(Received for publication, November 1, 1994)
From the
Amyloid- peptide (A
) consists of a hydrophobic
C-terminal domain (residues 29-42) that adopts
-strand
conformation and an N-terminal domain (amino acids 10-24) whose
sequence permits the existence of a dynamic equilibrium between an
-helix and a
-strand. In this paper we analyzed the effect of
the alternate N-terminal conformations on amyloid fibril formation
through the study of the analogous A
peptides containing single
amino acidic substitutions. The single mutation of valine 18 to alanine
induces a significant increment of the
-helical content of A
,
determined by Fourier transform infrared spectroscopy and circular
dichroism and dramatically diminishes fibrillogenesis, measured by
turbidity, thioflavine T binding, Congo red staining, and electron
microscopic examination. In hereditary Dutch cerebral hemorrhage with
amyloidosis (a variant of Alzheimer's disease), the substitution
of glutamine for glutamic acid at position 22 decreased the propensity
of the A
N-terminal domain to adopt an
-helical structure,
with a concomitant increase in amyloid formation. We propose that
A
exists in an equilibrium between two species: one
``able'' and another ``unable'' to form amyloid,
depending on the secondary structure adopted by the N-terminal domain.
Thus, manipulation of the A
secondary structure with therapeutical
compounds that promote the
-helical conformation may provides a
tool to control the amyloid deposition observed in Alzheimer's
disease patients.
Alzheimer's disease (AD) ()is the most common
form of dementia in adults. AD is characterized neuropathologically by
amyloid deposition in the form of neuritic plaques and congophilic
angiopathy as well as by the formation of neurofibrillary
tangles(1) . The main component of amyloid is the 4.3-kDa
amyloid
-peptide (A
), which is part of a much longer
precursor protein (APP) codified in chromosome 21(2) .
Amino
acid sequence analyses of the A peptide by the Chou-Fasman (3) and Garnier-Osguthorpe-Robson (4) methods indicate
that the probability of finding
-strand conformation in A
is
high within the C-terminal region after residue 28. The region between
amino acids 10 and 24 presents a high and similar probability to
display
-helix or
-strand conformation(5) . There are
also two probable
-turns between residues 6 and 8 and between
residues 24 and 29. Using synthetic peptides and spectroscopic
techniques, the secondary structure assignments obtained by predictive
methods have been
confirmed(6, 7, 8, 9) .
While the
hydrophobic segment in the C-terminal domain of A invariably
adopts a
-strand structure in aqueous solutions, independently of
pH or temperature conditions, the N-terminal domain can show different
conformations and solubilities depending on environmental
conditions(8, 9, 10) . In fact, the
N-terminal domain exists as a soluble monomeric
-helical structure
at pH 1-4 and pH greater than 7. However, at pH 4-7 it
rapidly precipitates into an oligomeric
-sheet structure.
Furthermore, it adopts an
-helical conformation in a
membrane-mimicking solvent that promotes intramolecular hydrogen
bonding(8) . Thus, A
exists in two alternative
conformations depending on the secondary structure adopted by the
N-terminal domain. These alternative conformations have different
solubility properties and may determine changes in the rate of amyloid
fibril formation. We had hypothetized that the structure adopted by the
N-terminal domain of A
is important in amyloid fibril
formation(5) . In the present work we have evaluated this
possibility by analyzing the ability of A
sequences containing
single mutations in its N-terminal domain to form amyloid fibrils.
Specifically we studied amyloid formation using peptides containing the
mutation of valine to alanine at residue 18, and glutamic acid to
glutamine at position 22 (Dutch variant of A
).
Figure 2: Aggregation and amyloid formation by SP40 and SP40A. The aggregation level obtained with different concentrations of SP40 (squares) and SP40A (circles) was studied (A). The peptides were incubated for 48 h at 25 °C in 0.1 M sodium acetate buffer, pH 5.0, at various peptide concentrations, from 1 µg/ml to 4 mg/ml. The aggregation was measured by the turbidity at 405 nm, as described in ``Experimental Procedures''. The value shown represent the mean between 2 different experiments. The amyloid formation was quantified through the ThT method (B). Aliquots of each peptide in a concentration of 1 mg/ml (0.25 mM) were incubated for 15 h in 0.1 M Tris-HCl, pH 7.5, at room temperature. Then the amyloid formation was quantified as described under ``Experimental Procedures''. The protein concentration for BSA and ubiquitin was 1 mg/ml. The graph shows the fluorescence emission, in arbitrary units, of ThT bound to the amyloid formed in the presence of the peptides indicated in the x axis. The value shown correspond to the average ± standard deviation of three different experiments made in duplicated.
