(The FASEB Journal. 2000;14:1255-1263.)
© 2000 FASEB
Protein aging hypothesis of Alzheimer disease
JOZEF ORPISZEWSKI*1,
NORBERT SCHORMANN
,
BARBARA KLUVE-BECKERMAN,
JURIS J. LIEPNIEKS and
MERRILL D. BENSON,
* Aprot Corporation, Carmel, Indiana 46082-3813, USA;
Department of Pathology and Laboratory Medicine, Indiana University School of Medicine, Indianapolis, Indiana 46202, USA; and
Richard L. Roudebush Veterans Affairs Medical Center, Indianapolis, Indiana 46202, USA
1Correspondence: Aprot Corporation, P.O. Box 3813, Carmel, IN 46082-3813, USA. E-mail: aprot{at}excite.com
 |
ABSTRACT
|
|---|
Alzheimer disease (AD), the most common form of aging-related
neurodegenerative disorders, is associated with formation of fibrillar
deposits of amyloid ß-protein (Aß). While the direct involvement of
Aß in AD has been well documented, the relations between Aß
production, amyloid formation, and neurodegeneration remain unknown. We
propose that AD is initiated by a protein aging-related structural
transformation in soluble Aß. We hypothesize that spontaneous
chemical modification of aspartyl residues in Aß to transient
succinimide induces a non-native conformation in a fraction of soluble
Aß, rendering it amyloidogenic and neurotoxic. Conformationally
altered Aß is characterized by increased stability in solution and
the presence of a non-native ß-turn that determines folding of Aß
in solution and the structure of Aß subunits incorporated into
amyloid fibrils. While the soluble ‘non-native’ Aß is both the
factor triggering the neurodegenerative cascade and the precursor of
amyloid plaques, these two events result from interaction of Aß with
different sets of cellular components and need not coincide in space
and time. Extensive literature data and experimental evidence are
provided in support of this hypothesis.—Orpiszewski, J., Schormann,
N., Kluve-Beckerman, B., Liepnieks, J. J., Benson, M. D.
Protein aging hypothesis of Alzheimer disease.
Key Words: amyloid ß-protein • succinimide • isoaspartate • amyloidosis
|
INTRODUCTION
|
|---|
ALZHEIMER DISEASE (AD) is the most common type of
aging-related neurodegenerative disorder and is characterized by the
presence of extracellular neuritic amyloid plaques, cerebrovascular
amyloid deposits, and intraneuronal neurofibrillary tangles. The
identification by Glenner and Wong in 1984 (1)
of amyloid
ß-protein (Aß) as the major component of neuritic amyloid plaques
opened a new era of intensive research on neurodegenerative diseases
dominated by the view that this 40–42 amino acid long peptide is the
causative agent in AD (Fig. 1
). Subsequent research confirmed the primary function of Aß in the
development of the disease, but the exact role played by this peptide
has remained enigmatic (2
, 3)
.
View larger version (12K):
[in this window]
[in a new window]
|
Figure 1. Amino acid sequence of amyloid ß-protein 1–42 (Aß42). Aß
contains three aspartyl residues (highlighted), two of which have been
demonstrated to be highly isomerized and racemized in amyloid plaques
(underlined).
|
|
For over a decade, the neurodegenerative character of Aß protein has
been attributed to its propensity to oligomerize and form amyloid
fibrils. The ‘amyloid cascade hypothesis’, which still dominates the
field, postulates that Aß amyloid formation is the primary event in
AD triggering a series of cellular events that lead to formation of
neurofibrillary tangles, neurodegeneration, and dementia (3
, 4)
. Although several alternative hypotheses have been proposed
over the years, none has approached the completeness and universality
of the amyloid hypothesis. However, the controversy over amyloid as a
mediator of Aß neurotoxicity intensified in the last 2 years in light
of several reports that transgenic animals overproducing Aß peptide
show neurodegeneration that preceded or occurred completely without
amyloid formation (5
6
7)
. Ironically, these studies also
provided additional strong evidence for direct involvement of Aß in
AD. These contradictory observations underscore an urgent need for a
new hypothesis that would address the association between Aß
production and neurodegeneration, one that would explain formation of
Aß amyloid plaques and their correlation with the progression of AD
in humans, and most important, one that could be tested.
Potential contributions of posttranslational modifications of aspartyl
residues to Aß amyloidosis have been addressed in several studies
initiated after the work of Shapira et al. (8)
and Roher
et al. (9
, 10)
showed that Aß isolated from amyloid
deposits has an unusually high content of racemized and isomerized Asp.
Roher and co-workers (9
, 11)
comprehensively discussed the
potential consequences of Asp modifications, suggesting they may affect
Aß production and resistance to proteolysis, complement cascade
activation, and amyloid nucleation and stabilization. They suggested
that the accumulation of modified aspartyl residues may result from
decreased protein turnover or from its sequestration from cytoplasmic
isoAsp repair enzyme, suggestive that the majority of modifications may
occur after the initial deposition of amyloid in the extracellular
space. Subsequent studies by other research groups have built on this
excellent work and addressed specific questions raised by Roher et al.
The work of Fukuda et al. (12)
and the research groups of
Drs. Roher (13)
, Otvos (14
15
16)
, and Tenner
(17)
showed that formation of isoAsp could have a
tremendous impact on Aß conformation, toxicity, and fibrillogenicity.
In addition, other groups showed that racemization of aspartate might
contribute to Aß amyloidosis (18
, 19)
. Even though these
studies indisputably demonstrated that modifications of aspartyl
residues could affect development of AD, they have not led to any
consistent conclusions or tenable hypothesis on the mechanism of Aß
pathogenicity. We proposed that a comprehensive evaluation of the
effect of posttranslational modifications on Aß toxicity and
amyloidogenicity required expanding the research on succinimide, which
is the product of Asp dehydration and the direct precursor of
isomerized and racemized residues (20)
. Our model studies
and a careful analysis of the literature have led to the conclusion
that an effect of succinimide on AD may far exceed that expected and
observed in vitro for isoAsp, D-Asp, or D-isoAsp, and have
prompted us to propose a testable mechanism of succinimide-induced
pathogenicity of Aß. We believe that the contribution of succinimide
may form the basis for a rational design of novel therapeutics aimed at
early events in AD.
