title: 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.
authors.affiliation: Department of Animal Sciences, Rutgers University, New Brunswick, New Jersey 08903-0231, USA.
authors.forename: P R
authors.initials: PR
authors.lastname: Strack
authors.forename: L
authors.initials: L
authors.lastname: Waxman
authors.forename: J M
authors.initials: JM
authors.lastname: Fagan
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002645:Chickens; D002729:Chloromercuribenzoates / Q000494:pharmacology; D003374:Coumarins / Q000494:pharmacology; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004912:Erythrocytes / Q000201:enzymology; D006861:Hydrogen Peroxide / Q000494:pharmacology; D006868:Hydrolysis; D007501:Iron / Q000494:pharmacology; D049934:Isocoumarins; D008297:Male; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D016877:Oxidants / Q000494:pharmacology*; D010455:Peptides / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex; D011487:Protein Conformation / Q000187:drug effects; D011506:Proteins / Q000302:isolation & purification / Q000494:pharmacology; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D020245:p-Chloromercuribenzoic Acid
chemical_list: D002729:Chloromercuribenzoates; D003374:Coumarins; D004791:Enzyme Inhibitors; C045076:Fenton's reagent; D049934:Isocoumarins; D009097:Multienzyme Complexes; D016877:Oxidants; D010455:Peptides; D011506:Proteins; D015842:Serine Proteinase Inhibitors; C046235:3,4-dichloroisocoumarin; D020245:p-Chloromercuribenzoic Acid; D006861:Hydrogen Peroxide; D007501:Iron; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex