From the Center for Neurodegenerative Disease Research, Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104
Received for publication, September 29, 2000, and in revised form, October 27, 2000
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ABSTRACT |
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Neuronal and oligodendrocytic aggregates of
fibrillar Intriguingly, although Expression and Purification of Western Blotting--
Proteins were resolved on slab gels by
SDS-PAGE (25) and electrophoretically transferred onto nitrocellulose
membranes (Schleicher & Schuell, Keene, NH) in buffer containing 48 mM Tris, 39 mM glycine, and 10% methanol.
Membranes were blocked with a 1% solution of powdered skimmed milk
dissolved in Tris-buffered saline-Tween (50 mM Tris, pH
7.6, 150 mM NaCl, 0.1% Tween 20), incubated with antibodies to Filament Assembly and Centrifugal Sedimentation--
Native and
hybrid synuclein proteins were assembled into filaments by incubation
at 37 °C in 100 mM sodium acetate, pH 7.0, with
continuous shaking. Samples were centrifuged at 100,000 × g for 20 min, and SDS sample buffer (10 mM Tris,
pH 6.8, 1 mM EDTA, 40 mM dithiothreitol, 1%
SDS, 10% sucrose) was added to pellets and supernatants, which were
heated to 100 °C for 15 min. Synuclein proteins were resolved by
SDS-PAGE, stained with Coomassie Blue R-250, and quantified by densitometry.
Negative Staining Electron Microscopy--
Assembled synuclein
filaments were absorbed to 300-mesh carbon-coated copper grids, stained
with 1% uranyl acetate, and visualized with a Joel (Peabody, MA) 1010 transmission electron microscope. Images were captured with a Hamamatsu
(Bridgewater, MA) digital camera using AMT (Danvers, MA) software. For
diameter determination, the width of 100-120 filaments was measured
using Image-Pro Plus software (Media Cybernetics, Del Mar, CA).
Protease Protection Analysis--
Sixty µg of synuclein
proteins in 30 µl of 100 mM sodium acetate, pH 7.0, were
digested with 4.2 µg of proteinase K at 37 °C. At different time
points, the reaction was stopped by adding SDS-sample buffer and
heating to 100 °C for 10 min. Samples from each time point were
resolved on 15% SDS-polyacrylamide gels.
Circular Dichroism Spectrometry--
Synuclein proteins prepared
at a concentration of 5 mg/ml in 100 mM phosphate buffer,
pH 7.0, were analyzed by circular dichroism either immediately or after
incubation at 37 °C for 2 days with constant shaking. Proteins were
diluted 1:20 in distilled water and transferred to a 0.1-cm quartz
cuvette, and the spectra between 190 and 260 nm were collected with a
Aviv (Lakewood, NJ) 62DS spectrophotometer.
Antibodies--
The production and epitope mapping of
anti- Synthetic Peptide--
The peptide VTGVTAVAQKTA corresponding to
amino acids 71-82 in human Generation of Mutant and Hybrid Synuclein cDNAs--
The
A76R and A76E Human The Requirement of Hydrophobic Residues
71VTGVTAVAQKTV82 in
The possibility that Effects of Introducing Charged Residues within the Hydrophobic
Residues 71VTGVTAVAQKTV82 of The Hydrophobic Region Containing Residues
71VTGVTAVAQKTV82 of The Synthetic Peptide 71VTGVTAVAQKTV82 Can
Self-assemble and Induce the Assembly of Human Our study used a number of different assays to show that amino
acid residues 71-82 within the middle of the hydrophobic domain of
We sought to explain the dramatic difference in assembly properties of
human Protection of unassembled and assembled To further confirm the role of the hydrophobic region in the
polymerization of The inefficiency of A detailed understanding of the protein motifs required in the
fibrillogenesis and subsequent formation of pathological inclusions is
critical to plan potentially effective therapeutic strategies targeted
at reducing the formation of pathological -synuclein define several diseases of the nervous system.
