The particular conformation of PrPSc confers characteristics very different to its normally folded counterpart:
In contrast to PrPC, the solubility of PrPSc is diminished in non-ionic detergent (Caughey and Raymond, 1991; Meyer et al., 1986), a trait, which renders its purification and
A
B
Protein PrPSc PrPC
Structure globular stretched
Resistance to proteases yes no
Production of fibrils yes no
Turnover of protein days hours
Table 3: Comparison of PrPSc and PrPC
The high content of !-sheets of PrPSc renders the protein more stable and resistent to digestion by proteases, especially by proteinase K (PK). In contrast to PrPC, which is completely digested by PK, only the N-terminal part of PrPSc (specifically the first 67 aa) are hydrolyzed. This hydrolysis leaves a large part of the protein undigested and results in an electrophoretic product that migrates now at the characteristic height of 27-30 kDa and is therefore called PrPres (res for resistant to digestion) or PrP 27-27-30. This intrinsic resistance to digestion with PK is a hallmark of PrPSc that distinguishes it from PrPC (Fig. 16).
Fig. 16: PrPSc is partly resistant to digestion with PK. Upon addition of PK (+PK) a N-terminal truncated PrP-form remains undigested in the case of PrPSc, migrating on a polyacrylamide gel at a height of 27-30 kDa. Please note that while PrPC is completely digested, PrPSc gives three bands, which remain nearly undiminished in terms of intensity but shifted to a lower molecular weight. The three distinct bands represent the three glycosylated forms of PrP (un-, mono and diglycosylated). (from (Riesner, 2003))
III.9: The conformational change in vivo
Two models were proposed to explain how PrPSc is propagated by conversion of endogenous PrPC (Fig. 17 and (Come et al., 1993; Harper and Lansbury, 1997; Prusiner, 1991; Prusiner, 1998)).
Fig. 17: Models for the conformational conversion of PrPC to PrPSc. A. Depicted in A is the
“template-directed refolding model” also termed “template assistance”, which hypothesizes an interaction between the exogenously introduced PrPSc and the endogenously produced PrPC. This interaction induces PrPC to adopt the malconformation of its interactor and turn itself into PrPSc. A high energetic barrier prevents the spontaneous transconformation of PrPC to PrPSc. B. The “seeded nucleation” model also termed “nucleation-polymerization model” proposes a reversible thermodynamic equilibrium between PrPC and PrPSc in cells. Only when PrPSc monomers organize themselves into a so-called “seed” do PrPSc -molecules reach an increased stability and become capable of recruiting new PrPSc monomers in order to form an amyloidal structure. The fragmentation of aggregates of PrPSc increases the number of seed-nuclei, which once again can recruit, and stabilize PrPSc-monomers and thereby achieve a replication of the agent (from (Aguzzi and Polymenidou, 2004)).
The “refolding” or “template-directed” model (Fig. 17, A) proposes that PrPSc is a template or matrix for the conversion of PrPC into new PrPSc monomers (Prusiner, 1991). On the other hand the “seeded nucleation model” (Fig. 17, B), posits that PrP can
equilibrium. Under physiological conditions the PrPC-conformation is highly favoured, due to the high activation energy, which is necessary for the acquisition and maintenance of the PrPSc-conformation (Come et al., 1993; Harper and Lansbury, 1997).
The presence of a PrPSc-aggregate shifts this equilibrium towards PrPSc by stabilizing this conformational form and achieves a rapid accumulation of new monomers to the aggregate (Jarrett and Lansbury, 1992).
This hypothesis fits well with the experimental finding that the conversional activity and infectivity are specifically associated with the aggregates and not with the monomers of PrPSc (Weissmann, 2004). Indeed, it was reported that disaggregation and denaturation of PrPSc (but not the reversible loss of conformation) coincided with the loss of conversional activity and infectivity (Morillas et al., 2001). As a consequence, while the process of spontaneous misfolding of PrPC might be a slow event, the pathological conversion with the help of a PrPSc-molecule would be a quick process, suggesting that it is the aggregates, which induce the formation of prions. The occasional fragmentation of aggregates could also explain the exponential growth of PrPSc during infection (Orgel, 1996). In addition more recently it has been proposed that oligomeric PrPSc might be just as infectious and toxic as bigger aggregates (Chiesa and Harris, 2001; Novitskaya et al., 2006; Silveira et al., 2005; Simoneau et al., 2007).
