From the Department of Molecular Biology, Max Planck
Institute for Biophysical Chemistry, Am Fassberg 11, Goettingen
D-37077, Germany and the
Institute of Molecular Genetics,
Russian Academy of Sciences, Kurchatov's Square, Moscow 123182, Russia
Received for publication, August 12, 2002, and in revised form, October 11, 2002
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ABSTRACT |
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The cellular polyamines putrescine, spermidine,
and spermine accelerate the aggregation and fibrillization of
Alpha-synuclein, a 14-kDa protein abundantly expressed in various
parts of the brain, is the major component of Lewy bodies, the
fibrillar proteinaceous cytosolic inclusions associated with Parkinson's disease (PD).1
Based on the amino acid sequence, three distinct domains have been
identified in In this work we studied the aggregation of Polyamines are present in neuronal cells (33, 34). Although systematic
function studies of their role are lacking, they are known to play a
role in the interaction with neurotransmitter receptors,
e.g. the N-methyl-D-aspartate
receptor, and ion channels, e.g. K+ (35, 36).
The polyamine levels in the substantia nigra of diseased human brains
of individuals exhibiting neurological disorders, including PD, are not
lowered, suggesting that the regulation of the substances is well
maintained even in degenerating cells (37). Spermine may serve to
protect neuronal cells from oxidative stress (38), a condition known to
induce aggregation of the amyloid A Expression and Purification of Aggregation Studies--
CD Spectroscopy--
CD spectra were recorded on a Jasco 720 spectropolarimeter equipped with a Peltier temperature controller. 15 µl of the protein solution, incubated with stirring in buffer alone
or in buffer supplemented with polyamines at 37 °C, was diluted in
10 mM Tris-HCl, pH 8.0, to a final volume of 200 µl for
CD measurements in cuvettes of 1-mm path length. These diluted samples
were not stirred further. The spectra were buffer-subtracted, and three
scans were averaged.
Fluorescence Measurements (Thioflavin T Binding
Assay)--
After incubation, protein solutions were diluted 40-fold
with 20 µM thioflavin T (thio T) (Sigma) in 10 mM Tris-HCl, pH 8.0. Fluorescence was measured on a Varian
Cary Eclipse spectrofluorometer in 1-cm path length quartz cuvettes.
Emission spectra (470-650 nm) were recorded for excitation at 450 nm,
using a 5-nm band-pass for both excitation and emission. The
contribution of unbound thio T to the fluorescence was measured on a
thio T sample at the same concentration but in the absence of
Electron Microscopy--
An aliquot was withdrawn from the
incubation mixture and placed onto a glow-discharged carbon film
attached to an EM grid. Carbon films, 3- to 4-nm thick, were pretreated
by glow discharge in the presence of pentylamine vapor (residual
pressure, ~150 millitorr; discharge current, 2-3 mA; duration of
discharge, 30 s) as described elsewhere (45). The adsorption
continued for 1-2 min, after which the grids were rinsed with a few
drops of 2% aqueous uranyl acetate, blotted with filter paper, and
dried. The samples were examined with a Philips CM12 electron
microscope. The negatives were scanned with a DuoScan T2500 scanner
(Agfa) at 1200 dots per inch. Micrographs were measured using
Image software (National Institutes of Health) modified for Windows.
For printing, images were flattened using a high pass filter with a
radius of 250 pixels and subsequently adjusted for contrast/brightness
using Adobe Photoshop.
