Loss of KEAP1 causes an accumulation of nondegradative organelles

KEAP1 is a cytoplasmic protein that functions as an adaptor for the Cullin-3-based ubiquitin E3 ligase system, which regulates the degradation of many proteins, including NFE2L2/NRF2 and p62/SQSTM1. Loss of KEAP1 leads to an accumulation of protein ubiquitin aggregates and defective autophagy. To better understand the role of KEAP1 in the degradation machinery, we investigated whether Keap1 deficiency affects the endosome-lysosomal pathway. We used KEAP1-deficient mouse embryonic fibroblasts (MEFs) and combined Western blot analysis and fluorescence microscopy with fluorometric and pulse chase assays to analyze the levels of lysosomal-endosomal proteins, lysosomal function, and autophagy activity. We found that the loss of keap1 downregulated the protein levels and activity of the cathepsin D enzyme. Moreover, KEAP1 deficiency caused lysosomal alterations accompanied by an accumulation of autophagosomes. Our study demonstrates that KEAP1 deficiency increases nondegradative lysosomes and identifies a new role for KEAP1 in lysosomal function that may have therapeutic implications

Delay of EGF-Stimulated EGFR Degradation in Myotonic Dystrophy Type 1 (DM1)

Funding Information: This research was supported by the Isabel Gemio Foundation (P18–13) and was also partially supported by the “Fondo Europeo de Desarrollo Regional” (FEDER) from the European Union. E.A.-C. was supported by a pre-doctoral fellowship of Valhondo Calaff Foundation. S.C.-C. and E.U.-C. were supported by FPU fellowships (FPU19/04435 and FPU16/00684, respectively) from the Ministerio de Ciencia, Innovación y Universidades, Spain. M.P.-B. and A.G.-B. received fellowships from the “Plan Propio de Iniciación a la Investigación, Desarrollo Tecnológico e Innovación (Universidad de Extremadura). M.N.-S. was supported by the “Ramon y Cajal” Program (RYC-2016–20883), and P.G.-S., was funded by “Juan de la Cierva Incorporación” Program (IJC2019–039229-I), Spain. S.M.S.Y.-D. was supported by the Isabel Gemio Foundation and CIBERNED (CB06/05/0041). J.M.F received research support from the Isabel Gemio Foundation and the “Instituto de Salud Carlos” III, CIBERNED (CB06/05/0041). Publisher Copyright: © 2022 by the authors.Myotonic dystrophy type 1 (DM1) is an autosomal dominant disease caused by a CTG repeat expansion in the 3′ untranslated region of the dystrophia myotonica protein kinase gene. AKT dephosphorylation and autophagy are associated with DM1. Autophagy has been widely studied in DM1, although the endocytic pathway has not. AKT has a critical role in endocytosis, and its phosphorylation is mediated by the activation of tyrosine kinase receptors, such as epidermal growth factor receptor (EGFR). EGF-activated EGFR triggers the internalization and degradation of ligand–receptor complexes that serve as a PI3K/AKT signaling platform. Here, we used primary fibroblasts from healthy subjects and DM1 patients. DM1-derived fibroblasts showed increased autophagy flux, with enlarged endosomes and lysosomes. Thereafter, cells were stimulated with a high concentration of EGF to promote EGFR internalization and degradation. Interestingly, EGF binding to EGFR was reduced in DM1 cells and EGFR internalization was also slowed during the early steps of endocytosis. However, EGF-activated EGFR enhanced AKT and ERK1/2 phosphorylation levels in the DM1-derived fibroblasts. Therefore, there was a delay in EGF-stimulated EGFR endocytosis in DM1 cells; this alteration might be due to the decrease in the binding of EGF to EGFR, and not to a decrease in AKT phosphorylation.publishersversionpublishe

Changes in Liver Lipidomic Profile in G2019S- LRRK2 Mouse Model of Parkinson's Disease

