Received for publication, June 3, 2002, and in revised form, September 27, 2002
The interaction of A
peptides with the lipid
matrix of neuronal cell membranes plays an important role in the
pathogenesis of Alzheimer's disease. By using EPR and CD spectroscopy,
we found that in the presence of Cu2+ or
Zn2+, pH, cholesterol, and the length of the peptide chain
influenced the interaction of these peptides with lipid bilayers. In
the presence of Zn2+, A
40 and A
42 both inserted into
the bilayer over the pH range 5.5-7.5, as did A
42 in the presence
of Cu2+. However, A
40 only penetrated the lipid bilayer
in the presence of Cu2+ at pH 5.5-6.5; at higher pH there
was a change in the Cu2+ coordination sphere that inhibited
membrane insertion. In the absence of the metals, insertion of both
peptides only occurred at pH < 5.5. Raising cholesterol to 0.2 mol fraction of the total lipid inhibited insertion of both peptides
under all conditions investigated. Membrane insertion was accompanied
by the formation of
-helical structures. The nature of these
structures was the same irrespective of the conditions used, indicating
a single low energy structure for A
in membranes. Peptides that did
not insert into the membrane formed
-sheet structures on the surface of the lipid.
 |
INTRODUCTION |
Alzheimer's disease
(AD)1 is a neurodegenerative
disorder affecting the memory and cognitive functions of the brain. A
characteristic central nervous system histologic marker in patients
with AD is accumulation of the 39-43-residue amyloid
-peptide
(A
) in morphologically heterogeneous neuritic plaques and
cerebrovascular deposits (1). A
is derived from a proteolytic
cleavage of the
-amyloid precursor protein (APP). This is a highly
conserved and widely expressed integral membrane protein with a single
membrane-spanning domain. Possible mechanisms of A
toxicity include
formation of plasma membrane channels (2) and generation of
H2O2 through Cu2+ reduction by the
peptide, which make cells more responsive to oxidative stress (3). The
mechanisms may be related because reactive oxygen species may cause
lipid peroxidation that leads to alterations in the order and fluidity
of the bilayer lipids (4) and may also lead to an increase of membrane
permeability. The common change observed in cell membrane permeability
is an increased intracellular calcium level (5, 6) that could occur
either indirectly through A
modulating an existing Ca2+
channel or directly through cation-selective channels formed by
A
.
The supramolecular structure of membrane-associated A
, either as an
ion channel or a fusogen, is unknown, although Durell et al.
(7) have developed theoretical models for the structure of ion channels
formed by the membrane-bound A
40. Recently, Bhatia et al.
(8) and Lin et al. (9) used atomic force microscopy, laser
confocal microscopy, and fluorescent calcium imaging to examine in real
time the acute effects of fresh and globular A
40, A
42, and
A
25-35 on cultured endothelial cells. A
peptides caused morphological changes within minutes after treatment and led to eventual cellular degeneration. Cellular morphological changes were
most sensitive to A
42, being seen at nanomolar concentrations and
accompanied by an elevated intracellular calcium level. Atomic force
microscopy of A
42 reconstituted in a planar lipid bilayer showed
multimeric channel-like structures. Biochemical analysis showed that
the predominantly monomeric A
peptides in solution formed stable
tetramers and hexamers after incorporation into lipid membranes.
In our initial study using the EPR technique (10), we found that, in
the presence of Cu2+ and Zn2+, A
42 formed
allosterically ordered
-helical structures that penetrated
negatively charged membranes. This paper extends that study to include
the influence of pH, metal concentration, peptide length, and
cholesterol on the conformation and incorporation of the peptide into
the lipid bilayer. We show that penetration of the bilayer is closely
related to conditions favorable to oligomerization of the peptide. The
significance of this finding is that metals play an important part in
the formation of amyloid plaques, the end point of oligomerization.
