Deuterium incorporation for peptic fragments derived from MM1 rPrP^Sc (red) and MM2 rPrP^Sc(blue).

<p>(a) 5 min incubation in D₂O. (b) 240 h incubation in D₂O. Error bars indicate standard deviation (3 independent experiments). *, P<0.05; **, P<0.02.</p

Structural Determinants of Phenotypic Diversity and Replication Rate of Human Prions

<div><p>The infectious pathogen responsible for prion diseases is the misfolded, aggregated form of the prion protein, PrP^Sc. In contrast to recent progress in studies of laboratory rodent-adapted prions, current understanding of the molecular basis of human prion diseases and, especially, their vast phenotypic diversity is very limited. Here, we have purified proteinase resistant PrP^Sc aggregates from two major phenotypes of sporadic Creutzfeldt-Jakob disease (sCJD), determined their conformational stability and replication tempo <i>in vitro</i>, as well as characterized structural organization using recently emerged approaches based on hydrogen/deuterium (H/D) exchange coupled with mass spectrometry. Our data clearly demonstrate that these phenotypically distant prions differ in a major way with regard to their structural organization, both at the level of the polypeptide backbone (as indicated by backbone amide H/D exchange data) as well as the quaternary packing arrangements (as indicated by H/D exchange kinetics for histidine side chains). Furthermore, these data indicate that, in contrast to previous observations on yeast and some murine prion strains, the replication rate of sCJD prions is primarily determined not by conformational stability but by specific structural features that control the growth rate of prion protein aggregates.</p></div

Sedimentation velocity, conformational stability, and seeding potency of isolated sCJD prions.

<p>(a) Distinct sedimentation velocity profiles of MM1 and MM2 prions. The samples were fractionated by ultracentrifugation in sucrose gradient and fractions were collected from the bottom of the tubes and analyzed for rPrP^Sc by CDI. The bars represent average ± SEM; CDI was performed on each sCJD sample in triplicate. (b) The conformational stability of MM1 and MM2 rPrP^Sc. The curves represent best fit to a sigmoidal function. The values of apparent fractional change (Fapp) are mean ± SEM obtained from four batches of purified MM1 and MM2 prions, each determined in triplicate measurements. (c) Amplification of MM1 and MM2 sCJD prions by QuIC using recombinant human PrP(23–231, 129M) substrate and by sPMCA using brain homogenate of Tg mice expressing human PrP^C (129M). The amplification index is the ratio between the concentration of PrP^Sc before and after PMCA measured with CDI. The data points represent results of six QuIC and three sPMCA experiments, each measured in triplicate with CDI. The mean values are indicated by horizontal lines. *** P<0.001, ** P<0.005 determined by ANOVA.</p

Deuterium incorporation for peptic fragments derived from MM1 rPrP^Sc (red) and MM2 rPrP^Sc(blue).

<p>(a) 5 min incubation in D₂O. (b) 240 h incubation in D₂O. Error bars indicate standard deviation (3 independent experiments). *, P<0.05; **, P<0.02.</p

Histidine H/D exchange for monomeric PrP^C (black), MM1 rPrP^Sc (red) and MM2 rPrP^Sc (blue).

<p>The parameter t_1/2 represents the half-time of exchange reaction for individual His residues. Error bars indicate standard deviation (3 independent experiments). **, P<0.01; ***, P<0.001.</p

Source, biophysical characteristics, and replication rate of Type 1 and Type 2 sCJD prions.

<p>Source, biophysical characteristics, and replication rate of Type 1 and Type 2 sCJD prions.</p

Schematic representation of PK-resistant fragments in rPrP^Sc corresponding to Type 1 (MM1) and Type 2 (MM2) sCJD prions and molecular characteristics of purified human rPrPSc used in structural studies.