The ‘Protein Aging Hypothesis of Alzheimer Disease’ presented below
is based on recent advances in the fields of Alzheimer Aß protein,
protein aging, and protein structure and folding. It is aimed at
explaining and linking many contradictory observations made in the
field of AD by various research groups. It significantly extends the
current concept of Aß involvement in AD and is not intended to
contradict existing hypotheses, but rather to provide a strong
alternative point of view for analysis and interpretation (and possibly
reinterpretation) of experimental results.
|
PROTEIN AGING HYPOTHESIS OF ALZHEIMER DISEASE
|
|---|
At the heart of the Protein Aging Hypothesis are widely accepted
notions that Aß protein plays a central role in AD and that the
conformational changes in Aß protein determine its pathogenicity
(2)
. We postulate that a fraction of total soluble Aß
undergoes spontaneous chemical modification of aspartyl residues as a
result of protein aging, and such modifications increase the
probability of structural transition in soluble Aß to a pathogenic
conformation with potentially neurotoxic and amyloidogenic properties.
Specifically, we make the following new postulates:
1. Spontaneous cyclization of an aspartyl residue to a succinimide in
soluble Aß protein is the initial event in AD.
2. Succinimide changes conformational preferences of Aß,
destabilizing the existing tertiary structure and increasing the
likelihood of a non-native conformation through stabilization of a
non-native ß-turn. Although only a portion of succinimide-containing
soluble Aß undergoes such structural transition, the non-native
conformer of Aß shows increased solubility (likely as a dimer or a
tetramer) and lower vulnerability to nonspecific aggregation. The
concentration of the non-native conformer is directly proportional to
the total concentration of Aß.
3. Soluble non-native Aß is the actual factor triggering the cascade
leading to neurodegeneration and is also a precursor of amyloid
plaques. However, neurodegeneration and amyloidogenesis occur
independently and do not need to coincide in space and time, because
they result from interaction of soluble non-native Aß with different
sets of cellular components. Neurodegeneration is the result of
cellular mis-recognition of non-native structural elements such as a
type II' ß-turn or a ß-hairpin. Fibrillogenesis is likely
facilitated by well-defined and very specific ß-sheets geometry
determined by the succinimide-induced type II' ß-turn.
|
FORMATION OF SUCCINIMIDE FROM ASPARTYL RESIDUE
|
|---|
As AD is associated with advanced age, several known aging-related
protein modifications such as oxidation, glycation, isomerization, and
racemization have been studied intensively in relation to AD and
amyloid formation. In most cases, they have been shown to increase
ß-sheet content and/or in vitro fibrillogenesis.
Nonetheless, proteins isolated from amyloid deposits, although highly
proteolyzed, do not generally show signs of any extensive modifications
and seem to be chemically identical to precursor proteins. An important
exception is the high level of isomerization and racemization of
aspartate detected in Aß protein isolated from Alzheimer cerebral
plaques and vascular deposits (9
, 10)
.
Isomerization and racemization of aspartate as well as deamidation of
asparagine constitute the most common types of aging-related protein
damage. These reactions proceed through a common pathway involving
formation of a transient cyclic succinimide intermediate (Fig. 2
) (for review, see refs 21
, 22
). Formation of the
succinimide from aspartate is a result of an intramolecular
nucleophilic attack of the peptide amide-nitrogen on the side chain
carbonyl group of Asp. Hydrolysis of succinimide leads to accumulation
of stable isoaspartyl sites (isoAsp) in which the peptide bond is
formed by the side chain carboxyl of aspartic acid. As this is the only
known reaction leading to isoAsp, every time an isoaspartyl site is
detected in proteins it provides direct evidence that cyclization of
aspartate to succinimide has occurred. In an alternative pathway,
succinimide may undergo racemization prior to hydrolysis, resulting in
formation of D-Asp or D-isoAsp residues.
View larger version (17K):
[in this window]
[in a new window]
|
Figure 2. Formation of succinimide through spontaneous cyclization of aspartyl
residue and products of its hydrolysis. Succinimide is formed by
intramolecular reaction between protonated carboxyl group of Asp and
unprotonated amide-nitrogen. Hydrolysis of succinimide yields a mixture
of L-Asp and L-isoAsp usually in the ratio of 1:3. In addition,
racemization of succinimide at the  -carbon can lead to formation of
D-Asp and D-isoAsp. L-isoAsp can undergo intracellular methylation by
protein L-isoaspartyl methyltransferase to unstable methyl ester, which
readily cyclizes to succinimide.
|
|
Several unique features of aspartate cyclization support its
involvement in the pathogenic processes in AD. As an intramolecular
reaction, it is independent of protein concentration and the presence
of other cellular components (as long as they do not influence the
structure of the polypeptide). Moreover, it is only slightly dependent
on pH and buffer composition, which means that cyclization of Asp to
succinimide can occur in any environment, both intra- and
extracellularly.
In vivo, intracellularly formed L-isoaspartyl sites are
subject to repair mediated by the widely distributed enzyme, protein
L-isoaspartyl methyltransferase (PIMT), which is particularly abundant
in brain tissue (21
, 23)
. In the first step of the repair
pathway, L-isoAsp is enzymatically methylated to form isoaspartyl
methyl ester, which readily cyclizes to the succinimide and is further
metabolized as described above (Fig. 2)
. Repeated action of PIMT leads
eventually to restoration of the original aspartyl residue. However,
proteins located in the extracellular space escape this repair and
several brain extracellular proteins have been shown to accumulate high
levels of isoAsp in vivo (24
25
26)
.
