Water-soluble tripeptide Aβ (9-11) forms amyloid-like fibrils and exhibits neurotoxicity

Article abstract of DOI:10.1021/ol8007217

(Chemical Equation Presented) A water-soluble, hydrophilic tripeptide GYE, having sequence identity with the N-terminal segment of amyloid peptides Aβ(9-11), upon self-association exhibits amyloid-like fibrils and significant neurotoxicity towards the Neu

Full text of DOI:10.1021/ol8007217

ORGANIC
LETTERS
2008
Vol. 10, No. 13
2625-2628
Water-Soluble Tripeptide Aꢀ (9-11)
Forms Amyloid-Like Fibrils and Exhibits
Neurotoxicity
Jishu Naskar,‡ Michael G. B. Drew,§ Ishani Deb,| Sumantra Das,| and
Arindam Banerjee*,†,‡
Chemistry DiVision, Indian Institute of Chemical Biology, JadaVpur, Kolkata 700032,
India, Department of Biological Chemistry, Indian Association for the CultiVation of
Science, JadaVpur, Kolkata 700 032, India, School of Chemistry, The UniVersity of
Reading, Whiteknights, Reading RG6 6AD, U.K., and Cell Biology and Physiology
DiVision, Indian Institute of Chemical Biology, JadaVpur, Kolkata 700032, India
arindam@iicb.res.in
Received March 31, 2008
ABSTRACT
A water-soluble, hydrophilic tripeptide GYE, having sequence identity with the N-terminal segment of amyloid peptides Aꢀ(9-11), upon self-
association exhibits amyloid-like fibrils and significant neurotoxicity towards the Neuro2A cell line. However, the tripeptides GFE and GWE,
in which the centrally located tyrosine residue has been replaced by phenylalanine or tryptophan, fail to show amyloidogenic behavior and
exhibit little or no neurotoxicity.
The aggregation of normally soluble proteins into well-
ordered amyloid fibrils is associated with a number of
diseases including Alzheimer’s disease, Parkinson’s disease,
type II diabetes, prion-related diseases, and others.1 Among
these diseases, Alzheimer’s disease (AD) is the most
prevalent and progressive neurodegenerative disease associ-
ated with deposition of ꢀ-sheet-rich protein aggregates in
specific regions of the human brain as amyloid fibrils,2 which
consist mainly of amyloid peptides like Aꢀ(1-40) and Aꢀ(1-
42).3 Aꢀ peptides are generated from highly regulated and
sequential cleavage of the amyloid precursor protein (APP)
by proteases designated as ꢀ- and γ-secretases and are readily
detected in human CSF4 as a range of isoforms between 38
and 43 amino acids in length. They normally exist as soluble
random coils. However, in a diseased condition they misfold
and form self-assembled oligomers which further self-
associate to form amyloid fibrils.5 The molecular structure
of full-length Aꢀ fibrils is still not completely clear because
of the difficulty of growing good quality crystals that can
diffract well enough to obtain crystal structures. Low
solubility and noncrystallinity of amyloid fibrils obtained
from full-length Aꢀ peptide preclude the possibility of getting
† Chemistry Division, Indian Institute of Chemical Biology.
‡ Indian Association for the Cultivation of Science.
§ The University of Reading.
| Cell Biology and Physiology Division, Indian Institute of Chemical
Biology.
(3) Glenner, G. G.; Wong, C. W. Biochem. Biophys. Res. Commun. 1984,
120, 885–890.
(1) (a) Dobson, C. M. Trends Biochem. Sci. 1999, 24, 329–332. (b)
Rochet, J. C.; Lansbury, P. T. J. Curr. Opin. Struct. Biol. 2000, 10, 60–68.
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K. S. Science 1992, 256, 780–783.
