RESUMEN
An integral aspect of innate immunity is the ability to detect foreign molecules of viral origin to initiate antiviral signaling via pattern recognition receptors (PRRs). One such receptor is the RNA helicase retinoic acid inducible gene 1 (RIG-I), which detects and is activated by 5'triphosphate uncapped double stranded RNA (dsRNA) as well as the cytoplasmic viral mimic dsRNA polyI:C. Once activated, RIG-I's CARD domains oligomerize and initiate downstream signaling via mitochondrial antiviral signaling protein (MAVS), ultimately inducing interferon (IFN) production. Another dsRNA binding protein PACT, originally identified as the cellular protein activator of dsRNA-activated protein kinase (PKR), is known to enhance RIG-I signaling in response to polyI:C treatment, in part by stimulating RIG-I's ATPase and helicase activities. TAR-RNA-binding protein (TRBP), which is â¼45% homologous to PACT, inhibits PKR signaling by binding to PKR as well as by sequestration of its' activators, dsRNA and PACT. Despite the extensive homology and similar structure of PACT and TRBP, the role of TRBP has not been explored much in RIG-I signaling. This work focuses on the effect of TRBP on RIG-I signaling and IFN production. Our results indicate that TRBP acts as an inhibitor of RIG-I signaling in a PACT- and PKR-independent manner. Surprisingly, this inhibition is independent of TRBP's post-translational modifications that are important for other signaling functions of TRBP, but TRBP's dsRNA-binding ability is essential. Our work has major implications on viral susceptibility, disease progression, and antiviral immunity as it demonstrates the regulatory interplay between PACT and TRBP IFN production.
Asunto(s)
Proteínas Portadoras/fisiología , Proteína 58 DEAD Box/fisiología , Proteínas de Unión al ARN/fisiología , Receptores Inmunológicos/fisiología , Transducción de Señal/fisiología , Transporte Activo de Núcleo Celular , Adenosina Trifosfato/metabolismo , Animales , Fibroblastos , Genes Reporteros , Células HEK293 , Humanos , Inmunidad Innata , Factor 3 Regulador del Interferón/genética , Factor 3 Regulador del Interferón/metabolismo , Interferones/fisiología , Ratones , Modelos Biológicos , Mutación , Fosforilación , Poli I-C/farmacología , Unión Proteica , Dominios Proteicos , Procesamiento Proteico-Postraduccional , ARN Bicatenario/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
Dystonia 16 (DYT16) is caused by mutations in PACT, the protein activator of interferon-induced double-stranded RNA-activated protein kinase (PKR). PKR regulates the integrated stress response (ISR) via phosphorylation of the translation initiation factor eIF2α. This post-translational modification attenuates general protein synthesis while concomitantly triggering enhanced translation of a few specific transcripts leading either to recovery and homeostasis or cellular apoptosis depending on the intensity and duration of stress signals. PKR plays a regulatory role in determining the cellular response to viral infections, oxidative stress, endoplasmic reticulum (ER) stress, and growth factor deprivation. In the absence of stress, both PACT and PKR are bound by their inhibitor transactivation RNA-binding protein (TRBP) thereby keeping PKR inactive. Under conditions of cellular stress these inhibitory interactions dissociate facilitating PACT-PACT interactions critical for PKR activation. While both PACT-TRBP and PKR-TRBP interactions are pro-survival, PACT-PACT and PACT-PKR interactions are pro-apoptotic. In this study we evaluate if five DYT16 substitution mutations alter PKR activation and ISR. Our results indicate that the mutant DYT16 proteins show stronger PACT-PACT interactions and enhanced PKR activation. In DYT16 patient derived lymphoblasts the enhanced PACT-PKR interactions and heightened PKR activation leads to a dysregulation of ISR and increased apoptosis. More importantly, this enhanced sensitivity to ER stress can be rescued by luteolin, which disrupts PACT-PKR interactions. Our results not only demonstrate the impact of DYT16 mutations on regulation of ISR and DYT16 etiology but indicate that therapeutic interventions could be possible after a further evaluation of such strategies.