It is likely that these inclusions impair vital metabolic processes and
compromise vialibity of affected cells. Here, we report that a 12-amino
acid stretch (71VTGVTAVAQKTV82) in the
middle of the hydrophobic domain of human
-synuclein is necessary
and sufficient for its fibrillization based on the following
observations: 1) human
-synuclein is highly homologous to
-synuclein but lacks these 12 residues, and it does not assemble into filaments in vitro; 2) the rate of
-synuclein
polymerization in vitro decreases after the introduction of
a single charged amino acid within these 12 residues, and a deletion
within this region abrogates assembly; 3) this stretch of 12 amino
acids appears to form the core of
-synuclein filaments, because it
is resistant to proteolytic digestion in
-synuclein filaments; and
4) synthetic peptides corresponding to this 12-amino acid stretch
self-polymerize to form filaments, and these peptides promote
fibrillization of full-length human
-synuclein in vitro.
Thus, we have identified key sequence elements necessary for the
assembly of human
-synuclein into filaments, and these elements may
be exploited as targets for the design of drugs that inhibit
-synuclein fibrillization and might arrest disease progression.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-synuclein (
-syn)1
is the major component of several pathological lesions diagnostic of
specific neurological disorders, including Parkinson's disease, the
Lewy body (LB) variant of Alzheimer's disease, dementia with LB,
multiple system atrophy, and neurodegeneration with brain iron
accumulation type 1 (formerly known as Hallervorden-Spatz disease;
Refs. 1-11). In neurons, abnormal
-syn can form LBs, Lewy neurites,
neuronal cell inclusions, and axonal spheroids. Moreover,
oligodendrocytic aggregates of
-syn, known as glial cytoplasmic
inclusions, are found abundantly in multiple system atrophy, but
it is unknown how they cause disease (6, 8-10).
-Syn cellular inclusions comprise fibrils formed by polymerized
-syn. This notion is supported by the intense labeling of these
fibrils by antibodies specific to
-syn in situ (1, 4, 6,
9, 10) as well as by the partial purification of
-syn immunoreactive
filaments from multiple system atrophy and dementia with LB brains (5,
8). Furthermore, in vitro,
-syn has an intrinsic
propensity to polymerize into fibrils that resemble authentic filaments
in pathological lesions (12-16).
-Syn is a small, 14-kDa protein that has sequence homology to three
other proteins termed
-synuclein (
-syn),
-synuclein (
-syn),
and synoretin (17, 18). These proteins are more highly homologous
within the amino-terminal half, which encompasses 5-6 degenerate
KTKEGV repeats. Of these proteins,
-syn has the greatest sequence
homology to
-syn. Furthermore, the expression and intracellular localization of
- and
-syn are very similar in that both proteins are predominantly expressed in neurons of the central nervous system
and are concentrated at the presynaptic terminal (19-22). In contrast,
-syn is predominantly expressed in neurons of the peripheral nervous
system, although it is also found at low levels in brain but diffusely
distributed throughout the cytoplasm (23). Synoretin is similar to
-syn with respect to amino acid sequence and intracellular
distribution, but synoretin is mainly expressed in the retina (17).
