From the
Center for Neurodegenerative Disease
Research and the Department of Pathology and Laboratory Medicine, the
¶Stokes Research Institute Children's Hospital of
Philadelphia and the Department of Biochemistry and Biophysics, University of
Pennsylvania Medical Center, Philadelphia, Pennsylvania 19104
Received for publication, December 6, 2002 , and in revised form, May 2, 2003.
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Recent studies support a role for oxidative and/or nitrative stress in
-syn modification and/or aggregation. For example, our previous work
showed that
-syn protein was nitrated in pathological lesions such as
LBs and GCIs (23). Other
studies also demonstrated that nitrating agents such as
peroxynitrite/CO2 or
myeloperoxidase/H2O2/nitrite can nitrate and oxidize
-syn at tyrosine (Tyr) residues, resulting in the formation of highly
stable o,o'-dityrosine oligomers
(25). Significantly,
cross-linking, or the formation of covalent bonds between amino acids of the
same or different
-syn molecules, of pre-formed
-syn filaments
by nitrating agents confers filament stability to chaotropic agents,
supporting a role for nitrating agent-induced modifications in the
stabilization of
-syn inclusions
(25).
-Syn
self-oligomerization into an SDS-resistant ladder can also be induced by
oxidative agents such as copper and hydrogen peroxide, and this
oligomerization appears to be dependent upon the acidic C terminus of the
-syn protein (26). In
contrast, oxidation by dopamine appeared to inhibit selectively the conversion
of protofibrils to fibrils, causing accumulation of the
-syn
protofibrils (27). Thus, the
contribution of oxidative versus nitrative species on the aberrant
conversion of
-syn from a highly soluble monomeric state to soluble
oligomers and finally to insoluble filamentous
-syn inclusions remains
unclear (23,
25,
28). Because Tyr residues are
potential targets of oxidation and we previously showed that all four Tyr
residues in
-syn are targets for nitration
(25), we investigated the role
of each of the four Tyr residues (at amino acid positions 39, 125, 133, and
136) in
-syn fibrillization and fibril stabilization under oxidative
and/or nitrative conditions. Using WT and Tyr (Tyr
Phe) mutant
-syn recombinant proteins in vitro and stably transfected
cultured cells, we showed that the presence of one or more Tyr residues is
required for nitrating species-induced
-syn protein cross-linking and
filament stabilization. Surprisingly,
-syn protein cross-linking and
fibril stabilization induced by transition metal-mediated oxidation does not
require Tyr residues in
-syn. These data suggest that independent
pathogenic mechanisms are involved in oxidative-versus
nitrative-induced
-syn fibrillogenesis.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
AntibodiesMonoclonal antibodies LB509, Syn 208, and Syn
204, which are specific for human -syn
(8,
29,
30), as well as monoclonal
antibodies that specifically recognize nitrated Tyr residues in
-syn
were used in this study (23).
The specificity of the latter antibodies is as follows: nSyn 12 recognizes a
complex epitope containing
-syn nitrated at Tyr-125 and Tyr-136; nSyn
14 recognizes
-syn nitrated at Tyr-39; and nSyn 24 recognizes
-syn nitrated at Tyr-125 and Tyr-133.
Exposure of -Syn Proteins to the Nitrating Agent,
Peroxynitrite Peroxynitrite (ONOO-) was synthesized
from sodium nitrite and acidified H2O2, and excess
H2O2 was removed by treatment with manganese dioxide
(31). Treatment of
-syn
proteins with ONOO- was carried out as described previously
(25). Briefly,
-syn
proteins were diluted into nitration buffer containing 100 mM
potassium phosphate, 25 mM sodium bicarbonate, pH 7.4, and 0.1
mM diethylenetriamine pentaacetic acid. The concentration of
ONOO- was determined spectrophotometrically at 302 nm in 2
N NaOH (
302 = 1670 M-1
cm-1). Peroxynitrite was added in 2 boluses of 10-fold molar excess
of the protein. After treatment, proteins were boiled in SDS sample buffer (10
mM Tris, pH 6.8, 1 mM EDTA, 40 mM
dithiothreitol, 1% SDS, 10% sucrose) and resolved by SDS-PAGE. Proteins were
either stained with Coomassie Blue R-250 for quantification by densitometry or
analyzed by immunoblotting using monoclonal antibodies directed against WT
-syn and nitrated
-syn followed by incubation with an anti-mouse
horseradish peroxidase-conjugated antibody (Jackson ImmunoResearch
Laboratories, West Grove, PA) and detected with enhanced chemiluminescence
reagents (PerkinElmer Life Sciences).
