RESUMEN
Secretory pathway proteins are cotranslationally translocated into the endoplasmic reticulum (ER) of metazoan cells through the protein channel, translocon. Given that there are far fewer translocons than ribosomes in a cell, it is essential that secretory protein-translating ribosomes only occupy translocons transiently. Therefore, if translocons are obstructed by ribosomes stalled or slowed in translational elongation, it possibly results in deleterious consequences to cellular function. Hence, we investigated how translocon clogging by stalled ribosomes affects mammalian cells. First, we constructed ER-destined translational arrest proteins (ER-TAP) as an artificial protein that clogged the translocon in the ER membrane. Here, we show that the translocon clogging by ER-TAP expression activates triage of signal sequences (SS) in which secretory pathway proteins harboring highly efficient SS are preferentially translocated into the ER lumen. Interestingly, the translocon obstructed status specifically activates inositol requiring enzyme 1α (IRE1α) but not protein kinase R-like ER kinase (PERK). Given that the IRE1α-XBP1 pathway mainly induces the translocon components, our discovery implies that lowered availability of translocon activates IRE1α, which induces translocon itself. This results in rebalance between protein influx into the ER and the cellular translocation capacity.Key words: endoplasmic reticulum, translocation capacity, translocon clogging, IRE1, signal sequence.
Asunto(s)
Endorribonucleasas , Proteínas Serina-Treonina Quinasas , Animales , Endorribonucleasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Señales de Clasificación de Proteína , Triaje , Estrés del Retículo Endoplásmico , Mamíferos/metabolismoRESUMEN
There are diverse links between macroautophagy/autophagy pathways and unfolded protein response (UPR) pathways under endoplasmic reticulum (ER) stress conditions to restore ER homeostasis. Phosphorylation of EIF2S1/eIF2α is an important mechanism that can regulate all three UPR pathways through transcriptional and translational reprogramming to maintain cellular homeostasis and overcome cellular stresses. In this study, to investigate the roles of EIF2S1 phosphorylation in regulation of autophagy during ER stress, we used EIF2S1 phosphorylation-deficient (A/A) cells in which residue 51 was mutated from serine to alanine. A/A cells exhibited defects in several steps of autophagic processes (such as autophagosome and autolysosome formation) that are regulated by the transcriptional activities of the autophagy master transcription factors TFEB and TFE3 under ER stress conditions. EIF2S1 phosphorylation was required for nuclear translocation of TFEB and TFE3 during ER stress. In addition, EIF2AK3/PERK, PPP3/calcineurin-mediated dephosphorylation of TFEB and TFE3, and YWHA/14-3-3 dissociation were required for their nuclear translocation, but were insufficient to induce their nuclear retention during ER stress. Overexpression of the activated ATF6/ATF6α form, XBP1s, and ATF4 differentially rescued defects of TFEB and TFE3 nuclear translocation in A/A cells during ER stress. Consequently, overexpression of the activated ATF6 or TFEB form more efficiently rescued autophagic defects, although XBP1s and ATF4 also displayed an ability to restore autophagy in A/A cells during ER stress. Our results suggest that EIF2S1 phosphorylation is important for autophagy and UPR pathways, to restore ER homeostasis and reveal how EIF2S1 phosphorylation connects UPR pathways to autophagy.Abbreviations: A/A: EIF2S1 phosphorylation-deficient; ACTB: actin beta; Ad-: adenovirus-; ATF6: activating transcription factor 6; ATZ: SERPINA1/α1-antitrypsin with an E342K (Z) mutation; Baf A1: bafilomycin A1; BSA: bovine serum albumin; CDK4: cyclin dependent kinase 4; CDK6: cyclin dependent kinase 6; CHX: cycloheximide; CLEAR: coordinated lysosomal expression and regulation; Co-IP: coimmunoprecipitation; CTSB: cathepsin B; CTSD: cathepsin D; CTSL: