From the Department of Chemistry and Biochemistry, University of California, Santa Cruz, California 95064
Received for publication, October 3, 2002, and in revised form, February 13, 2003
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ABSTRACT |
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Parkinson's disease is the second most
common neurodegenerative disorder, and the cause is unknown;
however, substantial evidence implicates the aggregation of
The N-terminal region (approximately residues 1-95) of The existing literature on the nature of the interactions of
Our previous studies have demonstrated that the fibril formation of
Expression and Purification of Protein--
Recombinant
Materials--
Thioflavin T (ThT) was obtained from Sigma.
1,2-Dipalmitoyl-sn-glycero-3-phosphate (PA),
1,2-dipalmitoyl-sn-glycero-3-phosphocholine (PC),
1,2-dipalmitoyl-sn-glycero-3-phospho-RAC-(1-glycerol) (PG), dissolved in chloroform, were purchased from Avanti Polar Lipids, Inc.
Preparation of SUVs and LUVs--
Sonicated SUVs of PA/PC (molar
ratio 1:1), PG/PC (molar ratio 1:1), and PC were prepared as described
previously (9). LUVs of PA/PC, PG/PC (molar ratios 1:1), and PC were
prepared by 10 cycles of freeze-thaw and extruded through a
0.1-µm polycarbonate membrane. CD Measurements--
Far-UV CD spectra were obtained with an
AVIV 60DS spectrophotometer (Lakewood, NJ) in a 0.1-cm path length
cell. The concentration of protein was kept at 14 µM,
with the mass ratio of protein to phospholipid varying from 5:1 to
1:10. The final spectra were obtained by calculating the mean of five
individual scans and subtracting the background with samples consisting
of buffer and vesicles without protein. The percentage of AFM Measurements of Kinetic Measurements of Binding of PA/PC SUVs or LUVs Induced
Fig. 2 shows that helix was induced on binding to the acidic
phospholipid vesicles regardless of vesicle size (SUVs or LUVs). However, the amount of helix induced was less for LUVs than for the
corresponding SUVs (Table I). Negatively stained preparations examined
by EM indicated that the large unilamellar vesicles had sizes from 60 to 200 nm (average 120 nm) (data not shown), and SUVs had diameters of
20-25 nm (9). Experiments with LUVs of PA/PC show that
To determine whether there were differences in the interaction of
To determine whether the tendency to induce Fibrillation of
We found that the fibrillation of
Increasing the relative PA/PC concentration in the incubation solution
led to a decrease in rate of fibril formation, as shown by the
increased lag time (Fig. 5). Under these conditions, CD spectra also
showed a change in secondary structure from partially unfolded
intermediate to mostly helical structure. There was no significant
difference in the kinetics of fibrillation between SUVs containing
intravesicular protein or non-intravesicular protein.
When the mass ratio of protein to lipid was increased from 5:1 to 1:5,
the helical structure content increased from 7-8 to 60-70% for
LUV-bound
PC vesicles, with their neutral head groups, were examined to address
the possible effects of charge and crowding on protein association and
fibrillation. PC vesicles had no effect on the fibrillation of
Our previous results demonstrated that fibril formation of
Interestingly, the effects of the vesicles on The effect of the vesicles on the fibrillation rate depends on the
vesicle size. In the case of SUVs, regardless of whether or not the
protein was in the vesicle core (lumen), fibril formation is inhibited
completely if the helix content is above 70%. However, with LUV-bound
On the other hand, with intravesicular protein, We have previously shown that formation of the Molecular crowding has been shown to dramatically stimulate the
fibrillation of We therefore conclude that the -synuclein as a critical factor in the etiology of the disease.
