RESUMEN
Tauopathies are a group of neurodegenerative diseases characterized by the presence of tau inclusions. We have developed over fifty anti-tau single-domain antibodies (sdAbs) derived from phage display libraries of a llama immunized with recombinant and pathological tau immunogens. We examined the therapeutic potential of four of these sdAbs in a Drosophila tauopathy model following their transgenic expression either in all neurons or neuronal subtypes. Three of these sdAbs showed therapeutic potential in various assays, effectively clearing pathological tau and attenuating or preventing tau-induced phenotypes that typically manifest as defects in neuronal axonal transport, neurodegeneration, functional impairments, and shortened lifespan. Of these three, one sdAb was superior in every assay, which may at least in part be attributed to its tau-binding epitope. These findings support its development as a gene therapy for tauopathies.
Asunto(s)
Anticuerpos de Dominio Único , Tauopatías , Proteínas tau , Proteínas tau/metabolismo , Proteínas tau/inmunología , Animales , Tauopatías/inmunología , Tauopatías/patología , Anticuerpos de Dominio Único/inmunología , Anticuerpos de Dominio Único/farmacología , Humanos , Modelos Animales de Enfermedad , Drosophila , Animales Modificados Genéticamente , Neuronas/metabolismo , Neuronas/patología , Camélidos del Nuevo Mundo/inmunología , Drosophila melanogaster/inmunologíaRESUMEN
The integrated stress response (ISR) refers to signaling pathways initiated by stress-activated eIF2α kinases. Distinct eIF2α kinases respond to different stress signals, including amino acid deprivation and mitochondrial stress. Such stress-induced eIF2α phosphorylation attenuates general mRNA translation and, at the same time, stimulates the preferential translation of specific downstream factors to orchestrate an adaptive gene expression program. In recent years, there have been significant new advances in our understanding of ISR during metabolic stress adaptation. Here, I discuss those advances, reviewing among others the ISR activation mechanisms in response to amino acid deprivation and mitochondrial stress. In addition, I review how ISR regulates the amino acid metabolic pathways and how changes in the ISR impact the physiology and pathology of various disease models.
Asunto(s)
Adaptación Fisiológica , Aminoácidos , Factor 2 Eucariótico de Iniciación , Estrés Fisiológico , Animales , Humanos , Aminoácidos/deficiencia , eIF-2 Quinasa/metabolismo , eIF-2 Quinasa/genética , Factor 2 Eucariótico de Iniciación/metabolismo , Mitocondrias/metabolismo , Fosforilación , Biosíntesis de Proteínas , Transducción de SeñalRESUMEN
Unfolded protein response (UPR) is the mechanism by which cells control endoplasmic reticulum (ER) protein homeostasis. ER proteostasis is essential to adapt to cell proliferation and regeneration in development and tumorigenesis, but mechanisms linking UPR, growth control, and cancer progression remain unclear. Here, we report that the Ire1/Xbp1s pathway has surprisingly oncogenic and tumor-suppressive roles in a context-dependent manner. Activation of Ire1/Xbp1s up-regulates their downstream target Bip, which sequesters Yorkie (Yki), a Hippo pathway transducer, in the cytoplasm to restrict Yki transcriptional output. This regulation provides an endogenous defensive mechanism in organ size control, intestinal homeostasis, and regeneration. Unexpectedly, Xbp1 ablation promotes tumor overgrowth but suppresses invasiveness in a Drosophila cancer model. Mechanistically, hyperactivated Ire1/Xbp1s signaling in turn induces JNK-dependent developmental and oncogenic cell migration and epithelial-mesenchymal transition (EMT) via repression of Yki. In humans, a negative correlation between XBP1 and YAP (Yki ortholog) target gene expression specifically exists in triple-negative breast cancers (TNBCs), and those with high XBP1 or HSPA5 (Bip ortholog) expression have better clinical outcomes. In human TNBC cell lines and xenograft models, ectopic XBP1s or HSPA5 expression alleviates tumor growth but aggravates cell migration and invasion. These findings uncover a conserved crosstalk between the Ire1/Xbp1s and Hippo signaling pathways under physiological settings, as well as a crucial role of Bip-Yki interaction in tumorigenesis that is shared from Drosophila to humans.
