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OCR for page 43
Colloquium
Studies of the aggregation of mutant proteins
in vitro provide insights into the genetics of
amyloicl diseases
Fabrizio Chiti*t, Martino Calamai*, Niccolo Taddei*, Massimo Stefani*, Giampietro Ramponi*,
and Christopher M. Dobsont:
*Dipartimento di Scienze Biochimiche, Universita degli Studi di Firenze, Viale Morgagni 50, 50134 Florence, Italy; and "Department of Chemistry, University
of Cambridge, Lensfield Road, Cambridge CB2 1EW, United Kingdom
Protein aggregation and the formation of highly insoluble amyloid
structures is associated with a range of debilitating human condi-
tions, which include Alzheimer's disease, Parkinson's disease, and
the Creutzfeldt-Jakob disease. Muscle acylphosphatase (AcP) has
already provided significant insights into mutational changes that
modulate amyloid formation. In the present paper, we have used
this system to investigate the effects of mutations that modify the
charge state of a protein without affecting significantly the hy-
drophobicity or secondary structural propensities of the polypep-
tide chain. A highly significant inverse correlation was found to
exist between the rates of aggregation of the protein variants
under denaturing conditions and their overall net charge. This
result indicates that aggregation is generally favored by mutations
that bring the net charge of the protein closer to neutrality. In light
of this finding, we have analyzed natural mutations associated
with familial forms of amyloid diseases that involve alteration of
the net charge of the proteins or protein fragments associated with
the diseases. Sixteen mutations have been identified for which the
mechanism of action that causes the pathological condition is not
yet known or fully understood. Remarkably, 14 of these 16 muta-
tions cause the net charge of the corresponding peptide or protein
that converts into amyloid deposits to be reduced. This result
suggests that charge has been a key parameter in molecular
evolution to ensure the avoidance of protein aggregation and
identifies reduction of the net charge as an important determinant
in at least some forms of protein deposition diseases.
A range of debilitating human diseases is known to be
Associated with the formation of stable highly organized
protein aggregates known as amyloid fibrils. These diseases
include cerebral conditions such as Alzheimer's disease, Parkin-
son's disease and Creutzfeldt-Jakob disease, and also a series of
systemic amyloidoses in which amyloid deposition occurs in a
wider variety of organs within the body (1, 24. In each of these
pathological conditions, a specific peptide or protein that is
normally soluble is deposited, either intact or in fragmented
form, into insoluble fibrils which accumulate in one or more
types of tissue. Some amyloid diseases are rare and are associ-
ated with specific mutations involving the peptide or protein
associated with amyloid deposition. Examples are familial amy-
loidotic polyneuropathy (3), hereditary renal amyloidosis (4, 5),
and the apoA1 amyloidosis (6, 7~. Other diseases, such as
Alzheimer's disease, frontotemporal dementia, and Parkinson's
disease, are largely sporadic, with hereditary cases involving only
a limited fraction of the patients suffering from the conditions
in question (8-10~. The identification of specific mutations
associated with familial forms of a disease that is otherwise
sporadic and the investigation of the mechanism by which the
mutations result in pathological behavior have proved to be of
www.pnas.org/cgi/doi/10. 1 073/pnas.21 2527999
fundamental importance for identifying specific genes associated
with the disease and for exploring the molecular basis of the
underlying pathology (8, 10, 11~.
The amino acid sequences and native structures of the proteins
associated with amyloid diseases have been found to be highly
variable, but structural studies have revealed that amyloid fibrils
from different sources share a common ultrastructure (12~.
Electron microscopy has shown that amyloid fibrils are typically
straight and unbranched and are formed from an assembly of
protofilaments 2-5 nm wide (12~. X-ray fiber diffraction studies
indicate a characteristic structure in which the polypeptide
chains form ,8 strands oriented perpendicular to the long axis of
the fibril, resulting in ,B-sheets propagating in the direction of the
fibril (124.
It is increasingly recognized that the ability to form amyloid
fibrils is not a property restricted to the relatively few amino acid
sequences associated with specific diseases but is a generic
phenomenon of polypeptide chains (13~. A considerable number
of proteins, including several that adopt cr-helical structures
under native conditions such as myoglobin and cytochrome cs52,
have been shown to form amyloid fibrils in vitro, provided
appropriate conditions are selected (14-17~. Fibril formation
involving globular proteins occurs when the native structure is at
least partially unfolded under conditions in which the ability to
form noncovalent interactions is retained (13, 15~. Importantly,
aggregates formed from such nondisease-related proteins have
been found, at least in some cases, to be highly toxic to both
neuronal and nonneuronal cells (184. Although the ability to
form amyloid structures appears generic, the propensities of
different protein sequences to aggregate under given conditions
can differ very substantially (19-25~.
