Prion protein-Semisynthetic prion protein (PrP) variants with posttranslational modifications.

Deciphering the pathophysiologic events in prion diseases is challenging, and the role of posttranslational modifications (PTMs) such as glypidation and glycosylation remains elusive due to the lack of homogeneous protein preparations. So far, experimental studies have been limited in directly analyzing the earliest events of the conformational change of cellular prion protein (PrPC ) into scrapie prion protein (PrPSc ) that further propagates PrPC misfolding and aggregation at the cellular membrane, the initial site of prion infection, and PrP misfolding, by a lack of suitably modified PrP variants. PTMs of PrP, especially attachment of the glycosylphosphatidylinositol (GPI) anchor, have been shown to be crucially involved in the PrPSc formation. To this end, semisynthesis offers a unique possibility to understand PrP behavior invitro and invivo as it provides access to defined site-selectively modified PrP variants. This approach relies on the production and chemoselective linkage of peptide segments, amenable to chemical modifications, with recombinantly produced protein segments. In this article, advances in understanding PrP conversion using semisynthesis as a tool to obtain homogeneous posttranslationally modified PrP will be discussed.

Highly efficient protein misfolding cyclic amplification.

Protein misfolding cyclic amplification (PMCA) provides faithful replication of mammalian prions in vitro and has numerous applications in prion research. However, the low efficiency of conversion of PrP(C) into PrP(Sc) in PMCA limits the applicability of PMCA for many uses including structural studies of infectious prions. It also implies that only a small sub-fraction of PrP(C) may be available for conversion. Here we show that the yield, rate, and robustness of prion conversion and the sensitivity of prion detection are significantly improved by a simple modification of the PMCA format. Conducting PMCA reactions in the presence of Teflon beads (PMCAb) increased the conversion of PrP(C) into PrP(Sc) from ∼10% to up to 100%. In PMCAb, a single 24-hour round consistently amplified PrP(Sc) by 600-700-fold. Furthermore, the sensitivity of prion detection in one round (24 hours) increased by 2-3 orders of magnitude. Using serial PMCAb, a 10¹²-fold dilution of scrapie brain material could be amplified to the level detectible by Western blotting in 3 rounds (72 hours). The improvements in amplification efficiency were observed for the commonly used hamster 263K strain and for the synthetic strain SSLOW that otherwise amplifies poorly in PMCA. The increase in the amplification efficiency did not come at the expense of prion replication specificity. The current study demonstrates that poor conversion efficiencies observed previously have not been due to the scarcity of a sub-fraction of PrP(C) susceptible to conversion nor due to limited concentrations of essential cellular cofactors required for conversion. The new PMCAb format offers immediate practical benefits and opens new avenues for developing fast ultrasensitive assays and for producing abundant quantities of PrP(Sc)in vitro.

Semisynthetic murine prion protein equipped with a GPI anchor mimic incorporates into cellular membranes.

Conversion of cellular prion protein (PrP(C)) into the pathological conformer (PrP(Sc)) has been studied extensively by using recombinantly expressed PrP (rPrP). However, due to inherent difficulties of expressing and purifying posttranslationally modified rPrP variants, only a limited amount of data is available for membrane-associated PrP and its behavior in vitro and in vivo. Here, we present an alternative route to access lipidated mouse rPrP (rPrP(Palm)) via two semisynthetic strategies. These rPrP variants studied by a variety of in vitro methods exhibited a high affinity for liposomes and a lower tendency for aggregation than rPrP. In vivo studies demonstrated that double-lipidated rPrP is efficiently taken up into the membranes of mouse neuronal and human epithelial kidney cells. These latter results enable experiments on the cellular level to elucidate the mechanism and site of PrP-PrP(Sc) conversion.

Characterization of prion protein (PrP)-derived peptides that discriminate full-length PrPSc from PrPC.

On our initial discovery that prion protein (PrP)-derived peptides were capable of capturing the pathogenic prion protein (PrP(Sc)), we have been interested in how these peptides interact with PrP(Sc). After screening peptides from the entire human PrP sequence, we found two peptides (PrP(19-30) and PrP(100-111)) capable of binding full-length PrP(Sc) in plasma, a medium containing a complex mixture of other proteins including a vast excess of the normal prion protein (PrP(C)). The limit of detection for captured PrP(Sc) was calculated to be 8 amol from a approximately 10(5)-fold dilution of 10% (wt/vol) human variant Creutzfeldt-Jakob disease brain homogenate, with >3,800-fold binding specificity to PrP(Sc) over PrP(C). Through extensive analyses, we show that positively charged amino acids play an important, but not exclusive, role in the interaction between the peptides and PrP(Sc). Neither hydrophobic nor polar interactions appear to correlate with binding activity. The peptide-PrP(Sc) interaction was not sequence-specific, but amino acid composition affected binding. Binding occurs through a conformational domain that is only present in PrP(Sc), is species-independent, and is not affected by proteinase K digestion. These and other findings suggest a mechanism by which cationic domains of PrP(C) may play a role in the recruitment of PrP(C) to PrP(Sc).

Formation of native prions from minimal components in vitro.

The conformational change of a host protein, PrP(C), into a disease-associated isoform, PrP(Sc), appears to play a critical role in the pathogenesis of prion diseases such as Creutzfeldt-Jakob disease and scrapie. However, the fundamental mechanism by which infectious prions are produced in neurons remains unknown. To investigate the mechanism of prion formation biochemically, we conducted a series of experiments using the protein misfolding cyclic amplification (PMCA) technique with a preparation containing only native PrP(C) and copurified lipid molecules. These experiments showed that successful PMCA propagation of PrP(Sc) molecules in a purified system requires accessory polyanion molecules. In addition, we found that PrP(Sc) molecules could be formed de novo from these defined components in the absence of preexisting prions. Inoculation of samples containing either prion-seeded or spontaneously generated PrP(Sc) molecules into hamsters caused scrapie, which was transmissible on second passage. These results show that prions able to infect wild-type hamsters can be formed from a minimal set of components including native PrP(C) molecules, copurified lipid molecules, and a synthetic polyanion.

Cyclic amplification of protein misfolding: application to prion-related disorders and beyond.

Diverse human disorders, including the majority of neurodegenerative diseases, are thought to arise from the misfolding and aggregation of protein. We have recently described a novel technology to amplify cyclically misfolded proteins in vitro. This procedure, named protein misfolding cyclic amplification (PMCA), is conceptually analogous to DNA amplification by PCR and has tremendous implications for research and diagnosis. The PMCA concept has been proved on the amplification of prions implicated in the pathogenesis of transmissible spongiform encephalopathies. In this article we describe the rational behind PMCA and some of the many potential applications of this novel technology.