Cells are continuously exposed to a variety of physiological and environmental stresses that can lead to protein aggregation and/or denaturation, and eventually cell death. In order to ensure survival, cells have evolved a stress response that monitors, detects, and responds to changes within the cellular environment. The stress response is characterized by the up-regulation of heat shock protein (hsp) genes whose products can mediate the assembly and/or degradation of misfolded or aggregated proteins within the cell. This stress-induced upregulation of heat shock protein encoding genes is under the regulation of heat shock transcription factor 1 (HSF1) and its associated proteins that together form what is known as the HSF1 heterocomplex. In eukaryotic cells, HSF1 exists as a non-DNA binding monomer in the absence of stress. Upon exposure to stress, HSF1 undergoes trimerization and acquires the ability to bind heat shock elements (HSEs) located upstream of all hsp genes and after further modification, can become converted into a transcriptionally active form. Following prolonged stress or after removal of stress, HSF1 loses its ability to bind DNA and transcription ceases in a process termed attenuation. <p>Several studies have suggested that the DNA-binding and transcriptional activities of HSF1 are regulated by phosphorylation and dephosphorylation events and by chaperone-based folding mechanisms similar to those involved in the regulation of glucocorticoid receptors. Protein phosphatase 5 (PP5) has been identified as a member of the glucocorticoid receptor chaperone complex and its phosphatase activity has been shown to regulate the maturation and activation of the receptor. It has been suggested that PP5 may regulate HSF1 in a manner similar to that of glucocorticoid receptors however it has not yet been determined how PP5 interacts with the HSF1 heterocomplex or if PP5 functions to regulate HSF1-DNA binding and/or HSF1 transactivation.<p>Utilizing the Xenopus model system, I tested the hypothesis that PP5 regulates the DNA binding and transcriptional activities of HSF1 through interactions with the HSF1 heterocomplex. Increasing the activity of PP5, either through the elevation of PP5 protein levels or by activating endogenous PP5, resulted in decreased HSF1-DNA binding as well as accelerated attenuation after the removal of stress. Conversely, inhibiting the phosphatase activity of PP5 using okadaic acid or by immunotargetting, where an antibody recognizing PP5 was microinjected into the nuclei of oocytes, resulted in delayed HSF1 attenuation. Transcription assays performed using activated PP5 also demonstrated that PP5 acts to decrease HSF1-mediated transcription. Immunoprecipitation and gel mobility supershift assays were also used to show that PP5 interacts with the HSF1 heterocomplex and PP5-HSP90 binding mutants illustrated that PP5 may exert its repressive effects independently of binding directly to HSP90.
| Identifer | oai:union.ndltd.org:USASK/oai:usask.ca:etd-10222003-140425 |
| Date | 22 October 2003 |
| Creators | McLoughlin, Christine Louise |
| 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-10222003-140425/ |
| 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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