<p>(a) Outline of classification of Type 1 and Type 2 human prions based on proteolytic fragmentation of PrP^Sc [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004832#ppat.1004832.ref005" target="_blank">5</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004832#ppat.1004832.ref052" target="_blank">52</a>]. Major cleavage sites by PK are indicated by arrows; GLP—glycolipid; CHO- complex N-glycosylation chains. The codes above light blue brackets represent monoclonal antibodies used in differentiation of various domains of human prions, and the numbers below these brackets indicate linear epitopes recognized by these antibodies. (b) Distinct glycosylation patterns and electrophoretic mobilities of purified human Type 1 (MM1) and Type 2 (MM2) sCJD rPrP^Sc (homozygous for methionine (M) in codon 129) used in structural studies. To differentiate Type 1, Type 2 prions, and their C-terminal fragments, Western blots of purified rPrP^Sc (fraction 8; F8) from the brain homogenate (BH) of type MM1 and MM2 sCJD were developed with mAb 12B2 (epitope residues 89–93) [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004832#ppat.1004832.ref053" target="_blank">53</a>], mAb 3F4 (epitope residues 107–112) [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004832#ppat.1004832.ref054" target="_blank">54</a>], and rabbit polyclonal antibody 2301 (epitope residues 220–231) [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004832#ppat.1004832.ref055" target="_blank">55</a>]. The lower panels correspond to prolonged exposure of the same WB to detect less abundant low molecular weight fragments of rPrP^Sc. (c) Distinct fragmentation patterns of purified MM1 and MM2 sCJD prions in silver stained SDS-PAGE before and after deglycosylation with PNGase F. The symbols (*) and (#) indicate bands corresponding to PK and PNGase F, respectively. The molecular weights of marker proteins are in kDa.</p

Small Protease Sensitive Oligomers of PrP^Sc in Distinct Human Prions Determine Conversion Rate of PrP^C

<div><p>The mammalian prions replicate by converting cellular prion protein (PrP^C) into pathogenic conformational isoform (PrP^Sc). Variations in prions, which cause different disease phenotypes, are referred to as strains. The mechanism of high-fidelity replication of prion strains in the absence of nucleic acid remains unsolved. We investigated the impact of different conformational characteristics of PrP^Sc on conversion of PrP^C in vitro using PrP^Sc seeds from the most frequent human prion disease worldwide, the Creutzfeldt-Jakob disease (sCJD). The conversion potency of a broad spectrum of distinct sCJD prions was governed by the level, conformation, and stability of small oligomers of the protease-sensitive (s) PrP^Sc. The smallest most potent prions present in sCJD brains were composed only of∼20 monomers of PrP^Sc. The tight correlation between conversion potency of small oligomers of human sPrP^Sc observed in vitro and duration of the disease suggests that sPrP^Sc conformers are an important determinant of prion strain characteristics that control the progression rate of the disease.</p> </div

Relationship between conversion potency and the conformational stability of PrP^Sc, rPrP^Sc, and sPrP^Sc in MM1 (n = 10) and MM2 (n = 10) sCJD cases.

<p>The (<b>A</b>) conformational stability of MM1 PrP^Sc before (red diamonds) and after (red circles) PK digestion; the (<b>B</b>) conformational stability of MM2 PrP^Sc before (blue triangles) and after (blue squares) PK digestion; and the (<b>C</b>) fractional change in stability of PrP^Sc conformers induced by PK in individual sCJD samples (filled red circles) Type 1 PrP^Sc(129M), and (filled blue squares) Type 2 PrP^Sc(129M). The (<b>D</b>) inverse relationship between stability of total PrP^Sc and amplification index; (<b>E</b>) no correlation between stability of rPrP^Sc and amplification index; and (<b>F</b>) direct correlation between PK-induced change in the stability of PrP^Sc (Δ Fapp) and amplification index. The stability of prion and conversion potency of PrP^Sc was determined by CDI and expressed as Gdn HCl_1/2 or stability change (Δ Fapp) induced by PK. Each symbol represents an average of triplicate experiment followed by triplicate measurement ± SEM with CDI.</p

Impact of protease treatment on stability of PrP^Sc monitored with CDI.

<p>Typical dissociation and unfolding of (<b>A</b>) Type 1 PrP^Sc(129M) and (<b>B</b>) Type 2 PrP^Sc(129M) followed by CDI before (blue squares) and after (red circles) PK treatment; the differences in Fapp values before and after PK treatments are in triangles (green). The curves are the best fit with sigmoidal transition model to determine the midpoint (GdnHCl_1/2 value) of the curve <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002835#ppat.1002835-Kim1" target="_blank">[4]</a>. The differential values are fitted with the Gaussian model and the peak maximum corresponds to the mean stability of sPrP^Sc as described previously <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002835#ppat.1002835-Kim1" target="_blank">[4]</a>. The values of apparent fractional change (Fapp) of each sample aliquot are mean ± SEM obtained from triplicate measurements.</p