From the Department of Biochemistry and ¶ Centro
Interdipartimentale di Biologia Applicata, University of Pavia, Via
Taramelli 3b, §§ Department of Chemistry,
University of Pavia, Via Taramelli 12, and § Biotechnology
Laboratories Istituto di Ricovero e Cura a Carattere Scientifico
Policlinico S. Matteo, P. le Golgi 2, 27100 Pavia, Italy and
** Department of Biochemistry, University of Cambridge,
Tennis Court Rd. 80, Cambridge CB2 1GA, United Kingdom
Received for publication, May 16, 2002, and in revised form, September 27, 2002
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ABSTRACT |
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The N-terminal portion of apolipoprotein A-I
corresponding to the first 93 residues has been identified as the main
component of apolipoprotein A-I fibrils in a form of systemic
amyloidosis. We have been able to characterize the process of
conformational switching and fibrillogenesis in this fragment of
apolipoprotein A-I purified directly from ex vivo amyloid
material. The peptide exists in an unstructured form in aqueous
solution at neutral pH. The acidification of the solution provokes a
collapse into a more compact, intermediate state and the transient
appearance of a helical conformation that rapidly converts to a stable,
mainly Amyloid fibrils derived from apolipoprotein A-I
(apoA-I)1 have been found in
patients with autosomal dominant systemic amyloidosis who display
particular mutations in the gene encoding apoA-I (Table I) as well as in elderly humans (10) and
aged dogs (11) expressing the wild type protein. In all cases of
apoA-I-associated amyloidosis, the main constituent of the fibrils is
an N-terminal fragment of the protein, 80-93 residues long. A
growing number of proteins and peptides have been shown to form amyloid
fibrils in vivo and in vitro (12). The native
structures of these polypeptides represent the spectrum of polypeptide
conformation, from natively unstructured peptides through all -structure in the fibrils. The transition from helical to
sheet structure occurs concomitantly with peptide self-aggregation, and
fibrils are detected after 72 h. The
-helical conformation is
induced by the addition of trifluoroethanol and phospholipids. Interaction of the amyloidogenic polypeptide with phospholipids prevents the switching from helical to
-sheet form and inhibits fibril formation. The secondary structure propensity of the
apolipoprotein A-I fragment appears poised between helix and the
-sheet. These findings reinforce the idea of a delicate balance
between natively stabilizing interactions and fatally stabilizing
interactions and stress the importance of cellular localization and
environment in the maintenance of protein conformation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,
mixed helix and
, to mainly helical proteins. Several
apolipoproteins including apoA-I (1), apolipoprotein A-IV (13), murine
and human apolipoprotein A-II (14), apo E (15), and serum amyloid A
(16) have been shown to form amyloid or to be implicated in
amyloidogenic diseases, and as such, they form a unique group of
proteins, sharing structural and sequence similarities and an apparent
propensity to form amyloid fibrils.
List of the apoA-I amyloidogenic mutations so far reported and the
sequence limits of the corresponding polypeptides isolated from
natural fibrils
The plasma apolipoproteins play a critical role in lipid metabolism,
and for this reason the sequences and structures of lipoproteins have
been extensively studied. Apolipoprotein A-I is the major protein
component of high density lipoprotein particles and plays a key role in
the solubilization of lipids in these particles, the activation of
lecithin cholesterol acyltransferase, the binding of high density
lipoprotein to cell surfaces, and the promotion of cholesterol efflux
from cells (17). Sequence analysis of apoA-I and other lipoproteins has
indicated that these proteins contain repeated amphipathic helices, and
this was confirmed in the crystal structure of truncated human
apolipoprotein A-I (residues 44-243, i.e. 43) (18).
These helices, 11 or 2 × 11 residues in length, make up the lipid
binding regions of the proteins. The pattern of hydrophobic residues in
the lipoprotein helical repeats is similar to the canonical
heptad repeat of
-helical coiled coil proteins (19). It has
been known for some time that apoA-I undergoes major structural changes
when going from the free to the lipid-bound forms (20), and it has been
suggested that the N-terminal 98 residues are responsible for
maintaining a stable, lipid-free structure of apoA-I and that a
conformational switch in residues 1-43 reveals a latent lipid binding
domain (21). The shared ancestry of the lipoproteins and the
conformational plasticity required for activity may underlie the
observed common amyloidogenic nature of the lipoproteins.
