Fig. 2
Biochemical analysis of spontaneously generated misfolded rec-PrPres to determine the fulfillment of key characteristics expected for bona fide prions. A Evaluation of self-propagation ability by serial PMSA passages. The spontaneously generated PMSA product, complemented with 1 mm zirconia silicate beads, was used to seed a fresh PMSA substrate at a 1:10 dilution. This was followed by a 24 h PMSA round. A total of 10 PMSA rounds, using a 1:10 dilution of the previous round as the seed, were performed. All reactions included 1 mm zirconia silicate beads, which promote propagation without spontaneous misfolding. The samples were concentrated approximately 50 times for improved detection and visualized by total protein staining. A stable and successful propagation was shown by the preservation of the original electrophoretic migration profile after PK digestion. B Evaluation of self-propagation ability by serial dilutions in PMSA. The spontaneously generated PMSA product was serially diluted from 10–1 to 10–9 in fresh substrate, supplemented with 1 mm zirconia silicate beads, and subjected to a single 24 h PMSA round. An unseeded sample served as a negative control. The samples were concentrated approximately 50 times for improved detection and visualized through total protein staining. After PK digestión, PrPres was detectable up to a dilution of 10–7, indicating high efficiency in in vitro propagation. C, D Evaluation of the proteinase K resistance of the spontaneously generated rec-PrPres. The resistance to proteinase K (PK) digestion of the spontaneously generated misfolded rec-PrP by PMSA was assessed using the product obtained from the serial PMSA propagation. The samples were digested with increasing concentrations of PK, ranging from 25 to 3000 µg/ml, at 42 °C and at room temperature (RT) for 1 h. The rec-PrPres propagated from the spontaneously generated seed exhibited high resistance, withstanding up to 2500 µg/ml at 42 °C and up to 3000 µg/ml at RT. This fulfilled another characteristic commonly observed in brain-derived prions. Additionally, the same sample underwent PK digestion with 100 µg/ml at 42 °C for 6 and 24 h, revealing resistance up to 6 h, with 10 kDa fragments remaining resistant even after 24 h of digestion. E Assessment of the capacity of the recombinant misfolded PrP to induce misfolding of PrPC from brain in vitro. To predict the potential in vivo infectivity of the spontaneously generated recombinant misfolded PrP (referred to as Spon. rec-PrPres) in PMSA, the capacity to induce misfolding of PrPC in brain homogenates of TgVole 1 × animals was evaluated using PMCA. The aggregates were purified through ultracentrifugation using a density gradient, resulting in two distinct visible halos of proteic aggregate (referred to Halo 1, h1, and Halo 2, h2). These purified fractions exhibited indistinguishable biochemical properties to the original product, as demonstrated by proteinase K digestion and retention of the same electrophoretic pattern. These purified fractions were used to seed a PMCA substrate based on TgVole 1 × brain homogenate at dilutions of 1:10 (10–1 and 10–2 dilutions), and single a 24 h PMCA reaction was performed. An additional seeded tube was included for each halo at the same 1:10 dilution but was not subjected to PMCA, as control of the signal of the seed. Two unseeded tubes were also included as control for spontaneous misfolding or cross contamination. After a 24 h PMCA round, PrPSc detection was carried out by proteinase K digestion (85 µg/ml, for 1 h at 42 °C) and Western blotting using mAb Sha31 at a dilution of 1:4000. Both fractions showed the capacity to induce PrPC misfolding up to dilution 10–2, resulting in a classical three-banded PrPSc pattern, indicative of potential in vivo infectivity. Furthermore, we assessed the propagation capacity of the same fractions on rec-PrP-based PMSA substrate using PMSA. Both halos were used as seeds at a 1:10 dilution on a substrate supplemented with zirconium silicate beads. Following a single 24 h PMSA round at 39 °C and 700 rpm, the presence of rec-PrPres was evaluated through proteinase K digestion, electrophoresis, and total protein staining. Both fractions were able to propagate in PMSA, exhibiting no detectable differences and maintaining the original electrophoretic patterns of the PMSA product. Uns.: Unseeded. rec-PrPSc: L-seeded-PMSA recombinant prion strain used as control [38]. rec-PrP: Untreated substrate. NBH control: Normal brain homogenate from TgVole 1x. Mw: Molecular weight