Differential Impact of Tetratricopeptide Repeat Proteins on the Steroid Hormone Receptors

Tetratricopeptide repeat (TPR) motif containing co-chaperones of the chaperone Hsp90 are considered control modules that govern activity and specificity of this central folding platform. Steroid receptors are paradigm clients of Hsp90. The influence of some TPR proteins on selected receptors has been described, but a comprehensive analysis of the effects of TPR proteins on all steroid receptors has not been accomplished yet.We compared the influence of the TPR proteins FK506 binding proteins 51 and 52, protein phosphatase-5, C-terminus of Hsp70 interacting protein, cyclophillin 40, hepatitis-virus-B X-associated protein-2, and tetratricopeptide repeat protein-2 on all six steroid hormone receptors in a homogeneous mammalian cell system. To be able to assess each cofactor's effect on the transcriptional activity of on each steroid receptor we employed transient transfection in a reporter gene assay. In addition, we evaluated the interactions of the TPR proteins with the receptors and components of the Hsp90 chaperone heterocomplex by coimmunoprecipitation. In the functional assays, corticosteroid and progesterone receptors displayed the most sensitive and distinct reaction to the TPR proteins. Androgen receptor's activity was moderately impaired by most cofactors, whereas the Estrogen receptors' activity was impaired by most cofactors only to a minor degree. Second, interaction studies revealed that the strongly receptor-interacting co-chaperones were all among the inhibitory proteins. Intriguingly, the TPR-proteins also differentially co-precipitated the heterochaperone complex components Hsp90, Hsp70, and p23, pointing to differences in their modes of action.The results of this comprehensive study provide important insight into chaperoning of diverse client proteins via the combinatorial action of (co)-chaperones. The differential effects of the TPR proteins on steroid receptors bear on all physiological processes related to steroid hormone activity

XAP2 inhibits glucocorticoid receptor activity in mammalian cells

AbstractXAP2 is member of a protein family sharing the TPR protein interaction motif. It displays close homology to the immunophilins FKBP51 and FKBP52 that act via the Hsp90 folding machinery to regulate the glucocorticoid receptor (GR). We show that XAP2 inhibits GR by reducing its responsiveness to hormone in transcriptional activation. The effect of XAP2 on GR requires its interaction with Hsp90 through the TPR motif. The PPIase-like region turned out to be enzymatically inactive. Thus, PPIase activity is not essential for the action of XAP2 on GR, similarly to FKBP51 and FKBP52

GR and MR activities in the presence of different TPR-proteins.

<p>A-C, SK-N-MC cells in 96 well plates were transfected with the MMTV-Luc, Gaussia-KDEL control plasmid, a plasmid expressing one of the HA-tagged steroid hormone receptor (GR in A and B, MR in C and D) and constant amounts (200 ng) of a plasmid expressing one of the FLAG-tagged TPR-proteins. After transfection, the cells were cultivated for 24 h in the presence of hormone or vehicle as indicated. Relative receptor activity represents firefly data normalized to Gaussia activities and presented as relative stimulation to control + S.E.M. of at least four independent experiments performed in duplicate. Control cells were transfected with cloning plasmid instead of the TPR protein expressing plasmid. Lower panels of A and C, immunoblot of cell extracts, probed with anti-HA antibody visualizing steroid receptor expression, the same membrane probed with FLAG antibody demonstrating expression of TPR proteins and with actin antibody as loading control. D, After transfection, cells were cultivated in 0.1% or 10% SF-FCS containing media for 24 h in the presence of 0.03 nM fludrocortisol, or EtOH as vehicle control. Firefly luciferase data were normalized to Gaussia luciferase activities and are presented as relative stimulation + S.E.M. of three independent experiments performed in triplicate. * denotes <i>p-</i>values ≤0.001.</p

TPR proteins are significantly enhanced upon ectopic expression and change Hsp90 heterocomplex composition.

