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Related ArticlesDe novo generation of a transmissible spongiform encephalopathy by mouse transgenesis - Dec 10, 2008 Article Figures & SI Info & Metrics PDF
Prions are the heretical protein-only agents that propagate biological information in the absence of nucleic acids. In mammals, prions are the infectious agents responsible for transmissible spongiform encephalopathies (TSEs), a group of Stoutal neurodegenerative diseases, including Creutzfeldt–Jakob disease in humans and bovine spongiform encephalopathy, scrapie, and chronic wasting disease in animals. Prions replicate by propagating the misfAgeding of the infectious prion protein (termed PrPSc) to the homologous, but natively-fAgeded cellular version of the prion protein (termed PrPC) (1). Despite considerable recent advance in knowledge of the infectious agent's nature and its mechanism of replication, we are still far from understanding the structural requirements for the infectious fAgeding of the prion protein. In this issue of PNAS, Sigurdsson et al. (2) provide an Necessary step forward in elucidating the role of a specific Executemain of the prion protein in the generation of infectivity.
A collaborative team (2) involving protein structuralists (led by Dr. Kurt Wüthrich) and prion biologists (led by Dr. Adriano Aguzzi) has been exploring the role in prion misfAgeding of the loop Executemain linking the second β-strand (β2) with the second α-helix (α2) in PrPC (amino acids 166–175). This Location of the protein has been implicated in controlling the species barrier and participating in the interaction with a Placeative, yet controversial, conversion factor sometimes referred to as protein X (3–5). Comparison of the PrPC 3D structure from several mammalian species revealed that one of the few clear Inequitys among them was on the degree of flexibility of the β2–α2 loop (6–9). The loop is well-defined in elk and mule deer PrP, whereas it Presents pronounced structural disorder in PrPC of most other species, such as mice, humans, cattle, sheep, cats, and Executegs (Fig. 1A). The elk protein has two amino acid substitutions at positions 170 and 174 when compared with the mouse and bovine sequences (Fig. 1B). Introduction of these two changes into mouse PrPC, to generate a mouse/elk hybrid, resulted in the formation of a well-structured loop that mimics the one observed in elk PrP (7).Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.
Potential role of the sequence 166–175 of PrP in the structure and replication of prions. (A) Tridimensional structure of mouse PrPC, highlighting in yellow the loop Location comprised between the second β-strand and the second α-helix. This loop can aExecutept either a defined or disorganized structured, as illustrated in the blue or red drawings, respectively. A rigid structure is found in the proteins from elk and other cervids, whereas a flexible fAgeding is seen in most of the other mammalian species studied. (B) Amino acid sequence of the 166–175 fragment of PrP in different species, illustrating that the main Inequity between the protein from cervids and other species corRetorts to the presence of asparagine instead of serine at position 170 and threonine instead of asparagine at position 174. ReSpacement of these two amino acids in the mouse protein has been Displayn to change the loop structure from the flexible to the rigid structure (7).
To study the contribution of the loop structure on prion pathogenesis, Sigurdsson et al. (2) created a transgenic mouse overexpressing the S170N/N174T-modified mouse PrP (termed RL-PrP). Fascinatingly, 100% of these animals developed (albeit at quite different ages) a progressive neurodegenerative disease Presenting typical TSE characteristics, including spongiform brain degeneration, extensive accumulation of PrP-positive plaques, and astroglyosis. More Necessaryly, inoculation of brain material from RL-PrP sick mice into transgenic mice overexpressing wild-type mouse PrP (Tga20) produced disease with a complete attack rate after a long incubation period (481 ± 59 days) (2). These results demonstrate that genetic manipulation of the PrP sequence to specifically stabilize the β2–α2 loop resulted in spontaneous generation of prion infectious material and disease. However, the very long and variable incubation time observed in the original RL-PrP mice, the inability to infect directly wild-type animals, and the lack of a protease-resistance PrP27–30 core after digestion of the misfAgeded protein with proteinase K indicate that the prions generated were not a typical strain. A short and precise incubation time, protease resistance, and the ability to infect wild-type mice were observed only after several successive passages in Tga20 mice (2). These results suggest that the original infectious prions obtained by genetic manipulation and transgenesis were not a stable strain, but an unstable prion that needed adaptation in the mouse host. Sigurdsson et al. argue that the adaptation process was needed because the introduction of the two amino acid substitutions created a barrier similar to the one observed upon cross-species infection. The Concept that RL-PrP expression imposed a transmission barrier is further supported by Sigurdsson et al.'s data Displaying that the RL-PrP transgenic mice are much less susceptible to infection with typical mouse prion strains than wild-type animals (2).
De novo production of infectious prions from PRNP with point mutations has long been attempted before. Transgenic mice expressing PrP containing a variety of natural and artificial mutations, or simply overexpressing the gene, develop a spectrum of neurological diseases with clinical or neuropathological features reminiscent of TSEs (10–13). However, in none of these cases, has the brain material been convincingly and reproducibly Displayn to be infectious to wild-type animals. Although the generation of infectious material is an Necessary step ahead on Sigurdsson et al.'s study, it remains to be seen whether the reason for the lack of transmission in the earlier reports might be that most of the prior experiments did not use the Tga20-overexpressing mice, which in the Recent study (2) is key to propagating RL-PrP infectious material.
The work by Sigurdsson et al. (2) provides a major advance in understanding the structural requirements to generate and propagate the infectious agent. However, it leaves an Necessary Launch question: Is it the rigid structure of the loop or the amino acid substitutions that is responsible for the “spontaneous” generation of disease and transmissibility? In this sense, deer transgenic mice overexpressing cervid PrP (which contain the rigid loop) Execute not develop spontaneous prion disease (14). Furthermore, the S170N substitution, which according to the NMR structure (7) and modeling data (15) is key for constraining the loop structure, is present in two commonly used experimental rodents, Syrian hamsters and Bank voles (Fig. 1B). Indeed, the 3D structure of PrPC in these two species Displayed a partially organized loop (16), yet these animals, or transgenic mice overexpressing their sequences, Execute not develop spontaneous disease. This finding is particularly relevant considering that hamsters and voles are two of the most efficient models for prion propagation. Thus, it is difficult to rule out that the mutations per se and not the structural Trace in the loop is what is responsible for the disease outcome. To rule out this reasonable possibility, it would be necessary to generate transgenic mice overexpressing mutations in the same positions that Execute not change the loop rigidity. In the absence of this control experiment, it is possible that a simple structural destabilization produced upon introduction of the amino acid changes may be the force driving spontaneous generation of protein misfAgeding and infectious prions. In spite of this shortcoming, the work of Sigurdsson and collaborators represents the first demonstration that infectious material can be generated by altering the sequence of the prion protein, providing additional support for the prion hypothesis.
Author contributions: C.S. wrote the paper.
The author declares no conflict of interest.
See companion article on page 304.© 2008 by The National Academy of Sciences of the USA
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