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INTRODUCTION |
A multisubunit complex, termed the proteasome, manages protein
turnover in the body. Proteins can be either degraded directly by the
proteasome, or they can be tagged with an 8-KD protein, termed ubiquitin. Three different forms of the proteasome exist in a cell: the 20 S ubiquitin-independent proteasome, the 26 S
ubiquitin-independent proteasome, and the 26 S
ubiquitin-dependent proteasome. The 20 S particle forms the
core of each form of proteasome (1, 2). Both the 26 S
ubiquitin-dependent and -independent proteasomes contain
the 20 S particle plus an additional smaller cap, which has a
sedimentation coefficient of 19 S. Although smaller than the 20 S
particle, the 19 S particle is also a multisubunit structure. The
protein subunits that compose the 19 and 20 S particles are all known.
The 19 S cap contains at least 18 different subunits, whereas the 20 S
particle contains 28 subunits (1, 2). The protein S6' (also known as
tat binding protein 1 and Rpt5) is in the 19 S cap and is of particular
interest because it was recently shown to directly bind ubiquitinated
proteins, which suggests that it is required for
ubiquitin-dependent proteasomal function (3).
Although the 26 S proteasome is responsible in both
ubiquitin-dependent and -independent protein degradation
(4, 5), the 20 S proteasome functions only in ubiquitin-independent
protein degradation and is involved in 70-80% of the selective
recognition and degradation of mildly oxidized proteins in the cytosol
(4, 5). The 26 S proteasomal ubiquitin-dependent pathway
degrades all ubiquitinated proteins within the cell and is the primary degradation pathway of the cell. The E1 ubiquitin-activating enzyme forms a thiodiester bond with mono-ubiquitin. The E2
ubiquitin-conjugating enzyme displaces the E1 enzyme and allows for
conjugation of multiple ubiquitin moieties with one another. The E3
ubiquitin ligase enzyme binds both to the substrate targeted for
degradation and to the E2 enzyme. The E2 and E3 enzymes are displaced,
leaving a multichained ubiquitinated substrate protein that is targeted
to the 26 S proteasome. This is both an ATP- and
ubiquitin-dependent pathway (6-13).
Recent studies suggest that protein aggregates cause toxicity by
inhibiting proteasomal function. Extended polyglutamine repeats, such
as occur in mutant forms of huntingtin associated with Huntington's disease, aggregate readily (14-16). Polyglutamine aggregates inhibit ubiquitin-dependent proteasomal function (17). Aggregates
of other proteins, such as the cystic fibrosis transmembrane receptor, also inhibit ubiquitin-dependent proteasomal function in
cell culture (17). Many other proteins with hydrophobic domains also aggregate, and overexpressing the aggregation-prone domains of these
proteins is toxic (18). The mechanism of toxicity for most aggregates
is unknown.
Blockade of proteasome activity is toxic to many cell types and appears
to be potentially important to many neurodegenerative diseases.
Proteasomal inhibition causes apoptosis in many cell lines and is being
tested as a potential chemotherapy (19). Although proteasomal
inhibition causes rapid toxicity in cell culture, the slow accumulation
of protein aggregates in neurodegenerative diseases might produce a
correspondingly slow inhibition of the proteasome.
-Synuclein is the major component of Lewy bodies, which are
intracellular inclusions that form in Parkinson's disease
(PD)1 (20, 21). The
association of
-synuclein with Lewy bodies suggests that protein
aggregation represents an important aspect of the pathophysiology of
-synuclein and of PD. The link between
-synuclein and protein
aggregation has been strengthened by the discovery of mutant forms of
-synuclein, A53T and A30P, that are associated with rare cases of
familial PD (22, 23). Both mutations accelerate aggregation of
-synuclein (24-27). The link between
-synuclein and aggregation
suggests that understanding the mechanism of toxicity induced by
protein aggregates could provide important insights into the mechanism
of cell death in PD.
Native
-synuclein has been shown to bind both fatty acids and many
different proteins, including phospholipase D, G proteins, synphilin-1,
protein kinase C, 14-3-3 protein, parkin, and the dopamine transporter
(28-34). In addition, rat
-synuclein has been shown to bind to rat
S6' (35). Of these proteins, only synphilin-1 and parkin have been
identified in Lewy bodies (33, 36, 37). Perhaps because of the
pleiotropic binding properties of native
-synuclein, overexpressing
it in cells produces multiple cellular effects.
