The focus of our research is towards understanding the mechanisms that underlie ATP-dependent protein remodeling carried out by molecular chaperone machines and the role of chaperones in ATP-dependent proteolysis. Molecular chaperones function during non-stress conditions to facilitate folding of newly synthesized proteins, to remodel protein complexes, and to target regulatory proteins and misfolded proteins for degradation. During cell stress, chaperones play an essential role in preventing folding intermediates from becoming irreversibly damaged and forming protein aggregates. They promote recovery from stress by disaggregating and reactivating proteins, a process once thought to be impossible. They are also involved in delivering damaged proteins to compartmentalized proteases. Protein aggregation, misfolding and premature degradation are major contributors to a large number of human diseases, including cancer, Alzheimer?s, Parkinson?s, type II diabetes, cystic fibrosis, and prion diseases. The goal of our research is to understand how chaperones function and to provide the foundation for discovering preventions and treatments for diseases involving protein misfolding. One goal of our research is to elucidate the mechanism of action of Hsp90. The Hsp90 family of heat shock proteins represents one of the most abundantly expressed and highly conserved families of molecular chaperones. Eukaryotic Hsp90 is known to control the stability and the activity of more than 200 client proteins, including receptors, protein kinases and transcription factors. Hsp90 is also important for the growth and survival of cancer cells and drugs targeting Hsp90 are currently in clinical trials. To gain insight into the mechanism of action of Hsp90 chaperones, we are studying Hsp90 from Escherichia coli, Hsp90Ec. Using a novel phenotype associated with Hsp90Ec overexpression in E. coli, we selected for Hsp90Ec mutants with defective chaperone function. We identified a functional region of Hsp90Ec that contains surface-exposed residues from the middle and C-terminal domains. To understand the role of this region, we purified the Hsp90Ec mutant proteins and characterized them in vitro. In contrast to wild type Hsp90Ec that reactivates heat-denatured luciferase in collaboration with the DnaK chaperone system, the Hsp90Ec mutants were defective for chaperone function. The mutant proteins hydrolyzed ATP, however, unlike wild type Hsp90Ec, the ATPase activities of the mutant proteins were poorly stimulated by client proteins suggesting that they bound clients weakly. Results from protein binding assays demonstrated that the Hsp90Ec mutant proteins were defective for client protein binding. Thus, our results define a functional region in E. coli Hsp90 that is important for substrate binding. We extended our conclusions to eukaryotic Hsp90 by reconstructing homologous mutations in S. cerevisiae Hsp82 and testing the mutants for chaperone activities, including yeast viability and activation of several client proteins. We discovered that this region is important for Hsp90 chaperone function in yeast. A second goal is to understand the mechanism of protein remodeling and disaggregation by Clp/Hsp100 molecular chaperones, including ClpB of prokaryotes and its yeast homolog, Hsp104. ClpB/Hsp104 dissolves insoluble aggregated proteins in combination with a second molecular chaperone system, DnaK in E. coli and Hsp70 in yeast. In the absence of the DnaK/Hsp70 system, ClpB and Hsp104 have the intrinsic ability to disaggregate soluble aggregates in vitro. Conformational changes in ClpB/Hsp104 driven by ATP binding and hydrolysis promote substrate binding, unfolding and translocation. Conserved pore tyrosines in both nucleotide-binding domain-1 (NBD-1) and 2 (NBD-2) that reside in flexible loops extending into the central pore of the ClpB/Hsp104 hexamer, bind substrates. When the NBD-1 pore loop tyrosine is substituted with alanine (Y251A), ClpB can collaborate with the DnaK system in disaggregation, although activity is reduced. The N-domain has also been implicated in substrate binding