Membranes as Catalysts for Prion Conversion

1
Fibrillization of the Prion Protein at a Membrane
Surface Formation of Spongiform Prions
Philip J Robinsona, Svetla Stoilova-McPhiea,
Ilya Reviakineb and Teresa JT Pinheiroa aDepartmen
t of Biological Sciences, University of Warwick,
Gibbet Hill Road, Coventry, CV4 7AL, UK bCIC
biomaGUNE, Paseo Miramón 182,20009 San Sebastián,
Gipuzkoa, SPAIN
Membranes as Catalysts for Prion Conversion The
transition from the ?-helical to the ?-sheet-rich
isoform of the prion protein is the central
misfolding event associated with prion diseases.
This transition is separated by an energy barrier
that is due to unfolding and oligomerization of
the protein. Our hypothesis is that cellular
membranes have properties that enable them to
lower this energy barrier and therefore act as
catalysts that promote prion conversion.
Association of the Prion Protein with Membranes
Induces Insertion and Structural Change
Binding of PrP(90-231) to vesicles as monitored
by the blue shift (??) in tryptophan
fluorescence. Binding increases as the POPS
content of the membrane increases.
POPSPOPC 11
POPSPOPC 28
Working Hypothesis
POPC Alone
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On incubation with vesicles that contain
POPCPOPS 82 the change in intensity of the
signal and shape of the CD spectra shows a clear
structural change. This change shows
characteristics of unfolding and an increase in
?- sheet structure.
1.The prion protein interacts with cellular
membranes. 2.The hydrophobic membrane environment
induces unfolding and structural
rearrangement of the prion protein. 3.This
misfolded form is then susceptible to
oligomerization and fibrillization.
Nucleation and Growth of Prion Aggregates on
Supported Phospholipid Bilayers (SPBs) Uniform
circular, clusters of prion aggregates form on
SPBs that contain POPC alone. These clusters grow
laterally across the membrane until they fuse. On
the SPBs that contain POPS, circular spongiform
aggregates grow on localised areas of the
bilayer. These structures are highly uniform in
height and contain holes that make them sponge
like in appearance.
AFM images showing the nucleation and latteral
growth of aggregates consisting of PrP(90-231) on
POPC/POPS SPBs. Each panel shows a SPB with the
addition of (A) 1 ?M PrP(90-231) (B ) a further 1
?M PrP(90-231) (C) Incubation following the
second injection of protein. The scan size is 25
?m and the height scale is 70 nm. The inset shows
cross section analysis across the black line.
Each panel shows an AFM image of a POPC SPB with
increasing concentrations of PrP(90-231) added as
you move from panels A to D. The scan size of
panels A to D is 8 ?m and the height scale is 30
nm. Panels A to D are zoomed in versions of
panels A to D. The scan size of panels A to D
is 2.5 ?m and the height scale is 30 nm.
Conclusions Tryptophan fluorescence and circular
dichroism reveal an interaction between
PrP(90-231) and vesicles that contain the
phospholipid POPS. Cryo-EM shows the formation of
junctions and a collapse of vesicles as a result
of this interaction. The disruption of the
bilayer is a possible mechanism of prion induced
cell death. In contrast with extruded vesicles in
solution PrP(90-231) interacts with POPC on
supported lipid bilayers. This may be explained
in terms of the difference in curvature between
the vesicles and the flat supported bilayer. Both
planar and curved membranes support neuronal
cells and their intracellular compartments and
therefore both represent biologically relevant
models of what may occur within the cell. Overall
this data gives further insights into the
nucleation and mechanism of growth of aggregates
on membranes that may influence prion conversion
and development of disease in vivo.
Interactions of Prion Protein with Lipid
Membranes Promotes the Formation of Junctions
B
A
100 nm
100 nm
Cryo-electron microscopy of vesicles with
POPSPOPC of 28 (A) Vesicles alone, (B) Vesicles
following addition of PrP(90-231). The prion
protein induces the collapse of vesicles and the
formation of junctions that link the vesicle
membranes. This suggests that the junctions
contains concentrated misfolded protein.