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Proteasomal inhibition by αααα-synuclein filaments and oligomers.
Lindersson, E.1¶, Beedholm, R.2¶, Højrup, P.3, Moos, T.4, Gai, W.-P.5, Hendil, K.B.6 and Jensen, P.H. 1
1Departments of Medical Biochemistry, 2Molecular Biology, University of Aarhus, Denmark 3Institute
of Molecular Biology, University of Southern Denmark, Denmark. 4Department of Medical Anatomy,
University of Copenhagen, Denmark, 5Department of Physiology, Flinders Medical Centre, Australia ,
6August Krogh Institute, University of Copenhagen, Denmark.
Correspondence to: Poul Henning Jensen, Dept. Med. Biochemistry, University of Aarhus, DK-8000
Aarhus-C, Tel. +4589422856, email [email protected]
JBC Papers in Press. Published on January 7, 2004 as Manuscript M306390200
Copyright 2004 by The American Society for Biochemistry and Molecular Biology, Inc.
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A unifying feature of many neurodegenerative disorders is the accumulation of poly-ubiquitinated
protein inclusions in dystrophic neurons, e.g. containing α-synuclein, which is suggestive of an
insufficient proteasomal activity. We demonstrate that α-synuclein and 20S proteasom components co-
localise in Lewy bodies and show that subunits from 20S proteasome particles, in contrast to subunits
of the19S regulatory complex, bind efficiently to aggregated filamentous but not monomeric α-
synuclein. Proteasome binding to insoluble α-synuclein filaments and soluble α-synuclein oligomers
result in marked inhibition of its chymotrypsin-like hydrolytic activity through a non-competitive
mechanism that is mimicked by model amyloid-Aβ peptide aggregates. Endogenous ligands of
aggregated α-synuclein like heat shock protein 70 and glyceraldehyde-6-phosphate dehydrogenase
bind filaments and inhibit their anti-proteasomal activity. The inhibitory effect of amyloid aggregates
may thus be amenable to modulation by endogenous chaperones, and possibly accessible for
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RUNNING TITLE
α-synuclein inhibits the 20S proteasome
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INTRODUCTION
The development of polyubiquitin-containing intracellular inclusions in cell bodies and dystrophic
neurites of nerve cells is a key feature of several neurodegenerative disorders e.g. in Alzheimer’s
disease (AD1), Parkinson’s disease (PD), dementia with Lewy bodies (DLB) and Huntington’s disease
(1). The poly-ubiquitination of proteins represents the result of the cellular system that recognises
misfolded or unwanted proteins and tags them for degradation by the proteasome via the sequential
action of three enzymes (for a recent review on the ubiquitin-proteasomal system, see 2). First the
ubiquitin activating enzyme E1 activates ubiquitin in an ATP dependent process, after which the
activated ubiquitin is passed onto the E2 class of ubiquitin conjugases. Then E3 ubiquitin ligases
recognise the misfolded proteins and mediate covalent binding of ubiquitin to them. Normally,
polyubiquitinated proteins are recognised by the 19S regulatory complex of the 26S proteasome that
unfolds the substrates and feeds them to the 20S proteolytic core particle of the 26S proteasome. The
proteasome comprises 3 hydrolytic activities; the trypsin-like, the chymotrypsin-like and the caspase-
like hydrolytic activities that combined assure the complete degradation of substrates. Accumulation of
ubiquitinated proteins is an indicator for an imbalance between the production and ubiquitin-labeling of
misfolded proteins and the catabolic capacity of the proteasome.
A group of neurodegenerative disorders, e.g. PD and DLB, are characterised by the
filamentous Lewy body-type of cytoplasmic inclusions in the degenerating cells (1). Aggregates of
misfolded α-synuclein (AS) form the filamentous core of the Lewy bodies and Lewy neurites (3) and
the aggregates presumably play a role in the degeneration for more reasons. First, the association of
missense mutations in AS with autosomal dominant PD evidences a direct gain of toxic function for AS
in the degenerative process where the dopaminergic neurons of the substantia nigra preferentially are
lost (4, 5). Second, the pathogenic AS mutations, A30P and A53T increase AS propensity for
aggregation (6, 7). Likewise, aggregated insoluble AS has been demonstrated in tissue affected by
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sporadic PD, DLB and multiple system atrophy (8-10). Third, the AS filaments in brain tissue resemble
those formed in vitro from recombinant human AS that are rich in β-folded structures and both bind
Congo Red and Thioflavin-S (11-13). Accordingly, AS aggregates bear the characteristics of amyloid-
type filaments accumulating in other diseases, e.g. formed from tau peptides in AD and huntingtin in
Huntington’s disease (3). The employment of animal models corroborates the involvement of
aggregated AS in the neurodegenerative process where over expression of AS leads to abnormal AS
accumulation, misfolding, neuronal dysfunction and overt degeneration (10, 14, 15). A compromised
proteasomal function in the substantia nigra in PD has been reported (16-20) and this may be due to a
direct effect of AS on the proteasome as transgenic cellular expression of AS inhibits the proteasome
(21-23). Importantly, aggregated AS displayed a strongly increased inhibitory activity toward the
proteasome in vitro as compared to monomeric AS (24). Such a proteasomal inhibition may have an
impact on the dopaminergic neurons that seem to be especially vulnerable to the stress of unfolded
proteins (23).
We demonstrate that AS, ubiquitin and 20S proteasomal components co-localise in Lewy
bodies and show that aggregated, but not monomeric AS binds efficiently to the 20S proteasome part of
the 26S proteasome. The proteasome binding results in an efficient and selective non-competitive
inhibition of the chymotrypsin-like proteasomal activity of the 20S proteolytic particle. This inhibition,
which is mimicked by Aβ amyloid filaments, is reverted by the amyloid targeting substances
Thioflavin-S and Congo Red. Moreover, heat shock protein 70 (HSP70) that protects neurons towards
AS-mediated toxicity in transgenic models (25), and glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) bind to the AS filaments and abrogate their anti-proteasomal activity. Finally, subjecting
cells to heat shock renders their cytosolic proteasomes less sensitive to the toxicity of aggregated AS
thus demonstrating a potential for modulation of this toxicity by endogenous factors. The proteasomal
inhibition by aggregated AS may represent a common neurodegenerative mechanism in diseases like
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PD, DLB and multiple system atrophy where the degenerating cells accumulate AS aggregates.
Abolition of the inhibition may open for new neuroprotective strategies.
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METHODS
Miscellaneous and proteins. All chemicals were of analytical grade if not otherwise stated. Thioflavin
S and Congo Red were from Sigma (St. Louis, MO.). Human 20S proteasome was isolated from human
erythrocytes by affinity chromatography on immobilised MCP21 monoclonal antibody (26). The
specific activities of the 20S proteasome were: chymotrypsin-like activity, 4.0 nmol/min/mg protein
using Suc-Ala-Ala-Phe-7-amido-4-methyl-coumarine; trypsin-like activity, 5.1 nmol/min/mg protein
using Z-Ala-Ala-Arg-7-amido-4-methyl-coumarine; caspase-like activity, 1.9 nmol/min/mg protein
using Z-Leu-Leu-Glu-β-naphylamide. The Aβ-(1-40) peptide was from (Schaefer-N., Copenhagen
Denmark). Recombinant human HSP70 was from Stressgen, (Victoria, B.C., Canada) and GAPDH
from Sigma. Both GAPDH and HSP70 were centrifuged for 30 min at 120,000 rpm at 4˚C in a TLA
120.1 rotor in a Optima TLX centrifuge (Beckman Instruments) to remove any aggregated materials
prior to pull-down experiments. Recombinant human full-length AS-(1-140), C-terminally truncated
AS-(1-95) and β-synuclein were expressed in E. coli and purified as described previously (27, 28),
followed by an additional reverse phase HPLC purification step on a Jupiter C18 column
(Phenomenex, CA) in 0.1% trifluoroacetic acid with an acetonitril gradient. The proteins were
subsequently aliquoted, lyophilized and stored at -80oC.
