Amino acid, peptide, and protein hydroperoxides and their decomposition products modify the activity of the 26S proteasome

TY - JOUR

T1 - Amino acid, peptide, and protein hydroperoxides and their decomposition products modify the activity of the 26S proteasome

AU - Gracanin, Michelle

AU - Lam, Magdalena A

AU - Morgan, Philip E

AU - Rodgers, Kenneth J

AU - Hawkins, Clare Louise

AU - Davies, Michael Jonathan

N1 - Copyright © 2010 Elsevier Inc. All rights reserved.

PY - 2011/1/15

Y1 - 2011/1/15

N2 - 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.

AB - 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.

KW - Amino Acids

KW - Animals

KW - Cells, Cultured

KW - Electron Spin Resonance Spectroscopy

KW - Humans

KW - Hydrogen Peroxide

KW - Macrophages

KW - Mice

KW - Oxidants

KW - Oxidation-Reduction

KW - Peptide Fragments

KW - Proteasome Endopeptidase Complex

KW - Proteins

U2 - 10.1016/j.freeradbiomed.2010.11.019

DO - 10.1016/j.freeradbiomed.2010.11.019

M3 - Journal article

C2 - 21111806

SN - 0891-5849

VL - 50

SP - 389

EP - 399

JO - Free Radical Biology & Medicine

JF - Free Radical Biology & Medicine

IS - 2

ER -