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Abstract
The 20 S proteasome has been suggested to play a critical role in mediating the degradation of abnormal proteins under conditions of oxidative stress and has been found in tight association with the molecular chaperone Hsp90. To elucidate the role of Hsp90 in promoting the degradation of oxidized calmodulin (CaM(ox)), we have purified red blood cell 20 S proteasomes free of Hsp90 and assessed their ability to degrade CaM(ox) in the absence or presence of Hsp90. Purified 20 S proteasome does not degrade CaM(ox) unless Hsp90 is added. CaM(ox) degradation is sensitive to both proteasome and Hsp90-specific inhibitors and is further enhanced in the presence of 2 mm ATP. Irrespective of the presence of Hsp90, we find that unoxidized CaM is not significantly degraded. Direct binding measurements demonstrate that Hsp90 selectively associates with CaM(ox); essentially no binding is observed between Hsp90 and unoxidized CaM. These results indicate that Hsp90 in association with the 20 S proteasome can selectively associate with oxidized and partially unfolded CaM to promote degradation by the proteasome.
Highlights
Summary—We have demonstrated an important role for Hsp[90], in association with the 20 S proteasome, in mediating the selective degradation of an oxidized signaling protein (i.e. CaM)
Significant degradation of CaMox by the 20 S proteasome occurs only in the presence of Hsp[90] (Fig. 3), indicating that the commonly observed co-purification of 20 S proteasome with Hsp[90] from tissue is likely to be a reflection of their close association in vivo
The maximal rates of CaMox degradation by the 20 S proteasome are observed upon the addition of a 4-fold molar excess of Hsp[90] (Fig. 4), consistent with a high affinity association between two Hsp[90] dimers and the proteasome
Summary
Materials—ATP, AMPPNP, bovine brain Hsp90␣/, carbobenzoxyLeu-Leu-Glu--naphthylamine (Cbz-LLE-Na), fluorescamine, geldanamycin, MES, MG132, MOPS, novobiocin, N-succinyl-Leu-Leu-Val-Tyr7-amido-4-methylcoumarin (Suc-AAF-Amc), N-t-Boc-Gly-Lys-Arg-7 amido-4-methylcoumarin (t-Boc-GKR-Amc), PIPES, and Tris were purchased from Sigma. CaM Proteolysis by the 20 S Proteasome—The rates of proteolysis using oxidized CaM as a substrate for the proteasome were determined using two different assays, essentially as described previously (13, 50) These assays involved (i) monitoring the disappearance of the integrated intensity of CaM bands on SDS-polyacrylamide gels and (ii) measurement of the initial release of peptides generated by proteasome cleavage using fluorescamine, which forms a fluorescent adduct with the amino termini of peptides (13, 53). Identical experimental conditions were used for both assays, which involved the incubation of native or oxidized CaM (12 M) with the 20 S proteasome (0.6 M) in the absence and presence of Hsp[90] (2.5 M) at 37 °C in 0.1 M KCl, 10 mM MgCl2, 0.1 mM EGTA, and 50 mM of one of the following buffers: MES (pH 6.0), PIPES (pH 6.5), MOPS (pH 7.0), or Tris (pH 7.5). Evaluation of the curves was carried out by Origin’s nonlinear fitting function (MicroCal software, Northampton, MA)
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