Asunto(s)
Trastornos Distónicos/genética , Factor 2 Eucariótico de Iniciación/genética , Mutación Missense/genética , Procesamiento Proteico-Postraduccional/fisiología , Apoptosis/genética , Apoptosis/fisiología , Factor 2 Eucariótico de Iniciación/metabolismo , Humanos , Estrés Oxidativo/genética , Estrés Oxidativo/fisiología , Unión Proteica , Procesamiento Proteico-Postraduccional/genética , Proteínas de Unión al ARN/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , eIF-2 Quinasa/metabolismoRESUMEN
Protein Activator (PACT) activates the interferon (IFN)-induced double-stranded (ds) RNA-activated protein kinase (PKR) in response to stress signals. Oxidative stress and endoplasmic reticulum (ER) stress causes PACT-mediated PKR activation, which leads to phosphorylation of translation initiation factor eIF2α, inhibition of protein synthesis, and apoptosis. A dominantly inherited form of early-onset dystonia 16 (DYT16) has been identified to arise due to a frameshift (FS) mutation in PACT. To examine the effect of the resulting truncated mutant PACT protein on the PKR pathway, we examined the biochemical properties of the mutant protein and its effect on mammalian cells. Our results indicate that the FS mutant protein loses its ability to bind dsRNA as well as its ability to interact with PKR while surprisingly retaining the ability to interact with PACT and PKR-inhibitory protein TRBP. The truncated FS mutant protein, when expressed as a fusion protein with a N-terminal fluorescent mCherry tag aggregates in mammalian cells to induce apoptosis via activation of caspases both in a PKR- and PACT-dependent as well as independent manner. Our results indicate that interaction of FS mutant protein with PKR inhibitor TRBP can dissociate PACT from the TRBP-PACT complex resulting in PKR activation and consequent apoptosis. These findings are relevant to diseases resulting from protein aggregation especially since the PKR activation is a characteristic of several neurodegenerative conditions.
Asunto(s)
Apoptosis , Caspasas , Trastornos Distónicos , Mutación del Sistema de Lectura , Proteínas de Unión al ARN , Animales , Caspasas/genética , Caspasas/metabolismo , Trastornos Distónicos/genética , Trastornos Distónicos/metabolismo , Trastornos Distónicos/patología , Activación Enzimática , Células HeLa , Humanos , Ratones , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , eIF-2 Quinasa/genéticaRESUMEN
PACT is a stress-modulated activator of protein kinase PKR (protein kinase, RNA activated), which is involved in antiviral innate immune responses and stress-induced apoptosis. Stress-induced phosphorylation of PACT is essential for PACT's increased association with PKR leading to PKR activation, phosphorylation of translation initiation factor eIF2α, inhibition of protein synthesis, and apoptosis. PACT-induced PKR activation is negatively regulated by TRBP (transactivation response element RNA-binding protein), which dissociates from PACT after PACT phosphorylation in response to stress signals. The conserved double-stranded RNA binding motifs (dsRBMs) in PKR, PACT, and TRBP mediate protein-protein interactions, and the stress-dependent phosphorylation of PACT changes the relative strengths of PKR-PACT, PACT-TRBP, and PACT-PACT interactions to bring about a timely and transient PKR activation. This regulates the general kinetics as well as level of eIF2α phosphorylation, thereby influencing the cellular response to stress either as recovery and survival or elimination by apoptosis. In the present study, we evaluated the effect of specific mutations within PACT's two evolutionarily conserved dsRBMs on dsRNA-binding, and protein-protein interactions between PKR, PACT, and TRBP. Our data show that the two motifs contribute to varying extents in dsRNA binding, and protein interactions. These findings indicate that although the dsRBM motifs have high sequence conservation, their functional contribution in the context of the whole proteins needs to be determined by mutational analysis. Furthermore, using a PACT mutant that is deficient in PACT-PACT interaction but competent for PACT-PKR interaction, we demonstrate that PACT-PACT interaction is essential for efficient PKR activation.
