50
Proteasomal inhibition by α-synuclein filaments and oligomers. Lindersson, E. , Beedholm, R. , Højrup, P. 3 , Moos, T. 4 , Gai, W.-P. 5 , Hendil, K.B. 6 and Jensen, P.H. 1 1 Departments of Medical Biochemistry, 2 Molecular Biology, University of Aarhus, Denmark 3 Institute of Molecular Biology, University of Southern Denmark, Denmark. 4 Department of Medical Anatomy, University of Copenhagen, Denmark, 5 Department of Physiology, Flinders Medical Centre, Australia , 6 August 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. by guest on March 29, 2018 http://www.jbc.org/ Downloaded from

Proteasomal inhibition by α-synuclein filaments and oligomers

  • Upload
    haphuc

  • View
    219

  • Download
    0

Embed Size (px)

Citation preview

Page 1: Proteasomal inhibition by α-synuclein filaments and oligomers

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.

by guest on March 29, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Page 2: Proteasomal inhibition by α-synuclein filaments and oligomers

2

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

therapeutic intervention by guest on March 29, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Page 3: Proteasomal inhibition by α-synuclein filaments and oligomers

3

RUNNING TITLE

α-synuclein inhibits the 20S proteasome

by guest on March 29, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Page 4: Proteasomal inhibition by α-synuclein filaments and oligomers

4

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

by guest on March 29, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Page 5: Proteasomal inhibition by α-synuclein filaments and oligomers

5

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

by guest on March 29, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Page 6: Proteasomal inhibition by α-synuclein filaments and oligomers

6

PD, DLB and multiple system atrophy where the degenerating cells accumulate AS aggregates.

Abolition of the inhibition may open for new neuroprotective strategies.

by guest on March 29, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Page 7: Proteasomal inhibition by α-synuclein filaments and oligomers

7

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

by guest on March 29, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Page 8: Proteasomal inhibition by α-synuclein filaments and oligomers

8

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

by guest on March 29, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Page 9: Proteasomal inhibition by α-synuclein filaments and oligomers

9

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)

by guest on March 29, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Page 10: Proteasomal inhibition by α-synuclein filaments and oligomers

10

(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

by guest on March 29, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Page 11: Proteasomal inhibition by α-synuclein filaments and oligomers

11

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;

by guest on March 29, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Page 12: Proteasomal inhibition by α-synuclein filaments and oligomers

12

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

by guest on March 29, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Page 13: Proteasomal inhibition by α-synuclein filaments and oligomers

13

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

fluorogenic substrate. by guest on March 29, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Page 14: Proteasomal inhibition by α-synuclein filaments and oligomers

14

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

by guest on March 29, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Page 15: Proteasomal inhibition by α-synuclein filaments and oligomers

15

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.

by guest on March 29, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Page 16: Proteasomal inhibition by α-synuclein filaments and oligomers

16

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

by guest on March 29, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Page 17: Proteasomal inhibition by α-synuclein filaments and oligomers

17

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

by guest on March 29, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Page 18: Proteasomal inhibition by α-synuclein filaments and oligomers

18

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.

by guest on March 29, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Page 19: Proteasomal inhibition by α-synuclein filaments and oligomers

19

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

by guest on March 29, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Page 20: Proteasomal inhibition by α-synuclein filaments and oligomers

20

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

by guest on March 29, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Page 21: Proteasomal inhibition by α-synuclein filaments and oligomers

21

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

by guest on March 29, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Page 22: Proteasomal inhibition by α-synuclein filaments and oligomers

22

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

by guest on March 29, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Page 23: Proteasomal inhibition by α-synuclein filaments and oligomers

23

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

by guest on March 29, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Page 24: Proteasomal inhibition by α-synuclein filaments and oligomers

24

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

by guest on March 29, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Page 25: Proteasomal inhibition by α-synuclein filaments and oligomers

25

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

by guest on March 29, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Page 26: Proteasomal inhibition by α-synuclein filaments and oligomers

26

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-

by guest on March 29, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Page 27: Proteasomal inhibition by α-synuclein filaments and oligomers

27

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

by guest on March 29, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Page 28: Proteasomal inhibition by α-synuclein filaments and oligomers

28

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.

by guest on March 29, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Page 29: Proteasomal inhibition by α-synuclein filaments and oligomers

29

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.

by guest on March 29, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Page 30: Proteasomal inhibition by α-synuclein filaments and oligomers

30

References

1 Ellison, D. & Love, S. (1998) Neuropathology, Mosby International Ltd.

2 Glickman, M.H. & Ciechanover, A. (2001) Physiol. Rev. 82, 373-428.

3 Goedert, M., Spillantini, M.G. & Davies, S.W. (1998) Curr. Opin. Neurobiol. 8, 619-632.

4 Polymeropoulos, M.H., Lavedan, C., Leroy, E., Ide, S.E., Dehejia, A., Dutra, A., Pike, B., Root, H.,

Rubenstein, J., Boyer, R., Stenroos, E.S., Chandrasekharappa, S., Athanassiadou, A., Papapetropoulos,

T., Johnson, W.G., Lazzarini, A.M., Duvoisin, R.C., Di Iorio, G., Golbe, L.I. & Nussbaum, R.L. (1997)

Science 276, 2045-2047.

