ZIA BC 008710 (ZIA) | |||
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Title | Molecular Chaperones and DNA Replication | ||
Institution | NCI, Bethesda, MD | ||
Principal Investigator | Wickner, Sue | NCI Program Director | N/A |
Cancer Activity | N/A | Division | CCR |
Funded Amount | $1,007,355 | Project Dates | 00/00/0000 - 00/00/0000 |
Fiscal Year | 2017 | Project Type | Intramural |
Research Topics w/ Percent Relevance | Cancer Types w/ Percent Relevance | ||
Bioengineering (100.0%) Cancer (100.0%) |
Brain (10.0%) Nervous System (45.0%) |
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Research Type | |||
Normal Functioning Development and Characterization of Model Systems |
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Abstract | |||
Our research is focused on elucidating 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. 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. 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 aim is to understand 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 this important family of chaperones, we are studying Hsp90 from Escherichia coli, Hsp90Ec, and from yeast, Hsp82. We discovered that Hsp90Ec, and the Hsp70 chaperone system of E. coli, the DnaK system, act synergistically in protein reactivation in vitro. ATP hydrolysis by Hsp90Ec is required, showing that Hsp90Ec exhibits ATP-dependent chaperone activity. Our work shows that Hsp90Ec interacts directly with the E. coli Hsp70, DnaK. However, the binding is weak. Using an in vitro protein-protein interaction assay, we found that Hsp90Ec formed a significantly more stable ternary complex with DnaK and a client protein than with DnaK alone. Moreover, a J-domain co-chaperone, DnaJ, promoted further stabilization of Hsp90Ec-DnaK-client complex. Additional results using Hsp90Ec and DnaK mutants defective in client binding or ATP hydrolysis demonstrated that client binding as well as ATP hydrolysis by both DnaK and Hsp90Ec were necessary for ternary complex formation. To determine the region of Hsp90Ec that interacts with DnaK we developed a screen using a bacterial two-hybrid system to isolate Hsp90Ec mutants potentially defective in DnaK interaction. In vitro these mutants were shown to be defective in both functional and physical interaction with DnaK. From this work, we concluded that a region of Hsp90Ec in the middle domain of the protein is important for the interaction with DnaK. By using molecular docking we identified a probable region in the nucleotide-binding domain of DnaK that interacts with the middle domain of Hsp90Ec. We then made substitution mutants in DnaK residues predicted from the model to interact with Hsp90Ec. Most of the mutants were defective or partially defective in their ability to interact with Hsp90Ec in vivo in a bacterial two-hybrid assay and in vitro in a bio-layer interferometry assay. The mutants were also defective in their ability to function in protein reactivation in combination with DnaK and DnaK cochaperones. The region of DnaK we identified as important for interacting with Hsp90Ec overlaps with the region of DnaK that interacts with J-domain proteins. This work provides a better understanding of the regulation of the chaperone activity of Hsp90Ec by DnaK and offers new insight in improving inhibitor design at specific stages of the Hsp90 chaperone cycle. A second aim is to elucidate the mechanism of protein disaggregatio |