Oxidative modification and inactivation of the proteasome during coronary occlusion/reperfusion

Restoration of blood flow to ischemic myocardial tissue results in an incre ase in the production of oxygen radicals. Highly reactive, free radical spe cies have the potential to damage cellular components. Clearly, maintenance of cellular viability is dependent, in part, on the removal of altered pro tein. The proteasome is a major intracellular proteolytic system which degr ades oxidized and ubiquitinated forms of protein. Utilizing an in vivo rat model, we demonstrate that coronary occlusion/reperfusion resulted in decli nes in chymotrypsin-like, peptidylglutamyl-peptide hydrolase, and trypsin-l ike activities of the proteasome as assayed in cytosolic extracts. Analysis of purified 20 S proteasome revealed that declines in peptidase activities were accompanied by oxidative modification of the protein. We provide conc lusive evidence that, upon coronary occlusion/reperfusion, the lipid peroxi dation product 4-hydroxy-2-nonenal selectively modifies 20 S proteasome a-l ike subunits iota, C3, and an isoform of XAPC7. Occlusion/reperfusion-induc ed declines in trypsin-like activity were largely preserved upon proteasome purification. In contrast, loss in chymotrypsin-like and peptidylglutamyl- peptide hydrolase activities observed in cytosolic extracts were not eviden t upon purification. Thus, decreases in proteasome activity are likely due to both direct oxidative modification of the enzyme and inhibition of fluor ogenic peptide hydrolysis by endogenous cytosolic inhibitory protein(s) and /or substrate(s). Along with inhibition of the proteasome, increases in cyt osolic levels of oxidized and ubiquitinated protein(s) were observed. Taken together, our findings provide insight into potential mechanisms of corona ry occlusion/reperfusion-induced proteasome inactivation and cellular conse quences of these events.