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

National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rocky Mountain Laboratories, Laboratory of Persistent Viral Diseases, Hamilton, MT 59840, USA.
The EMBO Journal (Impact Factor: 10.75). 02/2010; 29(4):782-94. DOI: 10.1038/emboj.2009.392
Source: PubMed

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 (Sup35GPI) was expressed in neuronal cells. Treatment of cells with Sup35NM fibrils induced the GPI anchor-dependent formation of self-propagating, detergent-insoluble, protease-resistant, prion-like aggregates of Sup35GPI. Live-cell imaging showed intercellular spread of Sup35GPI aggregation to involve contact between aggregate-positive and aggregate-negative cells and transfer of Sup35GPI 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.

  • [Show abstract] [Hide abstract]
    ABSTRACT: In prion-infected hosts, PrPSc usually accumulates as non-fibrillar, membrane-bound aggregates. Glycosylphosphatidylinositol (GPI) anchor-directed membrane association appears to be an important factor controlling the biophysical properties of PrPSc aggregates. To determine whether GPI-anchoring can similarly modulate the assembly of other amyloid-forming proteins, neuronal cell lines were generated that expressed a GPI-anchored form of a model amyloidogenic protein, the NM domain of the yeast prion protein Sup35 (Sup35GPI). We recently reported that GPI-anchoring facilitated the induction of Sup35GPI prions in this system. Here, we report the ultrastructural characterization of self-propagating Sup35GPI aggregates of either spontaneous or induced origin. Like membrane-bound PrPSc, Sup35GPI aggregates resisted release from cells treated with phosphatidylinositol-specific phospholipase C. Sup35GPI aggregates of spontaneous origin were detergent-insoluble, protease resistant, and self-propagating, in a manner similar to that reported for recombinant Sup35NM amyloid fibrils and induced Sup35GPI aggregates. However, GPI-anchored Sup35 aggregates were not stained with amyloid-binding dyes such as Thioflavin T. This was consistent with ultrastructural analyses, which showed that the aggregates corresponded to dense cell surface accumulations of membrane vesicle-like structures and were not fibrillar. Together these results showed that GPI-anchoring directs the assembly of Sup35NM into non-fibrillar, membrane-bound aggregates that resemble PrPSc, raising the possibility that GPI anchor-dependent modulation of protein aggregation might occur with other amyloidogenic proteins. This may contribute to differences in pathogenesis and pathology between prion diseases, which uniquely involve aggregation of a GPI-anchored protein, versus other protein misfolding diseases.
    Journal of Biological Chemistry 03/2014; 289(18). DOI:10.1074/jbc.M114.556639 · 4.60 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Mammalian prions refold host glycosylphosphatidylinositol-anchored PrP(C) into β-sheet-rich PrP(Sc). PrP(Sc) is rapidly truncated into a C-terminal PrP27-30 core that is stable for days in endolysosomes. The nature of cell-associated prions, their attachment to membranes and rafts, and their subcellular locations are poorly understood; live prion visualization has not previously been achieved. A key obstacle has been the inaccessibility of PrP27-30 epitopes. We overcame this hurdle by focusing on nascent full-length PrP(Sc) rather than on its truncated PrP27-30 product. We show that N-terminal PrP(Sc) epitopes are exposed in their physiological context and visualize, for the first time, PrP(Sc) in living cells. PrP(Sc) resides for hours in unexpected cell-surface, slow moving strings and webs, sheltered from endocytosis. Prion strings observed by light and scanning electron microscopy were thin, micrometer-long structures. They were firmly cell associated, resisted phosphatidylinositol-specific phospholipase C, aligned with raft markers, fluoresced with thioflavin, and were rapidly abolished by anti-prion glycans. Prion strings and webs are the first demonstration of membrane-anchored PrP(Sc) amyloids.
    The Journal of Cell Biology 02/2014; 204(3):423-41. DOI:10.1083/jcb.201308028 · 9.69 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The cellular prion protein (PrP(C)) is an N-glycosylated GPI-anchored protein usually present in lipid rafts with numerous putative functions. When it changes its conformation to a pathological isoform (then referred to as PrP(Sc)), it is an essential part of the prion, the agent causing fatal and transmissible neurodegenerative prion diseases. There is growing evidence that toxicity and neuronal damage on the one hand and propagation/infectivity on the other hand are two distinct processes of the disease and that the GPI-anchor attachment of PrP(C) and PrP(Sc) plays an important role in protein localization and in neurotoxicity. Here we review how the signal sequence of the GPI-anchor matters in PrP(C) localization, how an altered cellular localization of PrP(C) or differences in GPI-anchor composition can affect prion infection, and we discuss through which mechanisms changes on the anchorage of PrP(C) can modify the disease process.
    Prion 02/2014; 8(1). DOI:10.4161/pri.27892 · 1.97 Impact Factor

Full-text (2 Sources)

Available from
May 30, 2014