Asunto(s)
Proteínas de Drosophila , Proteínas Serina-Treonina Quinasas , Animales , Carcinogénesis/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Retículo Endoplásmico/metabolismo , Endorribonucleasas , Vía de Señalización Hippo , Humanos , Proteínas Serina-Treonina Quinasas/genética , Respuesta de Proteína Desplegada , Proteína 1 de Unión a la X-Box/genética , Proteína 1 de Unión a la X-Box/metabolismoRESUMEN
Wildtype or mutant proteins expressed beyond the capacity of a cell's protein folding system could be detrimental to general cellular function and survival. In response to misfolded protein overload in the endoplasmic reticulum (ER), eukaryotic cells activate the Unfolded Protein Response (UPR) that helps cells restore protein homeostasis in the endoplasmic reticulum (ER). As part of the UPR, cells attenuate general mRNA translation and activate transcription factors that induce stress-responsive gene expression.UPR signaling draws research interest in part because conditions that cause chronic protein misfolding in the ER or those that impair UPR signaling underlie several diseases including neurodegeneration, diabetes, and cancers. Model organisms are frequently employed in the field as the UPR pathways are generally well-conserved throughout phyla. Here, we introduce experimental procedures to detect UPR in Drosophila melanogaster.
Asunto(s)
Drosophila , Estrés del Retículo Endoplásmico , Animales , Drosophila melanogaster/genética , Retículo Endoplásmico/metabolismo , Estrés del Retículo Endoplásmico/fisiología , Respuesta de Proteína DesplegadaRESUMEN
Metazoans have evolved various quality control mechanisms to cope with cellular stress inflicted by external and physiological conditions. ATF4 is a major effector of the integrated stress response, an evolutionarily conserved pathway that mediates adaptation to various cellular stressors. Loss of function of Drosophila ATF4, encoded by the gene cryptocephal (crc), results in lethality during pupal development. The roles of crc in Drosophila disease models and in adult tissue homeostasis thus remain poorly understood. Here, we report that a protein-trap Minos-mediated integration cassette insertion in the crc locus generates a Crc-GFP fusion protein that allows visualization of Crc activity in vivo. This allele also acts as a hypomorphic mutant that uncovers previously unknown roles for crc. Specifically, the crc protein-trap line shows Crc-GFP induction in a Drosophila model for retinitis pigmentosa. This crc allele renders flies more vulnerable to amino acid deprivation and age-dependent retinal degeneration. These mutants also show defects in wing veins and oocyte maturation. Together, our data reveal previously unknown roles for crc in development, cellular homeostasis and photoreceptor survival. This article has an associated First Person interview with the first author of the paper.
Asunto(s)
Proteínas de Drosophila , Degeneración Retiniana , Factor de Transcripción Activador 4/genética , Factor de Transcripción Activador 4/metabolismo , Alelos , Animales , Drosophila , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Humanos , Oogénesis/genética , Degeneración Retiniana/genética , Degeneración Retiniana/metabolismoRESUMEN
PERK is an endoplasmic reticulum (ER) transmembrane sensor that phosphorylates eIF2α to initiate the Unfolded Protein Response (UPR). eIF2α phosphorylation promotes stress-responsive gene expression most notably through the transcription factor ATF4 that contains a regulatory 5' leader. Possible PERK effectors other than ATF4 remain poorly understood. Here, we report that the bZIP transcription factor Xrp1 is required for ATF4-independent PERK signaling. Cell-type-specific gene expression profiling in Drosophila indicated that delta-family glutathione-S-transferases (gstD) are prominently induced by the UPR-activating transgene Rh1G69D. Perk was necessary and sufficient for such gstD induction, but ATF4 was not required. Instead, Perk and other regulators of eIF2α phosphorylation regulated Xrp1 protein levels to induce gstDs. The Xrp1 5' leader has a conserved upstream Open Reading Frame (uORF) analogous to those that regulate ATF4 translation. The gstD-GFP reporter induction required putative Xrp1 binding sites. These results indicate that antioxidant genes are highly induced by a previously unrecognized UPR signaling axis consisting of PERK and Xrp1.
