From ICBAS, Instituto de Ciências
Biomédicas Abel Salazar, Universidade do Porto, Largo Prof. Abel
Salazar n°2, 4099-003 Porto, Portugal, the § Instituto de
Biologia Molecular e Celular, Universidade do Porto, Rua do Campo
Alegre n°823, 4150 Porto, Portugal, and the ¶ European
Synchrotron Radiation Facility (ESRF), BP 220, F-38043 Grenoble
Cedex, France
Received for publication, October 22, 2002, and in revised form, January 17, 2003
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ABSTRACT |
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Transthyretin (TTR) amyloid fibrils are the main
component of the amyloid deposits occurring in Familial Amyloidotic
Polyneuropathy patients. This is 1 of 20 human proteins leading to
protein aggregation disorders such as Alzheimer's and
Creutzfeldt-Jakob diseases. The structural details concerning
the association of the protein molecules are essential for a better
understanding of the disease and consequently the design of new
strategies for diagnosis and therapeutics. Disulfide bonds are
frequently considered essential for the stability of protein aggregates
and since in the TTR monomers there is one cysteine residue, it is
important to determine unambiguously the redox state of sulfur present
in the fibrils. In this work we used x-ray spectroscopy to further
characterize TTR amyloid fibrils. The sulfur K-edge absorption spectra
for the wild type and some amyloidogenic TTR variants in the soluble
and fibrillar forms were analyzed. Whereas in the soluble proteins the
thiol group from cysteine (R-SH) and the thioether group from
methionine (R-S-CH3) are the most abundant forms, in
the TTR fibrils there is a significant oxidation of sulfur to the
sulfonate form in the cysteine residue and a partial oxidation of
sulfur to sulfoxide in the methionine residues. Further
interpretation of the data reveals that there are no disulfide bridges
in the fibrillar samples and suggest conformational changes in the TTR
molecule, namely in strand A and/or in its vicinity, upon fibril formation.
Amyloidoses are a group of protein misfolding diseases, which
include spongiform encephalopathies, Alzheimer's disease, and Familial
Amyloidotic Polyneuropathy
(FAP),1 all of them
characterized by extracellular deposits of an insoluble fibrillar
protein. Transthyretin (TTR) is the most abundant protein component of
amyloid fibrils in the case of FAP patients. It is a plasma protein,
with an extended The molecular mechanisms involved in amyloid fibril formation are not
yet well understood. Most of the proposed models refer to the
dissociation of the tetrameric protein into an intermediate species
that self-assembles leading to the insoluble fibril (2, 3). However,
some controversy remains regarding this intermediate structure and in
particular regarding its monomeric or dimeric conformational nature.
For a better understanding of amyloid assembly, mutant TTR proteins
containing disulfide bonds linking two monomers were designed, and
their ability to form fibrils was tested. While some mutants did not
form amyloid, which is consistent with the idea that the monomeric
species are necessary for fibril formation (4), others do form fibrils,
pointing to a dimeric nature of the intermediate species (5). Recently,
scanning transmission electron microscopy (STEM) clarified this
question because it revealed a mass-per-length of the protofilaments of
~0.47 kDa/Å, which is consistent with the model where the monomer is
the building block (3).
Insights into the mechanism of fibril formation were obtained by x-ray
diffraction analysis of FAP amyloid fibrils. It was shown that fibrils
exhibit a cross The possible role for the sulfur-containing amino acids in the fibril
remains unknown. Based on the fact that Cys-10 is more exposed to the
solvent in V30M-TTR, it was considered that amyloid fibrils could
result from association of TTR molecules through disulfide bridges (9).
This idea was supported by biochemical analysis of amyloid from
homozygous and heterozygous individuals with the V30M-TTR mutation
(10). However, it does not explain the formation of fibrils in the case
of amyloidogenic C10R-TTR variant.
