3-nitrotyrosine and 1-anilino-8-naphthalenesulfonate

Peroxynitrite is a powerful nitrating and oxidizing molecule and capable of modifying proteins' structure. Hyper-nitration of tyrosine residues has been seen in various pathological states, including autoimmune disorders like systemic lupus erythematosus (SLE) and rheumatoid arthritis. SLE, a chronic autoimmune disease, is primarily characterized by increased levels of autoantibodies, predominantly against ds-DNA. However, the initial antigenic stimulus for the disease etiopathogenesis has remained elusive. Carbonyl and nitrotyrosine have been extensively used as a biomarker of oxidative and nitrosative stress. In this study, commercially available H1 histone was exposed to increasing concentrations of peroxynitrite for 30 min. The peroxynitrite-mediated structural changes in histone were studied by ultraviolet & fluorescence spectroscopy, CD, HPLC, 1-anilinonaphthalene-8-sulfonic acid binding and polyacrylamide gel electrophoresis. Analysis of results revealed that carbonyl and nitrotyrosine contents were significantly increased in peroxynitrite-modified H1 compared to native H1. In experimental animal, peroxynitrite-modified H1 induced high titre antibodies as compared to native H1, and the immunogenicity was found to be directly proportional to nitrotyrosine content. Further, the induced antibodies showed specificity for the immunogen and appreciable cross-reactions with tyrosine rich nitrated proteins. Formation of high molecular weight immune complex with retarded mobility further supports the specificity of induced anti-peroxynitrite-modified H1 antibodies for the immunogen. Binding of SLE anti-DNA autoantibodies with peroxynitrite-modified H1 was analyzed by direct binding and competition ELISA. The data show preferential binding of SLE autoantibodies to peroxynitrite-modified H1 as compared to native H1 histone and native DNA. The results point towards the possible role of peroxynitrite-modified H1 histone in SLE etiopathogenesis.

Topics: Anilino Naphthalenesulfonates; Animals; Antibodies, Antinuclear; Antigen-Antibody Complex; Autoantibodies; Cross Reactions; Electrophoretic Mobility Shift Assay; Enzyme-Linked Immunosorbent Assay; Female; Fluorescent Dyes; Histones; Humans; Immunization; Immunoglobulin G; Lupus Erythematosus, Systemic; Peroxynitrous Acid; Rabbits; Tyrosine

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.

Topics: Anilino Naphthalenesulfonates; Animals; Blotting, Western; Brain; Caseins; Cattle; Cysteine Endopeptidases; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Free Radicals; Kinetics; Multienzyme Complexes; Oxygen; Peptides; Peroxynitrous Acid; Proteasome Endopeptidase Complex; Protein Binding; Protein Conformation; Spectrometry, Fluorescence; Thymus Gland; Tryptophan; Tyrosine