-Synuclein inhibits
protein kinase C activity, phospholipase D activity, and the activity
of the dopamine transporter, and
-synuclein has chaperone activity
(28, 29, 34, 38). Overexpressing
-synuclein also inhibits
proteasomal function (39). The link between
-synuclein and the
proteasome is intriguing but is not directly related to the
pathophysiology of Parkinson's disease, because overexpressed
-synuclein retains a native structure until the cell is subjected to
a stress, such as incubation with rotenone or ferrous chloride
(40-44). Thus, whether aggregated
-synuclein inhibits
proteasomal function is unknown, and the mechanism by which it
might inhibit the proteasome is also unknown.
In this study we examine the interaction of
-synuclein with the
three different types of proteasome and demonstrate that aggregated
-synuclein binds to S6' and inhibits ubiquitin-dependent proteasomal function.
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MATERIALS AND METHODS |
Cell Lines, Transfections, Chemicals, and Antibodies--
The
human cell line HEK 293 and the human neuroblastoma cell line BE-M17
were grown in OPTIMEM (Cell Grow) plus 10% fetal bovine serum
supplemented with 200 µg/ml G418 (Sigma), as needed. G418 was used
for selection. Transfections utilized FuGENE at a 3:1 ratio to DNA, 4 µg per 10-cm dish. Recombinant
-synuclein was generated using
wild-type
-synuclein inserted into a ProEX-His6 plasmid
(Invitrogen) as described previously (41). Antibodies used include
monoclonal anti-
-synuclein (1:1000 IB, Transduction Labs);
polyclonal anti-S6' (1:1000, Affiniti); monoclonal anti-S6' (1:1000,
Affiniti); polyclonal anti-PA700 (1:1000, Affiniti); monoclonal
anti-10b (1:1000, Affiniti); polyclonal anti-
subunit 20 S (1:1000,
Affiniti); and polyclonal anti-
-synuclein (against amino acids
116-131, 1:1000).
Pull-down Assay--
Brain samples were precleared with
nickel-agarose for one hour at 4 °C to eliminate proteins that
directly bind to nickel-agarose (Invitrogen). These samples were
incubated overnight with 5 µg of recombinant
-synuclein
(His-tagged), either aggregated or monomeric. Samples were incubated
with nickel-agarose for one hour to allow binding of the His-tagged
-synuclein (monomeric or aggregated), and then they were centrifuged
at 1000 rpm for 1 min. Samples were washed three times with
immunoprecipitation buffer (50 mM Tris-HCl, 10 mM EGTA, 100 mM NaCl, 0.5% Triton-X, 1 mM dithiothreitol, 1 mM protease inhibitor
mixture (Sigma), pH 7.4) and run on 8-16% SDS gradient polyacrylamide
gels (BioWhitaker).
Immunoprecipitations--
Protein concentration was determined
using BCA protein assay (Pierce), and 500 µg of each sample was used
per immunoprecipitation in immunoprecipitation buffer (50 mM Tris-HCl, 10 mM EGTA, 100 mM
NaCl, 0.5% Triton-X, 1 mM dithiothreitol, 1 mM
proteasome inhibitor mixture (Sigma), pH7.4). Samples were precleared
using protein G-Sepharose beads (Seize X, Pierce) for 1 h at
4 °C and incubated with antibody overnight at 4 °C while rocking.
Samples were washed three times with immunoprecipitation buffer,
resuspended in 2× dithiothreitol protein loading buffer, boiled for 5 min at 90 °C, and run on 8-16% SDS gradient polyacrylamide gels (BioWhitaker).
Aggregation of
-Synuclein--
Recombinant
-synuclein
incubated for 2 months at 37 °C in phosphate-buffered saline while
shaking at 800 rpm; aggregation was confirmed by performing immunoblot analysis.
Immunoblot Analysis--
Transfers to polyvinylidene difluoride
(BioRad) were done overnight at 4 °C at 0.1 A/gel in transfer
buffer. The immunoblot was blocked in 0.2% I-block (Tropix) in
Tris-buffered saline with 0.1% Tween 20 for one hour at room
temperature while shaking. We then incubated blots overnight at 4 °C
in primary antibody at appropriate concentration in 5% bovine serum
albumin in Tris-buffered saline/0.1% Triton X-100. Blots were
washed three times, 10 min each, and incubated three hours in secondary
antibody (1:5000, Jackson Laboratories) in I-block at room temperature.
