Heat and other stress, including exposure to a wide variety of noxious chemicals, activate/enhance expression of a small group of genes encoding so-called heat shock or stress proteins (Hsps). Many Hsps are known molecular chaperones. Prior activation of this stress response renders cells, tissues and organs more resistant to a subsequent stress, and blockage of the response results in increased stress susceptibility. Activation of the stress response has also been shown to prevent or mitigate ischemia and reperfusion injury, toxicity of chemotherapeutics and inflammatory responses as well as to enhance the responsiveness of the immune system. The most upstream, specific regulator of the stress response is a heat shock transcription factor (HSF), HSF1 in vertebrate cells. Our previous work showed that, in the unstressed mammalian cell, HSF1 forms a dynamic complex with Hsp90 and is incapable of binding DNA. Activation of HSF1 is a process that comprises two distinguishable steps. When the cell is stressed, the rate of protein unfolding dramatically increases, and nonnative proteins accumulate. These nonnative proteins compete with HSF1 for Hsp90 binding, and uncomplexed HSF1 rapidly homotrimerizes. Trimerization is accompanied by acquisition of DNA-binding activity and nuclear relocation of HSF1. In a reaction that is somewhat slower than the trimerization reaction, trimeric HSF1 is converted to the active transcription factor. Indirect evidence suggests that this second reaction involves or is triggered by stress-regulated phosphorylation of certain critical Ser/Thr residues in HSF1. Subsequent to a stressful event, active HSF1 is recycled. This proposal is elucidate the unknown mechanisms by which trimeric HSF1 is converted to the transcriptionally competent factor, and by which active HSF1 is deactivated and returned to the inactive, nontrimeric state. The absence of a functional stress response was noted in aging cells as well as in cells expressing prions. The mechanistic knowledge gained in this project will be applied to a systematic examination aimed at discovering the reason(s) underlying the defective stress response in aging and prion- infected cells.