RESUMEN
The Salmonella enterica effector SteD depletes mature MHC class II (mMHCII) molecules from the surface of infected antigen-presenting cells through ubiquitination of the cytoplasmic tail of the mMHCII ß chain. This requires the Nedd4 family HECT E3 ubiquitin ligase Wwp2 and a tumor-suppressing transmembrane protein adaptor Tmem127. Here, through a proteomic screen of dendritic cells, we found that SteD targets the plasma membrane protein CD97 for degradation by a similar mechanism. SteD enhanced ubiquitination of CD97 on K555 and mutation of this residue eliminated the effect of SteD on CD97 surface levels. We showed that CD97 localises to and stabilises the immunological synapse between dendritic cells and T cells. Removal of CD97 by SteD inhibited dendritic cell-T cell interactions and reduced T cell activation, independently of its effect on MHCII. Therefore, SteD suppresses T cell immunity by two distinct processes.
Asunto(s)
Proteínas Bacterianas/metabolismo , Células Dendríticas/inmunología , Sinapsis Inmunológicas/inmunología , Receptores Acoplados a Proteínas G/inmunología , Linfocitos T/inmunología , Animales , Presentación de Antígeno/inmunología , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos C57BL , Infecciones por Salmonella/metabolismo , Salmonella entericaRESUMEN
The Salmonella enterica effector SteD depletes mature MHC class II (mMHCII) molecules from the surface of infected antigen-presenting cells through ubiquitination of the cytoplasmic tail of the mMHCII ß chain. Here, through a genome-wide mutant screen of human antigen-presenting cells, we show that the NEDD4 family HECT E3 ubiquitin ligase WWP2 and a tumor-suppressing transmembrane protein of unknown biochemical function, TMEM127, are required for SteD-dependent ubiquitination of mMHCII. Although evidently not involved in normal regulation of mMHCII, TMEM127 was essential for SteD to suppress both mMHCII antigen presentation in mouse dendritic cells and MHCII-dependent CD4+ T cell activation. We found that TMEM127 contains a canonical PPxY motif, which was required for binding to WWP2. SteD bound to TMEM127 and enabled TMEM127 to interact with and induce ubiquitination of mature MHCII. Furthermore, SteD also underwent TMEM127- and WWP2-dependent ubiquitination, which both contributed to its degradation and augmented its activity on mMHCII.
Asunto(s)
Proteínas Bacterianas/fisiología , Antígenos de Histocompatibilidad Clase II/metabolismo , Proteínas de la Membrana/fisiología , Salmonella typhimurium/fisiología , Ubiquitina-Proteína Ligasas/fisiología , Ubiquitinación , Animales , Presentación de Antígeno , Sistemas CRISPR-Cas , Línea Celular , Células Dendríticas/inmunología , Células Dendríticas/microbiología , Femenino , Interacciones Huésped-Patógeno , Humanos , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Mutación , Unión Proteica , Infecciones por Salmonella/inmunología , Infecciones por Salmonella/microbiología , Linfocitopenia-T Idiopática CD4-Positiva/inmunología , Linfocitopenia-T Idiopática CD4-Positiva/microbiología , VirulenciaRESUMEN
Acetyl-CoA carboxylase 1 (ACC1) is a cytosolic enzyme catalyzing the rate limiting step in de novo fatty acid biosynthesis. There is mounting evidence showing that ACC1 is susceptible to dysregulation and that it is over-expressed in liver diseases associated with lipid accumulation and in several cancers. In the present study, ACC1 regulation at the translational level is reported. Using several experimental approaches, the presence of an internal ribosome entry site (IRES) has been established in the 5' untranslated region (5' UTR) of the ACC1 mRNA. Transfection experiments with the ACC1 5' UTR inserted in a dicistronic reporter vector show a remarkable increase in the downstream cistron translation, through a cap-independent mechanism. The endoplasmic reticulum (ER) stress condition and the related unfolded protein response (UPR), triggered by treatment with thapsigargin and tunicamycin, cause an increase of the cap-independent translation of ACC1 mRNA in HepG2 cells, despite the overall reduction in global protein synthesis. Other stress conditions, such as serum starvation and incubation with hypoxia mimetic agent CoCl2, up-regulate ACC1 expression in HepG2 cells at the translational level. Overall, these findings indicate that the presence of an IRES in the ACC1 5' UTR allows ACC1 mRNA translation in conditions that are inhibitory to cap-dependent translation. A potential involvement of the cap-independent translation of ACC1 in several pathologies, such as obesity and cancer, has been discussed.