Cherny et al. (11) have shown that administration of a
lipophilic Cu2+/Zn2+ chelator
(5-chloro-7-iodo-8-hydroxyquinoline) that crosses the blood-brain
barrier attenuates plaque formation in APP2576 transgenic mice
expressing human APP. Recently, the importance of Zn2+ in
plaque formation has been emphasized by the finding that the age and
female sex-related plaque formation characteristic of these mice was
greatly reduced if they lacked zinc transporter 3, which is required
for zinc transport into synaptic vesicles (12).
 |
EXPERIMENTAL PROCEDURES |
Materials and Methods--
Peptides were obtained from
AusPep (Melbourne, Australia) and from the W. M. Keck
Laboratory, Yale University. The acidic phospholipid spin label 16NPS
was synthesized according to Hubbell and McConnell (13). The
water-soluble spin label TCC was obtained from Molecular Probes. All
spin probes were checked for purity and to ensure that their number of
spins/mol were >90% of theory (14). Cholesterol and synthetic
POPC was purchased from Sigma, and POPS was purchased from Avanti Polar
Lipids (Pelham, AL). LUV were prepared by the method described by Hope
et al. (15). Peptides as a freeze-dried powder were added to
the desired concentration to a suspension of LUV in buffer, and the
mixture was vortexed under N2 for 10 min at 305 K in
polypropylene tubes. One hundred-millimolar stock solutions of
analytical reagent grade CuCl2 or ZnCl2 were prepared, and the desired amount was added to the LUV after addition of
the peptide. 0.05 M of the chelator EGTA was added to all
control solutions to counter the possible effects of any trace metals present. For copper EPR measurements, 99.99% pure 65Cu
(Cambridge Isotopes) was used. Metal concentrations were measured by
ICP-MS (model 700, Varian), and peptide concentrations were determined
by quantitative amino acid analysis. Uptake of peptide by the LUV was
estimated by density gradient centrifugation in MetrizamideTM (Nyegaard, Oslo, Norway)-Tris buffer (pH 7.0, 0.05 M) as described by Cornell et al. (16) and
then measuring the extinction at 210 nm of the sub-phase. The
protein concentration was estimated by using an
E
of 204 (17). It was
found that 10-15% of the added peptide was not taken up by the
LUV.
EPR Spectroscopy--
Continuous wave X-band EPR spectra were
obtained using a Bruker ECS106 spectrometer equipped with a temperature
controller and flow-through liquid nitrogen cryostat. Cu2+
spectra were collected at 110 K from samples contained in 4-mm inner
diameter "Suprasil" quartz EPR tubes (Wilmad). In order to
eliminate the possibility that any line broadening observed in the
spectra might be due to freezing-induced localized concentrations of
sample, 10% glycerol was added to aqueous peptide buffer solutions. Labeled lipid samples (25 µl) were contained in 0.8-mm inner diameter quartz capillaries (Wilmad) and handled as described by Gordon et
al. (18) to ensure reproducibility. Other procedures, including adding to the spin-labeled SUV a small amount of the water-soluble, non-membrane penetrant spin probe TCC to ensure x axis
reproducibility, have been described previously (10, 19).
The analysis of EPR spectra of peptide/lipid mixtures was carried out
using the spectral subtraction and addition methods described by Marsh
(20). As a check on the validity of these procedures as applied to our
system where an MRLC was observed, the lipid spin label spectra were
simulated using the modified Bloch equations as described by Davoust
and Devaux (21). Their model I was used in the simulations where the
unique director orientation in the fluid component, corresponding to
the nitroxide axes being aligned along the membrane normal in the MRLC,
was assumed to be preserved on exchange.