The soluble fraction of aged human brain cortex contains an average of
490 pmol isoAsp/mg of protein (27)
, most of which is
located in soluble extracellular chondroitin sulfate proteoglycans
(CSPG), predominantly phosphacan (24)
, which do not
undergo the repair process. Assuming that half of isoAsp is derived
from aspartyl residues, as has been observed in bovine calmodulin
(28)
, one can estimate that ~4% of all aspartyl
residues in soluble bovine phosphacan have undergone succinimide
formation in adult brain. Studies in rats showed that the newborn brain
already contained close to 10 pmol of isoAsp/mg of protein and the
content of damaged CSPG increased dramatically with age
(24)
. Intact succinimide has been demonstrated in a
neuropeptide N-acetylaspartylglutamine (NAAG) isolated from the murine
and bovine central nervous system (29)
. In rat spinal
cord, the succinimide-NAAG accumulated progressively with age,
increasing from below detectable level in newborn to 5 pmol/mg of wet
tissue at the age of 12 months.
Purified proteins and peptides also undergo aging and spontaneous
succinimide formation at appreciable rates (21
, 30
, 31)
.
For example, the very labile Asp101 of hen egg-white lysozyme cyclizes
at a rate of ~1%/h at 40°C in solution and 7%/wk at 20°C in the
crystalline state (31)
. Brain-derived proteins such as
tubulin (32)
and synapsin (33)
accumulate
isoaspartyl sites from both aspartate and asparagine at the rate of
2.4–8 mol % per day on incubation in vitro at
near-physiological conditions (pH 7.2–7.4, 37°C). A significant rate
of succinimide formation can also be expected in Aß peptides ‘aged’
in vitro (34)
.
Succinimide also forms during solid phase peptide synthesis,
particularly when strong acids are used to cleave side chain-protecting
groups in Boc chemistry (35)
. Under typical cleavage
conditions using HF-anisole (9:1) at 0°C, the rate constant of
succinimide formation from aspartyl ß-benzyl ester in a model
tetrapeptide is 73.6 x 10-6
s-1, consistent with 5–8% of succinimide
by-product after deprotection (35)
. Even though
chromatographic separation of the main synthetic product from
succinimide by-product is usually straightforward for short peptides,
it may be quite difficult in the case of long and hydrophobic peptides
such as Aß (J. Orpiszewski and M. D. Benson, unpublished
observations). Variation in the relative amounts of succinimide Aß
and unmodified Aß may be responsible for irreproducibility of some
experimental results that show high dependence on the source and lot
number of synthetic Aß (for example, see ref 36
).
|
SUCCINIMIDE AND ISOASPARTATE RESIDUES IN Aß
|
|---|
Although the presence of succinimide in soluble Aß in
vivo is yet to be shown, the high level of isomerization and
racemization of Asp in amyloid plaques has been well documented
(9
, 10
, 37)
. Roher and co-workers (9)
proved
that aspartates at positions 1 and 7 were isomerized and racemized.
They showed that the hydrolysate of cortical Aß contained 25% of
D-Asp (9
, 11)
. In addition, chromatographic separation of
tryptic peptides in conjunction with enzymatic detection of isoAsp
revealed that ~75% of Asp1 and Asp7 were isomerized. Considering
that the succinimide intermediate is usually hydrolyzed to a mixture of
Asp and isoAsp at a ratio of 1:3 and that formation of D-Asp results
predominantly from racemization of succinimide intermediate, one can
reasonably estimate that all Asp residues at positions 1 and 7 must
have undergone succinimide formation during the lifetime of Aß
protein found in Alzheimer plaques.
A high degree of isomerization and racemization of Asp7 detected in
amyloid plaques proves that this residue is particularly vulnerable to
cyclization. In fact, the Asp-Ser peptide bond is the second most prone
to cyclization in synthetic peptides, next only to Asp-Gly (for review,
see ref 21
). This makes it highly probable that
cyclization of Asp7 occurs in soluble Aß, and that the content of
isomerized sites in Aß and soluble extracellular CSPG is comparable.
Contrary to Asp1 and Asp7, isomerization of Asp23 of Aß protein has
not been reported. However, the relative flexibility of Aß in the
region of Glu22-Asp23, detected by nuclear magnetic resonance (NMR)
(38)
, and the direct proximity to the Glu22Gln mutation
associated with familial amyloidosis (39)
indicate that
the possibility and importance of succinimide formation at residue
Asp23 cannot be ignored. In any case, the concentration of succinimide
containing peptide will be directly proportional to the total
concentration of Aß.
|
STRUCTURAL EFFECT OF ASPARTATE CYCLIZATION
|
|---|
Cyclization of an aspartyl residue to succinimide introduces
dramatic changes in the polypeptide chain: 1) neutralization
of the negative charge of the carboxyl group, 2) change in
the volume of the residue, and 3) tremendous steric
constraint similar to the effect of a proline residue. It is likely
that formation of succinimide in an unstructured or temporarily
refolded part of Aß would prevent formation of hydrogen bonds or salt
bridges necessary for ‘normal’ folding and would prevent formation of
secondary structure elements normally occurring in such a polypeptide
fragment. Wood and co-workers (40)
showed that
substituting proline for any residue in the 17–23 fragment of Aß led
to complete loss of fibril formation and excellent peptide solubility.
A restriction in peptide flexibility as imposed by the cyclic ring of
Pro can also be expected in the case of the flat and rigid succinimide
ring.