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10.1021/ol8007217 CCC: $40.75
Published on Web 06/05/2008
2008 American Chemical Society

good crystals. Determination of the molecular basis of
recognition and self-assembly process that govern amyloid
fibril formation is therefore a very challenging task. So, short
model peptides, which provide valuable information about
the factors responsible for amyloid formation are of crucial
importance.
behavior, and these fibrils are toxic towards the Neuro 2A
cell line. We have also synthesized two mutant tripeptides
in which the centrally positioned amino acid residue has been
substituted by other proteinous aromatic amino acid phenay-
lalanine (Phe) or tryptophan (Trp) to examine whether these
tripeptides can form amyloid-like fibrils and exhibit neuro-
toxicity. Figure 1 shows chemical structures of reported
peptides 1, 2, and 3.
Westermark and co-workers have carried out pioneering
work on the use of model peptides for the study of amyloid
fibril formation.6 Serranno et al. have shown the residue-
specific tendency for amyloid fibrilation in a series of model
hexapeptides.7 Gazit et al. have illustrated the self-aggrega-
tion of short peptide fragments into amyloid fibrils and
established the role of an aromatic residue (phenylalanine)
in amyloidosis.8 Serpell et al. have described the self-
association of an amyloid-forming 12-residue peptide in the
solid state, and the structure illustrates the molecular ar-
rangement of the amyloid fibril forming peptide in crystals
showing a tentative model for side-chain packing within the
amyloid fiber.9 Mihara et al. have recently reported that a
series of designed short peptides with various hydrophobici-
ties based on the sequence of Aꢀ(14-23) can form amyloid-
like fibrils effectively by using mature Aꢀ(1-42) fibrils as
nuclei.10 Recent studies have also demonstrated that the
fundamental unit of amyloid-like fibrils is a steric zipper
formed by two tightly interdigited ꢀ-sheets.11
Figure 1. Chemical structures of peptides 1, 2, and 3.
Our group has been engaged in studying the self-assembly
of short model peptides which form supramolecular ꢀ-sheets
and amyloid-like fibrils upon self-association.12 It has been
found that C-terminal portion of the full length Aꢀ peptide
has a definitive role in amyloid fibril formation.13 So, there
is a need to test whether any fragment from N-terminal
hydrophilic region can from amyloid-like fibrils or not. Gazit
and his co-workers have made a seminal contribution in the
self-assembly of very short peptide unit Phe-Phe.14 In this
report, we present the self-assembly of the water soluble
tripeptide having sequence identity with the N-terminal
segment of Aꢀ peptide Aꢀ(9-11), GYE, peptide 1, which
forms intermolecularly hydrogen-bonded supramolecular
ꢀ-sheet structure in crystals and in solution. It also forms
straight, unbranched nanofibrils that exhibit amyloid-like
FT-IR spectroscopic studies were carried out to obtain
structural information in the solid state and in solution. The
FT-IR spectra of peptide 1 in the solid state shows a well-
defined CdO stretching band (amide I) at 1637.5 cm-1 and
NH-stretching band at 3280 cm-1, typical of intermolecularly
hydrogen-bonded ꢀ-sheet structure in the solid state (Figure
S8).15a Moreover, peptide 1 shows a medium-intensity band
at 1678 cm-1 indicating the formation of an antiparallel
ꢀ-sheet structure in the solid state. N-H bending frequencies
of this peptide appear at 1515 cm-1 suggesting also the
formation of a ꢀ-sheet structure.15b On the other hand,
peptides 2 and 3 show a sharp CdO stretching band at
1666.4 and 1670 cm-1, respectively, which clearly indicates
that peptides 2 and 3 do not form the ꢀ-sheet structure in
the solid state.16 FTIR spectra of solutions contaning peptides
1 and 3, aged over 7 days, shows a sharp CdO stretching
(amide I) band at 1617 and 1620 cm-1, which indicates the
H-bonded supramolecular ꢀ-sheet conformation in solution
(Figure S9). On the other hand, a similarly aged solution of
peptide 2 shows a characteristic CdO stretching band at 1653
cm-1, indicating its random coil conformation.17
(6) (a) Westermark, P.; Engstro¨m, U.; Johnson, K. H.; Westermark,
G. T.; Betsholtz, C. Proc. Natl. Acad. Sci. U.S.A. 1990, 87, 5036–5040. (b)
Ha¨ggqvist., B.; Na¨slund, J.; Sletten, K.; Westermark, G. T.; Mucchiano,
G.; Tjernberg, L. O.; Nordstedt, C.; Engstro¨m, U.; Westermark., P. Proc.
