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INTRODUCTION |
Although many neurodegenerative diseases are characterized by
specific patterns of neuronal loss, distinct clinical presentations, and unique histopathological features, many of the diseases including Parkinson's, Alzheimer's, trinucleotide repeat diseases, and
spongiform encephalopathies display proteinaceous aggregates,
inclusions, or plaques as characteristic or defining features upon
pathologic examination of involved tissue (1). These plaques or
inclusions are composed of neuronal proteins or fragments thereof,
which have adopted altered conformations that have resulted in the
formation of fibrillar amyloid plaques or inclusions having a high
degree of
-pleated sheet secondary structure.
Parkinson's disease (PD)1 is
the second most common neurodegenerative disease with a prevalence of
about 2% of those 65 years of age and older. The etiology of PD is at
least partly genetic (2, 3), and kindreds having autosomal dominant and
autosomal recessive forms of PD have been identified (4).
In rare families, either of two identified mutations in the
-synuclein gene can cause autosomal dominant early onset PD.
-synuclein was initially identified as an abundant nerve terminal protein in the Torpedo electric organ and thought to be
transiently associated with neuronal membranes (5, 6). Since then it has been "rediscovered" in a variety of assays (7-9).
Heightened interest in
-synuclein developed when
-synuclein gene
mutations were found to be responsible for some cases of autosomal
dominant Parkinson's disease (10-12), and the protein was found to be
a major component of Lewy bodies (LBs) (13-15), the fibrillar neuronal
proteinaceous inclusions that are a histologically defining feature of
Parkinson's disease.
Ultrastructurally, Lewy bodies have a dense fibrillar core, and the
fact that they stain with thioflavin S (16, 17) indicates that the
constituent proteins have a high degree of
-pleated sheet secondary
structure. The fibrillar core contains synuclein (18, 19). LBs are seen
in sporadic and familial PD, and in vitro evidence exists
that PD-associated synuclein mutations increase the rate of synuclein
fibrilization (20-23).
Intriguingly,
-synuclein-containing glial cytoplasmic inclusions are
found in oligodendrocytes in multiple system atrophy (24-26), and in
neurodegeneration with brain iron accumulation type I, neurons in the
globus pallidus display Lewy body-like
-synuclein containing
inclusions (27). Taken together, these findings suggest that altered
synuclein conformation is linked to neurodegeneration.
Overexpression of human
-synuclein as a transgene in mice (28, 29)
or in Drosophila (30) results in formation of
synuclein-containing inclusions that in many respects resemble Lewy
bodies, in alterations of dopamine neurons, and in movement-related
phenotypes. Deletion of the
-synuclein gene in mice does not result
in a phenotype resembling PD. Instead, dopaminergic neurons prepared
from
-synuclein-deficient mice display altered dopamine release in
one experimental paradigm of synaptic plasticity (31). It therefore
appears more likely that the synuclein mutations are associated with a
gain of a toxic function, and it is not exclusively the loss of
synuclein function that causes PD in the affected patients. One effect
of PD mutations may be to increase the propensity to form fibrils,
again suggesting that synuclein conformations are related to PD.
The most striking feature of the primary amino acid sequence of
-synuclein is the repetition 8 times of an 11-amino acid motif that is predicted to form amphipathic
-helices (7). Contrary to the predictions, recombinant or purified
-synuclein is
considered "natively unfolded" and has little identifiable periodic
secondary structure as assessed by circular dichroism (32, 33) or NMR
spectroscopy (34). However, when incubated with small unilamellar
liposomes of certain lipid (mimicking those found in synaptic
vesicles), SDS micelles, or certain helix-forming solvents, the protein
undergoes a conformational change, becoming largely helical, and
associates with the liposomes (33, 35, 36). An NMR study showed that it
is indeed the N-terminal portion of the protein that can become helical
and membrane-associated (34). Despite these findings and the synaptic
localization of
-synuclein (5), immunoelectron (37) and
immunofluorescence microscopic and cell fractionation experiments (7,
38) have failed to demonstrate a definite or stable association with
synaptic vesicles or any other subcellular membrane. At this point, it is not clear which subcellular membranes
-synuclein may associate with.
Although purified
-synuclein initially has little in the way of
periodic secondary structure, in vitro studies with purified or recombinant synuclein have demonstrated that synuclein can spontaneously form fibrils having a high degree of antiparallel
-pleated sheet structure (16, 21, 39) and that PD-associated synuclein mutations accelerate fibril formation (20, 22, 23, 40).
Given the apparent paradox that pure synuclein spontaneously forms
fibrils or, in the presence of certain lipid membranes, becomes largely
helical and binds to membranes but that in cells most synuclein appears
to be natively unfolded and not membrane bound, we hypothesize that
synuclein conformation and/or membrane association may be regulated
by synuclein-binding proteins.
Several proteins have been proposed to be synuclein interactors (41,
42). In transfection experiments, synphillin (43), an
-synuclein
interactor identified in a yeast two-hybrid screen, promoted formation
of NAC (a peptide derived from synuclein) aggregates. Also, by
interacting with
-synuclein,
-synuclein may inhibit the ability
of
-synuclein to form fibrils (44).
Although much work has begun to elucidate the mechanism by which
purified
-synuclein forms fibrils and of some stimuli that may
trigger aggregation of
-synuclein, relatively little is understood about how or whether
-synuclein interactors alter fibril formation.
We employed a photocross-linking strategy (45) to detect proteins in
brain extracts present at endogenous levels and in direct contact with
-synuclein. We purified and identified one such protein as
calmodulin (CaM). We show that CaM and
-synuclein interact in a
calcium-dependent manner both in vitro and
in vivo and that activated CaM accelerates the kinetics of
synuclein fibrilization.