In order to find out the role of sulfur in the aggregation mechanism
leading to fibril formation it is important to determine unambiguously
its oxidation state when the protein is in a soluble form and when it
forms the amyloid fibrils. Sulfur K-edge x-ray absorption spectroscopy
provides the analytical tool needed for that purpose. The chemical
shift in the x-ray absorption edge and the energy of maximum absorption
depend on the sulfur coordination and environment in the sample. The
potential of this technique in the analysis of biological materials is
enormous because no chemical pretreatment is needed, and consequently
there is no perturbation of the sulfur redox state. In fact, the
oxidation state of sulfur in the human blood, plasma, and erythrocytes
was examined for the first time in 1998 using this methodology (11). Later, preliminary studies concerning the acid/base equilibrium of the
thiol group of papain, In this work we use sulfur K-edge spectroscopy to examine the sulfur
oxidation state of TTR in the soluble and polymerized forms of the
protein in order to determine the possible role of the sulfur in intra
and intermolecular interactions, namely through the presence or absence
of disulfide bridges. Additionally, x-ray diffraction was used to
confirm the fibrillar structures of the samples under study and to
improve our knowledge about their molecular structure.
Preparation of the Protein Samples--
All TTR mutants were
produced in an Escherichia coli expression system, and the
periplasmic space contents were obtained by osmotic shock. The
supernatant was passed through DEAE-Sephadex, and the TTR-containing
peaks were dialyzed overnight against water and then freeze-dried.
Further purification was achieved by preparative gel electrophoresis.
Finally, TTR solutions were dialyzed against water for 24 h.
Protein concentration was determined using the Lowry method.
In vitro amyloid fibrils, except for the L55P-TTR, were
formed by acidification. The proteins (2 mg/ml) were incubated with 0.05 M sodium acetate/0.1 M KCl, pH 3.6, for
48 h at room temperature. Then, the fibrils were sedimented by
centrifugation at 15,000 × g for 20 min in a
microcentrifuge, and the pellets were resuspended in MilliQ water and
incubated at 37 °C. Fibril formation was achieved for all the TTR
mutants except in the case of C10S-TTR, for which no significant amount
of fibrillar material was produced using these conditions.
The amyloidogenic properties of the purified L55P-TTR (concentration 6 mg/ml in water, pH ~7.0) were tested with the thioflavine-T binding
assay, and immediately after the purification procedure thioflavine did
not bind TTR. Then part of the protein sample was immediately stored at
Thioflavine-T Binding Assay--
Solutions of 100 µg of
amyloid fibrils were prepared as described above. To each solution was
added ThT, final concentration 30 µM, in 50 mM glycine/NaOH buffer, pH 9.0, in an assay volume of 1 ml.
Excitation spectra were recorded by spectrofluorimetry (FP-770; JASCO)
at 25 °C. Excitation and emission slits were set at 5 and 10 nm,
respectively. The excitation spectra (400-500 nm) were taken with
emission collected at 482.0 nm.
Sulfur K-edge XANES--
Sulfur K-edge spectra were
recorded at room temperature using synchrotron radiation, at ESRF,
beamline ID21: a double Si (111) crystal monochromator allows to
achieve an energy resolution of 10
The reference compounds cysteine, cystine, methionine, methionine
sulfoxide, and anthraquinone-2-sulfonic acid were of reagent grade,
purchased from Sigma and used as received. They were in the form of
powder, and a small amount was dispersed in a very thin layer. Also,
solutions of the reference compounds were prepared, and their spectra
measured. No differences between the spectra of the compounds in the
powder form and in solution were observed.
The protein samples (5-10 µl) were introduced in a special cell for
liquids. The soluble proteins were used at a concentration of 5-10
mg/ml. The fibril solutions were left to sediment for a few hours, and
a fraction of the pellet was used to record the spectra.
In order to ensure that there was no radiation damage, the complete
procedure adopted for protein spectroscopy was the following: a fast
spectrum in the region from 2.470 keV to 2.485 keV was recorded with a
step size of 0.0005 keV, then two full spectra in the region from 2.450 keV to 2.530 keV were obtained and compared with the initial spectrum
to confirm that they were all similar. In the two full spectra, a step
size of 0.00017 keV was taken near the edge region (2.470-2.485 keV),
and the time of radiation exposure was chosen to have between 5000 ct/s
and 10000 ct/s at the maximum of the spectrum. The integration time was
always between 4 and 10 s per point.