Primary antibodies. Sheep anti-AS was affinity purified as described (29) and obtained from Abcam,
UK. Rabbit ASY-1 IgG was raised against full-length human recombinant AS (28). Rabbit FILA-1
IgG, specific for aggregated AS, was prepared by immunising rabbits with sucrose density gradient
purified AS-(1-140) filaments. The immune serum was subsequently passed several times through a
column with immobilised AS. This assured the removal of all detectable immunoreactivity toward
monomeric AS as determined by dot blotting. The serum lacking the monomer binding antibodies was
then incubated with density gradient purified AS filaments and the aggregate-binding antibodies were
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isolated by gradient co-sedimentation with the filaments. The antibodies were subsequently eluted from
the filaments by incubation in 100 mM glycine pH 2.5 followed by gradient centrifugation to separate
the insoluble AS filaments from the FILA-1 IgG. The IgG was finally concentrated by protein A
chromatography. The monoclonal antibodies, used to detect 20S proteasom subunits were: MCP72 (α7
subunit); MCP196 (α5 subunit); MCP20 (α6 subunit); MCP231 (α2, α3, α6, α7 subunits); MCP421
(β1 subunit) (30). The monoclonal antibodies to subunits of the 19S regulatory proteasome complex
were: p31-38 (S14/Rpn12 subunit); S5a-18 (S5a/Rpn10 subunit); TBP1-19 (S6´/Rpt5 subunit) (31). A
rabbit polyclonal antibody to S6’(Affinity Bioreagens) was also used. As were affinity purified rabbit
anti-ubiqutin (Dako, Denmark) and monoclonal antibodies against HSP70 (clone BRM-22) and β-actin
(both from Sigma, St. Louis, MO., USA).
Immunohistochemistry. Samples of substantia nigra from sporadic PD (n =4), DLB (n =3) and
control subjects (n = 4) were obtained from the Netherlands Brain Bank (Amsterdam, the Netherlands)
and fixed in 10 % neutral buffered formalin and embedded in paraffin. Control samples were obtained
among patients without neurological disease and with no pathological changes in sections of the
substantia nigra pars compacta (SNpc). The diagnosis of PD in the human autopsies was based on loss
of neuromelanin pigment and excessive number of Lewy bodies in remaining SNpc neurons in eosin
stained section. The paraffin sections were cut at 2 µm and subjected to immunohistochemistry as
previously described (32). Complementary sections were demelanized using successive incubations
with 0.25 % potassium permanganate for 20 min followed by 1 % oxalic acid and 1 % potassium
bisulfite for 4 min (33). The sections were incubated overnight at 4oC with polyclonal rabbit-anti-
human ubiquitin diluted 1:500 (Dako, DK), sheep-anti-human AS diluted 1:200 (Abcam, UK), or anti-
human 20S proteasome antibody MCP20. The sections were developed using 3,3-diaminobenzidine
tetrahydrochloride as chromogen. To examine the extent of non-specific binding, non-immune sera was
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substituted for the primary antibody. The relative ratio between the presence of AS and 20S proteasome
in Lewy bodies was estimated by staining four pairs of sections per autopsy for AS and 20S
proteasome, which were counted for their content of positive Lewy bodies.
Immunolabeling of isolated Lewy bodies. Immunohistochemical staining was conducted in isolated
Lewy bodies from fresh frozen brains of patients with DLB. The cingulate cortex from 5 DLB cases
were analysed. The isolation procedure was modified from (28, 29, 34) with the Lewy body smears
prepared by washing the Lewy body enriched fraction 3 times in Tris-buffered saline (TBS, 0.1M Tris-
HCl, 0.9% NaCl, 5 mM EDTA, and protease inhibitors) followed by smearing on gelatine-coated glass
slides and air-drying for 2 hours at room temperature. The smears were fixed in 4% formaldehyde in
TBS for 10 minutes, and then incubated with 3% H2O2 in 50% methanol-TBS for 10 minutes to bleach
endogenous peroxidase activity. Following 3 x 5 minutes rinse in TBS, the smears were blocked with
20% normal horse serum for 30 minutes, and then incubated for 60 minutes in primary antibody
solution containing monoclonal antibody MCP72 (1:300), or MCP196 (1:300). To facilitate
recognizing Lewy bodies, the primary antibody solution also included sheep anti-AS (1:300), or anti-
ubiquitin (1:300). The smears were rinsed 3 times in TBS, then labeled for 60 minutes with Cy2
conjugated donkey anti-mouse IgG, in combination with Cy3 conjugated donkey anti-sheep IgG or Cy3
conjugated anti-rabbit IgG (all used 1:100 dilution, all from Jackson ImmunoResearch Labs Inc, PA,
USA). The primary and secondary antibody dilutions used above were predetermined by serial
titrations before formal experiments. Controls for antibody specificity included omitting primary or
secondary antibodies, and preabsorption the primary antibody with ubiquitin or AS. Lewy body
staining was not detected in these control experiments. The smears were examined using a Bio-Rad
confocal laser scanning microscope and software package (Bio-Rad MRC 1024, Richmond, CA, USA)
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(34) with the scanned image representing an approximately 1µm thick slice through the centre or
equatorial plane of the Lewy body.
Preparation of AS aggregates. AS aggregates were made by resuspending lyophilised AS in 20 mM
Tris, 150 mM NaCl, pH 7.5, 0.02% NaN3 at 7 mg/ml followed the ultracentrifugation to remove
insoluble aggregates (28). Incubation of β-synuclein (5.5 mg/ml) and Aβ-(1-40) peptide (4mg/ml) was
performed by analogous procedures. The supernatant was incubated at 37oC on a shaker for
approximately 14 days. Insoluble AS aggregates were isolated for proteasome activity assays and
electronmicroscopy by sedimentation through a 40% sucrose cushion (28) followed by resuspension in
the buffer of choice. Soluble AS aggregates were isolated by gelfiltration of the soluble supernatant,
obtained after ultracentrifugation of the aggregated AS sample, on a 24 x 1 cm Biogel A 1.5 M column
(Biorad) in 150 mM NaCl, 10 mM NaH2PO4, pH 7.4 (PBS) at 0.5 ml/min. The buffer was changed to 1
M Hepes, pH 8.0 when isolating soluble oligomers for the proteasome activity assay. The elution of the
aggregated oligomers was determined by dot-blotting of the eluted fractions using the FILA-1 antibody
and the total AS was monitored by probing the membrane with the ASY-1 antibody.
Electron microscopy. Electron microscopy was used to analyse the insoluble AS filaments and to
visualize the binding of purified 20S proteasomes to the filaments. Filaments isolated from 30 µl
aggregated AS (7mg/ml) were resuspended in 30 µl PBS supplemented 20 mM NaN3 after pelleting
through the sucrose cushion as described above. Purified 20S proteasome was centrifuged for 10 min at
100,000 rpm at 4˚C in a TLA 120.1 rotor in a Optima TLX centrifuge (Beckman Instruments) to
remove aggregated materials. Soluble 20S proteasome (40 µl at 0.18 mg/ml) was incubated together
with 5 µl resuspended AS filaments (5mg/ml) for 2h at 25˚C. Non-bound proteasome was removed by
sucrose gradient centrifugation of the filaments with bound 20S proteasome. The sedimented filaments
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with attached 20S proteasome was resuspended in 70 µl of distilled water. As a control was used
filaments not incubated with 20S proteasomes before the centrifugation and soluble 20S proteasomes
diluted to 0.16 mg/ml in PBS before direct application to the grid. All samples were pipetted onto
carbon-coated nickel grids (3 µl for each grid) and allowed to stand for 2 min. The samples were
stained with 1% aqueous uranyl acetate for 1 min. The grids were then air-dried, examined in a
Morgagni 268, 80kV electron microscope and photographs were taken at 14,000, 18,000 or 22,000
times magnification.
For immuno-gold labelling, grids with samples were blocked with blocking buffer (PBS
with 0.05 M glycine and 0.1% milk protein), for 10 min and incubated with the primary antibody
MCP72 (0.07 mg/ml) for 1h. The grids were then washed three times for 5 min with blocking buffer
and then incubated with goat anti-rabbit IgG conjugated to 5 nm diameter gold particles (Amersham)
diluted 1:100 in PBS with 1% fish gelatin, 0.06% polyethylenglycol and 0.1% milk protein for 1 h. The
grids were then washed twice for 5 min in PBS with 0.1% milk protein and then twice for 5 min in
water. The grids were finally stained and examined as the non-immunolabeled samples.
