PROTECTION FROM OXIDATIVE INACTIVATION OF THE 20 S PROTEASOME BY HEAT-SHOCK-PROTEIN-90

Heat-shock protein 90 (Hsp 90) has been implicated in both protection against oxidative inactivation and inhibition of the multicatalytic pr oteinase (MCP, also known as 20 S proteasome). We report here that the protective and inhibitory effects of Hsp 90 depend on the activation state of the proteasome. Hsp 90 (and also alpha-crystallin) inhibits t he N-Cbz-Leu-Leu-Leu-MCA-hydrolysing activity (Cbz = benzyloxycarbonyl ; MCA = 7-amido-4-methylcoumarin) when the rat liver MCP is in its lat ent form, but no inhibitory effects are observed when the MCP is in it s active form. Metal-catalysed oxidation of the active MCP inactivates the Ala-Ala-Phe-MCA-hydrolysing (chymotrypsin-like), N-Boc-Leu-Ser-Th r-Arg-MCA-hydrolysing (trypsin-like; Boc = t-butyloxycarbonyl), N-Cbz- Leu-Leu-Glu-beta-naphthylaminehydrolysing (peptidylglutamyl-peptide hy drolase) and N-Cbz-Leu-Leu-Leu-MCA-hydrolysing activities, whereas the se activities are actually increased when the MCP is in its latent for m. Hsp 90 protects against oxidative inactivation of the trypsin-like and N-Cbz-Leu-Leu-Leu-MCA-hydrolysing activities of the MCP active for m, and alpha-crystallin protects the trypsin-like activity. The specif icity of the Hsp 90-mediated protection was assessed by a quantitative analysis of the two-dimensional electrophoretic pattern of MCP subuni ts before and after oxidation of the MCP, in the presence or absence o f Hsp 90. Treatment of the FAO hepatoma cell line with iron and ascorb ate was found to inactivate the MCP. Hsp 90 overexpression obtained by challenging the cells with iron was associated with a decreased susce ptibility to oxidative inactivation of the MCP trypsin-like activity. Depletion of Hsp 90 by using antisense oligonucleotides resulted in an increased susceptibility to oxidative inactivation of the MCP trypsin -like activity, providing evidence for the physiological relevance of Hsp 90-mediated protection of the MCP.