15 páginas, 4 figurasThe identification of Parkinson's disease (PD) biomarkers has become a main goal for the diagnosis of this neurodegenerative disorder. PD has not only been intrinsically related to neurological problems, but also to a series of alterations in peripheral metabolism. The purpose of this study was to identify metabolic changes in the liver in mouse models of PD with the scope of finding new peripheral biomarkers for PD diagnosis. To achieve this goal, we used mass spectrometry technology to determine the complete metabolomic profile of liver and striatal tissue samples from WT mice, 6-hydroxydopamine-treated mice (idiopathic model) and mice affected by the G2019S-LRRK2 mutation in LRRK2/PARK8 gene (genetic model). This analysis revealed that the metabolism of carbohydrates, nucleotides and nucleosides was similarly altered in the liver from the two PD mouse models. However, long-chain fatty acids, phosphatidylcholine and other related lipid metabolites were only altered in hepatocytes from G2019S-LRRK2 mice. In summary, these results reveal specific differences, mainly in lipid metabolism, between idiopathic and genetic PD models in peripheral tissues and open up new possibilities to better understand the etiology of this neurological disorder.This research was supported by “Instituto de Salud Carlos III”, “Fondo de Investigaciones Sanitarias” (PI15/0034), “CIBERNED-ISCIII” (CB06/05/0041 and 2015/03), and partially supported by “European Regional Development Fund (ERDF)” from the European Union. J.M.B.-S.P. is funded by “Ramon y Cajal Program” (RYC-2018-025099-I) and supported by Spain’s Ministerio de Ciencia e Innovación (PID2019-108827RA-I00). Y.C.N. and L.M.G. are funded by Community of Madrid (CT5/21/PEJ-2020-TL/BMD-17685 and CT36/22-41-UCM-INV respectively). S.M.S.Y.-D. was supported by CIBERNED-ISCIII. P.M.-C. is funded by the MINECO Spanish Ministry (FPI grant, PRE2020-092668). M.N.-S. was funded by “Ramon y Cajal Program” (RYC-2016-20883). E.U.-C. and S.C.-C. were supported by an FPU predoctoral fellowship (FPU16/00684) and FPU19/04435), respectively, from “Ministerio de Educación, Cultura y Deporte”. M.P-B was funded by a University of Extremadura fellowship. E.A-C was supported by a Grant (IB18048) from Junta de Extremadura, Spain. J.M.F. received research support from the “Instituto de Salud Carlos III”; “Fondo de Investigaciones Sanitarias” (PI15/0034) and CIBERNED-ISCIII (CB06/05/0041 and 2015/03). A.P.-C. was supported by MINECO (SAF2014-52940-R and SAF2017-85199-P). J.P.-T. received funding from CIBERNED-ISCIII (CB06/05/1123 and 2015/03). G.K. is supported by the Ligue contre le Cancer (équipe labellisée); Agence National de la Recherche (ANR)—Projets blancs; ANR under the frame of E-Rare-2, the ERANet for Research on Rare Diseases; AMMICa US/CNRS UMS3655; Association pour la recherche sur le cancer (ARC); Association “Le Cancer du Sein, Parlons-en!”; Cancéropôle Ile de-France; Chancelerie des universités de Paris (Legs Poix), Fondation pour la Recherche Médicale (FRM); a donation by Elior; European Research Area Network on Cardiovascular Diseases (ERA-CVD, MINOTAUR); Gustave Roussy Odyssea, the European Union Horizon 2020 Project Oncobiome; Fondation Carrefour; High-end Foreign Expert Program in China (GDW20171100085), Institut National du Cancer (INCa); Inserm (HTE); Institut Universitaire de France; LeDucq Foundation; the LabEx Immuno-Oncology (ANR-18-IDEX-0001); the RHU Torino Lumière; the Seerave Foundation; the SIRIC Stratified Oncology Cell DNA Repair and Tumor Immune Elimination (SOCRATE); and the SIRIC Cancer Research and Personalized Medicine (CARPEM).Peer reviewe

Análisis: Filter trap

Para realizar el lisado celular o de tejido pueden usarse dos fracciones: soluble e insoluble. En las fracciones solubles, se alisan las muestras con el volumen que corresponda de NP40 0,5 x; se incuban 10-15 minutos en hielo; se centrifuga a 13.000 rpm entre 15-60 minutos; se recoge el sobrenadante en eppendorfs rotulados con la palabra “Soluble”. En las fracciones insolubles, los pasos son: resuspender el pellet con un volumen adecuado de SB1x; sonicar; calentar 10 minutos en el termobloque y dar un spin. Por último, mantener en el mismo eppendorf rotulado con el término “Insoluble”.Two fractions can be used for cell or tissue lysate: soluble and insoluble. For soluble fractions, samples are lysed with an appropriate volume of NP40 0.5 x; incubated 10-15 minutes on ice; centrifuged at 13,000 rpm for 15-60 minutes; the supernatant is collected in eppendorfs labelled 'Soluble'. For insoluble fractions, the steps are: resuspend the pellet with an appropriate volume of SB1x; sonicate; heat for 10 minutes in the thermoblock and spin. Finally, keep in the same eppendorf labelled "Insoluble"

