Oxidized and phosphorylated synthetic peptides corresponding to the second and third tubulin‐binding repeats of the τ protein reveal structural features of paired helical filament assembly

Publisher Website
Google Scholar
Abstract
The microtubule-associated protein τ of normal brains is attached to tubulin through its 18-amino-acid repeat units. In the paired helical filaments (PHF) of Alzheimer's disease, however, τ is oligomerized in an abnormally hyperphosphorylated form (PHF-τ). τ contains two cysteine residues in repeat units 2 and 3, but only the R3-R3 homodimer is present in PHF-τ. A serine residue two amino acids downstream of the R3 cysteine is a major phosphate acceptor site for protein kinase C. In the work reported here, we used synthetic peptides corresponding to R2, R3 and phosphorylated R3 to determine the binding of the τ repeat peptides to a peptide fragment corresponding to the C-terminal domain of ß-tubulin and to study the kinetics of homo- and heterodimer formation. Additionally, we studied two major biochemical properties of the peptides that distinguish between normal τ and PHF-τ: conformation and metabolic stability. All R2 and R3 peptides bound specifically to the tubulin peptide regardless of the state of phosphorylation or dimerization. The reverse-turn conformation of the τ repeat peptides in the presence of the tubulin peptide remained unaffected. Phosphorylation slightly loosened the turn structure of the monomeric and dimeric peptides, and did not univocally affect the serum stability of the peptides or the ability of the peptides to form dimers. The isolated R2 and R3 units formed homodimers approximately in the same rate. When the two peptides were mixed, however, the R2-R3 heterodimer was formed preferentially over the homodimers. The dimers were generally more stable in human serum than the monomers. Our results with the synthetic peptide fragments of τ indicate that neither oxidation nor phosphorylation of the repeat units is able to generate extended structure such as that found in PHF-τ. Additionally, phosphorylation of Ser324 does not appear to modulate the kinetics of oligomerization of τ, and in general biochemistry terms, does not affect disulfide bridge formation nearby. In agreement with studies at the full-protein level, the formation of homodimers of the peptides, a model of the self-association of τ, is not preferred. If the dimers are formed, however, their clearance is considerably slower than that of the monomers, explaining the remarkable protease resistance of PHF-τ in the affected brains. © Munksgaard 1997.