The background was removed from the spectra by subtracting the medium
value of the pre-edge horizontal region. Normalization was achieved by
dividing the spectra by the medium value of the far post-edge flat region.
The proportion of the different forms of sulfur in the samples was
estimated by simulation of the protein spectra using a linear
combination of the spectra of the reference samples. The sum of the
squares of the residuals between the original and the simulations was
calculated in the region from 2.470 keV to 2.485 keV and divided by the
number of experimental points measured in that region. The obtained
value was designated by relative error. The percentages of the
reference compounds found to give the minimum relative error were
judged to be the best fit of the data.
X-ray Fiber Diffraction--
Glass capillary tubes of 0.5-mm
diameter were treated with a siliconizing reagent. A fibril solution of
L55P-TTR (6 mg/ml) was drawn up into the siliconized glass capillary
tubes that were then sealed at the top, placed into a 2-T magnet, and
allowed to dry at room temperature. As soon as a thin disk was formed, the capillary tubes were sealed with wax in order to retain some hydration of the sample. The x-ray diffraction patterns were only collected for the samples showing birefringence under cross-polarized light.
The x-ray diffraction data were collected at ESRF, beam line ID13,
using a 0.975 Å wavelength beam and a beam size of 5 µm. Patterns
were recorded on a MAR research imaging plate during exposure times of
10-60 s. The background was recorded and subtracted to the diffraction
pattern for elimination of the air scattering effects, using the
software package FIT2D (13). The sample to detector distance was
calibrated with silver behenate.
X-ray Fiber Diffraction--
All the samples were examined by
x-ray diffraction, and the data always revealed a cross-
The meridional reflections, 4.85 and 4.15 Å, may be indexed as the
6th and 7th order of a pseudo-period of 29.1 Å. In previous FAP fiber x-ray diffraction studies was calculated a
29-Å minimum-repeating distance and based on that it was proposed that
the monomer was the building block of amyloid fibrils (8). Later, it
was confirmed by STEM that transthyretin fibrillogenesis entails the
assembly of monomers (3). However, the 4.85 and 4.65 Å reflections may
also represent sampling of a 4.75 Å reflection, corresponding to the
backbone separation of the neighboring main chains. The 4.15 Å reflection could be the second order of an 8.3 Å reflection, also
observed in the poly-L-glutamine peptide and Sup35 peptide
(14). Recently EPR measurements of spin-labeled TTR mutants suggest a
distance of ~8 Å between subunits interfaces in the fibrils (15)
that could originate that reflection.
The three observed meridional reflections indexed as Bragg reflections,
fit a repeating unit of 116.3 Å ± 0.1, close to the 115.5 Å already
proposed (7). This 4× higher period (4 × 29 = 116 Å), can
be due to a twist in the Sulfur K-edge XANES--
The K-edge spectra of cysteine, cystine,
methionine, methionine sulfoxide, and anthraquinone-2-sulfonic acid are
shown in Fig. 2. The energies of maximum
absorption for these compounds are 2.4730, 2.4723, 2.4731, 2.4759, and
2.4809 keV, respectively. The scans were run with a step size of
0.00017 keV near the edge region, and the measured values are in
agreement with those found in the literature (11, 16). The shapes of
the spectra are clearly different except for cysteine and methionine
that look very similar. Moreover, as described in previous studies
(16), it can be observed that the higher the energy of maximum
absorption the greater is the peak area.
The sulfur K-edge spectra of the WT-, V30M-, L55P-, and C10S-TTR
samples are shown in Fig. 3. Some
conclusions arise immediately from the observation of the spectra.
There are no S-S bonds present in the samples as neither spectra shows
the cystine characteristic peak. The three energies of maximum
absorption observed are 2.473 keV, 2.476 keV, and 2.481 keV indicating
the presence of cysteine and/or methionine, methionine sulfoxide, and
sulfonated cysteine. Although the shapes of the spectrum of TTR
variants are roughly similar, there are pronounced differences between
the non-polymerized and the polymerized forms: in the fibrillar samples
there is a large amount of oxidized sulfur (stronger peak at 2.4759 keV
from the S-sulfoxide and stronger peak at 2.4809 keV from
the S-sulfonated).