Análisis Seahorse

Para medir el metabolismo celular a partir de análisis Seahorse, hay que resuspender las células antes de contarlas y sembrarlas, y determinar la concentración ideal de siembra. Si las células no son adherentes, se puede pre-tratar la placa con polilisina o colágeno. Preparar más volumen de medio + células para utilizar la pipeta multicanal y no utilizar volúmenes demasiado pequeños y así, evitar errores. Después de 2 horas, dejar con 80 μl de medio para evitar mover las células. Éstas no estarán pegadas y se pueden ir a los bordes. Si se hidrata con H2O miliQ hay que cambiarlo, al menos una hora antes de realizar el experimento. Si se hidrata directamente con el tampón de calibrado, no debe estar más de 24 horas. Si las células no estuvieran el día 2, se cambia el agua y se rehidrata con el tampón 1 hora antes del experimento. El volumen del tratamiento es de 100 μl/pocillo. Preparar el medio Seahorse adicionando glutamina 1X, glucosa 10 mM y sodio piruvato 1X. Antes de añadir ningún inhibidor, meter y sacar varias veces el cartucho del líquido de calibrado para eliminar cualquier burbuja que se haya podido generar. Añadir de izquierda a derecha apoyando la punta de la pipeta multicanal en la pared y de forma muy lenta para que resbale poco a poco. Luego, meter las placas en el aparato sin las tapas. Al finalizar el calibrado, el aparato pide la placa de las células y devuelve la placa con el líquido de calibrado. La placa de las células estará no menos de 45 minutos en la estufa. Comenzará con una etapa de equilibrado. Tras 13 minutos, ofrecerá las medidas basales y las inyecciones. Al terminar todo el proceso, cuya duración dependerá del kit utilizado y de las modificaciones hechas, pedirá que se finalice el programa y la máquina devolverá el cartucho y la placa de las células.To measure cell metabolism from Seahorse assays, resuspend the cells before counting and seeding, and determine the ideal seeding concentration. If the cells are not adherent, the plate can be pre-treated with poly-L-lysine or collagen. Prepare more volume of medium + cells to use the multichannel pipette and do not use too small volumes to avoid errors. After 2 hours, leave with 80 μl of medium to avoid moving the cells. The cells will not be stuck together and may go to the edges. If hydrated with H2O miliQ, change the medium at least one hour before the experiment. If hydrated directly with the calibration buffer, it should not be more than 24 hours. If the cells were not present on day 2, the water is changed and rehydrated with the buffer 1 hour before the experiment. The treatment volume is 100 μl/well. Prepare Seahorse medium by adding 1X glutamine, 10 mM glucose and 1X sodium pyruvate. Before adding any inhibitor, dip the cartridge in and out of the calibration liquid several times to remove any bubbles that may have been generated. Add from left to right with the tip of the multichannel pipette resting on the wall and very slowly so that it slides off little by little. Then place the plates in the apparatus without the lids. At the end of the calibration, the apparatus asks for the cell plate and returns the plate with the calibration liquid. The cell plate shall remain in the oven for at least 45 minutes. It will start with a balancing step. After 13 minutes, it will provide the basal measurements and injections. At the end of the whole process, the duration of which will depend on the kit used and the modifications made, it will ask for the programme to be terminated and the machine will return the cartridge and the cell plate

Análisis Pulse-Chase

Se siembran células en placas de 6 pocillos (130184, Biolite) a la densidad adecuada. Dejar un pocillo más de control. Se procede al Marcaje con L-[14C]-Valina. Se aplica Pre-Chase por espacio de una hora, mientras se preparan los distintos tratamientos a realizar en el experimento, utilizando el medio con L-Valina. Tras ese tiempo, se cogen las placas, se resuspende muy bien el contenido de los pocillos y se recoge el volumen que haya en los eppendorfs rotulados como Rc. Las muestras en los eppendorfs pueden quedarse a temperatura ambiente hasta su análisis. Se dejan rotulados los tubos de centelleo, como ha sucedido con los eppendorfs.Seed cells in 6-well plates (130184, Biolite) at the appropriate density. Leave an extra control well. Proceed to L-[14C]-Valine labelling. Pre-Chase is applied for one hour, while the different treatments to be carried out in the experiment are prepared, using the medium with L-Valine. After this time, take the plates, resuspend the contents of the wells very well and collect the volume in the eppendorfs labelled Rc. The samples in the eppendorfs can remain at room temperature until analysis. The scintillation tubes are left labelled, as with the eppendorfs

Microscopia electrónica

Protocolo de procesamiento de muestras de cultivos celulares en microscopia electrónica. Se aplica a la investigación de enfermedades neurodegenerativas en un laboratorio de neurociencias.Protocol for processing cell culture samples in electron microscopy. Applied to the investigation of neurodegenerative diseases in a neuroscience laboratory