Asunto(s)
Antioxidantes/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimología , Glutatión Transferasa/metabolismo , Discos Imaginales/enzimología , eIF-2 Quinasa/metabolismo , Animales , Animales Modificados Genéticamente , Sitios de Unión , Proteínas de Unión al ADN/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/embriología , Drosophila melanogaster/genética , Estrés del Retículo Endoplásmico , Factor 2 Eucariótico de Iniciación/metabolismo , Regulación del Desarrollo de la Expresión Génica , Glutatión Transferasa/genética , Discos Imaginales/embriología , Sistemas de Lectura Abierta , Fosforilación , Rodopsina/genética , Rodopsina/metabolismo , Transducción de Señal , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Respuesta de Proteína Desplegada , eIF-2 Quinasa/genéticaRESUMEN
Rhodopsins are light-detecting proteins coupled with retinal chromophores essential for visual function. Coincidentally, dysfunctional Rhodopsin homeostasis underlies retinal degeneration in humans and model organisms. Drosophila ninaEG69D mutant is one such example, where the encoded Rh1 protein imposes endoplasmic reticulum (ER) stress and causes light-dependent retinal degeneration. The underlying reason for such light-dependency remains unknown. Here, we report that Drosophila fatty acid binding protein (fabp) is a gene induced in ninaEG69D/+ photoreceptors, and regulates light-dependent Rhodopsin-1 (Rh1) protein clearance and photoreceptor survival. Specifically, our photoreceptor-specific gene expression profiling study in ninaEG69D/+ flies revealed increased expression of fabp together with other genes that control light-dependent Rh1 protein degradation. fabp induction in ninaEG69D photoreceptors required vitamin A and its transporter genes. In flies reared under light, loss of fabp caused an accumulation of Rh1 proteins in cytoplasmic vesicles. The increase in Rh1 levels under these conditions was dependent on Arrestin2 that mediates feedback inhibition of light-activated Rh1. fabp mutants exhibited light-dependent retinal degeneration, a phenotype also found in other mutants that block light-induced Rh1 degradation. These observations reveal a previously unrecognized link between light-dependent Rh1 proteostasis and the ER-stress imposing ninaEG69D mutant that cause retinal degeneration.
Asunto(s)
Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila/genética , Drosophila/metabolismo , Células Fotorreceptoras de Invertebrados/metabolismo , Rodopsina/genética , Rodopsina/metabolismo , Animales , Mutación/genética , Fenotipo , Retina/metabolismo , Degeneración Retiniana/metabolismo , Transcriptoma/genéticaRESUMEN
Ire1 is an endoplasmic reticulum (ER) transmembrane RNase that cleaves substrate mRNAs to help cells adapt to ER stress. Because there are cell types with physiological ER stress, loss of Ire1 results in metabolic and developmental defects in diverse organisms. In Drosophila, Ire1 mutants show developmental defects at early larval stages and in pupal eye photoreceptor differentiation. These Drosophila studies relied on a single Ire1 loss of function allele with a Piggybac insertion in the coding sequence. Here, we report that an Ire1 allele with a specific impairment in the RNase domain, H890A, unmasks previously unrecognized Ire1 phenotypes in Drosophila eye pigmentation. Specifically, we found that the adult eye pigmentation is altered, and the pigment granules are compromised in Ire1H890A homozygous mosaic eyes. Furthermore, the Ire1H890A mutant eyes had dramatically reduced Rhodopsin-1 protein levels. Drosophila eye pigment granules are most notably associated with late endosome/lysosomal defects. Our results indicate that the loss of Ire1, which would impair ER homeostasis, also results in altered adult eye pigmentation.