Since more detailed interpretations demand a quantitative analysis, the
experimental protein spectra were simulated by linear combination of
the spectra from the reference samples. The simulated spectra are shown
in Fig. 4, and the results of the fits
and corresponding relative errors are presented in Table
II.
TTR is a homotetramer with 127 amino acids per monomer. Cys-10 and
Met-13 are the only sulfur-containing amino acids in the wild-type
protein and L55P-TTR. V30M-TTR contains one cysteine and two
methionines and variant C10S-TTR contains only one methionine.
In the non-polymerized protein samples there is a higher fraction of
cysteines oxidized to the sulfonated form (24-28%) then methionines
oxidized to the sulfoxide form (10% or less), which is consistent with
Cys-10 being more exposed to the solvent then Met-13 as revealed by the
x-ray crystallographic structures of these variants (9, 17, 18).
According to the crystal structure of V30M-TTR variant, the extra
methionine, Met-30, is buried in the molecule and not exposed to the
solvent. Therefore in this case, the methionine sulfoxide probably
results only from the partial oxidation of Met-13 as it will be the
case for the wild type and the other variant proteins. The presence of
the S-sulfonated in C10S-TTR variant is residual (2%) and
most improbable since no cysteines are present in the protein. The
corresponding peak, which is very small, should arise from the multiple
scattering effects at the high energy end of the spectra (11).
The data concerning the cysteine residue show that there is a slight
increase in the S-sulfonated content from the wild-type protein (24%) to V30M-TTR (27%) to L55P-TTR (28%). In fact, it was
already reported that sulfur K-edge spectroscopy detects changes of
~5% in the thiol-to-disulfide ratio (11). In our experiments, we
were able to obtain a higher accuracy in the thiol-to-sulfonate ratio,
~2%, because the energy peaks were not so close to each other.
However, the observed differences are near the sensitivity of the
experimental method.
To further understand the accuracy of the simulated spectra and their
relative errors the experimental spectrum of the soluble L55P-TTR was
compared with the spectra simulated with the parameters that best fit
the results of the four TTR mutants in the soluble form. These spectra
and corresponding errors are presented in Fig.
5. When the L55P-TTR spectrum was
simulated with the parameters obtained for the best fit of WT-TTR (84%
reduced sulfur, 4% sulfoxide, and 12% sulfonate), V30M-TTR (86%
reduced sulfur, 5% sulfoxide, and 9% sulfonate) and C10S-TTR (97%
reduced sulfur, 1% sulfoxide, and 2% sulfonate), the relative errors
(0.0083, 0.019, and 0.059, respectively) were over three times the
error associated with the spectrum that represents the best fit for
L55P-TTR, which is 0.0024.
The results suggest that L55P-TTR, the most aggressive variant
described in the literature until now, is the variant with the higher
content of sulfonated sulfur (28%).
Analysis of the fibrillar material reveals that the percentage of
sulfur from the methionine residues that are in the sulfoxide form
(V30M-TTR: 28%, L55P-TTR and WT-TTR: 32%) are approximately 3-4×
higher then the corresponding value in the soluble proteins (V30M-TTR:
8%, L55P-TTR and WT-TTR: 10%). The cysteines are even more oxidized:
in V30M-TTR variant 93% are in the sulfonated form, in L55P-TTR 76%
and in WT-TTR protein 48% of the cysteines are oxidized. It is worth
mentioning that L55P-TTR variant was not polymerized by acidification.
This indicates that cysteine and methionine oxidation is not an
artifact resulting from partial denaturation due to acidification but
results from structural alterations during or upon polymerization.
Thioflavine-T Binding Assay--
In order to improve our
understanding about the role of sulfur oxidation in the self-assembly
of TTR protein, the susceptibility to amyloid formation of reduced
WT-TTR was monitored using the thioflavine-T binding assay.
