A hallmark of most TSEs is the conversion of the physiological PrPC into its pathophysiological form, PrPSc. This process either occurs on the outer leaflet of the plasma membrane (Paquet et al., 2007) or intracellularly, after endocytosis of PrPSc (Peters et al., 2003). In cell culture experiments, the interaction between the cellular and the scrapie form of PrP is thereby dependent on accessory factors such as the laminin receptor and heparan sulfates (HS) (Figure 7). These factors directly bind to PrP and mediate the conversion as well as possibly the internalization of PrPSc into the cell (Gauczynski et al., 2001b; Horonchik et al., 2005;
Hundt et al., 2001; Paquet et al., 2007).
Figure 7: Prion conversion, assisted by co-factors
The conversion of PrP is mediated by accessory factors, such as heparan sulfates (grey), expressed by proteoglycans (green). These glycosaminoglycans bind both PrP conformations, thereby supporting PrPC and PrPSc interaction during the conversion process and also prion uptake into the cell.
From: (Horonchik et al., 2005)
16 During the conversion of PrPC into PrPSc, a portion of the -helices and the coil structure is refolded into β-sheets (Figure 8). Whereas PrPC consists of 42% α-helices and only 3% β-sheets, PrPSc is composed of 30% α-helical and 43% β-sheet structures (Pan et al., 1993).
Figure 8: Structural changes during the prion conversion process
Upon prion conversion, PrPC (a), mainly consisting of helical content, adopts a predominantly helical structure, becoming PrPSc (b). It should be noted that the structure shown in panel b is hypothetical.
From: http://www.sciscape.org/articles/madcow/
These structural changes render the protein partially resistant to proteinase K (PK) digestion, leading to an indigestible fragment of 27-30 kDa (= PrP 27-30). By contras the first 67 amino acids of the N-terminus remain digestible by proteinase K (Basler et al., 1986; Bolton et al., 1984; Oesch et al., 1985). The change in the tertiary structure of PrP is also responsible for other PrPSc-specific properties like detergent insolubility or loss of accessibility of the GPI-anchor to phosphatidylinositol-specific phospholipase C (PI-PLC) (Castle et al., 1987; Harris, 1999).
Of particular significance seems to be the folding of an N-terminal unstructured region between amino acids 90-120, comprising STE and HC, into β-sheet structure (Huang et al., 1996b; Peretz et al., 1997). Deletions in this region abrogate the convertibility of PrPC, and when overexpressed in cell culture, deletion mutants of the hydrophobic core exhibit
trans-Introduction
17 dominant inhibition of PrPSc accumulation in vitro (Hölscher et al., 1998; Norstrom &
Mastrianni, 2005). The importance of this region for PrP conversion is also supported by a truncated protein version, consisting of aa 105-125, which features PrPSc like properties (Singh et al., 2002).
This structural flexible hydrophobic core region may adopt short-lived intermediate conformations, which need further environmental influences in order to be stabilized (Liu et al., 1999). One possible intermediate conformation is the formation of -helical content between amino acids 90-124, including the hydrophobic core, which is maintained by the dimerization of two PrPC molecules (Kaimann et al., 2008). Due to its structural flexibility however, this region does not remain in one conformation, it rather swaps from a structured, -helical state into a variable, partially denatured state back and forth. The partial denaturing of the N-terminus turns PrPC into a preamyloid state, consequently lowering its activation barrier, which facilitates the conversion into a more stable -sheet structure (Stohr et al., 2008). This structural conversion of a predominantly -helical PrPC into a -sheet rich PrPSc needs two major steps: First, PrPC has to bind to a PrPSc molecule. This interaction leads in a second step to a structural change of PrPC resulting in the formation of a new PrPSc molecule.
These two steps could either occur at the same site, or PrPSc binding and the actual conversion might take place at two distinct regions of PrPC (Horiuchi et al., 2000). Fragments of PrP, inhibiting PrPSc binding downstream of HC, in combination with antibody binding and PrP-deletion mutant experiments, suggest that the first step, PrPSc binding, occurs not directly to but in the vicinity of the hydrophobic core, whereas this region is responsible for a major structural change upon prion conversion (Hölscher et al., 1998; Horiuchi & Caughey, 1999; highlighting the HC as a focal point of prion conversion. Furthermore, these deletion mutants exhibit a dominant negative inhibition of the conversion process in persistently infected cell cultures (Holscher et al., 1998; Norstrom & Mastrianni, 2005). Next to this N-terminal region, the C-terminal globular domain also contributes to the conversion process. Different inherited
18 point mutations are located within or near the α-helices, thus leading to a destabilization of these motifs (Huang et al., 1994; Riek et al., 1996), and deletions in either of the three α-helical PrP elements lead to a resistance to PrPSc infection, indicating that PrPSc formation is dependent on structural changes within these regions (Muramoto et al., 1996). In addition, a single point mutation, Q218K, is sufficient to abrogate PrPSc formation in cell culture and in vivo (Kaneko et al., 1997; Perrier et al., 2002).
The abrogation or at least the slowdown of the conversion process by dominant negative mutants of the prion protein has made these constructs a possible therapeutic agent for prion diseases. The mutant Q218K has been used in lentiviral gene transfer (Crozet et al., 2004) and for the intracerebroventricular administration, which significantly prolonged the survival of infected mice (Furuya et al., 2006).
As shown for mutant prion proteins causing heritable CJD, PrPSc does not acquire its specific properties in a single step - it is rather a multilevel process whereby PrPSc is converted in three steps acquiring one special feature after another, thus passing through different intermediate states until “maturation” to PrPSc is complete.
At first, PrPC becomes PI-PLC resistant. Secondly, the prion protein gets detergent insoluble (ca. 1h after synthesis), which presumably correlates with PrP aggregation, and lastly, 1-6 hours after synthesis, the protein acquires PK-resistance, thus becoming a “mature” PrPSc. Whereas the first step might already occur within the cell, possibly during protein traveling through ER and Golgi, the last two steps happen either in lipid rafts on the cell membrane, where PrPC and PrPSc can both be co- localized, or in endosomal compartments after endocytosis.
The nature of this multilevel process suggests that besides PrPSc, additional proteins are involved in the conversion that direct the conversion process towards the “maturation” of the scrapie protein (Caughey & Raymond, 1991; Daude et al., 1997; Naslavsky et al., 1997).
Introduction
19