Asunto(s)
Motivo de Unión al ARN Bicatenario/fisiología , ARN Bicatenario/metabolismo , Animales , Apoptosis/genética , Apoptosis/fisiología , Células COS , Chlorocebus aethiops , Motivo de Unión al ARN Bicatenario/genética , Células HeLa , Humanos , Fosforilación/genética , Fosforilación/fisiología , Unión Proteica/genética , Unión Proteica/fisiología , ARN Bicatenario/genética , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Técnicas del Sistema de Dos Híbridos , eIF-2 Quinasa/genética , eIF-2 Quinasa/metabolismoRESUMEN
Human immunodeficiency virus type 1 (HIV-1) has evolved various measures to counter the host cell's innate antiviral response during the course of infection. Interferon (IFN)-stimulated gene products are produced following HIV-1 infection to limit viral replication, but viral proteins and RNAs counteract their effect. One such mechanism is specifically directed against the IFN-induced Protein Kinase PKR, which is centrally important to the cellular antiviral response. In the presence of viral RNAs, PKR is activated and phosphorylates the translation initiation factor eIF2α. This shuts down the synthesis of both host and viral proteins, allowing the cell to mount an effective antiviral response. PACT (protein activator of PKR) is a cellular protein activator of PKR, primarily functioning to activate PKR in response to cellular stress. Recent studies have indicated that during HIV-1 infection, PACT's normal cellular function is compromised and that PACT is unable to activate PKR. Using various reporter systems and in vitro kinase assays, we establish in this report that interactions between PACT, ADAR1 and HIV-1-encoded Tat protein diminish the activation of PKR in response to HIV-1 infection. Our results highlight an important pathway by which HIV-1 transcripts subvert the host cell's antiviral activities to enhance their translation.
Asunto(s)
Adenosina Desaminasa/genética , Factor 2 Eucariótico de Iniciación/genética , VIH-1/genética , Biosíntesis de Proteínas , Proteínas de Unión al ARN/genética , eIF-2 Quinasa/genética , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genética , Adenosina Desaminasa/metabolismo , Animales , Factor 2 Eucariótico de Iniciación/metabolismo , Fibroblastos/metabolismo , Fibroblastos/virología , Regulación de la Expresión Génica , Genes Reporteros , Células HEK293 , VIH-1/metabolismo , Células HeLa , Interacciones Huésped-Patógeno , Humanos , Luciferasas/genética , Luciferasas/metabolismo , Ratones , Proteínas de Unión al ARN/metabolismo , Transducción de Señal , eIF-2 Quinasa/metabolismo , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
PACT is a stress-modulated activator of the interferon-induced double-stranded RNA-activated protein kinase (PKR). Stress-induced phosphorylation of PACT is essential for PACT's association with PKR leading to PKR activation. PKR activation leads to phosphorylation of translation initiation factor eIF2α inhibition of protein synthesis and apoptosis. A recessively inherited form of early-onset dystonia DYT16 has been recently identified to arise due to a homozygous missense mutation P222L in PACT. To examine if the mutant P222L protein alters the stress-response pathway, we examined the ability of mutant P222L to interact with and activate PKR. Our results indicate that the substitution mutant P222L activates PKR more robustly and for longer duration albeit with slower kinetics in response to the endoplasmic reticulum stress. In addition, the affinity of PACT-PACT and PACT-PKR interactions is enhanced in dystonia patient lymphoblasts, thereby leading to intensified PKR activation and enhanced cellular death. P222L mutation also changes the affinity of PACT-TRBP interaction after cellular stress, thereby offering a mechanism for the delayed PKR activation in response to stress. Our results demonstrate the impact of a dystonia-causing substitution mutation on stress-induced cellular apoptosis.
Asunto(s)
Apoptosis , Distonía , Mutación Missense , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , eIF-2 Quinasa/metabolismo , Sustitución de Aminoácidos , Distonía/genética , Distonía/metabolismo , Distonía/patología , Femenino , Células HeLa , Humanos , Masculino , eIF-2 Quinasa/genéticaRESUMEN
BACKGROUND: RNA interference (RNAi) is an important molecular tool for analysis of gene function in vivo. Daphnia, a freshwater microcrustacean, is an emerging model organism for studying cellular and molecular processes involved in aging, development, and ecotoxicology especially in the context of environmental variation. However, in spite of the availability of a fully sequenced genome of Daphnia pulex, meaningful mechanistic studies have been hampered by a lack of molecular techniques to alter gene expression. A microinjection method for gene knockdown by RNAi has been described but the need for highly specialized equipment as well as technical expertise limits the wider application of this technique. In addition to being expensive and technically challenging, microinjections can only target genes expressed during embryonic stages, thus making it difficult to achieve effective RNAi in adult organisms. RESULTS: In our present study we present a bacterial feeding method for RNAi in Daphnia. We used a melanic Daphnia species (Daphnia melanica) that exhibits dark pigmentation to target phenoloxidase, a key enzyme in the biosynthesis of melanin. We demonstrate that our RNAi method results in a striking phenotype and that the phenoloxidase mRNA expression and melanin content, as well as survival following UV insults, are diminished as a result of RNAi. CONCLUSIONS: Overall, our results establish a new method for RNAi in Daphnia that significantly advances further use of Daphnia as a model organism for functional genomics studies. The method we describe is relatively simple and widely applicable for knockdown of a variety of genes in adult organisms.