Natl. Acad. Sci. U.S.A. 1999, 96, 8669–8674.
(7) Paz, Manuela.; Serranno, L. Proc. Natl. Acad. Sci. U.S.A. 2004, 101,
87–92.
(8) Azriel, R.; Gazit, E. J. Biol. Chem. 2001, 276, 34156–34161.
(9) Makin, O. S.; Atkins, E.; Sikorski, P.; Johansson, J.; Serpell, L. C.
Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 315–320.
(10) Sato, J.; Takahashi, T.; Oshima, H.; Matsumura, S.; Mihara, H.
Chem. Eur. J. 2007, 13, 7745–7752.
The conformational analysis of peptide 1 in solid state
from FT-IR spectroscopy was further supported by a single-
crystal X-ray diffraction study.18 Colorless, needle-shaped
(11) Sawaya, M. R.; Sambashivan, S.; Nelson, R.; Ivanova, M. I.;
Sievers, S. A.; Apostol, M. I., Thompson, M. J.; Balbirnie, M.; Wiltzius,
J. J. W.; McFarlane, H. T.; Madsen, A. Ø.; Riekel, C, Eisenberg, D., Nature
2007, 447, 453-457.
(12) (a) Maji, S. K.; Drew, M. G. B.; Banerjee, A. Chem. Commun.
2001, 1446–1447. (b) Banerjee, A.; Maji, S. K.; Drew, M. G. B.; Halder,
D.; Das, A. K.; Banerjee, A. Tetrahedron 2004, 60, 5935-5944. (c) Ray,
S.; Das, A. K.; Drew, M. G. B.; Banerjee, A. Chem. Commun. 2006, 4230–
4232.
(15) (a) Mazor, Y.; Gilead, S.; Benhar, I.; Gazit, E. J. Mol. Biol. 2002,
322, 1013–1024. (b) Moretto, V.; Crisma, M.; Bonora, G. M.; Toniolo, C.;
Balaram, H.; Balaram, P. Macromolecules 1989, 22, 2939–2944.
(16) Kenndy, D. F.; Crisma, M.; Toniolo, C.; Chapman, D. Biochemistry
1991, 30, 6541–6548.
(13) Jarrett, T. Joseph.; Berger, P. Elizabath.; Lanbury, T. Peter.
Biochemistry 1993, 32, 4693–4697.
(17) Hu, X.; Kaplan, D.; Cebe, P. Macromolecules 2006, 39, 6161–
6170.
(14) Raches, M.; Gazit, E. Science 2003, 300, 625–627.
2626
Org. Lett., Vol. 10, No. 13, 2008

single crystals of peptide 1, suitable for X-ray analysis were
obtained by slow evaporation at room temperature from
water-methanol (95:5) solution (500 mg per 15 mL). The
molecular conformation of peptide 1 shows that there are
two independent molecules (A and B), present in the
asymmetric unit (Figure S10) and upon self-association they
from a complex quaternary supramolecular ꢀ-sheet structure
(Figure S11). Backbone torsion angles (Table S2) of the two
molecules present in the asymmetric unit fall mostly within
the extended region of the Ramachandan plot.22 Unfortu-
nately, we were unable to grow crystals of peptides 2 and 3
using a wide range of solvents and thereby preventing the
valuable insight on their organization in the solid state.