- and
-syn have sequence homology and very
similar biological properties,
-syn, but not
-syn, is found in
fibrillar pathological lesions (1, 4, 5, 7-9, 24). To determine the
reason for this difference, we compared the primary sequences of human
- and
-syn, and we recognized that a hydrophobic stretch of amino
acids within the middle hydrophobic region of
-syn is lacking in
-syn and that there is a significant sequence divergence within the
carboxyl-terminal region of these two proteins (Fig. 1). Deletion of
amino acids 71-82 within the hydrophobic region abrogated the ability
of human
-syn to polymerize, whereas the introduction of charged
residues within the region significantly reduced the rate of filament
formation. Conversely, substituting the carboxyl-terminal region of
-syn for that of
-syn did not affect polymerization. Proteolytic
digestion of assembled
-syn as well as coassembly experiments with a
synthetic peptide corresponding to the hydrophobic region further
confirmed the key role of this region in fibrillogenesis.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-Synuclein--
All synuclein
cDNAs were subcloned into the NdeI and
HindIII restriction sites of the bacterial expression vector
pRK172, and the respective proteins were expressed in Escherichia
coli BL21 (DE3). Bacterial pellets were resuspended in high-salt
buffer (0.75 M NaCl, 50 mM Tris, pH 7.4, 1 mM EDTA) containing a mixture of protease inhibitors,
heated to 100 °C for 10 min, and centrifuged at 70,000 × g for 30 min. The supernatants were applied onto a Superdex
200 gel filtration column (Amersham Pharmacia Biotech) and separated by
size exclusion using high-salt buffer. The fractions were assayed for
the presence of the synuclein proteins by SDS-polyacrylamide gel
electrophoresis (PAGE) followed by Coomassie Blue R-250 staining. The
proteins were concentrated using Centriprep-10 (Millipore Corp.,
Bedford, MA), dialyzed against 10 mM Tris, pH 7.5, applied to a Mono Q column (Amersham Pharmacia Biotech), and eluted with a
0-0.5 M NaCl gradient. Protein concentration was
determined using the bicinchoninic acid protein assay (Pierce) and
bovine serum albumin as a standard.
-syn and
-syn, followed by either goat anti-mouse or goat anti-rabbit antibodies conjugated to horseradish peroxidase, developed with Renaissance Enhanced Luminol Reagents (NEN Life Science
Products, Inc.) and exposure onto X-Omat Blue XB-1 films (Eastman Kodak
Co.).
-syn antibodies LB509, SNL-4, SNL-1, and Syn 208 and
anti-
-syn antibody Syn 207 were reported previously (26, 27). The
rabbit anti-
-syn antibody NAC-1 was raised to a synthetic
polypeptide (amino acids 75-91 in human
-syn) conjugated to
maleimide activated keyhole limpet hemocyanin (Pierce).
-syn (Fig.
1) was synthesized and purified on
reverse phase high performance liquid chromatography by the
Biotechnology Resource Center at Yale University (New Haven,
CT).
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Fig. 1.
Schematic depicting the sequence alignment of
human - and
-syn and
mutants of
-syn. The amino acids
identical between the two proteins are illustrated in a black
background. The arrowhead indicates the position where
the carboxyl-terminal domain of
-syn was exchanged for the
carboxyl-terminal domain of
-syn to generate the protein
/
-syn. The bar between amino acids 71-82 depicts the
residues deleted in the protein
71-82
-syn. The
asterisk indicates the position of the Ala residue mutated
to either an Arg or Glu.
