Generating new prions by targeted mutation or segment duplication

Abstract

Yeasts contain various protein-based genetic elements, termed prions, that result from the structural conversion of proteins into self-propagating amyloid forms. Most yeast prion proteins contain glutamine/asparagine (Q/N)-rich prion domains that drive prion activity. Here, we explore two mechanisms by which new prion domains could evolve. First, it has been proposed that mutation and natural selection will tend to result in proteins with aggregation propensities just low enough to function under physiological conditions and thus that a small number of mutations are often sufficient to cause aggregation. We hypothesized that if the ability to form prion aggregates was a sufficiently generic feature of Q/N-rich domains, many nonprion Q/N-rich domains might similarly have aggregation propensities on the edge of prion formation. Indeed, we tested four yeast Q/N-rich domains that had no detectable aggregation activity; in each case, a small number of rationally designed mutations were sufficient to cause the proteins to aggregate and, for two of the domains, to create prion activity. Second, oligopeptide repeats are found in multiple prion proteins, and expansion of these repeats increases prion activity. However, it is unclear whether the effects of repeat expansion are unique to these specific sequences or are a generic result of adding additional aggregation-prone segments into a protein domain. We found that within nonprion Q/N-rich domains, repeating aggregation-prone segments in tandem was sufficient to create prion activity. Duplication of DNA elements is a common source of genetic variation and may provide a simple mechanism to rapidly evolve prion activity.

Yeast Short-Lived Actin-Associated Protein Forms a Metastable Prion in Response to Thermal Stress

“…The Ross group previously demonstrated that the prion-forming activity could be artificially generated with as few as two mutations, suggesting that many non-prion Q/N-rich proteins may be just a small number of mutations from acquiring aggregation or prion activity (Paul et al, 2015). Our data, coupled with phylogenetic analysis of Lsb proteins, clearly demonstrates that such an acquisition has indeed happened in the process of yeast evolution.…”

Effects of Mutations on the Aggregation Propensity of the Human Prion-Like Protein hnRNPA2B1

“…Prion formation assays were performed as previously described (64). Briefly, cells expressing a given hnRNPA2-Sup35 fusion as the sole copy of Sup35 were transformed with either an empty vector (pKT24 [65]) or a plasmid expressing the matching PrLD-GFP fusion under the control of the GAL1 promoter.…”

The effects of glutamine/asparagine content on aggregation and heterologous prion induction by yeast prion-like domains

“…Foci formation assays were performed as described previously. 27 Briefly, yeast strains yER632, yER1019, and yER1615 were transformed with TRP1 plasmids expressing each PrLD-GFP from the GAL1 promoter. Strains were grown in galactose/raffinose dropout medium lacking tryptophan for 24 hours, and then imaged by confocal microscopy.…”

“…23,24 PAPA scans proteins for regions of intrinsic disorder, and scores the prion propensities of these regions. 25 PAPA has proven effective at predicting the prion-like activity of Q/N-rich PrLDs; 14 designing mutations to modulate the aggregation activity of PrLDs; [26][27][28] designing synthetic prion-forming domains; 24 and predicting the effects of some disease-associated mutations in human PrLDs. 29 However, PAPA still has substantial limitations.…”