Báo cáo khoa học: Synthetic constrained peptide selectively binds and antagonizes death receptor 5 ppt

Synthetic constrained peptide selectively binds and
antagonizes death receptor 5
Johanna Vrielink
1,
*, Mariette S. Heins
1,
*, Rita Setroikromo
1
, Eva Szegezdi
2
, Margaret M. Mullally
1
,
Afshin Samali
2
and Wim J. Quax
1
1 Department of Pharmaceutical Biology, University of Groningen, the Netherlands
2 Department of Biochemistry, Cell Stress and Apoptosis Research Group, National Centre for Biomedical Engineering Science,
National University of Ireland, Galway, Ireland
Introduction
Tumour necrosis factor-related apoptosis-inducing
ligand (TRAIL ⁄ Apo2L), a member of the tumour
necrosis factor (TNF) ligand family, is well known for
its ability to induce apoptosis in many cancer cells but
not in most untransformed cells [1]. This quality makes
TRAIL an interesting and promising target for cancer
therapy and the main focus of TRAIL research has
therefore been on its role in cancer. Less attention has
been paid to the role of TRAIL in neurodegenerative
diseases. Normally, mature neurones will last the lifespan

Keywords
apoptosis; DR5; phage display; R2C16;
TRAIL
Correspondence
W. J. Quax, Department of Pharmaceutical
Biology, University of Groningen, Antonius
Deusinglaan 1, 9713 AV, Groningen, the
Netherlands
Fax: +31 50 363 3000
Tel: +31 50 363 2558
E-mail:
*These authors contributed equally to this
work
(Received 12 October 2009, revised 17
December 2009, accepted 25 January
2010)
doi:10.1111/j.1742-4658.2010.07590.x
Apoptosis or programmed cell death is an inherent part of the development
and homeostasis of multicellular organisms. Dysregulation of apoptosis is
implicated in the pathogenesis of diseases such as cancer, neurodegenera-
tive diseases and autoimmune disorders. Tumour necrosis factor-related
apoptosis-inducing ligand (TRAIL) is able to induce apoptosis by binding
death receptor (DR)4 (TRAIL-R1) and DR5 (TRAIL-R2), which makes
TRAIL an interesting and promising therapeutic target. To identify
peptides that specifically interact with DR5, a disulfide-constrained phage
display peptide library was screened for binders towards this receptor.
Phage-displayed peptides were identified that bind specifically to DR5 and
not to DR4, nor any of the decoy receptors. We show that the synthesized
peptide, YCKVILTHRCY, in both monomeric and dimeric forms, binds
specifically to DR5 in such a way that TRAIL binding to DR5 is inhibited.

Surface plasmon resonance studies showed higher affinity towards DR5 for
the dimeric form then the monomeric form of the peptide, with apparent
K
d
values of 40 nm versus 272 nm, respectively. Binding studied on cell
lines by flow cytometry analyses showed concentration-dependent binding.
Upon co-incubation with increasing concentrations of TRAIL, the peptide
binding was reduced. Moreover, both the monomeric and dimeric forms of
the peptide reduced TRAIL-induced cell death in Colo205 colon carcinoma
cells. The peptide, YCKVILTHRCY, or its derivates, may be a useful
investigative tool for dissecting signalling via DR5 relative to DR4 or could
act as a lead peptide for the development of therapeutic agents in diseases
with dysregulated TRAIL-signalling.
Abbreviations
DcR, decoy receptor; DR, death receptor; EAE, experimental autoimmune encephalomyelitis; FACS, fluorescence-activated cell sorting;
HRP, horseradish peroxidase; OPG, osteoprotegerin; PE, phycoerythrin; pfu, plaque forming unit; RU, response unit; sTRAIL, soluble TRAIL;
TMB, tetramethylbenzidine; TNF, tumour necrosis factor; TRAIL, TNF-related apoptosis-inducing ligand; TRAIL-R, TRAIL-receptor.
FEBS Journal 277 (2010) 1653–1665 ª 2010 The Authors Journal compilation ª 2010 FEBS 1653
of the organism. However, in neurodegenerative
diseases, the balance is shifted towards death and the
major cause of neuronal loss is apoptosis. TRAIL is
present in the central nervous system and can induce
apoptosis in brain cells. Studies have shown the
involvement of TRAIL in neurodegenerative diseases
such as HIV-1-associated dementia, Alzheimer’s
disease and multiple sclerosis [2–4]. Molecules that
counteract the dysregulation of apoptosis could com-
prise effective therapeutic agents in these degenerative
disorders.
TRAIL induces apoptosis by binding to the cyste-