The ability of a wide range of natural proteins to form amyloid
fibrils in vitro provides a large variety of systems with which to
study the underlying nature of the conversion from the soluble
to the aggregated state of proteins. This opportunity allows the
fundamental principles of a process central to human disease to
be investigated using a set of proteins that can be chosen to have
particular advantages for their study. Human muscle acylphos-
phatase (AcP) is a model system that has proved to be partic-
ularly suitable for studies of misfolding and aggregation (15,
25-28 ). It is a relatively simple protein with 98 residues consisting
of a five-stranded antiparallel ,B-sheet packed against two par-
This paper results from the Arthur M. Sackier Colioquium of the Nationai Acaclemy of
Sciences, "Self-Perpetuating Structurai States in Biology, Disease, and Genetics," held
March 22-24, 2002, at the Nationai Acaclemy of Sciences in Washington, DC.
Abbreviations: A,B, amyioicl ,B peptide; AcP, muscle acyiphosphatase; apoAl, apolipo-
protein A1; TFE, 2,2,2, trifluoroethanoi; ThT, thioflavine T.
tTo whom reprint requests shouicl be adciressed. E-maii: cmd44~?cam.ac.uk.
PNAS 1 December 10, 2002 1 vol. 99 1 suppl. 4 1 16419-16426
OCR for page 44
Lys88
Glu90
Serg
Arg23
-
Ser21 ~
Ser43
GluS5
Fig. 1. Structure of AcP in its native state. Residues that have been mutated
in the present study are labeled and their side chains shown. The various
amino acid substitutions are listed in Table 1.
allel or-helices (Fig. 14. In addition to possessing a simple and well
defined fold with no disulphide bridges or other complications
such as bound cofactors, the normal folding behavior of AcP has
been studied in a great detail at a residue-specific level (29, 30~.
This is an important issue, because folding and aggregation are
potentially competitive events for a polypeptide chain in a
biological environment. AcP has been shown to form readily, in
the presence of moderate concentrations of denaturant such as
2,2,2, trifluoroethanol (TEE), amyloid fibrils structurally similar
to those associated with disease (15~. Importantly, a key step of
the aggregation process, the initial formation of insoluble spher-
ical and elongated protofibrils from soluble states, can be readily
followed for AcP with a variety of biophysical techniques, and its
rate is reproducible and easily measurable (25~. Monitoring the
first steps in the aggregation process leading to formation of
prefibrillar aggregates is gaining in importance, because it is
increasingly recognized that low-molecular weight oligomers
that precede formation of mature amyloid fibrils, often referred
to as protofibrils, represent the fundamental pathogenic species
in at least many of the amyloid diseases (11, 18, 31-33~.
In our initial studies of AcP, the propensity to form fibrils was
investigated for a series of mutants under conditions in which the
native states of the various protein variants were substantially
populated, although significantly destabilized (26~. The propen-
sity to aggregate was found to correlate inversely with the
confirmational stability of the native state of the protein in the
different mutants (26~. Consistent with this finding, stabilization
of the native state of AcP by ligand binding inhibits amyloid
formation (27~. These results show that the stability of the native
state is a major factor preventing the conversion of a globular
protein into amyloid fibrils under nonpathological conditions.
This conclusion is in accord with studies that have shown that
destabilization of the native state is a primary mechanism by
which naturally occurring mutations promote their pathogenic
16420 1 www.pnas.org/cgi/doi/10.1073/pnas.212527999
effect in at least some hereditary amyloid diseases (34-36~. The
overriding significance of the native state in preventing protein
aggregation also suggests that strategies aimed at stabilizing the
native states of amyloidogenic proteins could be of major value
in the prevention of amyloid diseases (27, 37~.
In a second study, the rates of protofibril formation of more
than 30 mutants, with conservative amino acid substitutions
spread throughout the sequence of the protein, were determined
under conditions in which the native states of all mutants are
fully destabilized (25~. This approach has allowed different
regions of the AcP sequence to be probed for their degree of
involvement in promoting the aggregation process from an
ensemble of partially denatured conformations. All mutations
found to perturb significantly the rate of aggregation were found
to be located in two specific regions of the protein sequence,
residues 16-31 and 87-98. This result indicates that aggregation
of AcP can be nucleated by specific regions of the protein
sequence that are consequently directly involved in the rate-
determining steps of this process (25~. The measured rates of
Arg77 aggregation were found to correlate with changes in the hydro-
phobicity and in the propensity to convert from X-helical to
3-sheet structure of the regions of the protein in which the
mutations are located (25, 28~. Interestingly, natural mutations
of the prion protein that leave the confirmational stability of the
cellular form of the protein unaltered (38, 39) increase either the
,B-sheet propensity or hydrophobicity of the prion sequence.
In this paper, we have extended the protein engineering
approach to investigate the role that charged residues play in the
process of aggregation and amyloid formation. Electrostatic
interactions have been suggested to be important in the modu-
lation of the aggregation behavior of a number of specific
disease-related proteins (14, 20, 40-42~. Nevertheless, a system-
atic investigation of the importance in aggregation of the total
charge state of a polypeptide chain or of particular electrostatic
interactions involving specific residues within its sequence has
not yet been reported. Furthermore, it is not yet clear whether
electrostatic interactions play a key role in protein aggregation
generally or whether they are particularly important for a limited
number of protein systems. In addition to providing new infor-
mation on the driving forces of protein aggregation, this study is
also designed to give additional insights into the origin of
heritable amyloid diseases, because a number of these conditions
are associated with amino acid replacements that alter the
charge state of the aggregating polypeptide chains.