Scanning Force Microscopy--
SFM images were acquired on a
Digital Instruments Nanoscope III microscope. A 2.5 µM
solution of Thioflavin T Assay--
Thioflavin T is a weakly fluorescent dye
in the free state but strongly fluorescent when bound to amyloidogenic
proteins in their aggregated state (25, 46). The fluorescence changes accompanying the binding of thio T to
The variation of fluorescence emission intensity of thio T
in the presence of
A qualitative comparison of the efficiency of different polyamines to
promote Circular Dichroism Spectroscopy--
The CD spectrum of native
After 8-h incubation with 10 mM putrescine, the sign of the
ellipticity of the band in the 200-nm region changed from negative to
positive, with a concurrent increase in negative ellipticity at 220 nm,
indicating that a significant fraction of the protein was converted to
a
Incubation of
The CD spectral changes associated with the aggregation of
Electron Microscopy--
The structures of Scanning Force Microscopy--
The structure of protein fibrils is
associated with a range of morphologies differing in structural
parameters such as fibril length, width, height, twist, and helical
periodicity (8, 47, 48). SFM constitutes a powerful tool for
visualizing and studying fibril morphology (47, 49). In the absence of
polyamines and after a long incubation time (48 h), Polyamines Modulate the Kinetics of
The model suggested by Uversky et al. (18) for metal
ion-induced fibrillization of
Formation of Amorphous Aggregates and Protofibrils Precedes
The increase in thio T fluorescence and the CD spectral changes
corresponding to the transition to the
Alternatively, the rapid aggregation at high polyamine concentration
may reflect the formation of insoluble small amorphous aggregates and
the corresponding turbidity at early time points (8 h) as observed for
Morphological Difference in the Fibrillar/Amorphous
Aggregates of Polyamines in Neurodegeneration and Neuroprotection--
The
concentrations of polyamines are maintained during neurodegeneration
associated with PD and AD (37), although the activities of ornithine
decarboxylase and spermidine/spermidine acetyl transferase, the key
enzymes in the biosynthetic pathway of polyamines, increase significantly (39, 60). Polyamines are scavengers for free radicals and
protect cells from free radical-induced oxidative damage, a process
that promotes aggregation of
We have demonstrated in this study that small molecules like polyamines
can modulate the kinetics of fibrillization of -synuclein, the major protein component of Lewy bodies associated
with Parkinson's disease. Circular dichroism and fluorometric
thioflavin T kinetic studies showed a transition of
-synuclein from
unaggregated to highly aggregated states, characterized by lag and
transition phases. In the presence of polyamines, both the lag and
transition times were significantly shorter. All three polyamines
accelerated the aggregation and fibrillization of
-synuclein to a
degree that increased with the total charge, length, and concentration of the polyamine. Electron and scanning force microscopy of the reaction products after the lag phase revealed the presence of aggregated particles (protofibrils) and small fibrils. At the end of
the transition phase,
-synuclein formed long fibrils in all cases,
although some morphological variations were apparent. In the presence
of polyamines, fibrils formed large networks leading ultimately to
condensed aggregates. In the absence of polyamines, fibrils were mostly
isolated. We conclude that the polyamines at physiological
concentrations can modulate the propensity of
-synuclein to form
fibrils and may hence play a role in the formation of cytosolic
-synuclein aggregates.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Synuclein has also been implicated in the pathogenesis of
Alzheimer's disease (AD), multiple system atrophy, diffuse Lewy body
disease, and amyotrophic lateral disease (1-5). The protein is
"natively unfolded" (6) but undergoes aggregation leading to
fibrillar structures, in which it adopts a
-sheet secondary
structure. The conformational transition of
-synuclein into a
-sheet structure and the fibrillization process are believed to
occur concurrently (7), although the mechanism is as yet unclear. The
formation of fibrils with an ordered
-sheet structure from monomeric
protein components is associated with many other diseases, including
AD, Huntington's disease, diabetes, and prion diseases (8-12).
Therefore, understanding the mechanism of fibrillization and the
factors modulating the aggregation process is essential for devising
therapeutic strategies against these diseases. Depending on the
structure of the aggregates formed, they are classified either as
amorphous, lacking ordered structures, or as fibrils, exhibiting a
-sheet secondary structure. Both types of aggregates occur in Lewy
bodies (13, 14), and their morphology is influenced significantly by
the solution conditions (13, 15).
-synuclein: (i) the N-terminal amphipathic region,
rich in amino acids with a high propensity for
-helix formation, and
known to be involved in binding to cell membranes and lipids, (ii) the
central hydrophobic region (amino acids 61- 95), and (iii) the acidic
C-terminal region in which most of the negatively charged amino acids
are located (16, 17). The aggregation of
-synuclein is promoted by a
variety of agents, including metal ions, lipids, pesticides, and
conditions that generate oxidative stress (18-22). Two mutations in
the
-synuclein gene (A53T and A30P) occur in about 10% of familial
PD cases and have been shown to accelerate the fibrillization of the
protein (23-25).
