Báo cáo khoa học: PA700, the regulatory complex of the 26S proteasome, interferes with a-synuclein assembly pptx

PA700, the regulatory complex of the 26S proteasome,
interferes with a-synuclein assembly
Medeva Ghee
1
, Ronald Melki
2
, Nadine Michot
3
and Jacques Mallet
1
1 Laboratoire de Ge
´
ne
´
tique Mole
´
culaire de la Neurotransmission et des Processus Neurode
´
ge
´
ne
´
ratifs, Centre National de la Recherche
Scientifique, Ho
ˆ
pital de la Pitie
´
Salpe
ˆ
trie
`

re, Paris, France
2 Laboratoire d’Enzymologie et Biochimie Structurales, Centre National de la Recherche Scientifique, France
3 Protein Production, Aventis Pharma, Vitry, France
The abnormal accumulation of insoluble cytoplasmic
protein aggregates is a factor common to neurodegene-
rative diseases, including Parkinson’s disease (PD). PD
is clinically characterized by resting tremor, brady-
kinesia and muscular rigidity. Neuropathologically, it
is defined by Lewy bodies (LBs), neuronal protein-
aceous cytoplasmic inclusions, which accompany a
selective degeneration of the dopaminergic neurons of
the substantia nigra [1]. Although the majority of PD
cases are sporadic, rare familial forms of PD have been
reported with the identification of mutations in the
genes encoding parkin, ubiquitin C-terminal hydrolase
L1, DJ-1, a-synuclein and most recently PINK-1 that
result in impairment of the ubiquitin–proteasome sys-
tem, mitochondrial impairment, oxidative stress and
protein misfolding [2]. Whereas altered expression of
these proteins contribute to the pathogenesis of PD, a
recent report indicates that overexpression of wild-type
Keywords
a-synuclein; aggregation; PA700;
proteasome; tat binding protein
Correspondence
M. Ghee, Laboratoire de Ge
´
ne
´
tique

Mole
´
culaire de la Neurotransmission et des
Processus Neurode
´
ge
´
ne
´
ratifs, Centre
National de la Recherche Scientifique,
UMR7091, Ba
ˆ
timent CERVI, Ho
ˆ
pital de la
Pitie
´
Salpe
ˆ
trie
`
re, 83, boulevard de l’Ho
ˆ
pital,
75013 Paris, France
Fax: +33 1 42 17 75 33
Tel: + 33 1 42 17 75 42
Email:
R. Melki, Laboratoire d’Enzymologie et

Biochimie Structurales, Centre National de
la Recherche Scientifique, 91198 Gif-sur-
Yvette Cedex, France
Fax: +33 1 69 82 35 04
Tel: + 33 1 69 82 35 03
E-mail:
(Received 28 February 2005, revised
20 April 2005 accepted 16 May 2005)
doi:10.1111/j.1742-4658.2005.04776.x
Parkinson’s disease is characterized by the loss of dopaminergic neurons
in the nigrostriatal pathway accompanied by the presence of intracellular
cytoplasmic inclusions, termed Lewy bodies. Fibrillized a-synuclein forms
the major component of Lewy bodies. We reported a specific interaction
between rat a-synuclein and tat binding protein 1, a subunit of PA700, the
regulatory complex of the 26S proteasome. It has been demonstrated that
PA700 prevents the aggregation of misfolded, nonubiquinated substrates.
In this study, we examine the effect of PA700 on the aggregation of wild-
type and A53T mutant a-synuclein. PA700 inhibits both wild-type and
A53T a-synuclein fibril formation as measured by Thioflavin T fluores-
cence. Using size exclusion chromatography, we present evidence for a sta-
ble PA700–a-synuclein complex. Sedimentation analyses reveal that PA700
sequesters a-synuclein in an assembly incompetent form. Analysis of the
morphology of wild-type and A53T a-synuclein aggregates during the
course of fibrillization by electron microscopy demonstrate the formation
of amyloid-like fibrils. Secondary structure analyses of wild-type and A53T
a-synuclein assembled in the presence of PA700 revealed a decrease in the
overall amount of assembled a-synuclein with no significant change in pro-
tein conformation. Thus, PA700 acts on a-synuclein assembly and not on
the structure of fibrils. We hypothesize that PA700 sequesters a-synuclein
oligomeric species that are the precursors of the fibrillar form of the pro-

tein, thus preventing its assembly into fibrils.
Abbreviations
AAA, ATPase-associated-with-different-cellular-activities family; HIF1a, hypoxia-inducible factor 1 alpha; LB, Lewy body; PD, Parkinson
disease; TBP1, Tat binding protein 1; pVHL, Von Hippel–Landau; WT, wild type.
FEBS Journal 272 (2005) 4023–4033 ª 2005 FEBS 4023
(WT) a-synuclein resulting from a genomic triplication
of the region containing a-synuclein in an Iowan kind-
red is also responsible for PD pathology [3].
a-Synuclein is an abundant brain presynaptic pro-
tein consisting of 140 amino acid residues. It has been
established that the WT a-synuclein assembles in vitro
into elongated filaments [4]. Moreover, the two a-synu-
clein mutations associated with PD, Ala53Thr [5] and
Ala30Pro [6], further accelerate the aggregation process
[7–9]. A third a-synuclein variant was recently des-
cribed with the substitution Glu46 fi Lys [10]. Its
assembly properties are not yet fully documented. Bio-
physical studies analyzing the in vitro aggregation
behavior of a-synuclein suggests that fibril formation
occurs via a nucleation-dependent mechanism [11] with
the rate-limiting step being the transformation of the
protein from the monomer to a prefibrillar oligomer.
It has been suggested that the prefibrillar oligomer, or
protofibril, may be the toxic species of the protein.
Protofibrillar forms of a-synuclein may transiently per-
meabilize vesicular membranes, predisposing these cells
to undergo apoptosis [12]. Moreover, it has been
reported that a-synuclein forms adducts with dopam-
ine in vitro, stabilizing potential toxic a-synuclein pro-
tofibrils [13].