Asunto(s)
Daphnia/genética , Técnicas de Silenciamiento del Gen/métodos , Modelos Biológicos , Interferencia de ARN , Animales , Daphnia/microbiología , Escherichia coli/genética , Melaninas/metabolismo , Monofenol Monooxigenasa/metabolismoRESUMEN
Mutations in Prkra gene, which encodes PACT/RAX cause early onset primary dystonia DYT-PRKRA, a movement disorder that disrupts coordinated muscle movements. PACT/RAX activates protein kinase R (PKR, aka EIF2AK2) by a direct interaction in response to cellular stressors to mediate phosphorylation of the α subunit of the eukaryotic translation initiation factor 2 (eIF2α). Mice homozygous for a naturally arisen, recessively inherited frameshift mutation, Prkra lear-5J exhibit progressive dystonia. In the present study, we investigate the biochemical and developmental consequences of the Prkra lear-5J mutation. Our results indicate that the truncated PACT/RAX protein retains its ability to interact with PKR, however, it inhibits PKR activation. Furthermore, mice homozygous for the mutation have abnormalities in the cerebellar development as well as a severe lack of dendritic arborization of Purkinje neurons. Additionally, reduced eIF2α phosphorylation is noted in the cerebellums and Purkinje neurons of the homozygous Prkra lear-5J mice. These results indicate that PACT/RAX mediated regulation of PKR activity and eIF2α phosphorylation plays a role in cerebellar development and contributes to the dystonia phenotype resulting from this mutation.
RESUMEN
BACKGROUND: HIV-1 translation is modulated by the activation of the interferon (IFN)-inducible Protein Kinase RNA-activated (PKR). PKR phosphorylates its downstream targets, including the alpha subunit of the eukaryotic translation Initiation Factor 2 (eIF2α), which decreases viral replication. The PKR Activator (PACT) is known to activate PKR after a cellular stress. In lymphocytic cell lines, HIV-1 activates PKR only transiently and not when cells replicate the virus at high levels. The regulation of this activation is due to a combination of viral and cellular factors that have been only partially identified. RESULTS: PKR is transiently induced and activated in peripheral blood mononuclear cells after HIV-1 infection. The addition of IFN reduces viral replication, and induces both the production and phosphorylation of PKR. In lymphocytic Jurkat cells infected by HIV-1, a multiprotein complex around PKR contains the double-stranded RNA binding proteins (dsRBPs), adenosine deaminase acting on RNA (ADAR)1 and PACT. In HEK 293T cells transfected with an HIV-1 molecular clone, PACT unexpectedly inhibited PKR and eIF2α phosphorylation and increased HIV-1 protein expression and virion production in the presence of either endogenous PKR alone or overexpressed PKR. The comparison between different dsRBPs showed that ADAR1, TAR RNA Binding Protein (TRBP) and PACT inhibit PKR and eIF2α phosphorylation in HIV-infected cells, whereas Staufen1 did not. Individual or a combination of short hairpin RNAs against PACT or ADAR1 decreased HIV-1 protein expression. In the astrocytic cell line U251MG, which weakly expresses TRBP, PACT mediated an increased HIV-1 protein expression and a decreased PKR phosphorylation. In these cells, a truncated PACT, which constitutively activates PKR in non-infected cells showed no activity on either PKR or HIV-1 protein expression. Finally, PACT and ADAR1 interact with each other in the absence of RNAs. CONCLUSION: In contrast to its previously described activity, PACT contributes to PKR dephosphorylation during HIV-1 replication. This activity is in addition to its heterodimer formation with TRBP and could be due to its binding to ADAR1. HIV-1 has evolved to replicate in cells with high levels of TRBP, to induce the expression of ADAR1 and to change the function of PACT for PKR inhibition and increased replication.
