Similarly, Trx1, Trx2, and Grx1 protein levels were also downregulated in aA crystallin KO retina

compete with HSF1 for binding to Hsp90, leading to the release of free HSF1. Therefore, we proposed that Hsf1 is present in an equilibrium with Hsp90, constantly associating with and dissociating from Hsp90. At elevated temperatures the protein kinase that phosphorylates Hsf1 becomes activated , and this leads to the subsequent activation of an inhibitor I which inactivates K. The identities of the Hsf1 kinase and Hsf1 phosphatase are currently unknown. The active K binds free Hsf1, forming the complex Hsf1K, mediating Hsf1 phosphorylation to form Hsf1P. Activated Hsf1 induces the transcription of HSP90 mRNA via heat shock elements within promoter regions, and subsequently induces the synthesis of new Hsp90. The model also accounts for the degradation of HSP90 mRNA. The transcriptional activity of Hsf1P can be repressed through the binding of Hsp90 and the formation of the 20952447” complex Hsf1Hsp90. Thus Hsf1 is assumed to be negatively regulated by Hsp90 in the model. During heat shock, Hsp90 binds unfolded and/or damaged proteins, preventing their aggregation and helping them to refold . This is considered a reversible process. In addition, both the Hsp90Complex and Hsp90 can be degraded. The degradation of Hsp90 protein and HSP90 mRNA are not explicitly regulated by heat shock in the model. However, the increased formation of Hsp90Complex due to a temperature up-shift indirectly promotes Hsp90 degradation by affecting the equilibrium between free and Hsf1-bound Hsp90. The initial conditions, the ODEs that define this model, and the parameter values are presented in Dynamics of heat shock adaptation in C. albicans Having constructed the model, it was parameterised to fit the experimentally determined dynamics of thermal adaptation in C. albicans. These included the kinetics of Hsf1 phosphorylation, and the temporal induction of HSP90 mRNA Chebulinic acid levels during 30uC37uC and 30uC42uC heat shocks. Replicate time series measurements of Hsf1 phosphorylation were completed for both 30uC37uC and 30uC42uC heat shocks. Protein extracts were prepared, subjected to ” western blotting, and Hsf1 phosphorylation levels quantified. Lambda phosphatase controls were run routinely to confirm band-shifts representing Hsf1 phosphorylation. Low levels of Hsf1 phosphorylation were reproducibly detected during a 30uC37uC heat shock. These subtle band-shifts were resolvable by lambda phosphatase at 2, 5 and 10 minutes post heat shock, but no band-shifts were observed after 10 minutes indicating that by 20 minutes Hsf1 phosphorylation levels had returned to basal levels equivalent to the no heat shock controls. Hsf1 phosphorylation levels were assayed up to 120 minutes post heat shock, but no detectable phosphorylation was observed after 20 minutes. In contrast, cells that received a 30uC42uC heat shock routinely displayed strong levels of Hsf1 phosphorylation within two minutes, the activation continuing to increase up to 20 minutes post heat shock before starting to decline again. Hsf1 phosphorylation levels had returned to low levels by the 120 minute time point. Once again, the band-shifts corresponding to Hsf1 phosphorylation were confirmed by the lambda phosphatase controls. These observations were reproducible in multiple independent experiments. HSP90 mRNA levels were also measured experimentally. During a 30uC37uC heat shock, HSP90 mRNA levels increased approximately three-fold relative to the internal ACT1 mRNA control, whereas HSP90 mRNA levels increased approximate