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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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TPR-proteins differently interact with GR heterocomplexes.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.
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pone-0011717-g007: TPR-proteins differently interact with GR heterocomplexes.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.

Mentions: For GR, the receptor IP revealed CHIP, FKBP51 and TPR2 as strong binders to the heterocomplex (Fig. 7). The FLAG-IPs targeting the TPR proteins revealed a similar binding pattern. We observed that Cyp40 exhibited weak interaction with the heterocomplex, which may account for its inability to compete the inhibitory effect of FKBP51. Notably, the strongest binders all were strongly inhibitory proteins. On the other hand, PP5, which also significantly reduced GR activity, in comparison displayed only moderate interaction.


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)

TPR-proteins differently interact with GR heterocomplexes.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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2908686&req=5

pone-0011717-g007: TPR-proteins differently interact with GR heterocomplexes.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.
Mentions: For GR, the receptor IP revealed CHIP, FKBP51 and TPR2 as strong binders to the heterocomplex (Fig. 7). The FLAG-IPs targeting the TPR proteins revealed a similar binding pattern. We observed that Cyp40 exhibited weak interaction with the heterocomplex, which may account for its inability to compete the inhibitory effect of FKBP51. Notably, the strongest binders all were strongly inhibitory proteins. On the other hand, PP5, which also significantly reduced GR activity, in comparison displayed only moderate interaction.

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