High Throughput Screens Against Heat Shock Protein 70 (Hsp 70)
dc.contributor.author | Chang, Chia Yin | en_US |
dc.date.accessioned | 2011-09-15T17:11:03Z | |
dc.date.available | NO_RESTRICTION | en_US |
dc.date.available | 2011-09-15T17:11:03Z | |
dc.date.issued | 2011 | en_US |
dc.date.submitted | en_US | |
dc.identifier.uri | https://hdl.handle.net/2027.42/86340 | |
dc.description.abstract | The molecular chaperone Hsp70 plays important roles in protein quality control. Moreover, Hsp70 has been linked to diseases of protein misfolding, such as neurodegenerative disorders, suggesting that it could be a promising new drug target. However, the molecular mechanisms that link Hsp70 to these diseases remain unclear. We hypothesized that one way to better understand the roles of Hsp70 would be to develop chemical probes that disrupt its specific functions. In this thesis, we developed the first high throughput screens for the ATPase activity of Hsp70. Using this platform, we explored the idea of “gray-box” screening, in which multiple components of the Hsp70 chaperone system are reconstituted in vitro to better approximate the biochemical properties of the physiological complexes. We wanted to understand whether this approach would provide a compromise between “black box” cell-based screens and assays that rely on individual purified proteins. Accordingly, we screened over 50,000 compounds and natural product extracts and reported a number of new Hsp70 inhibitors, including myricetin. Interestingly, we found that many of these inhibitors blocked the protein-protein interactions between the Hsp70, DnaK, and its important co-chaperone, DnaJ. Thus, we expect these compounds to be powerful probes for exploring the biological roles of the DnaK-DnaJ complex. Finally, we explored whether other Hsp70 chaperone functions, such as substrate binding or refolding, might also be useful targets for high throughput screening. Towards that goal, we generated point mutants in DnaK and human Hsc70 and studied how their different in vitro biochemical activities correlated with cellular functions. We found that luciferase refolding activity, not ATPase rate, was more predictive of certain cellular chaperone activities, such as heat shock rescue and effects on tau stability. These results suggest the need for multiple primary and secondary assays in searching for Hsp70 inhibitors. Together, these studies have provided important insights into the Hsp70 chaperone system and they have discovered molecules that could be used to further validate Hsp70 as a drug target. | en_US |
dc.language.iso | en_US | en_US |
dc.subject | Hsp70 | en_US |
dc.subject | High Throughput Screening | en_US |
dc.subject | DnaK | en_US |
dc.subject | ATPase | en_US |
dc.subject | DnaJ | en_US |
dc.subject | Tau | en_US |
dc.title | High Throughput Screens Against Heat Shock Protein 70 (Hsp 70) | en_US |
dc.type | Thesis | en_US |
dc.description.thesisdegreename | PhD | en_US |
dc.description.thesisdegreediscipline | Chemical Biology | en_US |
dc.description.thesisdegreegrantor | University of Michigan, Horace H. Rackham School of Graduate Studies | en_US |
dc.contributor.committeemember | Gestwicki, Jason E. | en_US |
dc.contributor.committeemember | Mapp, Anna K. | en_US |
dc.contributor.committeemember | Palfey, Bruce A. | en_US |
dc.contributor.committeemember | Xu, Zhaohui | en_US |
dc.subject.hlbsecondlevel | Biological Chemistry | en_US |
dc.subject.hlbtoplevel | Science | en_US |
dc.description.bitstreamurl | http://deepblue.lib.umich.edu/bitstream/2027.42/86340/1/taiwlyra_1.pdf | |
dc.owningcollname | Dissertations and Theses (Ph.D. and Master's) |
Files in this item
Remediation of Harmful Language
The University of Michigan Library aims to describe library materials in a way that respects the people and communities who create, use, and are represented in our collections. Report harmful or offensive language in catalog records, finding aids, or elsewhere in our collections anonymously through our metadata feedback form. More information at Remediation of Harmful Language.
Accessibility
If you are unable to use this file in its current format, please select the Contact Us link and we can modify it to make it more accessible to you.