Blots were washed three times and developed using a chemiluminescent
reaction (PerkinElmer Life Sciences).
Sucrose Gradients--
10-30% linear sucrose gradients were
prepared using Hoefer SG 15 gradient maker (Amersham Biosciences)
following the manufacturer's recommendations. Approximately 10 mg of
monomeric or aggregated
-synuclein, as determined by BCA protein
assay (Pierce), was added to the top of the gradient; they were
centrifuged using a SW41 rotor for 16 1/2 hours at 40,000 rpm at
20 °C. After centrifugation, 0.5-ml fractions were collected; 20 µl of each fraction was run on an 8-16% gradient gel, and immunoblotted.
In Vitro 20 S Ubiquitin-independent Chymotryptic Proteasomal
Activity Assay--
We incubated aggregated or monomeric
-synuclein
at various concentrations with purified 20 S proteasome (human
erythrocytes, BioMol) for 30 min and then added a fluorogenic substrate
(Suc-LLVY-AMC, BioMol). Ten minutes later, the samples were analyzed
with a GeminiXS SpectraMax fluorescent spectrophotometer (Amersham
Biosciences) using an excitation wavelength of 360 nm and an emission
wavelength of 460 nm.
In Vitro 20/26 S Ubiquitin-independent Chymotryptic
Proteasomal Activity Assay--
Aggregated or monomeric
-synuclein
at various concentrations was incubated with 250 µg of HEK 293 cell
lysates, as determined by BCA protein assay (Pierce) in assay buffer
(10 mM Tris-HCl, pH 7.8, 0.5 mM dithiothreitol,
5 mM MgCl2, and 5 mM ATP) for 30 or
60 min at 37 °C while shaking at 800 rpm. We then added a
fluorogenic substrate (Suc-LLVY-AMC, BioMol) and incubated samples an
additional 30 min at 37 °C while shaking at 800 rpm. Solutions were
analyzed using an excitation wavelength of 360 nm and an emission
wavelength of 460 nm with the GeminiXS SpectraMax spectrophotometer
(Amersham Biosciences).
In Vitro 26 S Ubiquitin-dependent Proteasomal
Activity Assay--
Substrates were generated with an in
vitro transcription and translation of substrate proteins using a
T7 promoter in Escherichia coli lysate (Promega),
supplemented with [35S]methionine, and then partially
purified by high-speed centrifugation and ammonium sulfate
precipitation as described (45). The protease substrate for ClP assays
was derived from barnase, which is a ribonuclease from Bacillus
amyloliquefaciens; the protease substrate for proteasomal assays
was derived from E. coli dihydrofolate reductase (DHFR) (45,
46). A ubiquitin moiety was added to the N terminus of the substrate
proteins via a 4-amino acid linker from the E. coli lac
repressor (45). Substrate proteins were constructed in pGEM-3Zf (+)
vectors (Promega) and were verified by sequencing. The reaction was
resuspended in 40 µl of buffer (25% (v/v) glycerol, 25 mM MgCl2, 0.25 mM Tris/HCl,
pH7.4) to which 5 µl of the in vitro reaction containing
the radiolabeled ubiquitinated substrate protein was added with 35 µl
of rabbit reticulocyte lysate (Green Hectares, containing 1 mM dithiothreitol) that is ATP-depleted as described (45).
We incubated the reactions with and without monomeric or aggregated
-synuclein. Concentration of
-synuclein was determined by BCA
protein assay (Pierce). We incubated at 37 °C for 7 min to allow
initial cleavage of substrate proteins. Ubiquitination and degradation
was initiated by the addition of ATP and an ATP-regenerating system
(0.5 mM ATP, 10 mM creatine phosphate, 0.1 mg/ml creatine phosphokinase, final concentrations). Reactions were
incubated at 37 °C. At designated time points (15, 30, 45, 60, 90, 120, 150, and 180 min), small aliquots were removed and transferred to
ice-cold 5% trichloroacetic acid. The trichloroacetic
acid-insoluble fractions were analyzed by 10% SDS-PAGE and quantified
by electronic autoradiography.
Statistics--
All statistics were performed using a
multifactorial analysis of variance analysis using the Statview
statistical package.
