Prion seeded conversion

Since the development of the first PrP in vitro conversion reaction [2], much more efficient, continuous, and sensitive prion-seeded conversion assays have been developed. These techniques have been used to investigate prion composition and propagation mechanisms as well as prion strain and transmission barrier phenomena. Moreover, these reactions serve as bases for ultra-sensitive prion detection that should facilitate TSE diagnostic tests and screening assays for medical, agricultural and environmental prion contamination. 

The fact that so far the infectivity of PrP in in vitro conversion products as been shown to be lower than that of bona fide PrPSc suggests that we are still missing important information about the conversion process and how to create an infectious prion. The prion-seeded conversion reactions described in this chapter provide valuable tools to investigate these issues. 

Fig. 5 Models of prion replication, (a) The template assistance model predicts that a PrPSo monomer is more stable than PrPc, but is kinetically inaccessible. In the rare event that a PrPSo monomer is created spontaneously (or provided exogenously), it can template the misfolding of another PrPc molecule by direct interaction. The dashed line shows that the newly created PrPSc monomer can act as another seed to formation of PrPSc. (b) The nucleation polymerization model predicts that barrier to prion protein conversion is the formation of a nucleus in which the protein adopts a PrPSo-like structure. The formation of such a low order aggregate is not favored however, once it has formed, polymerization from a pool of PrPc molecules can take place efficiently. Fragmentation of the polymer increases the number of ends for the recruitment of PrPc monomers...

Lee S, Eisenberg D (2003) Seeded conversion of recombinant prion protein to a disulfide-bonded oligomer by a reduction-oxidation process. Nat Struct Biol 10 725-730... 

Atarashi R, Moore RA, Sim VL et al (2007) Ultrasensitive detection of scrapie prion protein using seeded conversion of recombinant prion protein. Nat Methods 4 645-650... 

Like any other protein, the molecular structure of the prion is subject to conformational flexibility and to various thermal-induced fluctuations between varying conformational states. However, if these fluctuations permit the PrP conformation to be attained, then this abnormal conformer promotes the widespread conversion of PrP to PrP , leading to the precipitous deposition of the abnormal protein throughout the brain (mirrored by the rapid and relentlessly downhill clinical course). This pathological self-propagating shape conversion of a-helical PrP to P-sheet PrP may in principle be initiated by a seed PrP molecule in the neurotoxic conformation. This explains the transmissibility of prion diseases and accounts for how susceptible humans exposed to beef from an animal with mad cow disease develop variant Creutzfeldt-Jakob disease. 

A hard-to-understand aspect of the "protein-only" theory of prion diseases is the existence of various "strains" of prion proteins. These do not involve differences in amino acid sequence but differences in the conformations of the PrPSc forms and in the glycosylation patterns. dmw How can there be several different conformations of the same protein, all of which seed the conversion of normal PrP into differing insoluble forms In spite of this puzzle, support for the explanation of strain differences comes from a yeast prion system, which involves transcription termination factor eRF3.x z In this system, which involves a prion whose insoluble form can be redissolved by guanidine hydrochloride,aa differing strains have also been described.ybb cc Nevertheless, the presence of the various strains of animal prions, as well as observed vaccination of inbred mice against specific strains,dd may be more readily understood if the disease is transmitted by an unidentified virus rather than by a pure protein.1/U ee/ff In fact, the diseases have not been successfully transmitted by truly virus-free proteins synthesized from recombinant DNA.ee... 

The ability of PrPres to induce the conversion of PrPc to PrPres was initially demonstrated in cell-free reactions in which brain-derived PrPres was incubated with radioactively labeled PrPc, which, under suitable conditions, bound to the PrPres and became similarly partially protease-resistant [2], These first generation cell-free conversion (CFC) reactions were shown to be highly specific in ways that correlated with prion transmission barriers [3-7] and strains [8]. However, the newly generated PrPres was usually substoichiometric relative to the initial PrPres seed, and was not demonstrably associated with new infectivity [9]. As a result, these CFC reactions (reviewed in [10]) were not suitable for sensitive detection of PrPres or prions. 

In the simplest model of [PS7] metabolism, de novo conversion from the [psi to the PS/+ state involves two discrete steps initiation and propagation. During initiation, the first replicon (e.g., seed or nucleus) is formed. During propagation, newly synthesized protein is influenced by the replicon to convert to the [PS7+] state. Since the [PS7+] read-out is a colony-based assay (reversion to prototrophy), these steps are nearly impossible to separate. However, recent studies of [PS7+] interaction with other proteins, chemical agents, and prions have provided our first glimpses into the multistep process of prion induction (Fig. 1). 

The central issue in the progression of disease is how PrP = propagates itself from the source of exposure (food) to the brain. In the examples provided by kuru, the recent mad cow epidemic in the United Kingdom, and the resultant transmission of vCJD to humans, the common link is that infectious prions in contaminated food reached the brain by some mechanism, and once in the brain, seeded the conversion of native host PrP to an infective PrP conformation. 

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