A model for HC function in the prion conversion process

The observed resistance of mice harboring Prnp 114-121 to PrP^Sc inoculation clearly demonstrates the dominant negative effect on prion conversion by the deletion of the hydrophobic core region in vivo. However, the underlying mechanisms remain enigmatic.

Upon transition of PrP^C into PrP^Sc, the protein adopts a significant change in its secondary adopted preferentially -sheet structure (Heller et al., 1996; Satheeshkumar & Jayakumar, 2003), and the peptide 105-125 aggregated into fibrils and exhibited cellular neurotoxicity (Ettaiche et al., 2000; Singh et al., 2002).

Besides the region around the hydrophobic core, also further C-terminal PrP segments are involved in the conversion process. A single point mutation, Q218K, within a proposed conformational discontinuous epitope, for example, led also to the inhibition of prion conversion by an altered interaction between PrP^C and PrP^Sc (Horiuchi & Caughey, 1999;

Horiuchi et al., 2001; Kaneko et al., 1997).

Interestingly, Q218K could only elicit its dominant negative effect when the flexible segment around HC was still present. Deletions of the N-terminal part of PrP prevented the inhibitory impact of Q218K on prion conversion (Zulianello et al., 2000). In contrast, mutations of the hydrophobic core exhibited a dominant negative effect on their own (Holscher et al., 1998;

Norstrom & Mastrianni, 2005). These results not only demonstrate the pivotal role of these

124 two regions in PrP^Sc conversion, they also indicate a possible interdependence of HC and the C-terminus for the conversion process.

Nordstrom and Mastrianni explained this phenomenon by the direct binding of PrP^Sc to HC and the discontinuous C-terminal epitopein a coordinate manner. The mutation of either site would thereby lead to an improper alignment of PrP^Sc and PrP^C for the conversion process. If both sites were missing, every possible interaction would have been canceled, leading to the loss of the dominant negative effect (Norstrom & Mastrianni, 2005).

In contrast to this model, recent studies demonstrated that the rate limiting and decisive step for prion conversion, as well as for the dominant negative effect, is not associated with the binding of PrP^C to PrP^Sc, but rather with the subsequent structural conversion (Horiuchi et al., 2000; Lee et al., 2007; Rigter & Bossers, 2005).

In addition, there are a couple of evidences arguing for the binding of PrP^Sc to the adjacent regions around HC but not to the hydrophobic core directly.

For example, peptides encompassing farther C-terminal PrP regions, such as PrP118-135 were able to perturb the conversion, probably by interfering with a binding site for PrP^Sc (Chabry et al., 1998; Chabry et al., 1999). Furthermore, the deletion of segments which precede HC, was already sufficient to abolish the dominant negative effect of Q218K, arguing for a second binding site of PrP^Sc ahead of the hydrophobic core, most probably between aa 107-111 (Zulianello et al., 2000). Strikingly, this region encompasses the restriction site for PrP -cleavage (aa 109-111), which might therefore exert a protective function against prion conversion (see also Chapter 5.1.5) by destroying an essential site for PrP^Sc binding (Figure 47b), similar to the protective effect of -cleavage in Alzheimer’s disease (Checler & Vincent, 2002; Chen et al., 1995).

Taken these different evidences together, the binding of PrP^Sc to PrP^C is not mediated by HC during the conversion process. The binding seems to occur at the N- and C-terminal adjacent regions, thus allowing the core a certain degree of flexibility. This structural flexibility of HC and its upstream N-terminal region (aa 90-120 altogether) rather enables this segment to perform the transformation into -sheet structure during the conversion process (Peretz et al., 1997). This major structural alteration is proposed to serve as a “nucleation point from which conformational change can disseminate to other parts of the protein” (Leclerc et al., 2001).

Discussion

125 Hence, the decisive impact of the flexible segment on prion conversion is based on its function as a structural “hinge” or “switch”, initiating structural changes of further essential protein parts, such as the conversion of the discontinuous epitope within the C-terminal globular domain (Horiuchi & Caughey, 1999; Kaneko et al., 1997).

The observation that PrP 114-121 indeed revealed a slight increase protein stability compared to PrP-wt (Thaa et al., 2007), further supports the model of a reduced structural flexibility of PrP^C by the deletion of HC as the decisive factor for the dominant negative effect of PrP 114-121, rather than the loss of a PrP^Sc binding site at the hydrophobic core:

The deletion of HC in the mutant PrP 114-121 eliminates a crucial part of the “hinge”, which is no longer able to pass on structural changes to the C-terminus, consequently leading to the abortion of the conversion process. Since PrP^Sc binding is not affected by the deletion of the hydrophobic core, whereas the subsequent conversion of PrP^C into a “mature” prionform is eliminated, the bound PrP^Sc becomes “trapped” by the inconvertible PrP 114-121 molecule, consequently leading to the inhibition of the conversion process (Figure 47a).

This dominant negative inhibitory effect on the prion conversion process by of PrP 114-121 further underlines the importance of the highly conserved hydrophobic core as a focal point of prion conversion.

126

Figure 47: Impact of HC on prion conversion

(a) Physiological PrP^C (see Chapters 5.4 and 5.5) elicits a protective receptor signal. PrP^Sc switches the N-terminal flexible “hinge” region, including HC, into a -sheet rich conformation, resulting in an intermediate “lethal”

state of PrP^C, termed PrP^L and in the activation of a toxic receptor signal. The initial structural alteration of the

“hinge” enables further C-terminal conformational changes, which leads to the loss of receptor binding. The liberated, “mature” PrP^Sc conformation in turn is able to bind and to convert further protective PrP-wt molecules.

The deletion of HC in PrP 114-121 renders the mutant in a resistant PrP^N conformation (see Chapter 5.4 and 5.5) with regard to prion conversion. PrP^Sc can still bind to PrP 114-121; however, it is no longer able to convert the mutant into a toxic PrP^L or an infectious PrP^Sc form, resulting in the “trapping” of PrP^Sc to PrP 114-121 and in a dominant negative inhibition of the conversion process (grey).

(b) PrP^C -cleavage destroys the “hinge” of PrP, leading to a similar dominant negative inhibition as the mutant PrP 114-121.

Discussion

127 5.3.5 Different disease phenotypes in inoculated mice

Interestingly, inoculated transgenic PrP 114-121 mice on the knockout background developed pathological symptoms when they reached old age (between 400 - 500 days post infection).

However, the characteristics of the observed pathology differed markedly from the disease phenotype in inoculated PrP-wt mice, manifested in an overall less severe vacuolation and astrocytic gliosis (Figure 40, Figure 41) and most explicit in the inverse expression pattern of activated astrocytes at the corpus callosum and the cerebral cortex (Figure 42; see also Chapter 5.5.3).

In addition, even non-inoculated transgenic and non-transgenic mice on the knockout background exhibited the same type of pathology, strongly indicating that these pathologic alterations were not caused by the conversion of the transgene into a toxic form by PrP^Sc. These observations substantiate previous results, reporting the same type of pathology in aged Prnp-knockout mice (Nazor et al., 2007). Furthermore, the overexpression of other mutations, which also exerted a strong dominant negative inhibitory effect on prion conversion, also led to prion inoculation independent neurodegenerative pathology (Perrier et al., 2002).

The observed pathology in inoculated transgenic and non-transgenic mice on the knockout background was therefore independent from the inoculation with prions; it was rather caused by the loss or decreased activity of the physiological function of the prion protein or by an intrinsic toxicity of PrP 114-121 – or both (see Chapter 5.5).