Oxidation of -Syn Proteins Using Transition Metals and
Peroxide The effects of oxidative damage on
-syn proteins
were examined in reaction mixtures of phosphate-buffered saline (137.5
mM NaCl, 2.5 mM KCl, 10 mM
Na2HPO4, 1.75 mM
KH2PO4, pH 7.5) in the presence of 500 or 50
µM CuCl, CuCl2, FeCl2, or FeCl3
and 300 µM H2O2. Where indicated,
reactions were incubated at 37 °C for 4 h and prepared for assembly
experiments as described below or diluted with SDS sample buffer and heated to
100 °C for 5 min.
Assembly and Polymerization of -Syn
ProteinsUntreated or ONOO--exposed
-syn proteins
at a concentration of 5 mg/ml were exchanged in 100 mM sodium
acetate buffer (pH 7.0) containing 0.04% sodium azide. Studies involving
copper and peroxide oxidation were similarly exchanged into acetate buffer,
and the oxidative reagents were added at a final concentration of 500
µM CuCl and 300 µM H2O2.
-Syn proteins were incubated either at 33 °C for 4 days or at 37
°C for 2 days with continuous shaking at 1000 rpm. Each assembly reaction
sample was overlaid with 4050 µl of mineral oil to prevent
condensation of samples and hence the alteration of results.
Direct Visualization of -Syn Fibril FormationFor the
direct ultrastructural inspection of filament formation,
-syn protein
samples were adsorbed to 300-mesh carbon-coated copper grids, stained with 1%
uranyl acetate, and visualized with a transmission electron microscope (Joel
1010, Peabody, MA) as described previously
(19).
Assessment of -Syn Polymerization by Centrifugal Sedimentation
AnalysisSamples were centrifuged at 100,000 x g for
20 min after assembly incubation. Supernatants and pellets were separated, SDS
sample buffer was added, and the samples were heated to 100 °C for 5 min.
-Syn proteins were resolved by SDS-PAGE, stained with Coomassie Blue
R-250, and quantified by densitometry. The kinetic consistency of the
experimental conditions used for fibril formation was determined by incubating
-syn proteins for varying lengths of time (3 to 96 h) at 37 °C and
1000 rpm using sedimentation analysis in addition to the K114 fluorescence
analysis (described below) to confirm that
-syn protein assembly
increases over time reproducibly.
Assessment of the Formation of Amyloidogenic Polymers Using the K114
AssayThe formation of -syn amyloidogenic polymers was
assessed using a novel amyloid-binding dye
(trans,trans)-1-bromo-2,5-bis-(4-hydroxy)styrylbenzene (or K114).
K114 recognizes amyloid fibrils in vitro and in pathological
inclusions and has distinct fluorescent properties that allow for the
monitoring of amyloid fibril formation in
solution.2 The K114
fluorescence assay was performed by adding 5 µl of each assembly sample (25
µg of
-syn protein) to 100 µl of K114 assay solution (100
mM glycine buffer, pH 8.5, 50 µM K114). Fluorescence
was measured using a SpectraMax GeminiXS fluorometer (Molecular Devices,
Sunnyvale, CA) and SoftMax Pro 4.0 software with a fixed excitation wavelength
of 380 nm and a fixed emission wavelength of 550 nm with a cutoff at 530
nm.
