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Chaperone ligand-discrimination by the TPR-domain protein Tah1.

Millson SH, Vaughan CK, Zhai C, Ali MM, Panaretou B, Piper PW, Pearl LH, Prodromou C - Biochem. J. (2008)

Bottom Line: Tah1 [TPR (tetratricopeptide repeat)-containing protein associated with Hsp (heat-shock protein) 90] has been identified as a TPR-domain protein.Amino-acid-sequence alignments suggest that Tah1 is most similar to the TPR2b domain of Hop (Hsp-organizing protein) which when mutated reduces binding to both Hsp90 and Hsp70.In the present study we also show that Tah1 can affect the ATPase activity of Hsp90, in common with some other TPR-domain proteins.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Biotechnology, The University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, UK.

ABSTRACT
Tah1 [TPR (tetratricopeptide repeat)-containing protein associated with Hsp (heat-shock protein) 90] has been identified as a TPR-domain protein. TPR-domain proteins are involved in protein-protein interactions and a number have been characterized that interact either with Hsp70 or Hsp90, but a few can bind both chaperones. Independent studies suggest that Tah1 interacts with Hsp90, but whether it can also interact with Hsp70/Ssa1 has not been investigated. Amino-acid-sequence alignments suggest that Tah1 is most similar to the TPR2b domain of Hop (Hsp-organizing protein) which when mutated reduces binding to both Hsp90 and Hsp70. Our alignments suggest that there are three TPR-domain motifs in Tah1, which is consistent with the architecture of the TPR2b domain. In the present study we find that Tah1 is specific for Hsp90, and is able to bind tightly the yeast Hsp90, and the human Hsp90alpha and Hsp90beta proteins, but not the yeast Hsp70 Ssa1 isoform. Tah1 acheives ligand discrimination by favourably binding the methionine residue in the conserved MEEVD motif (Hsp90) and positively discriminating against the first valine residue in the VEEVD motif (Ssa1). In the present study we also show that Tah1 can affect the ATPase activity of Hsp90, in common with some other TPR-domain proteins.

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Tah1 activation of Hsp90 and determination of the relative molecular mass of Tah1(a) ATPase activation of yeast Hsp90 by PreScission-cleaved Tah1; (b) gel-filtration chromatography of PreScission-cleaved Tah1; (c and d) analytical equilibrium sedimentation ultracentrifugation analyses (INVEQ, Mfit and NONLIN fits, top to bottom panels) of PreScission-cleaved Tah1 at 7.0 mg·ml−1 (c) and (d) 0.5 mg·ml−1. The broken line represents the fit to the data points. The results show that PreScission-cleaved Tah1 can weakly activate the ATPase activity of Hsp90, elutes with a Mr of 18500 on gel filtration, which is close to the values estimated by analytical centrifugation [M(z) by Mfit=15968 and 16106]. The results indicate that Tah1 is mostly a monomer under the conditions used.
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Figure 7: Tah1 activation of Hsp90 and determination of the relative molecular mass of Tah1(a) ATPase activation of yeast Hsp90 by PreScission-cleaved Tah1; (b) gel-filtration chromatography of PreScission-cleaved Tah1; (c and d) analytical equilibrium sedimentation ultracentrifugation analyses (INVEQ, Mfit and NONLIN fits, top to bottom panels) of PreScission-cleaved Tah1 at 7.0 mg·ml−1 (c) and (d) 0.5 mg·ml−1. The broken line represents the fit to the data points. The results show that PreScission-cleaved Tah1 can weakly activate the ATPase activity of Hsp90, elutes with a Mr of 18500 on gel filtration, which is close to the values estimated by analytical centrifugation [M(z) by Mfit=15968 and 16106]. The results indicate that Tah1 is mostly a monomer under the conditions used.

Mentions: TPR-domain-containing proteins have been implicated in the regulation of the ATPase activity of Hsp90. The first report of such regulation was for Sti1, which was shown to be a potent inhibitor of the ATPase activity of Hsp90 [35]. In contrast, the TPR-domain-containing co-chaperone, Cpr6 (cyclosporin-sensitive proline rotamase 6), has been shown to have a weak stimulatory affect on the ATPase activity of Hsp90 [45]. Using a 10-fold excess of Tah1 we were able to show in the present study a very weak stimulation of the ATPase activity of Hsp90 (Figure 7a).


