Oxidative modification and inactivation of the proteasome during coronary occlusion/reperfusion.
Restoration of blood flow to ischemic myocardial tissue results in an increase in the production of oxygen radicals. Highly reactive, free radical species have the potential to damage cellular components. Clearly, maintenance of cellular viability is dependent, in part, on the removal of altered protein. The proteasome is a major intracellular proteolytic system which degrades oxidized and ubiquitinated forms of protein. Utilizing an in vivo rat model, we demonstrate that coronary occlusion/reperfusion resulted in declines in chymotrypsin-like, peptidylglutamyl-peptide hydrolase, and trypsin-like 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 conclusive evidence that, upon coronary occlusion/reperfusion, the lipid peroxidation product 4-hydroxy-2-nonenal selectively modifies 20 S proteasome alpha-like subunits iota, C3, and an isoform of XAPC7. Occlusion/reperfusion-induced 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 evident upon purification. Thus, decreases in proteasome activity are likely due to both direct oxidative modification of the enzyme and inhibition of fluorogenic peptide hydrolysis by endogenous cytosolic inhibitory protein(s) and/or substrate(s). Along with inhibition of the proteasome, increases in cytosolic levels of oxidized and ubiquitinated protein(s) were observed. Taken together, our findings provide insight into potential mechanisms of coronary occlusion/reperfusion-induced proteasome inactivation and cellular consequences of these events.
Bulteau
A L
AL
Departments of Biochemistry and Biology, Université Denis Diderot-Paris 7, 75251 Paris Cedex 05, France.
Lundberg
K C
KC
Humphries
K M
KM
Sadek
H A
HA
Szweda
P A
PA
Friguet
B
B
Szweda
L I
LI
D000447:Aldehydes / Q000494:pharmacology; D000818:Animals; D015153:Blotting, Western; D003546:Cysteine Endopeptidases / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000737:chemistry; D005609:Free Radicals / Q000378:metabolism; D015227:Lipid Peroxidation; D008297:Male; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors* / Q000378:metabolism; D015425:Myocardial Reperfusion; D010100:Oxygen / Q000378:metabolism*; D010447:Peptide Hydrolases / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex; D020033:Protein Isoforms; D051381:Rats; D017207:Rats, Sprague-Dawley; D014357:Trypsin / Q000494:pharmacology; D014452:Ubiquitins / Q000378:metabolism
D000447:Aldehydes; D005609:Free Radicals; D009097:Multienzyme Complexes; D020033:Protein Isoforms; D014452:Ubiquitins; D010450:Endopeptidases; D010447:Peptide Hydrolases; D014357:Trypsin; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; C114177:peptidylglutamylpeptide hydrolase; C027576:4-hydroxy-2-nonenal; D010100:Oxygen