Asunto(s)
Ojo Compuesto de los Artrópodos/química , Ojo Compuesto de los Artrópodos/fisiología , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Endorribonucleasas/genética , Endorribonucleasas/metabolismo , Pigmentos Biológicos/análisis , Alelos , Animales , Ojo Compuesto de los Artrópodos/ultraestructura , Drosophila melanogaster , Retículo Endoplásmico/metabolismo , Estrés del Retículo Endoplásmico , Color del Ojo , Mutación , Fenotiazinas/análisis , Células Fotorreceptoras de Invertebrados/metabolismo , Pigmentación , Pteridinas/análisis , Rodopsina/metabolismoRESUMEN
Nutrient sensors allow animals to identify foods rich in specific nutrients. The Drosophila nutrient sensor, diuretic hormone 44 (DH44) neurons, helps the fly to detect nutritive sugar. This sensor becomes operational during starvation; however, the mechanisms by which DH44 neurons or other nutrient sensors are regulated remain unclear. Here, we identified two satiety signals that inhibit DH44 neurons: (1) Piezo-mediated stomach/crop stretch after food ingestion and (2) Neuromedin/Hugin neurosecretory neurons in the ventral nerve cord (VNC) activated by an increase in the internal glucose level. A subset of Piezo+ neurons that express DH44 neuropeptide project to the crop. We found that DH44 neuronal activity and food intake were stimulated following a knockdown of piezo in DH44 neurons or silencing of Hugin neurons in the VNC, even in fed flies. Together, we propose that these two qualitatively distinct peripheral signals work in concert to regulate the DH44 nutrient sensor during the fed state.
Asunto(s)
Proteínas de Drosophila/metabolismo , Tracto Gastrointestinal/fisiología , Glucosa/metabolismo , Canales Iónicos/metabolismo , Inhibición Neural/fisiología , Neuronas/metabolismo , Neuropéptidos/metabolismo , Respuesta de Saciedad/fisiología , Animales , Drosophila , Drosophila melanogaster , Conducta Alimentaria/fisiología , Tracto Gastrointestinal/inervación , Hormonas de Insectos , Mecanotransducción Celular/fisiología , Neuronas/fisiología , Estómago/inervación , Estómago/fisiologíaRESUMEN
The Integrated Stress Response (ISR) helps metazoan cells adapt to cellular stress by limiting the availability of initiator methionyl-tRNA for translation. Such limiting conditions paradoxically stimulate the translation of ATF4 mRNA through a regulatory 5' leader sequence with multiple upstream Open Reading Frames (uORFs), thereby activating stress-responsive gene expression. Here, we report the identification of two critical regulators of such ATF4 induction, the noncanonical initiation factors eIF2D and DENR. Loss of eIF2D and DENR in Drosophila results in increased vulnerability to amino acid deprivation, susceptibility to retinal degeneration caused by endoplasmic reticulum (ER) stress, and developmental defects similar to ATF4 mutants. eIF2D requires its RNA-binding motif for regulation of 5' leader-mediated ATF4 translation. Consistently, eIF2D and DENR deficient human cells show impaired ATF4 protein induction in response to ER stress. Altogether, our findings indicate that eIF2D and DENR are critical mediators of ATF4 translational induction and stress responses in vivo.
Asunto(s)
Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Estrés del Retículo Endoplásmico/genética , Factores Eucarióticos de Iniciación/genética , Biosíntesis de Proteínas , Factores de Transcripción/genética , Factor de Transcripción Activador 4/genética , Factor de Transcripción Activador 4/metabolismo , Animales , Animales Modificados Genéticamente , Sitios de Unión , Línea Celular , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Factor 2 Eucariótico de Iniciación/genética , Factor 2 Eucariótico de Iniciación/metabolismo , Factores Eucarióticos de Iniciación/metabolismo , Humanos , Mutación , Sistemas de Lectura Abierta , Interferencia de ARN , Degeneración Retiniana/genética , Factores de Transcripción/metabolismoRESUMEN
[This corrects the article DOI: 10.1016/j.csbj.2018.09.003.].