cathepsin L; DAPI: 4',6-diamidino-2-phenylindole dihydrochloride; DMEM: Dulbecco's modified Eagle's medium; DMSO: dimethyl sulfoxide; DTT: dithiothreitol; EBSS: Earle's Balanced Salt Solution; EGFP: enhanced green fluorescent protein; EIF2S1/eIF2α: eukaryotic translation initiation factor 2 subunit alpha; EIF2AK3/PERK: eukaryotic translation initiation factor 2 alpha kinase 3; ER: endoplasmic reticulum; ERAD: endoplasmic reticulum-associated degradation; ERN1/IRE1α: endoplasmic reticulum to nucleus signaling 1; FBS: fetal bovine serum; gRNA: guide RNA; GSK3B/GSK3ß: glycogen synthase kinase 3 beta; HA: hemagglutinin; Hep: immortalized hepatocyte; IF: immunofluorescence; IRES: internal ribosome entry site; KO: knockout; LAMP1: lysosomal associated membrane protein 1; LMB: leptomycin B; LPS: lipopolysaccharide; MAP1LC3A/B/LC3A/B: microtubule associated protein 1 light chain 3 alpha/beta; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MEFs: mouse embryonic fibroblasts; MFI: mean fluorescence intensity; MTORC1: mechanistic target of rapamycin kinase complex 1; NES: nuclear export signal; NFE2L2/NRF2: NFE2 like bZIP transcription factor 2; OE: overexpression; PBS: phosphate-buffered saline; PLA: proximity ligation assay; PPP3/calcineurin: protein phosphatase 3; PTM: post-translational modification; SDS: sodium dodecyl sulfate; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SEM: standard error of the mean; TEM: transmission electron microscopy; TFE3: transcription factor E3; TFEB: transcription factor EB; TFs: transcription factors; Tg: thapsigargin; Tm: tunicamycin; UPR: unfolded protein response; WB: western blot; WT: wild-type; Xbp1s: spliced Xbp1; XPO1/CRM1: exportin 1.
Asunto(s)
Endorribonucleasas , Proteínas Serina-Treonina Quinasas , Animales , Ratones , Proteínas Serina-Treonina Quinasas/metabolismo , Fosforilación , Endorribonucleasas/metabolismo , Factor 2 Procariótico de Iniciación/metabolismo , Autofagia/genética , Calcineurina/metabolismo , Degradación Asociada con el Retículo Endoplásmico , Dodecil Sulfato de Sodio/metabolismo , Fibroblastos/metabolismo , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Lisosomas/metabolismoRESUMEN
The Golgi apparatus is an organelle where membrane or secretory proteins receive post-translational modifications such as glycosylation and sulfation, after which the proteins are selectively transported to their final destinations through vesicular transport. When the synthesis of secretory or membrane proteins is increased and overwhelms the capacity of the Golgi (Golgi stress), eukaryotic cells activate a homeostatic mechanism called the Golgi stress response to augment the capacity of the Golgi. Four response pathways of the Golgi stress response have been identified, namely the TFE3, CREB3, HSP47, and proteoglycan pathways, which regulate the general function of the Golgi, apoptosis, cell survival, and proteoglycan glycosylation, respectively. Here, we identified a novel response pathway that augments the expression of glycosylation enzymes for mucins in response to insufficiency in mucin-type glycosylation in the Golgi (mucin-type Golgi stress), and we found that expression of glycosylation enzymes for mucins such as GALNT5, GALNT8, and GALNT18 was increased upon mucin-type-Golgi stress. We named this pathway the mucin pathway. Unexpectedly, mucin-type Golgi stress induced the expression and activation of TFE3, a key transcription factor regulating the TFE3 pathway, suggesting that the activated mucin pathway sends a crosstalk signal to the TFE3 pathway. We identified an enhancer element regulating transcriptional induction of TFE3 upon mucin-type Golgi stress, and named it the mucin-type Golgi stress response element, of which consensus was ACTTCC(N9)TCCCCA. These results suggested that crosstalk from the mucin pathway to the TFE3 pathway has an important role in the regulation of the mammalian Golgi stress response.Key words: Golgi stress, mucin, TFE3, organelle autoregulation, organelle zone.
