Oxidative Modification and Inactivation of the Proteasome during Coronary Occlusion/Reperfusion

Abstract

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 ␣-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.Restoration of coronary blood flow to previously ischemic cardiac tissue is often associated with declines in cardiac function, including myocardial stunning, ventricular arrhythmias, hemodynamic abnormalities, and, in the long term, development of heart failure (1, 2). This paradoxical phenomenon, broadly termed ischemia/reperfusion injury, is accompanied by dramatic increases in tissue levels of free radicals as well as byproducts of lipid peroxidation (1-9). Due to the high reactivity of these species (10 -12), it has been proposed that free radical events play a key role in myocardial ischemia/reperfusion injury. Nevertheless, mechanisms by which free radicals alter cardiac function have not been fully elucidated. Free radical modification of protein can alter and, in many cases, inhibit, protein activity (10 -15). In addition, oxidatively crosslinked protein(s) are resistant to proteolytic degradation (16 -20). Thus, the presence of oxidized protein could impact cellular function by changing the catalytic and/or regulatory properties of specific proteins and by placing abnormal demands on finite cellular volume. Clearly, the level of oxidatively modified protein reflects the balance between free radical damage and p...

Redox regulation of the proteasome in T lymphocytes during aging

“…Oxidative stress-induced modifications in proteins have been documented in different cell types [21,22,38]. Consistent with this observation, our results demonstrate that increased protein carbonylation seen in T lymphocytes from elderly donors could be mimicked in T cells from young donors following exposure to pro-oxidant.…”

“…Consistent with this observation, our results demonstrate that increased protein carbonylation seen in T lymphocytes from elderly donors could be mimicked in T cells from young donors following exposure to pro-oxidant. As the proteasome is largely implicated in the removal of oxidized proteins [18][19][20][21][22]39], accumulation of carbonylated proteins during aging and following pro-oxidant exposure suggests that proteasome may itself be a target of oxidative stress. Indeed, cross-linked proteins formed by prolonged oxidation and/or HNE modifications have previously been shown to inhibit the proteasome [16].…”

“…Increased accumulation of highly oxidized and cross-linked protein aggregates within the cell observed during aging has been attributed to decreased proteasome function [18,19]. Indeed, both 20S and 26S proteasomes show age-related decline in their enzymatic activities in most cell types ranging from T lymphocytes to neurons [4,10,[20][21][22]. Studies by Chondrogianni et al revealed that, while the inhibition of proteasome induced a senescent phenotype, stable expression of β 1 or β 5 catalytic subunits in WI38 immortalized fibroblasts and HL60 leukemic cell line results in increased proteasomal activities and cell survival following treatment with oxidants [23].…”

Age-dependent inhibition of proteasome chymotrypsin-like activity in the retina

“…Second, the three catalytic sites can be differentially affected in aged tissues, suggesting different mechanisms of inhibition. Exclusive inhibition of the chymotrypsin-like (Bulteau et al, 2000;Keller et al, 2000;Louie et al, 2002;Ponnapan et al, 1999), caspase-like (Anselmi et al, 1998;Conconi and Friguet, 1997) and trypsin-like (Hayashi and Goto, 1998;Radak et al, 2002) activities has been reported. Taken together, the tissuespecific differences in how aging affects proteasome function suggests exposure to environmental stressors (i.e., light, free radicals), protective mechanisms (i.e., heat shock proteins, antioxidants), or differences in proteasome subtypes (i.e., constitutive versus immunoproteasome) that are unique to each cell type can influence the extent and specificity of the effect.…”

Oxidized protein degradation and repair in ageing and oxidative stress

“…Moreover, treatment of kidney with ferryl nitriloacetate and brain ischemia-reperfusion were associated with decreased proteasome function and modification of the proteasome by the lipid peroxidation product 4-hydroxy-2-nonenal [56,57]. Specific modification of three proteasome subunits by 4-hydroxy-2-nonenal was also associated with inactivation of the trypsin-like activity upon cardiac ischemia-reperfusion [58]. In addition, we have recently found that in vitro treatment of proteasome purified from rat heart results in the preferential inactivation of the trypsin-like activity and the modification of restricted set of proteasome subunits that include those previously reported to be targeted upon ischemia-reperfusion, hence making 4-hydroxy-2-nonenal modification of proteasome as a likely mechanism for proteasome inactivation in vivo [59].…”