RESUMEN
We have derived single-chain variable fragments (scFv) from tau antibody hybridomas and previously shown their promise as imaging diagnostic agents. Here, we examined the therapeutic potential of anti-tau scFv in transgenic Drosophila models that express in neurons wild-type (WT) human tau (htau) or the human tauopathy mutation R406W. scFv expressing flies were crossed with the tauopathy flies and analyzed. Overall, the survival curves differed significantly (p < .0001). Control flies not expressing htau survived the longest, whereas R406W expressing flies had the shortest lifespan, which was greatly prolonged by co-expressing the anti-tau scFv (p < .0001). Likewise, htau WT expressing flies had a moderately short lifespan, which was prolonged by co-expressing the anti-tau scFv (p < .01). In addition, the htau expression impaired wing expansion after eclosion (p < .0001), and caused progressive abdomen expansion (p < .0001). These features were more severe in htau R406W flies than in htau WT flies. Importantly, both phenotypes were prevented by co-expression of the anti-tau scFv (p < .01-0.0001). Lastly, brain analyses revealed scFv-mediated tau clearance (p < .05-0.01), and its prevention of tau-mediated neurotoxicity (p < .05-0.001). In summary, these findings support the therapeutic potential of an anti-tau scFv, including as gene therapies, and the use of Drosophila models for such screening.
Asunto(s)
Anticuerpos de Cadena Única/metabolismo , Anticuerpos de Cadena Única/farmacología , Tauopatías/prevención & control , Proteínas tau/antagonistas & inhibidores , Animales , Modelos Animales de Enfermedad , Drosophila , Humanos , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Fenotipo , Tauopatías/metabolismo , Proteínas tau/metabolismoRESUMEN
Eukaryotic cells respond to an overload of unfolded proteins in the endoplasmic reticulum (ER) by activating signaling pathways that are referred to as the unfolded protein response (UPR). Much UPR research has been conducted in cultured cells that exhibit no baseline UPR activity until they are challenged by ER stress initiated by chemicals or mutant proteins. At the same time, many genes that mediate UPR signaling are essential for the development of organisms ranging from Drosophila and fish to mice and humans, indicating that there is physiological ER stress that requires UPR in normally developing animal tissues. Recent studies have elucidated the tissue-specific roles of all three branches of UPR in distinct developing tissues of Drosophila, fish and mammals. As discussed in this Review, these studies not only reveal the physiological functions of the UPR pathways but also highlight a surprising degree of specificity associated with each UPR branch in development.
Asunto(s)
Estrés del Retículo Endoplásmico , Retículo Endoplásmico/metabolismo , Respuesta de Proteína Desplegada , Animales , Drosophila , Peces , Regulación del Desarrollo de la Expresión Génica , Humanos , Especificidad de Órganos , Transducción de SeñalRESUMEN
Rhodopsins require retinoid chromophores for their function. In vertebrates, retinoids also serve as signaling molecules, but whether these molecules similarly regulate gene expression in Drosophila remains unclear. Here, we report the identification of a retinoid-inducible gene in Drosophila, highroad, which is required for photoreceptors to clear folding-defective mutant Rhodopsin-1 proteins. Specifically, knockdown or genetic deletion of highroad blocks the degradation of folding-defective Rhodopsin-1 mutant, ninaEG69D. Moreover, loss of highroad accelerates the age-related retinal degeneration phenotype of ninaEG69D mutants. Elevated highroad transcript levels are detected in ninaEG69D flies, and interestingly, deprivation of retinoids in the fly diet blocks this effect. Consistently, mutations in the retinoid transporter, santa maria, impairs the induction of highroad in ninaEG69D flies. In cultured S2 cells, highroad expression is induced by retinoic acid treatment. These results indicate that cellular quality-control mechanisms against misfolded Rhodopsin-1 involve regulation of gene expression by retinoids.