-Synuclein is a relatively abundant brain protein of unknown
function, and the purified protein is intrinsically unfolded. The amino
acid sequence has seven repeats with an apolipoprotein lipid-binding
motif, which are predicted to form amphiphilic helices. We have
investigated the interaction of
-synuclein with lipid vesicles of
different sizes and properties by monitoring the effects on the
conformation of the protein and the kinetics of fibrillation. The
nature of the interaction of
-synuclein with vesicles was highly
dependent on the phospholipid composition, the ratio of
-synuclein
to phospholipid, and the size of the vesicles. The strongest
interactions were between
-synuclein and vesicles composed of
1,2-dipalmitoyl-sn-glycero-3-phosphate/1,2-dipalmitoyl-sn-glycero-3-phosphocholine and
1,2-dipalmitoyl-sn-glycero-3-phospho-RAC-(1-glycerol)/1,2-dipalmitoyl-sn-glycero-3-phosphocholine and involved formation of helical structure in
-synuclein. A strong
correlation was observed between the induction of
-helix in
-synuclein and the inhibition of fibril formation. Thus, helical, membrane-bound
-synuclein is unlikely to contribute to aggregation and fibrillation. Given that a significant fraction of
-synuclein is
membrane-bound in dopaminergic neurons, this observation has significant physiological significance.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-Synuclein is an abundant 140-amino acid neuronal protein of
unknown function, which is enriched in the presynaptic terminals of
neurons (1, 2). It is also an intrinsically unfolded, or natively
unfolded, protein, meaning that in the purified form at neutral pH it
lacks an ordered secondary or tertiary structure.
-Synuclein is also
the major fibrillar component of Lewy bodies, a pathological hallmark
of Parkinson's disease (3). The molecular basis for the distribution
of
-synuclein within neurons, and the formation of Lewy bodies, is
not well understood. Recent studies have shown that
-synuclein is
associated with membranous compartments in cultured cells and brain
tissue (4-6). It has been demonstrated that
-synuclein specifically
binds to phospholipids with acidic head groups (7-9). Although most of
-synuclein is found in the free cytosolic fraction in the cell,
membrane-bound
-synuclein has been suggested to play an important
role in fibril formation (10). Therefore, it is likely that
differential affinity for specific phospholipids is responsible for the
protein location and perhaps fibril formation.
-synuclein
contains six 11-amino acid imperfect repeats with a highly conservative
hexamer motif (KTKEGV), resulting in a variation in hydrophobicity with
a strictly conserved periodicity of 11. Such a periodicity is
characteristic of the amphipathic lipid-binding
-helical domains of
apolipoproteins (11), which have been extensively studied and assigned
to subclasses according to their unique structural and functional
properties (12, 13).
-Synuclein shares the defining properties of
the class A2 lipid-binding helix, distinguished by clustered basic
residues at the polar-apolar interface, positioned ±100o
from the center of apolar face, a predominance of lysines relative to
arginines among these basic residues, and several glutamate residues at
the polar surface (12, 13).
-synuclein with membranes is somewhat contradictory. Previous reports have indicated that
-synuclein interacts with certain phospholipids, which may transform it into a helical conformation (8,
9, 14). It has been reported that small oligomeric forms of
-synuclein preferentially associated with lipid droplets and cell
membranes (15) and that
-synuclein binds preferentially to small
unilamellar vesicles (SUVs)1
containing acidic phospholipids (with the induction of a helical circular dichroism (CD) signal), but not to vesicles with a net neutral
charge (9). In contrast, strong binding of
-synuclein to large
unilamellar vesicles (LUVs) with either anionic or zwitterionic headgroups has also been reported (16). Membranes have been reported to
accelerate the fibrillation of
-synuclein (10), and a recent report
suggests that
-synuclein aggregation may occur on membrane surfaces
(15) and that membranes preferentially induce
-synuclein oligomers.
However, it has also been reported that
-synuclein binds tightly to
neutral and anionic membranes and that membranes inhibit fibrillation
(16).