Asunto(s)
VIH-1/fisiología , Interacciones Huésped-Patógeno , Proteínas de Unión al ARN/metabolismo , Replicación Viral , eIF-2 Quinasa/antagonistas & inhibidores , Adenosina Desaminasa/metabolismo , Línea Celular , Humanos , Fosforilación , Unión Proteica , Multimerización de Proteína , Procesamiento Proteico-PostraduccionalRESUMEN
DYT-PRKRA is a movement disorder caused by mutations in the PRKRA gene, which encodes for PACT, the protein activator of interferon-induced, double-stranded RNA (dsRNA)-activated protein kinase PKR. PACT brings about PKR's catalytic activation by a direct binding in response to stress signals and activated PKR phosphorylates the translation initiation factor eIF2α. Phosphorylation of eIF2α is the central regulatory event that is part of the integrated stress response (ISR), an evolutionarily conserved intracellular signaling network essential for adapting to environmental stresses to maintain healthy cells. A dysregulation of either the level or the duration of eIF2α phosphorylation in response to stress signals causes the normally pro-survival ISR to become pro-apoptotic. Our research has established that the PRKRA mutations reported to cause DYT-PRKRA lead to enhanced PACT-PKR interactions causing a dysregulation of ISR and an increased sensitivity to apoptosis. We have previously identified luteolin, a plant flavonoid, as an inhibitor of the PACT-PKR interaction using high-throughput screening of chemical libraries. Our results presented in this study indicate that luteolin is markedly effective in disrupting the pathological PACT-PKR interactions to protect DYT-PRKRA cells against apoptosis, thus suggesting a therapeutic option for using luteolin to treat DYT-PRKRA and possibly other diseases resulting from enhanced PACT-PKR interactions.
RESUMEN
PKR (protein kinase, RNA activated) is an interferon (IFN)-induced serine-threonine protein kinase and is one of the key mediators in IFN's cellular actions. Although double-stranded (ds) RNA is the most relevant PKR activator during viral infections, PACT acts as a stress-modulated activator of PKR and is an important regulator of PKR dependent signaling pathways in the absence of viral infections. Stress-induced phosphorylation of PACT is essential for PACT's association with PKR leading to PKR activation. PKR activation by PACT leads to phosphorylation of translation initiation factor eIF2α, inhibition of protein synthesis, and apoptosis. In the present study, we have investigated the functional significance of PACT-PACT interaction in mediating PKR activation in response to cellular stress. Our results suggest that enhanced interaction between PACT molecules when PACT is phosphorylated in response to stress signals on serines 246 and 287 is essential for efficient PKR activation. Using a point mutant of PACT that is deficient in PACT-PACT interaction, we demonstrate that PACT-PACT interaction is essential for efficient PKR activation.
Asunto(s)
Proteínas de Unión al ARN/metabolismo , eIF-2 Quinasa/metabolismo , Células HeLa , Humanos , Inmunoprecipitación , Fosforilación , Unión Proteica , Transducción de Señal/genética , Transducción de Señal/fisiología , Técnicas del Sistema de Dos HíbridosRESUMEN
Macromolecular damage leading to cell, tissue and ultimately organ dysfunction is a major contributor to aging. Intracellular reactive oxygen species (ROS) resulting from normal metabolism cause most damage to macromolecules and the mitochondria play a central role in this process as they are the principle source of ROS. The relationship between naturally occurring variations in the mitochondrial (MT) genomes leading to correspondingly less or more ROS and macromolecular damage that changes the rate of aging associated organismal decline remains relatively unexplored. MT complex I, a component of the electron transport chain (ETC), is a key source of ROS and the NADH dehydrogenase subunit 5 (ND5) is a highly conserved core protein of the subunits that constitute the backbone of complex I. Using Daphnia as a model organism, we explored if the naturally occurring sequence variations in ND5 correlate with a short or long lifespan. Our results indicate that the short-lived clones have ND5 variants that correlate with reduced complex I activity, increased oxidative damage, and heightened expression of ROS scavenger enzymes. Daphnia offers a unique opportunity to investigate the association between inherited variations in components of complex I and ROS generation which affects the rate of aging and lifespan.