A soluble WT-TTR sample (100 µg) was reduced with dithiothreitol (10 mM), and the procedure to amyloid fibril formation by acidification was followed. The reducing agent was removed from the
fiber solution just before the thioflavine-T binding assay in order to
avoid possible quenching of the spectra. The results show that the
reduced protein solution had strong susceptibility to fibril formation
(Fig. 6).
Although many aspects of amyloid diseases are known, a molecular
model describing the interactions between amyloidogenic units at high
resolution, e.g. revealing the contacts between different atoms that are responsible for a highly stable structure, as it is the
case of the amyloid fibrils, does not exist. This is a priority for
defining how to design a drug that either (i) directly binds to a
contact region and destroys the interaction or (ii) rather occupies the
binding site of one amyloidogenic unit, avoiding its contact with a
nearby unit inhibiting amyloid formation.
In this work we conclude that in TTR amyloid fibrils, Cys-10, the only
cysteine residue present in the TTR protein, does not form disulfide
bridges with other molecules and that sulfur-containing amino acids,
namely Cys-10 and Met-13, are significantly oxidized upon
polymerization. The oxidation of these amino acids may occur before
fiber formation or in the mature fiber. In fact, there are two
possibilities: oxidation of both amino acids leads to a more polar
region capable of forming hydrogen bonds with other molecules and this
leads to amyloid fibers; or aggregation leads to conformational changes
in the protein that expose Cys-10 and Met-13 to the solvent allowing
their subsequent oxidation. However, oxidation of both amino acids is
not required for fibril formation as revealed by the strong susceptibly
of reduced protein samples to form amyloid. Sulfur oxidation might
result as a consequence of structural alterations during protein
aggregation exposing the cysteines and methionines to the solvent.
Although cysteine-free TTR mutants are capable of amyloid fibril
formation (19), showing that disulfide linkages are not required for
fibrillogenisis, there are a number of studies relating the
amyloidogenic behavior of TTR with the oxidation state of sulfur from
their cysteine residues. This relation is not clear yet, since some
results point to an increased amyloidogenicity of sulfonated
transthyretin (20), while others indicate that the stability of the
tetramer is increased by binding of sulfite to Cys-10 (21).
Nevertheless, S-sulfonated transthyretin has been detected
in serum and it was reported that it is significantly more abundant in
diseased individuals (22, 23).
The data show that a significant fraction of the thiol group of
cysteines present in the soluble TTR samples is in the sulfonate form.
A quantitative study of the sulfinic and sulfonic acid content was
carried out in different proteins leading to the conclusion that a
significant number of cysteines is irreversibly oxidized. In fact, one
in four cysteines was estimated as being reactive to oxidants (24).
Examination of the protein models available in the Protein Data Bank
(25) shows that only 2.5% of the structures contain oxidized
cysteines. However, in a significant amount of the protein structures
the cysteines are buried and not exposed to the solvent. Furthermore,
in some models the cysteic acid was represented by three water
molecules around the sulfur, and therefore its oxidation state is not
immediately obvious. In the x-ray crystallographic structures of TTR
proteins, the oxidation of Cys-10 has not been detected. This might be
explained by the fact that in those structures the N-terminal is
disordered and Cys-10 is in most cases the first residue to be
observed. In fact large temperature factors were assigned to the sulfur
atom of that residue. In human serum, sulfated TTR as well as
cysteinylated TTR were detected using mass spectroscopy (22).
Further insight to the structure of amyloid fibrils can be obtained
from the crystallographic structure of the TTR variants and the results
here reported. Met-13 is not buried in the molecule but still partially
protected by Lys-15, Glu-54, His-56, Arg-104, and Thr-106. Cys-10 is at
the external surface of the molecule and also somehow protected,
although not as much as Met-13. It is flanked by the main
chain of Leu-12 and Met-13, the side chain of Arg-104, and by the main
chain between His-56 and Thr-60 as shown in Fig.
