Disulfide bond formation significantly accelerates the assembly of Ure2p fibrils because of the proximity of a potential amyloid stretch.
Aggregation of the Ure2 protein is at the origin of the [URE3] prion trait in the yeast Saccharomyces cerevisiae. The N-terminal region of Ure2p is necessary and sufficient to induce the [URE3] phenotype in vivo and to polymerize into amyloid-like fibrils in vitro. However, as the N-terminal region is poorly ordered in the native state, making it difficult to detect structural changes in this region by spectroscopic methods, detailed information about the fibril assembly process is therefore lacking. Short fibril-forming peptide regions (4-7 residues) have been identified in a number of prion and other amyloid-related proteins, but such short regions have not yet been identified in Ure2p. In this study, we identify a unique cysteine mutant (R17C) that can greatly accelerate the fibril assembly kinetics of Ure2p under oxidizing conditions. We found that the segment QVNI, corresponding to residues 18-21 in Ure2p, plays a critical role in the fast assembly properties of R17C, suggesting that this segment represents a potential amyloid-forming region. A series of peptides containing the QVNI segment were found to form fibrils in vitro. Furthermore, the peptide fibrils could seed fibril formation for wild-type Ure2p. Preceding the QVNI segment with a cysteine or a hydrophobic residue, instead of a charged residue, caused the rate of assembly into fibrils to increase greatly for both peptides and full-length Ure2p. Our results indicate that the potential amyloid stretch and its preceding residue can modulate the fibril assembly of Ure2p to control the initiation of prion formation.
Fei
Li
L
National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Chaoyang District, Beijing 100101, China.
Perrett
Sarah
S
D000682:Amyloid / Q000737:chemistry*; D001120:Arginine / Q000737:chemistry; D002942:Circular Dichroism; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides; D017353:Gene Deletion; D005979:Glutathione Peroxidase; D007700:Kinetics; D008854:Microscopy, Electron; D009154:Mutation; D016877:Oxidants / Q000494:pharmacology; D010455:Peptides / Q000737:chemistry; D011328:Prions / Q000737:chemistry / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*
D000682:Amyloid; D004220:Disulfides; D016877:Oxidants; D010455:Peptides; D011328:Prions; D029701:Saccharomyces cerevisiae Proteins; D001120:Arginine; D005979:Glutathione Peroxidase; C067020:URE2 protein, S cerevisiae; D003545:Cysteine