CD Spectroscopy--
CD spectra of peptides in the LUV were
obtained using a CD spectropolarimeter model 62DS (AVIV, Lakewood, NJ)
as described previously (10).
 |
RESULTS |
Effect of pH and Peptide Length on Interaction of A
Peptides
with LUV--
Fig. 1 shows the EPR
spectra recorded at 305 K of 16NPS in POPS/POPC (1/1) LUV and the same
LUV containing A
42 before and after the addition of Cu2+
(0.3/1 M peptide) at pH 7.5, 7.0, 6.5, 6.0, and 5.5. The
sharp lines due to the TCC marker and the curved base line due to the broad Cu2+ line have been subtracted as described
previously (10). It can be seen that adding Cu2+ to the
vesicles at all pH values caused the appearance of the MRLC (marked
with an arrow) that is characteristic of annular phospholipid surrounding a rigid peptide inserted into the bilayer (20). At pH 5.5 the spectrum of the non-Cu2+-treated
A
-containing LUV clearly shows the MRLC, at a slightly lower
intensity than in the Cu2+-treated sample at the same pH.
The control spectra (set A) were subtracted from those of
the Cu2+-treated LUV and from the pH 5.5 peptide-containing
LUV to give the difference spectra (set D). These
experiments were repeated over the same range of pH with
Zn2+ (4/1 M peptide) and A
42 and with
Cu2+ and Zn2+ at the respective 0.3 and 4.0 molar ratios with A
40. No MRLC was found with rat A
40 with both
metals over the whole pH range (data not shown). By performing the
subtraction with spectra of control LUV made at 286 K, as done
previously with both A
42 (10) and a viral peptide (19), it was shown
that the peptides did not perturb the bulk lipid, as suggested by
McIntyre et al. (22). All of the spectra could be simulated
over the range of peptide/lipid mixtures used, as described by Davoust
and Devaux (21), further discounting long range perturbation effects on
the bulk lipid by the peptide at high peptide/lipid. The on- and
off-rate constants were estimated from the simulation; the former were
in the range 6-5.5 × 106 (s
1), and the
latter were 5.5-5 × 106 (s
1). These
values are similar to those published for other lipid/protein systems
(23).

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Fig. 1.
EPR spectra of 16NPS in POPS/POPC LUV over
the pH range 5.5-7.5 (A), after the addition of
A 42 (1 M peptide/30 M lipid)
(B), after the addition of 0.3 M
Cu2+/1 M peptide to the samples in B
(C), and difference spectra obtained by subtracting
the spectra in A from the corresponding spectra in C
(D). Spectra were recorded at 305 K. Instrument
conditions are as follows: microwave frequency 9.237 MHz, modulation
frequency 100 KHz, modulation amplitude 3.2 G, microwave power 6 milliwatts, sweep time 400 s, and time constant 0.5 s. Scan
width was 100 G and mid-field 3,300 G.
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The proportion of MRLC to total lipid was calculated by
double-integrating the five sets of difference spectra thus obtained and expressing the number of spins as a percentage of the number of
spins in each experimental spectrum. The values are given in Table
I. It can be seen that, with A
42,
decreasing pH with Cu2+ and Zn2+ leads to an
increase in the MRLC. It is notable that there is a significant MRLC at
pH 5.5 in the absence of either metal. With A
40, however, there is a
marked difference between the behavior of Cu2+ with pH in
that there is negligible MRLC at pH 7.0 and 7.5 with the
Cu2+, although with the Zn2+ the proportion of
MRLC at all pH values is similar to that found with Zn2+
and A
42. Adding Zn2+ to the pH 7.0 and 7.5 A
40/Cu2+ samples (2 M Zn2+/0.3
M Cu2+) resulted in the appearance of the MRLC.
At the lipid/peptide ratio employed (30/1), the proportion of MRLC
corresponded approximately to four lipid molecules/peptide. This value
was previously observed to remain constant for A
42 (10) over a range
of lipid/peptide ratios, and it can be satisfied on structural grounds
by postulating that the basic membrane penetrant unit is a
-helical
hexamer surrounded by 24 lipids (10).
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Table I
Effect of pH on percentages of MRLC in EPR spectra and -strand and
-helix content of A 40 and A 42 in LUV
Figures at left margin of each column show % MRLC, figures in
parentheses show % -strand (italics) and -helix (boldface).
