title: Amyloid nucleation and hierarchical assembly of Ure2p fibrils. Role of asparagine/glutamine repeat and nonrepeat regions of the prion domains.
abstract: The yeast prion protein Ure2 forms amyloid-like filaments in vivo and in vitro. This ability depends on the N-terminal prion domain, which contains Asn/Gln repeats, a motif thought to cause human disease by forming stable protein aggregates. The Asn/Gln region of the Ure2p prion domain extends to residue 89, but residues 15-42 represent an island of "normal" random sequence, which is highly conserved in related species and is relatively hydrophobic. We compare the time course of structural changes monitored by thioflavin T (ThT) binding fluorescence and atomic force microscopy for Ure2 and a series of prion domain mutants under a range of conditions. Atomic force microscopy height images at successive time points during a single growth experiment showed the sequential appearance of at least four fibril types that could be readily differentiated by height (5, 8, 12, or 9 nm), morphology (twisted or smooth), and/or time of appearance (early or late in the plateau phase of ThT binding). The Ure2 dimer (h = 2.6 +/- 0.5 nm) and granular particles corresponding to higher order oligomers (h = 4-12 nm) could also be detected. The mutants 15Ure2 and Delta 15-42Ure2 showed the same time-dependent variation in fibril types but with an increased lag time detected by ThT binding compared with wild-type Ure2. In addition, Delta 15-42Ure2 showed reduced binding to ThT. The results imply a role of the conserved region in both amyloid nucleation and formation of the binding surface recognized by ThT. Further, Ure2 amyloid formation is a multistep process via a series of fibrillar intermediates.
authors: National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Chaoyang District, Beijing 100101, China.
authors: Yi
authors: Y
authors: Jiang
authors: Hui
authors: H
authors: Li
authors: Li
authors: L
authors: Zhu
authors: Jun-Mei
authors: JM
authors: Zhou
authors: Sarah
authors: S
authors: Perrett
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000682:Amyloid / Q000737:chemistry* / Q000378:metabolism; D001216:Asparagine / Q000737:chemistry; D052160:Benzothiazoles; D019281:Dimerization; D005973:Glutamine / Q000737:chemistry; D005979:Glutathione Peroxidase; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D018625:Microscopy, Atomic Force; D008969:Molecular Sequence Data; D009154:Mutation; D011328:Prions / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013844:Thiazoles / Q000737:chemistry; D013997:Time Factors
chemical_list: D000682:Amyloid; D052160:Benzothiazoles; D011328:Prions; D029701:Saccharomyces cerevisiae Proteins; D013844:Thiazoles; D005973:Glutamine; C009462:thioflavin T; D001216:Asparagine; D005979:Glutathione Peroxidase; C067020:URE2 protein, S cerevisiae