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Differential impact of tetratricopeptide repeat proteins on the steroid hormone receptors.

Schülke JP, Wochnik GM, Lang-Rollin I, Gassen NC, Knapp RT, Berning B, Yassouridis A, Rein T - PLoS ONE (2010)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Chaperone Research Group, Max Planck Institute of Psychiatry, Munich, Germany.

ABSTRACT

Background: 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.

Methodology and principal findings: 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.

Conclusion and significance: 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.

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GR and MR activities in the presence of different TPR-proteins.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 p-values ≤0.001.
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pone-0011717-g003: GR and MR activities in the presence of different TPR-proteins.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 p-values ≤0.001.

Mentions: The first observation we made in the reporter gene assays was that, in general, the changes in receptor activity upon co-expression of TPR cofactors were more pronounced for GR, MR, and PR than for AR, and even more than for the two ERs, which were almost not affected (Figs. 3 and 4). Strong inhibitors of GR were CHIP, FKBP51, PP5, TPR2, and XAP2, while CYP40 and FKBP52 showed virtually no effect (Fig. 3A). As shown for GR, at saturating concentrations of hormone the inhibitory effect was greatly diminished, even though TPR2, for example, still reduced GR's activity twofold (Fig. 3B). Similar observations were made with saturating concentrations of hormone at the other receptors (data not shown).


Differential impact of tetratricopeptide repeat proteins on the steroid hormone receptors.

Schülke JP, Wochnik GM, Lang-Rollin I, Gassen NC, Knapp RT, Berning B, Yassouridis A, Rein T - PLoS ONE (2010)

GR and MR activities in the presence of different TPR-proteins.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 p-values ≤0.001.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2908686&req=5

pone-0011717-g003: GR and MR activities in the presence of different TPR-proteins.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 p-values ≤0.001.
Mentions: The first observation we made in the reporter gene assays was that, in general, the changes in receptor activity upon co-expression of TPR cofactors were more pronounced for GR, MR, and PR than for AR, and even more than for the two ERs, which were almost not affected (Figs. 3 and 4). Strong inhibitors of GR were CHIP, FKBP51, PP5, TPR2, and XAP2, while CYP40 and FKBP52 showed virtually no effect (Fig. 3A). As shown for GR, at saturating concentrations of hormone the inhibitory effect was greatly diminished, even though TPR2, for example, still reduced GR's activity twofold (Fig. 3B). Similar observations were made with saturating concentrations of hormone at the other receptors (data not shown).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Chaperone Research Group, Max Planck Institute of Psychiatry, Munich, Germany.

ABSTRACT

Background: 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.

Methodology and principal findings: 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.

Conclusion and significance: 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.

Show MeSH
Related in: MedlinePlus