Asunto(s)
Daphnia/crecimiento & desarrollo , Daphnia/metabolismo , Estrés Oxidativo , Animales , Daphnia/genética , Complejo I de Transporte de Electrón/genética , Complejo I de Transporte de Electrón/metabolismo , Longevidad , Mitocondrias/enzimología , Mitocondrias/genética , Mitocondrias/metabolismo , NADH Deshidrogenasa/genética , NADH Deshidrogenasa/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
DYT-PRKRA (dystonia 16 or DYT-PRKRA) is caused by mutations in the PRKRA gene that encodes PACT, the protein activator of interferon (IFN)-induced double-stranded (ds) RNA-activated protein kinase (PKR). PACT participates in several cellular pathways, of which its role as a PKR activator protein during integrated stress response (ISR) is the best characterized. Previously, we have established that the DYT-PRKRA mutations cause enhanced activation of PKR during ISR to sensitize DYT-PRKRA cells to apoptosis. In this study, we evaluate if the most prevalent substitution mutation reported in DYT-PRKRA patients alters PACT's functional role in induction of type I IFNs via the retinoic acid-inducible gene I (RIG-I) signaling. Our results indicate that the P222L mutation augments PACT's ability to induce IFN ß in response to dsRNA and the basal expression of IFN ß and IFN-stimulated genes (ISGs) is higher in DYT-PRKRA patient cells compared to cells from the unaffected controls. Additionally, IFN ß and ISGs are also induced at higher levels in DYT-PRKRA cells in response to dsRNA. These results offer a new avenue for investigations directed towards understanding the underlying molecular pathomechanisms in DYT-PRKRA.
Asunto(s)
Trastornos Distónicos , Interferón Tipo I , Humanos , Interferón Tipo I/genética , Interferón Tipo I/metabolismo , Mutación , ARN Bicatenario/genética , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , eIF-2 Quinasa/genética , eIF-2 Quinasa/metabolismoRESUMEN
PACT is a stress-modulated activator of interferon (IFN)-induced double-stranded (ds) RNA-activated protein kinase (PKR) and is an important regulator of PKR-dependent signaling pathways. Stress-induced phosphorylation of PACT is essential for PACT's association with PKR leading to PKR activation. PKR activation by PACT leads to phosphorylation of translation initiation factor eIF2α, inhibition of protein synthesis, and apoptosis. In addition to positive regulation by PACT, PKR activity in cells is also negatively regulated by TRBP. In this study, we demonstrate for the first time that stress-induced phosphorylation at serine 287 significantly increases PACT's ability to activate PKR by weakening PACT's interaction with TRBP. A non-phosphorylatable alanine substitution mutant at this position causes enhanced interaction of PACT with TRBP and leads to a loss of PKR activation. Furthermore, TRBP overexpression in cells is unable to block apoptosis induced by a phospho-mimetic, constitutively active PACT mutant. These results demonstrate for the first time that stress-induced PACT phosphorylation functions to free PACT from the inhibitory interaction with TRBP and also to enhance its interaction with PKR.
Asunto(s)
VIH-1/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas Virales/metabolismo , eIF-2 Quinasa/metabolismo , Secuencia de Bases , Cartilla de ADN , Activación Enzimática , Células HeLa , Humanos , Fosforilación , Unión Proteica , Técnicas del Sistema de Dos HíbridosRESUMEN
Transactivation response element RNA-binding protein (TRBP or TARBP2) originally identified as a pro-viral cellular protein in human immunodeficiency virus (HIV) replication is also a regulator of microRNA biogenesis and cellular stress response. TRBP inhibits the catalytic activity of interferon-induced double-stranded RNA (dsRNA)-activated protein kinase (PKR) during viral infections and cell stress thereby regulating stress-induced signaling pathways. During cellular stress, PKR is catalytically activated transiently by its protein activator PACT and TRBP inhibits PKR to bring about a timely cellular recovery. We have previously established that TRBP phosphorylated after oxidative stress binds to and inhibits PKR more efficiently promoting cell survival. In this study, we investigated if phosphorylation of TRBP enhances its interaction with PACT to bring about additional PKR inhibition. Our data establishes that phosphorylation of TRBP has no effect on PACT-TRBP interaction and TRBP's inhibitory actions on PKR are mediated exclusively by its enhanced interaction with PKR. Cells lacking TRBP are more sensitive to apoptosis in response to oxidative stress and show persistent PKR activation. These results establish that PKR inhibition by stress-induced TRBP phosphorylation occurs by its direct binding to PKR and is important for preventing apoptosis due to sustained PKR activation.