-synuclein is mediated by a critical partially folded intermediate
(17). Here we report that acidic phospholipids induced the partially
folded conformation upon binding to phospholipid vesicles at relatively
low lipid concentrations, but an
-helix-rich structure was induced
at higher concentrations of lipid. Of particular importance is the
observation that the partially folded structure led to fibril
formation, whereas the
-helical structure inhibited fibril formation.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-synuclein was expressed in Escherichia coli and purified
as described previously (17).
-Synuclein was added to
solutions of the vesicles immediately before CD or kinetics
measurements for preparations of non-intravesicular protein and mixed
with phospholipid before preparation of SUVs or LUVs to obtain
intravesicular protein. Vesicles with intravesicular protein contained
-synuclein both in the lumen of the vesicles as well as in the bulk
solution (see Fig. 1).
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Fig. 1.
Model of SUVs and LUVs with and without
intra-vesicular -synuclein.
Dark gray circles represent lipid-bound
-synuclein;
white circles represent free (unbound)
-synuclein;
light gray represents the lumen of the vesicle.
-helix was
determined as described previously (18).
-Synuclein Aggregation--
Aliquots (5 µl) of incubation solution were transferred to freshly cleaved mica.
Samples adsorbed on to mica were washed with water to remove unbound
protein and dried with a stream of dry N2. AFM images were
obtained with an Autoprobe CP Multiple AFM (Park Scientific) in
tapping mode. Measurements were carried out using silicon cantilevers
with a spring constant of 50 newton/m (Park Scientific) and a
resonance frequency of 290-350 kHz.
-Synuclein Aggregation--
A
filtered protein sample (0.22 µm) was treated with 0.001 M NaOH for 15 min, then centrifuged for 15 min at 14,000 rpm to remove any preformed aggregates. Freshly prepared stock
solutions with concentrations less than 1.0 mg/ml were used within 1 day. Fibril growth experiments involved incubating 0.5 mg/ml purified protein and SUVs or LUVs with varying ratios of
-synuclein to phospholipid in 20 mM Tris-HCl buffer (pH 7.5), 0.1 M NaCl, containing 20 µM ThT at 37 °C with
agitation. Fluorescence intensities were recorded in situ at
intervals of 30 min using a fluorescence plate reader (Fluoroskan
Ascent, Thermo-Labsystems) with excitation at 450 nm and
emission at 485 nm. The sigmoidal kinetics curves for fibril formation
were analyzed as described previously (19). In control experiments we
have shown that the presence of ThT (up to at least 20 µM) has no effect on the kinetics of
-synuclein fibrillation.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-Helical
Structure in
-Synuclein--
Preparations of SUVs are relatively
homogeneous, with a more curved (smaller radius) surface compared with
LUVs, and the SUVs are of similar size to synaptic vesicles. The far-UV
CD spectrum of
-synuclein at pH 7.5 is typical of an unfolded
protein and has a significant negative molar ellipticity at 198 nm.
When
-synuclein was mixed with PA/PC (1:1) vesicles at a mass ration
of 5:1 (protein to PA/PC), the CD spectrum showed a decrease in molar
ellipticity at 198 nm, indicating an increase in secondary structure
(Table I). When the phospholipid
mass ratio was increased to 1:1
-synuclein/(PA/PC) the CD spectra
displayed significant negative molar ellipticity (
12.4 × 103 degree·cm2·dmol
1) at 222 nm, and a large positive molar ellipticity at 195 nm (Fig.
2), indicating formation of substantial
-helix (Table I). When the PA/PC content was increased to 1:5
(
-synuclein/lipid), lipid binding was accompanied by a further
increase in helical structure from 32 to 73% as the mass ratio of
lipid increased from 1:1 to 1:5.
-Helical content of
-synuclein bound to PA/PC
vesicles, and its correlation with fibrillation
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Fig. 2.