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EXPERIMENTAL PROCEDURES |
In Vitro Transcription/Translation and
Photocross-linking--
In vitro transcription using T7 or
T3 RNA polymerase (Promega) was as described (46). mRNAs were
translated for 15-40 min at 26 °C in a wheat germ translation
system supplemented with [35S]Met (Amersham Biosciences;
1100 Ci/mmol). Photocross-linking using trifluoromethyldiazirinobenzoic
acid (TDBA)-Lys-tRNA was as described (45). After translation, 500 ml
of buffer (20 mM HEPES, pH 7.4, 150 mM KCl) was
added to translation reactions (100-150 ml) containing His-tagged
-synucleins. 25 ml of TALON Co2+-agarose beads
(Clontech) equilibrated in binding buffer was
added, and binding was allowed to proceed for 1 h at room
temperature. Beads were washed free of the majority of the wheat germ
lysate protein with two 1-ml washes of binding buffer (20 mM HEPES, pH 7.4, 150 mM KCl). Beads were
resuspended in a volume equal to the original translation volume in
binding buffer. Typically 5 ml of this suspension was used for
each cross-linking assay. After cross-linking, beads were sedimented,
after which supernatants were aspirated. Laemmli sample buffer
containing 50 mM EDTA was added to the beads to elute bound
-synuclein and photocross-links. After boiling for 5 min,
samples were subjected to SDS-PAGE and fluorography.
In experiments not utilizing bead-isolated
-synuclein, components
indicated in the figure legends were added directly to 5-ml translation
reactions and subjected to photoactivation. After cross-linking, 10 volumes of 10% trichloroacetic acid was added, and proteins were
precipitated. Pellets were rinsed with acetone before being resuspended
in Laemmli sample buffer. After heating, samples were subjected to
SDS-PAGE and fluorography.
Mutagenesis of synuclein was achieved using the QuikChange kit
(Stratagene), and all mutations were confirmed by DNA sequencing.
Preparation of BBC--
Meninges and large vessels were peeled
from three calf brains (~1 kg of tissue). After rinsing in PBS,
brains were homogenized in a blender in 2 liters of 20 mM
HEPES, 150 mM KCl, pH 7.5. The homogenate was filtered
through cheese cloth three times and then centrifuged to remove
unbroken cells, nuclei, and mitochondria (Sorvall GS-3 rotor, 8500 rpm,
1 h, 4 °C). The resulting supernatant was clarified by
centrifugation (Beckman Ti 50.2 rotor, 37,000 rpm, 60 min, 4 °C,
~110,000 × gav). The protein
concentration was 4 mg/ml by the Bio-Rad DC protein assay kit using
bovine serum albumin as a standard.
Purification of 15-19-kDa Cross-links from Brain
Cytosol--
Bovine brain cytosol (500 ml) was heated to 70 °C in a
circulating water bath. 100-ml aliquots were heated in flasks for 5 min
after the thermometer in the sample reached 70 °C. Flasks were
rapidly cooled in liquid nitrogen and then kept on ice. Aggregated protein was removed by ultracentrifugation (Beckman Ti50.2 rotor, 37,000 rpm, 35 min, 4 °C). 400 ml of heat-treated cytosol was incubated with 10 ml of SP-Sepharose fast flow (Amersham Biosciences) previously equilibrated in 20 mM HEPES, 150 mM
KCl, pH 7.5, in a batch for 12 h at 4 °C. 360 ml of the
SP-Sepharose supernatant was added to a vessel containing 7 ml of Q
Sepharose FF (Amersham Biosciences) equilibrated in 20 mM
HEPES, 150 mM KCl, pH 7.5. After 1 h at room
temperature, the flow-through was removed, and the resin was washed
with 100 ml of 20 mM HEPES, 400 mM KCl. The resin was then packed into a column (7-ml bed volume) and was washed
with 20 ml of 20 mM HEPES, pH 7.4, 400 mM KCl.
Bound proteins were eluted with a linear gradient of KCl (0.5-1.0
M) in ~12 column volumes. 28 3.5-ml fractions were
collected. Fractions were assayed for the ability to cross-link to
-synuclein. Peak activity eluted at around 515 mM KCl.
Mass Spectrometry--
Bands were excised from Coomassie
Blue-stained gels and digested with trypsin and processed for mass
spectrometric analysis (47). Briefly, the peptide mixture was partially
fractionated on a Poros 50 R2 RP microtip, and resulting peptide pools
were analyzed by matrix-assisted laser-desorption/ionization reflectron time-of-flight MS using a Reflex III instrument (Brüker Franzen; Bremen, Germany), and by electrospray ionization tandem MS on an API
300 triple quadrupole instrument (PE-SCIEX; Thornhill, Canada),
modified with an ultrafine ionization source as described (48).
Selected mass values from the matrix-assisted laser desorption time-of-flight experiments were taken to search a protein nonredundant data base (NCBI, Bethesda, MD) using the PeptideSearch (49) algorithm.
Triple quadrupole MS/MS spectra were inspected for y" ion series, and
the information was transferred to the PepFrag (50) program for use as
a search string.
Production of Recombinant
-Synuclein--
-Synuclein
cDNAs were cloned into pET 21D (Novagen) and the plasmids were
expressed in BL21DE3 E. coli. Cultures of 750 ml were grown
to midlog phase and
isopropyl-1-thio-
-D-galactopyranoside was added to 0.4 mM. After 2 h, cells were pelleted, washed in PBS, and
finally resuspended in 50 ml of 20 mM HEPES, 100 mM KCl, pH 7.2. The resuspended bacteria were heated to
90 °C for 5 min. Aggregated protein was removed by centrifugation
(Beckman Ti 60 rotor, 32,000 rpm, 30 min, 4 °C). The supernatant was
at least 90% pure synuclein as assessed by SDS-PAGE. Contaminating
nucleic acids and proteins were removed from the lysate by ion exchange chromatography on Q-Sepharose Hi-Trap columns (Amersham Biosciences).