7. The crystal structure of
G53S/E54D/L55S-TTR, a constructed variant that polymerizes
spontaneously at physiological conditions, revealed a structural change
of the edge strand D exposing Met-13 (26). Also in the highly
amyloidogenic L55P-TTR variant, a movement of strand D leading to a
long loop between strands C and E was observed (18). This loop contains
the His-56-Thr-60 region that mediates the access of the solvent to
Cys-10 and to Met-13. The effect of the His-56-Thr-60 region over the
accessibility of Met-13 to the solvent was determined for the x-ray
crystallographic structures of WT- (PDB ID: 1F41) and L55P-TTR (PDB ID:
5TTR), using Turbo-Frodo (27). The accessible surface of Met-13 in the
WT-TTR is 26 Å2, half of the accessible surface of Met-13
in the L55P-TTR, which is 52 Å2. The reported values are
the averaged results for the accessible surfaces of all Met-13 in the
crystallographic asymmetric unit. The accessible surface of Cys-10 was
not calculated since the N-terminal in these TTR crystal structures is
disordered.
INTRODUCTION
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-sheet conformation, implicated in the transport of
thyroxine and vitamin A. Over 80 TTR variants are described in the
literature (1), most of them are pathogenic and related to amyloidosis.
While V30M-TTR is the most frequent amyloidogenic variant, L55P-TTR is
the variant associated with the most aggressive form of FAP described
in the literature.
-sheet structure (6), and two structural models were
proposed: a continuous
-sheet helix (7) or an association of units
with a structure close to the TTR monomer (8). The two models are
obviously different, and neither of them refers to the packing
interactions between the different subunits.
-amylase, and human serum albumin have been
reported (12).
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20 °C. This sample, designated by L55P-TTR protein solution, was
restored to room temperature immediately before the spectroscopy
experiment. The other part of the protein sample was incubated at
37 °C for 2 weeks. Then the sample had amyloidogenic behavior as
revealed by the thioflavine-T assay and was designated by L55P-TTR
fibril solution.
4. The sample
fluorescence is collected either by a silicon photodiode or by a high
purity germanium fluorescence detector.
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pattern,
characteristic of the amyloid material. Data from L55P-TTR is shown in
Fig. 1, with a sharp 4.85 Å reflection
on the meridian and a diffuse equatorial 10.3 Å reflection. The
spacing and relative intensities of the observed meridional and
equatorial reflections are listed in Table I.
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Fig. 1.
X-ray diffraction patterns from L55P-TTR
amyloid fibrils. Specimen-to-film distance was 245 mm and the
exposure time 30 s.
Spacing of equatorial and meridional reflections
-strands along the fibril axis, leading to
the 116 Å repeat.
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Fig. 2.
Sulfur K-edge XANES spectra for the reference
compounds.
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Fig. 3.
Sulfur K-edge XANES spectra of protein
solutions (a) and fibril solutions (V30M-, WT-, and
L55P-TTR) (b). V30M-TTR spectra are present in
blue, WT-TTR in black, L55P-TTR in
red and C10S-TTR in green.
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Fig. 4.
Experimental and simulated sulfur K-edge
XANES spectra for the TTR samples (thick lines,
experimental; thin lines, simulation). Soluble
WT-TTR (a), V30M-TTR (b), L55P-TTR
(c), and C10S-TTR (d); and fibrillar WT-TTR
(e), V30M-TTR (f), and L55P-TTR (g).
The percentages of the different forms of sulfur used in the
simulations are presented in Table II.
The percentage of the different forms of sulfur used to simulate the
sample spectra
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Fig. 5.
Sulfur K-edge XANES spectrum of soluble
L55P-TTR and the spectra generated by linear combination of the
reference sulfur forms spectra. The experimental and best fit of
the simulated spectra are presented in red (thick
and thin lines, respectively). The black,
blue, and green spectra are simulations using the
percentages of oxidation form of sulfur found in the WT-, V30M-, and
C10S-TTR soluble samples.
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Fig. 6.
Thioflavine-T binding assay for WT-TTR and
reduced WT-TTR amyloid fibrils formed by acidification.
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Fig. 7.
The L55P-TTR monomer (PDB ID: 5TTR) showing
the local electrostatic environment around Cys-10 and Met-13. In
salmon, the Gly-53-Gly-57 main chain conformation and side chain of
His-56 of WT-TTR. The figure was produced with SETOR (33).