Increased lipid concentration induces helical
structure in -synuclein. CD spectra of
-synuclein in the presence of acidic headgroup lipid vesicles are
shown: solid line,
-synuclein alone; dotted
line, PA/PC vesicles, with protein:lipid mass ratio = 5:1,
mass ratio = 1:1 (dashed line), or mass ratio = 1:5 (-··-). A, SUVs; B, SUVs with
intravesicular protein; C, LUVs; D, LUVs with
intravesicular protein. The spectra show the transformation of
-synuclein from the initially natively unfolded conformation
(solid line) to increasingly more helix as more lipid is
present.
-helical
structure is also induced in
-synuclein (Fig. 2); the helix content
increased only to 61% at a protein/lipid ratio of 1:5 (Table I),
demonstrating that the protein preferentially binds to vesicles of
smaller diameter (20-25 nm) as opposed to larger (120 nm) vesicles.
The contact area (ratio of surface area to volume for small vesicles is
larger than that for large vesicles, which may contribute to the
increase in helical content (see model in Fig. 1). An alternative
explanation for the increased binding to the smaller vesicles is that
the
-helical conformation of
-synuclein is better accommodated on
more tightly curved surfaces than less curved surfaces. This
possibility is supported by the results with intravesicular
-synuclein (see below). For example, the increased spacing of the
phospholipid headgroups in the SUVs may more easily accommodate
insertion of helices.
-synuclein with lipids between the inside and outside of the
vesicle, we prepared small (SUVs) and larger vesicles (LUVs) with
protein trapped inside the vesicles. Although it is hard to estimate
the encapsulation efficiencies of
-synuclein in lipid vesicles
without separating free
-synuclein from bound protein, it is well
known that the encapsulation efficiencies of LUVs are higher than those
of SUVs. We used CD to examine the conformational changes, with the
results shown in Fig. 2, while the
-helix content is given in Table
I. There was consistently more helix induced with the vesicles
containing intravesicular
-synuclein, but the effect with LUVs was
smaller. Thus, the helix content increased from 73 to 84% for SUVs,
and from 61 to 70% for LUVs, when the protein was present inside the
vesicle lumen. At low protein to lipid ratios the amount of helix was
more than doubled with the SUVs, consistent with greater induction of
helix to the more curved interior surface.
-helix is a general
property of phospholipids with acidic head groups, PG/PC, PS/PC, and PC
alone were also prepared and CD spectra were collected. An equivalent
phenomenon was observed when negatively charged PA/PC was replaced by
negatively charged PG/PC or PS/PC (data not shown) to form SUVs or
LUVs. However, binding to neutral lipid vesicles of PC only slightly
decreased the ellipticity at 198 nm (Fig. 4), and no helical structure
was observed.
-Synuclein Modulated by Phospholipid
Vesicles--
-Synuclein was incubated with small and large PA/PC
vesicles with various concentrations of protein and lipid. These
studies involved incubation of vesicles at 37 °C for 3 days. EM
images demonstrated that the basic structure of
the vesicles did not change (Fig. 3).
Fig. 4A shows the effect on
the kinetics of
-synuclein fibrillation monitored by ThT
fluorescence of the addition of 0.5 mg/ml
-synuclein to the
vesicles, together with a control solution in which
-synuclein was
added to the buffer in the absence of vesicles. ThT is a fluorescence
dye that binds to amyloid fibrils leading to a large increase in the
fluorescence intensity. The fibrillation of
-synuclein was shown by
the increase in ThT signal and then verified by EM and AFM images.
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Fig. 3.
Electron micrographs showing that binding
of -synuclein, in its helical protein
conformation, does not disrupt vesicles. SUVs of PA/PC with
(B) and without (A)
-synuclein present are
shown.
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Fig. 4.
PC vesicles do not induce helical structure
in -synuclein and inhibit fibrillation.
A, kinetics of
-synuclein fibrillation monitored by
thioflavin T fluorescence. Circles,
-synuclein alone;
inverted triangles, fibrillation in the presence of PC SUVs
at protein:lipid mass ratios of 1:5, 1:10 (squares), and
1:20 (diamonds). A small amount of inhibition is observed in
the presence of the PC vesicles. B, far-UV CD spectra of
-synuclein (solid line) binding to PC SUVs at
protein:lipid mass ratios of 5:1 (dotted line), 1:1
(dashed line), and 1:5 (dash-dotted line).