-Synuclein eluted at ~250 mM KCl. Synuclein-containing
fractions were pooled and chromatographed on a Superose 12 column
(Amersham Biosciences) in 20 mM HEPES, pH 7.4, 100 mM KCl. Peak fractions were identified by Western blotting.
Chemical Cross-linking with Disuccinimidyl Suberate (DSS) in
Intact Cells--
DSS (Pierce) (25 mM stock in
Me2SO) was prepared fresh for each experiment and was used
at 1 mM. Cross-linking was allowed to proceed for 15-20
min at 37 °C. The reaction was then quenched by the addition of 0.1 volumes of 1 M glycine buffered in 100 mM
NaHCO3, pH 8.
SK-N-SH neuroblastoma cells were rinsed three times with PBS containing
calcium to remove serum proteins and amino acids that would quench the
DSS. DSS (1 mM final), a calcium ionophore, A23187 (20 mM final from 2 mM stock in Me2SO),
and calmidizol (10-20 mM from 5 mM stock in
Me2SO, from Sigma) were added to prewarmed 10-cm dishes as
indicated in the figures. After 20 min, the cross-linker was quenched
as described above. After 10 min on ice, cells were rinsed with PBS,
and each dish was lysed in 200 ml of lysis buffer (20 mM
HEPES, pH 7.4, 150 mM NaCl, 1% Triton X-100, 0.1% SDS). Nuclei and debris were removed by centrifugation at 1500 × gav at 4 °C for 10 min. Anti-synuclein
monoclonal antibody (Transduction Laboratories) was added to 6 mg/ml
and incubated overnight at 4 °C. Immune complexes were collected by
the addition of 10 ml of Protein G-Sepharose. After 1 h,
immunoprecipitates were washed three times in lysis buffer and once in
water. Immunoprecipitated protein was eluted by the addition of Laemmli
sample buffer. Samples were electrophoresed through 12% gels and
transferred to nitrocellulose, and blots were probed with monoclonal
anti-CaM antibodies (Upstate Biotechnology, Inc., Lake Placid,
NY) at 1 mg/ml in 5% milk/PBS overnight at 4 °C. Blots were
developed using the ECL-Plus Chemiluminescence kit (Amersham
Biosciences).
CaM-Sepharose Chromatography--
Wild type
-synuclein (120 ml of 0.5 mM synuclein in PBS plus 0.1 mM
CaCl2) was incubated with 15 ml of CaM-Sepharose (Amersham Biosciences) for 30 min at room temperature. The flow-through was
collected, and beads were washed three times with 200 ml of binding
buffer. Specifically bound synuclein was eluted with 200 ml of PBS
containing 5 mM EDTA. Fractions were subjected to
trichloroacetic acid precipitation and synuclein content was determined
by Western blotting.
Dansyl Labeling of CaM and Fluorescence Measurements--
CaM
(1.5 mg) was dissolved in 400 ml of 0.2 M
NaHCO3, pH 9.0. 20 ml of 10 mg/ml dansyl chloride in
dimethyl formamide was added. After mixing, derivatization
was allowed to proceed for 1 h on ice. Unincorporated dye was
removed by gel filtration (PD-10 column (Amersham Biosciences)
developed in PBS) and then by dialysis. Spectra were obtained in a
PerkinElmer LS50B fluorimeter. Excitation was at 338 nm, and emission
spectra were collected from 425 to 600 nm with an excitation slit width
of 7.5 nm and an emission slit width of 5.0 nm unless otherwise stated.
Dansyl-CaM was used at approximately 1 mM (Fig.
6a) or 0.086 mM (Fig. 6, b and
c).
For measurements of dansyl-CaM binding to synuclein, synuclein was
added to cuvettes containing dansyl-CaM at a final concentration of 86 nM in PBS with 2.5 mM CaCl2.
Emission spectra were collected, and fluorescence intensity at 486 nm
was recorded for each sample. For construction of the Scatchard plot in
Fig. 6, the fluorescence measured at 486 nm of dansyl-CaM in the
absence of synuclein was subtracted from the reading obtained for each
sample containing synuclein. Neither the synuclein alone nor unlabeled
CaM showed any fluorescence at 486 nm. The fraction of bound dansyl-CaM
at each concentration was determined from the fractional fluorescence increase at 486 nm using the following relationship:
fb = Im
If/Ib
If, where fb represents
the fraction of dansyl-CaM bound, Im is the measured
fluorescence, and If is the fluorescence of dansyl-CaM when all is free (i.e. in the absence of
synuclein). Ib is dansyl-CaM fluorescence when all
is bound. Ib was measured at saturating
concentrations of
-synuclein. For the batch of dansyl-CaM used in
Fig. 6, b and c, saturating concentrations of
-synuclein resulted in about a 1.4-fold increase in dansyl-CaM fluorescence. Measuring the fraction of dansyl-CaM bound at each synuclein concentration allowed us to measure the concentration of
bound synuclein (fraction of dansyl-CaM bound × [dansyl-CaM]). The concentration of free synuclein was determined by subtracting the
bound concentration from the total synuclein concentration in each
sample. These numbers allowed for construction of a saturation curve
and a Scatchard plot from which the dissociation constant was estimated
(y =
0.0561x + 4.5944). The least squares
method was used to fit the data from the Scatchard plot to a straight line.
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RESULTS |
Experimental Strategy--
To identify
-synuclein-interacting
molecules, we employed a photocross-linking approach. The technology,
developed by investigators primarily interested in protein
translocation across membranes, has been successfully utilized to
identify proteins comprising the translocation pores in ER (45, 51) and
mitochondrial membranes (52). In addition, it has been useful in
tracing the temporal changes in molecular interactions that newly
synthesized proteins undergo as they execute their folding program
(53-55).
