TY - CONF

T1 - Seeding of in Vitro Amyloid Aggregation by Endogenous and Artificially Produced Aggregates from Yeast Cells

AU - Kulichikhin, Konstantin

AU - Orlov, Valentin

AU - Rubel, Aleksandr

AU - Chernoff, Yury

N1 - Conference code: 28

PY - 2017

Y1 - 2017

N2 - 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 comparedefficiencies 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 experimentswas 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 humansamples.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.

AB - 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 comparedefficiencies 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 experimentswas 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 humansamples.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.

KW - Saccharomyces cerevisiae

KW - Sup35NM

KW - PMCA

M3 - Abstract

SP - 227

EP - 228

T2 - 28th International Conference on Yeast Genetics and Molecular Biology

Y2 - 27 August 2017 through 1 September 2017

ER -