To test whether the aggregates formed by the peptides are
amyloidogenic, we studied the morphologies of the ag-
gregates by TEM. A solution (4 × 10-3 M) of peptide 1 in
PBS buffer (10 mM) at pH 7.3 aged over 7 days at 37 °C
gave a straight, unbranched, and rodlike nanofibrillar struc-
ture with diameters ranging from 10 to 15 nm, thus exhibiting
a typical feature of amyloid-like fibrils (Figure 2). Under
Fibrils of peptide 1 bind with CR and exhibit a typical
green gold birefringence indicating its amyloid-like nature.
By contrast, both peptides 2 and 3 fail to bind with CR under
similar conditions (Figure 3).
Figure 3. (a-c) CR binding assay of peptides 1-3.
Along with CR, ThT is believed to specifically interact in
some unknown way with the cross ꢀ-sheet structure of
amyloid fibrils, and this gives rise to an excitation (absorp-
tion) maxima at 450 nm and enhanced emission at 482 nm
as compared to excitation at 385 nm and emission at 445
nm for the free dye molecules of ThT.24 The aged solution
of peptide 1 interacts with ThT and gives rise to an emission
at 482 nm (Figure 4) with much higher intensity compared
Figure 2. (a-c) TEM images of the aggregates formed by peptides
1-3 having been dissolved in PBS buffer (10 mM) at pH 7.3 and
incubated at 37 °C for 7 days.
Figure 4. Peptide 1 binds with ThT and gives an emission band at
482 nm with much higher intensity than the mixture of ThT and
peptide 2/3.
similar conditions, peptide 2 forms only branch fibrillar
morphology and peptide 3 failed to show any fibrillar
aggregates (straight or branched).
to the emission maxima of the mixture of ThT and each of
the peptides 2 and 3. It clearly establishes that the aged
solution of peptide 1 binds with ThT showing an amy-
loidogenic property.
Toxicity towards neuronal cells is one of the important
characteristics of amyloid fibrils.25 The toxicity of aged
peptides solution on the viability of 96 well cultured neuronal
cells (Neuro2A) were investigated by MTT assays as
performed earlier.26 It was found that only aggregates of
peptide 1 induced both time- and dose-dependent neuronal
cell death. On the other hand, peptide 2 leads only 15% cell
Further Congo Red (CR) and Thioflavin T (ThT) binding
assays were performed to check whether these aggregates
formed by peptides 1, 2, and 3 were amyloidogenic.
CR binds to Aꢀ fibrils to provide a diagnostic test for
amyloid deposits, as the dye-stained plaques appears green
in color under cross-polarized light due to birefringence.23
(18) Crystal data: C16.5H25. 5N3O8.75, FW ) 405.9, triclinic, space group
P1, a ) 9.4682(13) Å, b ) 9.6899(15) Å, c ) 12.0583(15) Å, R )
109.199(13)°, ꢀ ) 99.525(11)°, γ ) 102.145(12)°, Z ) 2, dcalcd ) 1.365
gm/cm3, cryst size ) 0.02 × 0.02 × 0.23 mm3. Diffraction data were
measured with Mo KR (λ) 0.71073 Å) radiation at 150 K using an Oxford
Diffraction X-Calibur CCD system. Data analysis was carried out with the
Crysalis program.19 The structure was solved by direct methods using the
SHELXS-9720 program. Refinement was carried out with a full matrix least
squares method against F2 using SHELXL-97.21 The non-hydrogen atoms
were refined with anisotropic thermal parameters. The hydrogen atoms were
included in geometric positions and given thermal parameters equivalent
to 1.2 times those of the atom to which they were attached. The final R
values were R ) 0.1043 and wR2 0.2931 for 3443 data points with I >
2σ(I) for peptide 1. Crystallographic data have been deposited at the
Cambridge Crystallographic Data Centre with reference number CCDC
682269.
(21) Sheldrick, G. M. SHELXS-97 and SHELXL-97: Programs for
Crystallographic Solution and Refinement;UniVersity of Gottingen: Ger-
many, 1997.
