Abstract
Oligopeptide repeats appear in many proteins that undergo conformational conversions to form amyloid, including the mammalian prion protein PrP and the yeast prion protein Sup35. Whereas the repeats in PrP have been studied more exhaustively, interpretation of these studies is confounded by the fact that many details of the PrP prion conformational conversion are not well understood. On the other hand, there is now a relatively good understanding of the factors that guide the conformational conversion of the Sup35 prion protein. To provide a general model for studying the role of oligopeptide repeats in prion conformational conversion and amyloid formation, we have substituted various numbers of the PrP octarepeats for the endogenous Sup35 repeats. The resulting chimeric proteins can adopt the [PSI+] prion state in yeast, and the stability of the prion state depends on the number of repeats. In vitro, these chimeric proteins form amyloid fibers, with more repeats leading to shorter lag phases and faster assembly rates. Both pH and the presence of metal ions modulate assembly kinetics of the chimeric proteins, and the extent of modulation is highly sensitive to the number of PrP repeats. This work offers new insight into the properties of the PrP octarepeats in amyloid assembly and prion formation. It also reveals new features of the yeast prion protein, and provides a level of control over yeast prion assembly that will be useful for future structural studies and for creating amyloid-based biomaterials.
Highlights
Scrapie in sheep, bovine spongiform encephalopathy in cattle, and Creutzfeldt-Jakob disease (CJD) in humans [1]
To provide a new model for studying prion conformational conversion and to better understand the role of the oligopeptide repeats in amyloid formation, we explored the role of the PrP octarepeats in the context of the yeast prion protein Sup35
Chimeric Sup35 Proteins with PrP Repeats Can Support Both the Prion and Non-prion States—We first constructed chimeric proteins consisting of Sup35 with different numbers of PrP repeats substituted for the repeats of Sup35
Summary
PrPC, cellular prion protein; PrPSc, pathological isoform of cellular prion protein; EPR, electron paramagnetic resonance; YPD, yeast peptone dextrose medium; AFM, atomic force microscopy; ThT, Thioflavin T; GST, glutathione S-transferase; MOPS, 4-morpholinepropanesulfonic acid; AFM, atomic force microscopy; ThT, thioflavin-T; SOD, superoxide dismutase. Oligopeptide repeats are clearly a crucial feature of these amyloid-forming proteins, the exact structural and functional role of these repeats remains unclear Compared with these other oligopeptide repeats, the biophysical properties of the PrP octarepeats are well characterized. To provide a new model for studying prion conformational conversion and to better understand the role of the oligopeptide repeats in amyloid formation, we explored the role of the PrP octarepeats in the context of the yeast prion protein Sup. Facilitated by the powerful genetic and biophysical techniques developed for yeast prions, we were able to characterize how the PrP octarepeats influence the conformational conversion and amyloid formation of these chimeric prion proteins both in vivo and in vitro. This control will be useful for further functional and structural work and could provide a practical means of controlling assembly for biomaterial and biotechnology applications
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