Filament pull-down assay.
Cellular cytosol was prepared from primary cultures of normal human skin fibroblasts (Rattan 1998)
and from rat brain (36). Cells, at confluence, were trypsinized and collected by centrifugation (1000×
g, 10 min, 4° C). The cells were sonicated in 50 mM NaCl, 10 mM HEPES pH 8, 0.5 M sucrose, 1 mM
EDTA, 0.2% (v/v) Triton X 100, 0.2 mM phenylmethanesulfonyl fluoride, 0.05% β-mercaptoethanol
(5 ml for three 75 cm2 flasks) on ice. The supernatant was collected after centrifugation at 108,000 × g
for 30 min at 4°C giving a cytosol of 1 mg protein/ml. To demonstrate the binding to AS filaments, 250
µl cytosol were incubated with 70 µg AS filaments for 1.5h, 37°C; 30µg GAPDH were incubated with
9 µg AS filaments for 16h, 4°C; HSP70 (1.2 µg) were incubated with 21 µg AS filaments for 2h, 25°C;
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purified 20S (5.4 µg) were incubated with 70 µg AS filaments for 16h at 4°C. All volumes were
adjusted to 300µl with 20 mM Tris and 150 mM NaCl, pH 7.4 except for the HSP70, where the buffer
was 50 mM Tris, 100 mM NaCl, 1 mM dithioerythreitol (DTE) and 0.1 mM phenylmethanesulfonyl
fluoride, pH 7.2. As negative controls cytosol, GAPDH, HSP70, 20S proteasomes or filaments were
treated as above but without mixing the individual proteins. As positive controls cytosol, GAPDH,
HSP70, 20S proteasome or the filaments were loaded directly on the sodium dodecylsulphate (SDS)
polyacrylamide gel for electrophoresis. Excess of purified monomeric AS was supplemented to the
ligand-filament samples to determine if this would inhibit the association of the ligands to the filaments
or the interaction displayed a selectivity for the filaments as demonstrated by an unchanged amount of
ligand in the pellet.
The filament pull-down assay was performed by placing the protein samples on a cushion of 1.6 ml
40% sucrose, 13 mM MES and 1 mM EDTA, pH 7.0 followed by centrifugation at 55,000 rpm for 30
min in a TLS 55 swing out rotor at 4°C in a Optima TLX centrifuge (Beckman Instruments). The
pellets were either recovered for the proteasome assay by resuspension in 1M HEPES pH 8.0 or
solubilised in 30 µl 8 M urea with 4% SDS for 16h in 37°C. The latter to ensure complete
depolymerisation of the filaments. Non-bound proteins, remaining in the supernatant, were analysed by
the precipitation of 500 µl of the supernatant in trichloroacetic acid. The solubilised pellets and the
precipitated supernatants were dissolved in DTE-containing SDS-polyacrylamide gel electrophoresis
(PAGE) loading buffer, heated 95°C for 3 min and subjected to SDS-PAGE followed by analysis with
staining of the gel by silver or Coomassie blue or by immunoblotting (28).
Proteasome assay. Approximately 80% confluent fibroblasts were scraped of into 100 mM Tris/HCl
buffer, pH 7.5 (1ml per 75 cm2 flask). The cells were placed on ice for 15 min to allow lysis. The
cytosolic extract was subsequently harvested as the supernatant after a centrifugation at 11,500 × g for
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10 min at 4°C. For the proteasome assay, the cytosol was used at a concentration of 100 µg protein/ml
and the purified human erythrocyte 20S proteasome at a concentration of 2 µg protein/ml. The
hydrolytic activity for the chymotrypsin-like, trypsin-like and caspase-like hydrolytic activities were
determined as previously described (37). The coefficient of variation for the proteasome assay ranged
from 0.8 – 6.3% in 6 independent experiments with the mean being 3%. Proteasome enzyme activities
were calculated as the difference in activity measured in the absence and in the presence of the
proteasome inhibitor, lactacystin and in some experiments the peptide aldehyde Cbz-Leu-Leu-Leucinal
(MG132), both being potent inhibitors of primarily of the chymotrypsin-like activity. All the activity
measurements were done in duplicates or triplicates from independent samples, as stated in the legends.
Fluorescence of cleavage products from peptide substrate was measured on a Kontron SFM 25
spectrofluorometer. The effect of aggregated and non-aggregated AS on the proteasomal activity was
determined by incubating proteasomes with AS for 60 min at 37oC prior to the addition of the
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RESULTS
Localisation of 20S components in Lewy bodies
Lewy bodies denote the characteristic inclusion bodies of PD and DLB (38); they contain abundant
ubiquitinated material indicative of an insufficient proteasomal function (Fig. 1, panel A). AS in a
filamentous form comprise a main component of Lewy bodies (Fig. 1, panel B; 39). 20S proteasomes
were observed in the cytoplasm of both control and brain disease cases and the α6 subunit specific
antibody MCP20 also labeled Lewy bodies of both PD and DLB cases (Fig. 1C). Labeling of Lewy
bodies by the anti-ubiquitin, anti-AS, and anti-proteasome 20S antibodies were mainly confined to their
peripheral zone (Figs. 1, panels A-C). Immunolabeling was not observed in nigral neurons when the
primary antibody was omitted from the immunoreaction (Fig. 1, panel D). The number of stained
Lewy-bodies was lower with 20S proteasome antibodies than with AS.
We also used confocal laser scanning microscopy to analyse freshly isolated Lewy bodies
from post-mortem brains of 5 patients with DLB. This technique is more sensitive and allows more
detailed analysis of co-localisation of AS and the 20S proteasome components in Lewy bodies. Nearly
all Lewy bodies (90-95%) were positive for ubiquitin. Approximately 70% of Lewy bodies (range 58-
80%) were positively labeled by the antibodies MCP196 (Fig. 1, lower panel) and MCP72 (not shown)
specific for 20S proteasomal α5 and α7 subunits. There was no obvious difference in the number or
intensity of the labeled Lewy bodies between the two antibodies. In general, both the ubiquitin and
proteasome antibodies labeled the peripheral portion of Lewy bodies more intensely where the AS
immunoreactivity was also most intense (Fig. 1, lower panel). Compared to that of ubiquitin, the
labeling of proteasome subunits appeared punctately or granularly, and often extended further towards
the cental core of the Lewy bodies. Furthermore, while all Lewy neurites were also ubiquitin positive,
they were rarely labeled by the proteasome specific antibodies (not shown). A similar co-localisation
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was noted in AS positive filamentous inclusions in astrocytes, isolated from brain tissue affected by
multiple system atrophy (Gai and Jensen, data not shown).
Direct interaction between AS filaments and 20S proteasome particles
The co-localisation of 20S proteasomes and AS in the Lewy bodies provides a topological frame for an
interaction between the two molecular species. To examine a putative direct interaction between AS
filaments and proteasome we employed an AS filament binding-assay based on the use of filaments
formed from recombinant human AS (Fig. 2A, D), which serve as bait for proteasomes in human
fibroblasts cytosol. These filaments were readily depolymerised prior to SDS-PAGE by over night
incubation in 8 M urea, 4% SDS, as demonstrated in the lanes with pelleted filaments (Fig. 2A). This
contrast to the AS aggregates previously used by Snyder et al. (24) and demonstrates the aggregates are
devoid of uncharacterised covalent modifications e.g. by oxidative crosslinks. Evidently, a range of
cytosolic proteins was bound to the filaments as demonstrated by their presence in the pellet only when
co-incubated with the filaments (Fig. 2A). The presence of putative proteasomal proteins were
investigated by immunoblotting using monoclonal antibodies; the subunits S6´/Rpt5 and S14/Rpn12
from the 19S regulatory complex were dectected in the cytosol as were the subunits α2, α3, α6, α7 and
β1 subunits from the 20S proteasome core (Fig. 2, panel B). Densitometric analysis of the blots showed
that 5-10% of the subunits from 20S proteasomes in the cytosolic input were bound to the filaments. In
contrast, a negligible binding was found for the 19S regulatory complex subunits S6`/Rpt5 and
S14/Rpn12 although a faint band of both epitopes could be discerned. The S6’ antibody in Fig. 2B, top
panel was used by Snyder et al. (24). The 19S regulatory complex can dissociate into two sub-
complexes, the lid and the base (40). S6´/Rpt5 is found in the base whereas S14/Rpn12 is a component
of the lid. Thus, neither lid nor base binds avidly to AS filaments.