In predictions of the secondary structure of apoA-I, the N terminus has
proved the most difficult to assign, and this may reflect the
conformational switching that occurs upon lipid binding. A detailed
analysis by Nolte and Atkinson (22) assigned random coil to residues
1-8, helix to 9-13, -structure from 14 to 22, coil or turn from 23 to 33, possible helix to 34-40, and well defined helix between
residues 55 and 83. The crystal structure of the
43 truncated form
of the protein, which has been shown by biochemical and biophysical
studies to have an overall structure similar to lipid-bound apoA-I
(18), has coil structure between residues 44 and 49 and well defined
helical structure in the rest of the molecule.
The amyloidogenic N-terminal fragment of apoA-I forms long, straight
amyloid fibrils with a dominant cross--structure similar to that
seen in all other forms of amyloid (12). However, we have recently
shown that elements of ordered, oriented helical structure are present
in some preparations of apoA-I amyloid material, notably in material
purified from two different patients with amyloid composed of
apoA-I-(1-93) and expressing the L174S mutation (23). The
superstructure of apoA-I amyloid can apparently accommodate some
residual helical structure, whereas the dominant stabilizing structure
is
-sheet. The successful purification of fragment 1-93 from
ex vivo tissue has made possible the study of the process of
fibrillogenesis in this polypeptide. It has not been possible to
produce this fragment in recombinant expression systems to date as it
is very unstable and sensitive to bacterial
proteases.2 Here we have been
able to use material purified from patients to study the pathway of
fibril formation and to probe the environmental triggers that may
initiate the conformational switching. These results may shed light on
the process of amyloid formation in the whole family of lipoproteins
that appears to be particularly susceptible to the formation of these
stable aggregates.
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EXPERIMENTAL PROCEDURES |
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Purification of ApoA-I-(1-93) from Tissue-- Amyloid fibrils were extracted from patient PER by the water extraction procedure of Pras et al. (24) as described in Mangione et al. (23). The protein was then purified by gel filtration in the presence of 5 M guanidine hydrochloride. After the removal of denaturant by dialysis against water, the protein was lyophilized and then resolubilized in water in a concentrated stock solution. The polypeptide extracted from the fibrils was submitted to N-terminal sequencing and mass spectrometry as previously described (23).
Fluorescence Spectra-- Fluorescence spectra were measured with a PerkinElmer LS50 spectrofluorimeter at 20 °C with excitation at 295 nm. ApoA-I-(1-93) was dissolved at a concentration of 0.06 mg/ml in 3 mM glycine, 3 mM sodium acetate, and 3 mM sodium phosphate adjusted to a pH range from 3 to 8.
FT-IR Spectroscopy-- FT-IR spectra were recorded on a Nicolet Magna 560 spectrometer (Madison, WI) purged with N2 and equipped with a MCT/A detector cooled with liquid nitrogen. The samples, typically 250 µl, were loaded into an ATR cell prepared for liquid samples and sealed with Teflon stoppers. The internal reflection element accessory was a trapezoidal (50 × 2 × 20 mm) 25-reflection germanium plate from Grasby Specac (Kent, UK). A cavity for the sample was created using a 0.5-mm-thick Teflon spacer between the internal reflection element and the metal plate. Spectra were collected at room temperature and without a polarizer.
ApoA-I-(1-93) was dissolved at a concentration of 1 mg/ml in 10 mM d3-acetate pH* 7.4 (corrected for
deuterium effects) and incubated overnight to allow H/D exchange. The
pH* of solution was adjusted to 4, and 300 interferograms were
collected at a resolution of 1 cm1 (collection lasted
approximately 1 min), which were averaged and processed with zero
filling Happ-Genzel apodization. Data were collected at regular times
over a period of 16 h.
Natural full-length (lipid-free) apoA-I and apoA-I fibrils were both obtained from patient PER as previously described (23). Both specimens were dissolved in 10 mM d3-acetate pH* 7.0 (corrected for deuterium effects) at a concentration of 1 mg/ml and incubated overnight before the spectral analysis.
Electron Microscopy-- Electron micrographs were acquired using a Phillips CM100 electron microscope operating at 80 keV. The FT-IR sample at pH* 4 was centrifuged, the fibrils were resuspended in filtered Milli-Q water, and the suspension of fibrils was applied to a Formvar-coated copper grid. This was blotted, negatively stained with 1% phosphotungstic acid (w/v), pH 7, air-dried, and then examined.