ine-rich extracellular domain of death receptors DR4
(TRAIL-R1) [5] and DR5 (TRAIL-R2) [6–8]. Binding
of the trimeric ligand leads to clustering of the
cytoplasmic death domains of the receptors and
recruitment of signalling molecules to form the death-
inducing signalling complex, which activates the
caspase cascade and thus initiates apoptosis [9,10]. In
addition to DR4 and DR5, TRAIL is capable of bind-
ing to three decoy receptors (DcR): DcR1 ⁄ TRAIL-R3
[6,7], DcR2 ⁄ TRAIL-R4 [11] and osteoprotegerin
(OPG) [12]. DcR1 lacks a functional death domain,
DcR2 contains a truncated death domain and OPG is
a soluble receptor; therefore, they cannot trigger a pro-
apoptotic signal.
Although the crystal structure of TRAIL in complex
with DR5 is known, the exact mechanism of binding
and signal initiation is still not completely understood
[13–15]. The first step in signalling by members of the
TNF family is considered to comprise ligand-induced
trimerization of the receptor. However, the identifica-
tion of a pre-ligand assembly domain suggested that
receptors may already be pre-assembled as trimers
before ligand binding [16–18]. Another intriguing fea-
ture of DR5 is that it appears to be able to mediate
distinctly different cell signals depending on the inter-
action with different receptor agonists [19,20]. Further-
more, depending on the cell type, TRAIL can signal
apoptosis either via DR4 [21–23] or DR5, or both
[24–26]. The reasons for the differences between DR4
versus DR5 signalling are not yet fully understood. To

study the differences in mechanism of ligand binding
and subsequent intracellular signalling through DR4
versus DR5, receptor-selective agonists and antagonists
are necessary. Recently, we have described DR5 [25]
and DR4 [27] selective agonistic variants of TRAIL.
To identify an antagonist to address the differences in
DR4 versus DR5 signalling, we now select for a pep-
tide that binds specifically to DR5. Peptides and small
proteins were demonstrated to bind their targets with
high affinity and specificity and to have advantage
over antibodies [28]. Phage display, a sophisticated
technique that links genotype and phenotype, was used
to select for such ligand-mimicking peptides. Earlier
studies have shown that it is a practical method for
identifying peptides with either agonistic [29–32] or
antagonistic properties for various receptors [33–35].
In the present study, by screening a disulfide-con-
strained phage display peptide library, we report the
identification of a peptide that specifically interacts
with DR5 and blocks binding of TRAIL to DR5. The
identified DR5-binding phage-displayed peptides shows
a strong consensus sequence and the monomeric and
dimeric forms of one of these peptides, YCK-
VILTHRCY, were further characterized. Both the
monomeric and dimeric peptide show selective binding
to DR5 in vitro. To confirm the binding specificity of
the monomeric and dimeric peptides on the membrane
of intact cells, we show binding towards Jurkat cells
that can be competed with soluble TRAIL (sTRAIL).
Finally, we demonstrate that the peptides can reduce

TRAIL-induced apoptosis on Colo205 cells. Compared
to the monomeric form, the dimeric form has higher
affinity for DR5 and increased antagonistic activity.
The identified peptide, or its derivatives, can be a use-
ful tool for elucidating the mechanism of TRAIL
signalling or the mechanism of controlling differential
signalling through DR4 or DR5. In addition, this pep-
tide may act as a lead peptide for the development of
therapeutic agents in diseases with dysregulated
TRAIL-signalling.
Results
Identification of DR5-binding phages
To select for peptides able to bind to DR5 with high
affinity, we used a cystein-constrained heptamer pep-
tide phage library. After three rounds of selection
(as described in the Experimental procedures), 25 indi-
vidual clones were picked and sequenced (Table 1).
The binding ability of the phages displaying these
peptides to DR5 was analysed using ELISA. The wells
were coated with DR5-Fc, the extracellular domain of
DR5 fused to the Fc-portion of human IgG
1
, and the
bound phages were detected with an horseradish per-
oxidase (HRP)-antibody against the phage coat protein
g8p. The background signal measured for a well with
no receptor coated was subtracted. Fourteen out of 25
phages showed binding to DR5 (Table 1). These
peptides share a highly homologous consensus C(K ⁄
I ⁄ L)V(Y ⁄ I ⁄ A)LT(Q ⁄ H ⁄ L)(K ⁄ R)C. Phage 77-R2C16