The finding that filamentous a-synuclein is the major
component of LBs suggests that protein aggregation
and ⁄ or dysfunction of the ubiquitin ⁄ proteasomal sys-
tem play a role in the development of familial PD.
Aberrant aggregation of proteins is one of many sig-
nals that activates the ubiquitin–proteasomal system
to process damaged and toxic proteins. Elimination of
proteins targeted for degradation is mediated by the
26S proteasome, a multisubunit, intracellular protease
[14]. It contains a proteolytic core complex, The 20S
proteasome, a cylinder-shaped particle formed by the
axial stacking of four rings of seven a and b subunits,
and one or two 19S regulatory complexes (PA700)
which associate with the termini of the 20S core.
The PA700 complex can be dissociated into two sub-
complexes called the lid and the base. The lid serves to
recognize ubiquinated target proteins. The base con-
sists of six ATPases that belong to the [15]. The func-
tions proposed for the ATPases include: (a) the
hydrolysis of ATP to promote the assembly of the 26S
proteasome from the PA700 complex and 20S protea-
some; (b) the opening of the channel leading to the
20S proteasome; and (c) the binding and unfolding of
substrate proteins before translocating them into the
20S central chamber for subsequent proteolysis [14].
This latter function is reminiscent of molecular chaper-
ones. Indeed, it has been reported that PA700 has
chaperone-like activity [16]. Moreover, PA700 has been
shown to recognize and interact with misfolded, non-
ubiquinated substrates and inhibit their aggregation

[17]. Nonubiquinated a-synuclein can be degraded by
proteasomes in a pathway which does not have an
absolute requirement for ubiquination [18]. We first
provided evidence that a-synuclein is a substrate of
PA700 via a direct interaction with Tat Binding Pro-
tein 1 (TBP1), a subunit of the base complex [19]. The
interaction between a-synuclein and TBP1 led us to
investigate whether PA700 was capable of inhibiting
a-synuclein aggregation.
In the present study, we analyze the effect of PA700
on WT and A53T a-synucleins assembly in vitro.We
demonstrate that PA700 prevents fibril formation of
both WT and A53T a-synuclein. We characterize the
a-synuclein oligomeric species that form in the absence
and the presence of PA700 and show that PA700
sequesters a-synuclein in an assembly incompetent
form. These findings suggest a mechanism by which a
component of the 26S proteasome may contribute to
the processing and eventual degradation of misfolded
proteins.
Results
The effects of PA700 on WT and A53T mutant
a-synuclein assembly into fibrils
To determine whether PA700 affects the fibrillation
properties of both WT and A53T, a-synucleins, we
designed an in vitro assembly assay in which purified
recombinant a-synuclein was incubated in the presence
or absence of PA700 for 24 h at 37 °C under continu-
ous shaking. The kinetics of fibril formation was moni-
tored by the use of Thioflavin T fluorescence. As

shown in Fig. 1A and C, respectively, both WT and
A53T a-synucleins assemble into fibrils in a concentra-
tion-dependent manner. The aggregation kinetics is
triphasic, with an inital lag phase, followed by an
exponential growth phase and ending with a steady
state phase [4]. Figure 1A and C illustrate that a
decrease in protein concentration is accompanied by
an increase in the lag phase, a decrease in the slope of
the exponential growth phase and of fluorescence
intensity at steady state, which reflects a decrease in
fibril formation.
The effect of PA700 on the kinetics of a-synuclein
fibrillation was analyzed. Addition of increasing con-
centrations of PA700 to either WT or A53T a-synu-
clein decreased Thioflavin T fluorescence intensity at
steady state (Figs 1B and D, respectively). At the high-
est PA700 concentration used (218 nm) we observed an
approximate twofold decrease in Thioflavin T fluores-
PA700 interacts with a-synuclein oligomers M. Ghee et al.
4024 FEBS Journal 272 (2005) 4023–4033 ª 2005 FEBS
B
C
D
A
Fig. 1. PA700 inhibits a-synuclein fibril for-
mation. The assembly of WT and A53T
a-synucleins were monitored by Thioflavin T
binding. WT (A) and A53T (C) a-synucleins
at 280 l
M, 210 lM,140lM,70lM and

35 l
M were assembled at 37 °C. Kinetics of
fibril formation of WT (B) and A53T (D)
a-synucleins in the absence or presence of
109 n
M PA700 (2570 : 1), 218 nM PA700
(1284 : 1). Error bars indicate the standard
deviation in triplicate samples. Similar
results were obtained in independent
experiments.
M. Ghee et al. PA700 interacts with a-synuclein oligomers
FEBS Journal 272 (2005) 4023–4033 ª 2005 FEBS 4025
cence intensity at steady state for the WT and a three-
fold decrease for the A53T mutant. The fluorescence
intensities at steady states of the assembly reaction of
WT and A53T a-synucleins (280 lm) in the presence
of 218 nm PA700 (i.e. a 1284 : 1 ratio) is equivalent to
that of (140 lm) WT and A53T a-synuclein. The latter
results strongly suggest that PA700 inhibits a-synuclein
assembly by binding and sequestering the a-synuclein
species that are the precursors of the fibrillar form of
the proteins.
Evidences for a soluble, assembly incompetent
a-synuclein-PA700 complex
To determine whether PA700 forms a stable complex
with a-synuclein, samples containing WT and A53T
a-synucleins (280 lm) in the absence or the presence of
PA700 (218 nm) were incubated at 37 °C with orbital
shaking for 1 h and analyzed by size exclusion chroma-
tography as described in the Experimental procedures.