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We next examined the direct interaction between AS filaments and human 20S proteasome particles,
purified from human erythrocytes, to further support the idea of a direct binding of the AS filament to
the 20S particles. The purity of the 20S proteasome is demonstrated by Coomassie blue staining of the
characteristic subunits of about 25-30 kDa (Fig. 2C). Negative staining electronmicroscopy showed
approximately 11 nm structures on the AS filaments upon incubation with the 20S proteasomes (Fig.
2D, panels 3,4). The 11 nm structures resembled 20S proteasomes (Fig. 2D, panel 2). The identity of
the filament-associated structures as 20S proteasome was verified by immunogold labelling with the
MCP72 antibody (Fig. 2D, panels 5,6).
It was of pivotal importance to determine if the interaction could represent a novel
function of aggregated AS. Addition of up to an 8-fold excess of monomeric non-fibrillar AS had no
detrimental effect on the co-sedimentation of purified 20S proteasome particles with the filaments, thus
supporting a filament selectivity of the binding (Fig. 2E). Similar data was obtained using proteasomes
in cytosolic extracts (data not shown). Hence, proteasomal subunits co-localise with AS in Lewy
bodies, and 20S proteasomes bind directly to AS filaments without the need for auxiliary cytosolic
components in this reaction, which represents an apparent novel function of AS developed upon
aggregation.
AS filaments selectively inhibit the chymotrypsin-like activity of the 20S proteasome
The possible functional consequences of the interaction between AS filaments and the proteasome were
determined by measuring the ubiquitin-independent hydrolytic activity of the proteasome in human
fibroblasts cytosol and in purified 20S proteasomes isolated from human erythrocytes. The assay uses
small fluorogenic substrates for the individual hydrolytic activities of the 20S proteasome; the caspase-
like, the chymotrypsin-like and the trypsin-like activity. The fluorescence in the presence of the
proteasomal inhibitor lactacystin was subtracted as background in the assays shown in Fig. 3A. Similar
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background was obtained with the proteasome inhibitor MG132 and this inhibitor was used in
subsequent analyses. Fig. 3A compares the effect on the individual hydrolytic activities of
supplementing cytosolic proteasome and purified 20S proteasome with 10 nM monomeric and
filamentous AS and demonstrates a strong inhibition of the chymotrypsin-like activity by aggregated
but not monomeric AS. Noticeable, the effect on the individual activities in the cytosol and in the
purified 20S proteasome is highly similar with i) a strong filament selective inhibition of the
chymotrypsin-like activity, ii) the trypsin-like activity being marginally inhibited by both monomeric
and filamentous AS and iii) the caspase-like activity being hardly affected. The molar concentration of
the filaments cannot be expressed precisely due to their heterogeneous homopolymeric nature. We
expressed their concentration by their monomeric AS building blocks well aware the actual
concentration of the individual filaments is several fold lower than the concentration of their individual
subunits. By this approach, IC50 for the chymotrypsin-like activity with filaments was a more than 103
fold lower than with monomeric AS and corresponded to 0.5 nM, expressed as the equivalent of
monomeric AS (Fig. 3B). This means that the selectivity on a molar basis is even more pronounced and
likely in the range105 – 106 given that filaments are aggregates of 100-1000 monomers. The saturation
curves for unpurified cytosolic proteasomes and purified 20S proteasome for the inhibition by
monomeric and aggregated AS are nearly identical. The dose curve for the inhibition of the
chymotrypsin-like activity by the proteasome inhibitor MG132 demonstrates an approximately 10 fold
lower potency as compared to the AS filaments, based on their monomeric concentration, and thus
underscores the toxic potential of AS unleashed upon conversion into filaments (Fig. 3B). The mode of
inhibition by AS filaments was investigated by measuring the rate of chymotrypsin-like catalysis at
different substrate concentrations in the absence or presence of filaments. AS filaments inhibit the Vmax
(Fig. 3C), thus demonstrating a non-competitive mode of inhibition. The reduction of the Vmax
displayed a dose-dependent correlation to the concentration of the AS filaments (data not shown). The
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similar inhibitory pattern and saturation curves of the isolated 20S proteasome and cytosolic
proteasomes indicate a similar inhibitory mode by the aggregated AS that likely is mediated by a direct
effect on the 20S catalytic particle.
Amyloid-like characteristics are responsible for the 20S proteasomal inhibition by AS filaments
The structural characteristics of the aggregates responsible for the proteasomal inhibition were
investigated by antibodies, filaments assembled from C-terminally truncated AS and Thioflavin-S that
targets amyloid-structures (Fig. 4B). The FILA-1 antibody is specific for the filamentous form of AS
by more than 103 fold as compared to monomeric AS (Fig. 4A). This antibody abrogated the inhibitory
activity of the AS filaments just as potently as the ASY-1 antibody, raised against full length AS, thus
strengthening the requirement for a structural transition of the AS (Fig. 4B). The FILA-1 antibody
bound only poorly to isolated Lewy bodies or to tissue investigated by immunohistochemistry (data not
shown). This is likely due to the coating of the α-synuclein core fibrils by cytosolic proteins (41). The
aggregation of AS depends on structural determinants within its N-terminal 2/3, and this allowed for
the formation of filaments by the C-terminal truncated AS-(1-95) peptides. A similar potency was
observed of filaments formed from full length AS-(1-140) and AS-(1-95) (Fig. 4B). AS filaments
exhibit characteristics of the β-pleated structures present in amyloid-type filaments that can be targeted
by Thioflavin-S. Preincubation of the AS filaments with Thioflavin-S, prior to their density gradient
purification, inhibited their chymotrypsin-like inhibitory activity by about 60% without solubilising the
aggregates (Fig. 4B). This underscores the requirement for amyloid-like structures in the proteasomal
inhibition by AS filaments, which were further corroborated by the potent proteasomal inhibition by
the model amyloid filaments assembled from synthetic Aβ-(1-40) peptide (Fig. 4B). Accordingly, the
chymotrypsin-like inhibitory activity depends on amyloid-like structural elements that evolve during
the development of AS filaments.
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Soluble amyloid-like AS oligomers inhibit the 20S proteasome
Strategies aiming at abrogating filament formation can increase the content of oligomeric AS (42).
Therefore, it is a question of considerable interest whether the detrimental effect of AS aggregates is
due to the end-state of insoluble filamentous forms or whether soluble oligomers can partake in the
inhibitory process. AS oligomers were isolated by subjecting samples of aggregated AS to
ultracentrifugation to remove insoluble aggregates, followed by the separation of the soluble AS
species by gelfiltration. Soluble oligomers, reacting with the filament specific antibody FILA-1, eluted
from the column corresponding to molecular size markers larger than 150 kDA when aggregated AS
was used as starting material (Fig. 5A, upper panel). The neoepitope, formed when AS monomers
polymerise, is therefore shared by insoluble filaments and soluble oligomers. No detectable FILA-1,
but only ASY-1 immunoreactivity was present if non-aggregated AS was analysed by gel filtration
albeit some of these ASY-1 positive structures likely represents AS multimers (Fig. 5A, lower panel).
The soluble FILA-1 positive AS aggregates inhibited the proteasomal chymotrypsin-like activity, and
this inhibition could be dose-dependly neutralised by FILA-1 IgG (Fig. 5B). These oligomers likely
contains β-pleated amyloid-like characteristics as preincubation with 2.5 mM Congo Red abrogated
their inhitory activity (Fig. 5B). Congo Red is able to disrupt preformed amyloid-type oligomers (43).