Difference between 100% and sum of and was due to
conformational flexibility.
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We have shown previously (10) that the appearance of the MRLC in the
A
42 negatively charged LUV system was associated with an increase in
-helicity of the peptide. The %
-helix (bold type) and
-structure (italics) are shown alongside the MRLC data in Table I.
It can be seen that wherever significant MRLC is present, the peptide
showed increased
-helicity. Wherever the peptide does not show a
MRLC, there is a preponderance of
-structure.
Effect of pH on the Coordination Sphere of Cu2+ in Both
Peptides in the LUV--
EPR spectra taken at 110 K of
65Cu2+-A
28 in buffer over the pH range
5.5-7.5 (Cu2+/peptide 0.3/1 M) are shown in
Fig. 2A. Identical spectra
were obtained at the same pH values for A
40 and A
42 in the LUV
and for A
16 in buffer. All of the spectra are characteristic of type 2 square planar copper. However, the notable feature of the series is a
decrease in g
(marked with arrow in Fig.
2A) between pH 5.5 and 6.0 and a further decrease between pH
7.0 and 7.5 indicating a change in the 65Cu2+
coordination sphere over these two pH ranges. The spectral parameters are given in Table II. As observed
previously (10) for A
42, spectra for
65Cu2+-A
40 in 16NPS-LUV showed no signs of
line broadening due to dipolar-induced spin lattice relaxation by the
nitroxide-free radical (24), indicating that the
Cu2+-binding site of the peptide did not penetrate the
bilayer and come into the proximity of the nitroxide spin label.
However, Cu2+ molar ratio-dependent line
broadening due to Heisenberg exchange, the result of Cu2+
atoms being close in space, had been observed previously in A
complexes (10). Those spectra had been recorded of samples in pH 6.9 buffer. Fig. 2B shows the effect of a range of pH on this line broadening. It can be seen that, at the 0.7 Cu2+ molar
ratio used, line broadening was present from pH 6 to 7, much reduced at
pH 7.5, and eliminated at pH 8. The broadened spectrum at pH 5, which
is markedly different to that seen at pH 6-7, was due to the presence
of unbound Cu2+, indicating a weakening of the strong
binding to the metal observed at the higher pH values, and
corresponding to the protonation of the imidazole side chain of the
histidine residues. There were no peaks at g >4 (not shown
in Fig. 2B), indicating the absence of axial
Cu2+ dimers, nor was there any marked reduction of signal
intensity, diagnostic of the formation of EPR "silent" dicopper
(25).

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Fig. 2.
A, EPR spectra taken at 110 K of
Cu2+-A 28 in aqueous buffer over the pH range
5.5-7.5 (Cu2+/peptide 0.3/1 M). B,
EPR spectra taken at 110 K of Cu2+-A 28 in aqueous buffer
over the pH range 5.0-8.0 (Cu2+/peptide 0.7/1
M). Instrument settings are as follows: frequency
A, 9.7660 GHz; frequency B, 9.7655, modulation
frequency 100 KHz, modulation amplitude 1.011 G, microwave power 2 milliwatts, sweep time 83.886 s, time constant 10.240 ms. Spectra
averaged over 36 sweeps.
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Table II
EPR parameters of the type-2 Cu2+ A complexes at
different pH values
The hyperfine data are given for 65Cu.
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From Fig. 3 it can be seen that the
increase in the amount of MRLC at pH 6.0 with both peptides reaches a
maximum at a Cu2+ concentration of 1 mol/mol, after
which it remains constant. The effect of Zn2+ follows a
similar pattern, albeit at higher concentrations, requiring almost 3 eq
in keeping with the lower affinity of A
for Zn2+
(26).

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Fig. 3.
