From the Department of Chemistry and Biochemistry, University of California, Santa Cruz, California 95064
Received for publication, March 31, 2003 , and in revised form, May 13, 2002.
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ABSTRACT |
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INTRODUCTION |
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-Synuclein is a small (14 kDa), highly conserved presynaptic protein
that is abundant in various regions of the brain
(8,
9). Structurally, purified
-synuclein belongs to the rapidly growing family of intrinsically
unstructured or natively unfolded proteins
(10,
11), which have little or no
ordered structure under physiological conditions due to a unique combination
of low overall hydrophobicity and large net charge
(12).
-Synuclein
readily assembles into amyloid-like fibrils in vitro with
morphologies and staining characteristics similar to those extracted from
disease-affected brain (11,
1318).
Fibrillation occurs via a nucleation-dependent polymerization mechanism
(14,
17) with a critical initial
structural transformation from the unfolded conformation to a partially folded
intermediate (11).
The cause of PD is unknown, but considerable evidence suggests a
multifactorial etiology involving genetic susceptibility and environmental
factors. Recent work has shown that, except in extremely rare cases, there
appears to be no direct genetic basis of PD
(19). However, several studies
have implicated environmental factors, especially pesticides and metals
(20). In agreement with these
observations, it has been recently reported that direct interaction of
-synuclein with metal ions
(21) or pesticides leads to
accelerated fibrillation
(2224).
Oxidative injury is also suspected as another causative agent in the
pathogenesis of PD (25,
26). The existence of nitrated
-synuclein (i.e. protein containing the product of the
tyrosine oxidation, 3-nitrotyrosine) accumulation in Lewy bodies has been
demonstrated
(2729).
Accumulation of another product of tyrosine oxidation, dityrosine, has been
detected in vitro during experiments on the aggregation of
-synuclein in the presence of copper and H2O2
(30) or catecholamines
(31) and leads to accelerated
fibrillation of
-synuclein
(32). The methionine side
chain is the most readily oxidized amino acid in
-synuclein, and the
four methionines, Met-1, Met-5, Met-116, and Met-127, are easily oxidized
in vitro in the presence of H2O2.
Interestingly, however, oxidation of the methionine residues of
-synuclein to the sulfoxides, rather than accelerating fibrillation,
was found to prevent it (33).
Furthermore, and most importantly, the presence of the methionine-oxidized
-synuclein was found to completely inhibit fibrillation of the
unmodified protein at ratios of
4:1
(33). Given the potential role
of metals in the pathological aggregation of
-synuclein and the known
strong coordination of some metals to sulfoxides, we decided to investigate
the structural and fibrillation properties of Met-oxidized
-synuclein
in the presence of several metals to shed more light on the combined effect of
environmental factors (metals) and oxidative damage (methionine oxidation to
the sulfoxide) on
-synuclein.
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MATERIALS AND METHODS |
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Supplies and ChemicalsThioflavin T (ThT) was obtained from Sigma. ZnSO4 and CaCl2 (analytical grade) were from Fisher. Analytical grade Ti2(SO4)3, CuCl2, and Hg(CH3CO2)2 were from Aldrich, whereas AlCl3 and PbO2 were from Mallinckrodt Chemical Works and Matheson Coleman & Bell, respectively. All other chemicals were of analytical grade from Fisher. All buffers and solutions were prepared with nanopure water and stored in plastic vials.
Oxidation of -Synuclein by Hydrogen
PeroxideOxidation of
-synuclein by
H2O2 was performed as described previously
(33).
Circular Dichroism (CD) MeasurementsCD spectra were
recorded on an AVIV 60DS spectrophotometer (Lakewood, NJ) using
-synuclein concentrations of 1.0 mg/ml and a 0.1-mm path length cell.
Spectra were recorded from 250190 nm with a step size of 1.0 nm, with a
bandwidth of 1.5 nm and an averaging time of 10 s. For all spectra, an average
of five scans was obtained. CD spectra of the appropriate buffers were
recorded and subtracted from the protein spectra.
