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Abstract: Elucidation of the structure of scrapie prion protein (PrPSc), essential to understand the molecular mechanism of prion transmission, continues to be one of the major challenges in prion research and is hampered by the insolubility and polymeric character of PrPSc. Limited proteolysis is a useful tool to obtain insight on structural features of proteins: proteolytic enzymes cleave proteins more readily at exposed sites, preferentially within loops, and rarely in β-strands. We treated PrPSc isolated from brains of hamsters infected with 263K and drowsy prions with varying concentrations of proteinase K (PK). After PK deactivation, PrPSc was denatured, reduced, and cleaved at Cys179 with 2-nitro-5-thiocyanatobenzoic acid. Fragments were analyzed by nano-HPLC/mass spectrometry and matrix-assisted laser desorption/ionization. Besides the known cleavages at positions 90, 86, and 92 for 263K prions and at positions 86, 90, 92, 98, and 101 for drowsy prions, our data clearly demonstrate the existence of additional cleavage sites at more internal positions, including 117, 119, 135, 139, 142, and 154 in both strains. PK concentration dependence analysis and limited proteolysis after partial unfolding of PrPSc confirmed that only the mentioned cleavage sites at the N-terminal side of the PrPSc are susceptible to PK. Our results indicate that besides the “classic” amino-terminal PK cleavage points, PrPSc contains, in its middle core, regions that show some degree of susceptibility to proteases and must therefore correspond to subdomains with some degree of structural flexibility, interspersed with stretches of amino acids of high resistance to proteases. These results are compatible with a structure consisting of short β-sheet stretches connected by loops and turns. [Copyright &y& Elsevier]

Abstract: Alzheimer’s disease and prion diseases (e.g., Creutzfeldt–Jakob disease) display profound neural lesions associated with aberrant protein processing and extracellular amyloid deposits. However, the intracellular events in prion diseases and their relation with the processing of the amyloid precursor protein (APP) and β-amyloid generation are unknown. The adaptor protein Dab1 may regulate intracellular trafficking and secretase-mediated proteolysis in APP processing. However, a putative relationship between prion diseases and Dab1/APP interactions is lacking. Thus, we examined, in inoculated animals, whether Dab1 and APP processing are targets of the intracellular events triggered by extracellular exposure to PrP(106–126) peptide. Our in vitro results indicate that PrP(106–126) peptide induces tyrosine phosphorylation of Dab1 by activated members of the Src family of tyrosine kinases (SFK), which implies further Dab1 degradation. We also corroborate these results in Dab1 protein levels in prion-inoculated hamsters. Finally, we show that fibrillar prion peptides have a dual effect on APP processing and β-amyloid production. First, they block APP trafficking at the cell membrane, thus decreasing β-amyloid production. In parallel, they reduce Dab1 levels, which also alter APP processing. Lastly, neuronal cultures from Dab1-deficient mice showed severe impairment of APP processing with reduced sAPP secretion and Aβ production after prion peptide incubation. Taken together, these data indicate a link between intracellular events induced by exposure to extracellular fibrillar peptide or PrPres, and APP processing and implicate Dab1 in this link. [Copyright &y& Elsevier]