Effect of increasing metal concentration on
proportion of MRLC in spectra of 16NPS in POPS/POPC LUV at pH 6.5. Closed circles, A 42 + Cu2+; closed
squares, A 40 + Cu2+; open circles,
A 42 + Zn2+; open squares, A 40 + Zn2+. Peptide/lipid, 1/30.
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Effect of Cholesterol on the MRLC--
In the light of numerous
reports on the significance of cholesterol on A
membrane
interactions in both biological membranes and model systems (27-29),
the effect of adding cholesterol to the LUV on MRLC formation was
investigated. The results are summarized in Table
III where it can be seen that at each pH
value and metal where the component was observed in the absence of
cholesterol it was reduced to zero when cholesterol represented 0.2 mol
fraction of the total lipid, i.e. cholesterol reduced the
stability of the membrane-penetrant form of the peptide.
The Effect of Adding Cu2+- to Zn2+-A
40
in the Spin-labeled LUV--
Because it had been observed that the
appearance of MRLC in the spin-labeled LUV after the addition of
Zn2+-A
40 was independent of pH, and it was
pH-dependent after the addition of Cu2+-A
40
(Table I), the effect of adding Cu2+ to
Zn2+-A
40 LUV was explored. It can be seen (Fig.
4) that at pH 7.0 the addition of
Cu2+ markedly reduced the percent MRLC until none was
detectable at 1 M Cu2+/M of
peptide. This result indicates that Cu2+ competes with
Zn2+ for at least one of the His residues of A
40, but
with a coordination sphere such that the stability of the complex is to
too low to promote membrane insertion.

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Fig. 4.
The effect of adding Cu2+ to
Zn2+-A 40 in spin-labeled LUV at pH
7.0 on the percentage of MRLC. Lipid/peptide, 1/30
(M/M), metal/peptide, 4/1
(mol/mol), temperature 294 K.
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 |
DISCUSSION |
pH, Metal-induced Membrane Penetration--
The addition of metal
ion and pH are two conditions that govern the formation of the MRLC
indicating peptide penetration of the hydrocarbon core of the membrane
by A
peptides in LUV (Fig. 1), and these conditions closely parallel
those governing A
oligomerization (30). In the absence of metals and
in the presence of the chelating agent EGTA, both A
40 and A
42
penetrate the membrane at pH 5.5 but not at higher pH values. Both
peptides aggregate in the absence of either metal at this pH (30). In
the presence of Zn2+ both peptides penetrated the membrane
over the entire range from pH 5.5 to 7.5. Zn2+ aggregates
A
across this pH range (31). A
40 in the presence of
Cu2+ only penetrated the membrane at pH 6.5 and below, pH
values at which it has been reported to oligomerize (32). On the other hand, A
42 in the presence of Cu2+ penetrates and is
oligomerized in solution at all pH values. It should be noted that rat
A
40, which has a 2N2O coordination (10) and much-reduced
metal-induced oligomerization (33), did not produce an MRLC with either
metal at any pH. The significance of this result is that the
selectivity of human over rat A
40 is one of the key
metal-differentiating effects that argues strongly for metals being
germane to the pathophysiology of AD. The induction of MRLC by
copper-bound A
42 was saturated at 1 eq (Fig. 3); this parallels our
previous observation that the cooperative binding of Cu2+
to A
was also saturated at 1 eq (10).
Our results showed that membrane penetration was associated with an
increase in
-helicity as determined by CD spectroscopy, whereas the
CD spectra of peptide associated with liposomes showing no evidence of
MRLC indicated predominantly
-structure (Table I). These results
indicated that peptide association with the hydrocarbon region of the
lipid bilayer facilitated
-helix formation. Previously published CD
studies of the interaction between the A
peptides and lipid vesicles
of varying composition have suggested that the peptides associated with
the lipids are in the
-strand conformation (34-36). Most of these
studies were limited to A
25-35, a peptide that is too short to be
able to span the lipid bilayer in an
-helical conformation. On the
other hand, NMR structural studies in SDS micelle systems show that
residues 15-36 are
-helical with a kink in the 25-27 region (37,
38).
