In:
Proceedings of the National Academy of Sciences, Proceedings of the National Academy of Sciences, Vol. 109, No. 28 ( 2012-07-10)
Abstract:
In summary, our work shows that cofactor molecules play an essential role in determining all of the unique characteristics of mammalian prions, i.e., infectivity, PrP Sc structure, and strain properties (including selective neurotropism). The precise mechanism by which these characteristics are defined remains undetermined. The only two logical possibilities are either that cofactor molecules are integral components of infectious prions (rejecting the “protein only” hypothesis) or that cofactor molecules are necessary to exert a continuous selective pressure during the propagation of infectious prions (and specific strains) composed of only PrP Sc molecules. Additional considerations, such as the colocalization of fluorescently labeled PE molecules with PrP Sc molecules in prion aggregates, the effects of organic solvents on prion disinfection ( 2 ), and Anfinsen’s principle of protein folding ( 3 ), argue that cofactor molecules are likely essential structural components of infectious prions, but further studies are required to confirm this assertion. To test whether cofactor molecules can modulate strain properties, we changed only the cofactor component of a substrate mixture during the serial propagation of a purified recombinant prion. This single change caused dramatic changes in strain properties, as determined by clinical incubation time, neuropathology, and biochemical assays of PrP Sc structure in infected animals. Moreover, we found that three different prion strains all produced the same unique output strain when propagated in a substrate mixture containing only one available cofactor. The most remarkable aspect of these experiments is that the cofactor component of the reaction, rather than the input strains used as seed, was the active determinant of the output strain. Collectively, these results provide direct evidence that prion strain diversity cannot be maintained with a restricted cofactor repertoire and imply that specific sets of cofactor molecules may be responsible for generating specific prion strains ( Fig. P1 ). Here, we used a purified cofactor preparation containing only one available cofactor, PE, as a unique tool to test these possibilities in an in vitro prion propagation system. To test whether cofactor molecules are required to maintain prion infectivity, we serially propagated purified prions in substrate mixtures that either contained or lacked the cofactor. Interestingly, both sets of reactions produced self-propagating PrP Sc molecules, but, based on an enzymatic protein digestion assay, the shape of the “protein only” PrP Sc molecules appeared be slightly different from that of the cofactor PrP Sc molecules. We measured the specific infectivity of both types of PrP Sc molecules by inoculating wild-type mice with serial dilutions of each. PrP Sc molecules formed with the cofactor were at least 10 5 times more infectious than the “protein only” PrP Sc molecules, which failed to cause prion disease even at the highest concentration. Thus, cofactor molecules appear to be necessary to maintain the infectious PrP Sc structure in mammalian prions. We focused our efforts on generating purified infectious prions from chemically defined substrates in vitro. Using this approach, we discovered that molecules other than PrP C are needed to form infectious prions and that infectious prions are formed de novo from a substrate mixture containing PrP C and cofactor molecules. We also found that different types of cofactor molecules, including lipid and polyanionic molecules, are able to serve as prion cofactors. Most recently, we purified and identified phosphatidylethanolamine (PE) as a cofactor that, by itself, can facilitate the propagation of prions from a wide variety of animal species. These findings raise the possibility that cofactor molecules actually may play an essential role in maintaining infectious PrP Ss structure and that different classes of cofactor molecules may be responsible for enciphering strain diversity. There are two important reasons to doubt the “protein only” hypothesis for mammalian prions. First, despite efforts by many investigators, prions with significant levels of specific infectivity have not yet been produced with PrP alone. In contrast, prions with moderate levels of specific infectivity can be formed spontaneously from a mixture of PrP C and cofactor molecules ( 1 ). Second, prions exist as a variety of distinguishable “strains” that exhibit specific patterns of clinical symptoms, neuropathology, and PrP Sc biochemical characteristics. It is difficult to envision how a single protein, by itself, can encode such a diversity of infectious properties, especially the property of selective neurotropism by which prion strains can be differentiated by their preferential patterns of accumulation in specific regions of the brain. Mammalian prions are unorthodox infectious agents that cause fatal neurological diseases such as Creutzfeldt–Jakob disease, bovine spongiform encephalopathy (also known as “mad cow disease”), and scrapie. Prions lack informational nucleic acids; therefore, it is puzzling that they replicate within and cause specific patterns of disease in infected hosts. Prions are formed by the conversion of a normal cellular prion protein (PrP C ) into a misfolded shape called the “scrapie prion protein” (PrP Sc ). According to the popular “protein only” hypothesis, PrP Sc may be the only essential component of the infectious agent, possessing the ability to replicate and cause disease by inducing the conversion of PrP C molecules into additional PrP Sc molecules. Here, we demonstrate that cofactor molecules appear to be necessary to maintain PrP Sc structure and that specific sets of cofactor molecules may generate specific prion strains ( Fig. P1 ).
Type of Medium:
Online Resource
ISSN:
0027-8424
,
1091-6490
DOI:
10.1073/pnas.1206999109
Language:
English
Publisher:
Proceedings of the National Academy of Sciences
Publication Date:
2012
detail.hit.zdb_id:
209104-5
detail.hit.zdb_id:
1461794-8
SSG:
11
SSG:
12
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