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.. We characteri
Trang 1interferes with a-synuclein assembly
Medeva Ghee1, Ronald Melki2, Nadine Michot3and Jacques Mallet1
1 Laboratoire de Ge´ne´tique Mole´culaire de la Neurotransmission et des Processus Neurode´ge´ne´ratifs, Centre National de la Recherche Scientifique, Hoˆpital de la Pitie´ Salpeˆtrie`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, Baˆtiment CERVI, Hoˆpital de la
Pitie´ Salpeˆtrie`re, 83, boulevard de l’Hoˆpital,
75013 Paris, France
Fax: +33 1 42 17 75 33
Tel: + 33 1 42 17 75 42
Email: mghee@infobiogen.fr
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: melki@lebs.cnrs-gif.fr
(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-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.
Trang 2(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 Glu46fi 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 37C 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
Trang 3C
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 l M , 140 l M , 70 l M 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 n M PA700
(1284 : 1) Error bars indicate the standard
deviation in triplicate samples Similar
results were obtained in independent
experiments.
Trang 4cence 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 37C 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) of WT a-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.
Trang 5bly 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
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).
Trang 6affected 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 D2O 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 αα-synuclein+PA700 A53T α-synuclein+PA700
Elongation phase
Time 5h
Steady State phase
Time18h
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).
Trang 7Addition 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 0.2
0.15
0.1
0 0.05
Wavenumber [cm-1]
Abs
Abs Abs Abs
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.
Trang 8the 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-synua-synu-clein 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
Trang 9b-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 A600of 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 0C by addition of ammonium sulfate to a final concen-tration of 30% Following centrifugation, the ammonium sulfate concentration was adjusted to 50% at 0C 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
NH4HCO3 Eluates containing a-synuclein were incubated with 1 m (NH4)2SO4at 4C, loaded onto a butyl-sepharose column in Buffer A (50 mm K2HPO4, KH2PO4,pH 7.1) and eluted in Buffer B (100 mm Buffer A; 2 m (NH4)2SO4, 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
Trang 10reaction 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,
5 mm 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 37C 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 37C 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 8C in buffer H The column
was eluted at a flow rate of 0.5 mLÆmin)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 37C and centrifuged for 20 min at
160 000 g, 30C 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 D2O 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 D2O 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
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