Project Details
Description
9986189
Liu
HSF1, a member of the multigene Heat Shock Factor family, is the principal transcription factor that mediates the cellular transcriptional response to stress. In higher eukaryotes, HSF1 is constitutively expressed and exists as a latent monomer in the cytosol under unstressed conditions. Stress triggers a multistep process of modification of HSF1 including trimerization, nuclear translocation, and inducible phosphorylation that results in the activation of DNA-binding and transcription activities of HSF1. The foundation of the goal of this research is the concept that redox-dependent modification of cysteines provides a useful and versatile means of regulating protein structure and function. Thus, the goal is to determine whether cysteine thiol-disulfide exchange modulates the structure and function of HSF1 in human cells. The hypothesis is that cysteine disulfide bond formation is an off-switch for the activation and trimerization of HSF1. The PI has observed that the placement of three of the cysteine residues in hHSF1 is such that C3 is likely to be juxtaposed to C4 and C5 in the hHSF1 monomer and thus capable of forming disulfide crosslink(s). The PI reasoned that such modification(s) would affect both the structure and function of the protein. Disulfide crosslinks would yield a more compact hHSF1 when analyzed by gel electrophoresis or filtration. Such crosslinking would also stabilize the monomer form of hHSF1 and interfere with its trimerization. These possibilities will be evaluated experimentally using pharmacological, genetic, and biophysical means. Cysteine-directed reagents, including diamide, dithiothreitol, and nitric oxide will be used to modulate the redox state of cysteines of hHSF1 in vitro and in vivo. Conformational differences and relative abundance of the reduced versus oxidized forms of hHSF1 will be assessed using protocols and reagents which have been developed and that allow for the resolution, detection and quantitation of redox conformers of HSF1. The ability of the oxidatively modified hHSF1 to undergo transformation from an inert monomer to the active DNA-binding homotrimer will be evaluated in an in vitro activation system. The particular cysteine residue(s) involved in this regulation will be defined using cysteine site-specific mutants of hHSF1. The occurrence of cysteine disulfide crosslink, the identity of the cysteine(s), and the reversibility of this crosslink by dithiothreitol will be evaluated and confirmed by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry. It is anticipated that this research will provide important information about of the role of redox as a regulator of the structure and function of hHSF1. In so doing, these studies will contribute to the emerging concept that reactive oxygen and/or nitrogen species and redox modulation provide a versatile mechanism by which cells regulate important biological processes.
Status | Finished |
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Effective start/end date | 5/15/00 → 7/31/04 |
Funding
- National Science Foundation: $315,030.00