| Autor: |
Sanne H. Olesen, Donna J. Ingles, Jin-Yi Zhu, Mathew P. Martin, Stephane Betzi, Gunda I. Georg, Joseph S. Tash, Ernst Schönbrunn |
| Jazyk: |
angličtina |
| Rok vydání: |
2015 |
| Předmět: |
|
| Zdroj: |
Molecules, Vol 20, Iss 1, Pp 1643-1660 (2015) |
| Druh dokumentu: |
article |
| ISSN: |
1420-3049 |
| DOI: |
10.3390/molecules20011643 |
| Popis: |
The molecular chaperone Hsp90 is regulated by co-chaperones such as p50Cdc37, which recruits a wide selection of client protein kinases. Targeted disruption of the Hsp90-p50Cdc37 complex by protein–protein interaction (PPI) inhibitors has emerged as an alternative strategy to treat diseases characterized by aberrant Hsp90 activity. Using isothermal microcalorimetry, ELISA and GST-pull down assays we evaluated reported Hsp90 inhibitors and nucleotides for their ability to inhibit formation of the human Hsp90β-p50Cdc37 complex, reconstituted in vitro from full-length proteins. Hsp90 inhibitors, including the proposed PPI inhibitors gedunin and H2-gamendazole, did not affect the interaction of Hsp90 with p50Cdc37 in vitro. Phosphorylation of Hsp90 and p50Cdc37 by casein kinase 2 (CK2) did not alter the thermodynamic signature of complex formation. However, the phosphorylated complex was vulnerable to disruption by ADP (IC50 = 32 µM), while ATP, AMPPNP and Hsp90 inhibitors remained largely ineffective. The differential inhibitory activity of ADP suggests that phosphorylation by CK2 primes the complex for dissociation in response to a drop in ATP/ADP levels. The approach applied herein provides robust assays for a comprehensive biochemical evaluation of potential effectors of the Hsp90-p50Cdc37 complex, such as phosphorylation by a kinase or the interaction with small molecule ligands. |
| Databáze: |
Directory of Open Access Journals |
| Externí odkaz: |
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