Calculation of the annular lipid/peptide ratio allows an estimate of
the size of the molecule/complex penetrating the membrane. For all the
spectra exhibiting MLRC the same value of 4-4.5/1 was obtained,
consistent with an
-helical hexameric peptide structure inserted
into the bilayer in line with our previous results (10). The constancy
of this ratio over a range of peptide/lipid suggested that formation of
multimers occurs at relatively low value of the latter and that their
size remains constant with increasing proportion of peptide in the
bilayer. Factors influencing the interpretation of the stoichiometry of
lipid-protein interactions determined by EPR have been discussed
in detail by Marsh and Horváth (39).
It has been postulated that the ion channels that have been observed in
various membrane systems could consist of either strands arranged in a
-barrel conformation or as an
-helical assemblage, probably a
tetramer or hexamer (see Refs. 7-10 and the data presented here). It has been shown by Horváth et al. (23)
and Wolfs et al. (40), however, that the motion of the MRLC
observed with membrane penetrant segments of known
-barrel structure
is much more restricted than with
-helical segments. This is because of a slower on-off rate for the lipids in the former case. The on-off
rates calculated by simulation in our case are close to those observed
for other
-helical systems (20).
Adding metal ions or lowering the pH are two different methods of
aggregating A
. In the absence of lipid, metal ions induce amorphous
aggregates of A
, and these oligomers are different from those formed
in the pH-induced oligomerization of A
. These have a fibrillar
-sheet structure as shown by Congo Red birefringence (41). In lipid
systems, however, both methods of A
oligomerization give the same
membrane penetrating
-helical structure, suggesting a well defined
low energy structure for A
in membranes. The observation of an
-helical hexamer is consistent with the results of the atomic
force microscopy study by Lin et al. (9) that showed that
A
peptides were able to form hexameric channel-like structures. The
greater stability of A
42 in the lipid is also consistent with the
observation by Bhatia et al. (8) showing a higher tendency
by A
42 to form channel-like structures and a greater ability to
disrupt cellular Ca2+ regulation than A
40. The two extra
hydrophobic residues at the C terminus of A
42 would result in this
peptide having a higher preference for the hydrophobic core of the
lipid bilayer. The effect of the extra two hydrophobic residues is
amplified in the hexameric unit, as there are 12 extra hydrophobic
residues per unit for A
42 versus A
40.
We have shown that Zn2+ will induce A
penetration of the
lipid with the formation of structures that are similar to the channels proposed by Lin et al. (9). However, their results
showed that Zn2+ and an antibody directed against the N
terminus had an inhibitory effect on A
channel activity (9).
Although our results show that metal ions can induce oligomerization
and membrane penetration, the metal-binding site itself is outside the
lipid bilayer (10). The antagonistic action of Zn2+ may be
due to its ability to extensively cross-link the three histidine
residues of A
to form a cap over the channel, physically blocking it.