Figure 4: Aggregation and amyloid formation by SP40 and SP40Q. This figure was performed under the same experimental conditions described in Fig. 2. In the turbidity measurement (A) the time of incubation was 24 h. In the fluorescence studies (B) the incubation time was 15 h. Both experiments were performed in a peptide concentration of 1 mg/ml. The mean ± standard deviation of three different experiments made in duplicated is shown.
To study the relation between the secondary structure of
A and its ability to form amyloid fibers, we designed a mutation
that does not produce a significant change in the ionic or hydrophobic
properties of A
but is sufficient to increase the propensity of
the N-terminal domain of A
to adopt the
helical conformation.
A peptide in which valine at position 18 of A
was replaced by
alanine (SP40A) was synthesized. This modification considers that
valine at position 18 is the residue that has the highest probability
value to exist as a
-strand in the N-terminal region of A
.
Valine is an amino acid that destabilizes the
-helix, whereas
alanine is a very good helix-former in aqueous solution. The above
statement is supported by thermodynamic
considerations(17, 18, 19) , by comparative
studies of the helix-forming tendency in synthetic
peptides(20) , and by statistical
surveys(3, 4) . In fact, by comparative analysis of
the secondary structure of both peptides through FT-IR, we found that
the modified peptide contains a significant amount of
-helical
structure (Fig. 1A). A quantitative analysis of the
spectra gave the following percentage of secondary structure for SP40A:
32.8% of
-helix, 31.3% of
-sheet, 24.7% of random coil, and
11.2% of
-turn, while the percentage of secondary structure for
the control peptide, containing the 1-40 sequence of A
(SP40), was: 1.1% of
-helix, 60.4% of
-sheet, 31.4% of random
coil, and 7.1% of
-turn. The higher content of
-helix in the
modified peptide was additionally found through CD studies in the
presence of 20% TFE (Fig. 1B). The
-helical
content for SP40 and SP40A in this solvent was 16.6% and 40.9%,
respectively. The higher level of
-helix obtained in the last
experiment may be due to the presence of TFE, which is a solvent that
stabilizes the
-helical conformation (21) .
Figure 1:
Fourier
transform infrared and Circular dichroism studies of wild type and
mutant A peptides. In A is shown the FT-IR spectra taken
under the following conditions; peptide aliquots of 20 mg/ml (20
µl) prepared in 25 mM HEPES (disolved in
H
O), pD 7.5, were placed in CaF
cells and the spectra were collected at 2 cm
of resolution taking 500 scans. The spectra obtained with SP40 (dashedline) and SP40A (solidline) are shown. The arrows indicate the
position of the band in the amide I region corresponding to the
-helix (
1653 cm
),
-sheet (
1626
cm
),
-turn (
1696 cm
),
and random coil (
1644 cm
) structures. In B the CD spectra of SP40 (solid line), SP40A (dashed
line), and SP40Q (dotted line) is shown. Aliquots of
peptides were disolved in 20% TFE prepared in 10 mM sodium
phosphate, pH 7.4. The peptide concentration was 0.15 mg/ml in a final
volume of 0.3 ml. The spectra were recorded at room temperature as
described under ``Experimental
Procedures.''