5 Krüger, R., Kuhn, W., Muller, T., Woitalla, D., Graeber, M., Kosel, S., Przuntek, H., Epplen, J.T.,

Schols, L. & Riess, O. (1998) Nat. Genet. 18, 106-108.

6 Narhi, L., Wood, S.J., Steavenson, S., Jiang, Y., Wu, G.M., Anafi, D., Kaufman, S.A., Martin, F.,

Sitney, K., Denis, P., Louis, J.C., Wypych, J., Biere, A.L. & Citron, M. (1999). J. Biol. Chem. 274,

9843-9846.

7 Conway, K.A., Harper, J.D. & Lansbury, P.T. (1998) Nat. Med. 4, 1318-1320.

8 Baba, M., Nakajo, S., Tu, P.H., Tomita, T., Nakaya, K., Lee, V.M., Trojanowski, J.Q. & Iwatsubo, T.

(1998) Am. J. Pathol. 152, 879-884.

9 Dickson, D.W., Liu, W., Hardy, J., Farrer, M., Mehta, N., Uitti, R., Mark, M., Zimmerman, T.,

Golbe, L., Sage, J., Sima, A., D'Amato, C., Albin, R., Gilman, S. & Yen, S.H. (1999) Am. J. Pathol.

155, 1241-1251.

10 Kahle, P.J., Neumann, M., Ozmen, L., Muller, V., Odoy, S., Okamoto, N., Jacobsen, H., Iwatsubo,

T., Trojanowski, J.Q., Takahashi, H., Wakabayashi, K., Bogdanovic, N., Riederer, P., Kretzschmar,

H.A. & Haass, C. (2001) Am. J. Pathol. 159, 2215-2225.

11 Serpell, L.C., Berriman, J., Jakes, R., Goedert, M. & Crowther, R.A. (2000) Proc. Natl. Acad. Sci. U

S A 97, 4897-4902.

by guest on March 29, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Page 31: Proteasomal inhibition by α-synuclein filaments and oligomers

31

12 Spillantini, M.G., Crowther, R.A., Jakes, R., Hasegawa, M. & Goedert, M. (1998) Proc. Natl. Acad.

Sci. U S A. 95, 6469-6473.

13 Conway, K.A., Harper, J.D. & Lansbury, P.T. Jr. (2000) Biochemistry 39, 2552-2563.

14 Masliah, E., Rockenstein, E., Veinbergs, I., Mallory, M., Hashimoto, M., Takeda, A., Sagara, Y.,

Sisk, A. & Mucke, L. (2000). Science 287, 1265-1269.

15 Feany, M.B. & Bender, W.W. (2000) Nature 404, 394-398.

16 McNaught, K.S. & Jenner, P. (2001) Neurosci. Lett. 297, 191-194.

17 McNaught, K.S., Olanow, C.W., Halliwell, B., Isacson, O. & Jenner, P. (2001) Nat. Rev. Neurosci.

2, 589-594.

18 McNaught, K.S., Belizaire, R., Jenner, P., Olanow, C.W. & Isacson, O. (2002) Neurosci. Lett. 326,

155-158.

19 McNaught, K.S., Belizaire, R., Isacson, O., Jenner, P. & Olanow, C.W. (2003) Exp. Neurol. 179,

38-46.

20 Tofaris, G.K., Razzaq, A., Ghetti, B., Lilley, K. and Spillantini, M.G. (2003) J. Biol. Chem. Aug. 15

epublication.

21 Stefanis, L., Larsen, K.E., Rideout, H.J., Sulzer, D. & Greene, L.A. (2001) J Neurosci. 21, 9549-60.

22 Tanaka, Y., Engelender, S., Igarashi, S., Rao, R.K., Wanner, T., Tanzi, R.E., Sawa, A.L., Dawson,

V., Dawson, T.M. & Ross, C.A.. (2001) Hum. Mol. Genet. 10, 919-926.