In this approach (see Fig. 1), the
"bait" is translated in vitro in the presence of
[35S]Met and lysyl-tRNA, where the amino group of the
lysine residue has been selectively modified with the photoactivable
cross-linker TDBA. The result is that cross-linker-modified
lysyl residues are incorporated into the in vitro translated
protein, as dictated by the distribution of lysine codons in the
mRNA. After in vitro translation, covalent attachment of
the bait to interacting molecules is caused by UV light-induced
activation of the cross-linker. UV activation of the photoprobe
generates a highly reactive carbene that will react with nearly any
chemical bond. The carbene intermediate persists for ~1 ns and is
therefore diffusion-limited. If water is next to the cross-linker, it
will be quenched. If, on the other hand, a protein is in direct contact
with the cross-linker-modified lysine upon activation, the bait will be
cross-linked to the target molecule. The size of the probe is about 5 Å. After translation, cross-links are detected by SDS-PAGE followed by
fluorography. The molecular weight of the in vitro
translated bait is known, and cross-linking of proteins to the
radiolabeled bait results in decreased electrophoretic mobility on
SDS-PAGE from which the approximate molecular weight of the
cross-linked protein can be inferred.

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Fig. 1.
a, schematic illustration of
photocross-linking assay. b, domain structure of
-synuclein. The positions of lysyl residues where
cross-linker can be incorporated are shown. Also, the positions of the
Parkinson's disease-associated mutations and of the serine
phosphorylation sites are shown.
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A salient feature of the approach is that it allows for temporal and
spatial control of cross-linking. When compared with conventional
cross-linkers, this approach is more specific, and cross-linking cannot
occur through intermediate proteins. Compared with other methods of
detecting protein-protein interactions, this approach has the advantage
that low affinity or transient interactions can be detected, and the
biochemical milieu of the reaction can be varied. Additionally, the
assay does not rely on overexpression systems. Therefore, all proteins
in the system are present at endogenous levels in the
extracts used. Also, since the full repertoire of cellular protein is
present during the assay, it is less likely that nonspecific
interactions, such as those that can be detected when two purified
proteins are assayed together, will be detected.
Identification of
-Synuclein Cross-links in Brain
Extracts--
mRNAs encoding human
-synuclein where the
C-terminal 20 amino acids were replaced with eight histidine codons
were translated in a wheat germ lysate supplemented with
[35S]Met and TDBA-Lys-tRNA. After translation,
-synuclein was bound to Co2+-chelating agarose beads.
Beads were washed to remove the majority of the contaminating wheat
germ proteins. Bovine brain cytosol was added to the washed
-synuclein-containing beads. After incubation, samples were
subjected to cross-linking as indicated in Fig.
2. Beads were washed, and then both
cross-linked and uncross-linked
-synuclein were released from the
beads by the addition of Laemmli sample buffer containing EDTA.
Cross-links were analyzed by SDS-PAGE on 12% gels followed by
fluorography. The radiolabel is in
-synuclein, and bands migrating
more slowly than
-synuclein represent cross-links of brain cytosolic
proteins to
-synuclein. The uncross-linked
-synuclein runs near
the dye front at ~14 kDa. Three cross-links were seen with estimated
molecular masses of 30-35 kDa. After subtracting the mass of synuclein
itself, the cross-links represent proteins of ~16-19 kDa. The
cross-links were dependent on the addition of brain cytosol, the
presence of
-synuclein mRNA (not shown), and irradiation. In
human
-synuclein, the wild-type amino acid at position 53 is Ala,
whereas in rodents and birds, it is Thr. However, in humans, Thr at
position 53 is a Parkinson's disease-linked mutation. Whereas peptide
sequence from the bovine
-synuclein is known, the full cDNA
sequence is not known. Since it is therefore unclear whether residue 53 of the bovine protein is Ala or Thr, we tested both versions in our
assay. The cross-links were observed to the WT (Fig. 2,
lanes 1-3) and the mutant A53T (Fig. 2,
lanes 4-6)
-synuclein proteins.

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Fig. 2.
Detection of photocross-links between brain
cytosolic proteins and human -synuclein.
mRNAs encoding octahistidine tagged WT or A53T mutant human
-synucleins were translated in vitro in a wheat germ
lysate supplemented with TDBA-Lys-tRNA and [35S]Met.
After translation, synuclein with the photoprobes incorporated was
isolated on chelating Co2+-agarose beads as described under
"Experimental Procedures" (lanes 1-6), or
translation mixtures were used directly (lanes 7 and 8). Brain cytosol was added as indicated
(lanes 2 and 3, 5 and
6, and 7 and 8) either to washed
synuclein-containing beads or to crude translation mixtures
(lanes 7 and 8). After incubation for
5-10 min at room temperature, samples were irradiated to induce
cross-linking (lanes 1, 3,
4, 6, and 8). Synuclein and synuclein
photoadducts were released from the beads by the addition of Laemmli
sample buffer containing EDTA. Where no beads were used
(lanes 7 and 8), proteins were
trichloroacetic acid-precipitated and resuspended in sample buffer.
Cross-links were analyzed by fluorography after SDS-PAGE. Synuclein
runs near the dye from at ~14 kDa (all lanes), and cross-links
(indicated by asterisks) between brain proteins and
radiolabeled -synuclein are seen at ~35 kDa only in the presence
of cytosol and upon irradiation (lanes 3 and
6). Larger cross-links are also observed (lane
8) when synuclein is not bound to the beads.
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We bound our substrate to beads so that we can wash away the wheat germ
cytosol and replace it with brain cytosol. To determine whether
immobilization altered the spectrum of detectable cross-links, we
analyzed the cross-links to soluble synuclein (Fig. 2, lanes 7 and 8). After in vitro translation
of the
-synuclein mRNA in the presence of [35S]Met
and TDBA-Lys-tRNA, cycloheximide was added to 0.5 mM to stop translation. Next, 20 ml of bovine brain cytosol or buffer was
added directly to 5-ml aliquots from the translation as indicated in
Fig. 2. After 10 min at room temperature, samples were irradiated as
indicated. Samples were trichloroacetic acid-precipitated and resuspended in sample buffer, after which cross-links were analyzed by
fluorography after SDS-PAGE. As was the case with the experiment done
on beads, the ~30-35-kDa cross-links were also evident when synuclein was in solution. However, additional cross-links with apparent molecular masses of ~45 and ~85 kDa were also observed. The simplest explanation for the difference is that immobilization on
Co2+ chelating agarose beads sterically blocks these larger
proteins from cross-linking to
-synuclein.