It is possible that in the case of sporadic CJD and genetic TSEs, mutations in the gene prnp generate a protein whose structure is more favourable to conversion, while in the infectious form it is the contact with exogenous PrPSc, which leads to progressive misfolding.
Elegant in vivo and in vitro studies have shown that sequence homology plays a very relevant role for pathological conversion of PrPC to PrPSc. Mice co-expressing a murine and a hamster prion-form, but infected with mouse-prions produced only PrPSc containing mouse-prions, while infection of littermates with hamster-prions resulted in the production of PrPSc containing only hamster-prions (Prusiner et al., 1990).
Furthermore, co-expression of a nonconvertible PrP-deletion mutant in scrapie-producing MNB-cells lead to a reduction of the infectious protein in these cells (Holscher et al., 1998) underlining that protein homology is required for the process of conversion and infection.
Another variable influencing the conversion reaction is the speed of synthesis of PrPSc from PrPC: continuous exposure of infected scN2a cells to phosphatidylinositol-specific phospholipase C (PIPLC), an enzyme which cleaves all GPI-anchored proteins from the
cell membrane (including PrPC but not PrPSc) resulted in depletion of membranous PrPC, and leading to the cure of these cells (Enari et al., 2001). Charles Weissmann put these findings into perspective in his model of “dynamic susceptibility”, in which he proposes that the capacity of cells to sustain and propagate infection by prions depends on equilibrium between synthesis and degradation of PrPSc (Weissmann, 2004).
According to this model, if the rate of synthesis of PrPSc is not at least equal to or twice superior to its rate of degradation, prions will be eliminated from infected cells. Only when the synthesis rate is two times higher than its degradation rate, can it overcome its dilution by cell division and manage to accumulate in cells. Prions manage to propagate only by keeping this delicate balance. This model explains why certain cell systems are not susceptible to prion infections (Race et al., 1987; Rubenstein et al., 1984; Schatzl et al., 1997; Vilette et al., 2001; Vorberg et al., 2004a; Vorberg et al., 2004b) however this does not explain why some cell lines are immune to some prion strains but susceptible to others (Bosque and Prusiner, 2000; Nishida et al., 2000; Race et al., 1987).
III.10: Prion strains and the species barrier phenomenon
By the 1920’s the existence of two different scrapie-forms in sheep was described: one was called the “nervous” form while the other was termed the “pruritic” form. Even though the existence of prion strains is now quite well established (Bruce, 1993), this remains one of the most poorly understood phenomena of prion research and is especially hard to reconcile with the protein-only hypothesis. Work with different prion strains began in the early 1970’s (Dickinson et al., 1968), by infecting laboratory animals with infectious material derived from wild animals. This technique quickly established itself as the standard procedure for the study, propagation and ultimately characterization of prion strains: Wild-type prion strain isolates are inoculated into murine animal models and are successively passaged to genetically identical animals utilizing the same amount of infectious agent, until the properties of the strain stabilizes in this new host species.
The criteria serving to define a new prion strain are:
i) The time of incubation of the strain once stabilized. This information is linked to the nature of the strain, the genetic predisposition of the host mice and the amount of the inoculum.
ii) The lesion profile caused by the infection in the brain of the host animal. This corresponds to its spatial distribution (and deposition) in the brain, as well as to the severity of the vacuolization. It is worthwhile to mention that the same isolate can lead to the isolation of different strains. The most interesting example of this was found when an isolate from a mink hosting transmissible mink encephalopathy (TME) was inoculated into Syrian golden hamsters. Infection of littermate hamsters resulted in two clearly distinguishable syndromes, termed hyper (HY) and drowsy (DY). These two strains are distinguishable by several key features: incubation period (HY: 65 days; DY:
168), clinical signs (HY: hyperaesthesia; DY: lethargy), titres of infection (HY: 109,5 LD50/g of tissue; 107,4 LD50/g of tissue) and pathogenesis (only the DY strain retained virulenece in mink) (Bessen and Marsh, 1992).
These types of studies also enabled researchers to find similarities between BSE and new variant CJD (nvCJD), supporting the notion that these two strains are closely related and giving the opportunity to distinguish them from sporadic CJD (Bruce et al., 1997).
Nevertheless this approach also has some disadvantages. First, it is time consuming, since some strains require passaging for a few years until properly stabilized. Secondly, it is costly, since hundreds of mice have to be kept at a high security level and third it is also complex, since the isolation of strains and analysis of their properties are challenging tasks not necessarily giving consistent results.
Alternative methods, basing on a biochemical approach (see below), aim at a much faster and easier characterization of the different prion species.