Protein Misfolding Cycling Amplification (PMCA) is a cutting-edge technique for seeding aggregation of prions and other amyloidogenic proteins in vitro by aggregates produced in vivo. This technique can be applied to the detection of prions and other amyloids in biological samples, however successful application of PMCA needs careful adjustment of experimental protocol for each new protein. Here we develop the PMCA protocol for seeding amyloid aggregation of yeast Sup35NM (truncated form of the Sup35 prion protein) by lysates of yeast cells containing [PSI+], a prion form of the Sup35 protein. We compared
efficiencies of seeding in quiescent conditions, where the monomeric form of Sup35NM did not show any sign of spontaneous aggregation during the period of experiment, and in shaking conditions, where spontaneous aggregation does occur. Our data indicate that the lag period of aggregation reaction inversely correlates with the amount of the “seed”, added to the reaction mixture, thus allowing to detect presence of Sup35 amyloids in the extracts of yeast cells by PMCA. The major obstacle we have encountered in our PMCA experiments
was associated with the high proteolytic activity of yeast extracts, leading to degradation of the substrate Sup35NM protein. Approaches used to minimize the impact of proteolytic activity will be discussed. Finally, we extend the PMCA technique to detection of amyloids, formed by the chimeric constructs containing human amyloid beta (Abeta) peptide (associated with Alzheimer disease) in the yeast cells. The yeast-based protocol can be employed to optimize conditions for the PMCA-based detection of Abeta polymers in human
samples.
This work is supported by grant 14-50-00069 from Russian Science Foundation. Experiments were performed using equipment of the Resource Centers “Molecular and Cell Technologies” and “Biobank”, St. Petersburg State University.