Filament Stabilization StudiesFollowing -syn protein
assembly at 37 °C for 3 days with continuous shaking, samples were
centrifuged at 100,000 x g for 20 min. Pellets were resuspended
in nitration buffer and exposed to ONOO- at room temperature or
resuspended in phosphate-buffered saline and treated with 500 µM
CuCl and 300 µM H2O2 for 1 h at 37 °C.
Samples were then diluted into either H2O or 4 M urea,
incubated at room temperature for 10 min, and centrifuged at 100,000 x
g for 20 min. The supernatants and pellets were separated, and
samples were heated to 100 °C for 5 min in SDS sample buffer.
-Syn
proteins were resolved by SDS-PAGE, stained with Coomassie Blue R-250, and
quantified by densitometry.
In Vivo Oxidative/Nitrative Stress ExperimentsHuman
-syn cDNAs corresponding to the WT or the mutant protein with all four
Tyr residues mutated to Phe, i.e. Y39F,Y125F,Y133F,Y136F (referred to
as 4(Y
F)), were cloned into the pcDNA 3.1+ mammalian expression vector
(Invitrogen). HEK293 cells were maintained with Dulbecco's modified
medium-high glucose containing 10% fetal bovine serum, 1%
penicillin/streptomycin, and 2% L-glutamine. Cells were transfected
with WT and 4(Y
F)
-syn plasmids by a calcium phosphate
precipitation method buffered with BES
(32). Stably transfected
clones were selected for and maintained in culture medium supplemented with
300 µg/ml geneticin. Cells were plated at a density of 1 x
106/well in 35-mm tissue culture plates and exposed to PapaNO
(1-propananmine-3-(2-hydroxy-2-nitroso-1-propylhydrazine)) and/or paraquat as
described previously (33).
Briefly, cells were rinsed with warm media and then incubated in 2 ml of media
supplemented with 7.5 mM paraquat for 30 min at 37 °C.
Subsequently, PapaNO was added at a final concentration of 1.05 mM,
and cells were incubated for an additional 1.5 h.
Cells plated on coverslips were fixed in 4% paraformaldehyde and permeabilized in 0.2% Triton/phosphate-buffered saline. Cells on coverslips were incubated overnight at 4 °C with Syn 208 and Syn 204, rinsed the following morning, and incubated with secondary anti-mouse antibody Alexa 488 fluorescein isothiocyanate (Molecular Probes, Eugene, OR). Coverslips were mounted on slides using Vecta Shield containing 4,6-diamindino-2-phenylindole (Vector Laboratories, Burlingame, CA). Cells plated without coverslips were rinsed with phosphate-buffered saline and lysed in 2% SDS and 50 mM Tris, pH 6.8. Protein concentration was determined using the bicinchoninic acid protein assay kit. Samples were boiled with SDS sample buffer, and 7 µg of total protein were resolved by SDS-PAGE and immunoblotted with LB509.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
All Tyr Phe mutant proteins were capable of forming polymers to the
same extent as the WT
-syn protein as assessed by sedimentation
analysis (Fig. 1, B and
C). Kinetic analysis of WT and mutant
-syn
assembly using both sedimentation and K114 fluorescence analyses revealed that
amyloidogenic polymer formation increased consistently and reproducibly over
time, although the 4(Y
F) mutant
-syn protein was slower in
forming amyloidogenic fibrils than the WT
-syn protein
(Fig. 1D). Shaking at
slightly higher temperatures results in a higher percentage (8090%) of
the
-syn monomers converted to fibrils in both WT and mutant proteins
(data not shown). Furthermore, direct observation of fibrils by EM examination
failed to reveal any significant morphological difference between WT and
mutant
-syn fibrils, including width, length, or twisting and turning
of the fibrils (Fig.
1E). These results indicate that the hydroxyl functional
group on Tyr residues in
-syn proteins is not critical for the assembly
of the protein into fibrils under these in vitro conditions.
