OXIDIZED AND PHOSPHORYLATED SYNTHETIC PEPTIDES CORRESPONDING TO THE 2ND-TUBULIN-BINDING AND 3RD-TUBULIN-BINDING REPEATS OF THE TAU-PROTEIN REVEAL STRUCTURAL FEATURES OF PAIRED HELICAL FILAMENT ASSEMBLY

The microtubule-associated protein tau 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. tau is oligomerized in an abnormally hyperphosphorylated form (PHF-tau). tau contains two cysteine residues in repeat units 2 and 3, but only the R3-R3 homodime r is present in PHF-tau. A serine residue two amino acids downstream o f the R3 cysteine is a major phosphate acceptor site for protein kinas e C. In the work reported here, we used synthetic peptides correspondi ng to R2, R3 and phosphorylated R3 to determine the binding of the tau repeat peptides to a peptide fragment corresponding to the C-terminal domain of beta-tubulin and to study the kinetics of home-and heterodi mer formation. Additionally, we studied two major biochemical properti es of the peptides that distinguish between normal tau and PHF-tau: co nformation and metabolic stability, All R2 and R3 peptides bound speci fically to the tubulin peptide regardless of the state of phosphorylat ion or dimerization. The reverse-turn conformation of the tau repeat p eptides in the presence of the tubulin peptide remained unaffected. Ph osphorylation slightly loosened the turn structure of the monomeric an d dimeric peptides. and did not univocally affect the serum stability of the peptides or the ability of the peptides to form dimers, The iso lated 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 syn thetic peptide fragments of tau indicate that neither oxidation nor ph osphorylation of the repeat units is able to generate extended structu re such as that found in PHF-tau. Additionally, phosphorylation of Ser 324 does not appear to modulate the kinetics of oligomerization of tau , 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-asso ciation of tau, is not preferred, If the dimers are formed, however, t heir clearance is considerably slower than that of the monomers, expla ining the remarkable protease resistance of PHF-tau in the affected br ains. (C) Munkspaard 1997.