-synuclein was affected by PA/PC
vesicles in a concentration and size dependent fashion. With moderate
concentrations of PA/PC, which induced a partially folded
-synuclein
intermediate, as evidenced by the CD spectra (Fig. 2), the rate of
fibril formation of
-synuclein was accelerated, regardless of
vesicle size (Fig. 5). EM and AFM images
show the size and shape of fibrils formed in the presence of PA/PC
(Fig. 6); the fibrils had a typical
height of 7.6 nm and 28-nm periodic left-handed twisted structure. This
morphology is very similar to that of fibrils grown in lipid-free
solution, indicating a similar fibrillation pathway as in the absence
of lipid.
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Fig. 5.
The effect of vesicles on the kinetics
of -synuclein fibrillation. Fibrillation
was monitored by thioflavin T fluorescence. Circles,
-synuclein alone. A, in the presence of PA/PC vesicles in
the form of non-intravesicular protein SUVs; B,
intravesicular protein SUVs; C, non-intravesicular protein
LUVs; D, intravesicular protein LUVs at protein:lipid ratios
of 5:1 (inverted triangles), 1:1 (squares), and
1:5 (diamonds). High concentrations of SUVs completely
inhibited fibrillation (A and B).
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Fig. 6.
The morphology of
-synuclein fibrils grown in the presence of
vesicles is similar to that of fibrils grown in the absence of
lipid. Left panel, EM image of fibrils; right
panel, AFM image of an individual
-synuclein fibril in the
presence of PA/PC SUVs at a protein:lipid ratio of 1:1.
-synuclein. Concurrently, the lag time for fibrillation
increased from 8.8 ± 0.6 h to 29.2 ± 3.8 h for
non-intravesicular LUVs and from 7.5 ± 0.9 to 39.8 ± 4.5 h for intravesicular protein in the LUVs, correlating well
with the increased helix content (Table I). For the SUV-bound
-synuclein, the helix content increased to 70-80%, and fibril
formation was completely inhibited (Fig. 5, A and
B). No fibrils were formed under these conditions in 4 weeks. In control experiments with no lipid, fibrils formed in 14 h. Similar results were obtained with vesicles of PG/PC and PS/PC (data
not shown).
-synuclein when the protein to lipid radio was increased up to 1:10,
as shown in Fig. 4B. This demonstrates that fibril formation
was inhibited by the
-helical conformation and not by the presence
of the vesicles themselves.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-synuclein was initiated by a conformational change from the
natively unfolded structure to a partially folded,
-sheet-containing
conformation (17). The current investigation focused on fibril
formation by membrane-associated
-synuclein. We used circular
dichroism to estimate the secondary structure and thioflavin T assays
to monitor the fibril formation, and the results were compared with lipid-free protein. The local concentration of protein is significantly increased upon binding to the lipid vesicles (20).
-synuclein
fibrillation were very dependent on the ratio of lipid to protein (under all conditions there was a molar excess of lipid). For example,
at low mass ratios of PA/PC vesicles to
-synuclein, there was a
substantial increase in rate of fibrillation, as manifested by the
decrease in lag time from 14 ± 2 h for lipid-free protein to
4.5 ± 0.7 h for lipid-bound (SUVs)
-synuclein. This
acceleration of fibrillation coincided with a change in the secondary
structure of
-synuclein from natively unfolded to that of the
partially folded intermediate. We attribute this increase in
fibrillation rate to the formation of the partially folded intermediate
conformation. In contrast, when the lipid content increased, the
-synuclein conformation changed to 60-85%
-helix for both SUVs
and LUVs of PA/PC (and PG/PC and PS/PC). Since no fibrils were formed
under these conditions, we conclude that the helical conformation
prevents formation of fibrils. This observation may be of major
physiological significance; if under normal conditions
-synuclein is
predominantly bound to membranes in dopaminergic neurons, this would
minimize its chances of aggregation. On the other hand, factors leading to significant reduction in the membrane-bound form of
-synuclein could result in pathological effects emanating from the aggregation of
-synuclein.