Role of Tyr Residues in the Cross-linking of -Syn Proteins by
Nitrating AgentsPrevious data indicated that WT
-syn
protein exposed to nitrating agents forms oligomers through dityrosine
cross-linking (25). However,
the specific Tyr residues involved in the formation of dityrosine cross-linked
oligomers have not been identified. To address this question, WT and mutant
-syn proteins were exposed to the nitrating agent, ONOO-, in
the presence of CO2. The exposed
-syn proteins were resolved
by SDS-PAGE gels and analyzed by Coomassie Blue R-250 staining
(Fig. 2A) or Western
blotting using antibodies specific for the different nitrated Tyr residues
(Fig. 2B). Exposure of
WT, single, double, and triple Tyr
Phe mutant
-syn proteins to
ONOO- in the presence of CO2 induces nitration as well
as oxidation of Tyr residues to form dityrosine, which results in the
formation of SDS-stable dimers and oligomers
(Fig. 2, A and
B). These results suggest that all four Tyr residues are
potential targets for protein nitration as well as for dityrosine
cross-linking. As predicted, the mutant
-syn protein that lacks all
four Tyr residues (4(Y
F)) neither formed SDS-resistant dimers nor was
nitrated after exposure to the nitrating agent
(Fig. 2, A and
B). Decreasing the number of Tyr residues in the
-syn protein resulted in a reduction in SDS-stable oligomer, yet the
presence of a single Tyr residue in the
-syn protein
(Y125F,Y133F,Y136F; Y39F,Y133F,Y136F; Y39F,Y125F,Y133F; and Y39F,Y125F,Y136F)
is sufficient for the formation of dimers
(Fig. 2B).
|
The ability of WT and mutant -syn proteins to polymerize after
exposure to ONOO- in the presence of CO2 was determined
by sedimentation analysis (Fig. 2,
C and D). The formation of pelletable polymers
of WT and mutant
-syn proteins containing at least one intact Tyr
residue was inhibited by treatment with ONOO- in the presence of
CO2. The lack of
-syn fibrils from samples of
ONOO--exposed WT and mutant
-syn proteins containing at
least one intact Tyr residue was further confirmed by EM (data not shown),
which indicates that the nitration of any Tyr residue can contribute to the
prevention of fibril formation. The 4(Y
F)
-syn protein exposed
to ONOO- formed some pelletable oligomers
(Fig. 2, C and
D), but to a lesser extent than unexposed 4(Y
F)
-syn. This result indicates that other protein modifications, in
addition to 3-nitrotyrosine, contribute to the inhibition of the
polymerization of
-syn protein upon exposure to nitrating agents.
Indeed, nitrating agents such as peroxynitrite or nitrogen dioxide are higher
oxidation-state species of nitric oxide, formed by chemical reactions of
nitric oxide with superoxide or by the one electron oxidation of nitrite by
peroxidases and other heme proteins and hydrogen peroxide. Although these
reactive nitrogen species primarily nitrate Tyr residues, they are also
capable of oxidizing Tyr to form dityrosine cross-linked species. In addition,
other amino acid residues, such as cysteine, tryptophan, and histidine could
be oxidized. Therefore, exposure of
-syn protein to nitrating agents
results in both nitration and some oxidation
(25).
Tyr Residues of -Syn Proteins Are Not Critical for the
Oligomerization Induced by Oxidizing AgentsIt was previously shown
that WT recombinant
-syn protein forms SDS-stable cross-linked
oligomers upon exposure to oxidizing agents such as H2O2
and redox active metals (26,
35,
36). Consistent with these
previous reports, incubation of WT
-syn with CuCl plus
H2O2 resulted in the formation of SDS-stable oligomers
(Fig. 3A), whereas
incubation with transition metals or H2O2 separately did
not result in the formation of these oligomeric species (data not shown). In
contrast to the effects of nitration, all mutant
-syn proteins formed
cross-linked oligomers equally well, including the 4(Y
F) mutant
-syn protein (Fig.
