1. | Góral A.♦, Bieganowski P.♦, Prus W.♦, Krzemień-Ojak Ł.♦, Kądziołka B.♦, Fabczak H.♦, Filipek A.♦, Calcyclin Binding Protein/Siah-1 Interacting Protein Is a Hsp90 Binding Chaperone, PLOS ONE, ISSN: 1932-6203, DOI: 10.1371/journal.pone.0156507, Vol.11, No.6, pp.e0156507-1-18, 2016Abstract: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. Keywords:Chaperone proteins, Luciferase, Phosphatases, Enzyme-linked immunoassays, Recombinant proteins, Ligation assay, Plasmid construction, Luciferase assay Affiliations:Góral A. | - | other affiliation | Bieganowski P. | - | Mossakowski Medical Research Centre, Polish Academy of Sciences (PL) | Prus W. | - | other affiliation | Krzemień-Ojak Ł. | - | other affiliation | Kądziołka B. | - | other affiliation | Fabczak H. | - | Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL) | Filipek A. | - | Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL) |
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