Stress-induced heat shock protein (HSP) gene transcription is controlled primarily by the transcription factor heat shock factor 1 (HSF1). HSF1 activation involves trimerization, heat shock element (HSE)-binding, and transactivation. During prolonged stress or upon removal of stress HSF1 activity attenuates. The mechanism(s) regulating HSF1 activity are unknown. Some reports have suggested that HSF1 may be regulated in some manner by the HSP90 chaperone (Nadeau, K., 'et.al.', 1993; Nair, S., 'et.al.', 1996). Utilizing the 'Xenopus' oocyte model system I tested the hypothesis that the HSP90 chaperone machine, known to function in the folding and maturation of molecules such as steroid receptors, might also participate in HSF1 regulation. Characterization experiments illustrated that the 'Xenopus' oocyte was capable of responding to some but not all forms of stress at the level of HSF1-HSE binding illustrating that certain stress pathways may be absent or inactive in the oocyte. Through transcriptional assaysit was also shown that HSF1-DNA binding and transactivation are regulated by independent mechanisms in the oocyte. HSP90 was shown to interact with and regulate the activity of HSF1 in oocytes. HSP90-HSF1 associations were illustrated ' in vivo' and 'in vitro' by co-immunoprecipitation and gel supershift assays. Immunotargeting HSP90 caused activation of HSF1 under control conditions and delayed deactivation during recovery. These data support a role for HSP90 in the oligomeric changes associated with HSF1 activation/deactivation. Immunotargeting HSP90 also inhibited HSF1 dependent transcription, supporting a role for HSP90 in mediating HSF1 transcriptional activity. HSP90 does not regulate HSF1 alone. Gel supershift analysis showed that p23, HSP90 and FKBP52 exist in a complex with activated HSF1. Furthermore, elevating the levels of various co-chaperones through injection of protein or mRNA had various effects on HSF1 during recovery from stress. Immunotargeting HSP90 or p23 induced HSF1-DNA binding in the absence of stress indicating these proteins may act together to repress HSF1 'in vivo'. Furthermore, injection of HSP90, Hip, Hop, p23, FKBP51, and FKBP52 antibodies significantly delayed HSF1 deactivation supporting a role for these proteins in trimer disassembly. Therefore multiple components of the HSP90 chaperone complex function to regulate HSF1 during its activation and/or deactivation cycle.
| Identifer | oai:union.ndltd.org:USASK/oai:usask.ca:etd-10212004-002531 |
| Date | 01 January 2001 |
| Creators | Bharadwaj, Steven Charles |
| Contributors | Ovsenek, Nicholas (Nick) |
| Publisher | University of Saskatchewan |
| Source Sets | University of Saskatchewan Library |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://library.usask.ca/theses/available/etd-10212004-002531 |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to University of Saskatchewan or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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