3A). Replacing CuCl with FeCl2,
FeCl3, or CuCl2 in the presence of
H2O2 resulted in similar formation of protein oligomers
(data not shown). Furthermore, WT and 4(Y
F)
-syn proteins
exposed to CuCl and H2O2 were also able to assemble and
form fibrils after shaking at 1000 rpm for 2 days at 37 °C, and a time
course study was performed using centrifugal sedimentation analysis in
conjunction with the K114 fluorescence technique to ensure the reproducibility
of our results (Fig.
3B). WT and 4(Y
F)
-syn proteins assembled
to the same extent with or without CuCl and H2O2
treatment, irrespective of whether or not the protein was oxidized during the
assembly (Fig. 3, C and
D) or 4 h before the assembly (data not shown). These
data indicate that simple oxidation without nitration of the protein does not
target Tyr residues, and it does not render
-syn assembly incompetent.
EM analysis was conducted to determine the precise structural characteristics
of these oxidation-challenged
-syn samples. The micrographs depict the
samples as bundles of tightly associated filaments admixed with some
non-filamentous
-syn protein (Fig.
3E). The formation of both filamentous and
non-filamentous aggregates is likely because of the co-occurrence of filament
assembly and chemical cross-linking of non-fibrillar
-syn because of
oxidation. Taken together, these results suggest that Tyr nitration disrupts
the process of fibril formation, but oxidation without nitration of Tyr
residues does not prevent
-syn protein fibril formation and may
potentiate the formation of inclusions that are comprised of both fibrillar
and aggregated
-syn protein.
|
Role of Tyr Residues in the Stability of WT and 4(Y F)
-Syn ProteinsThe ability of nitrating and oxidizing agents
to stabilize the pre-assembled, filamentous WT and 4(Y
F)
-syn
proteins was evaluated by incubating pre-assembled proteins exposed to
nitrating and/or oxidizing agents with the chaotropic agent, 4 M
urea. The addition of 4 M urea to fibrillar
-syn disrupts
the majority of the fibrils so that most of the protein is recovered from the
100,000 x g soluble fraction
(Fig. 4A). EM
confirmed the disruption of
-syn fibrils such that after exposure to 4
M urea, short fibrils were only very rarely observed
(Fig. 4F). Consistent
with our previously report
(25), exposure of
pre-assembled WT
-syn protein to ONOO- resulted in the
formation of urea-stable polymers (Fig.
4A). Peroxynitrite-exposed assembled WT
-syn
protein is significantly more stable to treatment with chaotropic agents
compared with untreated WT
-syn protein, where the urea-stable
pelletable fraction increases from 23.6 to 97.5% upon treatment with
ONOO- (Fig. 4, A and
E). Consistently, EM assessment of samples of WT
-syn fibrils exposed to ONOO- followed by 4 M
urea revealed that, although the amount of fibrils was slightly reduced
compared with untreated/unsolubilized sample, there were still abundant
fibrils that were resistant to urea (Fig.
4F). By contrast, exposure of pre-assembled 4(Y
F)
mutant
-syn fibrils to ONOO- failed to render them resilient
to 4 M urea by sedimentation analysis
(Fig. 4, B and
E), and there were far fewer urea-resistant fibrils
observed by EM (data not shown). These data are consistent with the hypothesis
that dityrosine cross-linking is responsible for the stabilization of
-syn fibrils treated with chaotropic agents. Thus, Tyr residues are
primary sites of reactivity for nitrating agents with
-syn protein. By
contrast, exposure of both assembled WT and 4(Y
F)
-syn proteins
to oxidants (Cu/H2O2) caused an increase in the
urea-stable pelletable fraction from 11.2 to 73.55% for WT protein and 32.4 to
76.75% for 4(Y
F)
-syn protein
(Fig. 4, CE).
