Activation of the Multicatalytic Endopeptidase by Oxidants. Effects on Enzyme Structure

Abstract

It is well established that the functional properties of proteins can be compromised by oxidative damage and, in vivo, proteins modified by oxidants are rapidly degraded. It was hypothesized that oxidants may also affect the ability of proteases to hydrolyze peptides and proteins. We therefore examined the effect of oxidants on the endopeptidase activities of the 650 kDa 20S proteasome or multicatalytic endopeptidase (MCP), which is thought to play a central role in nonlysosomal protein breakdown. Treatment of the MCP with the oxidant system, FeSO4-EDTA-ascorbate, stimulated the peptidase activities of the MCP while H2O2 treatment showed little or no stimulation. However, treatment of the MCP with FeSO4-EDTA-ascorbate or H2O2 stimulated proteinase activity by 480% and 730%, respectively. An endogenous activator of the MCP, PA28, stimulated the acidic, basic, and hydrophobic peptidase activities of the MCP, but had no effect on proteolytic activity. Treatment of PA28 with oxidants in the presence of MCP or alone did not greatly affect PA28's ability to activate the peptidase activities of the MCP. Using nondenaturing polyacrylamide gel electrophoresis, structural alterations in the enzyme which may be responsible for the activation of peptidase and protease activities following exposure to oxidants were investigated. Treatment of the MCP with reagents that activate proteolysis, including H2O2, as well as the serine protease inhibitor 3,4-dichloroisocoumarin and the cysteine protease inhibitor p-(chloromercuri) benzenesulfonic acid, all caused dissociation of the 650 kDa MCP. However, exposure to FeSO4-EDTA-ascorbate resulted in little or no dissociation of the complex. The MCP complex dissociated by p-(chloromercuri) benzenesulfonic acid could be reassociated upon treatment with the reducing agent dithiothreitol, but dithiothreitol failed to completely reassociate 3,4-dichloroisocoumarin- or H2O2 treated MCP. Therefore, chemical modification of the MCP can cause activation with varying degrees of complex dissociation. These results suggest that metabolites, such as reactive oxygen species, in addition to endogenous proteins, such as PA28, are capable of modulating MCP activity.

Changes in the Expression and the Enzymic Properties of the 20S Proteasome in Sugar-Starved Maize Roots. Evidence for an in Vivo Oxidation of the Proteasome

“…On the other hand, further oxidation of already oxidized proteasome (i.e. T24-starved proteasome) resulted in partial inactivation of the three peptidic activities (Table V), as observed for proteasomes treated with increasing H 2 O 2 concentrations (Strack et al, 1996;Conconi et al, 1998;Reinheckel et al, 1998), and in a strong increase in carbonyl residues (Fig. 6).…”

“…Conflicting results have been reported about the chymotrypsin-like activity of 20S proteasome, which was found to be either activated (Strack et al, 1996) or not (Reinheckel et al, 1998) by in vitro oxidative treatment. It has been hypothesized that the activation by H 2 O 2 found by Strack et al (1996) was mediated via an interaction of the proteasome and the PA28 activator (Reinheckel et al, 1998). However, without excluding a possible interaction between the 20S proteasome and the PA28 activator in vivo, our data show that the purified proteasome may be activated by a mild oxidation treatment only, i.e.…”

“…As a consequence of the potential role of the 20S proteasome in the degradation of oxidized proteins, the effects of oxidative treatments on 20S proteasome structure and activities have also been investigated in vitro (Strack et al, 1996;Conconi et al, 1998;Reinheckel et al, 1998). However, whereas the 20S proteasome from chicken (Gallus gallus) erythrocytes has been report to be activated by mild oxidative treatment with either hydrogen peroxide (H 2 O 2 ) or FeSO 4 -EDTA-ascorbate (Strack et al, 1996), no change in activity was observed with the 20S proteasome from human erythrocyte (Reinheckel et al, 1998). Moreover, whether the proteasome is modified in vivo by oxidative conditions is not known.…”

A Model to Study Antioxidant Regulation of Endotoxemia-Modulated Neonatal Granulopoiesis and Granulocyte Apoptosis

“…H 2 O 2 has also been implicated in the induction of NF⑂B translocation by oxidized LDL in monocytes (11,12). In earlier work, we demonstrated that hydroxyl radicals (OH⅐) but not H 2 O 2 or superoxide is involved in phorbol ester-induced monocytic differentiation of HL-60 cells (13).…”

Interplay of Cu,Zn Superoxide Dismutase and Nitric Oxide Synthase in Neurodegenerative Processes

“…Proteasome activity is under the regulation of several factors and conditions among which oxidative stress plays a dual role: sustained oxidative insult is reported to inactivate proteasome by direct oxidation or by the formation of protein cross-linking and aggregates (34); but on the other hand exposure to mild oxidative injury has been demonstrated to stimulate proteasome activity, presumably through favoring conformation changes in the 20S proteasome complex (35,36). Accordingly, in our FALS model, where a mild oxidative stress is operative, an activation of proteasome was found in response to G93A Cu,Zn SOD expression in order to counteract ROS toxicity via degradation of oxidized proteins.…”