Figure 2A, showing data obtained for WT a-synuclein,
illustrates our findings. In the absence of PA700, WT
a-synuclein emerges from the column in a single peak
centered at 300 kDa. In the presence of PA700, WT
a-synuclein emerges from the column with a molecular
mass of 670 kDa, indicating a colocalization with
PA700. Identical results were obtained using the A53T
variant (data not shown). This finding strongly suggests
that WT and A53T a-synucleins interact with PA700
within stable soluble high molecular mass complexes.
The interaction of PA700 with WT and A53T a-synu-
cleins was further documented using sedimentation
analysis. Aliquots were withdrawn during the lag (time
1 h) and steady state (time 18 h) phases from assembly
reactions and subjected to ultracentrifugation. Both
WT a-synuclein and PA700 are found in the super-
natant of the sample corresponding to the lag phase
(Fig. 2B). Following assembly and in the absence of
PA700, WT a-synuclein is found in the pellet, whereas
over 80% of WT a-synuclein is in the supernatant
together with PA700 in samples where assembly was
carried out in the presence of PA700 (Fig. 2B). Identical
results were obtained for A53T a-synuclein (data not
shown). This clearly indicates that PA700 sequesters
a-synuclein in a soluble, assembly incompetent state.
Characterization of WT and A53T a-synuclein
oligomeric species in the absence and the
presence of PA700 by transmission electron
microscopy
WT and A53T a-synuclein oligomeric species that are

generated in the absence or presence of PA700 were
further characterized by electron microscopy. Aliquots
of WT and A53T a-synuclein (280 lm) assembly reac-
tions in the presence or absence of (218 nm) PA700
were withdrawn at time intervals, diluted in the assem-
A
B
Fig. 2. PA700 forms stable assembly incompetent complexes with
WT a-synuclein. (A) Size exclusion chromatography elution profiles
of WT a-synuclein in the absence (n) and the presence (m)of
PA700 are shown. PA700 is detected using its intrinsic fluores-
cence (d). The immunoreactivity (n,m)ofWTa-synuclein was
monitored by dot-blot as this polypeptide lacks tryptophan residues.
Arrows show the location of molecular size markers (thyroglobulin,
670 kDa; immunoglobulin G, 158 kDa; ovalbumin, 44 kDa and myo-
globin, 17 kDa) run under identical conditions on the same column.
(B) Sedimentation behavior of WT a-synuclein in the absence and
the presence of PA700. Aliquots of WT a-synuclein assembly reac-
tions in the absence (–) or the presence (+) of PA700, 1 h and 18 h
after the onset of the assembly reaction (lag and steady state
phases, respectively) were centrifuged (160 000 g) for 20 min. The
protein content of the supernatant (S) and pellet (P) fractions was
analyzed by SDS ⁄ PAGE (12% polyacrylamide gels). The molecular
mass markers (Mw) are shown. Identical results were obtained for
A53T a-synuclein.
PA700 interacts with a-synuclein oligomers M. Ghee et al.
4026 FEBS Journal 272 (2005) 4023–4033 ª 2005 FEBS
bly buffer and processed for electron microscopy analy-
sis. In the lag phase preceeding assembly (time 1 h),
globular and short curved oligomers are the unique

constituents of both WT and A53T a-synuclein solu-
tions (Fig. 3A and B). In the elongation phase (time
5 h), semiflexible, unbranched fibrils are observed. They
coexist with the globular and the short curved
oligomers observed at the earlier stages of assembly
(Fig. 3C and D). At late stages in WT and A53T
a-synuclein assembly reactions (time 18 h), long helical
fibrils are observed with very few oligomers remaining
in the solution (Fig. 3E and F).
We next characterized WT and A53T a-synuclein
oligomeric species in the presence of PA700. The oligo-
mers that form in the lag phase upon addition of
PA700 to WT and A53T a-synuclein are indistinguish-
able from that observed in the absence of PA700 (data
not shown). WT a-synuclein oligomeric species (glo-
bular, curved oligomers and fibrils) that form in the
absence (Fig. 3C and E) or the presence (Fig. 4A
and C) of PA700 are indistinguishable. In contrast, a
change in the morphology of A53T a-synuclein oligo-
meric species that form upon assembly is observed
upon addition of PA700. Compare Fig. 3D and F to
Fig. 4B and D. Indeed, very few fibrils are present in
the solution and the vast majority of the high mole-
cular mass oligomers that form are globular and short
curved oligomers (Fig. 4B). In addition, the few fibrils
observed after examining multiple fields on the elec-
tron microscopy grids appear less structured (Fig. 4D)
than those obtained in the absence of PA700 (Fig. 3F).
This result further suggests, particularly in the case of
the A53T variant, that PA700 sequesters a-synuclein

oligomers that are the precursors of the fibrils.
Secondary structure and quantitative analysis
of WT and A53T a-synuclein oligomers in the
absence and presence of PA700 by FTIR
Spectroscopy
To assess whether the characteristic polypeptide chain
arrangement of WT and A53T a-synuclein fibrils is
WT α
α
-synuclein A53T α-synuclein
A B
C
D
E F
Lag phase
Time 1h
Elongation phase
Time 5h
Steady state phase
Time 18h
Fig. 3. Electron micrographs of WT and
A53T a-synuclein. The oligomeric species of
WT and A53T a-synucleins (280 l
M) were
analyzed at the early stages of assembly,
i.e. the lag phase (A, B, respectively), during
the elongation phase (C, D, respectively)
and at steady state (E, F, respectively).
Representative fields on the grids are
depicted (scale bar, 0.5 lm).