The structural effect of succinimide on Aß may extend far beyond
restricting ‘normal’ folding. In fact, there are multiple examples of
mutations of acidic residues in amyloidogenic proteins leading directly
to amyloidosis. Several of these mutations introduce very minor
chemical modifications to acidic residues (Table 1
), such as replacement of aspartic acid with asparagine in gelsolin and
prion protein or glutamic acid with glutamine in transthyretin and Aß
(41
, 42)
. Although the exact mechanism of amyloidogenicity
of these mutations is not understood, it is likely they both
destabilize the ‘native’ fold and increase the probability of an
alternative folding either through a kinetic or thermodynamic effect,
or both. Studies using synthetic dodecapeptides showed that any kind of
chemical modification of aspartyl residues can change folding
preferences and fibrillogenic properties of the polypeptide chain
(20)
. In the case of succinimide, in addition to its
strong structure-destabilizing effect, there is a well-defined steric
preference that may affect folding of the whole protein.
|
ß-TURN PREFERENCE OF SUCCINIMIDE RESIDUE AND ITS EFFECT ON
PROTEIN FOLDING
|
|---|
The flat structure of the succinimide ring imposes strict
constraints on the torsion angles of adjoining bonds, which disfavor
extended conformation of the polypeptide chain. In fact, the geometry
of succinimide residue is identical to that predicted for the second
residue of a type II' ß-turn in a polypeptide chain
(22)
. This theoretical prediction has been widely
supported by molecular simulation and by NMR and crystallographic data
obtained for short succinimide-peptides that assume type II' ß-turn
conformation even in the absence of long-range stabilizing interactions
(Fig. 3
; see, for example, refs 43
44
45
).
View larger version (13K):
[in this window]
[in a new window]
|
Figure 3. Crystallographic structure of a succinimide-containing tetrapeptide in
type II' ß-turn conformation. Succinimide residue (Asu) strongly
favors type II' ß-turn with Asu in the second position of the turn
and an intramolecular hydrogen bond between amide groups after the
first and third residues. The crystal structure of the tetrapeptide
Boc-Pro-Asu-Gly-Ala-OMe is shown. The drawing was prepared using
crystallographic data from ref 44
.
|
|
The unusual conformation and the effect of type II' ß-turns on
protein structure have been well reviewed in the literature
(46
47
48)
. This rare turn is a ‘mirror image’ of type II
turn, which along with type I are the most common turns in globular
proteins. The rare occurrence of the type II' ß-turn is due to the
fact that natural L-amino acids strongly destabilize it. However, the
mirror image turns II' and I' have a unique advantage of promoting a
two-residue tight ß-hairpin structure due to their conformation,
which is fully compatible with the natural right-handed twist of
ß-sheet (Fig. 4
) (46
, 48
49
50
51)
. Systematic survey of protein crystal
structures revealed that two residue ß-hairpins in fact contain
almost exclusively the mirror image turns, and half of all identified
type II' ß-turns were found in ß-hairpins (46
, 47)
.
View larger version (36K):
[in this window]
[in a new window]
|
Figure 4. A ß-hairpin with type II' ß-turn in aspartic proteinase. Type II'
ß-turn stabilizes the ß-hairpin formed by residues 256–268 of
Endothia aspartic proteinase (PDB entry 2ER7). The conformation of type
II' ß-turn is fully compatible with natural twist of antiparallel
ß-sheet in two-residue ß-hairpins. In contrast, the most common
ß-turns, types I and II, destabilize such tight ß-hairpins.
|
|
The propensity of succinimide to form type II' ß-turn has strong
implication for Aß folding or refolding. First, cyclization of Asp to
succinimide changes local folding preferences of the polypeptide chain
and can induce a ß-turn where it would not be expected in the
unmodified peptide. Second, formation of type II' ß-turn highly
increases the probability of a ß-hairpin structure even if the
flanking residues do not create a perfect match for an antiparallel
ß-sheet (52
, 53)
. Third, locally formed ß-turn and
ß-hairpin structures may serve as nucleation sites for Aß
folding/refolding. The strong propensity of ß-hairpins to nucleate
cooperative folding of proteins has already been well documented
(54
55
56
57)
.
The potential effect of succinimide formation on Aß folding is
enormous due to high structural plasticity of Aß protein. Whereas
typical globular proteins fold into one well-defined native structure
even in the presence of different mutations (57)
, Aß is
known to exist in various structures ranging from prone-to-aggregation
ß-sheet, to random coil, to -helix. Due to low activation energy
for structural transition and thermodynamic equivalence of different
folding states, Aß easily interconverts between different folds on
slight changes in environmental conditions. However, such high
plasticity means that the population of well-defined ß-turn and
ß-hairpin structures in succinimide-containing Aß will not be high
and may remain unnoticed when secondary structure is monitored in
solution by circular dichroism or NMR, which provide averaged
structural information (52
, 53)
. In fact, even
specifically designed ß-hairpin peptides do not assume a single fold
and are in equilibrium with random coil-like conformations in aqueous
solutions (52
, 58
, 59)
.
On the other hand, in the presence of favorable tertiary and possibly
quaternary structural interactions, weakly defined ß-turns and
ß-hairpins become dominant structural elements dictating the folding
of longer polypeptide chains (54
, 59
, 60)
. In the case of
Aß, such interactions may include dimerization or tetramerization,
which have been shown to exist in vivo and improve peptide
stability (13
, 61
, 62)
. The presence of
succinimide-induced non-native ß-turn and ß-hairpin may also
contribute to peptide stability by discouraging aggregation (ref
54
; J. Orpiszewski and M. D. Benson, unpublished
observations), similar to the effect of proline substitutions in Aß
(40)
.
|
POTENTIAL NEUROTOXICITY OF SUCCINIMIDE-CONTAINING SOLUBLE Aß
|
|---|
The presence of a succinimide-induced, non-native structure in
Aß opens the possibility that the neurodegenerative cascade in AD is
triggered by mis-interaction of modified soluble Aß with other
cellular components. The precise mechanism of such mis-interaction is
difficult to predict, since little is known about events leading to
neurodegeneration. In fact, both extracellular and intracellular signal
transduction pathways have been proposed and candidate Aß binding
proteins of diverse nature have been identified (2
, 63
64
65)
. The extracellular receptor RAGE of the immunoglobulin
superfamily is one of these proteins that mediate oxidative stress and
neurotoxicity (63)
. As antigen protein–immunoglobulin
interactions often include ß-turn and loop epitopes
(66)
, it is likely that formation of non-native ß-turn
and ß-hairpin in Aß may significantly alter its antigenic
properties and lead to mis-recognition by immunoglobulin-type cellular
receptors. Moreover, conformationally altered Aß may contain
additional non-native epitopes recognizable by other types of extra- or
intracellular receptors.