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RESULTS |
Overexpressing
-Synuclein Inhibits Proteasomal
Degradation--
To begin analyzing how
-synuclein might interact
with the proteasome, wild-type
-synuclein was stably expressed in
human neuroblastoma BE-M17 cells by transient transfection, and
ubiquitin-dependent and -independent proteasomal activity
was quantified. Because
-synuclein does not form aggregates
spontaneously under these conditions, this experiment addresses whether
increased concentration of cellular
-synuclein inhibits proteasomal
activity. Immunoblotting of the cellular lysates demonstrated a
significant increase in the
-synuclein levels in the transfected
cells (Fig. 1A). Next, we
investigated whether overexpressing
-synuclein affects the steady
state levels of ubiquitin-conjugated proteins, which provides a measure
of the ubiquitin-dependent proteasomal system. The BE-M17 cells expressing vector or wild-type
-synuclein were immunoblotted with anti-ubiquitin antibody. The amount of ubiquitin-conjugated proteins did not differ among the groups of transfected cells (Fig. 1,
B and C).

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Fig. 1.
Effects of
-synuclein overexpression on the proteasomal
system. A and B, BE-M17 neuroblastoma cells
were transfected with vector or wild-type -synuclein and
immunoblotted with antibodies to -synuclein (A),
ubiquitin (B), and actin (C). No differences in
levels of ubiquitin-conjugated proteins were observed among cells
transfected with vector or wild-type -synuclein. This is a
representative immunoblot from experiments that have been repeated at
least three times. D, activity of the ubiquitin-independent
proteasomal system in cell lines expressing wild-type -synuclein
compared with untransfected cells (**, p < 0.0005).
These data represent the combined data from five experiments each
containing 5 data points for each sample.
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We also investigated how overexpressing
-synuclein affects
ubiquitin-independent proteasomal degradation. Previous studies report
that cell lines overexpressing
-synuclein exhibit lower ubiquitin-independent proteasomal activity (39). To investigate ubiquitin-independent proteasomal activity, we measured hydrolysis rates of fluorogenic peptide analogues in cells transiently or stably
overexpressing
-synuclein (Fig. 1D). No difference in activity was observed in cells transiently transfected with
-synuclein (data not shown). However, cell lines stably expressing
wild-type
-synuclein showed an approximately 50% reduction in
ubiquitin-independent proteasomal degradation, depending on the
transgene (39) (Fig. 1D). These data suggest that
-synuclein does affect ubiquitin-independent proteasomal function.
-Synuclein Inhibits the 20 S Proteasome--
An increasing
number of studies suggest that the state of
-synuclein aggregation
plays a key role in the pathophysiology of PD. To better understand how
-synuclein affects the proteasome, we generated recombinant
monomeric
-synuclein and aggregated
-synuclein. The aggregated
-synuclein was generated by aging recombinant
-synuclein at
37 °C for 2 months. Aggregation of
-synuclein was verified by
immunoblot analysis (Fig. 2A).
The aggregated protein ran as a smear with an average molecular weight of ~160,000 (Fig. 2A). The immunoblot of the aged,
aggregated
-synuclein also exhibited some reactivity at 16,000. This
could reflect either that the sample had some non-aggregated, monomeric
-synuclein remaining or that some of the
-synuclein could be dissociated from the aggregate by SDS. To examine this question, we
fractionated monomeric or aged
-synuclein (37 °C for 2 months) by
centrifugation in a sucrose gradient and immunoblotted each of the 25 fractions with anti-synuclein antibody. The initial sample used before
the fractionation is shown in the first lane (Fig. 2B,
labeled In). Monomeric
-synuclein had a low density and
was most abundant in fractions 1-6 (Fig. 2B,
top). In contrast, the aggregated
-synuclein sample
showed nothing in the early fractions (corresponding to a low density)
and migrated exclusively in the last fraction, suggesting a high
density (Fig. 2B, fraction 25,
bottom). Immunoblots of fraction 25 for the aggregated
sample showed a small amount of SDS-sensitive
-synuclein that
migrated as a monomer following exposure to SDS during the
immunoblotting. This SDS-dissociable
-synuclein was particularly
evident following longer exposures (Fig. 2B, fraction
25B, bottom). This suggests that aggregated
-synuclein contains SDS-sensitive and SDS-resistant aggregated
protein.

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Fig. 2.
