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

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.

that ThT fluorescence is not directly proportional to the mass concentration of amyloid fibrils 48 (Groenning, 2010;Saar et al., 2016). It has been also suggested that amyloid fibrils formed under 49 different solution conditions exhibit differences in the intensity of ThT fluorescence. To test 50 these possibilities, we utilized SEC to follow amyloid growth in the absence of ThT. We 51 performed a seeded growth experiment in a ThT-free condition, in which 18 µM monomer were 52 co-incubated with 10% seed fibrils under various concentrations of GuHCl. At different times in 53 the reaction period, aliquots were taken from the reaction mixture and subsequently subjected to 54 SEC analysis. As shown in Figures S2A and S2B, the peak area of monomer decreased as the 55 reaction proceeded, indicating that the monomer was consumed and converted into amyloid 56 fibrils. Importantly, when the rate of monomer consumption was compared with that of increase 57 in ThT fluorescence, a good correlation was observed between the two variables ( Figure S2C).

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A high concentration of PrP potentially induces the formation of an off-pathway oligomer 69 (incapable of converting to fibrils) and subsequently produces an apparent decrease in the rate of 70 amyloid formation (Souillac et al., 2002). To test this possibility, we performed a SEC 71 experiment in which monomeric PrP at a high protein concentration (150 µM) was incubated 72 with or without 2.5% seed fibrils for 2 h and subsequently subjected to SEC analysis. The result 73 illustrated that the major fraction of PrP was eluted at the monomer position (8-10 mL), and the

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Although it is generally assumed that ThT binding occurs more rapidly than amyloid formation, 84 it remains unclear what occurs at a higher protein concentration where the amyloid formation 85 becomes faster. If ThT binding is the rate-limiting step in the observed fluorescence changes, 86 then we would underestimate the rate of amyloid formation. To test this possibility, we 87 performed a seeded growth experiment without ThT in the presence of a high and low 88 concentration of monomer (150 and 18 µM, respectively). After various reaction times, 20 µL 89 aliquots were withdrawn from the reaction mixture and directly added to a cuvette containing 90 180 µL of a ThT solution for the fluorescence measurement. The rationale behind this ex situ 91 assay is that this method can measure the rate of amyloid growth independently of the rate of 92 ThT binding. As shown in Figure S7, the ex situ assay yielded essentially the same growth rate 93 as those measured by in situ assays in all conditions examined ( Figure S9). We therefore 94 concluded that the ThT binding occurs more rapidly than amyloid growth, and it is not a rate-95 limiting step in the observed fluorescence changes.

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To determine the absolute values for the rate constants of amyloid growth, it is necessary to 99 convert the unit from RFU/% s −1 to s −1 . To this end, we first estimated the average 100 polymerization degree of seed fibrils (n) by assuming a cylindrical shape as follows; In this equation, w and l are the width and length of the seed fibril, respectively, v is the specific 103 volume of seed fibrils; NA is Avogadro's number; MW is the molecular weight of the monomer 104 (23 kDa). We estimated w and l as 13 and 122 nm, respectively, based on the electron 105 microscopy observation ( Figure 1B). If a specific volume of 1.3 g/cm 3 is assumed for the seed amyloid fibrils with (empty red circles) or without (filled red circles) 2 h of incubation at 37°C.

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The solid lines are shown only to guide the eye.