(22) Ramachandan, G. N.; Sasisekharan, V. AdV. Protein Chem. 1968,
23, 284–438.
(23) Cohen, A. S. Int. ReV. Exp. Pathol. 1965, 4, 159–243.
(24) Levine, H. Protein Sci. 1993, 2, 404–410.
(25) Bucciantini, M.; Giannoni, E.; Chiti, F.; Baroni, F.; Formigili, L.;
Zurodo, J.; Taddel, N.; Ramponi, G.; Dobson, M. C.; Stefani, M. Nature
2002, 416, 507–511.
(19) Crysalis program, version 1.0; Oxford Diffraction, 2006.
(20) Sheldrick, G. M. Acta Crystallogr. A 1990, 46, 467–473.
(26) Joardar, A.; Sen., A. K.; Das, S. J. Lipid Res. 2006, 47, 571–581.
Org. Lett., Vol. 10, No. 13, 2008
2627

death after 48 h of treatment and peptide 3 failed to show
any significant neuronal toxicity (Figure 5).
labeling (TUNEL) assay suggested that the cell death was
due to apoptosis (Table 1).
Table 1. Effect of Peptide 1 on Apoptosis of Neuro 2A Cells
Using TUNEL Assaya
treatment
% of apoptotic cells
control
5.47 ( 1.17
peptide 1
88.79 ( 2.80*
a Apoptotic cells were positive for both fluorescein-dUTP and
propidium iodide. The percentage of apoptotic cells in each group were
expressed as percentage of total number of cells which is represented as
100%. Three fields were selected randomly for each experiment, and both
apoptotic and total number of cells were counted. Values are mean ( S.E.M.
of three experiments. The asterisk indicates p < 0.0001 from untreated
controls.
Figure 5. MTT assay for cell viability. Peptide 1 (GYE) shows
significant neuronal toxicity and toxicity is reduced in peptide 2
(GFE), whereas peptide 3 (GWE) has insignificant neuronal toxicity.
Here the asterisk indicates significant difference (p < 0.0001) from
untreated control. Assays were performed using 5 mM concentration
of each peptide.
This study has demonstrated that a water-soluble tripeptide
comprised of N-terminal hydrophilic region of Aꢀ peptide
Aꢀ(9-11), GYE self-assembles to form supramolecular
ꢀ-sheet structure and amyloid-like fibrils that exhibit toxicity
towards Neuro2A cell lines. However, replacement of the
centrally located amino acid residue (Tyrosine) by pheny-
lalanine (Phe) or tryptophan (Trp) abolishes amyloidogenic
behavior and toxicity of peptide 2 has been reduced whereas
peptide 3 exhibit insignificant neurotoxocity. The result
highlights on the self-aggregation behavior, formation of
ꢀ-sheet structure, and amyloid-like nature of a small hydro-
philic segment of Aꢀ peptide.
Interestingly, the toxicity induced by peptide 1 increased
with the dose of the compound (Figure 6) and at 0.014 M
concentrations, almost all the cells died. Further characteriza-
tion of the nature of cytotoxicity induced by peptide 1 using
terminal deoxynucleotidyl transferase-mediated nick-end
Acknowledgment. We thank the EPSRC and University
of Reading, U.K., for funds for the Oxford Diffraction
X-calibur CCD system. We wish to acknowledge the CSIR,
New Delhi, India, for financial assistance.
Supporting Information Available: Synthetic and ex-
1
perimental procedures, H NMR, mass, and FTIR spectra
Figure 6
.
Dose response curve of peptide 1 (GYE) on cell viability
for all new compounds, crystal data for peptide 1 in CIF
format, dose-dependent assay. This material is available free
of charge via the Internet at http://pubs.acs.org.
determined by MTT assay. Neuro 2A cells were treated for 48 h
in the presence of different concentration of peptide 1. The asterisk
indicates p < 0.0001 from untreated controls.
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Org. Lett., Vol. 10, No. 13, 2008