Asunto(s)
Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Aparato de Golgi/metabolismo , Mucinas/metabolismo , Elementos de Respuesta/genética , Aparato de Golgi/genética , Células HT29 , Células HeLa , Humanos , Mucinas/genética , Mutación PuntualRESUMEN
Organelles have been studied traditionally as single units, but a novel concept is now emerging: each organelle has distinct functional zones that regulate specific functions. The Golgi apparatus seems to have various zones, including zones for: glycosylphosphatidylinositol-anchored proteins; proteoglycan, mucin and lipid glycosylation; transport of cholesterol and ceramides; protein degradation (Golgi membrane-associated degradation); and signalling for apoptosis. The capacity for these specific functions and the size of the corresponding zones appear to be tightly regulated by the Golgi stress response to accommodate cellular demands. For instance, the proteoglycan and mucin zones seem to be separately augmented during the differentiation of chondrocytes and goblet cells, respectively. The mammalian Golgi stress response consists of several response pathways. The TFE3 pathway regulates the general function of the Golgi, such as structural maintenance, N-glycosylation and vesicular transport, whereas the proteoglycan pathway increases the expression of glycosylation enzymes for proteoglycans. The CREB3 and HSP47 pathways regulate pro- and anti-apoptotic functions, respectively. These observations indicate that the Golgi is a dynamic organelle, the capacity of which is upregulated according to cellular needs.
Asunto(s)
Estrés del Retículo Endoplásmico , Aparato de Golgi/metabolismo , Animales , HumanosRESUMEN
In research on cell biology, organelles have been a major unit of such analyses. Researchers have assumed that the inside of an organelle is almost uniform in regards to its function, even though each organelle has multiple functions. However, we are now facing conundrums that cannot be resolved so long as we regard organelles as functionally uniform units. For instance, how can cells control the diverse patterns of glycosylation of various secretory proteins in the endoplasmic reticulum and Golgi in an orderly manner with high accuracy? Here, we introduce the novel concept of organelle zones as a solution; each organelle has functionally distinct zones, and zones in different organelles closely interact each other in order to perform complex cellular functions. This Copernican Revolution from organelle biology to organelle zone biology will drastically change and advance our thoughts about cells.Key words: organelle zone, contact site, ER stress, Golgi stress, organelle autoregulation.
Asunto(s)
Orgánulos/metabolismo , Animales , Retículo Endoplásmico/metabolismo , Aparato de Golgi/metabolismo , HumanosRESUMEN
OSW-1 is a plant-derived natural product proposed to selectively kill cancer cells by binding to members of the oxysterol binding protein family, thereby disrupting lipid/sterol homeostasis. However, how these protein-ligand interactions mediate cell death signaling has remained elusive. Here, we discovered that OSW-1 selectively activates the Golgi stress response leading to apoptosis, providing a mechanistic basis for the anticancer activity of OSW-1.
