Cellular stress invokes a protective response in which heat shock factor 1 (HSF1) is activated to increase heat shock protein (Hsp) expression. HSF1 exists as a latent monomer in unstressed cells. Upon stress HSF1 forms homotrimers, increasing its affinity for the heat shock DNA element upstream of all Hsp genes. A second conformational change is required for HSF1 to gain transcriptional competence. During prolonged heat shock or following the resumption of normal conditions HSF1 DNA-binding and transcriptional activities are reduced and HSF1 returns to the monomeric state in a process called attenuation. During the activation/deactivation cycle HSF1 is modified by small ubiquitin-related modifier (SUMO-1) conjugation and undergoes several phosphorylation and dephosphorylation events that modulate HSF1 activity. Hyperphosphorylation of HSF1 is hypothesized to trigger HSF1 transcriptional activity. HSF1 also interacts with a dynamic series of Hsp90/Hsp70-based chaperone heterocomplexes that negatively regulate DNA-binding, and transcriptional activity, and promote attenuation. This thesis was aimed at characterizing the mechanisms regulating HSF1 DNA-binding, and transcriptional activity. Expression of human HSF1 in Xenopus oocytes altered the set-point of DNA-binding in response to heat indicating that both the cellular environment and innate properties of the molecule allow HSF1 to set its activation/deactivation set-point in response to stress in vivo. HSF1 DNA-binding but not transcription was activated in oocytes treated with a high temperature heat shock. Further characterization of this observation determined that HSF1 activated by a brief high temperature heat shock inhibited transcriptionally competent HSF1 from activating transcription. It was hypothesized that this phenomenon exists to ensure the eventual death of the cell due to the accumulation of excessive damage and potential mutation caused by severe stress. The most significant observation made in this thesis is that Hsp expression was detected in oocytes injected with reporter plasmid only during recovery from a high temperature heat shock. These results led to the proposal of a model in which HSF1 trimers are either assembled in a transcriptionally incompetent form or one that has the potential to become transcriptionally competent during stress, prior to DNA-binding. The identity of HSF1-binding proteins that interact with HSF1 at different stages of activation/deactivation was characterized in an effort to assign regulatory roles to these proteins. HSF1 was detected in a high molecular weight complex (350-600 kDa) during all phases of the activation/deactivation cycle. HSF1 at different stages of activation was tested for interaction with specific molecular chaperones by electrophoretic mobility supershift analysis. Hsp90, p23, FKBP52, Hip and Hop are all associated with transcriptionally active and inactive HSF1 suggesting that interaction of HSF1 with any of these molecules does not activate HSF1 transcriptional activity. These results do not exclude the possibility that the function of these molecular chaperones may change during activation of HSF1 transcription or that post-translational modifications may be the primary mechanism that drives HSF1 from a transcriptionally inactive to active form.

Cellular stress invokes a protective response in which heat shock factor 1 (HSF1) is activated to increase heat shock protein (Hsp) expression. HSF1 exists as a latent monomer in unstressed cells. Upon stress HSF1 forms homotrimers, increasing its affinity for the heat shock DNA element upstream of all Hsp genes. A second conformational change is required for HSF1 to gain transcriptional competence. During prolonged heat shock or following the resumption of normal conditions HSF1 DNA-binding and transcriptional activities are reduced and HSF1 returns to the monomeric state in a process called attenuation. During the activation/deactivation cycle HSF1 is modified by small ubiquitin-related modifier (SUMO-1) conjugation and undergoes several phosphorylation and dephosphorylation events that modulate HSF1 activity. Hyperphosphorylation of HSF1 is hypothesized to trigger HSF1 transcriptional activity. HSF1 also interacts with a dynamic series of Hsp90/Hsp70-based chaperone heterocomplexes that negatively regulate DNA-binding, and transcriptional activity, and promote attenuation. This thesis was aimed at characterizing the mechanisms regulating HSF1 DNA-binding, and transcriptional activity. Expression of human HSF1 in Xenopus oocytes altered the set-point of DNA-binding in response to heat indicating that both the cellular environment and innate properties of the molecule allow HSF1 to set its activation/deactivation set-point in response to stress in vivo. HSF1 DNA-binding but not transcription was activated in oocytes treated with a high temperature heat shock. Further characterization of this observation determined that HSF1 activated by a brief high temperature heat shock inhibited transcriptionally competent HSF1 from activating transcription. It was hypothesized that this phenomenon exists to ensure the eventual death of the cell due to the accumulation of excessive damage and potential mutation caused by severe stress. The most significant observation made in this thesis is that Hsp expression was detected in oocytes injected with reporter plasmid only during recovery from a high temperature heat shock. These results led to the proposal of a model in which HSF1 trimers are either assembled in a transcriptionally incompetent form or one that has the potential to become transcriptionally competent during stress, prior to DNA-binding. The identity of HSF1-binding proteins that interact with HSF1 at different stages of activation/deactivation was characterized in an effort to assign regulatory roles to these proteins. HSF1 was detected in a high molecular weight complex (350-600 kDa) during all phases of the activation/deactivation cycle. HSF1 at different stages of activation was tested for interaction with specific molecular chaperones by electrophoretic mobility supershift analysis. Hsp90, p23, FKBP52, Hip and Hop are all associated with transcriptionally active and inactive HSF1 suggesting that interaction of HSF1 with any of these molecules does not activate HSF1 transcriptional activity. These results do not exclude the possibility that the function of these molecular chaperones may change during activation of HSF1 transcription or that post-translational modifications may be the primary mechanism that drives HSF1 from a transcriptionally inactive to active form.

With the increasing threat of multidrug resistant bacteria, there is a growing need to invent new drug classes that combat untreatable infections. Small molecule antibiotics have been successful in the past, but humanity is now losing the arms race against previously treatable pathogens. However, the number of clinically approved drugs targeting traditionally undruggable targets in bacteria remains low. New targets of complex protein-target interactions must be targeted for future pharmacological development. In an effort to create clinically viable biologics, the Verdine lab has developed a class of therapeutics called hydrocarbon stapled α-helical peptides; these peptides are known to affect protein-protein interactions by retaining secondary structure in vivo. Although this class of molecules has been extensively researched in cancer and viral therapies, there has been little work in bacteria due to the proposed endocytic method of entry. Moreover, DNA-binding stapled peptides have not been extensively investigated due the complexities in designing a peptide with gene selectivity. In an attempt to study peptides in bacteria, two stapled peptides based on the RpoN domain of σ54 and the FtsZ C-terminus have been synthesized. σ 54 is a DNA-binding co-factor of RNA polymerase (RNAP) and has been shown to regulate virulence and nitrogen and carbon metabolism. FtsZ is the structural unit of the contractile Z-ring that induces cell division. By designing stapled α-helical peptides to target these untraditional PPIs, we anticipate that these molecules may be used for future antimicrobial pharmacological development that treat multidrug resistant bacteria.