Characterisation of Sup35NMp amyloid-like fibrils and their infectivity in vivo

Abstract

Introduction: Prion particles are associated with infectious prion diseases in mammals and inherited phenotypes in Saccharomyces cerevisiae. Sup35p, found in yeast, undergoes prion-like fibril formation and infects S. cerevisiae cells by inducing [PSI+] phenotype. To understand why aggregates differ in infectivity, their physical properties are currently being intensively studied. Aim: The aim of this research was to analyze the characteristics and formation of Sup35NMp fibrils in vitro and their infectivity in S. cerevisiae cell for a better understanding of prion diseases in mammals. Materials and methods: Recombinant Sup35NMp was purified using Ni2+ affinity chromatography followed by a SP-Sepharose IEX chromatography. Aggregation studies were carried out in several pH 7.4 buffer solutions. Fibril formation kinetics were monitored by measuring intensity fluorescence of ThT using Varian Cary Eclipse fluorescence spectrophotometer. Samples were imaged using a Dimension Icon Bruker AFM. The stability of fibrils was monitored using different GuHCl concentrations in Synergy H4 Hybrid MultiMode microplate reader. Sup35NMp fibrils were transformed into S. cerevisiae using electroporation. Results: Experimental analysis showed that Sup35NMp fibrils formation kinetics depend on temperature. The stability of formed fibrils was examined revealing differences of strains. Moreover, we have found that the synthetic Sup35NMp fibrils induce the [PSI+] prion phenotype in S. cerevisiae cells, while the induction efficiency depends on the length of the particles. Conclusion: This research revealed that the Sup35NMp fibril strains formed in vitro can be characterized by different aggregation rate and stability. Moreover, the infectivity of [PSI+] phenotype in vivo depends on the Sup35NMp fibril characteristics. A better understanding of the prion fibrils formation characteristics and their infectivity mechanisms will lead to an effective cure of mammalian and human prion diseases.