Asunto(s)
Antineoplásicos/uso terapéutico , Colestenonas/uso terapéutico , Aparato de Golgi/efectos de los fármacos , Saponinas/uso terapéutico , Antineoplásicos/farmacología , Colestenonas/farmacología , Humanos , Saponinas/farmacologíaRESUMEN
The Golgi stress response is a homeostatic mechanism that augments the functional capacity of the Golgi apparatus when Golgi function becomes insufficient (Golgi stress). Three response pathways of the Golgi stress response have been identified in mammalian cells, the TFE3, HSP47 and CREB3 pathways, which augment the capacity of specific Golgi functions such as N-glycosylation, anti-apoptotic activity and pro-apoptotic activity, respectively. On the contrary, glycosylation of proteoglycans (PGs) is another important function of the Golgi, although the response pathway upregulating expression of glycosylation enzymes for PGs in response to Golgi stress remains unknown. Here, we found that expression of glycosylation enzymes for PGs was induced upon insufficiency of PG glycosylation capacity in the Golgi (PG-Golgi stress), and that transcriptional induction of genes encoding glycosylation enzymes for PGs was independent of the known Golgi stress response pathways and ER stress response. Promoter analyses of genes encoding these glycosylation enzymes revealed the novel enhancer elements PGSE-A and PGSE-B (the consensus sequences are CCGGGGCGGGGCG and TTTTACAATTGGTC, respectively), which regulate their transcriptional induction upon PG-Golgi stress. From these observations, the response pathway we discovered is a novel Golgi stress response pathway, which we have named the PG pathway.Key words: Golgi stress, proteoglycan, ER stress, organelle zone, organelle autoregulation.
Asunto(s)
Aparato de Golgi/genética , Proteoglicanos/metabolismo , Elementos de Respuesta/genética , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Estrés del Retículo Endoplásmico/genética , Proteínas del Choque Térmico HSP47/metabolismo , Células HeLa , Humanos , Transcripción GenéticaRESUMEN
The endoplasmic reticulum (ER) is the organelle where secretory and membrane proteins are synthesized and folded. Unfolded proteins that are retained within the ER can cause ER stress. Eukaryotic cells have a defense system called the "unfolded protein response" (UPR), which protects cells from ER stress. Cells undergo apoptosis when ER stress exceeds the capacity of the UPR, which has been revealed to cause human diseases. Although neurodegenerative diseases are well-known ER stress-related diseases, it has been discovered that endocrine diseases are also related to ER stress. In this review, we focus on ER stress-related human endocrine disorders. In addition to diabetes mellitus, which is well characterized, several relatively rare genetic disorders such as familial neurohypophyseal diabetes insipidus (FNDI), Wolfram syndrome, and isolated growth hormone deficiency type II (IGHD2) are discussed in this article.
Asunto(s)
Enfermedades del Sistema Endocrino/etiología , Enfermedades del Sistema Endocrino/metabolismo , Estrés del Retículo Endoplásmico , Animales , Modelos Animales de Enfermedad , Enfermedades del Sistema Endocrino/diagnóstico , Enfermedades del Sistema Endocrino/terapia , Humanos , Mamíferos , Transducción de Señal , Respuesta de Proteína DesplegadaRESUMEN
The capacity of each organelle in eukaryotic cells is tightly regulated in accordance with cellular demands by specific regulatory systems, which are generically termed organelle autoregulation. The Golgi stress response is one of the systems of organelle autoregulation and it augments the capacity of Golgi function if this becomes insufficient (Golgi stress). Recently, several pathways of the mammalian Golgi stress response have been identified, specifically the TFE3, HSP47, and CREB3 pathways. This review summarizes the essential parts of the Golgi stress response from the perspective of the organelle autoregulation.
Asunto(s)
Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Aparato de Golgi/metabolismo , Proteínas del Choque Térmico HSP47/metabolismo , Estrés Fisiológico , Animales , HumanosRESUMEN
The Golgi stress response is a homeostatic mechanism that controls the capacity of the Golgi apparatus in accordance with cellular demands. When the capacity of the Golgi apparatus becomes insufficient (Golgi stress), transcription levels of Golgi-related genes encoding glycosylation enzymes, a Golgi structural protein, and components of vesicular transport are upregulated through a common cis-acting enhancer-the Golgi apparatus stress response element (GASE). Here, we identified the transcription factor MLX as a GASE-binding protein. MLX resides in the cytoplasm and does not bind to GASE in normal growth conditions, whereas MLX translocates into the nucleus and specifically binds to GASE in response to Golgi stress. Suppression of MLX expression increased transcriptional induction of target genes of the Golgi stress response, whereas overexpression of MLX reduced GASE-binding of TFE3 as well as transcriptional induction from GASE, suggesting that MLX is a transcriptional repressor of the mammalian Golgi stress response.