23 Petrucelli, L., O'Farrell, C., Lockhart, P.J., Baptista, M., Kehoe, K., Vink, L., Choi, P., Wolozin, B.,

Farrer, M., Hardy, J. & Cookson, M.R. (2002) Neuron 36, 1007-19

24 Snyder, H., Mensah, K., Theisler, C., Lee, J., Matouschek, A. & Wolozin. B. (2003) J Biol Chem.

278, 11753-9

25 Auluck, P.K., Chan, H.Y., Trojanowski, J.Q., Lee, V.M. & Bonini, N.M. (2002) Science 295, 865-

868.

by guest on March 29, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Page 32: Proteasomal inhibition by α-synuclein filaments and oligomers

32

26 Hendil, K.B. & Uerkvitz, W. (1991) J. Biochem. Biophys. Meth. 22, 155-165

27 Jensen, P.H., Højrup, P., Hager, H., Nielsen, M.S., Jakobsen, L., Olesen, O.F., Gliemann, J. &

Jakes, R. (1997) Biochem. J. 323, 539-546.

28 Jensen, P.H., Islam, K., Kenney, J., Nielsen, M.S., Power. J. & Gai, W.P. (2000). J. Biol. Chem.

275, 21500-21507.

29 Gai, W.P., Power J.H.T., Blumbergs, P.C. & Jensen, P.H. (1999) J. Neurochem. 73, 2093-2100.

30 Kristensen, P., Johnsen, A.H., Uerkvitz, W., Tanaka, K. & Hendil, K.B. (1995) Biochem Biophys

Res Commun 207, 1059

31 Hendil, K.B., Hartmann-Petersen, R. & Tanaka, K. (2002) J. Mol. Biol. 315, 627 – 636

32 Mirza, B., Hadberg, H., Thomsen, P. & Moos, T. (2000) Neuroscience 95, 425-432.

33 Leveugle, B., Faucheux, B.A., Bouras, C., Nillesse, N., Spik, G., Hirsch, E.C., Agid, Y. & Hof, P.R.

(1996) Acta Neuropathol. 91, 566-572.

34 Gai, W.P., Yuan, H.X., Li, X.Q., Power, J.T.H., Blumbergs, P.C. & Jensen, P.H. (2000) Exp.

Neurol. 166, 324-333.

35 Rattan, S. I. (1998) Biochem. Mol. Biol. Int. 45, 753-759.

36 Jensen, P.H., Nielsen, M.S., Jakes, R., Dotti, C.G. & Goedert, M. (1998) J. Biol. Chem. 273, 26292-

26294.

37 Fonager, J., Beedholm, R., Clark, B.F. & Rattan, S.I. (2002) Exp. Gerontol. 37, 1223-1228.

38 McKeith, I.G., Galasko, D., Kosaka, K., Perry, E.K., Dickson, D.W., Hansen, L.A., Salmon, D.P.,

Lowe, J., Mirra, S.S., Byrne, E.J., Lennox, G., Quinn, N.P., Edwardson, JA., Ince, P.G., Bergeron, C.,

Burns, A., Miller, B.L., Lovestone, S., Collerton, D., Jansen, E.N., Ballard, C., de Vos, R.A., Wilcock,

G.K., Jellinger, K.A. & Perry, R.H. (1996) Neurology 5, 1113-1124.

39 Spillantini, M.G., Schmidt, M.L., Lee, V.M., Trojanowski, J.Q., Jakes, R. and Goedert, M.. (1997)

Nature 388, 839-40.

by guest on March 29, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Page 33: Proteasomal inhibition by α-synuclein filaments and oligomers

33

40 Glickman, M.H., Rubin, D.M., Coux, O., Wefes, I., Pfeifer, G., Cjeka, Z., Baumeister, W.,,Fried,

V.A. & Finley D. (1998) Cell 94, 615-23

41 Gai, W.P., Pountney, D.L., Power, J.H., Li, Q.X., Culvenor, J.G., McLean, C.A., Jensen, P.H. and

Blumbergs, P.C. (2003) Exp. Neurol. 181, 68-78

42 Conway, K.A., Rochet, J.C., Bieganski, R.M. & Lansbury, P.T. Jr. (2001) Science 294, 1346-1349.

43 Sanchez, I., Mahlke, C. and Yuan, J. (2003) Nature 421, 373-9.

44 Giasson, B.I., Murray, I.V., Trojanowski, J.Q. & Lee, V.M. (2001) J. Biol. Chem. 276, 2380-2386.

45 Conway, K.A., Lee, S.J., Rochet, J.C., Ding, T.T., Williamson, R.E. & Lansbury, P.T. Jr. (2000)

Proc. Natl. Acad. Sci. 97, 571-576

46 Chan, H.Y., Warrick, J.M., Gray-Board, G.L. Paulson, H.L. & Bonini, N.M. (2000) Hum Mol Genet

9, 2811-2820.

47 Tatton, W.G., Chalmers-Redman, R.M., Elstner, M., Leesch, W., Jagodzinski, F.B., Stupak, D.P.,

Sugrue, M.M. & Tatton, N.A. (2000a) J. Neural. Transm. Suppl. 60, 77-100.

