Mammalian amyloidogenic proteins promote prion nucleation in yeast

Abstract

Fibrous cross-β aggregates (amyloids) and their transmissible forms (prions) cause diseases in mammals (including humans) and control heritable traits in yeast. Initial nucleation of a yeast prion by transiently overproduced prion-forming protein or its (typically, QN-rich) prion domain is efficient only in the presence of another aggregated (in most cases, QN-rich) protein. Here, we demonstrate that a fusion of the prion domain of yeast protein Sup35 to some non-QN-rich mammalian proteins, associated with amyloid diseases, promotes nucleation of Sup35 prions in the absence of preexisting aggregates. In contrast, both a fusion of the Sup35 prion domain to a multimeric nonamyloidogenic protein, and an expression of a mammalian amyloidogenic protein that is not fused to the Sup35 prion domain failed to promote prion nucleation, further indicating that physical linkage of a mammalian amyloidogenic protein to the prion domain of a yeast protein is required for the nucleation of a yeast prion. Biochemical and cytological approaches confirmed the nucleation of protein aggregates in the yeast cell. Sequence alterations antagonizing or enhancing amyloidogenicity of human β amyloid (Aβ, associated with Alzheimer disease) and mouse PrP (associated with prion diseases) respectively antagonized or enhanced nucleation of a yeast prion by these proteins. The yeast-based prion nucleation assay, developed in our work, can be employed for mutational dissection of amyloidogenic proteins. We anticipate that it will aid in the identification of chemicals that influence initial amyloid nucleation and in searching for new amyloidogenic proteins in a variety of proteomes.

The genetic landscape for amyloid beta fibril nucleation accurately discriminates familial Alzheimer’s disease mutations

“…To globally quantify the impact of mutations on the nucleation of Aß fibrils, we used an in vivo selection assay in which the nucleation of Aß is rate limiting for the aggregation of a second amyloid, the yeast prion [PSI+] encoded by the sup35 gene (Chandramowlishwaran et al 2018). Aggregation of Sup35p causes read-through of UGA stop codons, allowing growth based-selection using an auxotrophic marker containing a premature termination codon ( Figure 1A).…”

“…50ml of overnight E.coli culture was harvested to purify the Aβ plasmid library with a midi prep (Plasmid Midi Kit, Qiagen). The resulting library contained 29.9% of WT Aβ, 23.8 % of sequences with 1nt change and 21.8Saccharomyces cerevisiae[psi-pin-] (MATa ade1-14 his3 leu2-3,112 lys2 trp1 ura3-52) strain (also provided by the Chernoff lab) was used in all experiments in this study(Chandramowlishwaran et al 2018).Yeast cells were transformed with the Aβ plasmid library starting from an individual colony for each transformation tube. After an overnight pre-growth culture in YPDA medium at 30C, cells were diluted to OD 600 =0.3 in 175ml YPDA and incubated at 30C 200rpm for ~5h.…”

Proteolysis: a double-edged sword for the development of amyloidoses

“…For example, Chandramowlishwaran et al have reported a de novo nucleation system that detects the ability of mammalian amyloidogenic protein (Aβ and PrP etc.) to promote prion nucleation by attaching the mammalian amyloidogenic protein to prion domain of Sup35 [34]. Also, aggregation and toxicity of human polyQ diseases (HD etc.)…”

Protein Misfolding during Pregnancy: New Approaches to Preeclampsia Diagnostics

“…Amyloids can also be detected by some amyloid-specific antibodies [94], and by electron microscopy (EM) [95,96]. Notably, the ability to form an amyloid is controlled by an amyloid protein itself, as confirmed by the observation that amyloids are formed by mammalian amyloidogenic proteins expressed in heterologous systems, such as yeast [97,98].…”