As any protein biochemist will tell you, protein aggregation is a frequent and annoying byproduct of an otherwise elegantly planned and executed experimental scheme. Such problems are remedied by sedimenting the offending material in a centrifuge tube and discarding it. When proteins aggregate in living cells the consequences may be deadly. We now know that misfolding and aggregation of specific proteins is the underlying mechanism for several devastating diseases. My main focus is to understand how molecular chaperones function together to prevent or reverse aggregation using approaches that integrate cell and molecular biology and biochemistry.

Molecular chaperones are structurally diverse proteins that are grouped together into highly conserved families. By binding to incompletely-folded target proteins, molecular chaperones help them to complete folding, assemble into oligomeric structures, or translocate across a variety of intracellular membranes. Therefore, molecular chaperones play pivotal roles in normal protein metabolism in a cellular environment that is so densely packed with macromolecules that unchaperoned processes are virtually impossible. Under suboptimal conditions, such as encountered when applying mild heat to cells or when a protein possesses a defect that decreases it folding stability, the balance is tipped in favour of protein misfolding and aggregation. Cells respond to stress by upregulating the expression of a subset of genes encoding the so-called heat shock proteins. Not surprisingly the majority of heat shock proteins are molecular chaperones.

Hsp104 of Saccharomyces cerevisiae is the most potent protein thermotolerance factor known. Unlike other molecular chaperones that function by preventing protein aggregation, Hsp104 resolubilizes proteins that have already aggregated and helps them to refold to the native state. Reconstitution of the Hsp104-mediated disaggregation and refolding reaction in the test tube shows that Hsp104 requires the cooperation of other molecular chaperones with more conventional properties. Intriguingly Hsp104 is also a key modulator of the aggregation state of the yeast translation termination factor Sup35. Sup35 forms self-seeding highly ordered fibrillar aggregates in vitro with strikingly similar properties to aggregates formed by proteins associated with Creuztfeldt-Jakob (the human form of Mad Cow Disease) and Alzheimer's Disease.

Research Projects
My research focuses on understanding the function of molecular chaperones, both in living cells and in purified form, with a view to determining how they interact with their targets and function together to alter the fate of aggregation-prone proteins. Students may chose to work in one of a number of related project areas that are briefly described below.

Recognition and processing of substrates by Hsp104.
One of our main goals is to understand how Hsp104 recognizes which proteins are misfolded and aggregated and how it extracts these proteins from the aggregate for resolubilization and refolding. One approach we have taken is to use large arrays of peptides derived from proteins that can be refolded by Hsp104 to identify those segments of protein that are most likely detected by Hsp104 as it polices the cell for aggregated proteins. We have used selected peptides to probe the peptide binding properties of Hsp104 and hope to eventually understand how ATP-driven conformation changes in Hsp104 coupled to peptide interactions, lead to the extraction and refolding of aggregated proteins. We are using a similar approach to determine how Hsp104 may traffic through the nuclear pore complex during heat shock without the help of nuclear import receptors.

Interactions of chaperones with prions.
Prions are infectious proteins that are capable of propagating themselves by promoting the conversion of normal form of the same protein into the infectious form. The formation of protein aggregates is one of the hallmarks of the pathogenic proteins. In yeast, prions are responsible for a novel form of inheritance at the level of protein conformation rather than through normal, Mendelian inheritance of allelic genes. Not surprisingly, chaperones including Hsp104 influence the stability of prions. We are studying these interactions and have recently extended our interests to include mammalian prions that are implicated in Transmissible Spongiform Encephalopathies (TSEs) including Mad Cow Disease or BSE.

Protein-protein interactions in PrP function, biogenesis, and misfolding.
PrP is a glycophosphatidyl inositol-anchoured cell surface protein that in its native form has a neuroprotective effect in the brain. When a healthy animal is infected with prions— a misfolded, aggregated form of PrP called PrPSc— the infectious form templates the conversion of normal PrP to the abnormal form causing neurodegeneration. We are interested in taking new approaches to discovering novel proteins that may interact with PrP and influence its normal function and its folding stability at the cell surface or in other subcellular organelles. We are using yeast membrane two-hybrid screens to identify candidate interactors and then verifying the functional consequences of the identified interaction. As a complementary approach, we have engineered novel forms of PrP that accumulate in organelles that normal PrP visits only transiently. These are being used to identify critical interactions that may influence the folding of newly synthesized PrP or PrP that is in the process of recycling through endocytic compartments. These interactions may profoundly influence the function of PrP in health and disease.

