GPI anchoring facilitates propagation and spread of misfolded Sup35 aggregates in mammalian cells

Abstract

Prion diseases differ from other amyloid-associated protein misfolding diseases (e.g. Alzheimer's) because they are naturally transmitted between individuals and involve spread of protein aggregation between tissues. Factors underlying these features of prion diseases are poorly understood. Of all protein misfolding disorders, only prion diseases involve the misfolding of a glycosylphosphatidylinositol (GPI)-anchored protein. To test whether GPI anchoring can modulate the propagation and spread of protein aggregates, a GPI-anchored version of the amyloidogenic yeast protein Sup35NM (Sup35 GPI ) was expressed in neuronal cells. Treatment of cells with Sup35NM fibrils induced the GPI anchor-dependent formation of self-propagating, detergentinsoluble, protease-resistant, prion-like aggregates of Sup35 GPI . Live-cell imaging showed intercellular spread of Sup35 GPI aggregation to involve contact between aggregatepositive and aggregate-negative cells and transfer of Sup35 GPI from aggregate-positive cells. These data demonstrate GPI anchoring facilitates the propagation and spread of protein aggregation and thus may enhance the transmissibility and pathogenesis of prion diseases relative to other protein misfolding diseases.

Dual Conformation of H2H3 Domain of Prion Protein in Mammalian Cells

“…Prion-determining regions appear to be an unstructured stretch of ϳ70 -200 amino acids that are, apart from Het-s, rich in glutamine/asparagine residues and have a bias against polar residues (9,(11)(12)(13)(14). For instance, the prion domain of Sup35, called Sup35NM, has been characterized as sufficient for functional prion aggregation (15) and even for replication in mammalian cells, either in the cytosol (16) or attached to the membrane through a GPI anchor (17).…”

Glycosylphosphatidylinositol Anchoring Directs the Assembly of Sup35NM Protein into Non-fibrillar, Membrane-bound Aggregates

“…Generation of N2a Cell Clones Expressing Sup35 ConstructsThe procedure for construction and culture of cell lines stably expressing GFP-and mCherry (mC)-tagged proteins is described elsewhere (47). Stably transfected cells were subjected to multiple rounds of FACS sorting to select for high expressing cell populations.…”

“…Antibodies-Generation of anti-Sup35N domain antibody was described elsewhere (47). Other antibodies were obtained as follows: anti-GFP mouse monoclonal and anti-HA tag rat monoclonal (Roche Applied Science); anti-HA mouse monoclonal 16B12 (biotinylated and unlabeled versions) and control mouse monoclonal antibody directed against the 3F4 epitope of hamster prion protein (Covance); peroxidase-conjugated NeutrAvidin (Pierce); peroxidase-conjugated goat anti-mouse IgG F(ab) 2 secondary antibody (Jackson ImmunoResearch); Alexa Fluor 594-streptavidin FluoroNanogold TM and anti-mouse Alexa Fluor 594-FluoroNanogold secondary antibody (Nanoprobes); and rabbit anti-RFP (for mCherry; Rockland Immunochemicals).…”

“…Ure2p) form amyloid in the yeast cytosol (56). In previous studies, we and others reported that Sup35NM is able to propagate as a prion in mammalian cells (47,57,58) and that GPI anchoring facilitates aggregate propagation between N2a cells, resembling mammalian prion behavior (47). In the present work, we go on to characterize the ultrastructural and biochemical features of GPI-anchored Sup35NM aggregates.…”

“…We initiated these investigations using a model system consisting of a GPI-anchored form of the highly charged, glutamine-rich N-terminal and middle (NM) prion domain from the yeast prion protein Sup35p (referred to here as Sup35 GPI ), stably expressed in N2a cells (47). When expressed in Saccharomyces cerevisiae in its native, soluble form, the function of Sup35p is as a translation termination factor (48).…”