Materials and Methods
Design, Production, and Purification of AcP Mutants. The 15 mutants
were designed to perturb the or-helical and I3-sheet propensities
and the hydrophobicity of the protein sequence as little as
possible. All replacements involving substitution of or by hydro-
phobic residues (Val, Ile, Leu, Ala, Gly, Tyr, Phe, Trp, Cys, Met,
and Pro) were therefore discarded. To evaluate the ,B-sheet and
c-helical propensities of the protein sequence before and after
mutation, the scale of ,B-sheet propensities edited by Street and
Mayo and the AGADIR algorithm were used, respectively (43, 44~.
Mutagenesis was carried out by using the QuikChange Site-
Directed Mutagenesis kit (Stratagene). Protein expression and
purification of wild-type and mutated AcP molecules were
performed according to the procedures described previously
(45~. All proteins have the cysteine residue at position 21
replaced by a serine residue to avoid complexities arising from
a free sulfhydryl group (29~. DNA sequencing was used to ensure
the presence of the desired mutation. Protein concentration
was measured by UV absorption by using an 2~0 value of
1.49 ml mg-~ cm-~.
Equilibrium Unfolding Experiments. Equilibrium urea denaturation
curves were obtained for each AcP variant by measuring the
Ch iti et a/.
OCR for page 45
intrinsic fluorescence of 25-30 equilibrated samples containing
0.02 mg ml-i protein and urea concentrations ranging from O to
8.1 M, in 50 mM acetate buffer, pH 5.5, 28C. A Perkin-Elmer
LS 55 with excitation and emission wavelengths of 280 and 335
nm, respectively, was used for the measurements. The data were
analyzed according to the method of Santoro and Bolen (46) to
yield the free energy of unfolding in the absence of denaturant
(~\GU20), the dependence of l\GU F on denaturant concentration
(m value) and the urea concentration at which the protein is 50~o
denatured (Cm). To overcome the problems associated with
accurate determination of m values in individual experiments,
the conformational stabilities of all protein variants are ex-
pressed as A/\GU-F = (m) (Cm - Cm), where (m; is the average
m value of all mutants; Cm and Cm are the midpoints of
denaturation for the wild-type and mutated proteins, respec-
tively (474.
Kinetics of Aggregation. The aggregation process was monitored
as described previously (254. In brief, each AcP variant was
incubated at a concentration of 0.4 mgml-i in 25% (vol/vol)
TFE/50 mM acetate buffer, pH 5.5, 25C. At regular time
intervals, aliquots of 60 Al of this solution were mixed with 440
al of 25 mM phosphate buffer, pH 6.0, containing 25 ,uM
thioflavine T (ThT). The resulting ThT fluorescence was mea-
sured by using excitation and emission wavelengths of 440 and
485 nm, respectively. Kinetic plots were fitted to single expo-
nential functions to determine the aggregation rate constants
(kAGG) for the various proteins.
Results
Selection and Characteristics of the AcP Mutations. Fifteen variants
of AcP, all having single replacements of charged or hydrophilic
residues located on the surface of the protein, were purified for
the present study (Table 1, Fig. 1~. The mutations consist of
substitutions of neutral residues with residues carrying a charge
under the conditions of pH investigated here (S8H, S21R, S43E,
and S92H), substitutions of charged residues with uncharged
ones (R23Q, E29Q, E55Q, K88N, K88Q, and R97Qj, and
substitutions of charged residues with others of opposite sign
(E29K, E29R, R77E, E9OH, and R97E). All mutations involve
an increase or a decrease in the charge state of the protein by 1
or 2 units (Table 1~. The rate of aggregation of AcP from a
denatured ensemble of conformations was found in a previous
study to be sensitive to amino acid substitutions only when these
occur within the two regions of the sequence comprising residues
16-31 and 87-98 (25~. The majority of the mutations were
therefore designed within such regions. Four mutations at
positions outside these sequence regions were, however, also
produced to act as controls (S8H, S43E, ESSQ, and R77E).
The 15 mutations used here were chosen for their ability to
leave the c-helical and ,B-sheet propensities of the protein
sequence unchanged, because secondary structure-forming pro-
pensities have been shown to be major determinants of the
aggregation behavior of AcP (see Materials and Methods for
details) (25, 28~. For the same reason, mutations in which
hydrophilic residues are replaced by hydrophobic ones were not
considered, because changes of hydrophobicity have been shown
to modify considerably the aggregation rate of AcP (25~.
Through analysis of the 15 mutational variants selected here, we
have therefore endeavored to relate effects on the aggregation
process resulting from mutations simply to the changes in the
charge state of the protein at the mutated position by minimizing
mutational changes of hydrophobicity and secondary structural
propensity of the protein.