M. Ghee et al. PA700 interacts with a-synuclein oligomers
FEBS Journal 272 (2005) 4023–4033 ª 2005 FEBS 4027
affected by PA700 and to quantify the amount of
fibrils formed in the absence and the presence of
PA700, the FTIR spectra of WT, A53T a-synuclein
(280 lm) assembled in the absence or presence of
218 nm PA700 in buffer A and subsequently exten-
sively dialyzed against D
2
O were recorded. The spectra
presented in Fig. 5 showed very similar amide I
regions dominated by an absorption maxima at
1624 cm
)1
, demonstrating the presence of aggregated
b-sheets.
Fourier deconvolution and curve fitting of the spec-
tra permitted the quantitative analysis of the secon-
dary structure content of the different samples. The
results are summarized in Table 1. We observed no
significant change in the secondary structure content
of the fibrils upon addition of PA700 to WT or
A53T a-synucleins. Interestingly, however, the absorp-
tion intensities of the samples assembled in the pres-
ence of PA700 are lower than those assembled in the
absence of PA700 at constant WT and A53T a-synu-
clein concentrations (compare the absorption intensi-
ties in Fig. 5A,C, and Fig. 5B,D, respectively). FTIR
spectra of PA700 alone reveals a secondary structure
composed primarily of a-helices (Fig. 1D, inset).

These data strongly suggest that PA700 does not
change the conformation of a-synuclein within the
fibrils but that it sequesters the precursors of the
fibrils, thus leading to a smaller amount of polymers
at steady state.
Discussion
The aggregation of either WT or mutant a-synuclein
proteins in dopaminergic neurons of the substantia
nigra pars compacta is thought to be responsible for
the subsequent neurodegeneration of these neurons in
PD. The process through which a-synuclein is trans-
formed from a disordered monomer into a stable amy-
loid fibril involves several aggregation states, including
the natively unfolded protein, which oligomerizes to
form a partially folded, b-sheet rich oligomeric inter-
mediate. This aggregation-competent oligomer, or pro-
tofibril, has been proposed to be an important precursor
that favors the formation of a-synuclein fibrils.
We show that PA700 interacts with a-synuclein,
thereby generating a PA700–a-synuclein species that is
unable to polymerize into fibrils. Four observations
support this finding. First, the ability of PA700 to inhi-
bit a-synuclein assembly into fibrils as witnessed by the
decrease of the overall amount of fibrillar a-synuclein
at steady state in the presence of increasing concen-
trations of PA700. Second, the existence of a high
molecular mass, stable PA700–a-synuclein complex as
demonstrated by size exclusion chromatography.
Third, the presence of increased amounts of soluble
a-synuclein in the presence of PA700 in sedimentation

experiments. Finally, the decrease in the amount of
fibrils in the presence of PA700 as measured by quanti-
tative FTIR spectroscopy.
WT α
α
-synuclein+PA700 A53T α-synuclein+PA700
Elongation phase
Time 5h
Steady State phase
Time18h
A B
C D
Fig. 4. Electron micrographs of WT and
A53T a-synuclein incubated in the presence
of PA700. Recombinant WT and A53T
a-synuclein (280 l
M) oligomers were imaged
during the elongation (time 5 h) and steady
state (time 18 h) phases (A, B and C, D,
respectively) in the presence of PA700
(218 n
M). Representative fields on the grids
are depicted (scale bar, 0.5 lm).
PA700 interacts with a-synuclein oligomers M. Ghee et al.
4028 FEBS Journal 272 (2005) 4023–4033 ª 2005 FEBS
Addition of increasing concentrations of PA700 to
either WT or A53T a-synuclein significantly decreases
Thioflavin T fluorescence intensity at steady state, sug-
gesting that PA700 inhibits a-synuclein assembly by
binding and sequestering a-synuclein oligomeric species

that are the precursors of the fibrillar form of the pro-
teins. Strikingly, however, the lag phases of both WT
and A53T a-synucleins and the elongation rates remain
constant. One expects a dramatic increase of the lag
phase and a significant decrease of the elongation rate
if PA700 only sequesters assembly competent a-synu-
cleins leading to the decrease of the latter concentra-
tion as shown in Fig. 1A,C. Thus, PA700 appears to
have a complex effect on the assembly reaction. It
sequesters a-synuclein oligomers in an assembly incom-
petent form and at the same time favors the formation
of oligomeric species that act as nuclei in fibrils assem-
bly. This complex observation can be accounted for by
0.5
A
B
C
D
0.6
0.4
0.2
0.21
0.1
0
0
0.4
0.3
0.2
0.007
0.004

0.002
0
1700 1680 1660 1640 1620 1600
Wavenumber [cm-1]
1700 1680 1660 1640 1620 1600
Wavenumber [cm-1]
Wavenumber [cm-1]
Abs
0.009
0.006
0.004
0.004
0.003
0.002
0.001
0
1689.34 1660 1640 1610.27
0.002
0
Abs
Abs
0.1
0
1700 1680 1660 1640 1620 1600
1700 1680 1660 1640 1620 1600
1700
0.2
0.15
0.1
0