and like the NBD-1 pore loop tyrosine, is not essential for disaggregation activity. To further probe the function and interplay of the ClpB N-domain and the NBD-1 pore loop, we made a double mutant with an N-domain deletion and Y251A substitution. This ClpB double mutant is inactive in substrate disaggregation with the DnaK system, although each single mutant alone can function with DnaK. Our data suggest that this loss in activity is primarily due to a decrease in substrate engagement by ClpB prior to substrate unfolding and translocation, and indicate an overlapping function for the N-domain and NBD-1 pore tyrosine. Furthermore, the functional overlap seen in the presence of the DnaK system is not observed in the absence of DnaK. For innate ClpB unfolding activity, the NBD-1 pore tyrosine is required, and the presence of the N-domain is insufficient to overcome the defect of the ClpB Y251A mutant. A third goal is to investigate the mechanism of action of Clp chaperones in proteolysis. Clp proteases of prokaryotes have analogous structures, functions and mechanisms of action to the eukaryotic proteasome. They are composed of an ATP-dependent protein unfolding component and a protease component. We are currently studying ClpX, which associates with a proteolytic component, ClpP, forming the ClpXP ATP-dependent protease. We discovered that ClpXP participates in cell division of E. coli and further showed that in vitro and in vivo E. coli ClpXP degrades FtsZ, the major cytoskeletal protein in bacteria and a tubulin homolog. FtsZ is an essential cell division component, forming a dynamic ring where constriction occurs to divide the cell by polymerizing into fibers and bundles in a GTP-dependent manner. To identify regions of FtsZ important for recognition and degradation by ClpXP, we designed a set of FtsZ mutant proteins containing amino acid substitutions and deletions near the FtsZ C-terminus. We identified two discrete regions of FtsZ important for recognition and degradation by ClpXP. One region in FtsZ overlaps with the C-terminal recognition site for several other FtsZ-interacting proteins;and the second region is located within a 52 amino acid linker that is between the polymerization domain and the C-terminus. We determined that both regions of FtsZ facilitate degradation by ClpXP in the presence of GTP, the condition that promotes polymerization, and in the absence of GTP.
| Hoskins, Joel R; Wickner, Sue; Doyle, Shannon M (2018) Bacterial Hsp90 ATPase Assays. Methods Mol Biol 1709:199-207 |
| Kravats, Andrea N; Hoskins, Joel R; Reidy, Michael et al. (2018) Functional and physical interaction between yeast Hsp90 and Hsp70. Proc Natl Acad Sci U S A 115:E2210-E2219 |
| Kravats, Andrea N; Doyle, Shannon M; Hoskins, Joel R et al. (2017) Interaction of E. coli Hsp90 with DnaK Involves the DnaJ Binding Region of DnaK. J Mol Biol 429:858-872 |
| Johnston, Danielle M; Miot, Marika; Hoskins, Joel R et al. (2017) Substrate Discrimination by ClpB and Hsp104. Front Mol Biosci 4:36 |
| Doyle, Shannon M; Shastry, Shankar; Kravats, Andrea N et al. (2015) Interplay between E. coli DnaK, ClpB and GrpE during protein disaggregation. J Mol Biol 427:312-27 |
| Reidy, Michael; Sharma, Ruchika; Shastry, Shankar et al. (2014) Hsp40s specify functions of Hsp104 and Hsp90 protein chaperone machines. PLoS Genet 10:e1004720 |
| Camberg, Jodi L; Viola, Marissa G; Rea, Leslie et al. (2014) Location of dual sites in E. coli FtsZ important for degradation by ClpXP; one at the C-terminus and one in the disordered linker. PLoS One 9:e94964 |
| Markovski, Monica; Wickner, Sue (2013) Preventing bacterial suicide: a novel toxin-antitoxin strategy. Mol Cell 52:611-2 |
| Genest, Olivier; Reidy, Michael; Street, Timothy O et al. (2013) Uncovering a region of heat shock protein 90 important for client binding in E. coli and chaperone function in yeast. Mol Cell 49:464-73 |
| Doyle, Shannon M; Genest, Olivier; Wickner, Sue (2013) Protein rescue from aggregates by powerful molecular chaperone machines. Nat Rev Mol Cell Biol 14:617-29 |
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