Abstract
The conversion of globular proteins into amyloid fibrils is associated with a wide variety of human diseases. One example is the prion protein (PrP), which adopts an α-helical structure in the native state but its amyloid form is implicated in the pathogenesis of prion diseases. Previous evidence has suggested that destabilization of the native state promotes amyloid formation, but the underlying mechanism remains unknown. In this study, we report that the native state of PrP serves as a potent inhibitor in the formation of PrP amyloid fibrils. By monitoring the time courses of thioflavin T fluorescence, the kinetics of amyloid formation was studied in vitro under various concentrations of pre-formed amyloid, monomer, and denaturant. Quantitative analysis of the kinetic data using various models of enzyme kinetics suggested that the native state of PrP is either an uncompetitive or noncompetitive inhibitor of amyloid formation. This study highlights the significant role of the native state in inhibiting amyloid formation, which provides new insights into the pathogenesis of misfolding diseases.
Highlights
We first hypothesized that rollover stems from a decreased population of a non-native state under the stabilizing conditions that can be directly converted into amyloid fibrils[26]. It remains controversial whether a partially or a completely unfolded state is involved[27,28,29,30,31,32], we considered the completely unfolded state (“U”) as the direct precursor of amyloid fibrils in this particular case
To extend the one-step model (Fig. 2D) to cover the saturation kinetics, we considered a reaction that involves a transient complex consisting of U state and amyloid fibrils (UMi) (Fig. 3B)
After testing several alternative models, we developed two models that include the inihibitory effect of N state and reproduce the “up-and-down” behavior of amyloid growth (Fig. 5)
Summary
A systematic investigation of amyloid formation in vitro. we will briefly outline our experimental strategy for investigating amyloid formation by PrP. We performed a seeded growth experiment in the lower concentration range of GuHCl (1.3–2.0 M), where both the N and U states are populated (Fig. 2C), by varying the total concentration of the monomers Under these conditions the reaction rate first rose to a maximum value and declined as the total monomer concentration increased (Fig. 4, Figures S5 and S6). This is not the case in amyloid growth because the decline of reaction rate is less pronounced in destabilizing conditions which favor the formation of the substrate (U state), such as in V210A (bottom panels in Fig. 4) and at the high concentration range of GuHCl (Fig. 3A) This result indicates that the inhibitor of amyloid growth is not the U state but a more structured state which can be populated under stabilizing conditions. Solving the equilibrium and rate equations of these schemes gives the initial rates of the reaction
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