48 Kisselev, A.F. & Goldberg, A.L. (2001) Chemistry & Biology 8, 739-758

49 Tsubuki, S., Saito, Y. & Kawashima, S. (1994) FEBS Lett. 344, 229-33.

50 Conconi, M., Djavadi-Ohaniance, L., Uerkvitz, W., Hendil, K.B. & Friguet, B. (1999) Arch

Biochem Biophys. 362, 325-8.

51 Tanahashi, N., Murakami, Y., Minami, Y., Shimbara, N., Hendil, K.B. & Tanaka, K. (2000) J Biol

Chem. 275, 14336-45.

52 Lashuel, H.A., Petre, B.M., Wall, J., Simon, M., Nowak, R.J., Walz, T., Lansbury, P.T. Jr. (2002) J.

Mol. Biol. 322, 1089-102.

53 Volles, M.J., Lee, S.J., Rochet, J.C., Shtilerman, M.D., Ding, T.T., Kessler, J.C. & Lansbury, P.T.

Jr. (2001) Biochemistry 40, 7812-7819.

54 Tatton, N.A. (2000b) Exp. Neurol. 166, 29-43.

by guest on March 29, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Page 34: Proteasomal inhibition by α-synuclein filaments and oligomers

34

55 Hashimoto, M., Rockenstein, E., Mante, M., Mallory, M. & Masliah, E. (2001) Neuron 32, 213-223.

56 Bence, N.F., Sampat, R.M. & Kopito, R.R. (2001) Science 292, 552-555.

57 Lee, M.K., Stirling, W., Xu, Y., Xu, X., Qui, D., Mandir, A.S., Dawson, T.M., Copeland, N.G.,

Jenkins, N.A. & Price, D.L. (2002) Proc. Natl. Acad. Sci. U S A 99, 8968-8973.

58 Davies, S.W., Turmaine, M., Cozens, B,A., DiFiglia, M., Sharp, AH., Ross, CA., Scherzinger, E.,

Wanker, E.E., Mangiarini, L. & Bates, G.P. (1997) Cell 90, 537-548.

by guest on March 29, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Page 35: Proteasomal inhibition by α-synuclein filaments and oligomers

35

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

by guest on March 29, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Page 36: Proteasomal inhibition by α-synuclein filaments and oligomers

36

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

by guest on March 29, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Page 37: Proteasomal inhibition by α-synuclein filaments and oligomers

37

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).

by guest on March 29, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Page 38: Proteasomal inhibition by α-synuclein filaments and oligomers

38

(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

by guest on March 29, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Page 39: Proteasomal inhibition by α-synuclein filaments and oligomers

39

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.

by guest on March 29, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Page 40: Proteasomal inhibition by α-synuclein filaments and oligomers

40

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.

by guest on March 29, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Page 41: Proteasomal inhibition by α-synuclein filaments and oligomers

41

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 <

by guest on March 29, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Page 42: Proteasomal inhibition by α-synuclein filaments and oligomers

42

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.

by guest on March 29, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Page 43: Proteasomal inhibition by α-synuclein filaments and oligomers

Fig. 1

Ub aSyn 20S Ctl

by guest on March 29, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Page 44: Proteasomal inhibition by α-synuclein filaments and oligomers

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

by guest on March 29, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Page 45: Proteasomal inhibition by α-synuclein filaments and oligomers

A

C

B

Fig. 3

CAL

by guest on March 29, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Page 46: Proteasomal inhibition by α-synuclein filaments and oligomers

Fig. 4

A

B

40

by guest on March 29, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Page 47: Proteasomal inhibition by α-synuclein filaments and oligomers

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

by guest on March 29, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Page 48: Proteasomal inhibition by α-synuclein filaments and oligomers

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

by guest on March 29, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Page 49: Proteasomal inhibition by α-synuclein filaments and oligomers

Fig. 7

A

B

48483333

2525

1616

GAPDH

α-synuclein

HSP70

1 2 4 53

Co

ntro

l

Fila

me

nts

He

at sh

oc

kFi

lam

ents

0.00

1.25

1.00

0.75

0.50

0.25

CL-

ac

tivity

HSP70

Actin

C HSD

C

DC

L-a

ct. (C

ontro

l-c

om

pe

tito

rs)

0.25

0.50

0.75

1.00

0.00

Competitor:

Cont

rol

Filam

ent

Filam

ent +

HSP

70

HSP70

Filam

ent +

GAP

DH

GAP

DH

Re

lativ

e C

L-a

ctiv

ity

Non

e by guest on March 29, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Page 50: Proteasomal inhibition by α-synuclein filaments and oligomers

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:

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

by guest on March 29, 2018

http://ww

w.jbc.org/

Dow

nloaded from