Abstract
The heat shock response (HSR) is a cellular mechanism for counteracting acute proteotoxic stress. In eukaryotes, transcriptional activation of the HSR is regulated by heat shock factor 1 (HSF1). Activation of HSF1 induces the expression of heat shock proteins (HSPs) that function as molecular chaperones to fold and maintain the three-dimensional structure of misfolded proteins. The regulation of the degree and duration of the HSR is controlled by multiple biochemical mechanisms that include posttranslational modification of HSF1 and numerous protein-protein interactions. In this chapter, we describe a method to evaluate the activation and deactivation of the HSR at the transcriptional level using a short half-life luciferase reporter assay. This assay can be used to further characterize the HSR or as a screen for small-molecule inducers, amplifiers, or repressors.
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References
Vihervaara A, Sistonen L (2014) HSF1 at a glance. J Cell Sci 127(Pt 2):261–266
Dayalan Naidu S, Dinkova-Kostova AT (2017) Regulation of the mammalian heat shock factor 1. FEBS J 284(11):1606–1627
Brown SA, Kingston RE (1997) Disruption of downstream chromatin directed by a transcriptional activator. Genes Dev 11(23):3116–3121
Bunch H et al (2014) TRIM28 regulates RNA polymerase II promoter-proximal pausing and pause release. Nat Struct Mol Biol 21(10):876–883
Zou J, Guo Y, Guettouche T, Smith DF, Voellmy R (1998) Repression of heat shock transcription factor HSF1 activation by HSP90 (HSP90 complex) that forms a stress-sensitive complex with HSF1. Cell 94(4):471–480
Guettouche T, Boellmann F, Lane WS, Voellmy R (2005) Analysis of phosphorylation of human heat shock factor 1 in cells experiencing a stress. BMC Biochem 6:4
Xu YM, Huang DY, Chiu JF, Lau AT (2012) Post-translational modification of human heat shock factors and their functions: a recent update by proteomic approach. J Proteome Res 11(5):2625–2634
Mendillo ML et al (2012) HSF1 drives a transcriptional program distinct from heat shock to support highly malignant human cancers. Cell 150(3):549–562
Calderwood SK, Murshid A, Prince T (2009) The shock of aging: molecular chaperones and the heat shock response in longevity and aging—a mini-review. Gerontology 55(5):550–558
Parsian AJ et al (2000) The human Hsp70B gene at the HSPA7 locus of chromosome 1 is transcribed but non-functional. Biochim Biophys Acta 1494(1–2):201–205
Younis I et al (2010) Rapid-response splicing reporter screens identify differential regulators of constitutive and alternative splicing. Mol Cell Biol 30(7):1718–1728
Kim D, Kim SH, Li GC (1999) Proteasome inhibitors MG132 and lactacystin hyperphosphorylate HSF1 and induce hsp70 and hsp27 expression. Biochem Biophys Res Commun 254(1):264–268
Murshid A et al (2010) Protein kinase A binds and activates heat shock factor 1. PLoS One 5(11):e13830
Gibson DG et al (2009) Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods 6(5):343–345
Zhang JH, Chung TD, Oldenburg KR (1999) A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J Biomol Screen 4(2):67–73
Galam L et al (2007) High-throughput assay for the identification of Hsp90 inhibitors based on Hsp90-dependent refolding of firefly luciferase. Bioorg Med Chem 15(5):1939–1946
Beebe K et al (2013) Posttranslational modification and conformational state of heat shock protein 90 differentially affect binding of chemically diverse small molecule inhibitors. Oncotarget 4(7):1065–1074
Acknowledgments
This work was supported by the Mowad Endowment for New Discoveries at Geisinger Health Systems, the JSPS Research Fellowship for Japanese Biomedical and Behavioral Research at NIH, and the Intramural Research Program at the National Cancer Institute.
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Kijima, T., Eguchi, T., Neckers, L., Prince, T.L. (2018). Monitoring of the Heat Shock Response with a Real-Time Luciferase Reporter. In: Calderwood, S., Prince, T. (eds) Chaperones. Methods in Molecular Biology, vol 1709. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7477-1_3
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DOI: https://doi.org/10.1007/978-1-4939-7477-1_3
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