title: Peroxynitrite-induced nitrative and oxidative modifications alter tau filament formation.
abstract: Tau undergoes numerous posttranslational modifications during the progression of Alzheimer's disease (AD). Some of these changes accelerate tau aggregation, while others are inhibitory. AD-associated inflammation is thought to create oxygen and nitrogen radicals such as peroxynitrite (PN). In vitro, PN can nitrate many proteins, including tau. We have previously demonstrated that tau's ability to form filaments is profoundly affected by treatment with PN and have attributed this inhibition to tyrosine nitration. However, PN is highly reactive and unstable leading to oxidative amino acid modifications through its free radical byproducts. To test whether PN can modify other amino acids in tau via oxidative modifications, a mutant form of the tau protein lacking all tyrosines (5XY → F) was constructed. 5XY → F tau readily forms filaments; however, like wild-type tau the extent of polymerization was greatly reduced following PN treatment. Since 5XY → F tau cannot be nitrated, it was clear that nonnitrative modifications are generated by PN treatment and that these modifications change tau filament formation. Mass spectrometry was used to identify these oxidative alterations in wild-type tau and 5XY → F tau. PN-treated wild-type tau and 5XY → F tau consistently displayed lysine formylation throughout tau in a nonsequence-specific distribution. Lysine formylation likely results from reactive free radical exposure caused by PN treatment. Therefore, our results indicate that PN treatment of proteins in vitro cannot be used to study protein nitration as it likely induces numerous other random oxidative modifications clouding the interpretations of any functional consequences of tyrosine nitration.
authors.affiliation: Department of Cell and Molecular Biology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611, United States. laurelvana2010@u.northwestern.edu
authors.forename: Laurel
authors.initials: L
authors.lastname: Vana
authors.forename: Nicholas M
authors.initials: NM
authors.lastname: Kanaan
authors.forename: Kevin
authors.initials: K
authors.lastname: Hakala
authors.forename: Susan T
authors.initials: ST
authors.lastname: Weintraub
authors.forename: Lester I
authors.initials: LI
authors.lastname: Binder
mesh_terms: D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D009566:Nitrates / Q000378:metabolism; D010084:Oxidation-Reduction; D018384:Oxidative Stress / Q000187:drug effects; D030421:Peroxynitrous Acid / Q000494:pharmacology*; D055503:Protein Multimerization / Q000187:drug effects*; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D026361:Reactive Nitrogen Species / Q000378:metabolism / Q000494:pharmacology*; D017382:Reactive Oxygen Species / Q000378:metabolism / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016875:tau Proteins / Q000378:metabolism*
chemical_list: D050505:Mutant Proteins; D009566:Nitrates; D026361:Reactive Nitrogen Species; D017382:Reactive Oxygen Species; D011994:Recombinant Proteins; D016875:tau Proteins; D030421:Peroxynitrous Acid