Thus, oxidation of assembled
-syn proteins induced fibril stability to
disruption by chaotropic agents through a Tyr-independent oligomerization
mechanism.
|
Formation of WT, but Not 4(Y F),
-Syn Protein
Inclusions by Intracellular Nitrative InsultsOur previous studies
have demonstrated that treatment of HEK293 cells stably expressing WT
-syn protein with nitrating and oxidizing agents resulted in the
formation of cytoplasmic nitrated
-syn inclusions
(33). To assess the role of
Tyr residues in this process, we treated HEK293 cells stably expressing either
WT or 4(Y
F)
-syn protein with paraquat, an alkylating agent and
superoxide generator
(3739),
and PapaNO, a nitric oxide donor
(40,
41). The formation of small
-syn inclusions in the cytoplasm of cells was assessed by indirect
immunofluorescence (Fig.
5A). As expected, cells expressing WT
-syn protein
formed
-syn protein inclusions in
34% of the cells, whereas
cells expressing 4(Y
F)
-syn protein produced significantly less
of these
-syn inclusions (
0.2%)
(Fig. 5B). This
finding was not a result of protein expression levels as the 4(Y
F)
-syn cells express more
-syn protein than the WT
-syn
cells (Fig. 5C).
Moreover, the
-syn protein levels were not influenced by treatment with
paraquat and nitric oxide reagents (Fig.
5C). Untreated cells and cells treated with paraquat
alone or PapaNO alone did not show
-syn inclusion formation
(Fig. 5B). Although
cells treated under these nitrative and oxidative conditions were lysed and
fractionated to determine the formation of covalently linked oligomers, higher
Mr oligomers were not detected
(Fig. 5D), even after
prolonged exposure of Western blots (data not shown). These results indicate
that if cross-linking occurs, it must be at very low levels. Nevertheless, the
paucity of inclusions in cells expressing 4(Y
F)
-syn suggests
that the nitrative modification of Tyr residues in
-syn induces a
redistribution of the protein into focal accumulations.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
To test our hypothesis, we examined the critical role of Tyr residues in
-syn fibril formation and stabilization after exposure to nitrating
and/or oxidizing agents. Our results showed that Tyr residues are responsible
for the formation of covalently cross-linked
-syn dimers and oligomers
after exposure to ONOO-, because such dimers were not detected in
the 4(Y
F)
-syn mutant protein that lacks Tyr residues. By
contrast, Tyr residues are not responsible for the formation of covalently
linked dimers and higher order oligomers after treatment of
-syn with
pure oxidizing agents (e.g. H2O2 plus CuCl),
because these stable oligomers were detected in the 4(Y
F)
-syn
mutant protein. Furthermore, despite the formation of dityrosine dimers,
-syn proteins nitrated at one or more Tyr residues do not assemble into
fibrils, whereas both native and purely oxidized
-syn proteins are
capable of fibrillogenesis under the same experimental conditions. Because 4(Y
F)
-syn was able to fibrillize following exposure to nitrating
agents, albeit to a lesser extent compared with untreated control, this
suggests that nitration is at least partially responsible for the inhibition
of fibril formation. It is important to recognize that 4(Y
F)
-syn protein was affected somewhat by the ONOO- exposure, as
it did not fibrillize to control levels. This result may be accounted for by
other oxidative mechanisms associated with ONOO-, and these
alternative chemical modifications act synergistically in preventing the
polymerization of
-syn. Although oxidation caused by CuCl and
H2O2 does not affect the assembly of
-syn
proteins, the oxidation caused by ONOO- may in fact be of a
different mechanism and hence affect fibril formation. In this regard, our
observations with nitrated
-syn might be reminiscent of the recent
report demonstrating that dopamine-mediated oxidative alterations of
-syn prevent the formation of mature fibrils, leading to the
accumulation of structures consistent with protofibrils
(27). Because it is difficult
to identify and quantify protofibrils, we are not certain whether or not the
nitration-induced inhibition of
-syn fibrillogenesis acts through a
similar mechanism. Future studies will be needed to address this important
issue.