The comparative study of the solubility properties of SP40 and SP40A was performed by means of turbidimetric measurements. After 48 h of incubation, SP40A became aggregated to a lesser extent than SP40 at equal concentration (Fig. 2A). Thus, at a peptide concentration of 4.0 mg/ml, SP40A exhibits 80% less aggregation than the control. However, an increase in the turbidity indicates only aggregation, but not necessarily amyloid formation. To semi-quantify the amount of amyloid formed under each condition, we used a novel method based on the fluorescence emission by thioflavine T (ThT) bound to amyloid(14, 15) . ThT binds specifically to amyloid and this binding produces a shift in the emission spectra and a fluorescence enhancement proportional to the amount of the amyloid formed(15) . The ability to form amyloid, measured by the ThT method, was compared between SP40 and SP40A. The modified peptide showed very little fluorescence in comparison with SP40 at a concentration of 1 mg/ml (Fig. 2B). To further evaluate the ability of SP40A to form amyloid, we performed staining with Congo red. Light microscopic examination after Congo red staining showed that only SP40 (1 mg/ml), incubated 5 days, displayed a significant green birefringence under polarized light, while the modified peptide exhibited only a slight green birefringence under the same conditions (data not shown). Furthermore, the presence and the morphology of the fibrils formed by both peptides was studied by electron microscopy. Both SP40 (Fig. 3A) and SP40A (Fig. 3B) were able to form amyloid fibrils with the typical features described by these fibers(7, 16) . Although the morphology of the amyloid fibers was identical, the amount of this structure obtained in the electron microscopic grids was higher in SP40 than in SP40A, which strengthened the results described above.
Figure 3:
Electron micrographs of negative-stained
preparations of fibrils assembled from SP40 and SP40A. Aliquots of both
peptides, SP40 (A) and SP40A (B), were adsorbed onto
300-mesh Formvar-coated grids and negative-stained with 2% uranyl
acetate. The specimens were viewed for fibrils with a Phillips electron
microscope. Magnification, 66,000.
In order to
additionally study the influence of the secondary structure of the
N-terminal domain on the rate of amyloid formation, we analyzed the
effect produced by the glutamine/glutamic acid substitution at residue
22 of A. This substitution is found in hereditary cerebral
hemorrhage with amyloidosis, Dutch type(22) . Previous studies
with synthetic peptides have shown accelerated fibril formation in a
28-residue peptide homologous to the Dutch variant A
(23) .
Our CD studies indicates that the Dutch mutation diminishes the level
of
helical conformation in comparison to the wild-type human
peptide (Fig. 1B). In fact, the
-helical content
of a synthetic peptide containing the sequence 1-40 of A
but
bearing the Dutch mutation (SP40Q), in the presence of 20% TFE was only
5.9%, in comparison with the 16.6% present in SP40. This result is in
agreement with previous studies showing a higher amount of
-sheet
in the Dutch variant than the normal A
obtained by FT-IR and CD
spectroscopy(24, 25) . To evaluate the effect of this
change in the secondary structure, we compared the ability to form
amyloid between SP40 and a SP40Q. The turbidity measurements showed
that SP40Q became aggregated at a higher level than SP40 (Fig. 4A) after 24 h of incubation. The amyloid
quantification using the ThT method showed that SP40Q formed 100% more
amyloid than SP40 (Fig. 4B) after 15 h of incubation.
This result indicates that the 1-40 peptide of the Dutch variant
A
has accelerated amyloid fibril formation, as was previously
shown for shorter peptides consisting of residues 1-28 and
21-28(23) . No differences were detected in the
morphology of the amyloid fibrils formed for SP40Q, in comparison with
SP40 (data not shown), which is consistent with previous
studies(23, 24) .
In the present work we demonstrate that a single mutation of
valine 18 to alanine produces a homologous A peptide that is less
able to form amyloid in vitro. This substitution does not
significantly modify either the ionic or the hydrophobic properties of
A
. However, this modification increases the
-helical content
of A
as was observed through FT-IR and CD spectroscopy.
Considering the position of the substitution and the analysis by
secondary structure prediction, it is widely possible that the change
in conformation occurs in the N-terminal domain of A
, which in
SP40A would adopt mainly an
-helical structure. By contrast, in
the Dutch variant of A
, the glutamine for glutamic acid
substitution at residue 22 diminishes the propensity of the N-terminal
region of A
to adopt the
-helical conformation concomitantly
with an increase in amyloid formation. These findings suggest the
existence of a correlation between the secondary structure of the
N-terminal domain (amino acids 10-24) of A
and its ability
to form amyloid fibrils.
