No abstract

In previous studies we have demonstrated that prion protein (PrP) binds directly to tubulin and this interaction leads to the inhibition of microtubule formation by inducement of tubulin oligomerization. This report is aimed at mapping the regions of PrP and tubulin involved in the interaction and identification of PrP domains responsible for tubulin oligomerization. Preliminary studies focused our attention to the N-terminal flexible part of PrP encompassing residues 23-110. Using a panel of deletion mutants of PrP, we identified two microtubule-binding motifs at both ends of this part of the molecule. We found that residues 23-32 constitute a major site of interaction, whereas residues 101-110 represent a weak binding site. The crucial role of the 23-32 sequence in the interaction with tubulin was confirmed employing chymotryptic fragments of PrP. Surprisingly, the octarepeat region linking the above motifs plays only a supporting role in the interaction. The binding of Cu(2+) to PrP did not affect the interaction. We also demonstrate that PrP deletion mutants lacking residues 23-32 exhibit very low efficiency in the inducement of tubulin oligomerization. Moreover, a synthetic peptide corresponding to this sequence, but not that identical with fragment 101-110, mimics the effects of the full-length protein on tubulin oligomerization and microtubule assembly. At the cellular level, peptide composed of the PrP motive 23-30 and signal sequence (1-22) disrupted the microtubular cytoskeleton. Using tryptic and chymotryptic fragments of alpha- and beta-tubulin, we mapped the docking sites for PrP within the C-terminal domains constituting the outer surface of microtubule.

In previous studies we have demonstrated that prion protein (PrP) interacts with tubulin and disrupts microtubular cytoskeleton by inducing tubulin oligomerization. These observations may explain the molecular mechanism of toxicity of cytoplasmic PrP in transmissible spongiform encephalopathies (TSEs). Here, we check whether microtubule associated proteins (MAPs) that regulate microtubule stability, influence the PrP-induced oligomerization of tubulin. We show that tubulin preparations depleted of MAPs are more prone to oligomerization by PrP than those containing traces of MAPs. Tau protein, a major neuronal member of the MAPs family, reduces the effect of PrP. Importantly, phosphorylation of Tau abolishes its ability to affect the PrP-induced oligomerization of tubulin. We propose that the binding of Tau stabilizes tubulin in a conformation less susceptible to oligomerization by PrP. Since elevated phosphorylation of Tau leading to a loss of its function is observed in Alzheimer disease and related tauopathies, our results point at a possible molecular link between these neurodegenerative disorders and TSEs.