The conformational stabilities of all of the 15 AcP variants
were evaluated by means of equilibrium urea denaturation
measurements (Fig. 2~. The resulting parameters show that the
mutations induce a destabilization of the native state of the
Chiti et a/.
Table 1. Parameters describing the conformational stabilities and
aggregation rates of AcP mutants
Net charge of the
mutant* Cm, Mt
Wild-type
S8H
S21R
R23Q
E29K
E29Q
E29R
S43E
E55Q
R77E
K88N
K88Q
E9OH
S92H
R97E
R97Q
+4
+7
+6
+3
+4
+5
+6
+6
+4
+7
+6
+7
+4
+6
+3
+4
AA GU-F,
kJ mol-'t
4.0 + 0.1
1.8 + 0.1
3.8 + 0.1
3.5 + 0.1
3.3 + 0.1
4.1 + 0.1
3.3 + 0.1
4.0 + 0.1
3.3 + 0.1
1.8 + 0.1
3.7 + 0.1
3.9 + 0.1
3.0 + 0.1
3.8 + 0.1
3.7 + 0.1
3.6 + 0.1
-6.98 + 0.04
11.9 + 0.6 - 6.89 + 0.12
1.4 + 0.6 - 9.04 + 0.1 4
3.0 + 0.6 - 6.31 + 0.13
4.0 + 0.6 - 7.60 + 0.13
0.6 + 0.6 - 7.39 + 0.12
4.0 + 0.6 - 8.52 + 0.14
0.1 + 0.6 - 6.33 + 0.14
4.1 + 0.6 - 6.85 + 0.12
11.7 + 0.6 - 6.16 + 0.14
1.6 + 0.6 - 7.22 + 0.12
0.7 + 0.6 - 7.20 + 0.12
5.5 + 0.6 - 9.50 + 0.14
1.4 + 0.6 - 7.32 + 0.12
1.8 + 0.6 - 6.57 + 0.13
2.2 + 0.6 - 6.98 + 0.12
All experimental errors reported in the table are standard deviations unless
stated otherwise.
*Calculated at pH 5.5 by using standard pKa values for protein side chains.
Under the denaturing conditions used to study aggregation of AcP and its
mutants, the pKa values of the various residues of AcP are assumed not to
deviatesignificantlyfrom normal ranges, because these residuesare likelyto
be highly solvent exposed. At pH 5.5, residues of Asp, Glu, Arg, Lys, and His
are therefore assumed to be charged.
tConcentration of urea at which the mutant is 50% denatured.
ti\6GU F values were obtained by using I\AGU-F = (m)~(Cm - Cm), where (m) is
theaveragemvalueofall mutants((m) = 5.40 + 0.15 kJ.mol-~.M-'); Cmand
Cm are the midpoint of denaturation for the wild-type and mutated protein,
respectively. The A/\GU-F values correspond to those obtained by subtracting
the AGU F value of the mutant from that of the wild-type protein (6Gu-F =
21.7 + 0.8 kJ.mol-, for the wild type).
The best estimate and experimental error reported for the wild-type protein
are, respectively, the average value and standard error obtained from nine
independent measurements.
protein ranging from O to 12 1cJ mol-i (Table 1~. The two
mutations S8H and R77E resulted in the largest values of the
free energy of destabilization (A I`GU-F) of 11.9 and 11.7
~ I I I ,,,,, I,,,, I,,,, I,,,, I,,
~o~ ~ ~
o.oE
-,. ., I ,,,, 1, ,,, ~!
O 1 2 3 4 5 6 7 8
[urea] (M)
Fig. 2. Urea denaturation curves of representative AcP variants in 50 mM
acetate buffer, pH 5.5, 28C. Curves are normalized to the fraction of folded
protein and correspond to those of wild-type AcP (filled circles), K88Q (open
circles), R23Q (filled squares), E9OH (open squares), and S8H (open triangles)
mutants. The solid lines through the data represent the best fits of the data
points to the equation given by Santoro and Bolen (46). The resulting ther-
modynamic parameters for all protein variants are listed in Table 1.
PNAS 1 December 10, 2002 1 vol. 99 1 suppl. 4 1 16421
OCR for page 46
kJmol-i, respectively. Apart from these mutations, however,
none of the remaining 13 substitutions resulted in a loss of
stability of >6 kJ mold. A previous mutational study of AcP, in
which residues in the hydrophobic core of the protein weren't
replaced by other hydrophobic residues with a smaller size,
indicated far larger 1\AGU-F values than those observed here
(30~. This difference is illustrated by the average values of
/\AGU-F determined for the substitutions at hydrophobic core
positions and surface hydrophilic residues, which are 11.5 and 3.5
kJ mol-i, respectively. Charged or hydrophilic residues do not,
therefore, play as significant a role in AcP conformational
stability as residues in the hydrophobic core of the protein.