Training in Protein Folding: Principles and Disease and PrioNet Canada.
Students or postdocs in the Glover lab may be able to take advantage funding opportunities provided by the CIHR training program or through the PrioNat Canada NCE.

Insight into the molecular basis of HSP104-mediated curing of the [PSI+] prion. C.W. Helsen & J. R. Glover. Submitted, 2011.

Remodeling of protein aggregates by Hsp104. J. R. Glover & R. Lum. Protein Pept Lett 16: 587-597, 2009. PMID: 19519516 [Pubmed]

The SmpB-tmRNA tagging system plays important roles in Streptomyces coelicolor growth and development. C. Yang & J.R. Glover. PLoS One 4:e4459, 2009. PMID: 19212432 [Pubmed]

Peptide and protein binding in the axial channel of Hsp104: insights into the mechanism of protein unfolding. R. Lum, M. Niggemman, & J. R. Glover. J Biol Chem, 283: 30139-30150, 2008. PMID: 18755692 [Pubmed]

The C-terminal extension of Saccharomyces cerevisiae Hsp104 plays a role in oligomer assembly. R. G. MacKay, C. W. Helsen, J. M. Tkach and J. R. Glover. Biochemistry, 47:1918-1927 , 2008. PMID: 18197703 [Pubmed]

Nucleocytoplasmic trafficking of the molecular chaperone Hsp104 in unstressed and heat-shocked cells. J. M. Tkach & J. R. Glover. Traffic 9:39-56, 2008 PMID: 17973656 [Pubmed]

BAG5 modulates Parkin and enhances dopaminergic neuron degeneration. S.K. Kalia, S. Lee, L. Liu, S.J. Crocker, T.E. Thorarinsdottir, P.D. Smith, J.R. Glover, E.A. Fon, D.S. Park, & A.M. Lozano. Neuron 44:931-945, 2004 [Pubmed]

Saccharomyces cerevisiae Hsp104 enhances the chaperone capacity of human
cells and inhibits heat stress-induced proapoptotic signaling. Mosser DD, Ho S, Glover JR. Biochemistry 43(25): 8107-15, 2004. [Pubmed]

Amino acid substitutions in the C-terminal AAA+ module of Hsp104 prevent
substrate recognition by disrupting oligomerization and cause high temperature inactivation. Tkach JM, Glover JR. J Biol Chem. (in press), 2004. [Pubmed]

Evidence for an unfolding/threading mechanism for protein disaggregation by
Saccharomyces cerevisiae Hsp104. Lum R, Tkach JM, Vierling E, Glover JR. J
Biol Chem. 279: 29139-46, 2004. [Pubmed]

Defining a pathway of communication from the C-terminal peptide binding domain to the N-terminal ATPase domain in a AAA protein.  Cashikar AG, Schirmer EC, Hattendorf DA, Glover JR, Ramakrishnan MS, Ware DM, Lindquist SL. Mol Cell. 9: 751-60, 2002. [Pubmed]

Crowbars and ratchets: hsp100 chaperones as tools in reversing protein
aggregation. Glover JR, Tkach JM. Biochem Cell Biol. 79: 557-68, 2001. [Pubmed]

Hsp104, Hsp70, and Hsp40: a novel chaperone system that rescues previously aggregated proteins. Glover, J. R., and Lindquist, S., Cell 94: 73-82, 1998.[Pubmed]

Self-seeded fibers formed by Sup35, the protein determinant of [PSI+], a heritable prion-like factor of S. cerevisiae. Glover, J. R., Kowal, A. S., Schirmer, E. C., Patino, M. M., Liu, J. J., and Lindquist, S., Cell 89: 810-819, 1997. [Pubmed]

Support for the prion hypothesis for inheritance of a phenotypic trait in yeast. Patino, M. M., Liu, J. J., Glover, J. R., and Lindquist, S., Science 273: 622-626, 1996. [Pubmed]

HSP100/Clp proteins: a common mechanism explains diverse functions. Schirmer, E. C., Glover, J. R., Singer, M.A., and Lindquist, S., Trends Biochem Sci 21: 289-296, 1996. [Pubmed]