-synuclein the rate of fibrillation was decreased, but not totally
inhibited, with 60 to 70%
-synuclein in a helical conformation. The
most likely explanation is that less
-synuclein is bound to the
LUVs, compared with the SUVs (at the same lipid/protein mass ratios),
and thus some
-synuclein is not bound to the vesicles and is present
in the partial folded conformation and goes on to form fibrils. This
may be a reflection of the increased lipid cooperativity of LUVs
compared with SUVs.
-synuclein can
become locally concentrated within a single vesicle and form fibrils in
the core (lumen). We have demonstrated that fibrillar
-synuclein
will disrupt lipid membranes in a short
time.2 Agitation in the
presence of a Teflon bead led to further destruction of the membrane.
Thus the initially formed fibrils in the vesicle core can serve as
seeds for further fibrillation after they are released to the solution
outside the vesicles. This is confirmed by the observation of shorter
lag times for intravesicular protein in LUVs as opposed to
intravesicular protein in SUVs (Fig. 5).
-synuclein partially
folded intermediate leads to fibrillation, presumably due to
self-association driven by regions of exposed hydrophobic residues (17,
21-23). However, when
-synuclein is in its helical conformation it
preferentially binds to lipid. Thus, exposure of
-synuclein to lipid
vesicles leads to preferential binding to the membrane and induction of
helix, probably simultaneously, and prevention of protein
self-association. Therefore, the protein-lipid interaction induces and
stabilizes the helical structure and thus prevents aggregation.
-synuclein (24). Thus it is possible that there may
be some crowding effects on
-synuclein fibrillation at high lipid
vesicle concentrations. To determine whether this was a contributing
factor, we used PC vesicles, which have been reported not to bind
-synuclein (9). These vesicles had minimal effect on the
conformation of
-synuclein, presumably reflecting minimal binding to
the vesicles containing neutral head groups and no evidence for helix
formation was observed. The lag times for fibrillation, estimated from
kinetics curves shown in Fig. 5, increase slightly from 14 ± 2 h to 15 ± 2 (5:1), 17 ± 2 (1:1), and 19 ± 2 (1:5) h in a lipid concentration-dependent fashion. This
slight inhibition of fibrillation is probably due to some interaction
with the vesicles. The complete inhibition of fibril formation by PA/PC
SUV-bound
-synuclein thus results from the high helical structure
and not molecular crowding. This is also verified by the observation
that LUVs with the same amount of lipid, and thus the same crowding
conditions, result in only moderate fibril inhibition.
-helical conformation of
-synuclein does not fibrillate. This is also in agreement with
investigations of the effect of the osmolyte trimethylamine
N-oxide, which show that high concentrations of
trimethylamine N-oxide induce a helical conformation
in
-synuclein, and a corresponding lack of fibrillation (21).
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FOOTNOTES |
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* This work was supported by Grant NS39985 from The National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Chemistry and
Biochemistry, University of California, Santa Cruz, CA 95064. Tel.: 831-459-2744; Fax: 831-459-2935; E-mail:
enzyme@cats.ucsc.edu.
Published, JBC Papers in Press, March 5, 2003, DOI 10.1074/jbc.M210136200
2 M. Zhu, J. Li, and A. Fink, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are: SUV, small unilamellar vesicle; LUV, large unilamellar vesicle; ThT, thioflavin T; PA, 1,2-dipalmitoyl-sn-glycero-3-phosphate; PC, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine; PG, 1,2-dipalmitoyl-sn-glycero-3-phospho-RAC-(1-glycerol); AFM, atomic force microscopy; EM, electron microscopy.
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