Purification of the Cross-links from Bovine Brain Cytosol--
We
used the cross-linking assay to purify the 15-19-kDa proteins from
bovine brain cytosol using a standard biochemical approach. Since
immobilization of the synuclein on beads resulted in only detecting the
smaller cross-links, gave cleaner gels, and resulted in greater
cross-linking efficiencies, we used this version of the assay for our
purification. Aliquots from each step of the purification or each
column fraction were assayed for the ability of proteins to cross-link
to in vitro translated
-synuclein. Fig.
3a shows the results of the
cross-linking assay, whereas Fig. 3b shows the
Coomassie-stained gels of the same fractions. The 15-19-kDa proteins
remain soluble after heating the cytosol and did not bind to
SP-Sepharose but were quantitatively absorbed to Q-Sepharose. After
washing at 0.4 M KCl, the bound proteins were eluted with a
0.5-1.0 M KCl gradient. The peak of activity eluted at
500-525 mM KCl. Coomassie staining of the peak fractions revealed a major band in the 15-19-kDa range (Fig. 3b). The
band indicated by an asterisk was excised from the gel,
digested with trypsin, and processed for mass spectrometric analysis.
The protein was identified as calmodulin and was verified by comparing
the computer-generated fragment ion series of the predicted tryptic peptide with the experimental MS/MS data.

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Fig. 3.
Purification of
-synuclein cross-linking partners from brain
cytosol. a, aliquots taken from sequential steps from
the purification scheme were analyzed for the ability to cross-link to
synuclein. The three cross-linking partners co-purify and are found in
the supernatant after a heating step and did not bind to SP-Sepharose
but did bind to Q Sepharose and eluted at around 500-530
mM KCl. In lanes 3-7, aliquots
corresponding to 5 ml of starting material were assayed. In
lanes 8-13, 5-ml aliquots of 2.6-ml fractions
were assayed. b, Coomassie Blue-stained gel of fractions
shown in a. In lane 1, 30 mg of brain
cytosol was loaded, and the corresponding amount of material was loaded
in lanes 2-5. In lanes
6-11, 50 ml of 2.6-ml fractions was loaded after proteins
were concentrated by trichloroacetic acid precipitation. The major band
as marked by an asterisk in lane 7 was
excised from a duplicate gel and identified by mass spectrometry as
calmodulin. c, mass spectrum of trypsin digest of the
excised band.
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Purified CaM Cross-links to
-Synuclein in a
Calcium-dependent Manner--
To confirm that we had
excised and identified the protein that was giving rise to the observed
cross-links, we added commercially purified CaM (>98% pure) to
cross-linking assays. His-tagged WT
-synuclein was translated in the
presence of radiolabel and TDBA-Lys-tRNA and isolated on beads, and
purified CaM was added at the concentrations indicated in Fig.
4a prior to cross-linking.
Purified CaM indeed cross-links to
-synuclein and results in a
pattern of three cross-links similar to that seen when complete BBC is
assayed (Fig. 4a, lanes 3 and
4-7), suggesting that all three cross-links observed in the
brain cytosol arise from the interaction with CaM. Half-maximal cross-linking intensity occurred at ~0.5 mM CaM, although
cross-links were detectable at lower concentrations. It should be
pointed out that cross-linking could significantly underestimate the
apparent affinity of CaM for synuclein (see Fig. 6). Additionally, when the calmodulin inhibitor calmidizol is added prior to cross-linking, cross-links to CaM are not observed when using either crude BBC (lane 9) or purified CaM (lane
8). The CaM preparation used has a sufficient amount of the
protein in the calcium-bound conformation to allow for visualization of
the cross-link in the absence of added calcium (lane
10). However, the addition of Ca2+ to the
reaction resulted in a more intense cross-link (lanes 11-13).

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Fig. 4.
Purified CaM cross-links to
-synuclein. a, to verify that the band
excised from the gel and identified as CaM is indeed the protein giving
rise to the -synuclein cross-links, His-tagged WT -synuclein
mRNA was translated in vitro in the presence of
[35S]Met and TDBA-Lys-tRNA, and then synuclein was
isolated on beads. After washing, brain cytosol, commercially purified
bovine brain CaM, CaCl2, and calmidizol (a CaM antagonist)
were added as indicated. After 10 min at room temperature, samples were
subjected to irradiation to induce cross-linking as indicated. The
three cross-links coming from brain cytosol (lane
3) were also observed when CaM alone was added
(lanes 4-7). Calmidizol prevented detection of
the cross-links in brain cytosol (lane 8) and
when purified CaM was used (lane 9). Residual
Ca2+ in the CaM preparation allowed for detection of
cross-links in the absence of added CaCl2 (lane
10), but supplementation with exogenous CaCl2
resulted in a more intense cross-link (lanes
11-13; see text). b, calcium dependence
of CaM-synuclein interaction. Brain cytosol was treated with EGTA and
then dialyzed to remove EGTA and calcium. The treated cytosol was then
assayed for the ability of CaM to cross-link to bead-isolated
-synuclein. The CaM cross-links seen in lane 2 are not evident in the absence of calcium (lane
3). The readdition of CaCl2 to the depleted
cytosol restores the CaM-synuclein interaction (lane
4).
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Cross-linking to
-synuclein requires the presence of calcium. Fig.
4b shows that the CaM cross-links normally seen in BBC cytosol are not observed if the cytosol is EGTA-treated and subjected to dialysis prior to use in the cross-linking assay. The addition of
CaCl2 to the calcium-depleted cytosol restores the CaM
cross-links, showing that the interaction is
calcium-dependent.