As noted above, both WT and 4(Y F)
-syn proteins were
unaffected in their abilities to form cross-linked oligomers and fibrils when
exposed solely to oxidizing agents, i.e.
Cu/H2O2 (Fig.
3), indicating that this oxidation of Tyr residues and/or other
-syn amino acid residues does not affect the ability of the protein to
form fibrils. Uversky et al.
(43) reported that extensive
oxidation of all methionine residues in
-syn protein using 1.2
M H2O2 prevents fibril formation of the
protein, yet this oxidation process is likely to be of a different mechanism
or the treatment with 1.2 M H2O2 more
extensively oxidizes the protein compared with the system used in the studies
of this paper. Therefore, the degree of oxidation may also be critical for the
ability of WT
-syn to form fibrils.
Recently, Krishnan et al.
(44) reported that the
formation of dityrosine dimers can facilitate fibrillogenesis, and they
suggested that these dimers might be a critical step in this process. Our
studies with 4(Y F)
-syn protein demonstrate that
fibrillogenesis does not require the formation of dityrosine, but this does
not exclude that dityrosine dimers could potentially act as initiators of
polymerization (44). A major
difference between our studies and those of Krishnan et al.
(44) is the condition used to
promote fibrillogenesis. Our system involves constant agitation to increase
the rate of polymerization, which allows our data to be measured and analyzed
within hours. However, the authors of the aforementioned work used a
non-agitation technique, which requires much longer periods of time
(1520 days) for
-syn to fibrillize
(44). These differences in
assembly kinetics could be responsible for the apparent differences in the
requirement for dityrosine dimers to initiate filament formation. The most
parsimonious interpretation of these data is that filament formation does not
require dityrosine dimers, but the formation of these dimers can facilitate
fibril formation, especially under slow kinetic conditions.
Exposure of -syn to the nitrating agent, ONOO-,
stabilizes filaments assembled from WT but not 4(Y
F)
-syn
proteins. In contrast, oxidative modifications induced by CuCl and
H2O2 stabilize fibrils comprised of either WT or 4(Y
F)
-syn proteins. This result suggests that different mechanisms
must be responsible for the stabilization of
-syn fibrils by nitrative
and oxidative damage. Whereas Tyr residues are required for the stabilization
of
-syn filaments induced by ONOO-, the stabilization of
-syn fibrils induced by CuCl- and H2O2-mediated
oxidative insults is independent of Tyr residues. Further investigation into
the oxidative-induced modifications of
-syn will be needed to determine
which amino acid residues are affected in this process.
The involvement of Tyr residues in the formation of -syn inclusions
in vivo associated with nitrative and oxidative stress was assessed
in a cultured cell line. Previously we showed that exposure of HEK293 cells
expressing WT as well as the A53T and A30P
-syn mutant proteins to
nitric oxide- and superoxide-generating compounds such as paraquat, rotenone,
dopamine, and PapaNO resulted in the formation of small cytoplasmic
-syn inclusions (33).
These cellular inclusions contain nitrated
-syn, and ultrastructural
analysis showed evidence for the presence of
-syn filaments
(33), suggesting that
nitration may play a role in the stabilization of some small
-syn
oligomers present in HEK293 cells expressing very high levels of
-syn
protein. Because our transfected cells express high levels of
-syn
protein and it is known that
-syn protein has a tendency to assemble
into fibrils at high concentrations, a dynamic equilibrium may exist between
the soluble monomers of
-syn and protofibrils and small oligomers of
-syn intracellularly. The equilibrium may favor monomeric
-syn
in the absence of nitrating agents, but in the presence of nitrating agents,
oligomers may become stabilized by nitration and/or dityrosine cross-linking
and hence form a nidus for further
-syn inclusion formation. The
demonstration that HEK293 cells stably expressing 4(Y
F)
-syn
produce significantly fewer inclusions when challenged with nitric oxide and
superoxide generators (Fig. 5, A
and B) provides further support that nitration of Tyr
residues in
-syn is involved in the stabilization of these inclusions.