CD with Dihexanoylphosphatidylcholine (DHPC) and
TFE--
Circular dichroism spectra were collected on a JASCO 810 spectropolarimeter using cuvettes of 1-mm path length. Samples of apoA-I-(1-93) at a final concentration of 0.336 mg/ml in 3 mM glycine, 3 mM sodium acetate, and 3 mM sodium phosphate at pH 4 and 7 were incubated in aqueous
TFE solutions of different molar ratios between 0 and 40% and with a
range of DHPC concentrations from 0 to 15 mM. Spectra were
corrected for base line using the spectrum of appropriate buffer alone.
The mean residue ellipticity [] was calculated from the equation
[
] =
/(cnl/Mr) where
l is the path length in cm,
is the observed ellipticity
in millidegrees, c is the concentration of protein (mg/ml),
n is the number of amino acid residues, and
Mr is the molecular weight.
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RESULTS |
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Effect of pH on Compactness and Secondary Structure
Content--
The apoA-I-(1-93) polypeptide was purified from ex
vivo material and displayed the N-terminal sequence of mature
apoA-I and a molecular mass of 10720 Da. As previously shown, it adopts
a random coil conformation at neutral pH in aqueous buffer (23). Knowing that the polypeptide assumes a cross- conformation in the
amyloid fibrils, we initially probed the acquisition of more compact
structure by monitoring the total tryptophan fluorescence signal
arising from the three tryptophan residues at positions 8, 50, and 72. It is clear from Fig. 1a that
the pH of the solution has an effect on the protein conformation such
that the environment of the tryptophans is altered. The tryptophan
emission maximum of a protein usually shifts from shorter wavelengths
to around 350 nm, the fluorescence maximum of tryptophan in aqueous
solution, when proteins are unfolded. At neutral pH the fluorescence
emission maximum from apoA-I-(1-93) is at 347 nm, but this shifts to
close to 341 nm at pH 3, consistent with transfer of the tryptophan side chains into a hydrophobic environment and the development of a
more compact structure. Studies of full-length apoA-I in n-propyl alcohol buffered with Tris at pH 8.2 indicate that
in this helix-inducing solvent, mimicking the effect of lipids, the wavelength of maximum fluorescence occurs at 342 nm (only one other Trp
occurs outside of the N-terminal amyloidogenic fragment, at position
108). Leroy and Jonas postulate (20) that the similarities in the
spectroscopic properties observed in apoA-I in 30% n-propyl alcohol and in reconstituted high density lipoprotein were such that it
was likely that most of the Trp residues are in or near a helix but are
accessible to solvent. In the recombinant high density
lipoprotein complexes the phospholipids make contact with the
tryptophans and shield them from solvent. Strikingly, our CD data from
polypeptide 1-93 suggested that the structural collapse from a
predominantly unstructured form at neutral pH (Fig. 1b), presenting a minimum at 203 nm, was accompanied by acquisition of
helical structure at lower pH (Fig. 1c) in which the minima are at 208 and 222 nm. The helical conformation induced at pH 4 is not
stable, however (Fig. 2), and the
formation of insoluble material is observed after ~5 min. This is
accompanied by loss of CD signal. The polypeptide in a helical
conformation at pH 4 appears to undergo intermolecular association and
precipitation.
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The Process of Fibril Formation--
We have used FT-IR
spectroscopy to follow the formation of aggregates at low pH because
this technique can be used for monitoring secondary structure changes
in a mixture of soluble and insoluble material. The loss of helical
signal and the increase in -structure that accompanies the formation
of aggregates are illustrated in Fig.
3a. After the pH of the
solution was adjusted to 4, acquisition of spectra was initiated
rapidly and continued over a period of 16 h. After 5 min at pH 4, an increase in the signal at 1615 and 1684 cm
1 was
observed. The signal centered at 1615 cm
1 corresponds to
the contribution of
-structure, and the signal at 1684 cm
1 is specific for the presence of anti-parallel
-structure. After 12 h the process of conversion into
-sheet
is complete. This can be compared with the FT-IR spectra at pH 3 and 7 (Fig. 3b) and with the spectrum of amyloid fibrils purified
from patient PER (Fig. 3c). These are particularly
interesting because they contain a substantial residual helical element
(1655 cm
1), and this information is consistent with the
-helical component we have detected by x-ray diffraction in natural
fibrils (23). The search for fibrils in the aggregate was performed by
electron microscopy every 24 h, and apparently only after 72 h at 4 °C, under these acidic conditions, very long, straight
fibrils were detected by negative stain electron microscopy (Fig.
4). The material analyzed by electron
microscopy does not display fibrillar structure in the first 72 h.