Our assay utilizes a slightly truncated and His-tagged version of
synuclein. We therefore wanted to know whether full-length, nontagged
synuclein can interact with CaM. To this end, full-length WT
-synuclein was translated in a wheat germ lysate supplemented with
[35S]Met and TDBA-Lys-tRNA (Fig.
5a). After translation,
purified CaM was added to 1 mM prior to cross-linking. The
pattern of three CaM cross-links is indistinguishable from that
obtained with octahistidine-tagged and immobilized synucleins,
indicating that His tagging and bead binding did not introduce readily
apparent artifacts into our assay. Also, the cross-links were not
observed in the presence of calmidizol (not shown) and were blocked by
EGTA.

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Fig. 5.
CaM cross-links to
-synuclein mutants. a, WT
(lanes 1-4), A53T (lanes
5-8), or A30P (lanes 9-12)
full-length -synuclein mRNAs were translated in vitro
in the presence of [35S]Met and TDBA-Lys-tRNA in a wheat
germ translation extract. After 30 min at 26 °C, cycloheximide was
added to stop translation. CaM (1 mM final) was added as
indicated. Samples received either 1 mM CaCl2
(lanes 1-3, 5-7, and
9-11) or 5 mM EGTA (lanes
4, 8, and 12). After 10 min at room
temperature, samples were irradiated as indicated. Cross-links were
resolved by SDS-PAGE and detected by fluorography. CaM cross-linked to
all of the synucleins tested, and cross-links were
calcium-dependent. b, CaM cross-linked to both
S87A,S129A and S87D,S129D mutant synucleins.
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CaM Cross-links to Parkinson's Disease-associated Mutant
Synucleins--
The preceding experiments showed that CaM cross-links
to both WT and A53T
-synucleins (Fig. 2). Fig. 5a shows
that A30P
-synuclein also cross-links to CaM.
-Synuclein is a
phosphoprotein with Ser87 and Ser129 being
identified as the major sites of phosphorylation (56). Ser129 is the major target of GRK5, a kinase that
phosphorylates G-protein-coupled receptors and synucleins (9). Mutating
both residues to Ala to prevent phosphorylation or to Asp to mimic
phosphorylation did not alter the ability to cross-link to CaM (Fig.
5b).
Recombinant
-Synuclein Binds CaM--
To measure
synuclein-CaM binding independently of cross-linking, we used an
established fluorescence assay exploiting changes in the fluorescence
of dansyl-labeled CaM upon Ca2+ and substrate binding (57)
(Fig. 6a). The
yellow spectrum in Fig.
6a shows the emission spectrum of dansyl-CaM in
the presence of EDTA. Upon the addition of CaCl2
(dark blue), there is a slight increase in
emission intensity, and the emission maximum is blue-shifted from 505 to 490 nm. The addition of recombinant SS 87,129 DD synuclein (0.16 mM (pink), 0.5 mM
(purple), or 0.8 mM (red)) resulted
in increased fluorescence emission in a dose-dependent
manner. The addition of 25 mM calmidizol (a competitive CaM
inhibitor) but not 4 mM RNase A increased fluorescence
intensity (not shown), indicating that the increase in fluorescence
required a CaM substrate. When EDTA was added to a sample containing
0.16 mM synuclein (light blue), there
was a decrease in fluorescence and a reversal of the blue shifting of
the emission maximum.

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Fig. 6.
Recombinant synuclein binds CaM.
a, fluorescence emission spectra of dansyl-CaM in the
absence or presence of synuclein. The fluorescence emission maximum of
1 mm dansyl-Cam was blue-shifted, and the emission intensity increased
in the presence of calcium (yellow, dansyl-CaM + EDTA;
dark blue, dansyl-CaM + CaCl2). The
addition of increasing concentrations of purified -synuclein (0.16, 0.5, and 0.8 mM) results in increased fluorescence
intensity (pink, purple, and red
curves). The addition of EDTA to the sample containing 0.16 mM synuclein results in decreased emission intensity and a
red-shifted maximum (pale blue). Excitation was at 338 nm, and emission spectra were collected from 425 to 600 nm. Slit widths
were 7.5 nm (excitation) and 5.0 nm (emission). b,
-synuclein binds dansyl-CaM in a saturable manner. The indicated
concentrations of recombinant A53T -synuclein were added to cuvettes
containing dansyl-CaM at a final concentration of 86 and 2.5 mM CaCl2 in PBS. The fluorescence emission at
486 nm was measured. Data from triplicate samples were averaged and
plotted as F (fluorescence intensity with
synuclein)/Fo (fluorescence intensity in the absence
of synuclein) versus synuclein concentration. Excitation was
at 338 nm, and emission spectra were collected from 425 to 600 nm. Slit
widths were 10 nm. Error bars denote the S.E.
C, quantitative analysis of the binding of dansyl-CaM to
A53T -synuclein. The data from b above was used to create
a Scatchard plot. For each concentration of synuclein, the fraction of
dansyl-CaM bound to synuclein was calculated from the fractional
increase in fluorescence as described under "Experimental
Procedures." Data are expressed as the mean ± S.E. from three
independent experiments. We estimated the Kd to be
~20 nM from the negative reciprocal of the slope of the
Scatchard plot, and the number of binding sites was estimated to be 82 nM. d, WT -synuclein binds to CaM-Sepharose.
WT -synuclein (120 ml of 0.5 mM) in PBS plus 0.1 mM CaCl2 was incubated with 15 ml of
CaM-Sepharose (Amersham Biosciences) for 30 min at room temperature.
The flow-through was collected, and beads were washed three times with
200 ml of binding buffer. Specifically bound synuclein was eluted with
200 ml of PBS containing 5 mM EDTA. Fractions were
subjected to trichloroacetic acid precipitation, and -synuclein
content was determined by Western blotting.
