Unexpected Tolerance of α-Cleavage of the Prion Protein to Sequence Variations

Abstract

The cellular form of the prion protein, PrPC, undergoes extensive proteolysis at the α site (109K↓H110). Expression of non-cleavable PrPC mutants in transgenic mice correlates with neurotoxicity, suggesting that α-cleavage is important for PrPC physiology. To gain insights into the mechanisms of α-cleavage, we generated a library of PrPC mutants with mutations in the region neighbouring the α-cleavage site. The prevalence of C1, the carboxy adduct of α-cleavage, was determined for each mutant. In cell lines of disparate origin, C1 prevalence was unaffected by variations in charge and hydrophobicity of the region neighbouring the α-cleavage site, and by substitutions of the residues in the palindrome that flanks this site. Instead, α-cleavage was size-dependently impaired by deletions within the domain 106–119. Almost no cleavage was observed upon full deletion of this domain. These results suggest that α-cleavage is executed by an α-PrPase whose activity, despite surprisingly limited sequence specificity, is dependent on the size of the central region of PrPC.

Knockout of the prion protein (PrP)-like Sprn gene does not produce embryonic lethality in combination with PrP C -deficiency

“…However, it is also to be considered whether the original formulation of the π hypothesis is compatible with our current knowledge of PrP C chemistry. For example, the π hypothesis envisages two discrete, covalently linked binding sites on PrP C (one N terminal, one C terminal) simultaneously docking onto the L PrP protein, yet we now know that much of PrP C at steady-state is endoproteolysed to separate the N-and C-terminal domains (31,(41)(42)(43). Furthermore, other hypotheses that also seek to explain the properties of internal PrP deletions neither invoke a PrP paralog nor consider action in embryonic development (44).…”

PrPCHomodimerization Stimulates the Production of PrPCCleaved Fragments PrPN1 and PrPC1

“…The HD is essential for PrP C homodimerization and ␣-cleavage (Rambold et al, 2008;Bremer et al, 2010;Oliveira-Martins et al, 2010). We sought to directly test whether PrP C homodimerization could modulate the ␣-cleavage.…”

“…Dimerization-mediated ␣-cleavage of PrP C is independent of ADAM8, 10, and 17 The two metalloproteases ADAM10 and ADAM17 were initially proposed to be the proteases that are responsible for constitutive and PKC-regulated ␣-cleavage of PrP C , respectively, yet their involvement is currently a matter of debate (Vincent et al, 2001;Taylor et al, 2009;Oliveira-Martins et al, 2010;Altmeppen et al, 2011). Recent results suggest that ADAM8 regulates PrP C ␣-cleavage in skeletal muscle (Liang et al, 2012).…”

“…Overall, these studies point to a possible neuroprotective activity associated with ␣-cleavage of PrP C . In favor of this hypothesis, PrP Sc and toxic PrP mutants are impaired in their ␣-cleavage (Oliveira- Martins et al, 2010).…”

“…However, ␣-cleavage occurs normally in neuron-specific ADAM10 knock-out mice and the search for other proteases that have the ability to perform the ␣-cleavage continues (Altmeppen et al, 2011). The hydrophobic domain (HD) (amino acids 111-129) of PrP C is essential for the ␣-cleavage both in cellulo and in vivo (Bremer et al, 2010;Oliveira-Martins et al, 2010). Interestingly, the HD is also essential for PrP C homodimerization and the stress-protective activity of PrP C (Rambold et al, 2008).…”