The University of Sheffield
Department of Molecular Biology and Biotechnology

Stress response, Stress adaptation and Stress protein function.

Prof Peter W Piper

blank space
Our research focuses on Heat Shock Protein (Hsp90), a "chaperone" protein essential for the correct folding and stability of a number of the proteins important for eukaryotic cell function. It is one of the most promising cancer drug targets, since many of the oncoproteins that drive cancer progression have a high dependency upon this chaperone for their activity.

 We are unravelling how this Hsp90 operates in molecular detail. To this end we have developed a way of screening the thousands of proteins in a cell for the subset that interact with Hsp90 [4] (Fig. 1). Also, in collaboration with the group of Laurence Pearl at the University of Sussex and Cara Vaughan at UCL, London, are combining protein structure, biochemical and genetic approaches in order to unravel how Hsp90 puts the “finishing touches” to its “client” proteins [4-7].

 

Hsp 90 chaperone Figure 1 (from [4])
A. Schematic of Hsp90, a protein constitutively dimerised at its C-terminus (C) and transiently dimerised at its N-terminus (N) in response to the binding of ATP. It uses ATP binding and hydrolysis to assist the final folding/maturation step of its target, “client" protein (green). In Hsp90 the E33A mutation arrests the ATPase step, causing locked Hsp90-client complexes to accumulate in vivo.
B. Three sample plates from a 16-plate, 384 colony format array of 6000 yeasts, each expressing just one of the 6000 proteins of yeast as a two hybrid “prey” fusion; together with either the control two hybrid “bait” containing Hsp90 (Hsp82-BD) or the E33A mutant version of this "bait" (Hsp82(E33A)-BD). Protein-protein interaction is monitored as growth in the presence of 3 aminotriazole (3-AT). Note that just a few of the 384 colonies on each plate are exhibiting growth dependent on the E33A mutation in the two-hybrid “Bait”, the mutation which “freezes” Hsp90/client complexes at a late stage of the chaperone cycle (see A).
The cancerous state of the most intractable tumours is driven by a number of mutant proteins needing Hsp90 for their activity. When their Hsp90 is inhibited these oncogenic proteins are - in a concerted manner - inactivated and targeted for degradation. In this way, drugs that are highly selective inhibitors of Hsp90 can potentially modulate all the hallmark traits of malignancy. Clinical trials reveal these highly-selective Hsp90 inhibitors display a high selectivity for cancer versus normal cells and a therapeutic activity at doses that are well tolerated in patients.
However, resistance acquired by mutation of the drug target remains one of the most serious problems responsible for the failure of cancer chemotherapy. We have been using the yeast model system to investigate how readily resistance to Hsp90 drugs can arise by Hsp90 mutation [2]. The Hsp90 inhibitors in clinical trials all act by binding the N-terminal domain of Hsp90. Resistance should not readily arise by changes within this drug binding site as mutations that weaken drug binding would be predicted to severely compromise Hsp90 function, since the amino acids that facilitate drug binding are required for the essential ATP binding/ATPase steps of the Hsp90 chaperone cycle and tightly conserved. Despite this, partly by analysing the Hsp90 proteins of the microbes that make Hsp90-targetting antibiotics, we have identified mutations causing significant levels of Hsp90 inhibitor resistance [1-3] (Fig. 2). It is too early to know if such mutations could ever appear in the clinic.

 

 

RAD resistant mutant Figure 2. A leucine to isoleucine change makes Hsp90 from a mycoparasitic fungus partially resistant to the most potent natural product inhibitor of Hsp90, radicicol. The conformation and binding interactions of RAD within the N-terminal domain of wild type and L34I mutant Hsp90 are shown superimposed. RAD is in cyan - wild-type structure - or in yellow - LI to IV mutant structure. Amino acid residues from the wild type Hsp90 are in cyan while those from the LI to IV mutant protein are in green, except for the residues representing the LI to IV mutations which are in yellow. Hydrogen bonds are shown as red (wild type) and blue (LI to IV mutant) dotted lines; water molecules as red (wild type) and cyan (LI to IV mutant) spheres. Note that the LI to IV mutant structure has 3 extra tightly coordinated water molecules which weaken the binding of RAD (taken from [3]).

 

Our work is funded by Cancer Research UK and Yorkshire Cancer Research.

 

Selected Publications

[1] Millson SH, Chua C-S, Roe SM, Polier S, Solovieva S, Pearl LH, Sim T-S, Prodromou C, Piper PW. (2011) Features of the Streptomyces hygroscopicus HtpG reveal how partial geldanamycin resistance can arise by mutation to the ATP binding pocket of a eukaryotic Hsp90. FASEB J. (in press)
[2] Millson, S.H., Prodromou, C. and Piper, P.W. (2010) A simple yeast-based system for analyzing inhibitor resistance in the human cancer drug targets Hsp90α/β. Biochem. Pharmacol. 79, 1581-8.
[3] Prodromou, C, Nuttall, J.M., Millson, S.H., Roe, S.M., Sim, T.S., Tan, D., Workman, P., Pearl, L.H. and Piper, P.W. (2009) Structural basis of the radicicol resistance displayed by a fungal Hsp90. ACS Chem. Biol. 17, 289-297
[4] Vaughan, C.K., Mollapour, M., Smith, J., Truman, A., Hu, B. Good, V.M., Panaretou, B., Neckers, L., Clarke, P., Workman, P., Piper, P.W., Prodromou C., and Pearl. L.H. (2008) Hsp90-dependent activation of protein kinases is regulated by chaperone-targeted dephosphorylation of Cdc37. Mol. Cell 31, 886-5.
[5] Truman, A.W., Millson, S.H., Nuttall. J.M., King, V., Mollapour, M., Prodromou, C., Pearl, L.H. and Piper, P.W. (2006) Expressed in yeast, human ERK5 is a client of the Hsp90 chaperone and complements loss of the Slt2(Mpk1)p cell integrity stress-activated protein kinase. Eukaryot. Cell 5, 1914-24.
[6] Ali, M.M., Roe, S.M., Vaughan, C.K., Meyer, P., Panaretou, B., Piper, P.W., Prodromou, C. and Pearl, L.H. (2006) Crystal structure of an Hsp90-nucleotide-p23/Sba1 closed chaperone complex. Nature 440, 1013-7.
[7] Millson, S.H., Truman, A.W., King, V., Prodromou, C., Pearl, L.H. and Piper, P.W. (2005) Two-hybrid screening the yeast proteome for Hsp90 chaperone interactors uncovers a novel Hsp90 requirement in the activity of a stress-activated MAP kinase, Slt2p(Mpk1p). Eukaryot. Cell 4, 849-60.