Peroxynitrite-mediated tau modifications stabilize preformed filaments and destabilize microtubules through distinct mechanisms.
Alzheimer's disease (AD) is a progressive amnestic dementia typified by abnormal modifications of the microtubule (MT)-associated tau protein that promote its pathological self-assembly and displacement from the MT lattice. Previously, we showed that peroxynitrite (ONOO-) induces the oxidative 3,3'-dityrosine (3,3'-DT) cross-linking and site-selective nitration of tau monomers [Reynolds et al. (2005) Biochemistry 44, 1690-1700]. In the present study, we examined the effects of ONOO(-)-mediated modifications on two key elements of tau pathobiology: (1) the stability of preformed tau filaments and (2) the ability of monomeric tau to promote tubulin assembly. Here, we report that treatment of synthetic tau filaments with ONOO- generates heat-stable, SDS-insoluble aggregates with a significantly reduced mobility by SDS-PAGE compared to that of nontreated filaments. Ultrastructurally, these aggregates appear to be cross-linked via interfilament bridges. Using LC-MS/MS and HPLC with fluorescent detection, we demonstrate that covalent 3,3'-DT linkages are present within these higher-order aggregates. Similar to monomeric tau, filamentous tau exhibits a hierarchical pattern of nitration following ONOO- treatment with site selectivity toward the amino-terminal residues Tyr18 and Tyr29. Further, select nitration of residues Tyr18, Tyr29, Tyr197, and Tyr394, events known to stabilize the pathological Alz-50 conformation [Reynolds et al. (2005) Biochemistry 44, 13997-14009], inhibits the ability of monomeric tau to promote tubulin assembly. This effect is specific for the 3-NT modification, as mutant tau proteins pseudophosphorylated at each Tyr residue are fully competent to stabilize MTs. Collectively, our results suggest that ONOO(-)-mediated modifications stabilize tau filaments via 3,3'-DT bonding and destabilize MTs by site-selective nitration of tau monomers. Moreover, assumption of the Alz-50 conformation may be the mechanism through which tau nitration modulates MT stability.
Department of Cell and Molecular Biology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611, USA. m-reynolds@md.northwestern.edu
Matthew R
MR
Reynolds
Thomas J
TJ
Lukas
Robert W
RW
Berry
Lester I
LI
Binder
D000595:Amino Acid Sequence; D008870:Microtubules / Q000187:drug effects* / Q000378:metabolism; D016874:Neurofibrillary Tangles / Q000187:drug effects*; D030421:Peroxynitrous Acid / Q000494:pharmacology*; D020836:Protein Structure, Quaternary / Q000187:drug effects; D014443:Tyrosine / Q000737:chemistry; D016875:tau Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D016875:tau Proteins; D030421:Peroxynitrous Acid; D014443:Tyrosine