-Synuclein aggregation is accelerated by cationic
molecules, such as glycosyl amines and polylysine, and by the
multivalent metal ions Cu 2+, Fe2+,
Fe3+, Zn2+, and Al3+ (13, 18, 26).
These metal ions interact with the protein and may act by inducing
conformational transitions (18). The recent identification of dopamine
and its structural analogs as ligands for
-synuclein that inhibit
the formation of mature fibrils (27) offers new perspectives for the
utilization of small molecules to control the process of
-synuclein
aggregation/fibrillization.
-synuclein in the
presence of the biogenic polyamines putrescine
(H2N(CH2)4- NH2), spermidine
(H2N(CH2)3NH(CH2)4NH2),
and spermine
(H2N(CH2)3NH(CH2)4NH(CH2)3NH2). Polyamines are naturally occurring organic cations involved in a large
number of cellular functions, including DNA replication, transcription,
and protein synthesis (28, 29). The cellular concentration of spermine
is ~1 mM, with higher concentrations reported for
putrescine and spermidine (30-32). Due to their cationic nature,
polyamines interact with polyanionic molecules such as DNA and RNA and
induce structural changes dependent on the nucleotide sequence. In
addition to their positive charge, the hydrophobicity of the polyamines
is another important factor modulating their interaction with other macromolecules.
peptide, the major component of
amyloid deposits associated with Alzheimer's disease. It was also
shown in cultured neuronal cells that the A
peptide combined with
spermine is more toxic than the peptide alone (39). A possible role for
these cations in the aggregation of
-synuclein has not been
proposed. Although
-synuclein is present both in the nucleus and
cytoplasm of neuronal cells, insoluble protein aggregates form
exclusively in the cytoplasm (40, 41). Polyamines are present in the
cytoplasm at concentrations regulated by biosynthesis and by uptake
mediated by the polyamine transport proteins (42-44). Thus, an
intriguing possibility is that the subcellular co-localization of
-synuclein and polyamines in the cytoplasm may facilitate their
interaction. Indeed, our results suggest that polyamines at
physiologically relevant concentrations may serve to modulate the
aggregation and fibrillization of
-synuclein in vivo.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Synuclein--
The
recombinant plasmid pT7-7, encoding
-synuclein, was kindly provided
by the laboratory of Peter Lansbury. The plasmid was transformed into
Escherichia coli BL21(DE3), and
-synuclein expression was
induced with 1 mM
isopropyl-1-thio-
-D-galactopyranoside. The cell pellet
was collected by centrifugation at 4500 × g,
resuspended in lysis buffer (10 mM Tris-HCl, pH 8, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol), freeze-thawed three times, and sonicated. DNA was removed by precipitation with streptomycin (10 mg/ml) and centrifugation at 22,000 × g (rotor JA-20,
Beckman Avanti J-25 centrifuge) for 30 min at 4 °C. The
supernatant was collected, incubated in a boiling water bath for 20 min, and centrifuged at 22,000 × g.
-Synuclein was
precipitated by adding ammonium sulfate to the supernatant (final
concentration, 361 mg/ml) and centrifuged at 22,000 × g. The precipitate was resuspended in 25 mM
Tris-HCl, pH 8.0, applied to a Poros HQ column of a Biocad gel
perfusion chromatographic system (Applied Biosystems), and eluted with
a NaCl gradient (final concentration, 300 mM) in the same
buffer. The protein fractions were collected, dialyzed against 10 mM Tris-HCl, pH 8.0, and concentrated with Millipore
Centricon filters. The purity was >95% according to polyacrylamide
gel electrophoresis, electrospray ionization-mass spectrometry, and
analytical gel filtration. The protein was quantitated
spectroscopically using a molar extinction coefficient at 274 nm
of 5600 M
1 cm
1 (6).
-Synuclein (70 µM) was
incubated in 25 mM Tris-HCl, pH 7.5, at 37 °C with
vigorous stirring (magnetic bar) in glass vials. The polyamines
putrescine-2HCl, spermidine-3HCl, and spermine-4HCl were purchased from
Sigma. Concentrated stock solutions were prepared in distilled water
and diluted into the protein solutions in 25 mM Tris-HCl,
pH 7.5. The pH of the solution was measured after polyamine addition,
and no significant change was observed. Aliquots were removed from the
incubation mix at different time intervals and diluted to appropriate
concentrations for CD and fluorescence measurements.
