LaPointe (Ph.D. University of Toronto)
Department of Cell Biology
5-51 Medical Sciences Building
Phone: (780) 492-1804
Fax: (780) 492-0450 (fax)
- Alberta Innovates - Health Solutions Scholar (2010-2017)
Molecular mechanism of action of the Hsp90 chaperone
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 operates in the context of an extensive cohort of co-chaperone proteins that regulate its interaction with client proteins as well as the ATPase cycle that is associated with its action. Hsp90 appears to be involves in chaperoning the acquisition of otherwise thermodynamically inaccessible conformations in client proteins. 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 laboratory is concerned with exploring this question in vitro and in vivo using a combination of genetic, proteomic and biochemical techniques.
Theme 1: Genetic and Biochemical Characterization of the Hsp90 System in Yeast
The Hsp90 system is well 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. One of the more recently identified Hsp90 co-chaperones, 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 are exploring the effect of Aha1 knockout and over-expression on the phenotypes associated with various mutant forms of Hsp90, as well as the contents of Hsp90 complexes that form under these conditions. These studies are complemented with biochemical approaches - involving enzymatic and protein refolding assays with model substrates and other related co-chaperones - aimed at elucidating the molecular properties of Aha1 and its effect on Hsp90 function.
Theme 2: Structural and Conformational Characterization of the Hsp90 Cycle
The conformationally dynamic nature of the Hsp90 dimer has made comprehensive structural analysis of its functional cycle very difficult. Using mutants of Hsp90 or co-chaperones identified in our yeast system, we are employing NMR, crystallographic and mass spectrometry approaches to elucidate the nature of Hsp90:co-chaperone complexes as well as their moving parts.
As mentioned earlier, Hsp90 function is essential for the activity of critical oncogenes such as v-src. We are exploring the Hsp90:client interaction using genetic, biochemical and structural approaches to better understand the nature of Hsp90’s client specificity as well as the conformational changes that Hsp90 action brings about in these clients.
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.
Structural Basis for Cargo Regulation of COPII Coat Assembly. S.M. Stagg*, P. LaPointe*, A. Razvi, C. Gürkan, C. Potter, B. Carragher and W.E. Balch. (2008) Cell. 134(3):474-84.
Hsp90 co-chaperone Aha1 downregulation rescues misfolding of CFTR in cystic fibrosis. X. Wang*, J. Venable*, P. LaPointe*, D.M. Hutt*, A.V. Koulov, J. Coppinger, C. Gurkan, W. Kellner, J. Matteson, H. Plutner, J.R. Riordan, J.W. Kelly, J.R. Yates III and W.E. Balch. (2006) Cell. 127: 803-15.
Structure of the Sec13/31 COPII coat cage. S.M. Stagg, C. Gurkan, D.M. Fowler, P. LaPointe, T.R. Foss, C.S. Potter, B. Carragher and W.E. Balch. (2006) Nature. 439: 234-8.
Purification and properties of mammalian Sec23/24 from insect cells. P. LaPointe and W.E. Balch. (2005) Methods Enzymol. 404: 66-74.
A role for the protease-sensitive loop region of Shiga-like toxin 1 in the retrotranslocation of its A1 domain from the endoplasmic reticulum lumen. P. LaPointe, X. Wei and J. Gariépy. (2005) J Biol Chem. 280: 23310-8.
* equal contribution