0.05
1680 1660 1640 1620 1600
1700 1680 1660
Wavenumber [cm-1]
Abs
Abs
Abs
Abs
1640 1620 1600
Fig. 5. Secondary structure and quantitative
analysis of WT and A53T a-synuclein oligo-
mers in the absence and presence of
PA700. FTIR spectra of fibrillar WT and
A53T a-synucleins (280 l
M) in the absence
(A, B, respectively) or the presence of
PA700 (218 n
M) (C, D, respectively). The
FTIR spectra of the soluble forms of WT,
A53T and PA700 are shown as insets in A,
B and D, respectively. Curve fit spectra are
presented in each case as dotted lines.
Absorption intensities (Abs) and wave-
numbers are indicated.
M. Ghee et al. PA700 interacts with a-synuclein oligomers
FEBS Journal 272 (2005) 4023–4033 ª 2005 FEBS 4029
the following. It is reasonable to envisage that the
affinity of PA700 for a-synuclein oligomers is not
infinite. A proportion of a-synuclein oligomers is
therefore released in solution where they can assemble

into fibrils. Thus, our experimental observations may
result from the PA700 sequestering activity, on the one
hand, and of the PA700 mediated a-synuclein oligo-
merization activity, on the other. This is shown sche-
matically in Fig. 6. Alternatively, our experimental
observations could be accounted for by PA700 chaper-
one activity [16,17]. Indeed, following the interaction
of native unfolded, assembly incompetent, a-synuclein
with PA700, partially folded, assembly competent,
a-synuclein may be produced. This favors nucleation
and assembly. PA700 could therefore bind a subset of
a-synuclein oligomers in an assembly incompetent,
native state, thus keeping off assembly track these
oligomers and reducing the amount of fibrils formed at
steady state. A recent report by Dedmon and col-
leagues demonstrate that the molecular chaperone,
Hsp70, preferentially binds to cytotoxic prefibrillar
a-synuclein species and consequently inhibits its fibril
formation [20].
The a-synuclein species that binds to PA700 is a
high molecular mass species. Indeed, the assembly
reactions of WT and A53T a-synuclein (280 lm) in the
presence of PA700 at a ratio of 1 : 1284 are super-
imposable to that of a-synucleins in the absence of
PA700 at 140 lm. If PA700 binds monomeric a-synu-
clein, the assembly kinetics in the absence or the pres-
ence of PA700 would be superimposable as the
concentration of free monomeric a-synuclein in the
presence of PA700 would be at least 279.5 lm. Our
data clearly indicate that 140 lm a-synuclein are

sequestered by 218 nm PA700, suggesting that the
PA700 particle binds an a-synuclein oligomeric form.
This species is unable to assemble into fibrils as wit-
nessed by the increased amount of high molecular
mass PA700–a-synucleinin the supernatants of assem-
bly reactions containing WT or A53T a-synuclein and
PA700 and by the lower amounts of fibrils formed at
steady state as measured by FTIR spectroscopy.
The affinity of PA700 for a -synucleins is not infinite
as the complex dissociates on sizing columns (Fig. 2A).
Furthermore, the PA700–a-synuclein complex neither
binds Thioflavin T, nor has an increased b-sheet con-
tent as measured by FTIR. Indeed, in our kinetic
measurements, the formation of soluble high molecular
mass a-synuclein species that has an apparent mole-
cular mass of 300 kDa is not accompanied by an
increased Thioflavin T binding. Similarly, the PA700–
a-synuclein complex that forms in the lag phase and in
the presence of PA700 does not influence Thioflavin T
fluorescence. The FTIR measurements demonstrate
that the amount of fibrillar a-synuclein (280 lm) (e.g.
PA700
Intermediate
oligomer
Fibrils
A
B
Fig. 6. PA700 promotes a-synuclein oligomerization and sequesters
soluble oligomeric species in an assembly incompetent form. In the
absence of PA700 (A), monomeric a-synuclein oligomerizes prob-

ably in an isodesmic manner. These soluble oligomers are the pre-
cursors of the fibrillar form of the protein. PA700 interacts with a
subset of soluble a-synuclein oligomers (B). This interaction shifts
the equilibria between monomeric and ⁄ or low molecular mass
a-synuclein oligomers, a subset of which are the precursors of the
fibrils toward the formation of a PA700–a-synuclein species. This
prevents a-synuclein fibril formation. However, binding of a-synu-
clein to PA700 favors the oligomerization of a-synuclein. As the
affinity of PA700 for a-synuclein oligomers is not infinite, the oligo-
mers are released in solution where they can elongate. Thus,
PA700 facilitates the rate-limiting nucleation phase and at the same
time limits assembly by sequestering a proportion of soluble
a-synuclein oligomers.
Table 1. Secondary structure content of WT and A53T a-synuclein
oligomers in the absence and presence of PA700 estimated from
the deconvolution of the FTIR spectroscopy measurements presen-
ted in Fig. 5.
b-Sheet
(%)
Disordered
(%)
Loops
(%)
Structural Assignment 1624 cm
)1
1647 cm
)1
1663 cm
)1
WT a-synuclein 65 19 11

