Potential amyloidogenic hot-spots Epigenetics within the sequence 127?43. Thus immediate fibrillization of prion peptide mPrP(127?43) might happen due to the presence of amyloidogenic hot-spots within this region that can act as a nucleation site. While investigating the cross-seeding ability of mPrP(23?30) seed in the fibrillization of mPrP(107?43) monomer, the lag time was found to be shortened compared to the unseeded reaction, but not eliminated. This is in agreement with the `surface competition hypothesis’ that we proposed earlier [39]. Based on this hypothesis the process of seed induced fibrillization is a two steps phenomenon which involves initial 15481974 `docking’ – the association of monomer with the preexisting seed followed by `locking’ i.e., the Epigenetic Reader Domain formation of cross-b structure between incoming monomer and the seed. In presence of protein segments not involved in the formation of amyloid core, the probability of incorrect binding between incoming monomer and seed becomes fairly high enough that leads to a detectable lag time for the reason that incorrect binding cannot go through subsequent elongation step resulting in a delay of fibril growth. Pre-digestion of the fibrils with PK reduces the probability of incorrect binding and favors the association between the monomer and the seeding nucleus thus shortening the lag phase greatly. Hence our data infer that the residues not involved in the structural conversion process could contribute to the species barrier in prion transmission [39]. Bocharova et al. reported that the PK-digested mPrP(23?30) fibrils harbor an epitope of a monocolonal antibody D18, part of which spans amino acid residues 133?43. This result also supports ourMouse Prion Amyloid 1315463 Has Sequence 127?43 in CoreFigure 6. Amyloid fibril formation by mPrP(23?30) monomer cross-seeded with mPrP(107?26) seed or mPrP(127?43) seed. mPrP(23?30) (22 mM) in 1 M GdnHCl, 3 M urea in PBS, pH 6.0, was incubated at 37uC with vigorous shaking alone (in (A) as well as (B) denoted by closed, half-filled and open squares for three independent measurements) or in the presence of (A) 50 mL of mPrP(107?26) seed (denoted by closed, half-filled and open circles for three independent measurements) containing 1.8 nmoles of mPrP(107?26) per microliter or (B) 20 mL of mPrP(127?143) seed (denoted by closed, half-filled and open up triangles for three independent measurements) containing 43.5, 36 and 57 pmoles (for three independent measurements) of mPrP(127?43) monomer per microliter. doi:10.1371/journal.pone.0067967.gconclusion that sequence 127?43 is in the PK resistance core of amyloid fibrils derived from mPrP(23?30) [46]. Cross-seeding of mPrP(23?30) monomer with mPrP(107?26) seed failed to shorten the lag time, even though a very high amount of mPrP(107?26) seed was used, indicating the inability of the full-length monomer to interact with the seed. The recruitment of a new monomer to a preexisting nucleus/fibril is an essential step in amyloid propagation. Successful cross-seeding relies on conformational adaptability between monomer and seed. Although, theoretically, all peptides or proteins have the potential to form amyloid structure and many prion peptides have been reported to be able to form amyloid fibrils, the lack of conformational adaptability between a specific monomer/seed pair might impede the successful propagation of prion amyloid formed from that particular monomer. Amyloid generated from mPrP(107?26) appears to be inaccessible to mPrP(23.Potential amyloidogenic hot-spots within the sequence 127?43. Thus immediate fibrillization of prion peptide mPrP(127?43) might happen due to the presence of amyloidogenic hot-spots within this region that can act as a nucleation site. While investigating the cross-seeding ability of mPrP(23?30) seed in the fibrillization of mPrP(107?43) monomer, the lag time was found to be shortened compared to the unseeded reaction, but not eliminated. This is in agreement with the `surface competition hypothesis’ that we proposed earlier [39]. Based on this hypothesis the process of seed induced fibrillization is a two steps phenomenon which involves initial 15481974 `docking’ – the association of monomer with the preexisting seed followed by `locking’ i.e., the formation of cross-b structure between incoming monomer and the seed. In presence of protein segments not involved in the formation of amyloid core, the probability of incorrect binding between incoming monomer and seed becomes fairly high enough that leads to a detectable lag time for the reason that incorrect binding cannot go through subsequent elongation step resulting in a delay of fibril growth. Pre-digestion of the fibrils with PK reduces the probability of incorrect binding and favors the association between the monomer and the seeding nucleus thus shortening the lag phase greatly. Hence our data infer that the residues not involved in the structural conversion process could contribute to the species barrier in prion transmission [39]. Bocharova et al. reported that the PK-digested mPrP(23?30) fibrils harbor an epitope of a monocolonal antibody D18, part of which spans amino acid residues 133?43. This result also supports ourMouse Prion Amyloid 1315463 Has Sequence 127?43 in CoreFigure 6. Amyloid fibril formation by mPrP(23?30) monomer cross-seeded with mPrP(107?26) seed or mPrP(127?43) seed. mPrP(23?30) (22 mM) in 1 M GdnHCl, 3 M urea in PBS, pH 6.0, was incubated at 37uC with vigorous shaking alone (in (A) as well as (B) denoted by closed, half-filled and open squares for three independent measurements) or in the presence of (A) 50 mL of mPrP(107?26) seed (denoted by closed, half-filled and open circles for three independent measurements) containing 1.8 nmoles of mPrP(107?26) per microliter or (B) 20 mL of mPrP(127?143) seed (denoted by closed, half-filled and open up triangles for three independent measurements) containing 43.5, 36 and 57 pmoles (for three independent measurements) of mPrP(127?43) monomer per microliter. doi:10.1371/journal.pone.0067967.gconclusion that sequence 127?43 is in the PK resistance core of amyloid fibrils derived from mPrP(23?30) [46]. Cross-seeding of mPrP(23?30) monomer with mPrP(107?26) seed failed to shorten the lag time, even though a very high amount of mPrP(107?26) seed was used, indicating the inability of the full-length monomer to interact with the seed. The recruitment of a new monomer to a preexisting nucleus/fibril is an essential step in amyloid propagation. Successful cross-seeding relies on conformational adaptability between monomer and seed. Although, theoretically, all peptides or proteins have the potential to form amyloid structure and many prion peptides have been reported to be able to form amyloid fibrils, the lack of conformational adaptability between a specific monomer/seed pair might impede the successful propagation of prion amyloid formed from that particular monomer. Amyloid generated from mPrP(107?26) appears to be inaccessible to mPrP(23.
