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Rapid Generation of Amyloid from Native Proteins In vitro
Published on: December 5, 2013
Rapid formation of amyloid from alpha-monomeric recombinant human PrP in vitro
Abdessamad Tahiri-Alaoui1, William James
1Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK.
Abstract:
The infectious agent of prion diseases is identified with PrP(Sc), a beta-rich, amyloidogenic and partially protease resistant isoform of the cellular glycoprotein, PrP(C). To understand the process of prion formation in vivo, we and others have studied defined misfolding pathways of recombinant PrP in vitro. The low-level infectivity of the in vitro misfolded murine PrP amyloid has recently been reported. Here we analyze the in vitro kinetics of amyloid formation from recombinant human PrP(90-231) in vitro in the context of two common allelic forms of PrP found in human populations that are associated with differences in prion disease susceptibility and pathological phenotype. We show that human PrP amyloid forms readily from its PrP(C)-like state in vitro, that the lag time of the reaction can be further shortened by the presence of a "seed" of pre-formed PrP amyloid, and that amyloid propagation is more complex than a simple crystallization process. We further show that the kinetics of amyloid formation do not differ between the Met129 and Val129 allelomorphs of human PrP, and that amyloid from each functions as an equally effective seed in heterologous, as in homologous amyloid reactions. The results could illuminate the process of amyloid formation in vivo as well as help understanding prion pathogenesis.
Insights
Prion diseases involve misfolded prion proteins (PrP). This study shows human PrP amyloid forms readily in vitro, with kinetics unaffected by common genetic variations, offering insights into prion formation and pathogenesis.
Area of Science:
- Neuroscience
- Biochemistry
- Molecular Biology
Background:
- Prion diseases are linked to PrP(Sc), an abnormal isoform of cellular PrP(C).
- Understanding in vivo prion formation requires studying PrP misfolding pathways.
- In vitro studies of recombinant PrP misfolding are crucial for this understanding.
Purpose of the Study:
- To analyze the in vitro kinetics of human PrP(90-231) amyloid formation.
- To investigate the influence of common human PrP allelic forms (Met129 and Val129) on amyloid formation kinetics.
- To assess the seeding efficiency of amyloids from different allelic forms.
Main Methods:
- In vitro kinetic analysis of amyloid formation from recombinant human PrP(90-231).
- Utilized pre-formed PrP amyloid seeds to study reaction lag times.
- Compared amyloid formation kinetics between Met129 and Val129 PrP allelomorphs.
Main Results:
- Human PrP amyloid forms readily from a PrP(C)-like state in vitro.
- Amyloid formation lag time is reduced by pre-formed seeds, indicating complex propagation.
- Amyloid formation kinetics and seeding efficiency are similar for Met129 and Val129 PrP allelomorphs.
Conclusions:
- Human PrP amyloidogenesis in vitro is a robust process.
- Common human PrP genetic variations do not significantly alter in vitro amyloid formation kinetics.
- These findings may elucidate in vivo amyloid formation and prion pathogenesis.