Electron MicroscopyTransmission electron micrographs were collected using a JEOL JEM-100B microscope operating with an accelerating voltage of 80 kV. Typical nominal magnifications were x75,000. Samples were deposited on Formavar-coated 300-mesh copper grids and negatively stained with 1% aqueous uranyl acetate.
Fibril Formation AssayFibril formation of oxidized and
non-oxidized -synuclein in the presence of various metals was monitored
using the ThT assay in a fluorescence plate reader (Fluoroskan Ascent) as
described previously (33).
Standard conditions were 35 µM
-synuclein, pH 7.5, 20
mM Tris-HCl buffer, 37 °C, with agitation. ThT fluorescence was
excited at 450 nm, and the emission wavelength was 482 nm.
Estimation of Hydrodynamic DimensionsDynamic light scattering was used to determine the Stokes radii with a DynaPro Molecular Sizing Instrument (Protein Solutions, Lakewood, NJ) using a 1.5-mm path length 12-µl quartz cuvette. Prior to measurement, solutions were filtered with a 0.1-µm Whatman Anodisc-13 filter.
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RESULTS |
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Previously, we demonstrated that the interaction of metal cations with
natively unfolded -synuclein induced a partially folded conformation
(21). This transition was
attributed to the counter ion-induced neutralization of the coulombic
charge-charge repulsion within the very negatively charged protein at neutral
pH (21). In agreement with
this observation, Fig. 1 shows
that in the presence of metals, definite changes occur in the far-UV CD
spectra of both non-oxidized and oxidized forms of
-synuclein. In
particular, a decrease in the minimum at 196 nm was accompanied by an increase
in negative intensity around 222 nm, reflecting metal binding-induced
formation of secondary structure (Fig.
1). Significantly, Fig.
1 shows that binding of the metals induced comparable structural
changes in both oxidized and unmodified proteins, most probably reflecting the
stabilization of identical partially folded conformations. Thus, Met-oxidized
-synuclein is slightly more unfolded than non-oxidized protein, but in
the presence of metal ions, it adopts a similar partially folded conformation.
Our previous studies have shown that formation of such a partially folded
conformation correlates with accelerated fibrillation, as is seen with the
effect of metals on non-oxidized
-synuclein
(21).
The Effect of Metal Binding on Fibrillation of Methionine-oxidized
-SynucleinNext, we determined the effect of the metals
on the fibrillation of Met-oxidized
-synuclein. ThT is a fluorescent
dye that interacts with amyloid fibrils, leading to an increase in the
fluorescence intensity in the vicinity of 480 nm
(34).
Fig. 2 compares fibrillation
patterns of non-oxidized (Fig.
2A) and oxidized
-synuclein
(Fig. 2B) in the
absence and presence of several metal cations monitored by ThT fluorescence.
Fibril formation for the non-oxidized
-synuclein at neutral pH was
characterized by a typical sigmoidal curve. In agreement with earlier studies
(24), the fibrillation rate
increased dramatically in the presence of all metal cations investigated
(Fig. 2A). The list of
the previously analyzed cations (Li+, K+,
Na+, Cs+, Ca2+,
Co2+, Cd2+,
Cu2+, Fe2+,
Mg2+, Mn2+,
Zn2+, Co3+,
Al3+, and Fe3+) has been extended
to consider the effect of Hg2+,
Pb2+, and Ti3+. Interestingly,
Hg2+ and Pb2+, which are of
particular relevance to environment-induced Parkinsonism, are among the most
effective accelerators of
-synuclein fibrillation. This underlines,
once again, a potential link between heavy metal exposure, enhanced
-synuclein fibrillation, and Parkinson's disease.