LA DISTROFIA MIOTÓNICA TIPO 1 Y EL RECICLAJE CELULAR

Resumen de la publicación de una participación en formato vídeo corto en las segundas jornadas del congreso Divulga NextGen que se celebrará online, de manera gratuita y en las redes sociales los días 28, 29 y 30 de noviembre de 2023.Fundación Valhondo, CIBERNED, la Fundación ISABEL GEMIO y FUNDESALU

Análisis de extracto proteico por Western blotting

Se estudia cómo hacer el extracto proteico a partir de Western blotting. Para ello, se prepara para la transferencia de cada gel, dos papeles Whatman (Extra Thick Blod PAPER Bio-Rad), el uso de dos esponjillas y una membrana de PVDF. Todo deberá estar equilibrado en su tampón de transferencia antes de proceder a realizar el proceso de transferencia. Estas piezas deben estar embebidas durante al menos 10 minutos en tampón de trasferencia. Se aplicará una tensión de 75 V y migrar durante 45 minutos en agitación continua y refrigeración en el caso de los geles de 18 pocillos (Criterion TGX). El tampón utilizado en la transferencia de geles de 18 pocillos es el tampón Tris Glicina Metanol. El anticuerpo secundario se diluye de 1:5.000 a 1:10.000 en 10 % de leche desnatada en una solución de TTBS. La elección del anticuerpo secundario entre monoclonal o policlonal depende siempre del anticuerpo primario. Finalmente, la membrana estará lista para ser reutilizada (al menos en dos o tres ocasiones más). Hay que tener en cuenta que antes del primer borrado, hay que incubar la membrana con los anticuerpos fosforilados de interés. Después se puede proceder al borrado de la membrana e incubar con los anticuerpos totales, específicos de los anticuerpos fosforilados.We study how to make the protein extract from Western blotting. To do this, two Whatman papers (Extra Thick Blod PAPER Bio-Rad), the use of two sponges and a PVDF membrane are prepared for the transfer of each gel. Everything must be balanced in its transfer buffer before proceeding with the transfer process. These parts shall be soaked for at least 10 minutes in transfer buffer. A voltage of 75 V shall be applied and migrate for 45 minutes under continuous agitation and cooling in the case of 18-well gels (Criterion TGX). The buffer used in the transfer of 18-well gels is Tris Glycine Methanol buffer. The secondary antibody is diluted 1:5,000 to 1:10,000 in 10 % skimmed milk in TTBS solution. The choice of monoclonal or polyclonal secondary antibody always depends on the primary antibody. Finally, the membrane is ready to be reused (at least two or three more times). Before the first blotting, the membrane must be incubated with the phosphorylated antibodies of interest. The membrane can then be blotted and incubated with the total antibodies specific to the phosphorylated antibodies

Extracción de proteínas

El estudio sobre la extracción de proteínas consiste en que, antes de usar, el reactivo A y reactivo B se complementaron con cóctel inhibidor de proteasa 10X (Sigma-Aldrich, P2714), ortovanadato de sodio al 0,5 M al 20 % (S6508, Sigma) y fluoruro de sodio al 0,1 M al 1 % (131675, Panreac). Las mitocondrias aisladas se lisaron en CHAPS al 2 % (C3023, Sigma). Las extracciones citosólicas y mitocondriales se analizaron mediante transferencia Western blotting. El protocolo B debe ser rápido, intentando no sobrepasar 30 segundos. Por otro lado, la adición de 1 μg/ml Leupeptina. 1 μg/ml Pepstatina. 1 μg/ml Aprotinina. 1 μg/ml Benzamidina. El PMSF se diluye en 1 ml de etanol absoluto filtrado. Si hay problemas con el método, probar modificando la concentración de digitonina.The protein extraction study consists of reagent A and reagent B supplemented with 10X protease inhibitor cocktail (Sigma-Aldrich, P2714), 0,5 M 20 % sodium orthovanadate (S6508, Sigma) and 0,1 M 1 % sodium fluoride (131675, Panreac) before use. Isolated mitochondria were lysed in 2 % CHAPS (C3023, Sigma). Cytosolic and mitochondrial extractions were analysed by Western blotting. Protocol B should be fast, trying not to exceed 30 seconds. On the other hand, the addition of 1 μg/ml Leupeptin. 1 μg/ml Pepstatin. 1 μg/ml Aprotinin. 1 μg/ml Benzamidine. PMSF is diluted in 1 ml of filtered absolute ethanol. If there are problems with the method, try modifying the digitonin concentration