Amino acid, peptide, and protein hydroperoxides and their decomposition products modify the activity of the 26S proteasome.
Proteins are major biological targets for oxidative damage within cells because of their high abundance and rapid rates of reaction with radicals and singlet oxygen. These reactions generate high yields of hydroperoxides. The turnover of both native and modified/damaged proteins is critical for maintaining cell homeostasis, with this occurring via the proteasomal and endosomal-lysosomal systems; the former is of particular importance for intracellular proteins. In this study we have examined whether oxidation products generated on amino acids, peptides, and proteins modulate 26S proteasome activity. We show that oxidation products, and particularly protein hydroperoxides, are efficient inhibitors of the 26S proteasome tryptic and chymotryptic activities, with this depending, at least in part, on the presence of hydroperoxide groups. Removal of these species by reduction significantly reduces proteasome inhibition. This loss of activity is accompanied by a loss of thiol residues, but an absence of radical formation, consistent with molecular, rather than radical, reactions being responsible for proteasome inhibition. Aldehydes also seem to play a role in the inhibition of chymotryptic activity, with this prevented by treatment with NaBH(4), which reduces these groups. Inhibition occurred at hydroperoxide concentrations of ≥1μM for oxidized amino acids and peptides and ≥10μM for oxidized proteins, compared with ca. 100μM for H(2)O(2), indicating that H(2)O(2) is a much less effective inhibitor. These data indicate that the formation of oxidized proteins within cells may modulate cell function by interfering with the turnover of native proteins and the clearance of modified materials.
Free Radical Group, The Heart Research Institute, Newtown, Sydney, NSW 2042, Australia.
Michelle
M
Gracanin
Magdalena A
MA
Lam
Philip E
PE
Morgan
Kenneth J
KJ
Rodgers
Clare L
CL
Hawkins
Michael J
MJ
Davies
D000596:Amino Acids / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D002478:Cells, Cultured; D004578:Electron Spin Resonance Spectroscopy; D006801:Humans; D006861:Hydrogen Peroxide / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology*; D008264:Macrophages / Q000166:cytology / Q000378:metabolism; D051379:Mice; D016877:Oxidants / Q000494:pharmacology; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*
D000596:Amino Acids; D016877:Oxidants; D010446:Peptide Fragments; D011506:Proteins; D006861:Hydrogen Peroxide; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease