From the Department of Medical Biochemistry and
Biophysics, Karolinska Institutet and the ¶ Department of Cell and
Molecular Biology, Medical Nobel Institute, Karolinska Institutet,
S-171 77 Stockholm, Sweden
Received for publication, November 16, 2000, and in revised form, December 22, 2000
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ABSTRACT |
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In Alzheimer's disease and spongiform
encephalopathies proteins transform from their native states into
fibrils. We find that several amyloid-forming proteins harbor an
Amyloid fibrils can be formed from different proteins and are
associated with severe diseases like the neurodegenerative Alzheimer's disease and the prion diseases Creutzfeld-Jakob in humans, scrapie in
sheep, and bovine spongiform encephalopathy, as well as other organ-specific and systemic amyloidoses (1). The ~20 proteins that
are associated with amyloid diseases have no obvious common properties
in amino acid sequence, three-dimensional structure, or function (2).
Despite these differences in native structures, the amyloid fibrils are
very similar irrespective from which protein they originate (3).
Amyloid fibrils are built up from Lung surfactant protein C (SP-C) is a 35-residue lipopeptide derived
from a larger precursor by proteolysis. SP-C isolated from natural
sources is composed of an Protein Data Set--
The May 1999 list of PDB_SELECT (17) was
initially used to obtain a nonhomologous set of proteins from the
Brookhaven data base (18). This list consists of 1106 sequences with
less than 25% residue identity in pairwise comparisons. During the
project a new version of PDB_SELECT was released (November 1999 set), and for completeness, proteins that were nonoverlapping with the May
list were added to the data set. Using FASTA (19), proteins in the
November 1999 set that are not homologous to any of the proteins in the
May set (expect value >0.1) were selected. This resulted in an
addition of 218 proteins, and the final data set thus consisted of 1324 nonhomologous proteins.
Experimentally Determined Secondary Structures--
The
secondary structure elements of the selected proteins were extracted
from the Protein Data Bank files by Defined Secondary Structure of
Proteins (20), as implemented in ICM (version 2.7, Molsoft LLC, San
Diego, CA) (21). This method defines eight secondary structure classes
from hydrogen bond patterns: Predicted Secondary Structures--
Secondary structures were
predicted using PHD (Profile network from HeiDelberg) (22), a system of
neural networks with an overall accuracy of about 72% (23). In 1978, Chou and Fasman (24) calculated the amino acid distributions in
helices, P Proteins Analyses and Electron Microscopy--
SP-C was purified
from porcine lungs (12) and the poly(Val Identification of
Among the proteins with
The correlation between We compared experimentally determined nonredundant protein
structures available from Protein Data Bank with secondary structures predicted for the same proteins using PHD and a Chou-Fasman approach. Thereby 37/1324 (2.8%) proteins were found to contain a Proteins that are not known to form fibrils in vivo can form
fibrils under in vitro conditions that favor
destabilization, like extremes of pH and presence of organic cosolvents
(41, 42). From these studies it has been suggested that amyloid
formation is not primarily dependent on the amino acid sequence but
could be a generic trait of destabilized polypeptides. The findings presented herein indicate that also under physiological conditions, fibril formation is more common than previously anticipated (Fig. 3).
On the other hand, SP-C polyvaline Helix 2 of PrP is discordant, whereas helices 1 and 3 of PrP were
predicted to be helical both with PHD and Chou-Fasman-based methods
(data not shown). Strikingly, in a previous prediction of PrP secondary
structure, contradictory results were obtained for the helix 2 region,
suggesting that alternative conformations of PrP can coexist (43). In
the discordant segment of PrP helix 2, the Cys is connected to helix 3 via a disulfide bond, and the Asn is glycosylated. The consequences of
these modifications for the Amyloid is believed to be associated with, and even responsible for,
the pathological changes seen during the course of the corresponding
diseases (1, 3, 48). This implies that obstruction of amyloid formation
may prevent the occurrence and/or progression of amyloidoses. Attempts
have been made to identify compounds that can abrogate fibril formation
by interfering with peptide-peptide contacts in fibrils or by
inhibiting proteolytic processing that leads to amyloidogenic peptides
(49). Our results suggest that -helix in a polypeptide segment that should form a
-strand
according to secondary structure predictions. In 1324 nonredundant
protein structures, 37
-strands with
7 residues were predicted in
segments where the experimentally determined structures show helices.
