- Alberta Innovates - Health Solutions Scholar (2010-2017)
Molecular mechanism of action of the Hsp90 chaperone and its role in proteostasis
All proteins inside of living cells are in dynamic and intimate association with cellular chaperone systems. The Hsp90 chaperone system is present in all organisms from bacteria to humans and is essential in almost all instances.
Hsp90 action is regulated by an extensive cohort of co-chaperone proteins. These co-chaperones regulate interaction with client proteins as well as progression through the ATP-dependent client activation cycle. Unlike other chaperone proteins that regulate the folding of nascent proteins, Hsp90 appears to be involved in chaperoning the acquisition conformational states that are required for client activation or assembly.
Kinases, transcription factors and hormone receptors are among the many proteins that require Hsp90 action to function properly. Hsp90 is a key regulator of proteins involved in disease such as oncogenic kinases in cancer and of the cystic fibrosis transmembrane conductance regulator (CFTR) protein in cystic fibrosis.
Despite its importance, Hsp90’s molecular mechanism of action remains a mystery. My research program is focused on characterizing the functional mechanics of Hsp90 both in vitro and in vivo using a combination of genetic and biochemical techniques.
Theme 1: Genetic and Biochemical Characterization of the Hsp90 System
The Hsp90 system is conserved from yeast to humans – with many of the components being functionally interchangeable. We employ the model organism Saccharomyces cerevisiae to manipulate the Hsp90 system and study its role in yeast and human client protein activation. We use strains expressing temperature sensitive mutants of Hsp90 to understand the relationship between Hsp90 and its many co-chaperones such as aha1p, hch1p, cdc37p, sti1p and sba1p. All of these co-chaperones have been implicated in the Hsp90 cycle but their precise relationship to Hsp90 is not well understood.
The co-chaperone Aha1, and the related co-chaperone Hch1, is a primary focus of our yeast work. Aha1p binds to Hsp90 and stimulates its very low ATPase activity. However, the biological role of this function remains controversial. Aha1 is conserved from yeast to humans and has been shown to be involved in the folding and export of CFTR, the function and stability of v-src and other client proteins of Hsp90. We have discovered that Aha-type co-chaperones act as nucleotide exchange factors in the Hsp90 ATPase cycle. We are interrogating the role of this activity in client folding in both yeast and mammalian systems.
The conformationally dynamic nature of the Hsp90 dimer has made comprehensive structural analysis of its functional cycle very difficult. We complement our enzymatic and genetic analysis of Hsp90 with a variety of structural approaches. Using mutants of Hsp90 or co-chaperones, we are employing NMR and dynamic fluorescence approaches to elucidate the nature of Hsp90:co-chaperone complexes and their moving parts.
Theme 2: Role of the Hsp90 system in regulating tumour immunogenicity
Hsp90 has been called a 'capacitor for morphological evolution' because of its ability to maintain the function of proteins that have been thermodynamically compromised by sequence alterations. Perturbations in cellular proteostasis results in the degradation of proteins that are particularly dependent on chaperones like Hsp90. A selection of peptides generated by proteasomal degradation of such proteins will be presented on the surface of cells on Major Histocompatibility Class I (MHC-I) for surveillance by the immune system. Presentation of neoantigens on the surface of cancer cells is the basis for antitumour immunity and a prerequisite for response to check point inhibitor immunotherapy.
My laboratory explores the effect of Hsp90 inhibition on MHC-I presentation in cancer cells and the effect of this on tumour immunogenicity.
The conserved NxNNWHW motif in Aha-type co-chaperones modulates the kinetics of Hsp90 ATPase stimulation. R. Mercier, A. Wolmarans, J. Schubert, H. Neuweiler, J.L. Johnson, P. LaPointe (2019). Nat. Commun. 10(1), 1273
The Hsp90 Chaperone: 1H and 19F Dynamic Nuclear Magnetic Resonance Sprectroscopy Reveals a Perfect Enzyme. B.L. Lee, S. Rashid, B. Wajda, A. Wolmarans, P. LaPointe, L. Spyracopoulos (2019). Biochemistry. 58(15), 1869-1877
A novel method for site-specific chemical SUMOylation:SUMOylation of Hsp90 modulates co-chaperone binding in vitro. A. Wolmarans, A. Kwantes, P. LaPointe (2018). Biol. Chem. doi: 10.1515/hsz-2018-0251
An Hsp90 co-chaperone protein in yeast is functionally replaced by site-specific posttranslational modification in humans. A.D. Zuehlke, M. Reidy, C. Lin, P. LaPointe, S. Alsomairy, D.J. Lee, G.M. Rivera-Marquez, K. Beebe, T. Prince, S. Lee, J.B. Trepel, W. Xu, J. Johnson, D. Masison, L. Neckers (2017) Nat Commun. 2017 May 24;8:15328.
The Mechanism of Hsp90 ATPase Stimulation by Aha1. A. Wolmarans, B. Lee, L. Spyracopoulos, P. LaPointe. (2016) Sci Rep. Sep 12;6:33179
A mutation in the catalytic loop of Hsp90 specifically impairs ATPase stimulation by Aha1p, but not Hch1p. N.K. Horvat, H. Armstrong, B.L. Lee, R. Mercier, A. Wolmarans, J. Knowles, L. Spyracopoulos, P. LaPointe. (2014) J Mol Biol. 426(12), 2379-92
Regulation of RNA interference by Hsp90 is an evolutionarily conserved process. Y. Wang, R. Mercier, T.C. Hobman, P. LaPointe. (2013) Biochim Biophys Acta 1833, 2673-2681.
Hsp90 cochaperones p23 and FKBP4 physically interact with hAgo2 and activate RNA interference-mediated silencing in mammalian cells. J.M. Pare, P. LaPointe, T.C. Hobman. (2013) Mol Biol Cell 24, 2303-10.
The co-chaperone Hch1 regulates Hsp90 function differently than its homologue Aha1 and confers sensitivity to yeast to the Hsp90 inhibitor NVP-AUY922. H. Armstrong, A. Wolmarans, R. Mercier, B. Mai, P. LaPointe. (2012) PLoS One. 7(11):e49322.
Fluorine- and rhenium-containing geldanamycin derivatives as leads for the development of molecular probes for imaging Hsp90.F. Wuest, V. Bouvet, B. Mai, P. LaPointe. (2012) Org Biomol Chem. Sep 7;10(33):6724-31.
Biological and structural basis for Aha1 regulation of Hsp90 ATPase activity in maintaining proteostasis in the human disease cystic fibrosis. A.V. Koulov*, P. LaPointe*, B. Lu*, A. Razvi, J. Coppinger, M.Q. Dong, J. Matteson, R. Laister, C. Arrowsmith, J.R. Yates III, W. E. Balch. (2010) Mol Biol Cell. 21(6):871-84.
Hsp90 regulates the function of argonaute 2 and its recruitment to stress granules and P-bodies. J.M. Pare, N. Tahbaz, J. López-Orozco, P. LaPointe, P. Lasko, T.C. Hobman. (2009) Mol Biol Cell. 20(14):3273-84.