The native state of prion protein (PrP) directly inhibits formation of PrP-amyloid fibrils in vitro

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.

Amyloid‐β Peptide Induces Prion Protein Amyloid Formation: Evidence for Its Widespread Amyloidogenic Effect

“…[9] To understand how Ab modulates the amyloidogenic propensity of PrP (enhancing or reducing it), the author examined amyloid formation by recombinant full-length human PrP (residues 23-230) in the presence of freshly disaggregated Ab peptide 1-42 (Ab42) using an in vitro conversion system previously described. [10] Under such experimental conditions, the time courses of ThTf luorescence exhibited as igmoidal transition both in the absence and presence of Ab42 (top panel in Figure 1A), consistent with at ypical nucleationdependent amyloid formation. This coaggregation experiment was performed in the presence of 3 m guanidine hydrochloride (GuHCl) at pH 6, in which the native structure of PrP was completely disrupted to facilitate amyloid formation.…”

“…[7] These findings raise the question as to whether Ab could act as ascaffold for the self-assembly of PrP molecules and modulate the amyloidogenic propensity of PrP.Although previous studies have established that PrP suppresses Ab aggregation in vitro, [8] thec onverse effect of Ab on PrPamyloid formation remains controversial. [10] To this end, ar eaction mixture containing al arge excess of PrP (25 mm) and as mall amount of Ab42 (0.1-2 mm)w as subjected to continuous shaking in a96-well plate reader,and PrP amyloid formation was monitored by measuring the change in thioflavin T( ThT) fluorescence.A ni ncrease in ThTf luorescence in the reaction mixture mainly reflects amyloid formation by PrP,b ecause 0.1-2 mm Ab42 gives rise to an egligible contribution to the total ThTf luorescence (less than 10 %; Supporting Information, Figure S1). [10] To this end, ar eaction mixture containing al arge excess of PrP (25 mm) and as mall amount of Ab42 (0.1-2 mm)w as subjected to continuous shaking in a96-well plate reader,and PrP amyloid formation was monitored by measuring the change in thioflavin T( ThT) fluorescence.A ni ncrease in ThTf luorescence in the reaction mixture mainly reflects amyloid formation by PrP,b ecause 0.1-2 mm Ab42 gives rise to an egligible contribution to the total ThTf luorescence (less than 10 %; Supporting Information, Figure S1).…”

Role of the Disulfide Bond in Prion Protein Amyloid Formation: A Thermodynamic and Kinetic Analysis

“…A 250-mM solution of protein was initially incubated under the reducing condition as described above, diluted to a final concentration of 10 or 25 mM in 5 mM HEPES buffer (pH 7.4) containing various concentrations of GuHCl with or without 1 mM TCEP, and incubated for 1 h at 37 C. The reaction mixture was then centrifuged at 62,900 Â g for 30 min to remove insoluble aggregates, and the protein concentration in the soluble fraction (i.e., supernatant) was determined by the UV absorbance analysis (ε 280 ¼ 16,640 cm À1 M À1 ). Soluble proteins with concentrations R1 mM in the supernatant were subsequently subjected to far-UV CD according to a previously published protocol (25).…”

Amyloid‐β Peptide Induces Prion Protein Amyloid Formation: Evidence for Its Widespread Amyloidogenic Effect

“…To understand how Aβ modulates the amyloidogenic propensity of PrP (enhancing or reducing it), the author examined amyloid formation by recombinant full‐length human PrP (residues 23–230) in the presence of freshly disaggregated Aβ peptide 1–42 (Aβ42) using an in vitro conversion system previously described . To this end, a reaction mixture containing a large excess of PrP (25 μ m ) and a small amount of Aβ42 (0.1–2 μ m ) was subjected to continuous shaking in a 96‐well plate reader, and PrP amyloid formation was monitored by measuring the change in thioflavin T (ThT) fluorescence.…”

“…An increase in ThT fluorescence in the reaction mixture mainly reflects amyloid formation by PrP, because 0.1–2 μ m Aβ42 gives rise to a negligible contribution to the total ThT fluorescence (less than 10 %; Supporting Information, Figure S1). This coaggregation experiment was performed in the presence of 3 m guanidine hydrochloride (GuHCl) at pH 6, in which the native structure of PrP was completely disrupted to facilitate amyloid formation . Under such experimental conditions, the time courses of ThT fluorescence exhibited a sigmoidal transition both in the absence and presence of Aβ42 (top panel in Figure A), consistent with a typical nucleation‐dependent amyloid formation.…”