Asunto(s)
Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Aparato de Golgi/metabolismo , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/antagonistas & inhibidores , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Northern Blotting , Inmunoprecipitación de Cromatina , Regulación de la Expresión Génica/genética , Genes Reporteros/genética , Aparato de Golgi/genética , Células HeLa , Humanos , Inmunohistoquímica , Microscopía Fluorescente , Unión Proteica , Interferencia de ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Elementos de Respuesta/genéticaRESUMEN
PURPOSE OF REVIEW: Recently, a number of papers have reported that endoplasmic reticulum (ER) stress is involved in the onset of various kidney diseases, but the pathological mechanisms responsible have not been clarified. In this review, we summarize recent findings on this issue and try to clarify the pathology of ER stress-induced kidney diseases. RECENT FINDINGS: ER stress is evoked in various kidney diseases, including diabetic nephropathy, renal fibrosis, inflammation or osmolar contrast-induced renal injury, ischemia-reperfusion, genetic mutations of renal proteins, proteinuria and cyclosporine A treatment. In some cases, chemical chaperones, such as 4-phenylbutyrate and taurodeoxycholic acid, relieve the symptoms, indicating that ER stress-induced apoptosis of renal cells is one of the major causes of certain kidney diseases. Actually, the ER stress response provides protection against some kidney diseases, although the PERK-ATF4-CHOP pathway of the ER stress response is proapoptotic in some kidney diseases. The disposal of unfolded proteins by autophagy is also protective for some ER stress-induced kidney diseases. SUMMARY: Because ER stress is a major cause of some kidney diseases, the ER stress response and autophagy, which deal with unfolded proteins that accumulate in the ER, are promising therapeutic targets in acute and chronic kidney diseases.
Asunto(s)
Autofagia/fisiología , Nefropatías Diabéticas/metabolismo , Estrés del Retículo Endoplásmico/fisiología , Enfermedades Renales/metabolismo , Riñón/metabolismo , Animales , Nefropatías Diabéticas/genética , Estrés del Retículo Endoplásmico/genética , Humanos , Enfermedades Renales/genética , Transducción de Señal/fisiologíaRESUMEN
Organelle autoregulation is a homeostatic mechanism to regulate the capacity of each organelle according to cellular demands. The endoplasmic reticulum (ER) stress response increases the expression of ER chaperones and ER-associated degradation factors when the capacity of the ER becomes insufficient, e.g. during cellular differentiation or viral propagation, and which can be restored through increased synthesis of secretory or membrane proteins. In the Golgi stress response, insufficient organelle capacity is responded to by augmentation of glycosylation enzyme expression and vesicular transport components. The mitochondrial stress response upregulates mitochondrial chaperone and protease expression in the mitochondrial matrix and intermembrane space when unfolded proteins accumulate in the mitochondria. The lysosome stress response is activated during autophagy to enhance the function of the lysosome by transcriptional induction of lysosome genes including cathepsins. However, many of the molecular mechanisms of organelle autoregulation remain unclear. Here, we review recent discoveries in organelle autoregulation and their molecular mechanisms.
