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

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: 10.43). 02/2010; 29(4):782-94. DOI: 10.1038/emboj.2009.392
Source: PubMed


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.

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    • "Until recently, cell-to-cell transmission of infectious protein entities and induction of self-propagating protein aggregates in the recipient cells had only been fully established for the mammalian prion protein, which is a glycosylphosphatidylinositol (GPI)anchored membrane protein. Although NM aggregates were shown to be able to propagate in murine neuroblastoma cells during cell division, similar to in yeast (Krammer et al., 2009b), Speare et al. had demonstrated that GPI anchoring facilitated spreading of NM from cell to cell (Speare et al., 2010). In contrast, the recent demonstration that cytosolic NM aggregates can also invade neighboring cells in primary cell culture and organotypic brain slices, and induce heritable self-perpetuating aggregates in the recipient cells, clearly shows that the mammalian cytosolic environment promotes prion propagation (Hofmann et al., 2013). "
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    • "phatidylinositol anchoring of PrP ( Klingeborn et al . 2011 ) . These findings raise the possibility that the unique glycosyl - phosphatidylinositol anchoring of PrP among amyloidogenic proteins might contribute to the differences in transmissibility of TSEs versus other protein misfolding diseases , especially via peripheral routes of infection ( Speare et al . 2010 ) . Advances in understanding the process of Ab - induced misfolding and further testing of prion - like mechanisms in AD will depend on the rigorous biochemical characterization of the seeding activity present in brain extracts . A number of initial experiments investigating the biochemical identity of the seeding activity have provide"

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    • "In this case, focusing on the uptake mechanism would also be an effective strategy, even if multiple downstream pathways are involved. Recent evidence suggests that the mechanism and machinery for prion propagation may be conserved between yeast and higher eukaryotes [62], [63], and the primary mechanism of pathogenesis in prion and amyloid diseases may have important common features, providing hope for development of broadly applicable therapeutics that will be effective against these devastating diseases. This study suggests that the use of well-characterized models, such as the yeast prion protein Ure2, may provide an important tool in achieving this goal. "
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    ABSTRACT: A number of amyloid diseases involve deposition of extracellular protein aggregates, which are implicated in mechanisms of cell damage and death. However, the mechanisms involved remain poorly understood. Here we use the yeast prion protein Ure2 as a generic model to investigate how amyloid-like protein aggregates can enter mammalian cells and convey cytotoxicity. The effect of three different states of Ure2 protein (native dimer, protofibrils and mature fibrils) was tested on four mammalian cell lines (SH-SY5Y, MES23.5, HEK-293 and HeLa) when added extracellularly to the medium. Immunofluorescence using a polyclonal antibody against Ure2 showed that all three protein states could enter the four cell lines. In each case, protofibrils significantly inhibited the growth of the cells in a dose-dependent manner, fibrils showed less toxicity than protofibrils, while the native state had no effect on cell growth. This suggests that the structural differences between the three protein states lead to their different effects upon cells. Protofibrils of Ure2 increased membrane conductivity, altered calcium homeostasis, and ultimately induced apoptosis. The use of standard inhibitors suggested uptake into mammalian cells might occur via receptor-mediated endocytosis. In order to investigate this further, we used the chicken DT40 B cell line DKOR, which allows conditional expression of clathrin. Uptake into the DKOR cell-line was reduced when clathrin expression was repressed suggesting similarities between the mechanism of PrP uptake and the mechanism observed here for Ure2. The results provide insight into the mechanisms by which amyloid aggregates may cause pathological effects in prion and amyloid diseases.
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