The role of the molecular chaperone, Hsp90, in the degradation of oxidized calmodulin by the 20S proteasome

The 20S proteasome has been suggested to play a critical role in mediating the degradation of abnormal proteins under conditions of oxidative stress; yet the crystal structure indicates that the structure is incapable of degradation without the assistance of a regulatory protein or proteins. The enzyme has been found in tight association with the molecular chaperone Hsp90. To determine the nature of the interaction between Hsp90 and the 20S proteasome, and how this interaction promotes the degradation of oxidized calmodulin (CaMox), we have purified red blood cell 20S proteasomes free of Hsp90 and assessed their ability to degrade CaMox in the absence or presence of Hsp90. The purified 20S proteasome does not degrade CaMox unless Hsp90 is added to the reaction. CaMox degradation is sensitive to both proteasome and Hsp90-specific inhibitors, and is further enhanced in the presence of intracellular concentrations of ATP. Irrespective of the presence of Hsp90, we find that wild-type CaM is not significantly degraded. Direct binding measurements demonstrate that Hsp90 selectively associates with CaMox; essentially no binding is observed between Hsp90 and wild-type CaM. Site-directed mutagenesis indicates that modification of Met144 and Met145 targets CaM for degradation by the 20S proteasome. Since oxidation has been previously shown to induce both global conformational changes and a reduction in helical content of CaM, these results suggest that Hsp90 in association with the 20S proteasome selectively associates with partially unfolded proteins to promote their degradation by the proteasome, indicating a regulatory role for the chaperone.