RESUMEN
The ubiquitin-proteasome system (UPS) is a specific, non-lysosomal pathway responsible for the controlled degradation of abnormal and short-half-life proteins. Despite its relevance in cell homeostasis, information regarding control of the UPS component gene expression is lacking. Data from a recent study suggest that the aryl hydrocarbon receptor (AHR), a ligand-dependent transcription factor, might control the expression of several genes encoding for UPS proteins. Here, we showed that activation of AHR by TCDD and ß-naphthoflavone (ß-NF) results in Ubcm4 gene induction accompanied by an increase in protein levels. UbcM4 is an ubiquitin-conjugating enzyme or E2 protein that in association with ubiquitin ligase enzymes or E3 ligases promotes the ubiquitination and 26S proteasome-mediated degradation of different proteins, including p53, c-Myc, and c-Fos. We also present data demonstrating increased c-Fos ubiquitination and proteasomal degradation through the AHR-mediated induction of UbcM4 expression. The present study shows that AHR modulates the degradation of proteins involved in cell cycle control, consistent with previous reports demonstrating an essential role of the AHR in cell cycle regulation.
Asunto(s)
Complejo de la Endopetidasa Proteasomal/efectos de los fármacos , Proteínas Proto-Oncogénicas c-fos/metabolismo , Receptores de Hidrocarburo de Aril/efectos de los fármacos , Enzimas Ubiquitina-Conjugadoras/biosíntesis , Ubiquitinación/efectos de los fármacos , Línea Celular , Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Humanos , Plásmidos/efectos de los fármacos , Dibenzodioxinas Policloradas/farmacología , Proteínas Proto-Oncogénicas c-fos/efectos de los fármacos , Transfección , Enzimas Ubiquitina-Conjugadoras/efectos de los fármacos , Enzimas Ubiquitina-Conjugadoras/genética , beta-naftoflavona/farmacologíaRESUMEN
The nonstructural rotavirus protein NSP1 binds specifically to viral mRNAs and to interferon regulatory factor 3 (IRF3), inducing IRF3 degradation through a proteasome-dependent pathway. By using a vaccinia virus expression system in mammalian cells, we found that the yield of NSP1 was 8- and 13-fold lower than the viral proteins VP2 or NSP3, respectively; while in the presence of proteasome inhibitors such difference could be reduced to 2- to 2.5-fold, respectively. The susceptibility of NSP1 to proteasome degradation was fully reversed in a dose-dependent manner by transfection with the full complement of 11 molecules of translation-competent rotavirus mRNAs, but this effect was abrogated by the protein synthesis inhibitor cycloheximide. These results demonstrate that NSP1 is degraded through a proteasome-dependent pathway, and viral proteins, alone or in combination with viral mRNAs, interfere with such degradation.
Asunto(s)
Regulación Viral de la Expresión Génica , Procesamiento Proteico-Postraduccional , Rotavirus/metabolismo , Proteínas no Estructurales Virales/metabolismo , Animales , Línea Celular , ARN Mensajero/metabolismo , Proteínas Recombinantes/metabolismo , Rotavirus/genética , Virus Vaccinia/genética , Proteínas no Estructurales Virales/genéticaRESUMEN
The activities of the enzymes involved in ammonium assimilation and glutamate biosynthesis were determined in wild-type and NADP-glutamate dehydrogenase (GDH) null mutant strains of Kluyveromyces lactis. The specific NADP-GDH activity from K. lactis was fivefold lower than that found in Saccharomyces cerevisiae. The glutamine synthetase (GS) and glutamate synthase (GOGAT) activities were similar to those reported in S. cerevisiae. The NADP-GDH null mutant was obtained by transforming the uraA strain MD2/1 with a linearized integrative yeast vector harbouring a 390 bp fragment of the NADP-GDH structural gene. This mutant grew as well as the parent strain on ammonium, but showed GS and GOGAT activities higher that those found in the wild-type strain, implying that the GS-GOGAT pathway could play a leading role in glutamate biosynthesis in K. lactis. Southern blotting analysis of K. lactis chromosomes separated by contour-clamped homogeneous electric field electrophoresis, indicated that the NADP-GDH structural gene is localized on chromosome VI.