-syn DNAs were engineered by creating the respective
nucleotide substitutions in the wild-type cDNA using complimentary
sets of synthetic single-stranded DNA containing the mutant
sequence (i.e. A76R primers, forward/reverse,
ACGGGTGTGACAAGAGTAGCCCAGAAGACAGTG/CACTGTCTTCTGGGCTACTCTTGTCACACCCGT; and A76E primers, forward/reverse,
ACGGGTGTGACAGAAGTAGCCCAGAAGACAGTG/CACTGTCTTCTGGGCTACTTCTGTCACACCCGT) and the QuikChange site-directed mutagenesis kit (Stratagene). The
deletion of the nucleotide sequence coding for residues 71-82 in
-syn cDNA (Fig. 1) was created using the ExSite polymerase chain
reaction site-directed mutagenesis kit (Stratagene) and oligonucleotides that specifically bind to the DNA sequence adjacent to
the targeted deleted sequence (GAGGGAGCAGGGAGCATTGCAGC and CACTGCTCCTCCAACATTTGTCACTTG). The cDNA encoding the hybrid protein
/
-syn, which consist of amino acid residues 1-97 of
-syn and 87-134 of
-syn (Fig. 1), was created by polymerase chain reaction with oligonucleotides that amplified the two separate cDNA
fragments of
- and
-syn, respectively (
-syn, primer 1, CATATGGATGTATTCATGAAAGGAC; and primer 2, GAATTCCTCCTTTTTGACAAAGCCAGTGGCTG;
-syn, primer 3, TGTCAAAAAGGAGGAATTCCCTACTGATC; and primer 4, AGGGACAGAAGCTTGCTGCTGGTG). The inner oligonucleotides (primers 2 and 3) contain extended complimentary sequences that were used to unite both initial polymerase chain reaction products by subsequent polymerase chain reaction reactions with primers 1 and 4. The sequences of the constructs were
verified using T7 primer and an ABI Prism 377 DNA sequencer (PE
Biosystems, Foster City, CA) as a service provided by the Nucleic
Acid/Protein Research Core Facility at the Children's Hospital of Philadelphia.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-Syn Fails to Fibrilize in Vitro--
To assess whether
human
-syn is capable of self-assembly into filaments, we compared
the ability of
- and
-syn to form filaments using previously
established conditions for the assembly of
-syn (12). Centrifugal
sedimentation analysis and electron microscopy (EM) were used to
determine the assembly of human
- and
-syn. Both proteins were
found in the soluble fraction before incubation for assembly (Fig.
2A), but the majority of
-syn was present in the pelletable fraction after 2 days of
incubation, whereas
-syn remained largely soluble (Figs. 2,
B and C, and 3). EM
analysis showed that human
-syn filaments were abundant within 2 days (Fig. 4A), but no fibrils
could be detected with
-syn after incubation to induce fibrilization
(data not shown).
-Syn failed to assemble even after 9 days in
vitro, as demonstrated by quantitative sedimentation analysis of
-syn (Fig. 3). Further extensive EM inspection of grids coated with
-syn incubated under assembly conditions for up to 6 weeks did not
reveal the presence of filaments. Finally, circular dichroism studies
demonstrated that both proteins had random coiled conformations before
assembly, but only
-syn acquired a
-pleated sheet conformation
during assembly conditions (Fig. 5,
A and C).
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Fig. 2.
Centrifugal sedimentation analysis of the
assembly of recombinant synuclein proteins and derivatives
thereof. Assembly of protein was monitored by sedimentation at
100,000 × g for 20 min as described under "Materials
and Methods." Superntant (S) and pellet (P)
fractions were loaded on 12% polyacrylamide gels. A,
unassembled proteins at 5 mg/ml were predominantly in the superntant
fractions. B and C, syn proteins (2.5 or 5 mg/ml)
were incubated at 37 °C for 48 h with continuous shaking.
D and E, -syn incubated alone or with either
-syn or
71-82 at 37 °C for 48 h with shaking.
F, A76E and A76R mutants of
-syn were incubated alone or
with
-syn for 48 h at 37 °C with shaking. Proteins resolved
by SDS-PAGE were visualized with Coomassie Blue R-250 (CB)
or by Western blot analysis with antibodies specific to
-syn (LB
509) or
-syn (Syn 207). A total of 5 µg or 50 ng of protein was
loaded for each set of assembly experiments analyzed by Coomassie Blue
staining or Western blot analysis, respectively.
,
, and
/
,
-syn,
-syn, and
/
-syn, respectively; A76E and
A76R, respective mutant proteins.
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Fig. 3.
Quantification of synuclein filament assembly
as analyzed by centrifugal sedimentation. Proteins (5 mg/ml) in
100 mM acetate, pH 7.0 were incubated for 2, 4, and 9 days
at 37 °C with continuous shaking followed by sedimentation at
100,000 × g for 20 min. Supernatants and pellets were
resolved by SDS-PAGE and after Coomassie Blue R-250 staining quantified
by densitometry. The percentage of protein in the pellet is expressed
on the y axis. n = 4.