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Fig. 6b shows that A53T synuclein binds to dansyl-CaM in a
saturable manner (Fig. 6b). To estimate the dissociation
constant for the binding of dansyl-CaM to synuclein, increasing amounts of A53T
-synuclein were added to cuvettes containing dansyl-CaM at a
final concentration of 86 nM (Fig. 6, b and
c). The fraction of free and bound dansyl-CaM at each
concentration was determined as described under "Experimental
Procedures," and from these numbers a Scatchard plot (Fig.
6c) was constructed. With the caveats discussed below, we
estimate the Kd to be about 20 nM, and
the number of binding sites was estimated to be 82 nM, in good agreement with the 86 nM
dansyl-CaM concentration determined by Lowry protein assay. Ideally, we
would have used dansyl-CaM concentrations near or below the
Kd. However, we were not able to label the dansyl-CaM to high enough specific activity where we could use lower
concentrations and reliably detect changes in fluorescence. Similarly,
although we wished to include synuclein concentrations below 25 nM in the construction of the curves in Fig. 6,
b and c, we were unable to measure changes in
dansyl-CaM fluorescence reliably at synculein concentrations below 25 nM.
Fig. 6d shows that recombinant WT
-synuclein binds to
CaM-Sepharose in a calcium-dependent manner. Together,
these results show that synuclein is a CaM substrate and that
CaM-synuclein interaction can be detected by methods other than
cross-linking.
Synuclein and CaM Interact in a Calcium-dependent
Manner in Intact Cells--
Next we addressed the question of whether
-synuclein and CaM expressed at endogenous, physiologic levels in
nontransfected cells interact in intact cells in a
calcium-dependent manner. In preliminary experiments, we
found that DSS, a membrane-permeable amine-reactive homobifunctional
cross-linker, cross-links CaM to recombinant synuclein in a
calcium-dependent manner (not shown). Therefore, SK-N-SH
neuroblastoma cells were treated with DSS in the absence or presence of
a calcium ionophore and with and without calmidizol, a CaM antagonist.
After incubation with the cross-linker, the reaction was quenched with
an excess of buffered glycine before cell lysis. Synuclein
immunoprecipitates containing both uncross-linked and cross-linked
-synuclein were made and subjected to SDS-PAGE, and, after transfer
to nitrocellulose, blots were probed with an anti-CaM monoclonal
antibody. Two cross-links immunoreactive for both
-synuclein and CaM
were observed in a DSS- and ionophore-dependent manner
(Fig. 7, lane 3).
Note that these cross-links were not observed in the presence of a
calmodulin antagonist (lane 4). The ~37-kDa
band migrates at the correct position for the Syn-CaM cross-link. One
other cross-link migrating more slowly was also observed at ~66 kDa
(marked by an asterisk). Since DSS potentially cross-links
several proteins together, this cross-link could represent an a
synucein-CaM complex of multiple stoichiometries. Alternatively, as yet
unidentified proteins could be present in the complex, thereby giving
rise to the larger cross-link.

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Fig. 7.
CaM and -synuclein
interact in intact cells. SNKSH neuroblastoma cells expressing
endogenous CaM and synuclein were rinsed in PBS before the addition of
calcium ionophore A 23187 (20 mM), DSS (1 mM),
and calmidizol (20 mM) as indicated. After incubation at
37 °C, the cross-linker was quenched with buffered glycine. Next,
cells were lysed and subjected to immunoprecipitation with
anti-synuclein antibodies. Immunoprecipitates were probed for the
presence of CaM by Western blotting. The positions of CaM and of
CaM-synuclein adducts are indicated. The figure shows that
CaM-synuclein adducts were only detected in the presence of
calcium and cross-linker and not observed in the presence of the CaM
antagonist. Note also that noncross-linked CaM co-immunoprecipitated
with synuclein or synuclein adducts (lane 3) (see
"Results"). The cross-link marked by an asterisk
is discussed under "Results."
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Notice that uncross-linked CaM was co-immunoprecipitated with
-synuclein (lane 3). While it is not
surprising that this only occurred in the presence of ionophore, the
presence of CaM in the synuclein IP also required DSS and was not
observed in the presence of ionophore (lane 1) or
DSS (lane 2) alone. Calmidizol prevented the
co-precipitation (lane 4).
There are at least two potential explanations for why DSS could
increase the co-immunoprecipitation efficiency. First, whereas synuclein and CaM alone can interact, CaM might bind with increased efficiency or affinity to synuclein in the presence of a third factor
or may bind better to oligomers of synuclein. Perhaps DSS stabilized
either of these apparently labile complexes, thereby providing a better
or more abundant CaM substrate. Whereas DSS may have entrapped some CaM
in these complexes (perhaps giving rise to the band marked by an
asterisk), CaM that bound but did not cross-link was
recovered as co-precipitating CaM.
A more trivial explanation for why DSS allows for
co-immunoprecipitation of synuclein is that, before lysis, DSS
derivatized synuclein and that this results in creation of an
enhanced CaM substrate. We ruled out this possibility by showing that
DSS-derivatized synuclein does not bind to CaM-Sepharose and in fact is
a worse CaM substrate (not shown). Thus,
-synuclein and CaM can
interact in intact cells in a calcium-dependent manner.
Calcium-activated CaM Accelerates Synuclein Fibril
Formation--
Although the function of
-synuclein is unknown, its
conformational status is closely linked to Parkinson's disease.
Purified
-synuclein assembles into amyloid fibrils that are
morphologically and biochemically similar to those found in Lewy
bodies. Because CaM often induces conformational changes in its
substrates and because synuclein's secondary structure changes when it
forms fibrils, we assessed whether Ca2+/CaM could alter the
kinetics of
-synuclein fibril formation.