It is unclear whether or not dityrosine formation is involved, because we were
unable to detect any cross-linked species of
-syn
(Fig. 5D). However,
because only a small number of cells (34%) developed
-syn
aggregates and because it is likely that only a small amount of
-syn is
modified by nitration and/or dityrosine cross-linking, it is not surprising
that we were unable to detect dityrosine by Western blot analyses. Future
development of a specific antibody to dityrosinated
-syn may help to
resolve this issue.
Recently, Fujiwara et al.
(45) reported the extensive
phosphorylation of Ser-129 in urea-soluble monomeric -syn protein
extracted from LBs in human disease brain. However, these authors did not find
any modification at Tyr residues 133 and 136 (the sequence data did not cover
Tyr residues 39 or 125). With the use of specific anti-nitrated
-syn
antibodies, we have shown that LBs are specifically labeled with antibodies
recognizing Tyr-39 as well as Tyr residues 125, 133, and 136
(23). The discrepancy between
our data and that of Fujiwara et al.
(45) could be explained as
follows. We recovered the majority of nitrated
-syn in the
urea-insoluble fraction, but Fujiwara et al.
(45) did not analyze this
insoluble fraction. Also, because nitrating agents would also covalently
cross-link
-syn, nitrated
-syn molecules would be present in
both monomeric and cross-linked oligomeric protein, but this also was not
examined in the aforementioned work.
Our studies demonstrate for the first time that distinct oxidative and
nitrative mechanisms play a role in modulating -syn fibril formation
and stabilization. Fig. 6
illustrates the possible consequences of nitrative- and oxidative-induced
modifications on the formation of
-syn lesions. Tyr residues are
required for nitrating agent-induced
-syn covalent cross-linking and
nitration. Although dityrosine cross-linking could represent a critical step
in fibril formation, the nitration of Tyr residues appears to prevent
fibrillogenesis from soluble
-syn proteins. Once fibrils of
-syn
protein are formed, nitrating agents can stabilize these pre-formed filaments
through dityrosine cross-linking. These data suggest that the nitrated
filamentous
-syn protein detected in LBs, Lewy neurites, and GCIs is
likely to be a late event that occurs after
-syn fibrils already have
developed. On the other hand, oxidative stress could be an early or late event
because it can cause soluble
-syn to form covalently linked dimers and
higher Mr oligomers and hence allow for fibril formation
and stabilization. Our data suggest that this process is independent of Tyr
residues, but the exact amino acid residues involved in this process remain to
be elucidated. Thus, although the molecular mechanisms by which
-syn
protein polymerizes and forms inclusions within cells remain elusive, the data
presented here provide credence for the role of oxidative and nitrative stress
in the formation of stable
-syn inclusions in human disease.
|
![]() |
FOOTNOTES |
---|
Supported by a senior fellowship award from the Canadian Institute of
Health Research.
|| John H. Ware III professor of Alzheimer's research. To whom correspondence should be addressed: Center for Neurodegenerative Disease Research, Dept. of Pathology and Laboratory Medicine, 3600 Spruce St., Maloney 3, HUP, Philadelphia, PA 19104-4283. Tel.: 215-662-6427; Fax: 215-349-5909; E-mail: vmylee{at}mail.med.upenn.edu.
1 The abbreviations used are: -syn,
-synuclein; EM, electron
microscopy; GCIs, glial cytoplasmic inclusions; HEK, human embryonic kidney;
LBs, Lewy bodies; ONOO-, peroxynitrite; WT, wild type; 4(Y
F), Y39F,Y125F,Y133F,Y136F; BES,
N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid; PapaNO,
1-propananmine-3-(2-hydroxy-2-nitroso-1-propylhydrazine).
2 Crystal, A. S., Giasson, B. I., Crowe, A., Kung, M.-P., Zhuang, Z.-P.,
Trojanowski, J. Q., and Lee, V. M.-Y. (2003) J. Neurochem., in
press.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|