Asunto(s)
Acetil-CoA Carboxilasa/genética , Cobalto/farmacología , Estrés del Retículo Endoplásmico/efectos de los fármacos , Sitios Internos de Entrada al Ribosoma/genética , Biosíntesis de Proteínas , Regiones no Traducidas 5'/genética , Acetil-CoA Carboxilasa/metabolismo , Hipoxia de la Célula/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Medio de Cultivo Libre de Suero , Células Hep G2 , Humanos , Plásmidos/metabolismo , Biosíntesis de Proteínas/efectos de los fármacos , Empalme del ARN/genética , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Non-alcoholic fatty liver disease (NAFLD) is a chronic disease characterized by accumulation of lipid droplets in hepatocytes. Enhanced release of non-esterified fatty acids from adipose tissue accounts for a remarkable fraction of accumulated lipids. However, the de novo lipogenesis (DNL) is also implicated in the etiology of the NAFLD. Sterol Regulatory Element-Binding Protein-1 (SREBP-1) is a transcription factor modulating the expression of several lipogenic enzymes. In the present study, in order to investigate the effect of lipid droplet accumulation on DNL, we used a cellular model of steatosis represented by HepG2 cells cultured in a medium supplemented with free oleic and palmitic fatty acids (FFAs). We report that FFA supplementation induces the expression of genes coding for enzymes involved in the DNL as well as for the transcription factor SREBP-1a. The SREBP-1a mRNA translation, dependent on an internal ribosome entry site (IRES), and the SREBP-1a proteolytic cleavage are activated by FFAs. Furthermore, FFA treatment enhances the expression and the nucleus-cytosolic shuttling of hnRNP A1, a trans-activating factor of SREBP-1a IRES. The binding of hnRNP A1 to the SREBP-1a IRES is also increased upon FFA supplementation. The relocation of hnRNP A1 and the consequent increase of SREBP-1a translation are dependent on the p38 MAPK signal pathway, which is activated by FFAs. By RNA interference approach, we demonstrate that hnRNP A1 is implicated in the FFA-induced expression of SREBP-1a and of its target genes as well as in the lipid accumulation in cells.
Asunto(s)
Regiones no Traducidas 5' , Hepatocitos/metabolismo , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/metabolismo , Lipogénesis , Hígado/metabolismo , Enfermedad del Hígado Graso no Alcohólico/metabolismo , ARN Mensajero/metabolismo , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/biosíntesis , Sitios de Unión , Regulación de la Expresión Génica , Células Hep G2 , Hepatocitos/efectos de los fármacos , Hepatocitos/patología , Ribonucleoproteína Nuclear Heterogénea A1 , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/genética , Humanos , Sitios Internos de Entrada al Ribosoma , Lipogénesis/efectos de los fármacos , Lipogénesis/genética , Hígado/efectos de los fármacos , Hígado/patología , Enfermedad del Hígado Graso no Alcohólico/genética , Enfermedad del Hígado Graso no Alcohólico/patología , Ácido Oléico/farmacología , Ácido Palmítico/farmacología , Transporte de Proteínas , Interferencia de ARN , ARN Mensajero/genética , Transducción de Señal , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/genética , Factores de Tiempo , Transfección , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
The Unfolded Protein Response (UPR) is an intracellular signaling pathway which is activated when unfolded or misfolded proteins accumulate in the Endoplasmic Reticulum (ER), a condition commonly referred to as ER stress. It has been shown that lipid biosynthesis is increased in ER-stressed cells. The N(ε)-lysine acetylation of ER-resident proteins, including chaperones and enzymes involved in the post-translational protein modification and folding, occurs upon UPR activation. In both ER proteins acetylation and lipid synthesis, acetyl-CoA is the donor of acetyl group and it is transported from the cytosol into the ER. The cytosolic pool of acetyl-CoA is mainly derived from the activity of mitochondrial citrate carrier (CiC). Here, we have demonstrated that expression of CiC is activated in human HepG2 and rat BRL-3A cells during tunicamycin-induced ER stress. This occurs through the involvement of an ER stress responsive region identified within the human and rat CiC proximal promoter. A functional Unfolded Protein Response Element (UPRE) confers responsiveness to the promoter activation by UPR transducers ATF6α and XBP1. Overall, our data demonstrate that CiC expression is activated during ER stress through the binding of ATF6α and XBP1 to an UPRE element located in the proximal promoter of Cic gene. The role of ER stress-mediated induction of CiC expression has been discussed.
