Na+/K+-ATPase Is Present in Scrapie-Associated Fibrils, Modulates PrP Misfolding In Vitro and Links PrP Function and Dysfunction

Abstract

Transmissible spongiform encephalopathies are characterised by widespread deposition of fibrillar and/or plaque-like forms of the prion protein. These aggregated forms are produced by misfolding of the normal prion protein, PrPC, to the disease-associated form, PrPSc, through mechanisms that remain elusive but which require either direct or indirect interaction between PrPC and PrPSc isoforms. A wealth of evidence implicates other non-PrP molecules as active participants in the misfolding process, to catalyse and direct the conformational conversion of PrPC or to provide a scaffold ensuring correct alignment of PrPC and PrPSc during conversion. Such molecules may be specific to different scrapie strains to facilitate differential prion protein misfolding. Since molecular cofactors may become integrated into the growing protein fibril during prion conversion, we have investigated the proteins contained in prion disease-specific deposits by shotgun proteomics of scrapie-associated fibrils (SAF) from mice infected with 3 different strains of mouse-passaged scrapie. Concomitant use of negative control preparations allowed us to identify and discount proteins that are enriched non-specifically by the SAF isolation protocol. We found several proteins that co-purified specifically with SAF from infected brains but none of these were reproducibly and demonstrably specific for particular scrapie strains. The α-chain of Na+/K+-ATPase was common to SAF from all 3 strains and we tested the ability of this protein to modulate in vitro misfolding of recombinant PrP. Na+/K+-ATPase enhanced the efficiency of disease-specific conversion of recombinant PrP suggesting that it may act as a molecular cofactor. Consistent with previous results, the same protein inhibited fibrillisation kinetics of recombinant PrP. Since functional interactions between PrPC and Na+/K+-ATPase have previously been reported in astrocytes, our data highlight this molecule as a key link between PrP function, dysfunction and misfolding.

Mechanisms of Cell Death in the Transmissible Spongiform Encephalopathies

“…Other than PrP, various proteinaceous molecules appear specifically enriched in infectious prion fibrils (Giorgi et al, 2009, Moore et al, 2010, Petrakis et al, 2009) and recent data from our laboratory suggest that at least one such protein can enhance prion protein conversion efficiency (Graham et al, 2011). The most likely places for PrP C to encounter PrP Sc and for conversion to take place are on the cell surface or within the endocytic pathway and it would appear reasonable to expect cofactors to reside in these locations.…”

“…Results from experiments in cell lines supporting either location as a site for conversion have been published (Borchelt et al, 1992, Hooper, 2011. Recent data from our laboratories , Graham, et al, 2011 and others (Abid et al, 2010), suggest that the plasma membrane is a more likely source of cofactors modulating prion protein misfolding. It is plausible that specific compositions of lipid can modulate prion protein structure thereby creating conditions for strain specific misfolding.…”

Complex folding and misfolding effects of deer-specific amino acid substitutions in the β2-α2 loop of murine prion protein

“…The increase in ThT fluorescence was plotted against time and a sigmoidal curve fitted to the data. The lag time of the sigmoidal curve was calculated as described previously 47 .…”

Use of proteomics in the study of microbial diseases of small ruminants

“…Apolipoprotein E co-localization occurred in moderately mature lesions in prion diseases, where it may play a role in the aggregation of PrP sc after a conformational change from cellular PrP isoform to PrP sc . A biomarker for diagnosis of scrapie in sheep, which has been identified by using proteomics from the brain of mice with experimental disease, can be Na + /K + ATPase (Graham et al, 2011). Na + /K + ATPase enhanced the efficiency of diseasespecific conversion of recombinant PrP, suggesting that it may act as a molecular cofactor.…”