RESUMEN
The Blm10/PA200 family of proteasome activators modulates the peptidase activity of the core particle (20S CP). They participate in opening the 20S CP gate, thus facilitating the degradation of unstructured proteins such as tau and Dnm1 in a ubiquitin- and ATP-independent manner. Furthermore, PA200 also participates in the degradation of acetylated histones. In our study, we use a combination of yeast and human cell systems to investigate the role of Blm10/PA200 in the degradation of N-terminal Huntingtin fragments (N-Htt). We demonstrate that the human PA200 binds to N-Htt. The loss of Blm10 in yeast or PA200 in human cells results in increased mutant N-Htt aggregate formation and elevated cellular toxicity. Furthermore, Blm10 in vitro accelerates the proteasomal degradation of soluble N-Htt. Collectively, our data suggest N-Htt as a new substrate for Blm10/PA200-proteasomes and point to new approaches in Huntington's disease (HD) research.
Asunto(s)
Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Proteínas Nucleares/metabolismo , Complejo de la Endopetidasa Proteasomal/química , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Línea Celular Tumoral , Supervivencia Celular , Técnicas de Silenciamiento del Gen , Humanos , Proteína Huntingtina/química , Modelos Moleculares , Proteínas Nucleares/química , Proteínas Nucleares/genética , Péptidos/metabolismo , Complejo de la Endopetidasa Proteasomal/genética , Agregado de Proteínas , Proteolisis , Saccharomyces cerevisiae/enzimología , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
All-trans retinoic acid (ATRA) is a derivative of vitamin A that has many important biological functions, including the modulation of immune responses. ATRA actions are mediated through the retinoic acid receptor that functions as a nuclear receptor, either regulating gene transcription in the nucleus or modulating signal transduction in the cytoplasm. NLRP3 inflammasome is a multiprotein complex that is activated by a huge variety of stimuli, including pathogen- or danger-related molecules. Activation of the inflammasome is required for the production of IL-1ß, which drives the inflammatory responses of infectious or non-infectious sterile inflammation. Here, we showed that ATRA prolongs the expression of IL-6 and IL-1ß following a 2-, 6-, 12-, and 24-h LPS (100ng/mL) activation in human monocyte-derived macrophages. We describe for the first time that ATRA modulates both priming and activation signals required for NLRP3 inflammasome function. ATRA alone induces NLRP3 expression, and enhances LPS-induced expression of NLRP3 and pro-IL-1ß via the regulation of signal transduction pathways, like NF-κB, p38, and ERK. We show that ATRA alleviates the negative feedback loop effect of IL-10 anti-inflammatory cytokine on NLRP3 inflammasome function by inhibiting the Akt-mTOR-STAT3 signaling axis. We also provide evidence that ATRA enhances hexokinase 2 expression, and shifts the metabolism of LPS-activated macrophages toward glycolysis, leading to the activation of NLRP3 inflammasome.
Asunto(s)
Glucólisis/efectos de los fármacos , Inflamasomas/inmunología , Macrófagos/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Transducción de Señal/efectos de los fármacos , Tretinoina/farmacología , Hexoquinasa/metabolismo , Humanos , Interleucina-10/metabolismo , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Lipopolisacáridos/farmacología , Sistema de Señalización de MAP Quinasas/inmunología , FN-kappa B/sangre , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Factor de Transcripción STAT3/antagonistas & inhibidores , Transducción de Señal/inmunología , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Tretinoina/metabolismoRESUMEN
The conserved Blm10/PA200 activators bind to the proteasome core and facilitate peptide and protein turnover. Blm10/PA200 proteins enhance proteasome peptidase activity and accelerate the degradation of unstructured proteasome substrates. Our knowledge about the exact role of PA200 in diseased cells, however, is still limited. Here, we show that stable knockdown of PA200 leads to a significantly elevated number of cells in S phase after treatment with the ATP synthase inhibitor, oligomycin. However, following exposure to the complex I inhibitor rotenone, more PA200-depleted cells were in sub-G1 and G2/M phases indicative of apoptosis. Chromatin immunoprecipitation (ChIP) and ChIP-seq data analysis of collected reads indicate PA200-enriched regions in the genome of SH-SY5Y. We found that PA200 protein peaks were in the vicinity of transcription start sites. Gene ontology annotation revealed that genes whose promoters were enriched upon anti-PA200 ChIP contribute to the regulation of crucial intracellular processes, including proliferation, protein modifications and metabolism. Selective mitochondrial inhibitors induced PA200 redistribution in the genome, leading to protein withdrawal from some gene promoters and binding to others. Collectively, the results support a model in which PA200 potentially regulates cellular homeostasis at the transcriptional level, in addition to its described role as an alternative activator of the proteasome.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Mitocondrias/metabolismo , Neuroblastoma/genética , Neuroblastoma/patología , Proteínas Nucleares/metabolismo , Apoptosis/efectos de los fármacos , Apoptosis/genética , Ciclo Celular/efectos de los fármacos , Ciclo Celular/genética , Muerte Celular/efectos de los fármacos , Muerte Celular/genética , Línea Celular Tumoral , Supervivencia Celular/genética , Cromatina/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Mitocondrias/efectos de los fármacos , Oligomicinas/farmacología , Reproducibilidad de los Resultados , Rotenona/administración & dosificación , Rotenona/farmacologíaRESUMEN
Huntington's disease (HD) is an inherited neurodegenerative disorder, caused by an abnormal polyglutamine (polyQ) expansion in the huntingtin protein (Htt). Mitochondrial dysfunction and impairment of the ubiquitin-proteasome system (UPS) are hallmarks of HD neurons. The extraneural manifestations of HD are still unclear. We investigated the crosstalk between mitochondria and proteolytic function in skin fibroblasts from juvenile HD patients. We found reduced mitosis, increased cell size, elevated ROS and increased mitochondrial membrane potential in juvenile HD fibroblasts, while cellular viability was maintained. Mitochondrial OXPHOS analysis did not reveal significant differences compared to control. However, the level of mitochondrial fusion and fission proteins was significantly lower and branching in the mitochondria network was reduced. We hypothesized that juvenile HD fibroblasts counterbalance cellular damage and mitochondrial network deficit with altered proteasome activity to promote cell survival. Our data reveal that juvenile HD fibroblasts exhibit higher proteasome activity, which was associated with elevated gene and protein expression of parkin. Moreover, we demonstrate elevated proteasomal degradation of the mitochondrial fusion protein Mfn1 in diseased cells compared to control cells. Our data suggest that juvenile HD fibroblasts respond to mutant polyQ expansion of Htt with enhanced proteasome activity and faster turnover of specific UPS substrates to protect cells.