Asunto(s)
Factor de Transcripción Activador 6/genética , Proteínas Portadoras/metabolismo , Proteínas de Unión al ADN/genética , Transducción de Señal , Factores de Transcripción/genética , Respuesta de Proteína Desplegada/genética , Acetilcoenzima A/metabolismo , Factor de Transcripción Activador 6/metabolismo , Animales , Proteínas Portadoras/genética , Ácido Cítrico/metabolismo , Chaperón BiP del Retículo Endoplásmico , Estrés del Retículo Endoplásmico/genética , Células Hep G2 , Humanos , Regiones Promotoras Genéticas , Pliegue de Proteína , Procesamiento Proteico-Postraduccional , Ratas , Factores de Transcripción del Factor Regulador X , Proteína 1 de Unión a la X-BoxRESUMEN
A growing amount of evidence suggests the involvement of ER (endoplasmic reticulum) stress in lipid metabolism and in the development of some liver diseases such as steatosis. The transcription factor SREBP-1 (sterol-regulatory-element-binding protein 1) modulates the expression of several enzymes involved in lipid synthesis. Previously, we showed that ER stress increased the SREBP-1a protein level in HepG2 cells, by inducing a cap-independent translation of SREBP-1a mRNA, through an IRES (internal ribosome entry site), located in its leader region. In the present paper, we report that the hnRNP A1 (heterogeneous nuclear ribonucleoprotein A1) interacts with 5'-UTR (untranslated region) of SREBP-1a mRNA, as an ITAF (IRES trans-acting factor), regulating SREBP-1a expression in HepG2 cells and in primary rat hepatocytes. Overexpression of hnRNP A1 in HepG2 cells and in rat hepatocytes increased both the SREBP-1a IRES activity and SREBP-1a protein level. Knockdown of hnRNP A1 by small interfering RNA reduced either the SREBP-1a IRES activity or SREBP-1a protein level. hnRNP A1 mediates the increase of SREBP-1a protein level and SREBP-1a IRES activity in Hep G2 cells and in rat hepatocytes upon tunicamycin- and thapsigargin-induced ER stress. The induced ER stress triggered the cytosolic relocation of hnRNP A1 and caused the increase in hnRNP A1 bound to the SREBP-1a 5'-UTR. These data indicate that hnRNP A1 participates in the IRES-dependent translation of SREBP-1a mRNA through RNA-protein interaction. A different content of hnRNP A1 was found in the nuclei from high-fat-diet-fed mice liver compared with standard-diet-fed mice liver, suggesting an involvement of ER stress-mediated hnRNP A1 subcellular redistribution on the onset of metabolic disorders.
Asunto(s)
Estrés del Retículo Endoplásmico/genética , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/genética , Biosíntesis de Proteínas , ARN Mensajero/genética , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/genética , Animales , Sitios de Unión/genética , Northern Blotting , Western Blotting , Células Cultivadas , Dieta Alta en Grasa , Estrés del Retículo Endoplásmico/efectos de los fármacos , Expresión Génica , Células Hep G2 , Hepatocitos/citología , Hepatocitos/metabolismo , Ribonucleoproteína Nuclear Heterogénea A1 , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/metabolismo , Humanos , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Unión Proteica , Interferencia de ARN , ARN Mensajero/metabolismo , Ratas , Ribosomas/metabolismo , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/metabolismo , Tapsigargina/farmacología , Tunicamicina/farmacologíaRESUMEN
Este trabajo tiene por objetivo investigar si existe alguna asociación entre la Depresión y la sintomatología de los Trastornos de la Conducta Alimentaria, en una muestra no clínica de 700 adolescentes mujeres de 12 a 21 años de edad. Durante 2008, se aplicaron de manera auto-administrada 2 cuestionarios: el Beck Depression Inventory-BDI (Beck, 1972) y el Eating Attitude Test-EAT-26 (Garner, Olmsted, Bohr & Garfinkel, 1982) en escuelas públicas de un Distrito bonaerense. Los resultados arrojan una asociación entre ambos instrumentos. Se analiza la relación entre las variables Depresión-TCA, en cada fase de la adolescencia, (temprana, media y tardía); confirmándose correlaciones en los 3 segmentos etáreos. Finalmente, se rastrea qué ítems del test de Beck son los mejores predictores de cada escala del EAT-26 y del total de dicho cuestionario. Conclusiones: Los resultados hallados confirman la asociación entre ambos trastornos. Se sugiere ahondar en el estudio de la comorbilidad.
The aim of the work was to research of the association between Depression and Eating Disorders in a non clinical female sample of 700 adolescents between 12 and 21 years old. During 2008, 2 questionnaires were self-administered: the Beck Depression Inventory-BDI (Beck, 1972) and the Eating Attitude Test-EAT-26 (Garner, Olmsted, Bohr& Garfinkel, 1982) in state schools of a suburb of Buenos Aires. Results show a high correlation between the test of Beck and the Eating Attitude Test. The relation between both variables (depression and TCA) in each phase of the adolescence (early, middle and late) was also analyzed; confirming the correlations in the 3 segments. Finally it was tracked which items of the test of Beck were the best predictors of each scale of the EAT-26 and the total of this questionnaire. Conclusions: Results confirm association between both disorders. Suggestion of studying comorbidity is been made.