Effect of the Mutations on the Aggregation Process of AcP. Some of
the mutations analyzed here cause~a significant, albeit small,
destabilization of the native state as described in the previous
section. Amino acid replacements that destabilize the native
state of a protein are known to favor the process of aggregation
by populating unfolded or partially folded states that are more b
prone to aggregation than the fully native state; such effects have
been observed for AcP as well as for other protein systems (26,
34-36, 48~. To overcome this problem, we probed the rate of
aggregation of the polypeptide chain under conditions where the
native fold of the protein is substantially disrupted, i.e., in
aqueous solutions containing 25% (vol/vol) TEE. These condi-
tions were found to denature even the most stable AcP mutants
within a few seconds but still to allow aggregation to occur (15,
25~. This procedure permits any change in aggregation rate to be
attributed entirely to the intrinsic effect of the amino acid
substitutions on the aggregation process, without the complica-
tions of additional contributions arising from the destabilization
of the native state.
Fig. 3a describes the increase of ThT fluorescence resulting
from aggregation of wild-type AcP and some representative
mutants under these conditions. Such increases in ThT fluores-
cence reflect the formation of small aggregates, revealed by
electron microscopy to be spherical or elongated 4-nm-wide
protofibrils (15, 25~. The rate constants obtained by fitting the
data points to single exponential functions are reported in Table
1 for all of the protein variants studied here. The majority of the
mutations involving residues within the two regions of the
sequence 16-31 and 87-98 change the rate of aggregation to a
significant extent (Table 1~. In addition to having a relatively
high hydrophobicity and a considerable propensity to form `S
structure, these two regions are also characterized by a net
charge of zero. The 16-31 region contains six charged residues,
three with a positive charge and the other three with a negative
charge. The 87-98 region is also neutral, because it contains two
positively charged residues, one negatively charged residue, and
the negatively charged C terminus of the protein. If the neu-
tralities of these two regions were directly responsible for their
critical role in the process of aggregation, all substitutions of
charged residues located within these two regions would be
expected to disfavor the process of aggregation. This does not
appear to be the case, however, as two mutations (R23Q and
R97E), both of which generate a local negative charge in these
regions, accelerate the process of aggregation significantly
(Table 1~.
A trend of a different type, however, can be observed when the
aggregation rates reported in Table 1 are examined from a
different perspective. Mutations decreasing the aggregation rate
invariably involve the addition to the protein of an extra positive
charge and/or deletion of a negative one. Similarly, mutations
that increase the aggregation rate involve deletion of positively
charged residues and/or insertion of negatively charged groups.
This behavior can be accounted for by considering the overall
charge of AcP. Under these conditions of pH, the wild-type
protein has a net charge of +5 (AcP contains 9 lysines, 6
16422 1 www.pnas.org/cgi/doi/10.1073/pnas.212527999
a
100
ID _ 80
c' a)
~5
co
In
a) X
'I 0
_
-~ 20
o
-7.0 F
-8.0 ~
-9.0
0 5000
10000 15000 20000
time (s)
2 3
o
r =0.72
p = 0.002
o
I I I I I I I I I I T
4 5 6 7 8
net charge of AcP
. .
Fig. 3. (a) Aggregation of six representative AcP variants followed by ThT
fluorescence. Aggregation was initiated in each case in 25% TFE/50 mM
acetate buffer, pH 5.5, 25C. Aliquots were withdrawn at regular time inter-
vals for the ThT assay. The AcP variants shown are: wild-type (filled circles),
R23Q (open triangles), E29Q (crosses), E29R (open circles), S21R (filled
squares), and E9OH (diamonds). The solid lines through the data points rep-
resent the best fits to single exponential functions. The resulting rate constant
values are reported for all variants in Table 1. (b) Aggregation rate versus net
charge constructed with the data points of the wild-type protein and the 15
mutants. Changes of net charge on mutation are calculated at pH 5.5 assuming
standard pKa values for amino acid residues.
arginines, 6 glutamates, 4 aspartates, and no histidines). Muta-
tions that disfavor the process of aggregation therefore increase
the overall net charge, whereas those that favor aggregation
reduce the net charge. This finding can be rationalized on a
simple electrostatic argument, that self association will tend to
be disfavored if the electrostatic repulsion between distinct
molecules is increased, provided all other factors remain con-
stant. For example, the mutation of the glutamate residue at
position 29 to glutamine increases the net charge of the protein
by 1 unit, from 5 to 6. This change results in an increase of the
overall aggregation rate (Table 1~. When Glu-29 is mutated to
positively charged residues such as arginine or lysine, a more
dramatic increase of the aggregation rate is observed (Table 1).
The greater effect of the mutation to arginine relative to that to
lysine can be attributed to the higher hydrophilicity of arginine
as compared with lysine.
Unlike the hydrophobic interactions and secondary structural
preferences, the effects of the net charge of the protein are not
confined to local regions, again consistent with the simple
Chiti eta/.