-synuclein. The 40-fold diluted concentrations of polyamines in the
fluorescence assay did not exert an effect on the fluorescence
intensity of free thio T.
-synuclein in 10 mM Tris-HCl, pH 8.0, was
deposited onto a freshly cleaved mica surface. The SFM head equipped
with a fluid cell was placed on the top of the J-Scanner, and more
protein solution was added to the fluid cell. Imaging was performed in
liquid in tapping mode. Cantilevers (NP-S, Digital Instruments) with a
nominal spring constant of 0.32 newtons/m were used at an oscillation
frequency of ~9 kHz. For the imaging of
-synuclein fibrils, 0.5 mM MgCl2 was added to the incubation buffer
before imaging. Samples containing polyamines were imaged without added magnesium.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-synuclein before and after aggregation are depicted in Fig. 1. Thio
T fluorescence was very weak when the assay was performed immediately
upon initiating the incubation. After a lag time of ~22 h, the
intensity increased and reached a limiting value after ~52 h
(inset).
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Fig. 1.
Fluorescence emission spectra of thio T in
presence of native ( ) and aggregated (
)
-synuclein (52-h incubation). The excitation
wavelength was 450 nm. Inset, the variation of fluorescence
emission intensity at 480 nm with incubation time. 70 µM
-synuclein was incubated in 25 mM Tris-HCl, pH 7.5, at
37 °C and diluted to 1.75 µM in 10 mM
Tris-HCl, pH 8, for fluorescence measurements.
-synuclein incubated with putrescine, spermidine, and spermine is depicted in Fig. 2,
A, B, and C, respectively. Putrescine
was used at concentrations of 1, 5, and 10 mM. In the presence of 1 mM putrescine, the increase in initial
intensity occurred after 10 h and reached a plateau at ~18 h. At
higher putrescine concentration, the kinetics of fibrillization was
faster, reflected in a decrease in the lag time for the thio T
intensity signal (e.g. to 4 h for 10 mM
putrescine). Spermidine was more efficient than putrescine in promoting
-synuclein aggregation. An enhanced aggregation rate was observed at
a spermidine concentration as low as 100 µM (Fig.
2B). The lag time decreased with increasing polyamine
concentration. At 2 mM spermidine, the lag time was 6 h, and saturation in the binding profile occurred after 9 h with
the development of visible turbidity. Spermine was the most effective
of the polyamines in inducing the aggregation of
-synuclein (Fig.
2C). This tetra-cationic polyamine promoted aggregation at a
very low concentration (10 µM). For
-synuclein
incubated with 100 µM spermine, the lag time was ~2 h
and saturation in the binding profile occurred after 9 h.
Aggregation was visible at spermine concentrations
100
µM.
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Fig. 2.
Variation of fluorescence emission intensity
of thio T at 480 nm in presence of -synuclein
incubated with putrescine (A), spermidine
(B), and spermine (C). The
concentration of polyamines were, putrescine: 0 (
), 1 (
), 5 (
), and 10 (
) mM, spermidine: 0 (
), 100 µM (
), 500 µM (
), and 2 mM (
) and spermine: 0 (
), 10 (
), 25 (
), and 100 (
) µM. The excitation wavelength was 450 nm. The
concentrations of
-synuclein and thio T were 1.75 and 20 µM, respectively.
-synuclein aggregation, characterized by the lag time
t1, the time at which initial increase in thio T
intensity was observed, and transition time t2,
the incubation time at which the kinetic profile was saturated, is
given in Table I. Because the plateau
values in the kinetic curves of
-synuclein aggregated under
different conditions were different, t2 was
calculated from individual saturation values. In the absence of
polyamines, the increase in thio T fluorescence was complete at ~52 h
with the inception of aggregation at ~22 h. In the presence of 2 mM spermidine and 100 µM spermine, the lag
time decreased to ~6 and 2 h, respectively, with saturation in
the fluorescence signal at ~9 h.
Effect of increasing concentrations of polyamines on the
aggregation kinetics of -synuclein
-synuclein was characterized by a strong negative CD band in the
195- to 200-nm region, indicative of a disordered structure. In
contrast, the aggregated form had a positive band at ~200 nm, and a
negative band at ~220 nm corresponding to a
-sheet structure (Fig.