|
SUCCINIMIDE CONTRIBUTION TO AMYLOID FORMATION
|
|---|
While Aß amyloid plaques are the hallmark of AD, several studies
including those with transgenic animals indicate that plaque formation
does not need to correlate with neurotoxicity either in space or in
time (5
6
7)
. Although succinimide-containing Aß in
non-native conformation may be both the cause of neurodegeneration and
precursor for amyloid plaques, it is likely that different
conformational features of Aß and different environmental factors
contribute to these two processes.
We used synthetic dodecapeptides derived from
H2N-VTVKVDAVKVTV-CONH2 to
study the effect of modifications of the centrally located aspartyl
residue on peptide secondary structure and fibrillogenicity (ref
20
; J. Orpiszewski and M. D. Benson, unpublished
observations). We demonstrated that substitution of the aspartyl
residue with succinimide, asparagine, glutamic acid, aspartyl methyl
ester, or glutamyl methyl ester lead to increased ß-sheet structure
detectable by circular dichroism and to increased fibrillogenicity,
although to different degrees. Aspartyl and glutamyl methyl esters
showed the highest ß-sheet content, lowest solubility, and highest
tendency for aggregation at neutral pH (20)
. Well-defined
fibrils prepared from these peptides were relatively short and thick,
corresponding to an extended conformation of the polypeptide chain
within fibrils. In contrast, the peptide containing succinimide in
place of aspartic acid was much more soluble and stable in aqueous
solution, showed a lower tendency to nonspecific aggregation, and
showed a lower ß-sheet content (J. Orpiszewski and M. D.
Benson, unpublished observations). However, it also formed well-defined
fibrils under the same experimental conditions (Fig. 5
). Moreover, fibrils prepared from the succinimide peptide were narrow
and very long, suggesting that the polymerization process was rapid,
specific, and uninterrupted. Although CD measurements did not reveal
any significant content of ß-turn in solution, fibril diameter
measured on electron micrographs was consistent with a ß-hairpin
structure of the peptide. It seems likely that the succinimide-induced,
two-residue ß-hairpin with a rigorously determined twist of
antiparallel ß-sheet strictly defined the surface geometry of the
growing fibril. This prevented nonspecific interaction between
ß-sheets and early termination of fibril polymerization.
View larger version (119K):
[in this window]
[in a new window]
|
Figure 5. Electron micrograph of amyloid-like fibrils formed by a
succinimide-containing designed peptide. A dodecapeptide
(H2N-Val-Thr-Val-Lys-Val-Asu-Ala-Val-Lys-Val-Thr-Val-CONH2)
formed long and narrow fibrils on incubation at pH 7.3 at 37°C. The
diameter of the fibrils (1.7–3.2 nm) was consistent with a dimer of
the peptide in a ß-hairpin conformation as the smallest
fibril-building unit. Scale bar represents 200 x 4 nm.
|
|
We propose that succinimide has the same effect on the polymerization
of Aß protein. The presence of a non-native type II' ß-turn induces
a right-handed twist of ß-sheet with a well-defined angle between the
strands of ß-hairpin (Fig. 4)
. Such a strictly defined non-native
geometry of ß-sheet would likely prevent its nonspecific interaction
with other proteins, which could lead to early termination of fibril
growth; however, it would constitute a very specific nucleus for
uninterrupted polymerization of Aß protein.
In addition to initiation of amyloidogenesis, the non-native ß-turns
and ß-hairpins within amyloid fibrils or, more likely, within protein
subunits resolublized from amyloid (67
68
69)
, could be
responsible for recruiting microglia and initiating the inflammations
often associated with amyloid in Alzheimer brains.
|
TESTING OF PROTEIN AGING HYPOTHESIS OF ALZHEIMER DISEASE
|
|---|
The hypothesis presented here is not intended to contradict
existing hypotheses on the origin of AD and amyloidosis. We believe
that AD is a complex disease that is manifested only when multiple
cellular, conformational, and metabolic factors come into play at the
same time, allowing a small fraction of an otherwise normal protein to
escape its metabolic fate and become pathogenic. Moreover, there are
probably many different ways to make Aß protein pathogenic due to its
high structural plasticity, and the question is not which pathway is
the right one, but rather which dominates in a particular case.
There are many excellent approaches developed by different researchers
studying the pathogenesis of Aß protein and hereditary amyloidoses
that can be used directly to test the involvement of succinimide in AD.
However, due to the high propensity of aspartyl residue to succinimide
formation both in vitro and in vivo, particular
care must be taken in designing experiments and choosing reagents to be
able to clearly distinguish between effects caused by unmodified and by
succinimide-containing Aß peptides. Moreover, for the same reasons it
may sometimes be more appropriate to ask whether succinimide-induced
pathogenesis actually forms the main pathological pathway rather than
trying to prove or disprove that succinimide contributes to AD.