Since in the fibrils we observe a substantial increase in the number of
Cys-10 and Met-13 amino acids in the oxidized form, we propose that
this occurs due to a movement of the protein main chain in the edge
region His-56-Thr-60, as it happens in the case of L55P-TTR. In
2-microglobulin, another
-sandwich protein, it was reported that
a restructuring of a
bulge that separates two short
strands
leads to a protein with a longer strand located at the edge of the
molecule and increased amyloidogenic properties (28, 29). Besides this
rearrangement of the edge strand, the authors reported a rotation of
His-51 increasing the hydrogen bonding potential of that strand and
therefore allowing the formation of intermolecular interactions. In the
case of TTR, also His-56, located at the edge
-strand that we
believe is modified for fibril assembly, has a different conformation
in WT- and L55P-TTR (Fig. 7). Furthermore, a contact between this amino
acid and a glutamic acid of a nearby molecule was reported as an
abnormal interaction that might be important for amyloid formation
(18).
Recently hydrogen/deuterium exchange combined with NMR analysis was
used to identify the stable core of 2-microglobulin amyloid fibrils
and suggests a loss of structure at the edge of the native
-sheet
during self-assembly of the protein (30). In a different study, it was
revealed that six of the seven strands present in this
-sandwich
protein are protected from H/D exchange in the fiber structure
(31).
According to the CATH-Protein Structure Classification (32), TTR and
2-microglobulin belong to the main
-class of proteins, both have
a sandwich architecture and an immunoglobulin-like topology. Therefore,
a high level of structure similarity exists between the two proteins,
and it is most probable that, as in the case of TTR, the two
-sheet
cores are maintained in the fibrils. An assembly mechanism, where two
edge strands are moved away and the penultimate strands A and B are
exposed was recently proposed for TTR (15). This would certainly
explain oxidation of cysteine (near strand A) and methionine (on strand A).
The x-ray diffraction patterns of TTR amyloid fibrils revealed the
classic -structure with the hydrogen bonds within the
-sheets
parallel to the fiber axis. The presence of a broad reflection at 10 Å indicates that the fiber is composed of two
sheets (14) as
previously proposed. Along the fiber axis, the 29 Å repeating unit may
correspond to a monomeric species, although only the 6th and higher
order reflections are detected. The lower order reflections were not
seen and are not usually detected (7, 8), which may indicate that the
fundamental spacing of 29 Å includes an ordered region with the
-chains perpendicular to the fibril axis, the core of the native
protein, and a disordered domain (8) that may be due to the structural
alterations in the edge strands.
We conclude that in TTR amyloid fibrils the repeating units are
composed of two -sheets, resembling the TTR monomer, and that a
structural alteration of the protein D-strand and the DE-loop lead to a
disordered region that exposes Cys-10 and Met-13 to the solvent. We
believe that the newly exposed surface is responsible for promoting
protein aggregation. Furthermore, Cys-10 does not form disulfide
bridges, and therefore such bridges are not responsible for the highly
packed structure present in the fibrils. The thioflavine-T binding
assays also show that the reduced protein is susceptible to amyloid
formation by acidification. Therefore oxidation, at the native protein
state, does not seem to promote amyloid fibril formation. Future
studies involve the use of compounds that specifically bind the
sulfur-containing amino acids in order to test their role in protein aggregation.
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ACKNOWLEDGEMENT |
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We thank P. Moreira for excellent technical assistance.
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FOOTNOTES |
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* This work was supported by PRAXIS Grants POCTI-35735/99 and POCTI/SAU/14095/1998 and ESRF, beamlines ID13 and ID21.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.
To whom correspondence should be addressed. Tel.:
351-22-6074900; Fax: 351-22-6099157; E-mail: amdamas@ibmc.up.pt.
Published, JBC Papers in Press, January 21, 2003, DOI 10.1074/jbc.M210798200
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ABBREVIATIONS |
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The abbreviations used are: FAP, Familial Amyloidotic Polyneuropathy; TTR, transthyretin; WT, wild type; ESRF, European Synchrotron Radiation Facility; STEM, scanning transmission electron microscopy; XANES, x-ray absorption near-edge structure.
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