3A). This transition in
secondary structure to a
-sheet is a characteristic structural
feature of
-synuclein aggregation and fibrillization (6, 25). CD studies of native
-synuclein incubated with 10 mM
putrescine (Fig. 3B), 2 mM spermidine, or 100 µM spermine (Fig. 3C) were carried out at
three time points (0, 8, and 24 h). In all three cases, CD spectra
measured immediately after addition of the polyamine to the monomeric
protein (t = 0 data point) were indistinguishable from
the CD spectrum of the protein alone.
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Fig. 3.
Time-dependent CD spectra of
-synuclein alone (A) and in
presence of 10 mM putrescine (B) and
100 µM spermine
(C), measured at different incubation times. CD
spectra were measured immediately after initiation of incubation of
protein (t = 0) in buffer alone (A) or in
buffer supplemented with polyamines (B and C).
The incubations were continued and further spectra were acquired at the
time points indicated in the figures.
-sheet configuration. After 24 h, the positive ellipticity at
200 nm and the negative ellipticity at 220 nm increased further. No
additional CD spectral changes were detected at later times.
-synuclein with 2 mM spermidine for 8 h at 37 °C yielded a turbid solution. The CD signal was very weak
and noisy, suggesting that the protein had precipitated (data not shown). The spectral properties of the solution after 24 h were similar.
-synuclein in the presence of 100 µM spermine are
depicted in Fig. 3C. After incubation for 8 h, the
sample was visibly turbid, but the circular dichroism spectrum was
still characteristic of a disordered structure, albeit of lower
intensity. However, the CD spectrum measured after 24 h showed a
spectral pattern characteristic of a
-sheet structure, but with a
decreased absolute ellipticity compared with that of the
-sheet
conformation achieved in the absence of polyamines, suggesting an
increased solubility of the aggregates with time. We conclude that
visible aggregates formed at earlier time points may have acted as
nucleation centers for fibril formation or, alternatively, that the
formation of ordered structures from aggregates proceeded very slowly.
-synuclein
aggregates formed upon incubation with and without polyamines for
6 h, 3 days, and 7 days were examined by EM. Aggregates
formed in the absence of any polyamines after a 3-day incubation (Fig.
4A) were seen as long fibrils
(width ~13 nm), often forming large networks, and distributed rather uniformly on the surface of the carbon film. In the presence of polyamines, the structure of aggregates varied with polyamine nature
and incubation time. Fig. 4 (B and C) shows EM
images of
-synuclein after a 6-h incubation with 1 mM
spermidine and 0.5 mM spermine, respectively. In the
presence of spermidine,
-synuclein formed small aggregates of
variable size, whereas in the presence of spermine small aggregates
co-existed with short fibrils. At long incubation time (3 days), the
samples displayed a fibrillar structure regardless of the nature of the
polyamine; this structure was qualitatively similar to that formed in
the absence of polyamines. However, the nets were slightly larger and
more condensed, possibly indicative of an increased adhesiveness of the
fibrils. Two examples are shown in Fig. 4 (D and
E) corresponding to
-synuclein incubated with 5 mM putrescine and 10 µM spermine,
respectively. Incubation for 12 days in 1 mM spermidine led
to highly condensed aggregates, unevenly distributed over the carbon
surface (Fig. 4F). We speculate that these aggregates
represented stacks of individual fibrils.
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Fig. 4.
Electron microscopy images of
-synuclein aggregated in the presence and absence
of polyamines, at different time points. A,
-synuclein alone, 3 days; B, 1 mM spermidine,
6 h (dark field image); C, 500 µM spermine, 6 h (dark field image);
D, 5 mM putrescine, 3 days;
E, 10 µM spermine, 3 days; and F, 1 mM spermidine, 12 days. Scale bars, 100 nm.
-synuclein
formed isolated fibrils ~12 nm in height (Fig.
5A). In samples incubated with polyamines, protein aggregates were seen at time points as early as
4 h. The fibrils formed in the presence of spermidine were isolated, short entities with mean heights of ~12 nm (Fig.