Asunto(s)
Homeostasis/fisiología , Orgánulos/fisiología , Estrés Fisiológico , Animales , Retículo Endoplásmico/fisiología , Aparato de Golgi/fisiología , Humanos , Lisosomas/fisiología , Mitocondrias/fisiologíaRESUMEN
The Golgi stress response is a mechanism by which, under conditions of insufficient Golgi function (Golgi stress), the transcription of Golgi-related genes is upregulated through an enhancer, the Golgi apparatus stress response element (GASE), in order to maintain homeostasis in the Golgi. The molecular mechanisms associated with GASE remain to be clarified. Here, we identified TFE3 as a GASE-binding transcription factor. TFE3 was phosphorylated and retained in the cytoplasm in normal growth conditions, whereas it was dephosphorylated, translocated to the nucleus and activated Golgi-related genes through GASE under conditions of Golgi stress, e.g. in response to inhibition of oligosaccharide processing in the Golgi apparatus. From these observations, we concluded that the TFE3-GASE pathway is one of the regulatory pathways of the mammalian Golgi stress response, which regulates the expression of glycosylation-related proteins in response to insufficiency of glycosylation in the Golgi apparatus.
Asunto(s)
Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Aparato de Golgi/genética , Aparato de Golgi/metabolismo , Elementos de Respuesta , Transporte Activo de Núcleo Celular , Núcleo Celular/metabolismo , Glicosilación , Células HeLa , Humanos , Metabolismo de los Lípidos , Estrés Oxidativo/genética , Fosforilación , Proteoglicanos/metabolismo , Transcripción Genética , Activación TranscripcionalRESUMEN
Dominantly inherited isolated GH deficiency is mainly caused by a heterozygous donor site mutation of intron 3 in the GH1 gene. An exon 3 deletion in GH (del32-71 GH) is produced from a mutant allele, whereas wild-type GH is produced from the other allele. Several studies have demonstrated a dominant negative effect of del32-71 GH on wild-type GH secretion, but the precise molecular mechanisms remain unclear. We hypothesized that unfolded del32-71 GH accumulates in the endoplasmic reticulum (ER) and causes ER stress and apoptosis in somatotrophs, promoting GH deficiency. To evaluate del32-71 GH-mediated ER stress, we established GH4C1 cell lines with doxycycline (dox)-controlled del32-71 GH expression. In 20 of 23 dox-controlled cell lines, the concentration of wild-type GH in the culture medium significantly decreased with del32-71 GH induction, demonstrating the dominant negative effect of this mutant. Cell viability, mRNA abundance of ER stress-response genes, caspase activation, and DNA fragmentation were evaluated in 5 dox-controlled cell lines selected as cellular models. In 4 of the 5 cell lines, del32-71 GH induction decreased cell viability, increased expression of 3 major ER stress response pathways (PRKR-like endoplasmic reticulum kinase [PERK], activating transcription factor-6 [ATF6], and inositol requirement 1 [IRE1]), and induced caspase-3 and caspase-7 activation. In 1 of the 4 cell lines, DNA fragmentation was demonstrated. Finally, overexpression of XBP1(S), a nuclear transcription factor downstream of IRE1, completely reversed the observed caspase activation. These data suggested that del32-71 GH-mediated ER stress and apoptosis contributed to the decrease in wild-type GH secretion observed in GH deficiency due to the GH1 gene slice-site mutations.
