Abstract
At times, it can be difficult to discern if a lack of overlap in reported interactions for a protein-of-interest reflects differences in methodology or biology. In such instances, systematic analyses of protein-protein networks across diverse paradigms can provide valuable insights. Here, we interrogated the interactome of the prion protein (PrP), best known for its central role in prion diseases, in four mouse cell lines. Analyses made use of identical affinity capture and sample processing workflows. Negative controls were generated from PrP knockout lines of the respective cell models, and the relative levels of peptides were quantified using isobaric labels. The study uncovered 26 proteins that reside in proximity to PrP. All of these proteins are predicted to have access to the outer face of the plasma membrane, and approximately half of them were not reported to interact with PrP before. Strikingly, although several proteins exhibited profound co-enrichment with PrP in a given model, except for the neural cell adhesion molecule 1, no protein was highly enriched in all PrP-specific interactomes. However, Gene Ontology analyses revealed a shared association of the majority of PrP candidate interactors with cellular events at the intersection of transforming growth factor β and integrin signaling.
Highlights
Little is known about how interactions of a given protein differ across cell models
The parental wild-type cell lines are familiar to the prion research community due to their distinct properties with regard to prion protein (PrP): (1) NMuMG cells exhibit a more than five-fold increase in their PrP protein levels when epithelial-to-mesenchymal transition (EMT) was induced by the addition of Tgfb[119]; (2) C2C12 cells are the only muscle cell model currently known to be susceptible to prion infection[20,21]; (3) N2a neuroblastoma cells may be the most often used cell model in prion research and can readily be infected with mouse-adapted Rocky Mountain Laboratory (RML) prions; and (4) CAD5 catecholaminergic cells exhibit susceptibility to infection with several prion strains[22]
The side-by-side binning of peptide-to-spectrum matches on the basis of cross-correlation (X corr) values computed by the SEQUEST score function[24] revealed similar stratifications for the four PrP interactome analyses, indicating that the respective datasets were comparable in regards to a key quality control benchmark and their depths of coverage (Fig. 1c)
Summary
Little is known about how interactions of a given protein differ across cell models. PrPC is central to the pathogenesis of prion diseases[4] and has been proposed to act as a critical cell surface receptor in Alzheimer’s disease (AD)[5], raising the expectation that insights into the function of PrPC will provide useful angles for understanding the molecular underpinnings of these diseases To this end, the molecular interactions of PrPC have repeatedly been characterized[6,7,8] and many proteins have been reported to interact with PrPC in separate studies, including the laminin receptor precursor[9], the neural cell adhesion molecule 1 (Ncam1)[7], the amyloid precursor like protein-110, and the stress-inducible protein 111. Did PrP-deficiency in distinct cell models cause the levels of members of the Marcks protein family to shift in opposite ways but it prevented Ncam[1] polysialylation in one cell model, yet caused a robust increase of this specific posttranslational modification (PTM) in another model[17]
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