Azadiradione regulates Heat Shock Factor 1 function by interacting with its DNA-binding domain independent of the oligomerization domain

Heat shock factor 1 (HSF1) masters cellular proteostasis under stress by upregulating the expression of molecular chaperones that help refold or degrade the misfolded proteins. HSF1 activation involves a monomer-to-oligomer transition and binding to its recognition sequence, the heat shock elements (HSEs) on its target gene promoters. HSF1 activity declines with age as well as in neurodegenerative disorders (NDs) such as Parkinsons disease, highlighting the need for strategies to restore its function. Azadiradione (AZD), a limonoid isolated from Azadirachta indica seeds, directly activates HSF1 in cellular and preclinical ND models, unlike other small-molecule activators reported elsewhere. We investigated the molecular basis of AZD-mediated HSF1 activation using purified variants of this protein including those without its oligomerization and transactivation domain. Fluorescence polarization and dynamic light scattering assays revealed that AZD promotes the oligomerization of monomeric HSF1 to enhance its HSE-binding affinity by engaging with its DNA-binding domain (DBD). The oligomerization domain known to be required for stress-induced activation, appears redundant in AZD-mediated activation. Furthermore, evidence suggests AZD-induced conformational alterations in the HSE facilitate its binding to the HSF1 monomer. Notably, AZD reduces the DNA-binding ability of pre-assembled oligomeric HSF1 by triggering its amyloid-like aggregation. This finding also highlighted a potential anticancer effect of AZD, as cancer cells heavily depend on this HSF1 population for rapid proliferation and survival. Overall, these findings offer novel insights into the functional regulation of HSF1 and suggest a framework for developing small-molecule HSF1 activators with therapeutic potential for protein conformation disorders.