WT a-synuclein + PA700 61 24 8.5
A53T a-synuclein 61 23 8
A53T a-synuclein + PA700 58 29 8
PA700 interacts with a-synuclein oligomers M. Ghee et al.
4030 FEBS Journal 272 (2005) 4023–4033 ª 2005 FEBS
b-sheet rich form) in the presence of PA700 (218 nm)
is equivalent to half of that in the absence of PA700,
consistent with the sequestering of 140 lm a-synuclein
in a form lacking b-sheets. The latter may be assembly
incompetent because of its secondary structure. The
PA700-mediated inhibition of a-synuclein assembly
may be the consequence of a physical interaction
between a-synuclein oligomers and PA700, i.e. a basic
sequestering effect.
The ability of PA700 to inhibit a-synuclein fibril for-
mation in vitro may shed more light on how a-synuc-
lein is degraded in vivo. It has been previously reported
that a-synuclein is degraded by the 26S proteasome
[21], a process which does not seem to require ubiquiti-
nation [18] and via autophagy [22]. We first observed
an interaction between a-synuclein and TBP1, an
ATPase residing within the PA700 base subcomplex
[19]. TBP1 possesses a coiled-coil domain, a mitoch-
ondrial energy transfer domain and an ATPase domain
that is highly conserved among all members of the
AAA family [15]. Consistent with these functions,
TBP1 would participate in the unfolding of a-synuclein
and its translocation into the 20S proteolytic core for
rapid hydrolysis. Additional evidence supporting this
hypothesis demonstrates that TBP1 contributes to the

E3 ubiquitin ligase activity of the von Hippel–Lindau
(pVHL) protein in order to promote the degradation
of the hypoxia-inducible factor 1 alpha (Hif1a) for
oxygen-dependent proteolysis [23]. The authors sugges-
ted that TBP1 may function as a chaperone, tethering
pVHL–Hif1a complexes to the proteasome. To date,
no direct evidence of PA700 functioning in the absence
of the 20S proteasome in the cell has been reported.
Therefore, the chaperone-like properties of the PA700
complex may play an important role in the degrada-
tion of misfolded proteins.
The question as to how a-synuclein escapes the
ubiquitin–proteasome system has yet to be elucidated.
It has been reported that a-synuclein is capable of
inhibiting the 26S proteasome via its interaction with
the TBP1 subunit [24]. Alternatively, PA700 complexes
could be sequestered in LB resulting in a depletion of
PA700 in the cells. Our data show unequivocally that
PA700 binds a-synuclein, in particular its oligomeric
forms. A significant decrease in PA700 concentration
could therefore be responsible for the malfunction of
the 26S proteasome in a-synuclein degradation. In vivo
ubiquination of the proteasomal clients may affect
their binding and degradation properties. Thus, thera-
peutic approaches to synucleinopathies having as a tar-
get PA700 or the proteasome should not only integrate
our findings but also the behavior of ubiquitinated
a-synuclein.
Experimental procedures
Materials

PA700 was purified from bovine red blood cells as des-
cribed [25,26]. Thioflavin T was obtained from ICN Bio-
chemicals (Aurora, OH).
Expression and purification of recombinant
a-synuclein proteins
The bacterial expression construct pRK172 encoding WT
human a-synuclein was a gift from R. Jakes and M. Goed-
ert (MRC Cambridge, UK). The QuikChange site-directed
mutagenesis protocol (Stratagene Europe, Amsterdam, The
Netherlands) was used to generate the mutant construct,
pRK172 a-synuclein A53T. Mutagenesis was confirmed by
DNA sequencing.
The expression constructs were transformed into the BL21
(DE3) Escherichia coli strain, grown to an A
600
of 0.6–0.8,
induced with 0.44 mm isopropyl-1-thio-b-d-galactopyrano-
side and harvested 2 h later. The pellet was resuspended in
10 mm Tris, pH 8, 1 mm EDTA, 1 mm phenylmethanesulfo-
nyl fluoride, and lysed by freezing in liquid nitrogen followed
by thawing and probe sonication. Cell lysate was precipitated
at 0 °C by addition of ammonium sulfate to a final concen-
tration of 30%. Following centrifugation, the ammonium
sulfate concentration was adjusted to 50% at 0 °C and the
solution centrifuged at 5000 g. The resulting pellet was resus-
pended in 10 mm Tris pH 7.5 and the solution loaded onto a
DEAE column eluted by a gradient of 0–500 mm NaCl. The
fractions containing a-synuclein, eluted at 200 mm NaCl,
were concentrated in an Ultrafree-15, 5K MWCO filter (Mil-
lipore Corp., Bedford, MA, USA), loaded onto a Superdex

75 HiLoad 26 ⁄ 60 column (Amersham Biosciences Europe
GmbH, Orsay, France), equilibrated and eluted in 100 mm
NH
4
HCO
3
. Eluates containing a-synuclein were incubated
with 1 m (NH
4
)
2
SO
4
at 4 °C, loaded onto a butyl-sepharose
column in Buffer A (50 mm K
2
HPO
4
,KH
2
PO
4,
pH 7.1) and
eluted in Buffer B (100 mm Buffer A; 2 m (NH
4
)
2
SO
4
, pH 7).