The lag phase between the
- to
-structure conversion and the
fibril formation is not surprising, in general, for protein
fibrillogenesis. In the case of apoA-I in particular, the coexistence
of non-fibrillar aggregate and typical amyloid fibrils is noted in the
natural fibrils analyzed by atomic force microscopy in which the non
fibrillar aggregate has a globule like structure presenting a mean
height around 4.5 nm.3
Therefore, it is possible that the process of fibrillogenesis in
vitro requires a slow conversion of oligomeric aggregates into long fibrils, and it is likely that an equilibrium of the two states of
association exists in vivo. Unfortunately the relatively small amounts of material available have so far made the alignment of
fibrils for x-ray fiber diffraction and comparison of in
vitro and ex vivo fibrils impossible.
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Some fibrillar material was also detected after incubation at pH 7, although the fibrils formed under these conditions were not as long or
as straight as those produced at pH 4. Full-length apoA-I has also been
shown to form fibrils in vitro at pH 7, and at least some
fragments of apoA-I appear to be colocalized with A in senile
plaques (25).
Effect of Phospholipids on Secondary Structure--
The
apoA-I-(1-93) peptide was incubated with a range of concentrations of
DHPC at pH 4 and pH 7 to study the effect of a lipid environment on
this lipid-associated protein (Fig. 5).
This is a short (6-carbon) chain phospholipid that exhibits a critical micelle concentration of 16 mM (26) and behaves as a
detergent micelle in aqueous solution rather than forming lipid
bilayers. The low dielectric constant medium provided by the lipid acyl chains promotes the formation of -helical structure, possibly by
increasing intramolecular hydrogen bonding and electrostatic and
dipolar interactions and decreasing competition with water. At pH 4, the effect of low concentrations of DHPC (up to 7.5 mM) is
to induce some helical structure into the polypeptide, but this form is
not stable and, with time, signal is lost. With increasing DHPC
concentration (from 10 mM and above), the helical
conformation is stabilized to a point where no signal is lost over
3 h (Fig. 6). Increasing the DHPC
concentration beyond this point increases the strength of the helical
signal slightly. At pH 7, concentrations of DHPC up to 10 mM have only a limited effect on the conformation of the
peptide. Above 10 mM DHPC, a decrease in the ellipticity at
222 nm indicates that some helix is present, but the spectra suggest
that there is a substantial coil element present. This altered
conformation appears to be stable at pH 7. DHPC appears to be able to
induce some helical structure under favorable pH conditions and to mask
intermolecular interactions that can occur between molecules in the
helical form. Therefore, high concentrations of this short chain lipid
prevent fibril formation at low pH but do not induce helical
conformation at pH 7.
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Effect of TFE on Secondary Structure--
To study the role of the
helical conformation in apoA-I-(1-93) fibrillogenesis, the polypeptide
was incubated with a range of TFE concentrations at pH 4 and at pH 7 (Fig. 7). TFE can promote -helical
structure in proteins and polypeptides (27), but high concentrations of
TFE have been used to destabilize the native states of proteins and to
populate amyloidogenic forms of proteins such as acylphosphatase (28)
as well as to induce fibril formation in an amphipathic peptide
derivative of apo C-II (29). The presence of TFE induces a strong
helical signal from apoA-I-(1-93) at both pH 4 and 7, and this helical
form is stable in TFE, presumably because of increased intramolecular
hydrogen bonding and a reduced hydrophobic effect. More TFE is required
to induce or to stabilize equivalent amounts of helix at pH 4 than at
pH 7. Therefore there is an apparent dichotomy between a high intrinsic
tendency to populate a helical structure at low pH and the apparent
requirement for higher concentrations of TFE to produce equivalent
amounts of helical structure at pH 4 and at pH 7. This may be explained by the formation of intermolecular coiled-coil structure at low pH. As
discussed previously, the 1-93 peptide is predicted to form a
coiled-coil structure in the full-length protein. An analysis of the
relative ellipticity exhibited at 222 and 208 nm can distinguish between the coiled-coil structure and the
-helical structure (30). A
222:208 ellipticity ratio of ~1 indicates interhelical contacts,
whereas values around 0.9 suggest helix without interhelical contacts.