In view of the foregoing results, we
postulate that A exists in an equilibrium between two alternative
species in solution: one able to form amyloid, which presents a
-strand conformation in its N-terminal domain; and another one
unable to form amyloid, which adopts an
-helical structure in this
region (Fig. 5). In fact, our recent studies have shown that
after long incubation times, the non-sedimentable peptide fractions
contain a large amount of
-helical and random coil structure,
completely different to the structure of the sedimentable fraction,
which presents almost completely a
-sheet conformation. Recently,
it has been proposed that in another type of amyloidosis the conversion
of an
-helix into a
-strand is a feature of the
transformation of the normal cellular prion proteins into the
pathological scrapie prion proteins(26) .
Figure 5:
Schematic representation of the
conformational equilibrium between alternative structures for A,
indicating some possible regulators of the transition. The structure
shown at left should be the soluble form of A
, which is
released by normal cells and is detected as a soluble entity in
cerebrospinal fluid from normal and AD
individuals(32, 33) . On the other hand, the structure
shown at right should be the form of A
able to form
aggregates(34, 35) , once the effective local
concentration allows interactions between
chains.
The existence of
an equilibrium between alternatives conformations for A is
additionally supported by a recent study of the tridimensional
structures of A
and its Dutch variant through NMR(27) .
Furthermore, the finding that the aggregated A
is in equilibrium
with a non-sedimentable form of the peptide (7, 28) supports the idea that in solution there are
two species of A
differing in their abilities to form amyloid.
When the N-terminal segment of A
adopts a
-strand
conformation, A
may exist as an anti-parallel
-sheet capable
of interacting with other peptides forming a cross-
supersecondary
structure. This structure has been proposed for the amyloid fibril,
based on its x-ray diffraction pattern(29, 30) .
It
has been shown previously that A analogs in which hydrophobic
residues (phenylalanine 19 and 20) were substituted by non-hydrophobic
amino acids exhibit a markedly increased solubility as determined by
sedimentation assays(8) . Accordingly, it was proposed that the
formation of aggregates depends upon a hydrophobic effect that leads to
intra- and intermolecular interactions between hydrophobic parts of
A
. Although the effect obtained with SP40A could be explained by
modifications in the hydrophobic properties induced by the
substitution, this appears unlikely because the putative hydrophobicity
changes in this case are small. Another study showing the effect of
A
(1-28) with alanine substituted by lysine at position 16
has been reported previously(31) . In that case, the modified
peptide formed
-pleated sheet assemblies that were dissimilar to
those formed by non-modified A
(1-28) and enhanced the
packing of the sheets. However, in that work, the effect of the
substitution in the aggregation kinetics was not studied.
Our data
show that the N-terminal domain of A may be important for
modulation of peptide aggregation and amyloid fibril formation. This is
an alternative view to the current model in which the determinant role
for A
aggregation is placed on its C-terminal
segment(6, 7, 13, 28) .
Our
hypothesis may explain how the same amino acid sequence can exist in a
soluble (32, 33) and an insoluble
form(34, 35) . In fact, small perturbations in the
conformational equilibrium could determine big changes in the ability
of A to form amyloid fibrils. These modifications of the
equilibrium shown in Fig. 5could, for example, be caused by
local pH changes, alterations of environmental hydrophobicity, or
binding to other proteins, which could act as pathological chaperones
such as proteoglycans (36) and apolipoprotein E (37) .
The factors that eventually produce alterations in the conformational
equilibrium of A
could be important risk factors in the
development of AD, and could explain why not all persons shown the
disease.
Finally, if the amyloid deposition is sensitive to the
secondary structure adopted by the N-terminal domain of A, as
proposed in this work, compounds that promote the
-helical
conformation in this region could prevent amyloid fibril formation. In
fact, in the presence of 1,1,1,3,3,3-hexafluoro-2-propanol, an
-helix promoting solvent, A
is completely soluble up to a
concentration of 40 mg/ml (28) . Thus, the hypothesis proposed
in this paper opens a potential new avenue to search for therapeutic
agents that can promote
-helix formation in the N-terminal domain
of A
and could stop or delay the advance of AD.