The development of proteasomal inhibitory activity during the course of AS aggregation
was compared to the development of insoluble aggregates to determine if soluble AS oligomers could
play a significant role. The initial rate of development of proteasome inhibitory activity was rapid with
a 50% of the maximal inhibition reached within 1- 2 days where after the rate declined, although the
inhibition continued to increase during the 14-day period (Fig. 6A). This contrasted the development of
insoluble AS aggregates that are first detectable after 6 days on Coomassie blue stained gels (Fig. 6B).
β-synuclein was used as negative control for these experiments, because it is unable to form filaments
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due to the absence of an internal approximately 11 amino acid peptide stretch (44). Incubation of this
control protein neither developed proteasome inhibitory activity nor insoluble aggregates (Fig. 6A, B).
The pathogenic AS mutations A30P and A53T has been reported to enhance AS filamentformation
although the process at neutral pH and at the concentrations used in the present study takes several days
(45). Of special interest was the observation that the A30P mutation specifically acted by increasing the
formation of oligomers (45). Fig. 6C demonstrates the faster development of proteasomal inhibitory
activity in incubates of the A30P mutant as compared to the wild type and A53T mutant. Hence, the
proteasome inhibitory activity of a given AS sample cannot simply be determined by assessing the
amount of insoluble AS but may be more optimally reflected by the amount of FILA-1
immunoreactivity.
HSP70 and GAPDH attenuate the proteasomal toxicity of AS filaments.
HSP70 and GAPDH were discovered as AS-filament binding proteins in our search for brain proteins
that selectively bind to aggregated AS (data not shown). The direct interaction of isolated GAPDH and
HSP70 with insoluble AS filaments is demonstrated in Fig. 7A and 7B. HSP70 is of special interest as
it attenuates the toxicity of AS and the amyloid-like polyglutamine peptides in transgenic Drosophila
models of PD and Huntingtons disease (25, 46). Recombinant HSP70 reverted the inhibitory effect of
insoluble AS filaments on the chymotrypsin-like activity (Fig. 7C). In PD, GAPDH was reported to
relocate from the cytoplasm to Lewy bodies and the nucleus (47). Co-incubation of GAPDH with the
AS aggregates completely reverted the inhibitory effects on the chymotrypsin-like activity (Fig. 7C).
Accordingly, endogenous proteins carry the potential for modulating the toxic properties of aggregated
AS.
Heat shock treatment is known to induce the expression of various chaperones that assist
the cell in combating unfolded protein stress (47). Cytosolic proteasomes extracted from fibroblast cells
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after heat shock were more resistant to AS filaments than non-treated controls. HSP70 may participate
in this protection as its expression was increased in this cytosol (Fig. 7D, inset). Accordingly, cells
have evolved physiological systems for combating the detrimental effects of misfolded AS on the
proteasome
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DISCUSSION
This report describes a mechanistic link between two hallmarks of the neurodegenerative disorders PD,
DLB, Lewy body variant of PD and multiple system atrophy, namely the presence of aggregated AS
and the accumulation of polyubiquitinated proteins. The latter likely being due to an insufficient
proteasomal activity (17). The functional link is the highly increased proteasome inhibitory activity that
is developed upon aggregation of AS from its monomeric conformer. The increased inhibitory activity
is selective toward the ubiquitin-independent proteasomal chymotrypsin-like activity in both human
fibroblast cytosol and isolated human 20S proteasomes. By contrast, there is only a minor effect of AS
on the trypsin-like and caspase-like hydrolytic activities and these effects do not depend on the
aggregative state of AS. Interestingly, the chymotrypsin-like activity is far more important for
degradation of proteins than are the trypsin-like- and caspase-like activities (48). The trypsin-like
inhibition had an IC50 about 1 nM, so AS filaments are more potent than the proteasomal inhibitor
MG132. This is in agreement with recent data on the AS inhibitory effect on HEK 293 cell extracts and
the ubiquitin-dependent degradation by 26S proteasomes in rabbit reticulocyte lysates (24). The
neutralisation of the inhibition by the FILA-1 antibody, which selectively binds to aggregated AS,
corroborates the need for an aggregation-dependent structural change. This inhibitory activity depends
on structures related to amyloid-type aggregates, as demonstrated by the similar inhibition by Aβ-(1-
40) aggregates. However, it does not require the aggregates to be in the insoluble filamentous state but
merely requires a structural transition to a form present on both filaments and soluble oligomers, a
transition that correlate to emergence of the epitopes recognised by the FILA-1 antibody. The
inhibitory mode is non-competitive and thus resembles the inhibitory effect by HSP-90 (49). It likely
represents a structure change by the binding of the aggregated AS to the exterior of the β5 subunit,
responsible for the chymotrypsin-like activity, and relayed to its catalytic site on its interior surface of
the proteasomal barrel. This would be analogous to the effect of binding of monoclonal antibodies to
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the 20S proteasome (50). Alternatively, the effect might be allosteric by binding to another subunit that
subsequently changes the β5 subunit but this is less likely as the trypsin-like and caspase-like activities
hardly were affected. Such interactions are corroborated by the electronmicrographs showing direct
binding of purified 20S proteasomes to AS filaments.
The proteasome exists in cells predominantly as 26S proteasomes consisting of the 20S
proteolytic particles with 19S regulatory complexes attached to the ends of the 20S cylinder. However,
naked 20S proteasomes account for about 1/3 of the 20S proteasomal particles in mammalian cells (51)
and hydrid proteasomes with 19S particles attached to one end and the activator PA28 attached to the
other end represent about 25% of 20S proteasomes in Hela cells (51). All proteasomal hydrolysis is
carried out within the 20S proteasome particle and this can take two forms. Ubiquitin-dependent
protein degradation, representing the major fraction, relies solely on the 26S proteasomes and the
hybrid proteasomes. By contrast, ubiquitin-independent proteolysis of certain unfolded or modified
proteins can be carried out by the single 20S proteasome. Interestingly AS belongs to the proteins,
which can be degraded by the 20S proteasome without participation of ubiquitin or the 19S regulatory
complex (20).
An important difference between the data presented by Snyder et al. (24) and the present
report relates to the effect of the aggregated AS on the 20S proteasome. Our data are identical with
Snyder’s (24) regarding the inhibitory mode toward the 3 hydrolytic activities when analysing the
effect of AS on the ubiquitin-independent proteasomal activity in cytosol. Moreover, the IC50 of
approximately 1 nM and 103 nM for aggregated and monomeric AS are similar for the cytosolic
proteasomes. The discrepancy relates to the effect on the isolated 20S proteasome, which we find
behaves similar to the cytosolic proteasome. By contrast, Snyder et al. (24) reports an IC50 of
approximately 1000 nM and 10,000 nM for the inhibition of the chymotrypsin-like activity of their 20S
preparation by aggregated and monomeric AS. More experimental details could account for this
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discrepancy. First, the purified 20S proteasome Snyder et al. (24) was from a commercial source and
purified by ammonium sulphate precipitation and several chromatographic steps. In contrast, the 20S
proteasome preparation used in the present study was isolated by a milder procedure involving
immuno-affinity chromatography (26). We have experienced with one batch of purified 20S
proteasome stored for prolonged time at –20oC, that it lost the majority of its hydrolytic activity.
However, the remaining lactacystin-inhibitable chymotrypsin-like activity was not well inhibited by
aggregated AS (data not shown). In this context, it is also reassuring that we got similar results with
purified proteasomes and cell extracts.
Another reason for the discrepancy could be the different state of the aggregated AS. The
material used in this study contains filaments, as demonstrated by electron microscopy (Fig. 2), and it
can be readily depolymerised to the native monomeric AS as seen by SDS-PAGE (Fig. 2). By contrast,
the AS used by Snyder (24) was a hexa-histidine fusionprotein incubated at an unspecified
proteinconcentration and in an unspecified buffer and the aggregates formed was very old and not
depolymerised by SDS treatment. Hence, the latter material could contain uncharacterised
modifications e.g. oxidative crosslinks. However, the differences of the aggregates are likely of less
importance as similar effects of aggregated AS on the cytosolic proteasomal activity was measured in
the two reports. Our data support the interpretation of a direct effect of aggregated AS on the 20S
proteasome particle because of the similar inhibitory profile on the individual proteasomal hydrolytic
activities and the identical chymotrypsin-like inhibitory potency on purified 20S proteasome and
cytosolic proteasomes. This is further corroborated by the demonstration of a direct binding of isolated
20S proteasomes to AS filaments.