Asunto(s)
Carboxipeptidasas/metabolismo , Drosophila melanogaster/metabolismo , Retinoides/metabolismo , Rodopsina/metabolismo , Animales , Modelos Animales de Enfermedad , Mutación , Células Fotorreceptoras de Invertebrados , Rodopsina/genéticaRESUMEN
Bacterial infection often leads to suppression of mRNA translation, but hosts are nonetheless able to express immune response genes through as yet unknown mechanisms. Here, we use a Drosophila model to demonstrate that antimicrobial peptide (AMP) production during infection is paradoxically stimulated by the inhibitor of cap-dependent translation, 4E-BP (eIF4E-binding protein; encoded by the Thor gene). We found that 4E-BP is induced upon infection with pathogenic bacteria by the stress-response transcription factor ATF4 and its upstream kinase, GCN2. Loss of gcn2, atf4, or 4e-bp compromised immunity. While AMP transcription is unaffected in 4e-bp mutants, AMP protein levels are substantially reduced. The 5' UTRs of AMPs score positive in cap-independent translation assays, and this cap-independent activity is enhanced by 4E-BP. These results are corroborated in vivo using transgenic 5' UTR reporters. These observations indicate that ATF4-induced 4e-bp contributes to innate immunity by biasing mRNA translation toward cap-independent mechanisms, thus enhancing AMP synthesis.
Asunto(s)
Factor de Transcripción Activador 4/genética , Antiinfecciosos/farmacología , Proteínas de Drosophila/metabolismo , Unión Proteica/efectos de los fármacos , Proteínas Quinasas/metabolismo , Animales , Infecciones Bacterianas/genética , Proteínas Portadoras/metabolismo , Drosophila , Proteínas de Drosophila/efectos de los fármacos , Proteínas de Drosophila/genética , Factor 4E Eucariótico de Iniciación/genética , Péptidos y Proteínas de Señalización Intracelular/efectos de los fármacos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Fosforilación/fisiología , Unión Proteica/genética , Biosíntesis de Proteínas/fisiología , Proteínas Quinasas/efectos de los fármacos , Transducción de Señal/efectos de los fármacosRESUMEN
IRE1 mediates the unfolded protein response (UPR) in part by regulating XBP1 mRNA splicing in response to endoplasmic reticulum (ER) stress. In cultured metazoan cells, IRE1 also exhibits XBP1-independent biochemical activities. IRE1 and XBP1 are developmentally essential genes in Drosophila and mammals, but the source of the physiological ER stress and the relative contributions of XBP1 activation versus other IRE1 functions to development remain unknown. Here, we employed Drosophila to address this question. Explicitly, we find that specific regions of the developing alimentary canal, fat body and the male reproductive organ are the sources of physiological stress that require Ire1 and Xbp1 for resolution. In particular, the developmental lethality associated with an Xbp1 null mutation was rescued by transgenic expression of Xbp1 in the alimentary canal. The domains of IRE1 that are involved in detecting unfolded proteins, cleaving RNAs and activating XBP1 splicing were all essential for development. The earlier onset of developmental defects in Ire1 mutant larvae compared to in Xbp1-null flies supports a developmental role for XBP1-independent IRE1 RNase activity, while challenging the importance of RNase-independent effector mechanisms of Drosophila IRE1 function.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/metabolismo , Endorribonucleasas/metabolismo , Estrés Fisiológico , Animales , Proteínas de Unión al ADN/genética , Regulación hacia Abajo/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Tracto Gastrointestinal/metabolismo , Regulación del Desarrollo de la Expresión Génica , Ontología de Genes , Inmunidad Innata , Larva/metabolismo , Masculino , Mutación/genética , Transgenes , Regulación hacia Arriba/genéticaRESUMEN
The Integrated Stress Response (ISR) refers to a signaling pathway initiated by stress-activated eIF2α kinases. Once activated, the pathway causes attenuation of global mRNA translation while also paradoxically inducing stress response gene expression. A detailed analysis of this pathway has helped us better understand how stressed cells coordinate gene expression at translational and transcriptional levels. The translational attenuation associated with this pathway has been largely attributed to the phosphorylation of the translational initiation factor eIF2α. However, independent studies are now pointing to a second translational regulation step involving a downstream ISR target, 4E-BP, in the inhibition of eIF4E and specifically cap-dependent translation. The activation of 4E-BP is consistent with previous reports implicating the roles of 4E-BP resistant, Internal Ribosome Entry Site (IRES) dependent translation in ISR active cells. In this review, we provide an overview of the translation inhibition mechanisms engaged by the ISR and how they impact the translation of stress response genes. [BMB Reports 2017; 50(11): 539-545].
