Biochemical and Biophysical Research Communications
Shadoo binds lipid membranes and undergoes aggregation and fibrillization
Introduction
Prion diseases are fatal neurodegenerative disorders that include bovine spongiform encephalopathy in cattle, scrapie in sheep, and variant Creutzfeld–Jacob disease in human. They are associated with aggregation of the prion protein (PrP) into oligomers and amyloid fibers. Prior to aggregate, cellular PrP (PrPC), mainly composed of α-helix, undergoes an α-to-β transition into an abnormal scrapie isoform (PrPSc) [1], [2]. A domain of two α-helixes and/or the protein hydrophobic domain (HD) are reported to be involved in the conformational switch, which can act as an aggregation seed for the conversion [3]. In contrast, the non-structured N-terminal domain of PrP is proposed to play a role in early stages of polymerization by increasing local protein concentration on some cell membranes [4], [5].
Although PrP seems to be the major agent in prion pathologies, some other biomolecules were proposed to be essential to infectivity. Firstly, conversion from PrPC to PrPSc is thought to take place at the plasma membrane or on endosomes/lysosomes [6]. This suggests that some membrane lipids may be involved in the conversion. Secondly, prion protein family contains two paralogs of PrP: Doppel and Shadoo (Sho) [7]. Doppel is unable to undergo a characteristic α-to-β conversion and to attain misfolded and aggregated states. In contrast, Sho was showed to form amyloid-structured aggregates under physiological conditions [8]. Sho shows some sequence homology to HD and N-terminal domains of PrPC and is an unstructured protein in aqueous solutions [8].
The structural homologies and similar brain distribution of PrP and Sho suggested that these two proteins may share the same function in neuronal cells. Indeed, both PrP and Sho have some neuroprotective properties. Expression of Sho is down regulated in prion infected mice [9]. However, Sho levels are unchanged in the brains of adult mice lacking PrPC [10]. Finally, Sho overexpression does not influence the kinetics of prion replication in mice [11].
Since lipid–protein interactions can favor PrP fibrilization, we investigated Sho amyloid fiber formation upon binding to model lipid membranes. We found that Sho binds negatively charged but not zwitterionic liposomes. Upon binding, Sho destabilizes vesicle structure, undergoes aggregation and amyloid fiber formation.
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Reagents and antibodies
Phosphatidylserine (PS) and Phosphatidylcholine (PC) were purchased from Avanti Polar Lipids (AL, USA). Other reagents were purchased from Sigma–Aldrich (France). Following buffers were used: 20 mM citrate buffer, pH 3.35; 5 mM sodium-acetate buffer, pH 5; 5 mM ammonium-acetate buffer, pH 5; 20 mM MES buffer, pH 6; 20 mM MOPS acid buffer, pH 7 and 20 mM MOPS buffer, pH 8.
Plasmid constructs and expression of proteins
The gene encoding mouse Sho (Sho25–122) was cloned in pET-28 plasmid (Novagen) and expressed in SolBL21 Escherichia coli
Sho aggregation is pH dependent
Sho produced in E. coli was purified as described under “Materials and Methods” and subjected to SDS–PAGE analysis. Coomassie Bleu staining revealed a band of about 12 kDa that corresponded to the theoretical molecular mass of Sho (Fig. 1A). Detected protein was further submitted to high resolution mass spectrometry analysis, which identified the band as mouse Sho of 12.2 kDa (22 unique peptides corresponding to a sequence coverage of 45%).
After purification, Sho was desalted against various
Discussion
Lipid bilayers may act as an effective catalyst of protein fibrillogenesis [5], [11], [12], [13]. Studies to date have reported the importance of the membrane lipid composition on providing a specific environment where proteins assemble into profibrillar and fibrillar structures [14]. Here, we first showed that Sho protein spontaneously undergoes aggregation and polymerization at physiological and basic pHs. At acidic pH, Sho is a stable, monomeric protein deprived of ordered secondary
Acknowledgments
We thank Natalie Doude and David Westerway (University of Alberta) and Michel Brémont (INRA) for providing us with Sho cDNA and BL21Sol, respectively; Christine Longin (INRA) for TEM assistance. Stephane Biacchesi (INRA) for OM assistance and critical reading of the manuscript; Rachel Young (Institut Gustave Roussy) for critical reading of the manuscript.
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