<p>A, SK-N-MC cells were transfected with plasmid expressing one of the TPR proteins, lysed after 48 h and levels of the respective TPR protein was determined by Western blot analysis. B, HEK-293 cells were transfected with FLAG tagged Hsp90 along with FKBP52 expressing plasmid or control plasmid. Hsp90 was precipitated from lysates and the levels of co-precipitated cofactors were determined by Western blot.</p

Estrogen receptors display little sensitivity to the Hsp90 inhibitor geldanamycin.

<p>SK-N-MC cells were transfected with 0.25 µg of one of the plasmids expressing ERα (A), ERβ (B) or GR (C), together with either ERE-Luc (A,B) or MMTV-Luc (C) as reporter plasmid and the Gaussia-KDEL control plasmid. After transfection, the cells were cultivated for 24 h in the presence of hormone and 10 ng/ml GA as indicated. Relative receptor activity represents Firefly data normalized to Gaussia activities and is presented as relative stimulation to control + S.E.M. of at least four independent experiments performed in duplicate. Lower panels, analysis of receptor expression after GA treatment in the presence or absence of hormone (10 nM estrogen, 500 nM cortisol).</p

Differential interaction of TPR-proteins with AR heterocomplexes.

<p>HEK-293 cells were transfected as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011717#pone-0011717-g007" target="_blank">figure 7</a>, except that HA-MR was expressed instead of HA-GR. Cells were processed and protein interactions were analyzed also as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011717#pone-0011717-g007" target="_blank">figure 7</a>. In A, binding of TPR-proteins is presented relative to the mean of the normalized FLAG-eluate signals of CHIP, FKBP51, and TPR2. Quantification represents means of three independent experiments (two for XAP2) +S.E.M. In B, binding is normalized as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011717#pone-0011717-g007" target="_blank">figure 7</a>. C, FLAG- and HA-immunoblot signals of the cell extracts, demonstrating expression of TPR proteins and AR. Quantifications represent means of three independent experiments +S.E.M.</p

TPR-proteins differently interact with GR heterocomplexes.

<p>HEK-293 cells were transfected with 5 µg of a plasmid expressing HA-tagged GR together with 2-10 µg (to achieve similar expression levels) of one of the plasmids expressing a FLAG-tagged TPR protein. After 48-72 h cultivation in SF-FCS containing media, cells were harvested, lysed, and protein extracts prepared for immunoprecipitation of either the HA-tagged GR (A), or the FLAG-tagged TPR-proteins (B). A, Precipitation of HA-GR. Displayed is an example of an immunoblot that was probed with FLAG antibody to visualize co-precipitated TPR-proteins (upper right panel), and an immunoblot of the same membrane probed with HA antibody demonstrating precipitated GR (lower right panel). Left panel, quantification of the relative binding of the TPR-proteins to the steroid receptor heterocomplexes. FLAG- and HA-immunoblot signals of the eluates and FLAG immunoblot signals of the cell extracts, demonstrating expression of TPR proteins (C), were scanned and subjected to densitometry. The signal from the co-precipitated FLAG protein was corrected first by the amount of precipitated receptor and second by the amount of the TPR-protein present in the respective cell extract. Binding of TPR-proteins is presented relative to the mean of the normalized FLAG-eluate signals of CHIP, FKBP51, FKBP52, and PP5. Quantification represents the means of three independent experiments +S.E.M. B, precipitation of TPR proteins. Upper right panel, coomassie stained gel of eluates visualizing precipitated TPR-proteins (arrowheads) and co-precipitated Hsp90 and Hsp70. Lower right panel, immunoblots of eluates probed with HA antibody to demonstrate binding of GR to TPR-protein heterocomplexes. Left panel, quantification of the relative binding of co-precipitated proteins to the precipitated TPR-proteins. For quantification, signals were scanned and subjected to densitometry. Each HA immunoblot signal of the eluate was corrected by the amount of precipitated TPR-protein. Binding of steroid receptors is presented relative to the mean of the corrected HA eluate signals. Quantifications represent means of three independent experiments +S.E.M.</p