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Fig. 4.
Transmission electron microscopy of
negatively stained synuclein filaments. Wild-type human -syn
(A),
/
-syn (B), and A76R human
-syn
(C) were incubated for assembly as described under
"Materials and Methods" at a concentration of 5 mg/ml for 2 days.
D, under the same condition, filaments of A76E human
-syn
were visualized after 9 days. The peptide corresponding to residues
71-82 in human
-syn (864 µM) also assembled into
filaments (E), and at the same concentration it coassembled
with 2.5 mg/ml human
-syn (F). The average diameter of
each filament population is indicated below each respective micrograph.
Scale bar, 125 nm.
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Fig. 5.
Circular dichroism spectra of synuclein
protein before (A and B) and after
(C and D) assembly for 2 days at 37 °C with
continuous shaking as described under "Materials and
Methods."
-syn for Filament
Assembly--
Alignment of the primary sequences of human
- and
-syn showed two major regional differences between the proteins. A
stretch of hydrophobic amino acids in the middle of
-syn is absent
in
-syn (Fig. 1). Also, the carboxyl termini of the two proteins are
relatively divergent compared with the amino termini. To ascertain the
importance of the hydrophobic region and the carboxyl-terminal region
in driving filament assembly, two types of recombinant synuclein
protein were generated. First, amino acid residues 71-82 in human
-syn were deleted to generate a protein termed
71-82 (see Fig.
1). Second, a hybrid comprising the first 97 amino acids of human
-syn and the last 48 amino acids of human
-syn was generated,
termed
/
-syn (Fig. 1). Before assembly, both recombinant proteins
were recovered mainly in the supernatant after centrifugal sedimentation (Fig. 2A) and had a predominantly random coil
conformation (Fig. 5, A and B). On incubation,
the assembly rate of
/
-syn paralleled that of
-syn (Fig. 3),
and it formed filaments of similar diameter and appearance (Fig. 4,
compare A with B). The fibrillization of
/
-syn also was associated with a change into a
-pleated sheet
conformation (Fig. 5D). On the other hand,
71-82 did not
assemble under similar assembly conditions, as monitored by
sedimentation analysis (Figs. 2, B and C, and 3),
and transmission EM did not reveal the presence of filaments even after
prolonged incubation for up to 6 weeks (data not shown). Finally, the
lack of assembly was paralleled by a lack of conformational change, as
detected by circular dichroism (Fig. 5D).
-syn or
71-82 could either coassemble with
-syn or inhibit the assembly of
-syn was determined by coassembly
experiments. Neither
-syn nor
71-82 had any impact on the
assembly of
-syn, as assayed by sedimentation (Fig. 2, D
and E) and by EM (data not shown). Furthermore, neither
protein coassembled with
-syn. Although
-syn can be separated
from
-syn by SDS-PAGE because of its greater mobility, the identity
of the bands was also confirmed by Western blotting with an antibody specific to human
-syn (LB509) and an antibody specific to
-syn (Syn 207).
-Syn--
To
further confirm the importance of this hydrophobic region in
fibrillogenesis, a single residue in the middle of this region, Ala-76,
was substituted with either a positive (arginine) or a negative
(glutamic acid) charge residue. Before assembly, the A76R and A76E
mutations in recombinant
-syn proteins did not alter the normal
random coil conformation of human
-syn in solution (Fig.
5B), and both recombinant proteins were in the soluble
fraction when analyzed by centrifugal sedimentation (data not shown).
The A76R mutation reduced the rate of filament formation when compared with wild-type
-syn at 2 days after assembly, but by 4 days the majority of the A76R protein was assembled (Figs. 2F and 3).