The inhibitory effect on the 20S proteasome does not exclude an interaction with subunits
in the 19S regulatory complex as reported for the S6´/Rpt5 (24). However, the reported binding of the
S6´/Rpt5 subunit was not selective to AS in its aggregated state and the efficiency of the binding was
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not reported in terms of e.g. how much of the input was bound to the aggregated AS (24). We
performed filament pull-down experiments on fibroblast cytosol and compared the amount of the 19S
subunits S6´/Rpt5 and S14/Rpn12 with the amount of the 20S proteasome subunits α2, α3, α6, α7 and
β1 in the input with the filament-bound proteins using a panel of proteasome antibodies. By this
approach approximately 5-10% of all the 20S proteasome subunits bound to the filaments. The equal
binding of the individual subunits supports a binding of the 20S particle in toto. The binding of the
19S regulatory complex subunits was, by contrast, negligible although the binding of the S6´/Rpt5 was
verified in separate experiments optimised to detect low efficient binding. Accordingly, the effect of
the interaction between aggregated AS and the 20S proteasome particle is likely of primary
significance as compared the putative binding to 19S regulatory complex subunits. Especially because
the 19S regulatory complex is involved in the ubiquitin-dependent proteasomal functions and we and
Snyder et al. (24) demonstrate the filament specific effect on the ubiquitin-independent hydrolysis in
cell extracts.
The recent demonstration of inhibition of proteasomal function by cellular expression of
wild type AS or its mutant peptides may reflect different mechanisms (21-24). Tanaka et al (22)
demonstrated an inhibition of the ubiquitin-independent hydrolysis of all the three hydrolytic activities
by the A30P mutant but not the wild type peptide in cellular extracts. By contrast, Petrucelli et al. (23)
showed in living M17 neuroblastoma cells a decreased ubiquitin-dependent degradation of an unstable
degron fused to a green fluorescent protein. Stefanis et al. (21) demonstrated an accumulation of
ubiquitinated proteins in the PC12 cells with the highest expression of the A53T mutant, which
correlated to a significantly decreased chymotrypsin-like activity in cellular extracts. The interpretation
of the relation of these experiments to the mechanism described in this report is not clear given the
uncertainty of whether the cells had accumulated aggregated AS species. The use of antibodies like
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FILA-1 or a reversal of the inhibition by e.g. Congo Red (43) or expression of e.g. HSP70 may help to
resolve this issue.
The transition of AS monomers into insoluble aggregates denotes a nucleation-dependent
process wherein soluble oligomers represent a step in the pathway. The oligomers have recently been
characterised extensively by biophysical techniques (52), and they have been proposed to represent
pore-forming structures that may perturb the cellular ionic homeostasis (53). We are the first to
demonstrate that these structures, being approximately 200-800 kDa as demonstrated by gelfiltration,
share both structural and functional properties with the insoluble filaments represented by i) the binding
of the FILA-1 antibody and ii) the sensitivity of their inhibition of the proteasomal chymotrypsin-like
activity to Congo Red. However, whereas the development of filaments is slow (Conway 2000c), the
proteasome-inhibitory oligomers form within hours (Fig. 6A; 52). The process of oligomer formation is
not yet clear but it is stimulated by the A30P mutation (45) and oxidative conjugation of dopamine to
AS (42). Our demonstration of a faster development of proteasome inhibitory activity in the A30P
mutant (Fig. 6C), as compared to the A53T and the wild type AS, is thus compatible with biochemical
experiments on aggregation (45). However, a comparison between different experiments on
oligomerformation has to be exerted with great care, as the formation of these species is extremely
sensitive to the protein concentration. Lashuel (52) demonstrated they can develop in less than one hour
at AS concentrations of between 300-700 µM which is in the range of the current experiments.
Importantly, we demonstrate that endogenous ligands carry the potential of attenuating
the proteasomal inhibition by AS aggregates as shown for HSP70 and GAPDH. The HSP70-mediated
rescue of proteasomal function from AS toxicity may be the reason for its cytoprotective effects in vivo
when co-expressed with AS in Drosophila (25) and likely forms the basis for the protective effect of
heat shock treatment on the proteasomal sensitivity to AS aggregates (Fig. 7). GAPDH is considered as
a household enzyme in the glycolytic pathway but accumulating data support it may partake non-
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glycolytic processes (47). Its relocalisation into the nucleus and Lewy bodies in PD (54) suggest it may
be sequestered and rendered unable to form its protective function against aggregated AS species in the
cytosol. However, the current data does not allow unambiguously stating that the direct binding of
GAPDH and HSP-70 to the AS aggregates causes the attenuation of the inhibition. A binding of
GAPDH and HSP-70 to the 20S proteasome could in principle also cause the effect. Solving this issue
will require additional experimentation e.g. with the identification of GAPDH mutants that fail to bind
to the filaments. β-synuclein may represent another factor that modulates the AS toxicity, because it is
able to bind AS and inhibit its aggregation, and because the co-expression of AS and β-synuclein leads
to an attenuation of the pathology observed in AS single transgenic mice (55).
The amyloid-type characteristics are important for proteasomal inhibition, as
demonstrated by the effects of Congo Red and Thioflavin-S on the inhibitory effect of AS aggregates,
and the inhibitory effects of amyloid filaments of the Aβ peptide, raise the following hypothesis.
Diseases characterised by intracellular amyloid-type filaments are subject to an inhibition of the
chymotrypsin-like proteasomal activity, which contributes to the accumulation of polyubiquitinated
proteins and a general degeneration of the cellular homeostasis via an impaired protein catabolism.
Such inhibition of the chymotrypsin-like activity has been reported in substantia nigral tissue affected
by PD and DLB (16, 20). The cellular selectivity of the neurodegeneration displayed by specific
aggregating proteins, as observed in e.g. striatal neurons in Huntington’s disease (1), contrast the often-
wider expression of the proteins. This may be due to a cell specific expression of the attenuating
proteins. The latter binds to protein-specific structures in the aggregating proteins that are different
from the common amyloid structures and hereby revert the toxicity, as exemplified by HSP70 and
GAPDH. This hypothesis is corroborated by the demonstration of in vivo proteasome inhibitory
properties of aggregates of glutamine expanded huntingtin peptides (56) that can be attenuated by
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HSP70 (46), and the accumulation of ubiquitinated proteins in animal models expressing aggregation
prone AS and huntingtin (14, 57, 58).
The linkage of AS aggregation to proteasomal inhibition represents a paradigm to be
exploited for the protection against AS-mediated cellular degeneration. Structural insight into this
interaction may allow therapeutic strategies based on the targeting of the aggregates by small molecules
or endogenous proteins thereby preventing their binding to the proteasome. Such an approach may
complement the ongoing attempts to design inhibitors of AS aggregation, and potentially it may have a
wider applicability toward other intracellular amyloidosises.
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Acknowledgement
This work was supported by grants from the Danish Medical Research Council (9902995; 99000377),
The Lundbeck Foundation, NMMRC (102170, 187636), Fonden af 2. juli1984 til bekæmpelse af
Parkinsons sygdom, the PA Messerschmidt and Hustrus Fund, and the Mette and Mogens Mogensens
Fund. We thank Prof. B.F.C. Clark for invaluable support, prof. A. Maunsbach for valuable guidance
on the electronmicroscopy, and The Netherlands Brain Bank and the NHMRC Brain Bank for donating
human brain tissue.
Footnotes 1The abbreviations used are: AD, Alzheimer’s disease; AS, α-synuclein; CL, chymotrypsin-
like; DLB, dementia with Lewy bodies; DTE, dithioerythreitol; GAPDH, glyceraldehydes-6-phosphate
dehydrogenase; HSP70, heat shock protein-70; PA, post-acidic; PAGE, polyacrylamide gel
electrophoresis; PBS, phosphate buffered saline; PD, Parkinson’s disease; SDS, sodium dodecyl
sulphate; SNpc, substantia nigra pars compacta; TL, trypsin-like; TBS, Tris buffered saline.
