Two Functional Domains of Human Heat Shock Factor 1 Have Different Effects on Its DNA-binding Activi Article Abstract
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Published: October 08, 2007
Functional Domains of Human Heat Shock Factor 1 Have Different Effects on Its DNA-binding Activity through Redox Changes
by Ming Lu, Hee-Eun Kim, Chun-Ri Li, Yun-Jeong Hwang, Sol Kim, Im-Jung Kwak, Yun-Ju Lee, So-Sun Kim, Shin-Won Kang, Nam-Gyu Park, Dong-Kyoo Kim, Ho Sung Kang,and Jang-Su Park
Human heat shock transcription factor 1 (hHSF1) plays important roles in heat shock response. In unstressed human cells, HSF1 is present in an inactive monomer. After heat stimuli, hHSF1 forms homotrimer and gains the heat shock element (HSE)-binding activity.1-4 Three function domains, including DNA-binding domain (DBD), trimerization domain (TD) and transcription activation domain (TAD), exist in hHSF1.2 Previous studies verified that redox changes could influence hHSF1 DNA-binding.5,6 However, their roles of different domains remained unknown. Thus, we have built three kinds of hHSF1 proteins including: full-length hHSF1-α (amino acid 1-529), hHSF1-β (1-290) containing the DBD and TD, and hHSF1-γ (1-120) containing the DBD. Proteins were expressed in E. coli stains7 and purified by His-tagged and Mono Q HP columns.8 DNA-binding and trimerization activities were analyzed by electrophoretic mobility shift assay and electrophoresis gel in the presence of redox chemicals (diamide (DM), hydrogen peroxide (H2O2), dithiothreitol (DTT)). In Figure 1, hHSF1-α was treated with redox chemicals and heat-activated. As a result, the addition of increasing amounts of DTT, a reducing agent, gradually increased the formation of heat-induced hHSF1-HSE complexes and homotrimer (lanes 2-4 in Figure 1A; lanes 2-3 in Figure 1B). However, when treated with oxidizing agents DM or H2O2, hHSF1-α showed an inhibitory effect on the process of DNA-binding and trimerization (lanes 5-10 in Figure 1A; lanes 4-7 in Figure 1B). These results strongly suggested that heat-induced HSE binding and trimerization activities of hHSF1 were regulated by redox potential.
Bull. Korean Chem. Soc. 2007, Vol. 28, No. 9
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