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Ultra-efficient PrP(Sc) amplification highlights potentialities and pitfalls of PMCA technology.

ABSTRACT: In order to investigate the potential of voles to reproduce in vitro the efficiency of prion replication previously observed in vivo, we seeded protein misfolding cyclic amplification (PMCA) reactions with either rodent-adapted Transmissible Spongiform Encephalopathy (TSE) strains or natural TSE isolates. Vole brain homogenates were shown to be a powerful substrate for both homologous or heterologous PMCA, sustaining the efficient amplification of prions from all the prion sources tested. However, after a few serial automated PMCA (saPMCA) rounds, we also observed the appearance of PK-resistant PrP(Sc) in samples containing exclusively unseeded substrate (negative controls), suggesting the possible spontaneous generation of infectious prions during PMCA reactions. As we could not definitively rule out cross-contamination through a posteriori biochemical and biological analyses of de novo generated prions, we decided to replicate the experiments in a different laboratory. Under rigorous prion-free conditions, we did not observe de novo appearance of PrP(Sc) in unseeded samples of M109M and I109I vole substrates, even after many consecutive rounds of saPMCA and working in different PMCA settings. Furthermore, when positive and negative samples were processed together, the appearance of spurious PrP(Sc) in unseeded negative controls suggested that the most likely explanation for the appearance of de novo PrP(Sc) was the occurrence of cross-contamination during saPMCA. Careful analysis of the PMCA process allowed us to identify critical points which are potentially responsible for contamination events. Appropriate technical improvements made it possible to overcome PMCA pitfalls, allowing PrP(Sc) to be reliably amplified up to extremely low dilutions of infected brain homogenate without any false positive results even after many consecutive rounds. Our findings underline the potential drawback of ultrasensitive in vitro prion replication and warn on cautious interpretation when assessing the spontaneous appearance of prions in vitro.

SUBMITTER: Cosseddu GM

PROVIDER: S-EPMC3219717 | biostudies-literature | 2011 Nov

REPOSITORIES: biostudies-literature

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Ultra-efficient PrP(Sc) amplification highlights potentialities and pitfalls of PMCA technology.

Cosseddu Gian Mario GM Nonno Romolo R Vaccari Gabriele G Bucalossi Cecilia C Fernandez-Borges Natalia N Di Bari Michele Angelo MA Castilla Joaquin J Agrimi Umberto U

PLoS pathogens 20111117 11

In order to investigate the potential of voles to reproduce in vitro the efficiency of prion replication previously observed in vivo, we seeded protein misfolding cyclic amplification (PMCA) reactions with either rodent-adapted Transmissible Spongiform Encephalopathy (TSE) strains or natural TSE isolates. Vole brain homogenates were shown to be a powerful substrate for both homologous or heterologous PMCA, sustaining the efficient amplification of prions from all the prion sources tested. Howeve ...[more]

PMID: 22114554

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Project description:It is established that prion protein is the sole causative agent in a number of diseases in humans and animals. However, the nature of conformational changes that the normal cellular form, PrP(C), undergoes in its conversion to a self-replicating state is still not fully understood. The ordered C-terminus of PrP(C) proteins has three helices (H1-H3). Here, we use statistical coupling analysis (SCA) to infer covariations at various locations using a family of evolutionarily related sequences and the response of mouse and human PrP(C)s to mechanical force to decipher the initiation sites for the transition from PrP(C) to an aggregation-prone PrP* state. Sequence-based SCA predicts that the clustered residues in nonmammals are localized in the stable core (near H1) of PrP(C), whereas in mammalian PrP(C), they are localized in frustrated helices H2 and H3 where most of the pathogenic mutations are found. Force-extension curves and free energy profiles as a function of extension of mouse and human PrP(C) in the absence of a disulfide (SS) bond between residues Cys179 and Cys214, generated by applying mechanical force to the ends of the molecule, show a sequence of unfolding events starting first with rupture of H2 and H3. This is followed by disruption of structure in two strands. Helix H1, stabilized by three salt bridges, resists substantial force before unfolding. Force extension profiles and the dynamics of rupture of tertiary contacts also show that even in the presence of an SS bond the instabilities in most of H3 and parts of H2 still determine the propensity to form the PrP* state. In mouse PrP(C) with an SS bond, there are ?10 residues that retain their order even at high forces. Both SCA and single-molecule force simulations show that in the conversion from PrP(C) to PrP(SC) major conformational changes occur (at least initially) in H2 and H3, which because of their sequence compositions are frustrated in the helical state. Implications of our findings for the structural model for the scrapie form of PrP(C) are discussed.

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Ultra-efficient PrP(Sc) amplification highlights potentialities and pitfalls of PMCA technology.

Publications

Ultra-efficient PrP(Sc) amplification highlights potentialities and pitfalls of PMCA technology.

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