Fibrils comprising A76R
-syn (Fig. 4C) were similar to
those assembled of the wild-type protein (Fig. 4A). By
contrast, the A76E mutation had a much greater impact on the rate of
filament assembly. Even after 4 days of incubation, the mutant protein
was predominantly found in the soluble fraction after sedimentation
analysis (Figs. 2F and 3), and only very rare and short
fibrils were observed by EM (data not shown). However, after 9 days of
assembly, the A76E
-syn protein eventually polymerized into fibrils
that were morphologically indistinguishable from those generated from
the wild-type protein (Fig. 4, compare A with D).
The presence of either the A76R or A76E mutant in coassembly
experiments did not seem to inhibit the assembly of wild-type protein,
as assayed by sedimentation (Fig. 2F), and observation by EM
of grids coated with samples of both proteins coincubated for assembly
(data not shown).
-Syn Is Resistant to
Protease Digestion of
-Syn Filaments--
Digestion of
-syn
fibrils with proteinase K was used to identify regions that are
probably buried within and therefore are core elements of these
filaments. Digestion of unassembled human
-syn resulted in the rapid
proteolysis of the protein within 30 min (Fig.
6A). Conversely, several lower
Mr fragments, resistant to proteolysis, were
detected when assembled
-syn filaments were treated with proteinase
K for 0.5 h (Fig. 6B). The acquired resistance to
proteinase K was specific to assembled
-syn filaments, because two
other proteins that are assembly incompetent,
-syn and
71-82,
were completely digested even after assembly incubation (Fig.
6C). To identify the proteolytic resistant polypeptides, Western blot analysis was performed using antibodies with defined epitopes throughout the
-syn molecule (Fig. 6, D and
E), and the approximate sequences of the three major
polypeptides, designated F1-F3, were deduced on the basis of their
immunoreactivity with the different antibodies and their apparent
molecular mass on SDS-PAGE gels (Fig. 6, D and
E). All three peptides contain amino acid residues 71-82
within the middle hydrophobic region of
-syn.
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Fig. 6.
Within -syn
filaments, the central hydrophobic region is protected against
proteolytic digestion. Unassembled (A) or polymerized
(B) human
-syn was untreated (U) or digested
with proteinase K for the time indicated above each lane.
C,
-syn,
-syn, and
71-82 were incubated under
assembly conditions (100 mM sodium acetate, pH 7.0, at
37 °C for 2 days with shaking) and undigested (
) or digested (+)
with proteinase K for 30 min. Note that proteolytic resistant fragments
are only observed with
-syn. In A-C, proteins were
resolved on 15% SDS-polyacrylamide gels and visualized by staining
with Coomassie Blue R-250. D, the protected peptides were
identified by using antibodies mapped to specific epitopes (26).
Western blot analysis of assembled
-syn undigested (
) or digested
(+) with proteinase K was performed using the antibodies indicated
above each panel. The epitope of each antibody is also
indicated in parentheses above each panel.
E, Schematic summarizing the approximate location of the
three major fragments (F1-F3) resistant to proteolytic
digestion as determined by antibody recognition and apparent molecular
mass on SDS-PAGE. The black region defines amino acids
61-95, also known as NAC (see "Discussion"). The mobility of
molecular mass markers are indicated on the left.
-Syn--
To further
assess the importance of amino acids 71-82 in human
-syn for
fibrillization, we asked whether synthetic peptides corresponding to
this sequence self-assemble into fibrils. Fig. 4E shows that
peptide 71-82 polymerizes into filaments, but these filaments are much
straighter than those assembled from the full-length
-syn (Fig. 4,
compare A with E). However, when full-length
-syn was assembled in the presence of peptide 71-82, only the more serginous fibrils, typical of full-length
-syn, were observed, suggesting that this peptide was incorporated into the same fibrils as
full-length
-syn (Fig. 4F). On a molar basis, peptide
71-82 is less efficiently assembled than full-length
-syn;
nevertheless, its ability to self-assemble can also be observed by
light scattering (Fig. 7A).
