Characterization of a signal generated by oxidation of protein thiols that activates the heat shock transcription factor

The diazenecarbonyl derivative, diamide, was used to produce nonnative protein disulfides in Chinese hamster ovary cells in order to characterize the events that occur during thiol oxidation-induced denaturation that trigger induction of Hsp 70. We limit the term protein denaturation to a process involving a conformational rearrangement by which the ordered native structure of a protein changes to a more disordered structure. Protein thiol oxidation resulted in immediate destabilization of proteins, as assessed by differential scanning calorimetry (DSC). The DSC profile indicated both a decrease in the onset temperature for detection of denaturation and destabilization of a class of proteins with an average transition temperature (Tm) of 60 degrees C. Concomitant with destabilization was an increase in proteins associated with isolated nuclei. Thiol oxidation also induced heat shock transcription factor (HSF) binding activity, however, this was nearly undetectable immediately following diamide treatment: maximum activation occurred 3 hr following exposure. In contrast, heat shock denatured thermolabile proteins which exhibited a Tm of < or = 48 degrees C. Heat shock also resulted in a rapid increase in proteins associated with isolated nuclei and produced immediate and maximum activation of HSF binding. The accumulation of Hsp and Hsc 70 mRNA following thiol oxidation reflected the delay in HSF binding. Acquisition of HSF binding activity occurred immediately if diamide-treated cells were subsequently exposed to a heat shock, indicating that HSF was not inactivated by the diamide treatment. Ostensibly, the cellular system for detecting denatured/abnormal proteins failed to immediately recognize the signal generated by thiol oxidation. These results suggest that at least two processes are involved in the induction of Hsp 70 by nonnative disulfide bond formation: destabilization of protein structure resulting in denaturation and recognition of denatured protein.