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De novo [PSI ⁺] prion formation involves multiple pathways to form infectious oligomers.

ABSTRACT: Prion and other neurodegenerative diseases are associated with misfolded protein assemblies called amyloid. Research has begun to uncover common mechanisms underlying transmission of amyloids, yet how amyloids form in vivo is still unclear. Here, we take advantage of the yeast prion, [PSI ⁺], to uncover the early steps of amyloid formation in vivo. [PSI ⁺] is the prion form of the Sup35 protein. While [PSI ⁺] formation is quite rare, the prion can be greatly induced by overexpression of the prion domain of the Sup35 protein. This de novo induction of [PSI ⁺] shows the appearance of fluorescent cytoplasmic rings when the prion domain is fused with GFP. Our current work shows that de novo induction is more complex than previously thought. Using 4D live cell imaging, we observed that fluorescent structures are formed by four different pathways to yield [PSI ⁺] cells. Biochemical analysis of de novo induced cultures indicates that newly formed SDS resistant oligomers change in size over time and lysates made from de novo induced cultures are able to convert [psi ^-] cells to [PSI ⁺] cells. Taken together, our findings suggest that newly formed prion oligomers are infectious.

SUBMITTER: Sharma J

PROVIDER: S-EPMC5427932 | biostudies-literature | 2017 Mar

REPOSITORIES: biostudies-literature

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De novo [PSI +] prion formation involves multiple pathways to form infectious oligomers.

Sharma Jaya J Wisniewski Brett T BT Paulson Emily E Obaoye Joanna O JO Merrill Stephen J SJ Manogaran Anita L AL

Scientific reports 20170306 1

Prion and other neurodegenerative diseases are associated with misfolded protein assemblies called amyloid. Research has begun to uncover common mechanisms underlying transmission of amyloids, yet how amyloids form in vivo is still unclear. Here, we take advantage of the yeast prion, [PSI +], to uncover the early steps of amyloid formation in vivo. [PSI +] is the prion form of the Sup35 protein. While [PSI +] formation is quite rare, the prion can be greatly indu ...[more]

PMID: 28250435

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Project description:The formation of β-sheet rich prion oligomers and fibrils from native prion protein (PrP) is thought to be a key step in the development of prion diseases. Many methods are available to convert recombinant prion protein into β-sheet rich fibrils using various chemical denaturants (urea, SDS, GdnHCl), high temperature, phospholipids, or mildly acidic conditions (pH 4). Many of these methods also require shaking or another form of agitation to complete the conversion process. We have identified that shaking alone causes the conversion of recombinant PrP to β-sheet rich oligomers and fibrils at near physiological pH (pH 5.5 to pH 6.2) and temperature. This conversion does not require any denaturant, detergent, or any other chemical cofactor. Interestingly, this conversion does not occur when the water-air interface is eliminated in the shaken sample. We have analyzed shaking-induced conversion using circular dichroism, resolution enhanced native acidic gel electrophoresis (RENAGE), electron microscopy, Fourier transform infrared spectroscopy, thioflavin T fluorescence and proteinase K resistance. Our results show that shaking causes the formation of β-sheet rich oligomers with a population distribution ranging from octamers to dodecamers and that further shaking causes a transition to β-sheet fibrils. In addition, we show that shaking-induced conversion occurs for a wide range of full-length and truncated constructs of mouse, hamster and cervid prion proteins. We propose that this method of conversion provides a robust, reproducible and easily accessible model for scrapie-like amyloid formation, allowing the generation of milligram quantities of physiologically stable β-sheet rich oligomers and fibrils. These results may also have interesting implications regarding our understanding of prion conversion and propagation both within the brain and via techniques such as protein misfolding cyclic amplification (PMCA) and quaking induced conversion (QuIC).

Dataset Information

De novo [PSI ⁺] prion formation involves multiple pathways to form infectious oligomers.

Publications

De novo [PSI <sup>+</sup>] prion formation involves multiple pathways to form infectious oligomers.

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