Purification of monomeric a-synuclein was confirmed by
SDS ⁄ PAGE.
Purified WT and A53T a-synuclein samples were concen-
trated using the Ultrafree-15, 5-K MWCO filter. Proteins
were filtered through Microcon 100-kDa cutoff filters to
remove any oligomeric material that could have formed
during the concentration. Protein concentration was deter-
mined using the bicinchoninic acid protein assay (Pierce,
Rockford, IL) and BSA as a standard.
Aggregation assays
Fibril formation of WT and A53T mutant a-synuclein
recombinant proteins was performed using a GENIOS multi-
detection microplate reader (TECAN). The aggregation
M. Ghee et al. PA700 interacts with a-synuclein oligomers
FEBS Journal 272 (2005) 4023–4033 ª 2005 FEBS 4031
reaction mixture consists of 280 lm of either WT or A53T
recombinant a-synuclein proteins, 10 lm thioflavin T in buf-
fer H (20 mm Tris ⁄ HCl pH 7.5; 20 mm NaCl; 1 mm EDTA,
5mm 2-mercaptoethanol) and increasing concentrations of
PA700 as indicated. A total volume of 100 lL was aliquotted
per well of a 96-well plate containing a Teflon sphere in each
well. The samples were incubated at 37 °C with orbital sha-
king. A total of 97 fluorescence measurements were taken at
15-min intervals resulting in a 24-h incubation with excita-
tion at 450 nm and emission at 485 nm. Each experiment
was performed in triplicate. Measurements were corrected by
subtracting the background fluorescence.
Size exclusion chromatography and
sedimentation analysis
WT and A53T mutant a-synuclein (280 lm) were incubated

at 37 °C with orbital shaking for 1 h in the absence and the
presence of PA700 (218 nm). The different samples were loa-
ded on a Superose 6 HR10-30 gel filtration column (Amer-
sham) equilibrated and run at 8 °C in buffer H. The column
was eluted at a flow rate of 0.5 mLÆ min
)1
. The presence of
PA700 and a-synuclein in the fractions (0.5 mL) emerging
from the column was monitored using PA700 intrinsic fluor-
escence (excitation, 280 nm, emission 340 nm) and a-synuc-
lein immunoreactivity using a dot-blot assay, respectively.
The column was calibrated with the molecular size markers
(thyroglobulin, 670 kDa; immunoglobulin G, 158 kDa; ov-
albumin, 44 kDa; myoglobin, 17 kDa and vitamin B-12,
1.35 kDa, Bio-Rad Laboratories, Inc., Hercules, CA, USA).
Sedimentation analysis was carried out using a Beckman
TL100 ultracentrifuge. Aliquots (100 lL) of WT and A53T
a-synuclein (280 lm) in the absence or the presence of
PA700 (218 nm) were removed at time 1 h (lag phase) and
18 h (steady state) from the reaction mixture incubated
with orbital shaking at 37 °C and centrifuged for 20 min at
160 000 g,30°C. The resulting supernatants and pellets
were analyzed by SDS ⁄ PAGE [27].
Transmission electron microscopic analysis of
a-synuclein filaments assembled in vitro from
recombinant proteins
Aliquots of soluble (280 lm) and assembled WT and A53T
a-synuclein in the presence or absence of 218 nm PA700
were deposited on carbon-coated copper grids (200 mesh).
The grids were then negatively stained with 1% uranyl acet-

ate, and examined in a Philips EM 410 transmission elec-
tron microscope.
FTIR spectroscopy
WT and A53T a-synuclein were assembled into fibrils in
the presence or the absence of PA700 in buffer H. Soluble
and assembled WT and A53T a-synuclein and PA700 were
extensively dialyzed against D
2
O. The spectra of the soluble
and fibrillized forms of the aforementioned samples were
recorded on a JASCO 660 Plus FTIR spectrometer
equipped with an MCT detector using attenuated total
reflectance mode. The background consisted of D
2
O. A
total of 1024 interferograms were collected with a resolu-
tion of 2 cm
)1
. Second derivatives were calculated from
smoothed primary spectra. The data were fitted using a
Gaussian species model centered at 1624, 1647, 1655, 1663,
1677 and 1692 cm
)1
[28].
Acknowledgements
We thank G. DeMartino for the generous gift of
PA700 and P. Thomas and C. Liu for their helpful dis-
cussions and critical review of the manuscript. We
thank R. Jakes and M. Goedert for the pRK172 wild-
type a-synuclein plasmid. This work was supported

by the Fondation de France, Centre National de la
Recherche Scientifique, the Association Francaise con-
tre les Myopathies, Universite
´
Pierre et Marie Curie
Paris VI and Aventis Pharma.
References
1 Forno LS (1996) Neuropathology of Parkinson’s dis-
ease. J Neuropathol Exp Neurol 55, 259–272.
2 Greenamyre JT & Hastings TG (2004) Biomedicine.
Parkinson’s – divergent causes, convergent mechanisms.
Science 304, 1120–1122.
3 Singleton AB, Farrer M, Johnson J, Singleton A, Hague
S, Kachergus J, Hulihan M, Peuralinna T, Dutra A,
Nussbaum R, et al. (2003) alpha-Synuclein locus tripli-
cation causes Parkinson’s disease. Science 302, 841.
4 Uversky VN, Li J & Fink AL (2001) Evidence for a
partially folded intermediate in alpha-synuclein fibril
formation. J Biol Chem 276, 10737–10744.
5 Polymeropoulos MH, Lavedan C, Leroy E, Ide SE,
Dehejia A, Dutra A, Pike B, Root H, Rubenstein J,
Boyer R, et al. (1997) Mutation in the alpha-synuclein
gene identified in families with Parkinson’s disease.
Science 276, 2045–2047.
6 Kruger R, Kuhn W, Muller T, Woitalla D, Graeber M,
Kosel S, Przuntek H, Epplen JT, Schols L & Riess O
(1998) Ala30Pro mutation in the gene encoding alpha-
synuclein in Parkinson’s disease. Nat Genet 18, 106–108.
7 Giasson BI, Uryu K, Trojanowski JQ & Lee VM (1999)
Mutant and wild type human alpha-synucleins assemble