5B), whereas spermine induced formation of large aggregates
displaying a highly diffuse structure (Fig. 5C). Similar
aggregates formed in the presence of 1 mM spermidine but
required longer (24 h) incubation (Fig. 5D). In 5 mM putrescine
-synuclein formed large aggregates
consisting of clearly visible individual short fibrils (Fig. 5,
E and F, for 24-h and 4-day incubations,
respectively).
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Fig. 5.
Scanning force microscopy images of
-synuclein aggregated in the presence and absence
of polyamines, at different time points. A,
-synuclein alone, 48 h; B, 1 mM
spermidine, 4 h; C, 100 µM spermine,
4 h; D, 1 mM spermidine,
24 h; E, 5 mM putrescine, 24 h; and
F, 5 mM putrescine, 4 days. Scale
bars: 100 nm (A and B); 200 nm
(C-F).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Synuclein
Aggregation--
The aggregation of
-synuclein into ordered fibrils
is a kinetically slow process involving the formation of a
-sheet
structure by the natively unfolded protein (50). Our studies suggest
that biogenic polyamines may play a role in accelerating this process.
-Synuclein has 24 negatively charged residues, the majority of which
are located in the C-terminal domain. The presence of this acidic
region and the hydrophobic region located between the C and N termini
provide binding targets for cationic and hydrophobic molecules that
could enhance the aggregation process. The polyamines are multivalent
cations with aliphatic hydrocarbon chains separating the charges, and
thus could potentially bind to
-synuclein via both hydrophobic and
electrostatic interactions. The potency with which polyamines
facilitate aggregation correlates with their cationic charge and the
number of aliphatic carbon chains between the amino/imino groups. Thus,
the tetracationic spermine with three hydrocarbon chains is more potent
than the lower homologs spermidine and putrescine. Presumably, the
greater number of charges located further apart by flexible linkers
permit more effective interactions with different regions of the same
protein or between different proteins. Because hydrophobic interactions
are important for holding different
-sheets together and are thus
essential features of the aggregation process, molecules that can bind
to the hydrophobic region but do not form
-sheets by themselves are
being developed and screened as drugs against diseases characterized by
the formation of protein aggregates (51).
-synuclein is relevant to the present study. As in the case of multivalent metal ions, polyamines may act by
bridging the carboxylate groups from the same protein or from
different proteins, thereby promoting aggregation. However, the
aggregation pathways may be different because the metal ions induce a
partially folded conformation of
-synuclein, whereas no such
secondary structural transition is observed in the presence of
polyamines. The ability of spermine to induce the aggregation, at a
concentration of about 1/10 that of spermidine and 1/100 that of
putrescine, cannot be explained by simple electrostatic considerations.
As discussed above, hydrophobic interactions may be responsible for the
higher potential of spermine to promote
-synuclein aggregation at
physiologically relevant concentrations.
-Synuclein aggregation is a cooperative process characterized by a
significant lag time. In addition to reducing the lag time, polyamines
also enhance the cooperativity of the transition. Saturation in the
kinetic profile is reached ~7 h after inception for
-synuclein
incubated with 2 mM spermidine or 100 µM
spermine, compared with ~30 h for
-synuclein incubated alone (Fig.
2 and Table I). The enhancement of the aggregation rate in the presence of polyamines reported here may serve to devise faster aggregation assays, a major goal in the screening of large numbers of potential drug molecules for the prevention or inhibition of fibrillization. A
competition assay in the presence of a ligand like spermine could be
carried out in hours rather than the many days currently required for
-synuclein aggregation in the absence of added ligands.
-Synuclein Fibrillization--
The formation of fibrils from
natively unfolded protein is presumed to involve at least four species
in the aggregation pathway, which follows the
scheme,
where UN, I, P, and F represent the initial unfolded
state, initial nucleation sites containing proteins in a conformation suitable for protein-protein interactions, protofibrils, and the mature
fibrils, respectively (52, 53). The protofibrils are intermediate
structures in the protein fibrillogenesis, exhibited by many proteins,
and assume different shapes (spherical, annular, and chain
protofibrils) often with lengths extending up to 200 nm and 1-10 nm in
diameter (8, 47, 54). Mature fibrils are formed by the association of
protofibrils or by further growth of protofibrils by the attachment of
monomeric molecules (48, 55). The initial lag phase, the time required
to form the nucleation centers, and the propagation phase that leads to
the formation of the protofibrils, were affected by the interaction
with polyamines. Work on the development of a kinetic scheme
incorporating both amorphous and fibrillar aggregates is in progress.