These discordances include the prion protein (helix 2, positions
179-191), the Alzheimer amyloid
-peptide (A
, positions 16-23),
and lung surfactant protein C (SP-C, positions 12-27). In addition,
human coagulation factor XIII (positions 258-266), triacylglycerol
lipase from Candida antarctica (positions 256-266), and
D-alanyl-D-alanine transpeptidase from
Streptomyces R61 (positions 92-106) contain a discordant helix. These proteins have not been reported to form fibrils but in
this study were found to form fibrils in buffered saline at pH 7.4. By
replacing valines in the discordant helical part of SP-C with leucines,
an
-helix is found experimentally and by secondary structure
predictions. This analogue does not form fibrils under conditions where
SP-C forms abundant fibrils. Likewise, when A
residues 14-23 are
removed or changed to a nondiscordant sequence, fibrils are no longer
formed. We propose that
-helix/
-strand-discordant stretches are
associated with amyloid fibril formation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-strands perpendicular and
-sheets parallel to the fiber axis (4). Thus, amyloid-forming
proteins that mainly contain
-helical structures in their native
states must undergo
-helix
-strand conversions before or
during fibril formation. Tinctorial and spectroscopic studies indicate
that the conversion of the cellular form of the prion protein
(PrPC)1 to its
fibrillar scrapie counterpart (PrPSc) is accompanied by
reduction in
-helix content and increase in
-sheet structure (5).
Amyloid
-peptide (A
) fibril formation associated with
Alzheimer's disease also involves
-helix to
-strand conversion
(6). Amyloid diseases mostly occur without known precipitating factors
(7), and no explanation has yet been found to the occurrence of
de novo
conversion in amyloid-forming proteins.
This conversion may occur in partially denaturing cellular environments
(8), which, however, does not explain why only certain proteins form
amyloid. Destabilizing point mutations can cause fibril formation of an
otherwise stable protein (9), but point mutations related to inherited
forms of human prion diseases do not induce PrPSc in
vitro and are not generally destabilizing (10). The
A
-(1-42)-peptide is highly fibrillogenic, whereas peptides lacking
residues 14-23 are not (11). Proteins that give rise to amyloid may
harbor polypeptide segments that make them more prone to undergo
conversions than nonamyloidogenic proteins. A key question is whether specific helices of these proteins are predisposed to undergo
conversions.
-helix covering positions 9-34 (12).
Monomeric
-helical SP-C is thermodynamically unstable, and the
peptide irreversibly forms aggregates with
-sheet structure (13).
SP-C assembles into amyloid fibrils upon incubation in solution, and
fibrils composed of SP-C were isolated from a patient with the disease
pulmonary alveolar proteinosis (14). The
-helix of mature SP-C
contains a very long continuous stretch of valine residues, which is
unusual since valines are well known to be overrepresented in
-strands and underrepresented in helices. Intriguingly, synthetic
SP-C peptides are inefficient in helix formation but form insoluble
aggregates (15). In contrast, peptides where the polyvaline
segment of SP-C was replaced with a helical part of
bacteriorhodopsin or a polyleucine stretch, both with high statistical
helical propensities, readily form helices (15, 16). These features
suggest that
-helices for which
-strands are predicted may be
prone to undergo
-helix
-strand transition and amyloid
formation (14). Here we tested this hypothesis by searching for
discrepancies between experimentally determined
-helices and
predicted extended (
-strand) structures in 1324 nonredundant entries
in the Protein Data Bank. This revealed a striking correlation between
/
discordance and ability to form amyloid fibrils.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helix (H), 310-helix (G),
-helix (I), extended strand (E), isolated
bridge (B), turn (T),
bend (S), and coil (_). Here three classes of secondary structures were
used as follows: helix (H, G, and I), strand (E), and loop (B, T, S,
and _), since these three classes are employed by the method used for
secondary structure prediction.
, and
-strands, P
, based on
a set of 29 proteins. The P values are defined as the
frequency of each amino acid residue in
-helices (or
-strands)
divided by the average frequency of all residues in
-helices (or
-strands). We recalculated these values using stretches of at least
five consecutive residues of helix (H) or
-strand (E) in the Protein
Data Bank May data set described above. For this purpose transmembrane
proteins were removed, resulting in a set of 1091 proteins. The
resulting P
and P
values are given in
Table I.
Leu)-substituted SP-C
analogue (SP-C(Leu)) was synthesized as described (16).
D-Alanyl-D-alanine transpeptidase from
Streptomyces R61 was a kind gift from Drs. Frère and
Joris, University of Liege, Belgium, and triacylglycerol lipase from
Candida antarctica and human coagulation factor XIII were
purchased from Sigma. For fibrillation studies the latter three
proteins were dissolved in phosphate-buffered saline, pH 7.4, at
concentrations of 10-100 µM. SP-C and SP-C(Leu) were
dissolved at 100 or 250 µM in chloroform/methanol/0.1 M HCl, 32:64:5 (by volume), a solvent mixture in which SP-C
retains a structure very similar to that in phospholipid bilayers (25). The protein solutions were incubated at 37 °C for 3 days; thereafter the solutions were centrifuged at 20,000 × g for 20 min. SP-C and SP-C(Leu) contents in the supernatants at different time
points after solubilization were determined by amino acid analysis of triplicate samples. For analyses of fibrils, the pellets were suspended
in a small volume of water by low energy sonication for 5 s.
Aliquots of 8 µl were placed on electron microscopy grids covered by
a carbon-stabilized Formvar film. Excess fluid was withdrawn after
30 s, and after air-drying the grids were negatively stained with
2% uranyl acetate in water. The stained grids were examined and
photographed in a Philips CM120TWIN electron microscope operated at 80 kV.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Helices Predicted to Form
-Strands--
A
nonredundant set of protein sequences with known three-dimensional
structures (1324 proteins, total of 269,058 amino acid residues) was
submitted to PHD for secondary structure prediction. 37 of these
proteins contained a 7-residue or longer
-helix predicted (reliability index
5 on a scale from 1 to 9) to be in
-strand conformation (Fig. 1). This discrepancy
will be referred to as
-helix/
-strand discordance. Secondary
structure predictions based on
-helix and
-strand propensity
values (Table I) gave results in
excellent agreement with the PHD results (Fig.
2). The number of discordant stretches
increases steeply for
6-residue segments (Fig. 1), and only proteins
with
7-residue discordant segments were analyzed further. Fig. 2
shows amino acid sequences as well as determined and predicted
secondary structures for the 17 proteins with
9-residue discordant
segments. The 8-residue discordant segment of A
is also included in
Fig. 2. Moreover, the discordant segments of human and mouse PrP (26,
27) are shown, in addition to the discordant segment of Syrian hamster PrP (28) found by the initial search. The proteins that contain
/
-discordant segments represent a wide selection of structures (ranging from single helical peptides to large globular proteins with
complex
/
architectures), localizations (nuclear, cytosolic, integral and peripheral membrane proteins, as well as extracellular proteins), and species of origin (ranging from virus to human). The set
encompasses three previously known amyloidogenic proteins, i.e. the prion protein (13- or 15-residue discordant
segment, depending on species, corresponding to helix 2), A
(8-residue segment), and SP-C (16-residue segment) (Figs. 1 and 2). No
consensus pattern in the primary structures of the discordant segments
could be detected. Neither a recently proposed consensus sequence for amyloid-forming proteins (29) nor a binary pattern of hydrophobic and
hydrophilic residues found in fibrillating peptides by a combinatorial approach (30) could be observed.
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Fig. 1.
Occurrence of
-helical segments with high
-strand propensities. 1324 nonredundant amino
acid sequences with structures present in the Protein Data Bank
(versions of May and November 1999) were submitted to secondary
structure prediction according to the PHD algorithm. The number of
protein segments are plotted versus the lengths of the
segments for which experimentally determined
-helices coincide with
-strands predicted with a PHD reliability index
5 for all
residues. The Protein Data Bank codes are given for the proteins from
which the helices with
7 residues emanate. Codes in bold
identify proteins that form amyloid fibrils in vivo, and
italics denote proteins shown herein to form fibrils. Also
indicated are the outcome of predictions of prion proteins from human
(hPrP) and mouse (mPrP). The Protein Data Bank
codes represent, in alphabetical order, the following: 1aa0, fibritin
deletion mutant (bacteriophage T4); 1aura, carboxylesterase
(Pseudomonas fluorescens); 1b10, prion protein (Syrian
hamster, sPrP); 1b2va, heme-binding protein A (Serratia
marcescens); 1b5ea, dCMP hydroxymethylase (bacteriophage T4);
1b8oa, purine nucleoside phosphorylase (Bos taurus); 1ba6,
amyloid
-peptide (Homo sapiens); 1bct, bacteriorhodopsin
(Halobacterium halobium); 1bl1, parathyroid hormone receptor
(H. sapiens); 1cpo, chloroperoxidase (Leptoxyphium
fumago); 1cv8, staphopain (Staphylococcus aureus);
1ecra, replication terminator protein (E. coli); 1ggtb,
coagulation factor XIII (H. sapiens); 1h2as, Ni-Fe
hydrogenase (Desulfovibrio vulgaris); 1iab, astacin
(Astacus astacus); 1jkmb, brefeldin A esterase
(Bacillus subtilis); 1kpta, killer toxin (Ustilago
maydis); 1lml, leishmanolysin (Leishmania major);
1mhdb, smad MH1 domain (H. sapiens); 1mnma, transcription
factor MVM1 (S. cerevisiae); 1mtyd, methane monooxygenase
(Methylococcus capsulatus); 1nom, DNA polymerase
(Rattus norvegicus); 1noza, DNA polymerase (bacteriophage
T4); 1pbv, sec7 domain of exchange factor ARNO (H. sapiens);
1quta, lytic transglycosylase Slt35 (E. coli); 1spf, lung
surfactant protein C (SP-C) (Sus scrofa); 1sra, osteonectin
(H. sapiens); 1taha, lipase (Burkholdia glumae);
1tca, lipase B (C. antarctica, CALB); 1vns, chloroperoxidase
(Curvularia inaequalis); 1wer, Ras-GTPase-activating domain
of p120GAP (H. sapiens); 2erl, pheromone Er-1
(Euplotes raikovi); 2ifo, inovirus (Xanthomonas
oryzae); 2occk, cytochrome c oxidase (B. taurus); 2sqca, squalene-hopene cyclase (Alicyclobacillus
acidocaldarius); 3aig, adamalysin II (Crotalus
adamanteus); 3pte, transpeptidase (Streptomyces
R61).