Asunto(s)
Apoptosis , Estrés Oxidativo , ARN Bicatenario/metabolismo , Proteínas de Unión al ARN/metabolismo , eIF-2 Quinasa/metabolismo , Animales , Células HeLa , Humanos , Ratones , Fosforilación , ARN Bicatenario/genética , Proteínas de Unión al ARN/genética , Transducción de Señal , eIF-2 Quinasa/genéticaRESUMEN
PKR is an interferon (IFN)-induced protein kinase, which is involved in regulation of antiviral innate immunity, stress signaling, cell proliferation and programmed cell death. Although a low amount of PKR is expressed ubiquitously in all cell types in the absence of IFNs, PKR expression is induced at transcriptional level by IFN. PKR's enzymatic activity is activated by its binding to one of its activators. Double-stranded (ds) RNA, protein activator PACT and heparin are the three known activators of PKR. Activation of PKR in cells leads to a general block in protein synthesis due to phosphorylation of eIF2alpha on serine 51 by PKR. PKR activation is regulated very tightly in mammalian cells and a prolonged activation of PKR leads to apoptosis. Thus, positive and negative regulation of PKR activation is important for cell viability and function. The studies presented here describe human dihydrouridine synthase-2 (hDUS2) as a novel regulator of PKR. We originally identified hDUS2 as a protein interacting with PACT in a yeast two-hybrid screen. Further characterization revealed that hDUS2 also interacts with PKR through its dsRNA binding/dimerization domain and inhibits its kinase activity. Our results suggest that hDUS2 may act as a novel inhibitor of PKR in cells.
Asunto(s)
Oxidorreductasas/metabolismo , eIF-2 Quinasa/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Línea Celular , Secuencia Conservada , Células HeLa , Humanos , Datos de Secuencia Molecular , Oxidorreductasas/química , Estructura Terciaria de Proteína , ARN Bicatenario/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , Técnicas del Sistema de Dos Híbridos , eIF-2 Quinasa/antagonistas & inhibidores , eIF-2 Quinasa/químicaRESUMEN
HOXA9 is a homeobox transcription factor expressed in endothelial cells (EC) and its expression is rapidly downregulated during EC activation by inflammatory signals like tumor necrosis factor-alpha (TNF-alpha) and lipopolysaccharide (LPS). Recently, we have shown that HOXA9 overexpression prevents EC activation by inhibiting NF-kappaB activity, which suggests that HOXA9 downregulation is an essential event for EC activation. The present study is directed towards understanding the mechanism of HOXA9 regulation during EC activation. Here we show that nuclear factor-kappaB (NF-kappaB) activation is an essential step for HOXA9 downregulation. Deletion analyses of HOXA9 promoter in EC and NF-kappaB knockout cells have shown that NF-kappaB is a major transcription factor that is absolutely required for HOXA9 downregulation. Our 5' deletion analysis of HOXA9 promoter shows that NF-kappaB response element is localized within first 400 nucleotides, while minimal basal promoter is within 100 nucleotides upstream of its transcriptional start site. We demonstrate that HOXA9 regulates its own expression by positive feedback mechanism. To define mechanism by which HOXA9 autoregulates its expression, we show that HOXA9 DNA binding and transactivation domains are essential.