Asunto(s)
Factor 2 Eucariótico de Iniciación/metabolismo , Estrés Fisiológico/fisiología , Animales , Factor 2 Eucariótico de Iniciación/fisiología , Factor 4E Eucariótico de Iniciación/metabolismo , Regulación de la Expresión Génica/genética , Humanos , Fosforilación , Biosíntesis de Proteínas/genética , Biosíntesis de Proteínas/fisiología , Procesamiento Proteico-Postraduccional , Transducción de Señal , Estrés Fisiológico/genéticaRESUMEN
Reduced amino acid availability attenuates mRNA translation in cells and helps to extend lifespan in model organisms. The amino acid deprivation-activated kinase GCN2 mediates this response in part by phosphorylating eIF2α. In addition, the cap-dependent translational inhibitor 4E-BP is transcriptionally induced to extend lifespan in Drosophila melanogaster, but through an unclear mechanism. Here, we show that GCN2 and its downstream transcription factor, ATF4, mediate 4E-BP induction, and GCN2 is required for lifespan extension in response to dietary restriction of amino acids. The 4E-BP intron contains ATF4-binding sites that not only respond to stress but also show inherent ATF4 activity during normal development. Analysis of the newly synthesized proteome through metabolic labeling combined with click chemistry shows that certain stress-responsive proteins are resistant to inhibition by 4E-BP, and gcn2 mutant flies have reduced levels of stress-responsive protein synthesis. These results indicate that GCN2 and ATF4 are important regulators of 4E-BP transcription during normal development and aging.
Asunto(s)
Factor de Transcripción Activador 4/metabolismo , Envejecimiento/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Factores de Iniciación de Péptidos/metabolismo , Proteínas Quinasas/metabolismo , Factores de Transcripción/metabolismo , Factor de Transcripción Activador 4/genética , Envejecimiento/genética , Aminoácidos/deficiencia , Animales , Sitios de Unión , Línea Celular , Química Clic , Dieta con Restricción de Proteínas , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/crecimiento & desarrollo , Estrés del Retículo Endoplásmico , Genotipo , Péptidos y Proteínas de Señalización Intracelular/genética , Intrones , Longevidad , Mutación , Factores de Iniciación de Péptidos/genética , Fenotipo , Proteínas Quinasas/genética , Proteoma , Proteómica/métodos , Interferencia de ARN , Transducción de Señal , Factores de Tiempo , Factores de Transcripción/genética , Transcripción Genética , Transfección , Regulación hacia Arriba , eIF-2 Quinasa/metabolismoRESUMEN
A number of age-dependent degenerative diseases are caused by chronic endoplasmic reticulum (ER) stress in vital cells. In many cases, the afflicted cells suffer from ER stress since birth, but the death of irreplaceable cells occurs only late in life. Although our understanding of ER stress-induced cell death has advanced significantly, most of the known mechanisms involve pathways that signal within hours, and it remains unclear how these pathways regulate cell death that occurs only decades later. Here, I highlight the conceptual issues and suggest ways to make sense of the age-related effect of ER stress-induced cell death in degenerative diseases.
Asunto(s)
Muerte Celular/fisiología , Estrés del Retículo Endoplásmico/fisiología , Envejecimiento/patología , Envejecimiento/fisiología , Animales , Apoptosis/fisiología , Caspasas/metabolismo , Senescencia Celular/fisiología , Humanos , Modelos Biológicos , Transducción de Señal , Factores de Tiempo , Respuesta de Proteína DesplegadaRESUMEN
Drosophila has served as a particularly attractive model to study cell death due to the vast array of tools for genetic manipulation under defined spatial and temporal conditions in vivo as well as in cultured cells. These genetic methods have been well supplemented by enzymatic assays and a panel of antibodies recognizing cell death markers. This chapter discusses reporters, mutants, and assays used by various laboratories to study cell death in the context of development and in response to external insults.