¶ E.L and R.B contributed equally to this manuscript.
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Legends
Figure 1. Upper panel. Ubiquitin, AS and 20S proteasome co-localise in Lewy bodies in SNpc
neurons in PD and DLB. Lewy bodies are marked by arrowheads. A) Ubiquitin is seen densely in the
cytoplasm and in a Lewy body. The labeling of the peripheral zone of the Lewy body is higher than
within the center. B) AS in Lewy body in DLB tissue. Note the predominant labeling of the peripheral
zone of the Lewy body. C) 20S proteasome in a neuron from a PD case obtained from a section that
was subjected to demelanization before immunostaining. Note the labeling by the MCP20 antibody of
the Lewy body with the main labeling of the peripheral zone. D) Control section from a PD case that
was subjected to demelanization before immunstaining but without incubation with the primary
antibody. The bars represent 20 µm.
Lower panel. Co-localisation of AS, ubiquitin and 20S proteasome in isolated Lewy bodies.
Lewy bodies were isolated from cortical brain tissue affected by DLB. Lewy bodies were identified by
AS staining and the localisation herein of the 20S proteasomal α5 subunit by MCP196 was investigated
by confocal laser scanning microscopy. The upper left panel show ubiquitin, the lower left panel shows
20S subunits. The middle column shows AS and the merged images are shown in the right column. The
bar in lower middle image represents 10 µm and applies to all images in the lower panel.
Abbreviations: a-Syn = α-synuclein; ub = ubiquitin; Ctl = control.
Figure 2. AS filaments bind 20S proteasomes.
(A ) The binding of proteins from human fibroblast cytosol to in vitro formed AS filaments was
investigated by co-sedimentation analysis and SDS-PAGE. Centrifugation of the filaments resulted in a
pellet of the approximately 20 kDa AS protein, whereas a small amount of protein was sedimented
from the cytosol alone. However, co-incubation of the cytosol and filaments prior to centrifugation
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results in the detection of distinct proteins bands and a smear of proteins in the molecular range
between 25-66 kDa. Electrophoresis of 8 % of the input of cytosol and 25% of the input of filaments is
shown, whereas the total pellets are displayed. Molecular size markers in kDa are indicated to the right.
One of > 3 experiments is presented.
(B) The binding of subunits of the 19S regulatory complex (upper part) and 20S proteasomes (lower
part) from fibroblast cytosol to the AS filaments is demonstrated by immunoblotting. The total
cytosolic input (C-T), the cytosolic pellet (C-P) and pellet formed by incubating cytosol and filaments
(C+F-P) is shown to the left, middle and right and the antibodies used are indicated to the left. The
subunits subunit S6’/Rpt5 of the 19S regulatory complex was probed by a commercial rabbit antibody
S6’ and the TBP1-19 antibody with identical results. The S14/p31/Rpt5 subunit was detected with the
p31-38 antibody. A faint filament-associated band is detectable by the TBP1-19 and the p31-38
antibodies. The 20S proteasome subunits α7 and β1 were probed by MCP72 and MCP421, and the α-
subunits 2, 3, 6 and 7 by MCP231 and all antibodies demonstrated filament-associated
immunoreactivity corresponding to approximately the same proportion of the cytosolic input. One of 3
experiments is presented.
(C) The purity of purified human 20S proteasomes is demonstrated by a Coomassie Blue staining of 8
µg material resolved by reducing SDS-PAGE. Molecular size markers in kDa are indicated to the left.
(D) Negative staining electron microscopy of purified AS filaments (panel 1) and filaments after
incubation with purified 20S proteasomes (panels 3-6). Note the smooth appearance of the native
filaments (panel 1) and the presence of spherical, approximately 11 nm particles, associated to the
filaments upon incubation with the 20S proteasomes (panels 3-6). Immuno-electronmicroscopy with
the MCP72 antibody and 5 nm gold particles conjugated to the second antibody was performed to
verify the presence of the 20S proteasomes (panels 5-6). Panel 2 demonstrates the presence of
approximate 11 nm electron dense particles labeled by the MCP72 antibody and shows that the
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particles are of similar diameter as those associated to the filaments (panels 3-6). Bars representing 25
nm are presented in panels 1-6. One of > 3 independent analysis is presented
(E) Purified 20S proteasomes bind preferentially to AS in its filamentous form. Lane 1 shows 30% of
the input of 20S proteasome subunit α7. Negligible amounts of the subunit was present in pellets upon
sedimentation of the 20S proteasome without filaments (lane 2) and in the presence of monomeric AS
(lane 8). By contrast, proteasomes sedimented with filaments (lanes 3-7). In lanes 4 – 7, increasing
amounts of monomeric AS were added to the AS filaments. The molar ratio of monomeric to
filamentous AS is indicated below the lanes. The absent effect of increasing the ratio of
monomeric/filamentous AS prior to incubation with the 20S proteasome, demonstrates selective
binding of proteasomes to filaments. One of 3 similar experiments is presented.
Figure 3. AS filaments inhibit the proteasomal chymotrypsin-like activity.
(A) The individual proteasomal hydrolytic activities, chymotrypsin-like (CL), trypsin-like (TL)
and caspase-like (CAL) were measured in fibroblast cytosol and purified human 20S proteasomes
(hatched bars) by fluorogenic substrates in the absence (black column) and presence of 10 nM of
monomeric (M) AS (grey columns) and filamentous AS (F) (white columns). The samples were
incubated with the test substances for 60 min at 37oC prior to addition of the fluorogenic substrate. The
ordinate shows the ratio between the hydrolytic activity in the absence and presence of AS. The bars
show the mean 1 ± S.D. of 3 replicates. One of 3 similar experiments is presented. The chymotrypsin-
like activity was efficiently and selectively inhibited by the AS filaments, whereas the trypsin-like
activity displayed some inhibition by both monomeric and filamentous AS. The asterisks demonstrate
that only the chymotrypsin-like activity was significantly inhibited (P< 0.01, two sampled t-test,
unpaired data).
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(B) Concentration dependence of the inhibition of the chymotrypsin-like activity in fibroblast
cytosol (filled circles and triangles) and purified 20S proteasome (open circles and triangles) by
monomeric (triangles) and filamentous AS (circles) and of cytosol in the presence of the proteasomal
inhibitor MG132 (filled squares). The concentration of the inhibitors is displayed on the abscissa, and
the ratio between the chymotrypsin-like activity at the indicated concentration of inhibitor and the
chymotrypsin-like activity in the absence of inhibitor is displayed on the ordinate. The concentration of
the filaments is indicated as the concentration of its monomeric constituents. One of 3 representative
experiments is presented.
(C) Non-competitive inhibition of the chymotrypsin-like activity by AS filaments. The substrate
hydrolysis of increasing concentrations of substrate by cytosolic proteasomes was recorded in the
absence (filled circles) and presence of 35 µg/ml purified AS filaments (open circles) and the initial
rate of hydrolysis was measured. The abscissa shows the concentration of the fluorogenic substrate, and
the ordinate shows the initial rate of hydrolysis in arbitrary units. Note that the filaments inhibit the
maximal rate of hydrolysis, indicating a non-competitive mode of inhibition. One of 3 similar
experiments is presented.
Fig. 4. Characteristics of the filaments being responsible for the proteasomal inhibition.
Requirements of the aggregated AS needed for inhibiting the proteasome were investigated by several
approaches. (A) First the filament-specificity of the FILA-1 antibody was characterised by solid phase
binding assay as previously described for MAP-1B (28). The per cent binding of 125I-labeled FILA-1
IgG (50 pM) to immobilised filaments in the absence and presence of increasing concentrations of
soluble filamentous (filled circles) and monomeric AS (open circles) is displayed on the ordinate. The
abscissa displays the concentration of soluble filamentous and monomeric AS presented by their
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monomeric content. The points display the mean ± 1 S.D. of triplicates. One of 3 representative
experiments is displayed.