OCR for page 47
Table 2. Mutations associated with hereditary forms of amyloid disease involving changes in the net charge of the peptide or
protein associated with each disease
Effect of mutation
Mutation* Amyloid disease Protein/peptide involved on net charges Reference
E693Q Hereditary cerebral hemorrhage with amyloidosis A,B From -3 to -2t 57
E693K Hereditary cerebral hemorrhage with amyloidosis A,B From -3 to -1$ 58
E693G Early-onset Alzheimer's disease A,B From -3 to -2$ 24, 59
D694N Dementia with cerebral amyloid angiopathy A,B From -3to -2$ 23, 60
G26R apoA1 amyloidosis apoA1 From -9to -8 61, 62
W50R apoA1 amyloidosis apoA1 From -9to -8 63
L60R apoA1 amyloidosis apoA1 From -9to -8 64
Z\70-72 apoA1 amyloidosis apoA1 From -9to -8 40
A60-71~ apoA1 amyloidosis apoA1 From -9to -8 7
K257T Frontotemporal dementia with Parkinsonism ~ From +11 to +10ll 65
/\K280 Frontotemporal dementia with Parkinsonism ~ From +11 to +1011 66
K3691 Frontotemporal dementia with Parkinsonism ~ From +11 to +10 67
G389R Frontotemporal dementia with Parkinsonism ~ From +11 to +12ll 68
R406W Frontotemporal dementia with Parkinsonism ~ From + 11 to + 10ll 69
E526V Hereditary renal amyloidosis Fibrinogen c~chain From -3to -2** 4
R554L Hereditary renal amyloidosis Fibrinogen oechain From -3to -4** 5
This list does not include mutations for which a causative link with pathogenesis has been proposed (see text). The 17 mutations listed include only cases for
which this link is not yet established or is still under debate. A recently compiled database (70) has been utilized to identify some of the mutations listed in this
table.
*The numbering refers to the sequence of the intact proteins (~) or of the precursors (A,B, apolipoprotein A1, fibrinogen cY chain). The numbering of A,B and
refers to the longest isoform in each case.
"Calculated at physiological pH 7.4, when only Lys, Arg, Glu, and Asp are assumed to be charged (71).
$The net charge of -3 refers to the 40- or 42-residue form of the A,B peptide.
The net charge of -9 refers to the 93-residue form of apoA1, which is found in amyloid deposits. The charge is -6 if the alternative form of 82 residue is
considered.
~Consisting of the deletion of residues 60-71 and insertion of Val-Thr at the same position.
The net charge of + 11 refers to the four-repeat domain that forms the core of the amyloid fibrils by ~ (paired helical filaments) (72). The net charge becomes
+2 if the whole sequence of the longest ~ isoform is considered.
**The net charge of -3 refers to the fragment of the fibrinogen cr chain (residues 500-580) that has been extracted from ex vivo fibrils (5).
electrostatic argument. Thus, two of the four substitutions that
involve residues outside the two regions 16-31 and 87-98 result
in aggregation rates significantly different from that of wild-type
AcP (Table 14. The most marked rate change is for the R77E
mutation, a substitution that results in a decrease of the net
charge by 2 units. When all 15 variants are considered, a highly
significant negative correlation exists between the net charge
and the aggregation rate, with a linear correlation coefficient (r)
and P values of 0.72 and 0.002, respectively (Fig. 3b).
Despite showing a highly significant correlation, the plot
shown in Fig. 3b also indicates that mutations that cause the same
change in net charge can exhibit considerably different aggre-
gation rates. This variation may reflect a number of factors that
influence this analysis such as the fact that the hydrophobicities
and secondary structure preferences of the protein are not
completely unchanged by any of the mutations. Such effects are
expected to be particularly pronounced when the mutations are
located in the two regions previously identified as key nucleation
sites for aggregation. In accord with this expectation, the S21R
and E9OH mutations, both of which are located well inside the
two key regions, decelerate the aggregation process more than
do analogous replacements outside or at the edges of these
regions. In addition, specific electrostatic effects may contribute
to the aggregation rates, introducing further complications into
the analysis. It is notable, however, that the presence of charged
residues as such does not by itself act to inhibit aggregation. If
this were the case, replacements of negatively charged residues
by positive ones (E29K, E29R, E9OH) and of positively charged
residues by negative ones (R97E, R77E) would not be expected
to produce significant effects on the aggregation kinetics. By
contrast, these mutations result in substantial decelerations and
accelerations of aggregation, respectively (Table 1~.
Ch iti et a/.
Discussion
Net Charge, Hydrophobicity, and Secondary Structure Preferences in
Protein Aggregation. The mutational study described here indi-
cates that the total charge of the aggregation-prone state of a
protein strongly influences its propensity to aggregate. The
relevance of charged residues as "structural gatekeepers" against
aggregation does not, however, appear to be based only on the
ability of these residues to interrupt contiguous stretches of
hydrophobic residues, as suggested (21), but also on their ability
to generate electrostatic repulsions between protein molecules.