Analysis of monomeric and aggregated
-synuclein. A, fresh -synuclein
(lane 1) migrated at about 16,000, which is consistent with
a monomeric size. Aggregated -synuclein (lane 2)
exhibited both low and high molecular weight species following the
process of immunoblotting, which involved heating in 2% SDS for 5 min,
running on PAGE, and immunoblotting. This is a representative
immunoblot from an experiment that had been repeated three times.
B, samples of fresh (top) and aged
(bottom) -synuclein were fractionated on a 5-30%
sucrose gradient, and each fraction was immunoblotted. An immunoblot of
the non-fractionated starting material is shown in the first lane,
IN'. The monomeric -synuclein was present predominantly
in the early fractions, suggesting a low molecular weight; the aged
-synuclein was present in the last fraction, suggesting a high
molecular weight and little if any free monomeric -synuclein.
Because some SDS-sensitive aggregated -synuclein appeared to be
present in the aged Input sample, we overexposed the aged fractionated
sample to determine whether it also contained any SDS-sensitive aged
-synuclein. A long exposure of fraction 25 (lane 25B)
demonstrates the presence of 16,000 -synuclein, suggesting that some
of the aged, aggregated -synuclein can be dissociated by SDS. This
is a representative immunoblot from an experiment that had been
repeated three times.
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Next, we examined the activity of purified 20 S proteasome particles in
the presence of varying amounts of monomeric or aggregated
-synuclein using synthetic fluorescent peptides to monitor
proteasomal activity. Increasing doses of monomeric
-synuclein
progressively inhibited proteasomal activity (Fig.
3A). The IC50 for
inhibition of the proteasome by monomeric
-synuclein was ~16
µM, assuming
-synuclein could achieve complete
inhibition. Aggregated
-synuclein also inhibited the 20 S
ubiquitin-independent proteasomal activity, exhibiting a maximal
inhibition similar to that of monomeric
-synuclein (Fig.
3B).

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Fig. 3.
Effects of monomeric and aggregated
-synuclein on the 20 S proteasome.
A, inhibition of the 20 S proteasome by monomeric
-synuclein dose response. Five data points were used for each
sample. B, inhibition of the 20 S proteasome by aggregated
-synuclein dose response. Five data points were used for each
sample. C, substrate dependence of proteasomal inhibition by
-synuclein. Five data points were used for each sample.
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To determine whether the inhibition was at the level of the proteasome
or due to binding of the peptide substrate, we examined whether varying
the level of substrate affected the
-synuclein-dependent proteasomal inhibition. Inhibition of the 20 S proteasome by monomeric
-synuclein increased with increasing substrate concentration (Fig.
3C). Increased proteasomal inhibition by
-synuclein might occur because larger effects are possible at higher rates of substrate degradation. These data indicate that the proteasomal inhibition that
was observed did not result from substrate binding and substrate sequestration by
-synuclein. Thus,
-synuclein appears to inhibit the proteasomal activity via an interaction with the proteasome, rather
than by binding substrate peptide.
Aggregated
-Synuclein Inhibits the 26 S Proteasome--
Next,
we examined the effects of monomeric and aggregated
-synuclein on a
mixture of the 20 and 26 S proteasomes in HEK 293 cell lysates.
Monomeric
-synuclein inhibited the 20 S/26 S proteasome mixture only
partially, which could reflect greater inhibition of the 20 S
proteasome complex and less inhibition of the 26 S proteasome complex
(Fig. 4B). The concentration
producing maximal inhibition of the 20 S/26 S proteasome complex was
similar to that seen for the 20 S proteasome complex (> 10 µM), based on 50% maximal inhibition (Fig.
4B). Aggregated
-synuclein also inhibited the 20 S/26 S
ubiquitin-independent proteasomal mixture. Based on an estimated
molecular weight for aggregated
-synuclein of 160,000, we calculated
that the IC50 of aggregated
-synuclein for the 20 S/26 S
proteasome was 1 nM (Fig. 4A). The ability of aggregated
-synuclein to inhibit a mixture of the 26 S and 20 S
proteasomes, but not the 20 S proteasome, suggests that aggregated
-synuclein selectively inhibits the 26 S proteasome.

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Fig. 4.
Inhibition of ubiquitin-independent
proteasomal degradation by the 20 S/26 S proteasome with aggregated
(A) and monomeric (B)
-synuclein using HEK 293 lysates. The percent
inhibition was normalized to the inhibition produced by the proteasomal
inhibitor lactacystin (25 µM). *, p < 0.002 compared with no inhibitor.
