequences of fragments of the E2 and NS5A regions from 33 outpatients infected with genotype 3a, including patients that have responded (SVR) or not responded (NR) to treatment. HCV RNA was extracted and amplified with specific primers for the NS5A and E2 regions and the PCR products were then sequenced. The sequences obtained covered amino acids (aa) 636-708 in E2 and in NS5A [including the IFN sensitivity determining region (ISDR), PKR-binding domain and extended V3 region)]. In the E2 and NS5A regions, we did observe aa changes among patients, but these changes were not statistically significant between the SVR and NR groups. In conclusion, our results suggest that the ISDR domain is not predictive of treatment success in patients infected with HCV genotype 3a.