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In contrast, there was no evidence of fibril formation by
methionine-oxidized -synuclein at neutral pH
(Fig. 2B). Previously,
we showed that the inhibitory effect of methionine oxidation on
-synuclein fibrillation can be eliminated under conditions of low pH,
due to the formation of a partially folded intermediate reflecting protonation
of the carboxylate groups
(33). In view of this
observation, and the observation that metal cations induce partial folding of
oxidized
-synuclein (Fig.
1), one might expect that fibrillation of the methionine-oxidized
protein would occur in the presence of metals. In accord with this hypothesis,
methionine-oxidized
-synuclein readily formed fibrils in the presence
of certain metal ions, such as Ti3+,
Al3+, Zn2+, and
Pb2+ (Fig.
2B and Table
I). However, not all metals were able to accelerate the
fibrillation of methionine-oxidized
-synuclein: for example,
Hg2+, Cu2+, and
Ca2+, although able to induce the partially folded
conformation in the oxidized protein, did not induce its fibril formation (at
least not within the time scale examined). Moreover,
Fig. 2 and
Table I show that in the
presence of Zn2+ and Pb2+,
fibrillation of the oxidized
-synuclein was as accelerated as for the
non-oxidized protein, whereas Al3+ and
Ti3+ showed a less pronounced effect. The morphology of
the fibrillar material formed by the non-oxidized and oxidized
-synuclein in the presence of several metal cations was analyzed by
transmission electron microscopy, and both forms of
-synuclein formed
typical amyloid fibrils, as shown in Fig.
3.
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Dynamic Light Scattering Experiments to Monitor Hydrodynamic
SizeThere are a number of possible mechanisms whereby methionine
oxidation could inhibit -synuclein fibrillation. One of these would be
through stabilization of off-pathway oligomers, and another would be through
the capping nascent fibrils. To investigate these possibilities, we monitored
the association state of Met-oxidized
-synuclein during its incubation,
in the absence and presence of metal ions, using dynamic light scattering
(Fig. 4). Given the nature of
the experimental measurements, populations of oligomers of less than
510% are not considered significant. Since the data shown in
Fig. 4 are only for soluble
protein, the total concentrations may be different in the different panels of
the figure.
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Met-oxidized -synuclein remained monomeric for >100 h under
standard incubation conditions (35 µM
-synuclein, pH 7.5,
37 °C, with agitation), as shown in
Fig. 4D, indicating
that neither oligomers nor fibrils were formed in statistically significant
amounts. In contrast, unmodified
-synuclein remained predominantly
monomeric for the first 20 h (corresponding to the lag time) but then showed
dimers and higher oligomers at longer times (in addition to fibrils), as shown
in Fig. 4A. Thus, the
conversion of methionine to its sulfoxide must, in some way, prevent formation
of the critical partially folded intermediate conformation and subsequent
association into fibrils. In the presence of Zn2+, which
leads to fibril formation from the Met-oxidized
-synuclein, the monomer
is the only species initially present. However, at later times, in addition to
fibrils, soluble oligomers were detected, amounting to as much as 30% of the
total protein and having an Rs of
40 nm,
similar to the size of the oligomers observed with the unmodified protein. In
contrast, in the presence of Ca2+, which does not lead
to fibrils with Met-oxidized
-synuclein, only the monomer was detected
during the incubation. Since these two metals reflect the observed behavior of
the two types of metal ion-induced effects in the other properties
investigated, their behavior is considered representative of other metal
ions.
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DISCUSSION |
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Previously, we have shown that formation of a partially folded intermediate
is a critical initial step of the -synuclein fibrillogenesis
(11) and that
-synuclein fibrillation is accelerated under conditions that stabilize
such an intermediate state, e.g. at acidic pH or high temperature
(11,
36) or in the presence of
metal cations (21). A
contributing factor to the inhibition of methionine-oxidized
-synuclein
fibrillation is believed to be the slightly increased stabilization of the
natively unfolded conformation
(33).