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Aggregated Synuclein, but Not Monomeric Synuclein, Inhibits Protein
Degradation by the 26 S Proteasome--
The greater ability of
aggregated
-synuclein compared with monomeric
-synuclein in
inhibiting 26 S ubiquitin-independent proteasomal activity raises the
possibility that 26 S ubiquitin-dependent proteasomal
function might also be selectively inhibited by aggregated
-synuclein. To investigate this, we examined ubiquitin-mediated degradation of a fusion protein made up of barnase and E. coli dihydrofolate reductase that had been fused with an
N-terminal degradation tag (DHFR-U) (45). Prior studies show
that degradation of ubiquitinated DHFR-U by reticulocyte lysates is
mediated by the 26 S proteasome (45). We used this system to
investigate how monomeric and aggregated
-synuclein affect
ubiquitin-mediated proteasomal degradation. Degradation of
ubiquitinated DHFR-U was examined in the presence of monomeric or
aggregated
-synuclein (Fig.
5A). The half-life of DHFR-U
was 125 min both under basal conditions and in the presence of 5 µM monomeric
-synuclein (Fig. 5A,
gray bars). However, the half-life of DHFR-U greatly
increased in the presence of 500 nM aggregated
-synuclein (Fig. 5A, dotted bars). No
inhibition was seen with 50 nM aggregated
-synuclein (data not shown). These data indicate that 26 S
ubiquitin-dependent proteasomal degradation is selectively
inhibited by aggregated
-synuclein.

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Fig. 5.
Inhibition of ubiquitin-dependent
proteasomal degradation by aggregated
-synuclein. A, aggregated
-synuclein (0.5 µM) inhibits
ubiquitin-dependent degradation of DHFR-U by reticulocyte
lysates by the 26 S proteasome, whereas monomeric -synuclein (5 µM) does not inhibit degradation of DHFR-U by
reticulocyte lysates by the 26 S proteasome. The overall analysis of
variance was significant at p < 0.001. Stars show significance relative to DHFR-U in the absence of
added -synuclein. Each sample point was performed in triplicate.
B, lack of inhibition of Clp1 by monomeric or aggregated
-synuclein. Degradation of DHFR-U was not significantly different
between degradation of DHFR-U alone or in the presence of aggregated
(0.5 µM) or monomeric (5 µM) -synuclein.
Each sample point was performed in triplicate.
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To determine whether inhibition of ubiquitin-dependent
proteasomal degradation was specific to the 26 S proteasome, we also examined degradation of barnase that had been fused with a 65-amino acid N-terminal tag (DHFR-65) that allows the protein to be recognized and degraded by the bacterial proteasomal analog ClpAP (45). DHFR-65
was incubated with ClpAP alone or in the presence of 5 µM
monomeric
-synuclein or in the presence of 500 nM
aggregated
-synuclein, and the rate of degradation was monitored.
Neither monomeric nor aggregated
-synuclein inhibited degradation of DHFR-65 by ClpAP (Fig. 5B). This indicates that proteasomal
inhibition by aggregated
-synuclein is specific for the
ubiquitin-dependent 26 S proteasomal system.
Native and Aggregated
-Synuclein Bind S6'--
The ability of
aggregated
-synuclein to inhibit degradation mediated by the 26 S
proteasome could be explained by interaction between aggregated
-synuclein and a protein in the 19 S cap that is present in the 26 S
proteasome but not the 20 S proteasome. Studies with rat
-synuclein
suggest that
-synuclein binds the rodent 19 S proteasomal component
S6' (35). Based on this work, we investigated whether human
-synuclein interacts with S6'. His-tagged recombinant native or
aggregated
-synuclein was incubated overnight with substantia nigra
or cingulated cortex from normal human brain and then precipitated with
nickel-agarose. The precipitates were immunoblotted with antibodies to
S6'. A representative immunoblot with native
-synuclein is shown in
Fig. 6A, and a pull-down with native or aggregated
-synuclein is shown in Fig. 6B. Both
aggregated and monomeric
-synuclein associated with S6'. The term
`native' is used in this discussion because the overnight incubation
of recombinant
-synuclein with the lysates appeared to
promote formation of some recombinant
-synuclein dimer, in addition
to the more abundant
-synuclein monomer (Fig. 6B,
lower panel). Co-association of
-synuclein with S6' was
also observed by immunoprecipitating endogenous
-synuclein and
immunoblotting for S6' (Fig. 6C). To test the selectivity of
the association, we examined whether
-synuclein binds other
components of the 19 S proteasomal cap, such as Rpn12 and subunit
10b. Neither Rpn12 nor subunit 10b was observed to co-precipitate
with
-synuclein (Fig. 6D, immunoblot for 10b
shown). It was not possible to test the association of S6' with
-synuclein by immunoprecipitating S6', because none of the
antibodies to S6' that we tested were successfully able to precipitate
S6' (data not shown). Together, these data suggest that both monomeric
and aggregated
-synuclein bind S6'.