Chaperone ligand-discrimination by the TPR-domain protein Tah1.

Millson SH, Vaughan CK, Zhai C, Ali MM, Panaretou B, Piper PW, Pearl LH, Prodromou C - Biochem. J. (2008)

Tah1 activation of Hsp90 and determination of the relative molecular mass of Tah1(a) ATPase activation of yeast Hsp90 by PreScission-cleaved Tah1; (b) gel-filtration chromatography of PreScission-cleaved Tah1; (c and d) analytical equilibrium sedimentation ultracentrifugation analyses (INVEQ, Mfit and NONLIN fits, top to bottom panels) of PreScission-cleaved Tah1 at 7.0 mg·ml−1 (c) and (d) 0.5 mg·ml−1. The broken line represents the fit to the data points. The results show that PreScission-cleaved Tah1 can weakly activate the ATPase activity of Hsp90, elutes with a Mr of 18500 on gel filtration, which is close to the values estimated by analytical centrifugation [M(z) by Mfit=15968 and 16106]. The results indicate that Tah1 is mostly a monomer under the conditions used.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2865030&req=5

Figure 7: Tah1 activation of Hsp90 and determination of the relative molecular mass of Tah1(a) ATPase activation of yeast Hsp90 by PreScission-cleaved Tah1; (b) gel-filtration chromatography of PreScission-cleaved Tah1; (c and d) analytical equilibrium sedimentation ultracentrifugation analyses (INVEQ, Mfit and NONLIN fits, top to bottom panels) of PreScission-cleaved Tah1 at 7.0 mg·ml−1 (c) and (d) 0.5 mg·ml−1. The broken line represents the fit to the data points. The results show that PreScission-cleaved Tah1 can weakly activate the ATPase activity of Hsp90, elutes with a Mr of 18500 on gel filtration, which is close to the values estimated by analytical centrifugation [M(z) by Mfit=15968 and 16106]. The results indicate that Tah1 is mostly a monomer under the conditions used.
Mentions: TPR-domain-containing proteins have been implicated in the regulation of the ATPase activity of Hsp90. The first report of such regulation was for Sti1, which was shown to be a potent inhibitor of the ATPase activity of Hsp90 [35]. In contrast, the TPR-domain-containing co-chaperone, Cpr6 (cyclosporin-sensitive proline rotamase 6), has been shown to have a weak stimulatory affect on the ATPase activity of Hsp90 [45]. Using a 10-fold excess of Tah1 we were able to show in the present study a very weak stimulation of the ATPase activity of Hsp90 (Figure 7a).

Bottom Line: Tah1 [TPR (tetratricopeptide repeat)-containing protein associated with Hsp (heat-shock protein) 90] has been identified as a TPR-domain protein.Amino-acid-sequence alignments suggest that Tah1 is most similar to the TPR2b domain of Hop (Hsp-organizing protein) which when mutated reduces binding to both Hsp90 and Hsp70.In the present study we also show that Tah1 can affect the ATPase activity of Hsp90, in common with some other TPR-domain proteins.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Biotechnology, The University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, UK.

ABSTRACT
Tah1 [TPR (tetratricopeptide repeat)-containing protein associated with Hsp (heat-shock protein) 90] has been identified as a TPR-domain protein. TPR-domain proteins are involved in protein-protein interactions and a number have been characterized that interact either with Hsp70 or Hsp90, but a few can bind both chaperones. Independent studies suggest that Tah1 interacts with Hsp90, but whether it can also interact with Hsp70/Ssa1 has not been investigated. Amino-acid-sequence alignments suggest that Tah1 is most similar to the TPR2b domain of Hop (Hsp-organizing protein) which when mutated reduces binding to both Hsp90 and Hsp70. Our alignments suggest that there are three TPR-domain motifs in Tah1, which is consistent with the architecture of the TPR2b domain. In the present study we find that Tah1 is specific for Hsp90, and is able to bind tightly the yeast Hsp90, and the human Hsp90alpha and Hsp90beta proteins, but not the yeast Hsp70 Ssa1 isoform. Tah1 acheives ligand discrimination by favourably binding the methionine residue in the conserved MEEVD motif (Hsp90) and positively discriminating against the first valine residue in the VEEVD motif (Ssa1). In the present study we also show that Tah1 can affect the ATPase activity of Hsp90, in common with some other TPR-domain proteins.

Show MeSH
Related in: MedlinePlus