Prion amyloids, the infectious protein agent of mad cow disease (bovine spongiform encephalopathy, or BSE), and other transmissible spongiform encephalopathies, are protein aggregates with a flair for self-promotion. These abnormally folded, spontaneously propagating polymers enter cells and coax soluble cellular prion protein (PrP) to adopt their toxic conformation. The result is a widespread amyloidosis and neurodegenerative disease that leaves the brain with a characteristic sponge-like pathology. Two papers, published this week in Cell, describe how researchers used new techniques for propagating PrP fibers in vitro to illuminate the role of both amino acid sequence and protein conformation in prion infectivity. The results not only explain how infectious prions can break the species barrier, but also raise questions about the structure of non-infectious amyloids, as found in Alzheimer disease.
The likelihood that prion protein from one species will successfully infect another, as happened with the bovine-to-human transmission of BSE, depends on the amino acid similarities between each species’ PrP—the closer the match, the more likely that the infectious prion can whip the host protein into pathological shape. But amino acid sequence does not tell the whole story, because prions with identical amino acid sequences can adopt different conformations, and these different-shaped prion strains may coexist in a single host species. So what’s more important, primary sequence or secondary structure?
Both studies, one from Jonathan Weissman and colleagues at the University of California in San Francisco, and the other from Eric Jones and Witold Surewicz at Case Western Reserve University, Cleveland, Ohio, show that proteins with an identical primary amino acid sequence can be induced to form distinctly different fiber conformations. But in each case, it is the shape of the protein, not its amino acid sequence per se, that dictates whether it will jump the species barrier and catalyze fibril formation in a different host.
Jones and Surewicz’s work extends a study they published last year (Vanik at al., 2004) showing that soluble recombinant fragments of human, mouse, or hamster PrP are easily cajoled into forming amyloid fibrils if they are seeded with preformed amyloid fibrils in vitro. Using this assay, the researchers established species barriers for the seeding reaction; mouse prion could seed human and vice versa, while a hamster prion induced polymerization of mouse protein, but not human.
In the new paper, Jones and Surewicz describe the physical characteristics of these fibrils. Spectroscopic and microscopic techniques show that human and mouse PrP fibrils take on a similar string-of-beads shape, while hamster PrP yields a different conformation—smooth fibers. But their most intriguing results come from the cross-seeding experiments. When they used a small amount of hamster amyloid to seed mouse prion, the resulting fibrils, even though made almost entirely of mouse protein, had the appearance and physical properties of hamster fibrils. In addition, the new hamster-type mouse PrP fibrils had lost the ability to seed human PrP polymerization, proving that by cross-species seeding, the investigators had generated a new strain of mouse prion that inherited the secondary structure of its hamster template.
The species barrier-busting ability of the new mouse strain remains theoretical, since none of the recombinant prion fibers has been proven to be infectious in animals yet. But in the second paper, first author Motomasa Tanaka and the UCSF group get around that limitation by using yeast. In yeast, a transmissible, prion-like conformation of the protein Sup35 suppresses translation. As with mammalian prions, a strong species barrier normally prevents transmission from one strain of yeast to another. For example, Sup35 from Candida albicans (Ca) can only infect C. albicans and not Saccharomyces cerevisiae (Sc) and vice versa.
Is this barrier also based on secondary structure? Apparently so. The researchers showed that a truncated recombinant Sup35 from Sc formed different fibril conformations at different temperatures. One conformation in particular, formed at 4 degrees C, displayed the unusual ability to seed the polymerization of Ca Sup35. The fibrils generated from this seeding could themselves seed both Sc and Ca monomers, and could infect both strains of yeast Sup35. The species cross was all the more dramatic given that the Ca and Sc Sup35 proteins are fairly divergent—unlike the closely related mammalian PrPs used by Jones and colleagues, the Sup35s share only 40 percent amino acid similarity in the prion domain. Biophysical measurements on the fibrils supported the idea that attaining a permissive three-dimensional shape was the important determinant of successful infection.
Both studies, then, point to a model where the amino acid sequence of a PrP proscribes a set of possible conformations, and a species barrier can be breached when the sets of possible shapes for two PrP sequences overlap. This shape-shifting mechanism for new strain production provides a plausible explanation for the emergence of variant Cruetzfeldt-Jakob disease after infection of humans with the BSE prion. Moreover, the specter of the production of new prion strains in nature by repeated cross-species infection should raise a flag as prion diseases keep popping up among both domestic and wild animals.
These results may also have application to other amyloid disease, even where the protein aggregates are non-infectious, as in AD. In these cases, the California group speculates, different amyloid conformations adopted by the pathogenic protein could determine its ability to recruit other, heterologous proteins, possibly modulating the toxic effects of accumulating misfolded protein aggregates.—Pat McCaffrey
Pat McCaffrey is a freelance science writer in Newton, Massachusetts.
- Vanik DL, Surewicz KA, Surewicz WK. Molecular basis of barriers for interspecies transmissibility of mammalian prions. Mol Cell. 2004 Apr 9;14(1):139-45. PubMed.
- Collins SR, Douglass A, Vale RD, Weissman JS. Mechanism of prion propagation: amyloid growth occurs by monomer addition. PLoS Biol. 2004 Oct;2(10):e321. PubMed.
- Tanaka M, Chien P, Naber N, Cooke R, Weissman JS. Conformational variations in an infectious protein determine prion strain differences. Nature. 2004 Mar 18;428(6980):323-8. PubMed.
- Jones EM, Surewicz WK. Fibril conformation as the basis of species- and strain-dependent seeding specificity of mammalian prion amyloids. Cell. 2005 Apr 8;121(1):63-72. PubMed.
- Tanaka M, Chien P, Yonekura K, Weissman JS. Mechanism of cross-species prion transmission: an infectious conformation compatible with two highly divergent yeast prion proteins. Cell. 2005 Apr 8;121(1):49-62. PubMed.