Asunto(s)
Proteínas de Homeodominio/genética , FN-kappa B/metabolismo , Regulación hacia Abajo , Células Endoteliales/metabolismo , Proteínas de Homeodominio/metabolismo , Humanos , Regiones Promotoras Genéticas , Factor de Transcripción ReIA/metabolismo , Activación Transcripcional , Transfección , Factor de Necrosis Tumoral alfa/metabolismo , Regulación hacia ArribaRESUMEN
Transactivation response element RNA-binding protein (TRBP or TARBP2) initially identified to play an important role in human immunodeficiency virus (HIV) replication also has emerged as a regulator of microRNA biogenesis. In addition, TRBP functions in signaling pathways by negatively regulating the interferon-induced double-stranded RNA (dsRNA)-activated protein kinase (PKR) during viral infections and cell stress. During cellular stress, PKR is activated and phosphorylates the α subunit of the eukaryotic translation factor eIF2, leading to the cessation of general protein synthesis. TRBP inhibits PKR activity by direct interaction as well as by binding to PKR's two known activators, dsRNA and PACT, thus preventing their interaction with PKR. In this study, we demonstrate for the first time that TRBP is phosphorylated in response to oxidative stress and upon phosphorylation, inhibits PKR more efficiently promoting cell survival. These results establish that PKR regulation through stress-induced TRBP phosphorylation is an important mechanism ensuring cellular recovery and preventing apoptosis due to sustained PKR activation.
Asunto(s)
Proteínas de Unión al ARN/metabolismo , Estrés Fisiológico , eIF-2 Quinasa/metabolismo , Apoptosis , Línea Celular Tumoral , Supervivencia Celular , Humanos , Inmunoprecipitación , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Potencial de la Membrana Mitocondrial , Estrés Oxidativo , Fosforilación , Unión Proteica , Mapeo de Interacción de Proteínas/métodos , Proteínas de Unión al ARN/genética , Técnicas del Sistema de Dos HíbridosRESUMEN
PACT is a stress-modulated, cellular activator of interferon (IFN)-induced double-stranded (ds) RNA-activated protein kinase (PKR) and is an important regulator of PKR-dependent signaling pathways. The research presented here is aimed at understanding the regulation of PACT expression in mammalian cells. PACT is expressed ubiquitously in different cell types at varying abundance. We have characterized the sequence elements in PACT promoter region that are required for its expression. Using deletion analysis of the promoter we have identified the minimal basal promoter of PACT to be within 101 nucleotides upstream of its transcription start site. Further mutational analyses within this region, followed by electrophoretic mobility shift analyses (EMSAs) and chromatin immunoprecipitation (ChiP) analysis have shown that Specificity protein 1 (Sp1) is the major transcription factor responsible for PACT promoter activity.
Asunto(s)
Regulación de la Expresión Génica , Proteínas de Unión al ARN/genética , Factor de Transcripción Sp1/metabolismo , Secuencia de Bases , Sitios de Unión/genética , Inmunoprecipitación de Cromatina , Ensayo de Cambio de Movilidad Electroforética , Femenino , Células HeLa , Humanos , Luciferasas/genética , Luciferasas/metabolismo , Masculino , Datos de Secuencia Molecular , Mutación , Oligonucleótidos/genética , Oligonucleótidos/metabolismo , Regiones Promotoras Genéticas/genética , Unión Proteica , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Factor de Transcripción Sp1/genética , TransfecciónRESUMEN
PKR is a cellular kinase involved in the regulation of the integrative stress response (ISR) and pro-inflammatory pathways. Two N-terminal dsRNA Binding Domains (DRBD) are required for activation of PKR, by interaction with either dsRNA or PACT, another cellular DRBD-containing protein. A role for PKR and PACT in inflammatory processes linked to neurodegenerative diseases has been proposed and raised interest for pharmacological PKR inhibitors. However, the role of PKR in inflammation is subject to controversy. We identified the flavonoid luteolin as an inhibitor of the PKR/PACT interaction at the level of their DRBDs using high-throughput screening of chemical libraries by homogeneous time-resolved fluorescence. This was further validated using NanoLuc-Based Protein Complementation Assay. Luteolin inhibits PKR phosphorylation, the ISR and the induction of pro-inflammatory cytokines in human THP1 macrophages submitted to oxidative stress and toll-like receptor (TLR) agonist. Similarly, luteolin inhibits induction of pro-inflammatory cytokines in murine microglial macrophages. In contrast, luteolin increased activation of the inflammasome, in a PKR-independent manner. Collectively, these data delineate the importance of PKR in the inflammation process to the ISR and induction of pro-inflammatory cytokines. Pharmacological inhibitors of PKR should be used in combination with drugs targeting directly the inflammasome.