In addition to AD, protein aging and succinimide formation may also
underlie other disorders associated with conformational changes in
protein including prion diseases. However, at this time there are not
enough data available to justify extending the Protein Aging Hypothesis
beyond Alzheimer disease.
|
FOOTNOTES
|
|---|
Received for publication July 20, 1999. Revised for publication January 10, 2000.
|
REFERENCES
|
|---|
-
Glenner, G. G., Wong, C. W. (1984) Alzheimer’s disease: initial report of the purification and characterization of a novel cerebrovascular amyloid protein. Biochem. Biophys. Res. Commun. 120,885-890[Medline]
-
Selkoe, D. J. (1999) Translating cell biology into therapeutic advances in Alzheimer’s disease. Nature (London) 399,A23-A31[Medline]
-
Hardy, J. (1997) Amyloid, the presenilins and Alzheimer’s disease. Trends Neurosci 20,154-159[Medline]
-
Hardy, J. A., Higgins, G. A. (1992) Alzheimer’s disease: the amyloid cascade hypothesis. Science 256,184-185[Free Full Text]
-
Neve, R. L., Robakis, N. K. (1998) Alzheimer’s disease: a re-examination of the amyloid hypothesis. Trends Neurosci 21,15-19[Medline]
-
Chui, D. H., Tanahashi, H., Ozawa, K., Ikeda, S., Checler, F., Suzuki, H., et al (1999) Transgenic mice with Alzheimer presinilin 1 mutations show accelerated neurodegeneration without amyloid plaque formation. Nat. Med. 5,560-564[Medline]
-
Moechars, D., Lorent, K., Van Leuven, F. (1999) Premature death in transgenic mice that overexpress a mutant amyloid precursor protein is preceded by severe neurodegeneration and apoptosis. Neuroscience 91,819-830[Medline]
-
Shapira, R., Austin, G. E., Mirra, S. S. (1988) Neuritic plaque amyloid in Alzheimer’s disease is highly racemized. J. Neurochem. 50,69-74[Medline]
-
Roher, A. E., Lowenson, J. D., Clarke, S., Wolkow, C., Wang, R., Cotter, R. J., et al (1993) Structural alterations in the peptide backbone of ß-amyloid core protein may account for its deposition and stability in Alzheimer’s disease. J. Biol. Chem. 268,3072-3083[Abstract/Free Full Text]
-
Roher, A. E., Lowenson, J. D., Clarke, S., Woods, A. S., Cotter, R. J., Gowing, E., Ball, M. J. (1993) ß-Amyloid-(1–42) is a major component of cerebrovascular amyloid deposits: implications for the pathology of Alzheimer disease. Proc. Natl. Acad. Sci. USA 90,10836-10840[Abstract/Free Full Text]
-
Lowenson, J. D., Roher, A. E., Clarke, S. (1994) Protein aging: extracellular amyloid formation and intracellular repair. Trends Cardiovasc. Med. 4,3-8
-
Fukuda, H., Shimizu, T., Nakajima, M., Mori, H., Shirasawa, T. (1999) Synthesis, aggregation, and neurotoxicity of the Alzheimer’s Aß1–42 amyloid peptide and its isoaspartyl isomers. Bioorg. Med. Chem. Lett. 9,953-956[Medline]
-
Kuo, Y.-M., Webster, S., Emmerling, M. R., De Lima, N., Roher, A. E. (1998) Irreversible dimerization/tetramerization and post-translational modifications inhibit degradation of Aß peptides of Alzheimer’s disease. Biochim. Biophys. Acta 1406,291-298[Medline]
-
Fabian, H., Szendrei, G. I., Mantsch, H. H., Greenberg, B. D., Otvos, L., Jr (1994) Synthetic post-translationally modified human A ß peptide exhibits a markedly increased tendency to form ß-pleated sheets in vitro. Eur. J. Biochem. 221,959-964[Medline]
-
Szendrei, G. I., Fabian, H., Mantsch, H. H., Lovas, S., Nyeki, O., Schon, I., Otvos, L., Jr (1994) Aspartate-bond modification affects the major conformations of synthetic peptides. Eur. J. Biochem. 226,917-924[Medline]
-
Szendrei, G. I., Prammer, K. V., Vasko, M., Lee, V. M., Otvos, L., Jr (1996) The effects of aspartic acid-bond isomerization on in vitro properties of the amyloid beta-peptide as modeled with N-terminal decapeptide fragments. Int. J. Pept. Protein Res. 47,289-296[Medline]
-
Velazquez, P., Cribbs, D. H., Poulos, T. L., Tenner, A. J. (1997) Aspartate residue 7 in amyloid ß-protein is critical for classical complement pathway activation: implications for Alzheimer’s disease pathogenesis. Nat Med 3,77-79[Medline]
-
Mori, H., Ishii, K., Tomiyama, T., Furiya, Y., Sahara, N., Asano, S., Endo, N., Shirasawa, T., Takio, K. (1994) Racemization: its biological significance on neuropathogenesis of Alzheimer’s disease. Tohoku J. Exp. Med. 174,251-262[Medline]
-
Tomiyama, T., Asano, S., Furiya, Y., Shirasawa, T., Endo, N., Mori, H. (1994) Racemization of Asp23 residue affects the aggregation properties of Alzheimer amyloid ß-protein analogues. J. Biol. Chem. 269,10205-10258[Abstract/Free Full Text]
-
Orpiszewski, J., Benson, M. D. (1999) Induction of ß-sheet structure in amyloidogenic peptides by neutralization of aspartate: a model for amyloid nucleation. J. Mol. Biol. 289,413-428[Medline]
-
Aswad, D. W. (1995) Deamidation and isoaspartate formation in peptides and proteins. CRC Series in Analytical Bio/Technology CRC Press Boca Raton.
