Paci A, Liu XH, Huang H, Lim A, Houry WA, Zhao R
J. Biol. Chem. 2012 Dec;287(52):43205-14
Pih1 is an unstable protein and a subunit of the R2TP complex that, in yeast Saccharomyces cerevisiae, also contains the helicases Rvb1, Rvb2, and the Hsp90 cofactor Tah1. Pih1 and the R2TP complex are required for the box C/D small nucleolar ribonucleoprotein (snoRNP) assembly and ribosomal RNA processing. Purified Pih1 tends to aggregate in vitro. Molecular chaperone Hsp90 and its cochaperone Tah1 are required for the stability of Pih1 in vivo. We had shown earlier that the C terminus of Pih1 destabilizes the protein and that the C terminus of Tah1 binds to the Pih1 C terminus to form a stable complex. Here, we analyzed the secondary structure of the Pih1 C terminus and identified two intrinsically disordered regions and five hydrophobic clusters. Site-directed mutagenesis indicated that one predicted intrinsically disordered region IDR2 is involved in Tah1 binding, and that the C terminus of Pih1 contains multiple destabilization or degron elements. Additionally, the Pih1 N-terminal domain, Pih1(1-230), was found to be able to complement the physiological role of full-length Pih1 at 37 °C. Pih1(1-230) as well as a shorter Pih1 N-terminal fragment Pih1(1-195) is able to bind Rvb1/Rvb2 heterocomplex. However, the sequence between the two disordered regions in Pih1 significantly enhances the Pih1 N-terminal domain binding to Rvb1/Rvb2. Based on these data, a model of protein-protein interactions within the R2TP complex is proposed.
Song H, Fan P, Shi W, Zhao R, Li Y
J. Plant Physiol. 2010 Sep;167(14):1172-8
The genome of Arabidopsis thaliana contains seven Hsp90 family genes. Three organellar and two cytosolic AtHsp90 isoforms were characterized by functionally expressing them in a temperature-sensitive Hsp90 mutant and a conditional Hsp90-null mutant of Saccharomyces cerevisiae. The cytosolic AtHsp90-1 and AtHsp90-2 showed function similar to that of yeast in chaperoning roles; they could support the growth of yeast mutants at both permissive and non-permissive temperature. Neither the full-length nor mature forms of chloroplast-located AtHsp90-5, mitochondria-located AtHsp90-6 and endoplasmic reticulum (ER)-located AtHsp90-7 could complement the yeast Hsp90 proteins. The cytosolic AtHsp90s could stabilize the biomembrane of the temperature-sensitive Hsp90 mutant strains under stress conditions, while the organellar AtHsp90s could not protect the biomembrane of the temperature-sensitive Hsp90 mutant strains. Yeast two-hybrid results showed that either pre-protein or mature forms of organellar AtHsp90s could interact with cofactors cpHsp70, Hsp70, Hsp70t-2, Cyp40, p23 and a substrate protein of NOS, while cytosolic AtHsp90s could not interact with them. These results suggest that organellar and cytosolic AtHsp90s possibly work through different molecular mechanisms in forming chaperone complexes and performing their functional roles.
Zhao R, Leung E, Grüner S, Schapira M, Houry WA
PLoS ONE 2010;5(4):e9934
BACKGROUND: Hsp90 is an essential molecular chaperone that is also a novel anti-cancer drug target. There is growing interest in developing new drugs that modulate Hsp90 activity.
METHODOLOGY/PRINCIPAL FINDINGS: Using a virtual screening approach, 4-hydroxytamoxifen, the active metabolite of the anti-estrogen drug tamoxifen, was identified as a putative Hsp90 ligand. Surprisingly, while all drugs targeting Hsp90 inhibit the chaperone ATPase activity, it was found experimentally that 4-hydroxytamoxifen and tamoxifen enhance rather than inhibit Hsp90 ATPase.
CONCLUSIONS/SIGNIFICANCE: Hence, tamoxifen and its metabolite are the first members of a new pharmacological class of Hsp90 activators.