Abstract
Prions are infectious proteins that possess multiple self-propagating structures. The information for strains and structural specific barriers appears to be contained exclusively in the folding of the pathological isoform, PrPSc. Many recent studies determined that de novo prion strains could be generated in vitro from the structural conversion of recombinant (rec) prion protein (PrP) into amyloidal structures. Our aim was to elucidate the conformational diversity of pathological recPrP amyloids and their biological activities, as well as to gain novel insights in characterizing molecular events involved in mammalian prion conversion and propagation. To this end we generated infectious materials that possess different conformational structures. Our methodology for the prion conversion of recPrP required only purified rec full-length mouse (Mo) PrP and common chemicals. Neither infected brain extracts nor amplified PrPSc were used. Following two different in vitro protocols recMoPrP converted to amyloid fibrils without any seeding factor. Mouse hypothalamic GT1 and neuroblastoma N2a cell lines were infected with these amyloid preparations as fast screening methodology to characterize the infectious materials. Remarkably, a large number of amyloid preparations were able to induce the conformational change of endogenous PrPC to harbor several distinctive proteinase-resistant PrP forms. One such preparation was characterized in vivo habouring a synthetic prion with novel strain specified neuropathological and biochemical properties.
Highlights
Prion diseases or transmissible spongiform encephalopathies are fatal neurodegenerative disorders of humans and animals
Colby et al reported that proteasesensitive synthetic prions could be generated in vitro during polymerization of recombinant prion protein (PrP) into amyloid fibers [6]
To generate recombinant PrP (recPrP) amyloids with different conformations, we systematically altered the conditions for their formation, including denaturant concentrations, pH and buffer composition (S1 and S2 Tables)
Summary
Prion diseases or transmissible spongiform encephalopathies are fatal neurodegenerative disorders of humans and animals. During the course of these maladies the cellular prion protein (PrPC) converts into an abnormally folded isoform, PrPSc, which accumulates in the central nervous system (CNS), leading to the host death [1, 2]. The key molecular event of these disorders is the conformational change of PrPC from its physiological form into the pathological structural isoform noted as prion or PrPSc. The physiological PrPC is a glycosylphosphatidylinositol (GPI)-anchored protein present in all cell types. Differences in structural conformation lead to different biological characteristics: while PrPC is soluble in detergents and sensitive to proteinase K (PK) digestion, PrPSc is partially PK-resistant and insoluble in non-ionic detergents [3]. A sizeable fraction of PrPSc is sensitive to PK digestion [4] and is dispersed with detergent and/or sonication [5]. PK-sensitive and PK-resistant PrPSc were shown to share a common structure and phenotype despite the differences in resistance to PKdigestion, sediment and distribution of multimers [7, 8]
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