Abstract
The Hsp90 chaperone activity is tightly regulated by interaction with many co-chaperones. Since CacyBP/SIP shares some sequence homology with a known Hsp90 co-chaperone, Sgt1, in this work we performed a set of experiments in order to verify whether CacyBP/SIP can interact with Hsp90. By applying the immunoprecipitation assay we have found that CacyBP/SIP binds to Hsp90 and that the middle (M) domain of Hsp90 is responsible for this binding. Furthermore, the proximity ligation assay (PLA) performed on HEp-2 cells has shown that the CacyBP/SIP-Hsp90 complexes are mainly localized in the cytoplasm of these cells. Using purified proteins and applying an ELISA we have shown that Hsp90 interacts directly with CacyBP/SIP and that the latter protein does not compete with Sgt1 for the binding to Hsp90. Moreover, inhibitors of Hsp90 do not perturb CacyBP/SIP-Hsp90 binding. Luciferase renaturation assay and citrate synthase aggregation assay with the use of recombinant proteins have revealed that CacyBP/SIP exhibits chaperone properties. Also, CacyBP/SIP-3xFLAG expression in HEp-2 cells results in the appearance of more basic Hsp90 forms in 2D electrophoresis, which may indicate that CacyBP/SIP dephosphorylates Hsp90. Altogether, the obtained results suggest that CacyBP/SIP is involved in regulation of the Hsp90 chaperone machinery.
Highlights
The heat shock protein 90 (Hsp90) is a highly abundant and ubiquitous molecular chaperone that plays an essential role in the proper folding, stabilization and activation of a myriad of "client” proteins involved in various cellular processes
CacyBP/SIP interacts with Hsp90 in the cell and under in vitro conditions
In this work we have shown that CacyBP/SIP interacts with Hsp90 and exhibits chaperone properties
Summary
The heat shock protein 90 (Hsp90) is a highly abundant and ubiquitous molecular chaperone that plays an essential role in the proper folding, stabilization and activation of a myriad of "client” proteins involved in various cellular processes. Hsp is important for stress response and seems to be a key player in maintaining cellular homeostasis [1,2]. Hsp activity depends on ATP hydrolysis coupled with cyclic conformational rearrangements and is tightly regulated by sequential binding and release of different co-chaperones as well as by post-translational modifications [7,8,9,10,11]. Various co-chaperones can influence Hsp activity in different ways, including regulation of ATP hydrolysis and conformational
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