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Fig. 6.
-Synuclein binds S6'.
A, upper panel, immunoblot showing S6' in brain
lysate (lane 1, 10 µg) and precipitation of S6' by
monomeric His-tagged recombinant -synuclein (lanes 2 and
3). Lower panel, reprobe of the same immunoblot
with anti- -synuclein antibody. The arrow points to the
band corresponding to the S6' protein. The -synuclein band in lane 1 is lower than that in lanes 2 and 3 in the lower panel because the
protein in lane 1 is endogenous -synuclein, whereas the protein in
lane 2 and 3 is His-tagged protein. B, upper
panel, immunoblot showing S6' in brain lysate (lane 1,
30 µg), precipitation of S6' by aggregated (lane 2,
A-S) or native His-tagged recombinant -synuclein
(lane 3, N-S), and lack of precipitation of S6'
using Ni-agarose pull-down without recombinant -synuclein
(lane 4). The arrow points to the band
corresponding to the S6' protein. Lower panel, reprobe of
the same immunoblot with anti- -synuclein antibody. The
arrow points to monomeric -synuclein, and the
bar demonstrates the position of aggregated -synuclein.
The band at 36 kDa in lane 3 likely represents an -synuclein dimer
that might have been promoted by incubation of a large amount of
recombinant -synuclein with lysate but was not present in most other
experiments (for example, see panel C). C,
upper panel, immunoblot of S6' showing the association of
S6' with -synuclein following an immunoprecipitation of endogenous
-synuclein by anti-synuclein antibody. The arrow points
to the band corresponding to the S6' protein. Lane 1 (Lys), crude brain lysates that had not been subject to
immunoprecipitation. Lane 2 (IgG),
immunoprecipitation with nonspecific preimmune IgG antibody. Lane
3 (Syn), immunoprecipitation with anti- -synuclein
antibody. Lower panel, reprobe with anti- -synuclein
antibody. D, no association was observed between
-synuclein and other proteasomal components, such as the 19 S
subunit 10b. The arrow points to the band corresponding to
the 10b protein, which is present in the lysates (lane 2)
but not immunoprecipitated with -synuclein (lane 1) or
nonspecific IgG (lane 3). The abbreviations for this panel
are the same as for panel B. All immunoblots in this figure are
representative immunoblots from experiments that had been repeated at
least three times.
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DISCUSSION |
Proteasomal inhibition is known to be toxic to many cell types and
is thought to contribute to the pathophysiology of neurodegenerative diseases (17, 18, 47). Our data demonstrate that overexpressing
-synuclein inhibits 20 and 26 S proteasomal activity. The
relationship between overexpressed
-synuclein and the
pathophysiology of PD, though, is unclear. Overexpressing
-synuclein
in mammalian neurons does not lead to its spontaneous aggregation,
except after delays of 6-12 months (48-51). Because protein
aggregation is thought to play a critical role in the pathophysiology
of neurodegenerative diseases and aggregation of
-synuclein appears
to be important to the pathophysiology of PD, we sought to design
experiments that would allow analysis of the actions of aggregated
-synuclein. To investigate whether aggregated
-synuclein
interacts with the proteasome, we examined the behavior of
-synuclein that had been aggregated in vitro. We observed
that aggregated
-synuclein inhibits both
ubiquitin-dependent and -independent 26 S proteasomal
activity. The IC50 of aggregated
-synuclein for
ubiquitin-independent 26 S proteasomal activity was 1 nM,
which was over 1000-fold higher than the IC50 for 20 S
proteasomal activity. In contrast, monomeric
-synuclein inhibited 20 and 26 S proteasomal activity with an IC50 > 10 µM.
