La protéine de choc thermique (HSP 90 kD) : purification, caractérisation, modalités d'interaction avec le récepteur des glucocorticoïdes
(1993)
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- Steroid receptor proteins are direct transducers, binding specifically to cognate hormonal gene regulatory DNA sequences. The glucocorticoids receptor (GR) is a cytoplasmic protein in its hormone-free, untransformed, state; after hormone binding GR undergoes transformations (activation) which induces GR nuclear translocation, and the binding to specific DNA sequences termed glucocorticoids response elements. Analysis if cell extracts indicates that the untransformed GR is oligomecir, and that complexes contain non-receptor proteins. One of these is the 90 KDa a heat shochk protein, HSP 90. Whether ither factors are apart of untransformer receptor complexes is not well established, but HSP 90 kDA appears to associate with untransformed receptor even within intact cells, implying that this interaction may be physiologically relevant. HSP 90 kDa seems to be necessary to maintain the unliganded GR in a high affinity steroid binding conformation, and to repress its transcriptional activity. Upon hormone binding, HSP 90 kDa dissociates from GR and GR acquires an increases affinity for DNA. Whereas several regions involved in the formation of a stable complexes with HSP 90 kDa have been localized in the sequence of GR and other steroid hormone receptors, the corresponding regions of HSP 90 kDa still remain unidentified. To characterize these regions interacting with GR, we have, a first step, developed a fast and efficient method for the isolation of HSP 90 kDa, comprising a two-step high-performance anion-exchange and gel permeation column chromatography. The purification of this protein allowed us to determine its biochemical and physicochemical properties. As a second step, we have studied the highly negatively charged sequence region present in all eukaryotic HSP 90 kDa. Others have suggested that this region could interact with steroid receptor. Therefore, a peptide corresponding to the highly negatively charged sequence (232-266) from the mouse HSP 90 kDa β was synthesized. A polyclonal antibody (Ac232-266) was raised against this peptide. The antibody reacted with both the native and denatured forms of HSP 90 kDa, and cross-reacted with HSP 90 kDa from a broad range of species. Contrary to the monoclonal antibody Ac88 (which recognizes all eukaryotic HSP 90 kDa), our polyclonal antipeptide antibody did not interact with the untransformed rat glucocorticoid receptor. We demonstrated that epitopes recognized by these two antibodies are different and the one recognized by Ac88 is found in the part C-terminal of HSPs 90 kDa. More interestingly the addition of either Ac232-266 or peptide 232-266 to untransformed glucocorticoid receptor complexes affected steroid binding and/or physicochemical properties of the complexes. In the presence of Ac232-266 a time and antibody concentration dependent loss of steroid binding activity occurred. The same effect was observed in the presence of peptide 232-266 which was also able to induce a dissociation of the transformed from of the receptor to the DNA binding from. This effect was observed at 0-4°C and in the presence of the 10 mM molybdate. All these data strongly offred that the region extending from aminoacids 232 to 266 is directly involved in the interaction between HSP 90 kDa and the glucocorticoid receptor
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Tbarka, N. (1993). La protéine de choc thermique (HSP 90 kD) : purification, caractérisation, modalités d’interaction avec le récepteur des glucocorticoïdes. https://hdl.handle.net/2078.5/111194