(B) Inhibition of the proteasomal chymotrypsin-like activity by amyloid filaments and reversal
of the inhibition by antibodies and amyloid targeting agents. The proteasomal chymotrypsin-like
activity in fibroblasts cytosol was measured in the presence of purified filaments formed from 170 nM
full length AS-(1-140), 170 nM C-terminally AS-(1-95) and 170 nM Aβ-(1-40). AS-(1-140) aggregates
was preincubated with non-immune (NI) rabbit IgG, ASY-1 IgG, FILA-1 IgG, in a 300 fold molar
excess as compared to the AS, or 0.7% Thioflavin-S for 60 min at 20oC prior to their isolation. The
effect of the treatment on the proteasome assay was measured. The ordinate displays the difference
between the total chymotrypsin-like activity and the activity measured in the presence of the inhibitors
expressed as per cent of the decrease in activity measured in the presence of AS-(1-140). The columns
demonstrate the mean of duplicate samples from one of two similar experiments. The neutralisation of
the AS-(1-140) inhibitory activity marked by stars was significant (P < 0.01), two sampled t-test,
unpaired data).
Fig. 5. Soluble AS oligomers inhibit the chymotrypsin-like proteasome activity.
(A) AS oligomers were isolated by gelfiltration of high-speed supernatants to separate high
molecular weight oligomers from monomeric AS. Non-aggregated AS was treated in parallel as
control. Fractions were assayed by dotblotting with a filament-specific antibody (FILA-1) and an
antibody (ASY-1), which reacts with both soluble and filamentous AS. Row NI shows the reactivity
with a non-specific antibody. The lower panel shows the immunoreactivity of the eluate from the
monomeric sample analysed by the same antibodies. The elution of molecular size markers in kDa is
indicated above and the fraction numbers below. One of 3 similar experiments is presented.
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B) Samples from the eluted fractions shown in panel A were assayed for the effect on the
proteasomal chymotrypsin-like activity of fibroblast cytosol. Fractions from AS oligomers are
indicated by filled circles and fractions from non-aggregated AS by filled squares. The abscissa shows
the fraction number from the gel filtration and the ordinate shows the chymotrypsin-like activity of the
cytosol. The effect of supplementing the eluate with FILA-1 antibody (at 0.1 mg/ml (filled triangle)
and 0.025 mg/ml (open circle)) and 2.5 mM Congo Red (open triangle) is demonstrated. The elution of
molecular size markers in kDa is indicated above. One of 3 similar experiments is presented.
Fig. 6. Development of chymotrypsin-like inhibitory activity.
(A) The temporal development of chymotrypsin-like inhibitory activity upon incubation of 0.5
mM AS (filled circles) and β-synuclein (open circles). The abscissa shows the days of incubation, and
the ordinate shows the inhibition of the chymotrypsin-like activity by 7 µM AS samples with the
activity at day 14 subtracted. The points demonstrate the mean of duplicate samples in one of two
similar experiments.
(B) The development of insoluble aggregates in the AS and β-synuclein samples as
demonstrated in panel A is shown by Coomassie Blue staining of the protein remaining soluble (S) or
being pelleted (P) upon ultracentrifugation. Note the late development of insoluble AS aggregates as
compared to the rapid development of chymotrypsin-like inhibitory activity in panel A.
(C) Effect of AS mutations on the development of chymotrypsin-like inhibitory activity upon
incubation of 0.5 mM wild type (triangles), A30P (filled circles) and A53T AS (open circles). The
abscissa shows the hours of incubation, and the ordinate shows the inhibition of the chymotrypsin-like
activity by 7 µM AS samples with the activity by A30P at 36h subtracted. The points demonstrate the
mean of duplicate samples in one of three similar experiments.
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Fig. 7. HSP70 and GAPDH bind AS filaments and abrogate their chymotrypsin-like inhibitory activity.
(A) The binding of purified rabbit GAPDH to AS filaments was determined by the filament-
pull-down assay as described in Fig. 2. The panel shows a silver stained SDS PAGE gel where lane 1
demonstrates the input of 6 µg GAPDH. Lanes 3-5 show pellets recovered after centrifugation of
GAPDH alone (lane 3), AS filaments alone (lane 4) and GAPDH incubated with AS filaments (lane 5).
The molecular size marker in kDa is shown to the left and the positions of AS and GAPDH to the right.
One of 3 similar experiments is presented.
(B) The binding of recombinant human HSP70 to AS filaments was analysed analogous to the
above-described binding of GAPDH but visualised by immunoblotting, using a monoclonal anti-
HSP70 antibody and enhanced chemilumenescense. Lane 1, input of 0.3 µg recombinant HSP70. Lanes
3 and 5 shows HSP70 sedimented in the absence and presence of AS filaments. One of 3 similar
experiments is presented.
(C) Reversal of the AS dependent inhibition of the chymotrypsin-like activity of purified 20S
proteasome by HSP70 and GAPDH. The ordinate demonstrates the chymotrypsin-like activity in
arbitrary units with the activity without filaments being one and in the presence of filaments being zero.
The effects of HSP70 and GAPDH alone and in a 100 fold excess as compared to AS filaments are
shown. The reversal of the inhibitory effects of the filaments by HSP70 and GAPDH are significant as
marked by asterisk (P < 0.01) when measured by the two-sample t-test of unpaired data. One of 3
similar experiments is presented.
(D) Heat shock treatment of fibroblast increases the resistance of their cytosolic proteasome
toward the inhibition by AS filaments. The ordinate shows the chymotrypsin-like activity in arbitrary
units and the columns show the chymotrypsin-like like proteasomal activity in the cytosol from cells
treated with or without heat shock, and absence (expressed as 1) or presence of AS filaments. The
filamentous inhibition of the heat shock treated cytosol differs significantly from untreated control (P <
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0.05) as marked by asterisks when measured by the two-sample t-test of unpaired data. The inset shows
the presence of HSP70 and actin in cells treated with (HS) or without (C) heat shock as determined by
immunoblotting. One of 3 similar experiments is presented.
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Fig. 1
Ub aSyn 20S Ctl
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Fig. 2
116
66
45
35
25
1814
11 22 44 6633 55 77
M/FM/F
C D
53
21
64
AA
8% C
8% C
25%
F
25%
FCC F F C
+ F
C +
F
InputInput PelletPellet
1161166666
45453535
25251818
MCP72MCP72
S6’S6’
P31-38P31-38
TBP1-19TBP1-19
C+F
-P
C+F
-P
C-P
C-P
20
S2
0S
19
S1
9S
C-T
C-T
MCP231MCP231
MCP421MCP421
A B
E
00 88442211
88
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A
C
B
Fig. 3
CAL
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Fig. 5
FilamentFila + ¼ Fila IgG
Fila + Congo redMonomer
Fila + Fila IgG
1 111098765432 120.2
0.3
0.8
0.6
0.4
CL-
ac
tivity
Fraction
45
0
24
0
15
0
68
45
Oligomers
Monomer
Antibody
Fila
Fila
ASY-1
NI
NI
ASY-1
1 111098765432 12Fraction
45
0
24
0
15
0
68
45A
B
Oligo
Oligo + Fila IgGOligo + ¼ Fila IgG
Oligo + Congo red
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Fig. 6
0 2 4 6 10 128 14
Days
α-synuclein
β-synuclein
0.25
0.00
0.50
0.75
1.00
1.25
DC
L-a
ctiv
ity
β-synuclein
α-synuclein
10 3 6 10 14
S P P PP P PS S S SS
B
A
C
DC
L-a
ctiv
ity
Hours
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Fig. 7
A
B
48483333
2525
1616
GAPDH
α-synuclein
HSP70
1 2 4 53
Co
ntro
l
Fila
me
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at sh
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ents
0.00
1.25
1.00
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tivity
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Competitor:
Cont
rol
Filam
ent
Filam
ent +
HSP
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HSP70
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DH
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Hendil and Poul H. JensenEvo Lindersson, Rasmus Beedholm, Peter Højrup, Torben Moos, Weiping Gai, Klavs B.
-synuclein filaments and oligomersαProteasomal inhibition by
published online January 7, 2004J. Biol. Chem.
10.1074/jbc.M306390200Access the most updated version of this article at doi:
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