Finally, the addition of peptide 71-82 was found to enhance the
assembly of full-length
-syn. After a 48-h incubation period, with
less vigorous agitation to reduce the rate of assembly, the majority
of
-syn when assembled alone was recovered in the supernatant,
whereas in the presence of peptide 71-82,
-syn was found primarily
in the pellet after sedimentation analysis (Fig. 7).
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Fig. 7.
The synthetic peptide 71-82 in human
-syn can self-assemble and promote the assembly of
the full-length
-syn. Fibrillization was
assayed by measuring light scattering at a wavelength of 400 nm
(A) or sedimentation at 100,000 × g for 20 min followed by Coomassie Blue R-250 staining of a 12% SDS-PAGE gel in
which supernatants (S) and pellets (P) were
loaded on separate lanes after incubation for 24 h (B).
Unassembled peptide 71-82 (864 µM) and
-syn (2.5 mg/ml) had an A400 of <0.02. Assembly was
conducted with less vigorous continuous shaking to reduce the rate of
assembly.
-syn was at a concentration of 2.5 mg/ml, whereas peptide
71-82 was used at either a 2.5-fold higher molar concentration (432 µM; low) or a 5-fold higher molar
concentration (864 µM; high).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-syn are necessary and sufficient for
-syn to polymerize into
filaments. The importance of residues 71-82 for filament formation was
realized when we found that human
-syn demonstrates an intrinsic
ability to assemble into filaments, whereas human
-syn cannot
polymerize into filaments even after prolonged (6 weeks) exposure to
assembly conditions. These results are consistent with a recent report
(28). However, it cannot be completely excluded that
-syn would
never form filaments under any circumstances, because another study
suggested that a small number of
-syn filaments were observed after
5 weeks of incubation (29). Although we did observe a slight increase
in the amount of pelletable
-syn after prolonged incubation (Fig.
3), we attributed this to nonspecific aggregation because of the lack
of visible filaments by EM. Taken together, these findings indicate
that
-syn is far less prone to polymerize into fibrils relative to
-syn.
- and
-syn by comparing their sequences, which revealed two
major differences: 1)
-syn lacks a hydrophobic amino acid stretch
found near the middle region of
-syn; and 2) the carboxyl termini of
- and
-syn are significantly divergent. The ability of a hybrid
protein containing the amino terminus of
-syn and the carboxyl
terminus of
-syn to assemble into filaments demonstrates that
differences within the carboxyl-terminal region of
- and
-syn do
not account for the paucity of filament formation by
-syn.
Conversely, a 12-amino acid deletion of the middle hydrophobic region
within
-syn (
71-82) obliterated polymerization. The importance of the hydrophobic region is further supported by the inhibitory influences of the substitution of a single uncharged amino acid to a
charged residue (either Glu or Arg). The introduction of either of
these single substitutions did not completely inhibit assembly, but it
did slow down the rate of filament assembly, especially the A76E
mutation. Thus, these results demonstrate that the introduction of a
single charged residue is not sufficient to disrupt the hydrophobic
intermolecular interactions driving filament formation.
-syn from proteinase K
digestion was used to screen for segments of the protein buried in the
core, because they are likely to be involved in filament assembly.
Three predominant proteolytic resistant fragments (F1-F3) were
observed. These fragments mapped to the middle hydrophobic region and
contained amino acids 61-95, which has also been termed the nonamyloid
component (NAC) of A
amyloid plaques (30). The NAC peptide was
initially purified from SDS-insoluble fractions enriched in amyloid
cores from Alzheimer's disease brains, and it was thought to be the
second most abundant component of plaques after
-amyloid. Whether
this peptide is actually an intrinsic component of amyloid plaques is
still a matter of contention (30-33). It may have been purified as a
contaminant of amyloid cores, since almost all brains from Alzheimer's
disease patients contain variable amounts of aggregated
-syn in LBs
and in Lewy neurites. The proteolytic resistance of this region within
fibrillar
-syn shown in our study may explain why the NAC peptide
was selectively recovered with amyloid cores, since both NAC and A
may be resilient to proteolysis by endogenous proteases during
purification or to the protease that was used in the final stage of purification.