Propensity values for -helices (P
) and
-strands (P
)
and P
values now
calculated from 1091 proteins are given without parentheses, while the
corresponding values derived originally from a set of 29 proteins are
given in parentheses. P values that differ
0.15 between
the two data sets are written in bold.
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Fig. 2.
Characteristics of long discordant
protein segments. Amino acid sequences, together with determined
and predicted secondary structure elements for all identified
9-residue discordant segments, and those of A
, mouse, and human
PrP. The proteins are grouped after the length of their discordant
stretch. The proteins for which amyloid formation has been demonstrated
are identified by full names or abbreviations, in addition to the
Protein Data Bank codes given for all entries. The bottom
row of each case shows the experimentally determined helical
segments as blue cylinders in which the amino acid sequences
are given. The sequence locations of the helices in the Protein Data
Bank entries (upper numbers) and in the full-length proteins
(lower numbers) are given. In the cases where only one
number is given, the Protein Data Bank entries correspond to the
full-length protein. In the middle row, the locations of the
-strands predicted by PHD are visualized by yellow
strands, where the reliability index for each residue is shown.
The propensity-based predictions, averaged for 6-residue segments and
plotted above residue 3 in each segment, are given in the
top row. In these predictions, E denotes
extended structures (i.e.
-strands) predicted
with high (average >1.0) and e denotes low (average
1.0) probability. H and h represent predicted
helical structures in an analogous manner.
7-residue discordant segments, five are
integral membrane proteins or parts thereof (bacteriorhodopsin, cytochrome c oxidase, SP-C, A
, and parathyroid hormone
receptor). The driving forces for
-helix formation in a membrane
environment differ from those in aqueous solution (31), and the
secondary structure prediction methods used here are based mainly on
soluble proteins. However, A
, which is derived from a trans- and
juxtamembrane region of its precursor protein, and SP-C, which is a
transmembrane peptide, form amyloid in vivo. The proteins
found cover a broad range of functions and many are enzymes (59%
compared with 47% in the starting data set) or other ligand-binding
proteins (Figs. 1 and 2). In several cases the discordant helices
harbor active site or ligand-interacting residues. For example, the
metalloproteases astacin (code 1iab) and adamalysin II (code 3aig) and
methane monooxygenase (code 1mty) harbor zinc- or iron-binding residues in their respective helix (32-34); the helix of heme-binding protein A
(code 1b2v) contains several residues important for heme binding (35);
in the Arf exchange factor ARNO (code 1pbv) the discordant segment is
involved in Arf binding (36); the helix of the light-driven ion pump
bacteriorhodopsin (code 1bct) binds the photo-sensitive retinal
(37); and the active-site serine of Streptomyces R61 transpeptidase is located in the discordant helix (38). Glockshuber et al. (39) observed that PrP shows similarities to signal
peptidases and that His-177 then is a putative active-site residue.
His-177 is located in the N-terminal part of PrP helix 2, now found to be discordant (Fig. 2).
/
-Discordant Segments Predispose to Amyloid Fibril
Formation--
Three of the proteins harboring long discordant
helices, i.e. transpeptidase from Streptomyces
R61, triacylglycerol lipase from C. antarctica, and human
coagulation factor XIII, with 15-, 11-, and 9-residue discordant
segments were available to us for fibrillation studies. All three
proteins were found to form amyloid fibrils in buffer at physiological
pH (Fig. 3). Thus, 6/37 proteins with
7-residue-long
/
-discordant segments and 4/10 with segments of
11 residues have been analyzed, and all form amyloid fibrils.
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Fig. 3.
Amyloid fibrils formed from proteins with
long /
-discordant
segments. Electron micrographs of fibrils formed after 3 days of
incubation in phosphate-buffered saline, pH 7.4, at 37 °C of
transpeptidase from Streptomyces R61 (A),
triacylglycerol lipase B from C. antarctica (B),
and human coagulation factor XIII (C).
/
discordance and fibril formation
suggests that there may be a causal connection, and experiments with
two of the discordant proteins support this. First, replacement of all
valine residues in the discordant segment of SP-C with leucine yields a
peptide, SP-C(Leu), with helical conformation as judged by circular
dichroism and infrared spectroscopy (16). SP-C(Leu) is in contrast to
the discordant native SP-C predicted to form an
-helical structure,
and the time-dependent aggregation of SP-C and SP-C(Leu)
shows striking differences (Fig. 4). SP-C starts to precipitate during the first hours of incubation and shows
extensive aggregation after 5-10 days, but SP-C(Leu) shows no signs of
precipitation during the same period. Consistently, SP-C(Leu) forms no
fibrils or only occasional fibrils after 3 days of incubation at 250 µM concentration (data not shown). SP-C, on the other
hand, forms abundant fibrils at 100 µM concentration after a few hours (14). Second, a synthetic analogue of A
-(1-42) that lacks residues 14-23 and thus is devoid of the
/
-discordant stretch (cf. Fig. 2) does not form detectable amyloid
fibrils under conditions where A
-(1-42) readily forms fibrils (11). Moreover, A
-(1-28) with alanine substitutions at positions 16, 17, and 20 does not form fibrils, whereas A
-(1-28) forms fibrils that
are similar to those formed by A
-(1-42) (40). Intriguingly, these
substitutions revert the discordance of A
, giving instead a
predicted helix between residues 15 and 21 (Fig.
5).
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Fig. 4.
Val Leu
substitutions in SP-C abolish
/
-discordance and reduce
amyloid formation. A, amino acid sequence and
predicted secondary structure by PHD and according to Chou-Fasman
for a polyleucine analogue of SP-C. As in Fig. 2, the PHD prediction
including reliability indices are given in the middle row
and the Chou-Fasman data in the top row, but in this case an
-helix is predicted by both methods, symbolized by a blue
cylinder for the PHD prediction. See Fig. 2 for the sequence of
native SP-C and its predicted secondary structure. The localization of
the
-helix of SP-C(Leu) is inferred from the NMR data of the native
peptide (12) and CD and Fourier transform infrared spectroscopic
analyses of the analogue (16). B, relative amounts, as
determined by amino acid analysis, of SP-C (filled circles)
and SP-C(Leu) (open triangles) that remain in solution after
centrifugation at 20,000 × g for 20 min at different
time points after solubilization. Peptide concentration at start of
incubation is 250 µM for SP-C(Leu) and 100 µM for SP-C. In the case of SP-C, fibrils are readily
detected in the 20,000 × g pellets already after a few
hours of incubation (14), whereas for SP-C(Leu) none or very few
fibrils were found even after 30 days of incubation.
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Fig. 5.
Ala substitutions reverts
/
-discordance of
A
. Predicted secondary structure of
A
-(1-28) and A
-(1-28, K16A, L17A, F20A) compared with the
experimentally determined structure of A
-(1-42). Symbols
as described for Figs. 2 and 4.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
7-residue
-helix that is predicted to form a
-strand. These proteins
include PrP associated with spongiform encephalopathies, A
involved
in Alzheimer's disease, and lung SP-C that forms amyloid in pulmonary alveolar proteinosis (Figs. 1 and 2). Lysozyme contains a
5-residue-long discordant helix (positions 30-34; data not shown), and
wild-type lysozyme does not form amyloid. However, two naturally
occurring human lysozyme variants, with point mutation Ile-56
Thr
or Asp-67
His, are both unstable and amyloidogenic (9). It remains to be investigated whether the discordant helix of lysozyme contributes to the amyloidogenic nature of the mutants. The remaining proteins known to be associated with amyloid diseases (1, 2) are either all
proteins or lack experimentally determined three-dimensional structures. Three proteins now found to be
/
-discordant,
i.e. human coagulation factor XIII, Streptomyces
R61 transpeptidase, and C. antarctica triacylglycerol
lipase, were investigated and found to form amyloid fibrils upon
incubation in aqueous solution at neutral pH (Fig. 3). We also show
that fibril formation and peptide aggregation is practically abolished
by converting the
/
-discordant stretch of SP-C to a helix
composed of residues that statistically favor helix formation (Fig. 4).
Likewise, fibril formation is abolished when the discordant stretch of
the A
peptide is removed or rendered nondiscordant by replacement of
three residues (11, 40) (Fig. 5). It is clear that there are a number
of ways that amyloid fibrils could be formed, and the data presented herein strongly suggest that long stretches of
-helix/
-strand discordance in proteins predict amyloid fibril formation. It appears reasonable that proteins with discordant helices can form intermediates where the helix has unfolded. The high
-strand propensities of these
regions indicate that they are less likely to refold into a helical
conformation than are regions for which helices are predicted.
-Strands in such species could form
-sheets via protein oligomerization, leading to fibril formation. This has been observed for SP-C; once unfolding of the SP-C helix occurs, refolding is not
observed by NMR (13) or mass
spectrometry,2 but the
peptide instead forms
-sheet aggregates and amyloid fibrils (13,
14).
polyleucine substitution and
K16A/L17A/F20A mutations of A
-(1-28) practically abolish fibril
formation, showing that at least in these cases the fibrillation process is sequence-dependent (40). The clear-cut
difference in aggregation between SP-C and its polyleucine analogue
(Fig. 4) contradicts side chain hydrophobicity as a general determinant of fibril formation. Further investigations of the fibril formation of
/
-discordant proteins, and site-directed mutants where the discordant helices have been modulated, may shed more light on general
principles that underlie amyloid fibril formation under physiological conditions.
/
-discordance are not known at the
present. No posttranslational chemical modifications responsible for
the conversion of PrPC to PrPSc have been found
(44). In many cases protein aggregation involves structured folding
intermediates (45). PrP can exist in multiple conformations, suggesting
that PrPC may be intrinsically flexible and prone to
structural transitions (46). Out of eight point mutations,
corresponding to polymorphic sites associated with inherited forms of
prion disease, T183A localized in the middle of helix 2 resulted in
extensive aggregation at a concentration 160-fold lower than that at
which the other mutants were soluble when introduced in recombinant
mouse PrP-(121-231) (10). Likewise, recombinant human PrP-(90-231)
D178N mutation (in helix 2) gave rise to aggregation, whereas P102L and
E200K mutants were soluble (47).
/
-discordant helices are involved
in the fibrillation process for some proteins and that stabilization of
these segments in helical conformation could prevent amyloid formation.
Such an approach appears worthwhile to explore, as the
/
-discordant segments represent small and specific regions, and
their localizations in the disease-associated proteins A
and PrP are
now identified.
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ACKNOWLEDGEMENTS |
---|
We are grateful to Prof. Kurt Wüthrich and Dr. Lars Tjernberg for constructive comments on this manuscript and to Erik Nordling for extracting the experimentally determined structures from the Protein Data Bank.
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FOOTNOTES |
---|
* This work was supported by the Swedish Medical Research Council, Gunvor and Josef Anérs Foundation, Magn. Bergvalls Foundation, the Swedish Heart Lung Foundation, the King Gustaf V 80th Birthday Fund, the European Union Grant Bio4-CT97-2123, the Åke Wiberg Foundation, and the Swedish Foundation for Strategic Research.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.
§ Associated with Stockholm Bioinformatics Centre, Karolinska Institutet, S-171 77 Stockholm, Sweden.
To whom correspondence should be addressed. Fax: 46-8-337462;
E-mail: jan.johansson@mbb.ki.se.
Published, JBC Papers in Press, December 27, 2000, DOI 10.1074/jbc.M010402200
2 M. Gustafsson, W. J. Griffiths, E. Furusjó, and J. Johansson, manuscript in preparation.
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ABBREVIATIONS |
---|
The abbreviations used are:
PrPC, cellular form of prion protein;
A, amyloid
-peptide;
PrP, prion protein;
PrPSc, scrapie form of prion protein;
SP-C, lung surfactant protein C;
SP-C(Leu), poly-Val
poly-Leu
substituted analogue of SP-C;
PHD, Profile network from
HeiDelberg.
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1. | Kelly, J. W. (1996) Curr. Opin. Struct. Biol. 6, 11-17[CrossRef][Medline] [Order article via Infotrieve] |
2. | Sipe, J. D. (1992) Annu. Rev. Biochem. 61, 947-975[CrossRef][Medline] [Order article via Infotrieve] |
3. | Dobson, C. M. (1999) Trends Biochem. Sci. 24, 329-332[CrossRef][Medline] [Order article via Infotrieve] |
4. |
Jiménez, J. L.,
Guijarro, J. I.,
Orlova, E.,
Zurdo, J.,
Dobson, C. M.,
Sunde, M.,
and Saibil, H. R.
(1999)
EMBO J.