We have measured the ellipticity ratio for apoA-I-(1-93) at pH 4, and
we obtain a ratio of 1.07 in the absence of TFE and 0.87 in the
presence of 40% TFE, suggesting interacting helices in the absence of
TFE but loss of these contacts in the presence of the solvent. At pH 7, the ratio falls to 0.78 at 20% TFE. We propose that there may be a
competing effect of the TFE at pH 4 to destabilize the coiled-coil
interactions on the one hand and to stabilize the
-helical structure
on the other, and higher concentrations are therefore required to
stabilize equivalent amounts of helical structure compared with pH 7. At pH 7, the helix induced by 20% TFE is stable. However, the same
proportion of TFE at pH 4 appears to accelerate the loss of signal and,
by inference, precipitation.
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These results appear to support the idea that the formation of these
amyloid aggregates requires the peptide to pass from its random
conformation through a helical conformation to the stable -sheet
structure. If all of the polypeptide material is stabilized in this
helical form, with masking of intermolecular interactions, then
aggregation and fibril formation are prevented. No development of
soluble
-structure was observed during fibril formation by this
apoA-I polypeptide, and this is different to what has been reported in
numerous amyloidogenic peptide systems and also in other lipoproteins
that form amyloid (31). Instead, the aggregating conformer that is
transiently populated below the pI (4.35) is mainly helical, and the
conformational switching to form fibrils with
-structure occurs
after precipitation.
Hydrophobicity, Charge Distribution, and the Effect of
Mutations--
The mean hydrophobicity and the net charge of apoA-I
full-length and truncated 1-93 were calculated as suggested by Uversky et al. (32) and are reported in Fig.
8. Through these parameters it was also
possible to calculate the "boundary" mean hydrophobicity value,
(H)boundary, by means of the equation (33)
(H)boundary = (R + 1.151)/2.785.
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This equation, in which R corresponds to the mean net charge of the polypeptide, predicts a natively unfolded state for proteins that have a mean hydrophobicity below the (H)boundary value. Both full-length apoA-I and the polypeptide 1-93 fulfil the requirements for natively unfolded proteins.
There is clearly an effect of pH on this amyloidogenic polypeptide; the
helical conformation is populated at pH values below the pI, and the
polypeptide has a random coil structure at neutral pH. The sequence of
apoA-I is very rich in charged residues. Of the 93 residues, 20 are
negatively charged (10 Asp and 10 Glu), and 11 are positively charged
(4 Arg and 7 Lys) (Fig. 8). Protonation of the acidic residues appears
to be the trigger for the transient helix formation and subsequent
intermolecular association and conversion to -structure. At neutral
pH the clusters of acidic residues may result in charge-charge
repulsion and a more random conformation, whereas neutralization of the
negative charges removes the local repulsion effects and allows helical
structure. The pKa values of glutamate and aspartate
suggest that lowering of the pH will result in protonation of the
glutamate residues first. These acidic residues are mainly situated in
the C-terminal part of the polypeptide (Fig. 8), so neutralization of
these side chains may allow the helical conformation to be adopted.
Several of the amyloidosis-associated mutations identified to date have had the effect of adding an extra positive charge to the sequence of
the N-terminal fragment. However, because of the large number of
charges in the sequence, the additional charge actually has a
relatively small effect on the pI of the polypeptide (increasing from
4.35 to 4.45). The mutations at 26, 50, and 60 will result in the
positioning of opposite charges separated by one residue. This may
favor extended
-sheet structure.
Many other amyloidogenic proteins have been shown to be affected by
acidification, notably transthyretin and lysozyme. Dobson (12) and
Kelly (34) propose that the lysosomes may play a key role in initiation
of fibrillogenesis, although the pH of the lysosome may not be as low
as the pH used to initiate amyloidosis in vitro. An acidic
environment may destabilize some protein structures and allow
conformational switching or it may allow the population, even to a
small extent, of a misfolded species that is aggregation prone. In the
case of the apoA-I fragment, the very large number of charged residues
in the N-terminal fragment that will be directly affected by pH changes
are almost certainly influential in the initiation of
conformational change. Our studies of the effect of
acidification by fluorescence indicated that reducing the pH caused
some conformational change, presumably collapse of the random coil
structure. At the relatively low protein concentrations used for the
fluorescence experiments, protein aggregation was not observed after
the pH was reduced. However, monitoring of the acidification process by
CD at higher protein concentrations indicated that aggregation
succeeded conformational change.