The data presented here are consistent with the conclusion that interaction
of methionine-oxidized -synuclein with certain metals modulates its
conformational properties and propensity for fibrillation. Whereas all the
metals studied are able to induce partial folding in this intrinsically
unstructured (natively unfolded) protein, not all cations are equal in their
abilities to eliminate the inhibitory effect of methionine oxidation on
-synuclein fibrillation. In particular, the fibrillation rates were
very close for oxidized and non-oxidized
-synuclein in the presence of
Zn2+ and Pb2+; however,
fibrillation was still inhibited in the presence of
Hg2+, Cu2+, and
Ca2+. This observation indicates that factors other than
electrostatic interactions must play an important role in overcoming the
inhibition of
-synuclein fibrillation caused by methionine oxidation.
One such factor is undoubtedly the known propensity for certain metals to
strongly coordinate with sulfoxides, leading to very stable complexes
(37). In particular, for some
metal ions, bridging between two sulfoxides is favored. Such intermolecular or
intramolecular coordination of two (or more) methionine sulfoxides could
significantly affect the fibrillation. In particular, we propose that stable
intermolecular bridging metal complexes would significantly promote
fibrillation: thus, the presence of Zn2+ or
Pb2+ leads to intermolecular cross-bridging, which
facilitates the association of Met-oxidized
-synuclein and leads to its
subsequent fibrillation. Metals such as Hg2+ and
Cu2+, which may also form sulfoxide bridges, may be
limited to intramolecular coordination due to different ligand bonding. The
results show that in those conditions where fibrillation occurs, large soluble
oligomers are present at the latter stages of the lag time and during the
fibril growth stage of the aggregation process.
With regard to the biological relevance of these observations, it is
becoming clear that many factors can affect the rate of -synuclein
fibrillation, suggesting that in dopaminergic neurons, there is a balance
between factors that can accelerate fibrillation and those that inhibit or
prevent it. It is likely that there are chaperones or chaperone-like species
that are important in minimizing
-synuclein aggregation under normal
conditions. In our earlier study, showing that the addition of Met-oxidized
-synuclein inhibited fibrillation of the non-oxidized form
(33), we suggested that the
methionine residues in
-synuclein may be used by the cells as a natural
scavenger of reactive oxygen species, since (a) methionine can react
with essentially all of the known oxidants found in normal and pathological
tissues; (b)
-synuclein is a very abundant brain protein;
(c) it has recently been shown that the concentration of
-synuclein could be increased significantly as a result of the neuronal
response to toxic insult (23);
and (d) methionine sulfoxide residues in proteins can be cycled back
to their native methionines by methionine sulfoxide reductase
(38), a process that might
protect other functionally essential residues from oxidative damage
(39). It should be noted,
however, that the efficiency of this regeneration system must take into
account the finding that methionine oxidation forms the sulfoxide in two
diastereoisomer forms and that stereoselective oxidation can sometimes occur,
dependent on both the structural restraints in the region of the methionine
molecule and on the oxidant itself
(40). Each methionine
sulfoxide isomer can be reduced back to its original methionine state,
provided that the corresponding complementary reductase is present and active
(41).
The balance between the protective antioxidant role of the methionine
residues that is enhanced by this recycling and the protective
antifibrillation effect of oxidized methionine residues in -synuclein
may fail under conditions of environmental pollution due to exposure of a
person to lead, aluminum, zinc, titanium, and other metals. We assume that in
the presence of the enhanced concentrations of such industrial pollutants,
toxic insult-induced up-regulation of
-synuclein may no longer play a
protective role; rather, it may represent a risk factor, leading to
metal-triggered fibrillation of the methionine-oxidized protein.
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FOOTNOTES |
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Present address: 307 Greene St., Mill Valley, CA 94941.
To whom correspondence should be addressed. Tel.: 831-459-2744; Fax:
831-459-2935; E-mail:
enzyme{at}cats.ucsc.edu.
1 The abbreviations used are: PD, Parkinson's disease; ThT, thioflavin T.
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REFERENCES |
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