L' endocytose (une fonction cellulaire qui est stimulée par les champs électromagnétiques et qui joue un rôle dans l'internalisation de la bléomycine)

La bléomycine (BLM) est une drogue utilisée en chimiothérapie, caractérisée par une faible incorporation cellulaire et un très fort pouvoir cytotoxique une fois qu'elle a atteint le cytosol d'où elle peut interagir avec sa cible (l'ADN). Nous avons montré sur différentes lignées cellulaires en culture que la cytotoxicité de la BLM nécessite sa liaison sur une protéine membranaire spécifique (récepteur), et qu'elle est corrélée à la vitesse d'endocytose. Cette cytotoxicité dépend de la surface de membrane incorporée mais pas du volume contenu dans les vésicules d'endocytose. Ces résultats permettent de conclure que les molécules de BLM exercent leur toxicité grâce à leur internalisation suivant un mécanisme de type endocytose médiée par récepteur. Nous avons aussi montré que l'endocytose en phase fluide, que est un processus cellulaire fondamental, est stimulée aussi bien par l'application des champs électromagnétiques de type GSM que par les champs électriques pulsés de basse fréquence qui leur correspndent...PARIS5-BU Saints-Pères (751062109) / SudocSudocFranceF

In vitro increase of the fluid-phase endocytosis induced by pulsed radiofrequency electromagnetic fields: importance of the electric field component.

Nowadays, due to the wide use of mobile phones, the possible biological effects of electromagnetic fields (EMF) become a public health general concern. Despite intensive research, there are no widely accepted theories about the interactions between EMFs and living cells, and the experimental data are often controversial. We examined the effects of mobile phones EMF (envelope frequency of 217 Hz, carrier frequency of 900 MHz and pulse duration of 580 micros) or its pure, low-frequency pulsed electric field component on fluid-phase endocytosis. In both cases, with exposures exceeding 10 min, an increase of the fluid-phase endocytosis rate was observed ( approximately 1.5-fold), on three different cell types. This increase is an all-or-nothing type of response that is occurring for threshold values comprised between 1.3 and 2.6 W/kg for the delivered EMF powers and between 1.1 and 1.5 V/cm for the electric fields intensities depending upon the cell type. The electric component of these EMFs is shown to be responsible for the observed increase. Variations of frequency or pulse duration of the electric pulses are shown to be without effect. Thus, EMF, via their electrical component, can perturb one of the most fundamental physiological functions of the cells-endocytosis

The human EKC/KEOPS complex is recruited to Cullin2 ubiquitin ligases by the human tumour antigen PRAME.

Contains fulltext : 103825.pdf (publisher's version ) (Open Access

OSGEP interacts with PRAME and Cul2 ubiquitin complex components.

<p>OSGEP interacts with PRAME and Cul2-EloBC ligases. Immunoblot analysis of TAG-PRAME and TAG-OSGEP protein complexes purified from K562 cells to verify the mass spectrometry data. Mock purification was performed on wild type cells. 0.8% of input and 33% of IP were separated on NuPage 4–12% gels. Tagged proteins were detected with mouse HA antibody (Covance, top panel); endogenous PRAME and TAG-PRAME were detected in the second panel with affinity-purified PRAME antibody after staining for Cul2; the other proteins were detected as indicated. Asterisks indicate protein A that dissociated from the beads after elution.</p

PRAME-interacting proteins identified by Mass Spectrometry of purified complexes from K562 and HeLaS3 cells.

<p>PRAME-interacting proteins identified by Mass Spectrometry of purified complexes from K562 and HeLaS3 cells.</p

Organization of human EKC complex.

<p>SF21 cells were co-infected with baculoviruses expressing the indicated proteins. Anti-FLAG immunoprecipitations were performed, and total cell lysates and eluates were analyzed by western blotting. Interactions detected are summarized in the diagram on the right. Panels showing anti-HA immunoblots are from the same exposure of the same blot, as are the panels showing anti-cMyc immunoblots. Asterisk indicates light chains of the antibodies used in the immunoprecipitation.</p

PRAME recruits Cul2-EloBC ligases to EKC.

<p>(A) PRAME does not require an intact BC-box to interact with EKC components. Coimmunoprecipitation assays as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042822#pone-0042822-g001" target="_blank">Fig.1A</a> with wild type and BC-box mutant M2 PRAME. (B) PRAME bridges Cullin2 ligases to EKC complex. Immunoblot analysis as in Fig. 2 of TAG-OSGEP or TAG-LAGE3 immunoprecipitates with or without knock down of endogenous PRAME. Asterisk indicates heavy chains of the antibodies used in the immunoprecipitation. (C) Models of the protein complexes architecture.</p

OSGEP protein levels are modulated by protein-protein interactions.

<p>(A) Multiple OSGEP moieties are present in the same complex. Constructs expressing the indicated proteins were transiently transfected in 293T cells. Total cell lysates and FLAG eluates were analyzed by western blotting. A plasmid expressing GFP was cotransfected to control for the transfection efficiency. (B) OSGEP levels are affected by protein-protein interactions. 293T cells were transfected with constructs expressing FLAG-OSGEP and the proteins indicated. The bar graph shows FLAG-OSGEP levels quantified by immunoblot with the Odyssey system (values are the average of two independent experiments). A plasmid expressing GFP was cotransfected to control for the transfection efficiency. (C) and (D) LAGE3-OSGEP interface mutants decrease OSGEP protein levels. Transient transfections in 293T cells with the mutant constructs indicated and immunoblot by Odyssey of total cell lysates. Graphs report intensities of FLAG-OSGEP quantified with Odyssey (A.U., arbitrary units).</p

PRAME interacts with OSGEP and LAGE3.

<p>(A) Coimmunoprecipitation assays verify the interaction of PRAME with the EKC subunits OSGEP and LAGE3. Constructs expressing the indicated proteins were transiently transfected in 293T cells. Anti-TAG immunoprecipitations were performed with rabbit HA antibody (Abcam). TAG-PRAME was detected with monoclonal anti-HA (Covance) and TTE-tagged proteins with monoclonal BB2 (Diagenode) which recognizes the TY1 tag. The lower panel shows a scheme of the tagged proteins used (tags and coding sequences are not on scale). (B-C) PRAME directly interacts with EKC complex through OSGEP and LAGE3. SF21 cells were co-infected with baculoviruses expressing the indicated proteins. Anti-FLAG immunoprecipitations were performed, and total cell lysates and eluates were analyzed by western blotting. Panels showing anti-HA immunoblots are from the same exposure of the same blot, as are the panels showing anti-cMyc immunoblots. Asterisks indicate light chains of the antibodies used in the immunoprecipitation.</p

The tumour antigen PRAME is a subunit of a Cul2 ubiquitin ligase and associates with active NFY promoters

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