Peroxynitrite-induced oxidation and its effects on isolated proteasomal systems.
The proteasomes are the major intracellular proteolytic systems involved in the removal of altered proteins. In this study, we examined different susceptibilities of constitutive (XYZ) and interferon-gamma inducible (LMP) 20S proteasomes, isolated from bovine brain and thymus, respectively, to peroxynitrite-mediated oxidation. Exposure of XYZ and LMP proteasomes to increasing amounts of peroxynitrite resulted in different levels, in the two enzymes, of 3-nitrotyrosine groups and tryptophan residues oxidation. 1-Anilino-8-naphtalene-sulfonic acid binding studies and quenching of tryptophan residues indicated that the LMP complex was more sensitive to peroxynitrite. Regarding the proteolytic activities, the XYZ proteasome showed an overall activation (even if the trypsin-like (T-L) component was 20% inhibited), with the peptidyl-glutamyl peptide-hydrolyzing (PGPH) and branched-chain amino acid-preferring (BrAAP) activities being the most stimulated. On the other end, the LMP proteasome was inhibited, especially the BrAAP activity, whereas the T-L activity was not affected. Furthermore, exposure to increasing amounts of peroxynitrite induced a gradual decrease of beta-casein degrading rate by the LMP proteasome, whereas it did not influence the constitutive complex. Our results indicated that peroxynitrite caused a mild modification of the XYZ complex, leading to activation of its catalytic activities. Differently, the LMP proteasome showed a more significant conformational change resulting in the inhibition of the proteolytic functions.
Amici
Manila
M
Department of Molecular, Cellular and Animal Biology, Postgraduate School in Clinical Biochemistry, University of Camerino, Camerino, Italy.
Lupidi
Giulio
G
Angeletti
Mauro
M
Fioretti
Evandro
E
Eleuteri
Anna Maria
AM
D000817:Anilino Naphthalenesulfonates / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D001921:Brain / Q000378:metabolism*; D002364:Caseins / Q000378:metabolism; D002417:Cattle; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D005609:Free Radicals; D007700:Kinetics; D009097:Multienzyme Complexes / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry; D030421:Peroxynitrous Acid / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D046988:Proteasome Endopeptidase Complex; D011485:Protein Binding; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013950:Thymus Gland / Q000378:metabolism*; D014364:Tryptophan / Q000378:metabolism; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000378:metabolism
D000817:Anilino Naphthalenesulfonates; D002364:Caseins; D005609:Free Radicals; D009097:Multienzyme Complexes; D010455:Peptides; D030421:Peroxynitrous Acid; C002744:3-nitrotyrosine; D014443:Tyrosine; C027132:1-anilino-8-naphthalenesulfonate; D014364:Tryptophan; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D010100:Oxygen