This idea is also supported by separate observations that shield-
ing of positively charged groups at low pH accelerates aggrega-
tion (14) and that aggregation of different proteins induced at
neutral pH by preformed fibrils of a positively charged peptide
correlates inversely with the isotonic point of the various proteins
tested (41~. Consistent with this view, proteins that are unfolded
under physiological conditions ("natively unfolded proteins")
generally have a total net charge that is significantly higher than
proteins that fold into globular structures (49~. This mechanism
is likely to be a strategy through which proteins that do not fold
into globular structures avoid aggregation and remain soluble in
the crowded environment of the cell.
Unlike the amino acid replacements that alter the hydropho-
bicity, or-helical or 13-sheet propensities of the AcP sequence,
mutations that modify the charge of the protein are able to alter
the aggregation rate even when these modifications occur out-
side the regions of sequence 16-31 and 87-98 that appear to be
primarily responsible for regulating the aggregation process.
Different regions may play a critical role at different stages of the
aggregation process and therefore produce a change in the
aggregation rate when mutated. However, the change of aggre-
gation rate resulting from such mutations correlates well with the
PNAS | December ~o, 2002 I vof.99 | supp~.4 1 36423
OCR for page 48
overall change in the charge of the protein, rather than with that
of specific regions of the sequence. The lack of sequence
specificity of the charge effects suggests that the formation of
specific electrostatic interactions is not an important determi-
nant of the rate of aggregation of AcP, at least under the
conditions examined in the present study. These data do not, of
course, rule out the possibility that formation of salt bridges
stabilizes the resulting aggregates; rather, they suggest that their
formation plays a minor role in determining the kinetics of
aggregation. The common ability of globular proteins to form
amyloid fibrils at acidic pH values is not inconsistent with these
general findings, because the primary effect of lowering the pH
is the destabilization of the protein native fold, a process
necessary for initiating aggregation (14, 50, 51~. Indeed, aggre-
gation of AcP is substantially slower at low pH than at neutral
pH values if TEE is added to both solutions such that the protein
remains partially denatured through this pH range (data not
shown).
As mentioned above, the aggregation rate is highly sensitive to
conservative mutations that alter the hydrophobicity or I3-sheet
propensity only when these occur within two relatively narrow
regions of the AcP sequence, both of which have intrinsically
high hydrophobicities and high propensities to form I3-sheet
structure (25~. Because hydrogen bonding within I3-structure
and hydrophobic interactions between side-chains are likely to
be the major stabilizing interactions within aggregates, increases
in the propensities for such interactions are likely to enhance the
rate at which aggregation occurs. As with the process of protein
folding, however, formation of such interactions appear to be
rather specific; indeed, both processes appear to involve well
defined groups of residues that have the capability of nucleating
the formation of either the intramolecular or intermolecular
interactions (25~. Interestingly, the residues that nucleate folding
and aggregation are located in different regions of the polypep-
tide sequence, indicating that the two processes are highly
distinct in natural proteins (25~.
Origins of Familial Amyloid Diseases. Nearly 20 human diseases so
far have been associated with amyloid deposition in either the
brain or other organs in the body (1, 2~. Hereditary forms have
been described for many of these diseases, with single-point
mutations often being the genetic changes that are responsible
for their onset. The mechanisms through which the pathogenic
effects of such naturally occurring mutations are mediated have
been well established in a number of cases. Most of the mutations
associated with early-onset Alzheimer's disease and related
pathologies, for example, have been demonstrated to alter the
efficiency or specificity of ,8- and y-secretases, the two proteases
that generate the amyloid I3 (Al3) peptide from the precursor
protein, APP (52-54~. Such alterations are responsible for
either an overproduction of the Al3 peptide or an increased
release of the more amyloidogenic form of the peptide con-
sisting of 42 rather than 40 residues. Alteration of proteolytic
processing is also thought to be crucial in the pathogenesis of
apolipoprotein A1 amyloidosis induced by the L174S mutation
of this protein (6~.
In other cases, destabilization of the native state of the
globular protein responsible for amyloid deposition has been
identified as the primary mechanism by which amino acid
substitutions give rise to disease; this mechanism is particularly
important in a number of systemic noncerebral amyloidoses such
as familial amyloid polyneuropathy, lysozyme amyloidosis, and
light chain amyloidosis (34-36~. The two mutations of gelsolin
associated with familial Finnish type amyloidosis (D187N and
D187Y) have also been found to destabilize the native state of
the protein, facilitating proteolytic attack that generates the
highly amyloidogenic fragment (55, 56~. An increased rate of
oligomerization and an increased ability to permeate the cyto-
16424 1 www.pnas.org/cgi/doi/10.1 073/pnas.212527999
plasmic membrane have been suggested to be possible mecha-
nisms by which the two natural mutations of a-synuclein, the
A30P and A53T substitutions, cause familial Parkinson's disease
(9, 111.