-
Clarke, S. (1987) Propensity for spontaneous succinimide formation from aspartyl and asparaginyl residues in cellular proteins. Int. J. Pept. Protein Res. 30,808-821[Medline]
-
Kim, E., Lowenson, J. D., MacLaren, D. C., Clarke, S., Young, S. G. (1997) Deficiency of a protein-repair enzyme results in the accumulation of altered proteins, retardation of growth, and fatal seizures in mice. Proc. Natl. Acad. Sci. USA 94,6132-6137[Abstract/Free Full Text]
-
David, C. L., Orpiszewski, J., Zhu, X.-C., Reissner, K. J., Aswad, D. W. (1998) Isoaspartate in chondroitin sulfate proteoglycans of mammalian brain. J. Biol. Chem. 273,32063-32070[Abstract/Free Full Text]
-
Johnson, B. A., Aswad, D. W. (1985) Identification and topography of substrates for protein carboxyl methyltransferase in synaptic membrane and myelin-enriched fractions of bovine and rat brain. J. Neurochem. 45,1119-1127[Medline]
-
Orpiszewski, J., Aswad, D. W. (1996) High mass methyl-accepting protein (HMAP), a highly effective endogenous substrate for protein L-isoaspartyl methyltransferase in mammalian brain. J. Biol. Chem. 271,22965-22968[Abstract/Free Full Text]
-
Johnson, B. A., Shirokawa, J. M., Geddes, J. W., Choi, B. H., Kim, R. C., Aswad, D. W. (1991) Protein L-isoaspartyl methyltransferase in postmortem brains of aged humans. Neurobiol. Aging 12,19-24[Medline]
-
Potter, S. M., Henzel, W. J., Aswad, D. W. (1993) In vitro aging of calmodulin generates isoaspartate at multiple Asn-Gly and Asp-Gly sites in calcium-binding domains II, III, and IV. Protein Sci 2,1648-1663[Abstract]
-
Brovia, V., Costa, A., Barbeito, L. (1996) N-acetylsuccinimidylglutamate, a cyclic imide form of the peptide N-acetylaspartylglutamate, is present in low micromolar concentrations in murine and bovine CNS. J. Neurochem. 67,382-388[Medline]
-
Hui, J. O., Chow, D. T., Markell, D., Robinson, J. H., Katta, V., Nixon, L., Chang, B. S., Rohde, M. F., Haniu, M. (1998) Identification of Asp95 as the site of succinimide formation in recombinant human glial cell line-derived neurotrophic factor. Arch. Biochem. Biophys. 358,377-384[Medline]
-
Noguchi, S., Miyawaki, K., Satow, Y. (1998) Succinamide and isoaspartate residues in the crystal structures of hen egg-white lysozyme complexed with tri-N-acetylchitotriose. J. Mol. Biol. 278,231-238[Medline]
-
Najbauer, J., Orpiszewski, J., Aswad, D. W. (1996) Molecular aging of tubulin: accumulation of isoaspartyl sites in vitro and in vivo. Biochemistry 35,5183-5190[Medline]
-
Paranandi, M. V., Aswad, D. W. (1995) Spontaneous alterations in the covalent structure of synapsin I during in vitro aging. Biochem. Biophys. Res. Commun. 212,442-448[Medline]
-
Pike, C. J., Walencewicz, A. J., Glabe, C. G., Cotman, C. W. (1991) In vitro aging of ß-amyloid protein causes peptide aggregation and neurotoxicity. Brain Res 563,311-314[Medline]
-
Tam, J. P., Riemen, M. W., Merrifield, R. B. (1988) Mechanisms of aspartimide formation: the effects of protecting groups, acid, base, temperature and time. Peptide Res 1,6-18
-
May, P. C. (1997) Commentary. Amyloid-ß deposits: can you take them to the bank?. Alzheimer’s Dis. Rev. 2,15-19
-
Fonseca, M. I., Head, E., Velazques, P., Cotman, C. W., Tenner, A. J. (1999) The presence of isoaspartic acid in ß-amyloid plaques indicates plaque age. Exp. Neurol. 157,277-288[Medline]
-
Coles, M., Bicknell, W., Watson, A. A., Fairlie, D. P., Craik, D. J. (1998) Solution structure of amyloid ß-peptide(1–40) in a water-micelle environment. Is the membrane-spanning domain where we think it is?. Biochemistry 37,11064-11077[Medline]
-
Levy, E., Carman, M. D., Fernandez-Madrid, I. J., Power, M. D., Lieberburg, I., van Duinen, S. G., Bots, G. T., Luyendijk, W., Frangione, B. (1990) Mutation of the Alzheimer’s disease amyloid gene in hereditary cerebral hemorrhage, Dutch type. Science 248,1124-1126[Abstract/Free Full Text]
-
Wood, S. J., Wetzel, R., Martin, J. D., Hurle, M. R. (1995) Prolines and amyloidogenicity in fragments of the Alzheimer’s peptide ß/A4. Biochemistry 34,724-730[Medline]
-
Benson, M. D. (1995) Amyloidosis. Scriver, C. R. Beandet, A. L. Sly, W. S. Valle, D. eds. Metabolic Basis of Inherited Disease ,4159-4191 McGraw-Hill Inc. New York.