-sheet secondary conformation occurred simultaneously during
-synuclein aggregation in the absence
and presence of putrescine. However, it was unclear from CD spectra
whether the aggregates formed in the presence of spermidine or spermine
at high concentration possessed a
-sheet structure. The development
of turbidity in the sample, and the decrease in CD signal intensity
were indicative of precipitation of the protein. Fibrillar structures
were seen by SFM as early as 4 h, in the presence of high
concentrations of polyamines (Fig. 5, B and C). Examination by EM of the samples obtained under similar conditions after 6 h revealed a significant fraction of aggregates
corresponding to that of protofibrils (Fig. 4, B and
C). Only larger sized fibrils were present after 3 days of
incubation, implying that most of the protofibrils aggregated into
mature fibrils. However, the thio T fluorescence did not increase after
8-10 h of incubation, revealing that the transition from protofibrils
to fibrils was not associated with structural changes leading to
additional thio T binding. A logical conclusion is that the fibrils
probably formed by association of protofibrils.
-synuclein incubated with 100 µM spermine (Fig.
3C). Upon further incubation (24 h), a substantial fraction of the protein adopts a more soluble
-sheet conformation, giving rise to the characteristic CD spectra. In the case of the amyloid A
aggregation, the conversion of amorphous to fibrillar structures and
the generation of nucleation sites from amorphous aggregates for the
formation of structured fibrils by addition of monomeric A
have been
proposed (56).
-Synuclein Formed in the Absence and Presence of
Polyamines--
Electron microscopy and SFM revealed differences in
the
-synuclein aggregates formed in the presence or absence of
polyamines. In the EM images, the fibrils of
-synuclein formed in
the absence of polyamines were evenly distributed on the surface of
carbon films, forming networks of individual fibrils, many of which
were twisted (Fig. 4). In contrast, the polyamine-mediated aggregates comprised a multitude of fibrils, containing many filaments assembled as large aggregates. The aggregate morphology may be important in
toxicity as shown for
-synuclein (13) and other amyloid aggregates
(57-59).
-synuclein (21, 61). Paradoxically, our
observation that the polyamines accelerate aggregation suggests that
they may also facilitate neurodegeneration. A similar effect has been
reported for dopamine, which promotes the formation of
-synuclein
protofibrils, considered by some (27) to be more neurotoxic than the
mature fibrils. In contrast, Levadopa, the precursor of
dopamine, is a major drug used against PD (62). An intriguing
possibility is that a balance is maintained between the cell protective
and degenerative functions of these molecules. This balance would be
disrupted upon degeneration by the failure or dysfunction of regulatory
mechanisms such as the degradation of aggregates or binding of
-synuclein to membranes, a state stabilizing the protein in an
-helical structure (63).
-synuclein. This
finding may be important for the development of more efficient bioassays and the identification of drug candidates that inhibit, delay, or reverse aggregation. More fundamental is the possibility that
these ubiquitous cations are directly involved in the development of
Parkinson's disease.
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FOOTNOTES |
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* 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.
§ Recipient of a postdoctoral fellowship from the Alexander von Humboldt Foundation.
¶ Supported by the Stiftung Stipendien-Fonds des Verbandes der Chemischen Industrie, Germany and the Bundesministerium für Bildung und Forschung, Germany.
** To whom correspondence may be addressed. Tel.: 49-551-201-1382; Fax: 49-551-201-1467; E-mail: tjovin@gwdg.de.
To whom correspondence may be addressed (present
address): Advanced Science and Technology Laboratory, AstraZeneca R&D
Charnwood, Bakewell Rd., Loughborough LE11 5RH, United Kingdom. Tel.:
44-1509-647152; Fax: 44-1509-645519; E-mail:
vinod.subramaniam@astrazeneca.com.
Published, JBC Papers in Press, November 14, 2002, DOI 10.1074/jbc.M208249200
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ABBREVIATIONS |
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The abbreviations used are: PD, Parkinson's disease; AD, Alzheimer's disease; CD, circular dichroism; EM, electron microscopy; SFM, scanning force microscopy; thio T, thioflavin T.
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