pH-induced Differences between A
40 and A
42--
There is a
significant reduction in Cu2+-facilitated membrane
insertion of A
40 at pH values of 7.0 and above, contrasting with A
42 that showed consistent membrane insertion across the pH range investigated. Our present and earlier EPR data (10) indicated that
Cu2+ formed type 2 square planar complexes with A
peptides. The spectra were independent of the peptide chain length and
the presence or absence of lipid, but were influenced by pH. For type 2 complexes the g
and
A
values correlated with the types of
equatorially coordinated atoms. The decrease in
g
(Fig. 2A and Table II) between
pH 6.0 and 5.5 and a further decrease between pH 7.0 and 7.5 indicated
a change in the 65Cu2+ coordination sphere over
these two pH ranges. This is further illustrated in Fig. 2B
where the Cu2+ molar fraction-dependent line
broadening attributed to Heisenberg exchange effects (10) diminishes
with increasing pH, suggesting a change in the coordination sphere such
that the Cu2+ atoms are no longer close in space. This
would be consistent with reduction in the extent of histidine bridging
between Cu2+ centers, although this was not eliminated
until pH 8. Such a change in the coordination mode reflects a change in
the Cu2+ from a coordination sphere that promotes peptide
oligomerization such as with a histidine residue bridging copper atoms
(a coordination sphere that resembles the active site of superoxide
dismutase) to a mode that does not. Miura et al. (42) and
Suzuki et al. (43) have shown by using Raman spectroscopy
that Cu2+ binds to the N
of the
three His residues of A
at pH 5.8, whereas at pH 6.6 and 7.4 it
could bind to the N
and an as yet
unidentified backbone amide nitrogen. Zn2+, on the other
hand, binds to the N
over the pH range
5.8-7.4. A change in the coordination mode of the Cu2+
bound to A
at higher pH that also reduces the oligomerization of the
peptides would explain the observed reduction of copper-induced MLRC by
A
40 at these pH values. These observations are consistent with the
reduced aggregation of A
by copper at higher pH (32). The extra
hydrophobic stabilization of A
42 versus A
40 is able to
shift the pH-induced change in equilibrium between the different coordination modes of Cu2+ back to the form that favors
membrane penetration and peptide oligomerization; this is a form that
resembles the active site of superoxide dismutase as we have shown
previously (10). Zn, which does not undergo a pH-induced change
in metal coordination, induces peptide insertion into the membrane with
both A
40 and A
42 over the pH range investigated. However, from
the data shown in Fig. 4 it appears that the 1 eq of copper at pH 7.0 will displace the zinc and inhibit A
40 insertion into the membrane.
Competition between Cu2+ and Zn2+ at this pH
for binding to at least one of the histidine residues would explain
these results. The different Cu2+ coordination modes and,
hence, lipid interactions may explain the increased toxicity normally
associated with A
42 because this peptide is more stable in the lipid environment.
The Effect of Cholesterol on Membrane Penetration--
Cholesterol
is a major component of mammalian cell membranes and is known to affect
the dynamics and thickness of the hydrocarbon region of the membrane.
It has been widely reported to modulate A
-membrane interactions, and
there is strong circumstantial evidence that it has a role to play in
AD (27-29). Increased membrane cholesterol reduces the membrane
disordering effects of A
, as shown by fluorescence polarization
techniques, and inhibits its effects on Ca2+ signaling
(35). Our results show that enhanced membrane cholesterol has the
marked effect of decreasing MRLC formation by A
. Penetration of the
membrane by the peptides would depend on a balance between the
rigidifying effects of cholesterol and the possible better match of the
hydrophobic region of the peptide by the increased bilayer thickness.
Because the
-helical hydrophobic regions of A
peptides span
22-25 residues (37), close to the 20-23-residue width of the
unexpanded bilayer, it is likely that the rigidifying effects will
predominate, resulting in peptide exclusion. Such exclusion in the
presence of cholesterol would explain the inhibitory effects of the
latter on Ca2+ signaling, because A
-induced channels
would be destabilized in lipid rich in cholesterol.
Overall, the data suggest the location and stability of A
peptides
within the membrane is delicately balanced, and as such, small changes
in pH and lipid composition would be able to affect the stability of
the A
-lipid interactions. Because neuronal cells are in a
dynamic environment, with respect to pH and lipid composition that
varies with different sub-cellular membrane fractions, it is not
surprising that other workers (44) have found wide variations in the
effects of A
on membrane order and fluidity in such fractions. A
challenge for the future will be to define the links between the
biophysical observations made in these diverse systems with the
pathophysiology of AD.
Published, JBC Papers in Press, November 14, 2002, DOI 10.1074/jbc.M205455200
1.
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Weinman, N. A.,
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3.
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Montessuit, S.,
Lewis, S.,
Martinou, I.,
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