-
Ghetti, B., Piccardo, P., Frangione, B., Bugiani, O., Giaccone, G., Young, K., et al (1996) Prion protein amyloidosis. Brain Pathol 6,127-145[Medline]
-
Capasso, S., Mattia, C. A., Mazzarella, L., Zagari, A. (1984) Preferred conformations of tert-butoxylcarbonyl-L-aminosuccinyl-glycyl-glycine methyl ester in the solid and solution state. Acta Cryst. C43,1607-1610
-
Capasso, S., Mazzarella, L., Sica, F., Zagari, A., Cascarano, G., Giacovazzo, C. (1992) Solid state conformations of aminosuccinyl peptides: structure of tert-butoxylcarbonyl-L-prolyl-L-aminosuccinyl-glycyl-L-alanine methyl ester (Boc-L-Pro-L-Asu-Gly-L-Ala-OMe). A case of pseudo-translational symmetry. Acta Cryst. B48,285-290
-
Capasso, S., Mazzarella, L., Zagari, A. (1995) Folding of aminosuccinyl peptides: thermodynamic data from temperature dependent circular dichroism measurements. Chirality 7,605-609[Medline]
-
Sibanda, B. L., Thornton, J. M. (1985) ß-Hairpin families in globular proteins. Nature (London) 316,170-174[Medline]
-
Wilmot, C. M., Thornton, J. M. (1988) Analysis and prediction of the different types of ß-turn in proteins. J. Mol. Biol. 203,221-232[Medline]
-
Zhou, H. X., Hoess, R. H., DeGrado, W. F. (1996) In vitro evolution of thermodynamically stable turns. Nat. Struct. Biol. 3,446-451[Medline]
-
Haque, T. S., Little, J. C., Gellman, S. H. (1994) ‘Mirror image’ reverse turns promote ß-hairpin formation. J. Am. Chem. Soc. 116,4105-4106
-
Haque, T. S., Gellman, S. H. (1997) Insights on ß-hairpin stability in aqueous solution from peptides with enforced type I' and type II' ß-turns. J. Am. Chem. Soc. 119,2303-2304
-
Blanco, F., Ramirez-Alvarado, M., Serrano, L. (1998) Formation and stability of ß-hairpin structures in polypeptides. Curr. Opin. Struct. Biol. 8,107-111[Medline]
-
Ramirez-Alvarado, M., Blanco, F. J., Serrano, L. (1996) De novo design and structural analysis of a model ß-hairpin system. Nat. Struct. Biol. 3,604-612[Medline]
-
de Alba, E., Jimenez, M. A., Rico, M., Nieto, J. L. (1996) Conformational investigation of designed short linear peptides able to fold into ß-hairpin structures in aqueous solution. Folding & Design 26,133-144
-
Neira, J. L., Fersht, A. R. (1996) An NMR study on the ß-hairpin region of barnase. Folding & Design 1,231-241[Medline]
-
Yang, A.-S., Hitz, B., Honig, B. (1996) Free energy determinants of secondary structure formation: III. ß-Turns and their role in protein folding. J. Mol. Biol. 259,873-882[Medline]
-
Fersht, A. R. (1997) Nucleation mechanisms in protein folding. Curr. Opin. Struct. Biol. 7,3-9[Medline]
-
Baldwin, R. L., Rose, G. D. (1999) Is protein folding hierarchic? II. Folding intermediates and transition states. Trends Biochem. Sci. 24,77-83[Medline]
-
Munoz, V., Thompson, P. A., Hofrichter, J., Eaton, W. A. (1997) Folding dynamics and mechanism of ß-hairpin formation. Nature (London) 390,196-199[Medline]
-
Sharman, G. J., Searle, M. S. (1997) Dissecting the effects of cooperativity on the stabilization of a de novo designed three stranded anti-parallel ß-sheet. Chem. Commun. 1997,1955-1956
-
Kortemme, T., Ramirez-Alvarado, M., Serrano, L. (1998) Design of a 20-amino acid, three stranded ß-sheet protein. Science 281,253-256[Abstract/Free Full Text]
-
Kuo, Y. M., Emmerling, M. R., Vigo-Pelfrey, C., Kasunic, T. C., Kirkpatrick, J. B., Murdoch, G. H., Ball, M. J., Roher, A. E. (1996) Water-soluble Aß (N-40, N-42) oligomers in normal and Alzheimer disease brains. J. Biol. Chem. 271,4077-4081[Abstract/Free Full Text]
-
Garzon-Rodriguez, W., Sepulveda-Becerra, M., Milton, S., Glabe, C. G. (1997) Soluble amyloid Aß-(1–40) exists as a stable dimer at low concentrations. J. Biol. Chem. 272,21037-21044[Abstract/Free Full Text]
-
Yan, S. D., Chen, X., Fu, J., Chen, M., Zhu, H., Roher, A., et al (1996) RAGE and amyloid-ß peptide neurotoxicity in Alzheimer’s disease. Nature (London) 382,685-691[Medline]
-
Yan, S. D., Fu, J., Soto, C., Chen, X., Zhu, H., Al-Mohanna, F., et al (1997) An intracellular protein that binds amyloid-ß peptide and mediates neurotoxicity in Alzheimer’s disease. Nature (London) 389,689-695[Medline]
-
Small, D. H. (1998) The role of the amyloid protein precursor (APP) in Alzheimer’s disease: does the normal function of APP explain the topography of neurodegeneration?. Neurochem. Res. 23,795-806[Medline]
-
Dyson, H. J., Wright, P. E. (1995) Antigenic peptides. FASEB J 9,37-42[Abstract]
-
Solomon, B., Koppel, R., Frankel, D., Hanan-Aharon, E. (1997) Disaggregation of Alzheimer ß-amyloid by site-directed mAb. Proc. Natl. Acad. Sci. USA 94,4109-4112[Abstract/Free Full Text]
-
Lambert, M. P., Barlow, A. K., Chromy, B. A., Edwards, C., Freed, R., Liosatos, M., et al (1998) Diffusible, nonfibrillar ligands derived from Aß1–42 are potent central nervous system neurotoxins. Proc. Natl. Acad. Sci. USA 95,6448-6453[Abstract/Free Full Text]
-
Schenk, D., Barbour, R., Dunn, W., Gordon, G., Grajeda, H., Guido, T., et al (1999) Immunization with amyloid-ß attenuates Alzheimer-disease-like pathology in the PDAPP mouse. Nature (London) 400,173-177[Medline]
This article has been cited by other articles:
|
|
|
|
 
S. Zirah, S. A. Kozin, A. K. Mazur, A. Blond, M. Cheminant, I. Segalas-Milazzo, P. Debey, and S. Rebuffat
Structural Changes of Region 1-16 of the Alzheimer Disease Amyloid beta-Peptide upon Zinc Binding and in Vitro Aging
J. Biol. Chem.,
January 27, 2006;
281(4):
2151 - 2161.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Yazaki, J. J. Liepnieks, J. R. Murrell, M. Takao, B. Guenther, P. Piccardo, M. R. Farlow, B. Ghetti, and M. D. Benson
Biochemical Characterization of a Neuroserpin Variant Associated with Hereditary Dementia
Am. J. Pathol.,
January 1, 2001;
158(1):
227 - 233.
[Abstract]
[Full Text]
[PDF]
|
|
|
TOP
ABSTRACT
INTRODUCTION