Asunto(s)
Apoptosis , Enanismo Hipofisario/genética , Estrés del Retículo Endoplásmico , Hormona de Crecimiento Humana/genética , Mutación , Sitios de Empalme de ARN , Somatotrofos/metabolismo , Alelos , Animales , Antibacterianos/farmacología , Apoptosis/efectos de los fármacos , Línea Celular , Supervivencia Celular/efectos de los fármacos , Células Clonales , Doxiciclina/farmacología , Enanismo Hipofisario/metabolismo , Estrés del Retículo Endoplásmico/efectos de los fármacos , Eliminación de Gen , Hormona de Crecimiento Humana/metabolismo , Humanos , Regiones Promotoras Genéticas/efectos de los fármacos , Ratas , Proteínas Recombinantes/metabolismo , Reproducibilidad de los Resultados , Transducción de Señal/efectos de los fármacos , Somatotrofos/efectos de los fármacos , Somatotrofos/ultraestructura , Regulación hacia Arriba/efectos de los fármacosRESUMEN
XBP1 is a key transcription factor regulating the mammalian endoplasmic reticulum (ER) stress response, which is a cytoprotective mechanism for dealing with an accumulation of unfolded proteins in the ER (ER stress). The expression of XBP1 is regulated by two different mechanisms: mRNA splicing and protein stability. When ER stress occurs, unspliced XBP1 mRNA is converted to mature mRNA, from which an active transcription factor, pXBP1(S), is translated and activates the transcription of ER-related genes to dispose of unfolded proteins. In the absence of ER stress, pXBP1(U) is translated from unspliced XBP1 mRNA and enhances the degradation of pXBP1(S). Here, we analyzed the regulatory mechanism of pXBP1(S) stability, and found that a SUMO-conjugase, UBC9, specifically bound to the leucine zipper motif of pXBP1(S) and increased the stability of pXBP1(S). Suppression of UBC9 expression by RNA interference reduced both the expression of pXBP1(S) and ER stress-induced transcription by pXBP1(S). Interestingly, overexpression of a UBC9 mutant deficient in SUMO-conjugating activity was able to increase pXBP1(S) expression as well as wild-type UBC9, indicating that UBC9 stabilizes pXBP1(S) without conjugating SUMO moieties. From these observations, we concluded that UBC9 is a novel regulator of the mammalian ER stress response.
Asunto(s)
Proteínas de Unión al ADN , Estrés del Retículo Endoplásmico/genética , Empalme del ARN/genética , Factores de Transcripción , Enzimas Ubiquitina-Conjugadoras , Animales , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/fisiología , Estrés del Retículo Endoplásmico/fisiología , Regulación de la Expresión Génica , Células HeLa , Humanos , Mutación , Pliegue de Proteína , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factores de Transcripción del Factor Regulador X , Saccharomyces cerevisiae/genética , Sumoilación/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Enzimas Ubiquitina-Conjugadoras/genética , Enzimas Ubiquitina-Conjugadoras/metabolismo , Proteína 1 de Unión a la X-BoxRESUMEN
The endoplasmic reticulum (ER) stress response is a cytoprotective mechanism that maintains homeostasis of the ER by upregulating the capacity of the ER in accordance with cellular demands. If the ER stress response cannot function correctly, because of reasons such as aging, genetic mutation or environmental stress, unfolded proteins accumulate in the ER and cause ER stress-induced apoptosis, resulting in the onset of folding diseases, including Alzheimer's disease and diabetes mellitus. Although the mechanism of the ER stress response has been analyzed extensively by biochemists, cell biologists and molecular biologists, many aspects remain to be elucidated. For example, it is unclear how sensor molecules detect ER stress, or how cells choose the two opposite cell fates (survival or apoptosis) during the ER stress response. To resolve these critical issues, structural and computational approaches will be indispensable, although the mechanism of the ER stress response is complicated and difficult to understand holistically at a glance. Here, we provide a concise introduction to the mammalian ER stress response for structural and computational biologists.
RESUMEN
The endoplasmic reticulum (ER) stress response is a cytoprotective mechanism against the accumulation of unfolded proteins in the ER (ER stress) that consists of three response pathways (the ATF6, IRE1 and PERK pathways) in mammals. These pathways regulate the transcription of ER-related genes through specific cis-acting elements, ERSE, UPRE and AARE, respectively. Because the mammalian ER stress response is markedly activated in professional secretory cells, its main function was thought to be to upregulate the capacity of protein folding in the ER in accordance with the increased synthesis of secretory proteins. Here, we found that ultraviolet A (UVA) irradiation induced the conversion of an ER-localized sensor pATF6α(P) to an active transcription factor pATF6α(N) in normal human dermal fibroblasts (NHDFs). UVA also induced IRE1-mediated splicing of XBP1 mRNA as well as PERK-mediated phosphorylation of an α subunit of eukaryotic initiation factor 2. Consistent with these observations, we found that UVA increased transcription from ERSE, UPRE and AARE elements. From these results, we concluded that UVA irradiation activates all branches of the mammalian ER stress response in NHDFs. This suggests that the mammalian ER stress response is activated by not only intrinsic stress but also environmental stress.