into elongated filaments with distinct morphologies
in vitro. J Biol Chem 274, 7619–7622.
8 Narhi L, Wood SJ, Steavenson S, Jiang Y, Wu GM,
Anafi D, Kaufman SA, Martin F, Sitney K, Denis P,
PA700 interacts with a-synuclein oligomers M. Ghee et al.
4032 FEBS Journal 272 (2005) 4023–4033 ª 2005 FEBS
et al. (1999) Both familial Parkinson’s disease mutations
accelerate alpha-synuclein aggregation. J Biol Chem 274,
9843–9846.
9 Conway KA, Harper JD & Lansbury PT (1998) Accel-
erated in vitro fibril formation by a mutant alpha-synu-
clein linked to early-onset Parkinson disease. Nat Med
4, 1318–1320.
10 Zarranz JJ, Alegre J, Gomez-Esteban JC, Lezcano E,
Ros R, Ampuero I, Vidal L, Hoenicka J, Rodriguez O,
Atares B, et al. (2004) The new mutation, E46K, of
alpha-synuclein causes Parkinson and Lewy body
dementia. Ann Neurol 55, 164–173.
11 Wood SJ, Wypych J, Steavenson S, Louis JC, Citron
M & Biere AL (1999) alpha-synuclein fibrillogenesis
is nucleation-dependent. Implications for the patho-
genesis of Parkinson’s disease. J Biol Chem 274,
19509–19512.
12 Volles MJ & Lansbury PT Jr (2003) Zeroing in on the
pathogenic form of alpha-synuclein and its mechanism
of neurotoxicity in Parkinson’s disease. Biochemistry 42,
7871–7878.
13 Conway KA, Rochet JC, Bieganski RM & Lansbury
PT Jr (2001) Kinetic stabilization of the alpha-synuclein
protofibril by a dopamine-alpha-synuclein adduct.

Science 294, 1346–1349.
14 Voges D, Zwickl P & Baumeister W (1999) The 26S
proteasome: a molecular machine designed for con-
trolled proteolysis. Annu Rev Biochem 68, 1015–1068.
15 Hoyle J, Tan KH & Fisher EM (1997) Localization of
genes encoding two human one-domain members of the
AAA family: PSMC5 (the thyroid hormone receptor-
interacting protein, TRIP1) and PSMC3 (the Tat-bind-
ing protein, TBP1). Hum Genet 99, 285–288.
16 Braun BC, Glickman M, Kraft R, Dahlmann B, Kloet-
zel PM, Finley D & Schmidt M (1999) The base of the
proteasome regulatory particle exhibits chaperone-like
activity. Nat Cell Biol 1, 221–226.
17 Strickland E, Hakala K, Thomas PJ & DeMartino GN
(2000) Recognition of misfolding proteins by PA700,
the regulatory subcomplex of the 26 S proteasome.
J Biol Chem 275, 5565–5572.
18 Tofaris GK, Layfield R & Spillantini MG (2001) alpha-
synuclein metabolism and aggregation is linked to ubi-
quitin-independent degradation by the proteasome.
FEBS Lett 509 , 22–26.
19 Ghee M, Fournier A & Mallet J (2000) Rat alpha-synu-
clein interacts with Tat binding protein 1, a component
of the 26S proteasomal complex. J Neurochem 75,
2221–2224.
20 Dedmon MM, Christodoulou J, Wilson MR & Dobson
CM (2005) Hsp70 inhibits a-synuclein fibril formation
via preferential binding to prefibrillar species. J Biol
Chem 280, 14733–14740.
21 Bennett MC, Bishop JF, Leng Y, Chock PB, Chase TN

& Mouradian MM (1999) Degradation of alpha-synu-
clein by proteasome. J Biol Chem 274, 33855–33858.
22 Webb JL, Ravikumar B, Atkins J, Skepper JN &
Rubinsztein DC (2003) Alpha-Synuclein is degraded by
both autophagy and the proteasome. J Biol Chem 278 ,
25009–25013.
23 Corn PG, McDonald ER, 3rd Herman JG & El-Deiry
WS (2003) Tat-binding protein-1, a component of the
26S proteasome, contributes to the E3 ubiquitin ligase
function of the von Hippel-Lindau protein. Nat Genet
35, 229–237.
24 Snyder H, Mensah K, Theisler C, Lee J, Matouschek A
& Wolozin B (2003) Aggregated and monomeric alpha-
synuclein bind to the S6¢ proteasomal protein and inhi-
bit proteasomal function. J Biol Chem 278, 11753–
11759.
25 Ma CP, Slaughter CA & DeMartino GN (1992) Identi-
fication, purification, and characterization of a protein
activator (PA28) of the 20 S proteasome (macropain).
J Biol Chem 267, 10515–10523.
26 McGuire MJ, McCullough ML, Croall DE & DeMar-
tino GN (1989) The high molecular weight multicataly-
tic proteinase, macropain, exists in a latent form in
human erythrocytes. Biochim Biophys Acta 995,
181–186.
27 Laemmli U.K. (1970) Cleavage of structural proteins
during the assembly of the head of bacteriphage T4.
Nature 227, 680–685.
28 Seshadri S, Khurana R & Fink AL (1999) Fourier
transform infrared spectroscopy in analysis of protein

deposits. Methods Enzymol 309, 559–576.
M. Ghee et al. PA700 interacts with a-synuclein oligomers
FEBS Journal 272 (2005) 4023–4033 ª 2005 FEBS 4033