Evidence that lipid rafts are involved in human pathologies abounds (reviewed in (Fantini et al., 2002; Simons and Ehehalt, 2002)). As shown for other pathogens it has been hypothesized that prions exploit lipid rafts for entering cells (Simons and Ehehalt, 2002; van der Goot and Harder, 2001) (Fig. 28 and Fig. 29).
It seems that lipid rafts are a key element for the formation of PrPSc. Although the exact mechanisms remain obscure and are debated by researchers, some models could be envisioned.
1) Lipid rafts could act as transport platforms for taking PrPC to specific intracellular compartments where it could encounter PrPSc (Fig. 29A). Data from our own laboratory show that in transfected cells, treatments that destabilize the association of PrPC with lipid rafts does not alter its exocytic transport to the plasma membrane (Sarnataro et al., 2002), but slows down its endocytosis (Sarnataro et al., in preparation). These data support the hypothesis that rafts could be involved in regulating PrPSc conversion during endocytosis.
2) Lipid rafts might contain machinery such as proteins or lipids that are indispensable for the formation of PrPSc (Fig. 29B). This would be supported by the factor X-hypothesis (see paragraph III.12).
3) Lipid rafts may facilitate the encounters between PrPC and PrPSc molecules (e.g. by clustering the molecules) and therefore favour conversion (Fig.
29C). In this hypothesis in which lipid rafts serve as a meeting place, is indirectly supported by work from Baron et al., which showed that PrPC could be converted into PrPSc only upon insertion into contiguous membranes (Baron et al., 2002).
4) Another alternative could be that the interaction of PrP with different lipids produces different kinds of conformations (Fig. 29D). In this model, the different lipids could act as lipochaperones and facilitate/preserve the unfolding of "-helical or the refolding into !-helical conformations.
The notion that lipid domains somehow participate in the conversion process came at first from reports showing that both PrPC and PrPSc are associated with DRMs (Baron and Caughey, 2003; Baron et al., 2002; Botto et al., 2004; Naslavsky et al., 1997;
Taraboulos et al., 1992; Taraboulos et al., 1995). Additionally it was shown that prion-aggregates contained low amounts of two sphingolipids (galactosylceramide and sphingomyelin) usually found in lipid rafts (Klein et al., 1998b). It is important to stress that in some cases different lipid rafts appear to be hosting PrPC versus PrPSc and that these can be separated by solubilization and density floatation techniques (Naslavsky et al., 1997). This suggests that the conversion process is not just a matter of refolding the participating PrPC-protein but that PrPSc actively influences the surrounding lipid
raft-compartment (Fig. 29A) or that the two proteins have distinguishable affinities for different DRMs (maybe due to their folding differences) (Fig. 29B).
One interesting piece of evidence hinting at the role of rafts in conversion was a report showing that a transmembrane form of PrPC does not localize to lipid rafts anymore.
This form was also resistant to the conversion process while co-expressed normal PrPC was not (Kaneko et al., 1997; Taraboulos et al., 1995). The destabilization of lipid rafts by depleting cholesterol in living cells also resulted in inhibition of PrPSc-production (Mange et al., 2000; Taraboulos et al., 1995); however depletion of sphingolipids resulted in an increase of PrPSc-replication (Naslavsky et al., 1997). This suggests that lipid rafts might support the transconformation process.
Fig. 29: Two proposed models of how PrPC and PrPSc could interact with lipid rafts. Depending on their conformation, PrP-proteins could interact with different lipid rafts. A) PrPC is normally sequestered in lipid rafts. Exogenous PrPSc interacts with PrPC inside of lipid rafts and converts it; this leads to a change of PrP-interaction with the lipids of the rafts and therefore gives rise to the formation of distinct lipid rafts. B) Exogenous PrPSc has an affinity for lipid rafts that differs from PrPC and therefore gives rise to different lipid rafts. Coalescence of these different lipid rafts leads to transfer of PrPSc, conversion and formation of a bigger PrPSc-specific lipid raft (adapted from (Campana et al., 2005)).