-Synuclein fibrils are
easily separated from soluble monomeric synuclein by
ultracentrifugation (23). Reactions were mixed according to the legend
for Fig. 8. The synuclein used was
precentrifuged to remove any preformed aggregates before the start of
the assay. At each time point, aliquots were removed, diluted, and
centrifuged to pellet fibrils. The amount of soluble synuclein
remaining in the supernatant fractions was detected by Western blotting
(Fig. 8a). The decrease in synuclein solubility proceeded
more rapidly in the presence of calcium-activated CaM (lanes
1-5) than in the presence of EGTA (lanes
6-10). Western blotting with CaM antibodies shows that the
CaM remained soluble. We were therefore not observing bulk protein
precipitation or protein degradation. Synuclein, including higher
molecular weight forms, was recovered from pellet fractions but was not
quantitatively solubilized with SDS and recovered on gels, a known
property of amyloid fibrils (not shown). Neither calcium (50 mM) nor EGTA (1 mM) alone had any effect on synuclein fibril formation under our conditions (not shown). Fig. 8b presents a quantitative analysis of the aggregation
assay. Synuclein fibril formation is a nucleation-dependent
reaction and proceeds through a protofibrillar intermediate. Further
work will be devoted to figuring out precisely where and how CaM
affects fibril formation.

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Fig. 8.
Calcium/CaM accelerates synuclein fibril
formation. a, assays contained 10 mM A53T
recombinant -synuclein and 20 mM CaM in 50 mM HEPES, pH 7.4, 100 mM NaCl, 0.05%
NaN3. Samples either received 50 mM
CaCl2 (lanes 1-5) or 1 mM EGTA (lanes 6-10). Samples were
incubated at 37 °C without agitation. At the indicated times,
aliquots were removed, diluted 10-fold, and centrifuged (Beckman TL100
rotor, 50,000 rpm, 4 °C, 15 min, 100,000 × gav) to sediment synuclein fibrils. The
fibril-depleted supernatants were analyzed for synuclein and CaM
content by Western blotting. The synuclein stock was precentrifuged to
remove any preformed fibrils or aggregates before the start of the
experiment. b, quantitation of fibril formation assay. The
amount of soluble synuclein remaining at each time point was
quantitated by densitometry using Image J software and normalized to
the initial amount of soluble synuclein present at the beginning of the
experiment. The mean values obtained from three experiments are shown.
The error bars denote the S.E.
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DISCUSSION |
We utilized a photocross-linking strategy to identify potential
-synuclein-binding proteins that may have eluded identification by
other methodologies. We show that several brain cytosolic proteins are
in contact with synuclein and identified CaM as an
-synuclein-binding protein. We employed a photocross-linking
technique heretofore not utilized in investigating neurodegenerative
diseases. With this technology, cross-links are detected only if the
two proteins are contacting one another during the instant the
cross-linker is active. An additional advantage is that biochemical
environment can be altered to assess what effect such alterations have
on the interaction under investigation. Furthermore, any interactions detected are occurring in extracts made from tissues expressing endogenous levels of all proteins in question. We show that the interaction between synuclein and CaM is calcium-dependent
and is unaffected by Parkinson's disease-related mutations in
synuclein. Also, the interaction could be revealed in intact cells
expressing endogenous levels of both CaM and
-synuclein in a
calcium-dependent manner and could be blocked with a CaM inhibitor.
Whereas one recent report has identified a low affinity calcium binding
domain in synuclein (58), this region, comprising the last ~30
residues of
-synuclein, is probably dispensable in our assay, since
our His-tagged version of synuclein, which binds CaM, lacks the
C-terminal 20 residues. Furthermore, there are no lysine residues in
this region to give rise to the cross-links (see Fig. 1). Although some
metal ions can alter synuclein secondary structure and propensity to
aggregate, the Lansbury laboratory (32) has published that low
millimolar concentrations (10 mM) of calcium ions do not
directly alter synuclein secondary structure as assessed by CD
spectroscopy. We have demonstrated that CaM and synuclein interact
directly in a calcium-dependent manner. The apparent
affinity constant of CaM for synuclein is ~20 nM. CaM and
synuclein are abundant enough to make this a physiologically relevant
interaction. Intracellular CaM concentrations have been estimated to be
1-10 mM, and CaM is estimated to be 0.1-1% of total protein in neurons (59). Since the BBC protein
concentration is 4 mg/ml (which is significantly diluted compared with
intracellular cytosol concentrations), the CaM concentration is likely
to be 0.004-0.04 mg/ml, which corresponds to at least 0.24-2.4
mM. Therefore, CaM concentrations are high enough to allow
for physiologically relevant interactions with
-synuclein.
Intriguingly,
-synuclein alters stimulation-dependent
dopamine release, and calcium levels modulate this function of
synuclein (31). Because synuclein is regulated by calcium and we
independently purified CaM as a cross-linking partner of synuclein, we
hypothesize that CaM may mediate the effect of calcium on the
previously observed ability of synuclein to alter dopamine release.
Synucleins are also substrates for GRK 5, a G-protein-coupled receptor
kinase (9). This kinase, which is involved in
ligand-dependent receptor desensitization, is inhibited by
CaM. Whereas CaM inhibits GRK5 autophosphorylation and phosphorylation
of other substrates in the absence of synuclein, CaM actually
stimulates kinase autophosphorylation and synuclein phosphorylation in
the presence of synuclein. Thus, synuclein may act as a switch to
convert CaM from an inhibitor to an activator of GRK.
The fact that we observed several species that were both CaM- and
synuclein-immunoreactive (Fig. 7) only with calcium and not when the
CaM inhibitor was used, indicates that CaM and synuclein may form
higher order complexes in the presence of Ca2+. It is
tempting to speculate that Ca2+/CaM drives assembly of
synuclein-containing multimeric complexes or perhaps regulates the
oligomerization status of synuclein. There is precedence for CaM
driving oligomeric assembly of subunits of small conductance
K+ channels (60). Our in vitro fibril formation
data is at least consistent with this idea. Further work will be
directed toward elucidating the mechanism by which CaM alters
synuclein's ability to change conformation.