Despite the progress made recently in understanding the
origins of a number of familial amyloid diseases, the mechanism
of action through which the pathogenic effects of many amino
acid replacements are mediated remains elusive. The conclusion
from the present study raises the possibility that some disease-
associated mutations act primarily through a reduction of the net
charge of the corresponding peptides or proteins. To explore this
possibility, we have examined pathogenic mutations involving
substitution, or introduction, of charged residues within the
proteins or peptides associated with amyloid diseases, and for
which the causative link with the pathogenic effects is not yet
established. The mutations of this type that we have been able
to identify are listed in Table 2. Remarkably, 14 of 16 mutations
listed in the table do indeed reduce the net charge of the
polypeptides involved.
Unlike other mutations of APP, and despite the common
assumption that all substitutions within the APP protein alter the
processing of the protein, none of the four mutations of the A,l3
peptide listed in the table have been found to act in this way (23,
24, 53~. By contrast, all four mutations have been shown to
increase the intrinsic propensity of A,8 to form fibrils or proto-
fibrils in vitro (20, 22-24~. That these mutations involve a
reduction of the charge of Al3 suggests this perturbation could
be the primary molecular basis for their pathogenic effect. In this
regard, it is interesting to observe that the charge-preserving
E693D substitution of APP is not pathogenic (20~. Similarly, the
six mutations of the apolipoprotein A1 listed in the table are not
thought to stimulate the release of the N-terminal 82 residue
fragment of the protein that is found in the fibrils, because they
are too distant from the site of proteolysis (6~. All six mutations,
however, have the effect of reducing the negative charge within
the amyloidogenic fragment (Table 24. It has, in fact, previously
been suggested that the pathogenic action of these mutations
could be related to alterations of the charge state (7, 404. This
conclusion is well supported by the overall analysis of charge
mutations described in the present work.
As far as the mutations associated with ~ pathologies are
concerned, a conflicting picture has emerged as to whether their
pathogenicity results primarily from an impairment of the
interactions of ~ with microtubules or an accelerated rate of
aggregation (19, 73, 74~. An exception to this situation is the
N279K variant of the protein, as this has been shown not to
perturb the interaction with microtubules (75~. The nucleotide
substitution that leads to this particular mutation, however, has
been shown to be pathogenic because of an alteration of the
RNA splicing, which results in the production of a more aggre-
gating variant (76, 77~. Among the remaining five mutations that
involve charge alterations within the ~ protein, four of these
reduce the net positive charge of the protein (K257T, AK280,
K369I, and R406W). A mechanism unrelated to the reduction of
charge must account for the pathogenicity of the mutation that
increases the charge (G389R). Finally, the two amino acid
substitutions of the fibrinogen cl-chain associated with heredi-
tary renal amyloidosis cause opposite effects on the net charge
(Table 2~; the E526V mutation reduces the charge of the protein,
but the R554L increases it. In both cases, however, highly
hydrophobic residues replace charged residues. The hydrophobic
effect of the R554L substitution may outweigh its effect on the
charge of the protein.
Overall, therefore, on the assumption that the pKa values are
comparable to those of model compounds, 14 of the 16 muta-
tions listed in Table 2 result in a reduction of the net charge of
the corresponding peptide or protein under physiological con-
ditions, whereas only two mutations increase it. This analysis
Chiti eta/.
OCR for page 49
does not consider mutations for which an alternative causative
link with pathogenesis, such as reduction of protein stability,
alterations of splicing, or proteolytic processes, has been pro-
posed; the 16 mutations discussed here include only cases for
which this link is not established or is still under debate. For
example, disease-related mutations involving globular proteins,
such as the prion protein or transthyretin, are not considered,
because in at least some cases they are likely to be pathogenic as
a consequence of their destabilizing effect on the native fold of
the precursor protein (34-36~. A preliminary analysis, however,
indicates that 12 of 13 and 19 of 25 mutations involving alter-
ations of charge do, in fact, cause a reduction in the total charge
for the prion protein and transthyretin, respectively (for the
prion protein, the 125-231 domain is considered). The present
analysis suggests that the mechanism of action through which the
deposition of amyloid fibrils and the onset of the disease are
mediated could be rather straightforward for the familial dis-
eases associated with these particular mutations. Indeed, these
mutations may stimulate pathological effects simply by increas-
ing the intrinsic propensity of the aggregating polypeptide chains
to self-assemble through a reduction in the electrostatic repul-
sion between the molecules.
Conclusion
Investigating the fundamentals of protein aggregation by using
a model system not linked to amyloid disease and then using any
principles that emerge to interpret the behavior of peptides and
proteins that form amyloid deposits in viva is a powerful
approach to gaining an understanding of the general principles
involved in the development of pathological conditions of this
type. Such an approach may reveal common features in the
process of protein aggregation in a particularly straightforward
manner. The studies carried out on AcP show that mutations can
favor aggregation either by destabilizing the native state, hence
allowing unfolded or partially unfolded species prone to aggre-
gation to be significantly populated, or by directly favoring the
process of self-assembly through an increase in hydrophobicity,
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Biological cells and extracellular spaces are highly crowded
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Fondazione Telethon-Italia (to F.C. ) and the Wellcome Trust (to
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Chiti et a/.
Representative terms from entire chapter:
native state
