Prion protein region 23-32 interacts with tubulin and inhibits microtubule assembly.
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.
Osiecka
Katarzyna M
KM
Department of Biochemistry, Nencki Institute of Experimental Biology, Warsaw, Poland.
Nieznanska
Hanna
H
Skowronek
Krzysztof J
KJ
Karolczak
Justyna
J
Schneider
Gabriela
G
Nieznanski
Krzysztof
K
D020816:Amino Acid Motifs / Q000502:physiology; D000595:Amino Acid Sequence; D001665:Binding Sites / Q000502:physiology; D002460:Cell Line; D003599:Cytoskeleton / Q000378:metabolism / Q000648:ultrastructure; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism; D006801:Humans; D046529:Microscopy, Electron, Transmission; D008870:Microtubules / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism; D011328:Prions / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D017384:Sequence Deletion; D014404:Tubulin / Q000378:metabolism* / Q000648:ultrastructure
D009842:Oligopeptides; D011328:Prions; D014404:Tubulin