On the other hand, other reports speak against this hypothesis and for a protective role
anchored PrPC is present in DRMs and resistant to conversion by PrPSc, unless its GPI-anchor is cleaved by PIPLC-treatment, which results in the release of PrPC from DRMs into the medium. Conversion of PrPC could also be achieved by promoting the fusion of different membranes containing PrPC or PrPSc by high levels of polyethylene glycol.
Interestingly this treatment also disrupts raft structures (Baron et al., 2002). Results obtained in our laboratory showed that cholesterol-depletion increased the amounts of misfolded PrPC (PK-resistant PrP) in the endoplasmatic reticulum and would therefore argue for a role of DRMs in stabilizing the tertiary structure of the cellular protein (Campana et al., 2006; Sarnataro et al., 2004).
Two independent reports suggested that lipids could act as lipochaperones and participate in the folding/misfolding of proteins (Bogdanov and Dowhan, 1999; Sanders and Nagy, 2000). Others reported that binding to membranes containing the monoganglioside GM1 supported the refolding of Alzheimer amyloid peptide A beta (1-40) towards a !-sheet enriched structure (Choo-Smith and Surewicz, 1997). The influence of lipids on conversion was also the subject of a recent report from Wang et al., who found that lipid interaction with recombinant PrP-protein initiated conversion of full-length "-helix-enriched protein to different forms, as well as to a !-sheet enriched form which was resistant to proteinase K digestion (Wang et al., 2007).
Recombinant PrP-protein was also utilized for studies on differences in lipid-affinity.
Critchley et al., showed that PrPC-resembling, "-helix-enriched PrP-forms ("-PrP) binds in decreasing order of affinity to membranes enriched in palmitoyl-phosphatidyl-glycerol (POPG), di-palmitoyl-phosphatidyl-choline (DPPC) and with lowest affinity lipids from lipid rafts (POPG > DPPC > lipids from lipid rafts), suggesting that at steady-state PrPC might be situated outside of rafts (Critchley et al., 2004). Other studies reported that binding of PrPC to lipid rafts stabilizes the "-helical structure of PrP, while interaction with negatively charged lipids (as found in higher amounts outside of lipid rafts) increases the content of !-sheets and even produces a disruptive effect on membranes (Sanghera and Pinheiro, 2002). The same group also reported that !-sheets enriched PrP is unfolded upon insertion into “lipid raft-like membranes” and is converted into fibrils (Kazlauskaite et al., 2003) arguing for a chaperone-like activity for lipids and a potentially protective role for lipid rafts in the conversion of PrPC -protein (discussed above).
It appears that the binding of PrPC to rafts could induce the folding of the unstructured N-terminal part of the protein and thereby stabilize the wild-type conformation of the protein. This notion is strengthened by findings that PrPC situated inside lipid rafts is more resistant to conversion to PrPSc (Baron et al., 2002) and also that the inhibition of sphingolipid-synthesis, and thereby perturbation of lipid raft composition, increases the production of PrPSc (Naslavsky et al., 1997). The appearance of exogenous PrPSc might negatively affect the stabilizing effect of lipid rafts or might be not available when PrPC exits lipid rafts during its endocytosis (Sunyach et al., 2003).
Data from our own laboratory in which lipid rafts were perturbed by cholesterol depletion, argue for a model in which lipid rafts act as a protective surrounding for PrPC and agrees well with the aforementioned reports that lipid rafts confer protection by stabilizing the "-helical folded form of the protein both in the case of PrPC and of some mutants (Campana et al., 2006; Sarnataro et al., 2004).
In contrast recombinant !-sheet-enriched protein has an altered affinity for lipid rafts and tends to produce fibrils therein (Kazlauskaite et al., 2003), showing that the role of lipid rafts in the pathogenesis is still unclear.
III.15: Exogenous prion-invasion and their dissemination in organisms