Asunto(s)
Estrés del Retículo Endoplásmico/efectos de la radiación , Retículo Endoplásmico/efectos de la radiación , Fibroblastos/efectos de la radiación , Regulación de la Expresión Génica/efectos de la radiación , Factor de Transcripción Activador 6/genética , Factor de Transcripción Activador 6/metabolismo , Células Cultivadas , Dermis/citología , Dermis/metabolismo , Dermis/efectos de la radiación , Retículo Endoplásmico/genética , Retículo Endoplásmico/metabolismo , Endorribonucleasas/genética , Endorribonucleasas/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Genes Reporteros , Humanos , Luciferasas , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Pliegue de Proteína/efectos de la radiación , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Rayos Ultravioleta , Respuesta de Proteína Desplegada/genética , eIF-2 Quinasa/genética , eIF-2 Quinasa/metabolismoRESUMEN
When increased production of secretory proteins overwhelms the capacity of the endoplasmic reticulum (ER) and the Golgi apparatus, eukaryotic cells expand their capacity to sustain secretory function. The capacity of the ER is enhanced by the mechanism called the ER stress response, but the mechanism regulating Golgi capacity (the Golgi stress response) has remained unclear. Here, we found that transcription of Golgi-related genes, including glycosylation enzymes as well as factors involved in post-Golgi vesicular transport and maintenance of Golgi structure, was upregulated upon treatment with monensin, an ionophore that disrupts the function of acidic organelles, including the Golgi apparatus and lysosomes by neutralizing their lumen. This transcriptional induction was found to be commonly regulated by a novel cis-acting element called the Golgi apparatus stress response element (GASE), whose consensus sequence is ACGTGgc. When the function of the Golgi apparatus was specifically disturbed by overexpression of GCP60, a Golgi-localized protein that binds to giantin, transcription from GASE was significantly induced. These results suggest that mammalian cells have the Golgi stress response, and that GASE regulates transcriptional induction involved in the Golgi stress response.
Asunto(s)
Aparato de Golgi/fisiología , Elementos de Respuesta/genética , Estrés Fisiológico/genética , Activación Transcripcional/genética , Secuencia de Bases , Aparato de Golgi/efectos de los fármacos , Células HeLa , Humanos , Monensina/farmacología , Elementos de Respuesta/efectos de los fármacos , Estrés Fisiológico/efectos de los fármacos , Activación Transcripcional/efectos de los fármacosRESUMEN
XBP1 is a key transcription factor that regulates the mammalian unfolded protein response. Its expression is regulated by unconventional mRNA splicing that is carried out by endonuclease IRE1 and a specific, as yet unknown, RNA ligase in response to the accumulation of unfolded proteins in the ER. Conventional mRNA splicing occurs only in the nucleus, but it has remained unclear whether unconventional splicing of XBP1 mRNA takes place in the nucleus, cytoplasm or both. Here, we show that the catalytic domain of IRE1 contains a nuclear exclusion signal to prevent IRE1 from mislocalizing to the nucleus. In addition, RNA ligase, which joins XBP1 exons cleaved by IRE1 was detected in the cytoplasm but not in the nucleus. Moreover, the cytoplasm contained large amounts of unspliced XBP1 mRNA compared with the nucleus. Most unspliced XBP1 mRNA was converted to spliced mRNA by unconventional splicing even if de novo transcription was blocked, suggesting that cytoplasmic XBP1 mRNA, not nuclear XBP1 mRNA, is a major substrate for unconventional splicing. From these observations, we concluded that unconventional splicing of XBP1 mRNA occurs predominantly in the cytoplasm.