18,
815-821 |
5. | Pan, K. M., Baldwin, M., Nguyen, J., Gasset, M., Serban, A., Groth, D., Mehlhorn, I., Huang, Z., Fletterick, R. J., Cohen, F. E., and Prusiner, S. B. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 10962-10966[Abstract] |
6. | Barrow, C. J., Yasuda, A., Kenny, P. T., and Zagorski, M. G. (1992) J. Mol. Biol. 225, 1075-1093[Medline] [Order article via Infotrieve] |
7. |
Lansbury, P. T., Jr.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
3342-3344 |
8. |
Kelly, J. W.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
930-932 |
9. | Booth, D. R., Sunde, M., Bellotti, V., Robinson, C. V., Hutchinson, W. L., Fraser, P. E., Hawkins, P. N., Dobson, C. M., Radford, S. E., Blake, C. C. F., and Pepys, M. B. (1997) Nature 385, 787-793[CrossRef][Medline] [Order article via Infotrieve] |
10. | Liemann, S., and Glockshuber, R. (1999) Biochemistry 38, 3258-3267[CrossRef][Medline] [Order article via Infotrieve] |
11. |
Tjernberg, L. O.,
Callaway, D. J.,
Tjernberg, A.,
Hahne, S.,
Lilliehook, C.,
Terenius, L.,
Thyberg, J.,
and Nordstedt, C.
(1999)
J. Biol. Chem.
274,
12619-12625 |
12. | Johansson, J., Szyperski, T., Curstedt, T., and Wüthrich, K. (1994) Biochemistry 33, 6015-6023[Medline] [Order article via Infotrieve] |
13. |
Szyperski, T.,
Vandenbussche, G.,
Curstedt, T.,
Ruysschaert, J.-M.,
Wüthrich, K.,
and Johansson, J.
(1998)
Protein Sci.
7,
2533-2540 |
14. | Gustafsson, M., Thyberg, J., Näslund, J., Eliasson, E., and Johansson, J. (1999) FEBS Lett. 464, 138-142[CrossRef][Medline] [Order article via Infotrieve] |
15. | Johansson, J., Nilsson, G., Strömberg, R., Robertson, B., Jörnvall, H., and Curstedt, T. (1995) Biochem. J. 307, 535-541[Medline] [Order article via Infotrieve] |
16. | Nilsson, G., Gustafsson, M., Vandenbussche, G., Veldhuizen, E., Griffiths, W. J., Sjövall, J., Haagsman, H. P., Ruysschaert, J.-M., Robertson, B., Curstedt, T., and Johansson, J. (1998) Eur. J. Biochem. 255, 116-124[Abstract] |
17. |
Hobohm, U.,
Scharf, M.,
Schneider, R.,
and Sander, C.
(1992)
Protein Sci.
1,
409-417 |
18. |
Berman, H. M.,
Westbrook, J.,
Feng, Z.,
Gilliland, G.,
Bhat, T. N.,
Weissig, H.,
Shindyalov, I. N.,
and Bourne, P. E.
(2000)
Nucleic Acids Res.
28,
235-242 |
19. | Pearson, W. R., and Lipman, D. J. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 2444-2448[Abstract] |
20. | Kabsch, W., and Sander, C. (1983) Biopolymers 22, 2577-2637[Medline] [Order article via Infotrieve] |
21. | Abagyan, R., and Totrov, M. (1994) J. Mol. Biol. 235, 983-1002[CrossRef][Medline] [Order article via Infotrieve] |
22. | Rost, B., and Sander, C. (1993) J. Mol. Biol. 232, 584-599[CrossRef][Medline] [Order article via Infotrieve] |
23. | Rost, B., and Sander, C. (1994) Proteins 19, 55-77[Medline] [Order article via Infotrieve] |
24. | Chou, P. Y., and Fasman, G. D. (1978) Adv. Enzymol. Relat. Areas Mol. Biol. 47, 45-148[Medline] [Order article via Infotrieve] |
25. | Johansson, J., Szyperski, T., and Wüthrich, K. (1995) FEBS Lett. 362, 261-265[CrossRef][Medline] [Order article via Infotrieve] |
26. |
Zahn, R.,
Liu, A.,
Luhrs, T.,
Riek, R.,
von Schroetter, C.,
Lopez,
Garcia, F.,
Billeter, M.,
Calzolai, L.,
Wider, G.,
and Wüthrich, K.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
145-150 |
27. | Riek, R., Hornemann, S., Wider, G., Billeter, M., Glockshuber, R., and Wüthrich, K. (1996) Nature 382, 180-182[CrossRef][Medline] [Order article via Infotrieve] |
28. |
James, T. L.,
Liu, H.,
Ulyanov, N. B.,
Farr-Jones, S.,
Zhang, H.,
Donne, D. G.,
Kaneko, K.,
Groth, D.,
Mehlhorn, I.,
Prusiner, S. B.,
and Cohen, F. E.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
10086-10091 |
29. | Kurochkin, I. G. (1998) FEBS Lett. 427, 153-156[CrossRef][Medline] [Order article via Infotrieve] |
30. |
West, M. W.,
Wang, W.,
Patterson, J.,
Mancias, J. D.,
Beasley, J. R.,
and Hecht, M. H.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
11211-11216 |
31. | Li, S. C., and Deber, C. M. (1994) Nat. Struct. Biol. 1, 368-373[Medline] [Order article via Infotrieve] |
32. | Bode, W., Gomis-Ruth, F. X., Huber, R., Zwilling, R., and Stocker, W. (1992) Nature 358, 164-167[CrossRef][Medline] [Order article via Infotrieve] |
33. |
Gomis-Ruth, F. X.,
Meyer, E. F.,
Kress, L. F.,
and Politi, V.