-syn, a synthetic peptide corresponding to amino
acids 71-82 in human
-syn was assayed for its ability to generate
filaments and to coassemble with full-length
-syn. Peptide 71-82
readily self-assembled under the same conditions used for
-syn, and
it further promoted the assembly of
-syn. Consistent with these
results and the notion that the hydrophobic region is essential for
assembly, it was reported that a peptide corresponding to amino acids
1-18 in NAC (i.e. 61-78 in
-syn) can also form filaments, but not a peptide corresponding to amino acids 19-35 in NAC
(i.e. 79-95 in
-syn; Ref. 34). Furthermore, a
carboxyl-terminal
-syn truncation mutant (1-89) not only formed
filaments, it actually assembled faster than full-length
-syn (29).
Collectively, these studies raise the possibility that aberrant
proteolysis of
-syn and the accumulation of a polypeptide
constituting the hydrophobic region may act together as a nidus for the
formation of large inclusions. Indeed, LBs isolated from dementia with
LB brains contain truncated
-syn fragments (1).
-syn to form filaments likely explains the
paucity of
-syn within pathological lesions defined by
-syn immunoreactivity. The only exception is a recent study (35) in which an
accumulation of both
- and
-syn was observed in mossy fibers
terminals that synapse on hilar neurons in patients with Parkinson's
disease or dementia with LB. However, this pathology is different from
other
-syn lesions, because it is not defined by the presence of
abundant fibrils as seen in LBs and glial cytoplasmic inclusions. The
accumulation of
- and
-syn in synaptic terminals is likely
attributable to the degeneration of presynaptic terminals independent
of
-syn fibrillogenesis, a notion that is consistent with the
accumulation of other synaptic proteins such as synaptophysin, synapsin, and synaptobrevin (35).
-syn aggregation. Candidates for such therapy may include small molecules that could interfere with the assembly process by binding to the hydrophobic region. However, as demonstrated by the peptide corresponding to amino
acids 71-82 in human
-syn, such treatments could produce an effect
opposite to that desired by increasing aggregation. Pharmacological
agents that could prevent
-syn from converting into a
-pleated
sheet conformation may be effective. Indeed, this structural alteration
is associated with filament formation in vitro, and
pathologically aggregated
-syn is also likely to be in a
-sheet
conformation, as suggested by the staining of pathological lesions with
thioflavin S (36). Further refinement of in vitro assembly
systems and the development of in vivo models are key to
developing and testing putative therapies for diseases characterized by
filamentous
-syn inclusions.
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ACKNOWLEDGEMENTS |
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We thank Drs. K. S. Reddy and C. Moser for assistance with circular dichroism studies and the Biochemical Imaging Core Facility of the University of Pennsylvania for assistance with the EM studies.
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FOOTNOTES |
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* This work was supported in part by grants from the NIA, National Institutes of Health, and a Pioneer Award from the Alzheimer's Association. B. I. G. and I. V. J. M. contributed equally to this study.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Recipient of a fellowship from the Human Frontier Science Program Organization.
§ Recipient of a fellowship from the Medical Research Council of Canada.
¶ To whom correspondence should be addressed: Center for Neurodegenerative Disease Research, Dept. of Pathology and Laboratory Medicine, Maloney 3, Hospital of the University of Pennsylvania, Philadelphia, PA 19104-4283. Tel.: 215-662-6427; Fax: 215-349-5909; E-mail: vmylee@mail.med.upenn.edu.
Published, JBC Papers in Press, November 1, 2000, DOI 10.1074/jbc.M008919200
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ABBREVIATIONS |
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The abbreviations used are: syn, synuclein; EM, electron microscopy; LB, Lewy body; NAC, nonamyloid component of amyloid plaques; PAGE, polyacrylamide gel electrophoresis.
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