RESUMO
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.
Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Complexo de Golgi/metabolismo , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/antagonistas & inibidores , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Northern Blotting , Imunoprecipitação da Cromatina , Regulação da Expressão Gênica/genética , Genes Reporter/genética , Complexo de Golgi/genética , Células HeLa , Humanos , Imuno-Histoquímica , Microscopia de Fluorescência , Ligação Proteica , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Elementos de Resposta/genéticaRESUMO
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.
Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Complexo de Golgi/genética , Complexo de Golgi/metabolismo , Elementos de Resposta , Transporte Ativo do Núcleo Celular , Núcleo Celular/metabolismo , Glicosilação , Células HeLa , Humanos , Metabolismo dos Lipídeos , Estresse Oxidativo/genética , Fosforilação , Proteoglicanas/metabolismo , Transcrição Gênica , Ativação TranscricionalRESUMO
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.
Assuntos
Proteínas de Ligação a DNA , Estresse do Retículo Endoplasmático/genética , Splicing de RNA/genética , Fatores de Transcrição , Enzimas de Conjugação de Ubiquitina , Animais , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/fisiologia , Estresse do Retículo Endoplasmático/fisiologia , Regulação da Expressão Gênica , Células HeLa , Humanos , Mutação , Dobramento de Proteína , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Transcrição de Fator Regulador X , Saccharomyces cerevisiae/genética , Sumoilação/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Enzimas de Conjugação de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/metabolismo , Proteína 1 de Ligação a X-BoxRESUMO
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.
Assuntos
Estresse do Retículo Endoplasmático/efeitos da radiação , Retículo Endoplasmático/efeitos da radiação , Fibroblastos/efeitos da radiação , Regulação da Expressão Gênica/efeitos da radiação , Fator 6 Ativador da Transcrição/genética , Fator 6 Ativador da Transcrição/metabolismo , Células Cultivadas , Derme/citologia , Derme/metabolismo , Derme/efeitos da radiação , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Endorribonucleases/genética , Endorribonucleases/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Genes Reporter , Humanos , Luciferases , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Dobramento de Proteína/efeitos da radiação , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Raios Ultravioleta , Resposta a Proteínas não Dobradas/genética , eIF-2 Quinase/genética , eIF-2 Quinase/metabolismoRESUMO
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.
Assuntos
Complexo de Golgi/fisiologia , Elementos de Resposta/genética , Estresse Fisiológico/genética , Ativação Transcricional/genética , Sequência de Bases , Complexo de Golgi/efeitos dos fármacos , Células HeLa , Humanos , Monensin/farmacologia , Elementos de Resposta/efeitos dos fármacos , Estresse Fisiológico/efeitos dos fármacos , Ativação Transcricional/efeitos dos fármacosRESUMO
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.
Assuntos
Citoplasma/genética , Proteínas de Ligação a DNA/genética , Dobramento de Proteína , Splicing de RNA/genética , Fatores de Transcrição/genética , Sequência de Aminoácidos , Domínio Catalítico , Endorribonucleases/química , Células HeLa , Humanos , Membranas Intracelulares/metabolismo , Dados de Sequência Molecular , Sinais de Exportação Nuclear , Proteínas Serina-Treonina Quinases/química , RNA Ligase (ATP) , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Transcrição de Fator Regulador X , Solubilidade , Frações Subcelulares/metabolismo , Transcrição Gênica , Proteína 1 de Ligação a X-BoxRESUMO
Upon the accumulation of unfolded proteins in the mammalian endoplasmic reticulum (ER), X-box binding protein 1 (XBP1) premessenger RNA (premRNA) is converted to mature mRNA by unconventional splicing that is mediated by the endonuclease inositol-requiring enzyme 1. The transcription factor protein (p) XBP1 spliced (S), which is translated from mature XBP1 mRNA, contains the nuclear localization signal and the transcriptional activation domain and activates the transcription of target genes, including those encoding ER chaperones in the nucleus. We show that pXBP1 unspliced (U) encoded in XBP1 pre-mRNA was constitutively expressed and markedly accumulated at the recovery phase of ER stress. pXBP1(U) contained the nuclear exclusion signal instead of the transcriptional activation domain and shuttled between the nucleus and the cytoplasm. Interestingly, pXBP1(U) formed a complex with pXBP1(S), and the pXBP1(U)-pXBP1(S) complex was sequestered from the nucleus. Moreover, the complex was rapidly degraded by proteasomes because of the degradation motif contained in pXBP1(U). Thus, pXBP1(U) is a negative feedback regulator of pXBP1(S), which shuts off the transcription of target genes during the recovery phase of ER stress.