Kosmotropic Anions Promote Conversion of Recombinant Prion Protein into a PrPSc-Like Misfolded Form

Abstract

Prions are self-propagating proteins involved in transmissible spongiform encephalopaties in mammals. An aberrant conformation with amyloid-like features of a cell surface protein, termed prion protein (PrP), is thought to be the essential component of the infectious particle, though accessory co-factor molecules such as lipids and nucleotides may be involved. The cellular co-factors and environmental conditions implicated in PrP misfolding are not completely understood. To address this issue, several studies have been done inducing misfolding of recombinant PrP (recPrP) into classical amyloid structures using partially denaturing conditions. In this work, we report that misfolding of recPrP into PrPSc-like aggregates can be induced by simply incubating the protein in the presence of kosmotropic salts at concentrations that are known to retain or increase the stability of the protein. We used a simple experimental reaction (protein, buffer and salts) submitted to agitation/incubation cycles at physiological temperature and pH. The formation of protease resistant-recPrP was time and salt-concentration dependent and required the presence of kosmotropic anions such as F− or SO4 −2. The molecular weights of the protease resistant recPrP fragments are reminiscent of those found in degradation assays of bona fide PrPSc. The aggregates also exhibited PrPSc-like ultrastructural features including rod-shape morphology under electron microscope, high beta-sheet content and thioflavin-T positive signal. The formation of recPrP aggregates with PrPSc biochemical features under conditions closer to physiological in the absence of organic co-factor molecules provides a simple setup that may prove helpful to understand the molecular mechanism of PrP misfolding.

Contributions of the Prion Protein Sequence, Strain, and Environment to the Species Barrier

“…Specifically, strongly hydrated anions (kosmotropes) initiate nucleation quickly and promote rapid fiber elongation, whereas poorly hydrated anions (chaotropes) delay nucleation and mildly affect the elongation rate (43,44). A similar effect of kosmotropes has also been observed by another group for the mammalian prion protein PrP (45). Moreover, amyloid formation by Sup35NM in the presence of different anions resulted in the generation of different spectra of prion strains, with kosmotropes favoring the formation of strong strains (characterized by smaller aggregate size and higher efficiency of fragmentation and proliferation), and chaotropes favoring the formation of weak strains (characterized by larger aggregate size and lower efficiency of fragmentation and proliferation) (43,44).…”

Ion-specific Effects on Prion Nucleation and Strain Formation

“…Similar to temperature, the ionic composition of the solution has been shown to affect aggregation kinetics in a number of amyloid-forming proteins, including ␣-synuclein (41), mouse prion protein PrP (42,43), PABPN1 (44), and Sup35NM (18); however, the effect of solution composition on amyloid structures and biological effects was not studied in detail. The present work relates the differences in aggregation kinetics of Sup35NM caused by different salts to the structures and propagation parameters of amyloids formed in respective conditions.…”

The Extent of Protease Resistance of Misfolded Prion Protein Is Highly Dependent on the Salt Concentration

“…Interestingly, calcium is known to be a highly destabilizing cation within the series, whereas in addition being an activator of PK activity (24). We previously found that NaF efficiently promoted the conversion of recPrP to a PrP Sc -like state, probably by salting-out effects (20). Although the concentrations used in that work were higher than those reported here, a similar saltbased stabilization mechanism may be operating with PrP Sc , and additional experiments are ongoing to explore this scenario.…”

“…In particular, the concentrations of sodium chloride and several metals can greatly change PrP Sc sensitivity to proteolysis (19). Kosmotropic salts and copper can also induce misfolding of recombinant PrP into PrP Sc -like species in vitro (20).…”