(1998)
Protein Sci.
7,
283-289 |
34. | Rosenzweig, A. C., Nordlund, P., Takahara, P. M., Frederick, C. A., and Lippard, S. J. (1995) Chem. Biol. 2, 409-418[CrossRef] |
35. | Arnoux, P., Haser, R., Izadi, N., Lecroisey, A., Delepierre, M., Wandersman, C., and Czjzek, M. (1999) Nat. Struct. Biol. 6, 516-520[CrossRef][Medline] [Order article via Infotrieve] |
36. | Cherfils, J., Menetrey, J., Mathieu, M., Le Bras, G., Robineau, S., Beraud-Dufour, S., Antonny, B., and Chardin, P. (1998) Nature 392, 101-105[CrossRef][Medline] [Order article via Infotrieve] |
37. | Barsukov, I. L., Nolde, D. E., Lomize, A. L., and Arseniev, A. S. (1992) Eur. J. Biochem. 206, 665-672[Abstract] |
38. |
Kelly, J. A.,
Knox, J. R.,
Moews, P. C.,
Hite, G. J.,
Bartolone, J. B.,
Zhao, H.,
Joris, B.,
Frere, J. M.,
and Ghuysen, J. M.
(1985)
J. Biol. Chem.
260,
6449-6458 |
39. | Glockshuber, R., Hornemann, S., Billeter, M., Riek, R., Wider, G., and Wütrich, K. (1998) FEBS Lett. 426, 291-296[CrossRef][Medline] [Order article via Infotrieve] |
40. |
Tjernberg, L. O.,
Näslund, J.,
Lindqvist, F.,
Johansson, J.,
Karlström, A. R.,
Thyberg, J.,
Terenius, L.,
and Nordstedt, C.
(1996)
J. Biol. Chem.
271,
8545-8548 |
41. |
Guijarro, J. I.,
Sunde, M.,
Jones, J. A.,
Campbell, I. D.,
and Dobson, C. M.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
4224-4228 |
42. |
Chiti, F.,
Webster, P.,
Taddei, N.,
Clark, A.,
Stefani, M.,
Ramponi, G.,
and Dobson, C. M.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
3590-3594 |
43. | Huang, Z., Gabriel, J.-M., Baldwin, M. A., Fletterick, R. J., Prusiner, S. B., and Cohen, F. E. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 7139-7143[Abstract] |
44. | Stahl, N., Baldwin, M. A., Teplow, D. B., Hood, L., Gibson, B. W., Burlingame, A. L., and Prusiner, S. B. (1993) Biochemistry 32, 1991-2002[Medline] [Order article via Infotrieve] |
45. | Wetzel, R. (1996) Cell 86, 699-702[Medline] [Order article via Infotrieve] |
46. | Zhang, H., Stockel, J., Mehlhorn, I., Groth, D., Baldwin, M. A., Prusiner, S. B., James, T. L., and Cohen, F. E. (1997) Biochemistry 36, 3543-3553[CrossRef][Medline] [Order article via Infotrieve] |
47. |
Swietnicki, W.,
Petersen, R. B.,
Gambetti, P.,
and Surewicz, W. K.
(1998)
J. Biol. Chem.
273,
31048-31052 |
48. |
Näslund, J.,
Haroutunian, V.,
Mohs, R.,
Davis, K. L.,
Davies, P.,
Greengard, P.,
and Buxbaum, J. D.
(2000)
J. Am. Med. Assoc.
283,
1571-1577 |
49. | Selkoe, D. J. (1999) Nature 399 (suppl.), 23-31[CrossRef] |