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DISCUSSION |
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In this study we have identified environmental conditions that
drive the conversion of the polypeptide 1-93 of human apolipoprotein A-I from random coil to a predominantly anti-parallel -sheet fibrillar aggregate, passing through a helical conformation. We have
shown that this polypeptide has chameleon properties (35). Under
certain conditions, random, helical, or
-sheet
conformations can be stabilized, with a barrier to reciprocal
conversion. Aqueous solutions at neutral pH favor the random coil
structure, trifluoroethanol and phospholipids stabilize the helical
conformation, and low pH conditions prime the self-aggregation into
-sheet structure. The persistence of a predominantly random coil
structure in aqueous solution at neutral pH is unusual for a
polypeptide as long as the apoA-I-(1-93) peptide but is not unique
among fibrillogenic proteins. Synuclein, one of the human proteins able
to create pathological fibrils in vivo, has been
demonstrated to behave in a similar manner.
-Synuclein is unfolded
in its native state (36), but the presence of an organic solvent like
hexafluoroisopropanol induces the acquisition of helical structure, and
in this conformation the protein is very sensitive to the heat-induced
aggregation (37). Phospholipids have a similar effect on synuclein and
apoA-I-(1-93); in fact both proteins undergo large conformational
changes and an increase in and stabilization of helical structure in
the presence of phospholipids (38). It is worth noting that the most
fibrillogenic portion of
-synuclein is the N-terminal polypeptide
1-87 (39); however, despite the similar behavior, the level of
sequence homology between
-synuclein and the apoA-I N terminus is
not significant. A comparison that may be more significant though is
the fact that both apoA-I-(1-93) and
-synuclein belong to the
category of natively unfolded proteins (32). The experimental data
presented here and previously performed NMR studies (23) are fully
consistent with the prediction of apoA-I-(1-93) being a natively
unfolded protein on the basis of the algorithm that considers the mean hydrophobicity and the mean net charge in aqueous solution at neutral
pH (32, 33). On the basis of these parameters full-length apoA-I in the
lipid-free state also fulfils the criteria for the inclusion in this
protein category. Conditions permissive for in vitro
fibrillogenesis of lipid-free full-length apoA-I were discovered by
Wisniewski et al. (25), but it is uncertain if this process
is relevant to the deposition in vivo. Biomedical data so
far available suggest that the cleavage of full-length apoA-I and the
release of the N-terminal polypeptide could represent the regulatory
step in the fibrillogenic pathway. The accuracy of the site of the
proteolytic cleavage is apparently crucial for any further metabolic
processing of apoA-I polypeptide. In fact, enhanced proteolytic
susceptibility is described for apoA-I variants associated with
metabolic abnormalities, but without amyloid disease. Of interest is
the metabolic pathway of apoA-I Finland associated with
hypoalphalipoproteinemia (40). In this case the mutation (L159R)
affects the protein secretion, reduces the half-life of circulating
apoA-I, and has a dominant negative effect on wild type apoA-I, a
behavior quite similar to that hypothesized for the apoA-I (L174S)
variant (23). In the case of the apoA-I Finland mutation, a
18-kDa N
terminal polypeptide (40) is released in plasma and
appears to prime a degradative, rather than a fibrillogenic, pathway.
The tissue compartment where the putative primary proteolytic cleavages
occur for both the wild type and amyloidogenic species awaits
definition; at present, the chemical environment and the nature of the
proteolytic enzyme(s) are largely unknown. We believe that new data in
this area will complement the data we have presented on the folding
dynamics of this apoA-I polypeptide and will allow the molecular
mechanism underlying this disease to be elucidated.
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ACKNOWLEDGEMENTS |
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We are very grateful to Giuseppina Ferri and Piero Pucci for continuous advice and support. The assistance of Alberto Bovera is also acknowledged.
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FOOTNOTES |
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* The work was supported by funds from the Ministero della Sanità (ricerca finalizzata sulla Malattia di Alzheimer code 020ALZ00/01), the MURST (Cofin 2000 protocol MM05221899), FIRB 2002 protocol RBNE01S29H, and Fondazione Telethon-Italia Grant 164-11477.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.:
39-0382-507783; Fax: 39-0382-423108; E-mail: vbellot@unipv.it.
Current address: School of Biological Sciences, Nanyang
Technological University, 637616 Singapore.
¶¶ Current address: School of Molecular and Microbial Biosciences, University of Sydney, NSW 2006, Australia.
Published, JBC Papers in Press, November 5, 2002, DOI 10.1074/jbc.M204801200
2 D. Booth, personal communication.
3 A. Relini et al., manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are: apoA-I, apolipoprotein A-I; CD, circular dichroism; DHPC, dihexanoylphosphatidylcholine; FT-IR, Fourier transform infrared spectroscopy; TFE, 2,2,2-trifluoroethanol.
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