The high affinity of aggregated
-synuclein for inhibiting 26 S
proteasomal activity could be explained by binding of aggregated
-synuclein to a protein in the 19 S cap, which is the proteasomal complex that binds to the 20 S proteasome and confers
ubiquitin-dependence, as discussed below (3). Consistent with this
hypothesis, we observed that
-synuclein binds to S6', which is a
subunit of the 19 S cap that was recently shown to bind
polyubiquitinated proteins (3). Both aggregated and monomeric
-synuclein bind the S6' protein. The interaction appears to be
selective for S6' because no association was observed with other 19 S
proteasomal proteins, such as Rpn12 or subunit 10b.
Binding of
-synuclein to S6' is consistent with prior publications.
Ghee et al. (35) demonstrated that rat S6' (also termed Tat
binding protein-1, TBP1) binds
-synuclein using the yeast two-hybrid
method. The association was confirmed by showing that an epitope-tagged
S6' could pull down
-synuclein following transfection of both
proteins into HEK 293 cells. However, this study did not demonstrate
interaction using the endogenous proteins and also did not investigate
whether human
-synuclein binds to human S6'. In addition, Ii
et al. (52) have documented the presence of proteasomal
proteins in Lewy bodies, which supports our observation that aggregated
-synuclein binds S6'. The information presented in this study
provides the functional relevance for these observations by showing
that binding of
-synuclein to the proteasome inhibits proteasomal function.
The function of S6' was recently identified and suggests a mechanism
explaining why aggregated
-synuclein might inhibit the activity of
the 26 S proteasome. The S6' protein appears to function in the 19 S
proteasomal cap as the docking protein for ubiquitin-conjugated proteins and is essential for binding of ubiquitin-conjugated proteins
by the proteasome (3). Because aggregated
-synuclein is much larger
than monomeric
-synuclein and often contains covalent cross-links,
binding to S6' might inhibit the function of the 19 S protein by
competing with binding of other ubiquitin-conjugated proteins. Bound
aggregated
-synuclein might occupy the unfolding proteins associated
with proteasomal degradation, and the aggregate might also physically
block the pore of the 19 S cap. This model provides an explanation for
the ability of aggregated
-synuclein to interfere with both the
ubiquitin-dependent and -independent 26 S proteasomal function.
Many other protein aggregates have been shown to be toxic to cells
(18). Both aggregated cystic fibrosis transmembrane receptor and
polyglutamine repeat exhibit toxicity that correlates with proteasomal
inhibition (17, 18, 47). This study focuses attention on the
interaction between S6' and protein aggregates. Whether S6' has a
particular affinity for
-synuclein or is a general target for all
protein aggregates remains to be determined. Inhibiting the
ubiquitin-dependent proteasomal system (UPS) is known to be
toxic, perhaps because it induces apoptosis (19). Inhibiting the UPS
causes the accumulation of many toxic proteins, such as Pael-R, which
was recently identified as a parkin substrate (53). Inhibiting the UPS
is also known to cause the accumulation of protein aggregates in the
endoplasmic reticulum (17, 47). Inhibiting the UPS could alter the
regulation of cell cycle proteins (54). Reduced degradation of cell
cycle proteins could account for the apparent abnormal activation of
the cell cycle proteins observed in many neurodegenerative processes
(55, 56).
Proteasomal inhibitors have recently been shown to induce degeneration
of the dopaminergic neurons of the substantia nigra and induce
-synuclein aggregation (57). The tendency of
-synuclein to
accumulate under conditions of proteasomal inhibition raises the
possibility that the accumulation of aggregated
-synuclein adds to
the proteasomal inhibition and increases the toxicity associated with
proteasomal inhibition.
The discordance between the rapid kinetics of cell death associated
with UPS inhibition in cell culture and the slow nature of degeneration
in PD is notable. This discordance might be explained by the slow
appearance of aggregated
-synuclein.
-Synuclein does not form
aggregates under basal conditions when transiently overexpressed, but
studies in transgenic mice show that overexpressing
-synuclein does
lead to a delayed accumulation of aggregated
-synuclein (48-51).
The slow rate of accumulation of aggregated
-synuclein could also
lead to a correspondingly gradual inhibition of the UPS during the
course of PD. Hence, progressive inhibition of the UPS by aggregated
-synuclein might be a gradual process in PD. Together these data
suggest a model in which the gradual accumulation of aggregated
-synuclein progressively inhibits S6' function, which leads to a
gradual but progressive inhibition of the UPS and the progressive
neurodegeneration that occurs in PD.