(CKVILTHRC) showed the highest affinity for DR5
and was selected for further investigation. Purified
phage 77-R2C16 showed a dose-dependent binding to
Inhibition of DR5 signalling J. Vrielink et al.
1654 FEBS Journal 277 (2010) 1653–1665 ª 2010 The Authors Journal compilation ª 2010 FEBS
DR5. Even when using increased amounts, no binding
to DR5 of control phage (i.e. a phage that displays
g3p without a C7C peptide) was observed (Fig. 1A).
Phage 77-R2C16 was also used to determine selectivity
towards DR5 using ELISA. Wells were coated with
different TNF family receptors (i.e. DR4-Fc, DR5-Fc,
DcR1-Fc, DcR2-Fc, OPG-Fc, mouse OPG-Fc, mouse
receptor activator of nuclear factor-jB-Fc and TNF-
receptor 1-Fc) and binding of 77-R2C16 or control
phage was measured. Phage 77-R2C16 exclusively
binds to DR5 and not to any of the other receptors
tested. The control phage demonstrated no binding to
any of the receptors, confirming that binding of
77-R2C16 to DR5 was via the displayed peptide, and
not via other regions of the phage (Fig. 1B). To assess
where the peptide binds to the receptor ⁄ ligand inter-
face of DR5, we tested whether sTRAIL competes for
DR5-binding with phage 77-R2C16. Phage 77-R2C16
was added to the wells at a concentration of 1 · 10
10
plaque forming units (pfu)ÆmL
)1
and the sTRAIL
concentration was increased. With an increasing
concentration of sTRAIL, the binding of the phage to

the receptor DR5 decreased, suggesting that phage
77-R2C16 attaches to a binding patch on DR5 over-
lapping with sTRAIL (Fig. 1C).
Competition studies with synthetic constrained
peptides
Because the phage-displayed peptide may have differ-
ent binding characteristics compared to the constrained
peptide alone, the corresponding constrained peptide
YCKVILTHRCY (peptide R2C16) was synthesized.
Tyrosine residues were added to the ends of this
hydrophobic peptide to increase its solubility. During
the synthesis, next to the monomer, a dimeric peptide
was also formed. This dimeric peptide was separated
from the monomeric peptide by HPLC. The mass of
the dimeric peptide and measurements using MALDI-
TOF indicated that all cysteines in the dimeric peptide
Table 1. Sequences of the 25 clones picked after three rounds of biopanning against DR5. The sequences are denoted by the single-letter
amino acid code. Of these 25 clones, 14 clones showed binding to DR5 with ELISA (indicated by an asteriak). These 14 clones show a
strong consensus sequence with valine at position 2, leucine at position 4, threonine at position 5 and a basic amino acid (arginine or lysine)
at position 7. The random residues are shown in bold; fixed cysteines and the preceding alanine in are shown in normal text; and consensus
residues are shaded grey.
Clone Sequence
77-R2C16 ACKVILTHRC *
89-R2C2 ACKVILTHRC *
77-R2C5 ACKVALTLRC *
77-R2C12 ACKVALTLRC *
77-R2C15 ACKVALTLRC *
77-R2C18 ACKVALTLRC *
77-R2C20 ACKVALTLRC *
77-R2C8 ACLVYLTQRC *

77-R2C19 ACLVYLTQRC *
89-R2C5 ACLVYLTQRC *
77-R2C2 ACIVYLTQKC *
77-R2C3 ACIVYLTQKC *
77-R2C13 ACIVYLTQKC *
77-R2C1 ACILYLTQKC *
77-R2C4 ACKLAMTMKC
77-R2C9 ACKLAMTMKC
89-R2C4 ACKLAMTMKC
77-R2C6 ACFLVMSQRC
77-R2C10 ACLWFPREQC
77-R2C14 ACLWFPREQC
89-R2C3 ACMLPLYFPC
77-R2C11 ACELPRSPSC
77-R2C7 ACTVPAFPAC
89-R2C1 ACTNSAMADC
77-R2C17 ACKHEPTPNC
Consensus ACKVYLTQRC
LA HK
II L
J. Vrielink et al. Inhibition of DR5 signalling
FEBS Journal 277 (2010) 1653–1665 ª 2010 The Authors Journal compilation ª 2010 FEBS 1655
were oxidized and that covalent bonds were formed
between the two monomers. This suggests that the two
monomers are linked to each other via disulfide
bridges. To determine the orientation of the two
monomeric peptides in relation to each other (i.e. par-
allel or symmetrical), the dimeric peptide was digested
with trypsin and measured using MALDI-TOF. The
analysis showed that the dimeric peptide sample con-

tained both parallel and antiparallel orientated mono-
mers (data not shown).
The monomeric and dimeric peptides were used in
competitive studies with ELISA. By adding increasing
concentrations of the peptide R2C16, a competition
with the phage 77-R2C16 (1 · 10
10
pfuÆmL
)1
) for
DR5-binding could be seen with ELISA. A known
TNFa antagonist peptidomimetic, WP9QY [36], was
used as a control peptide. When used at the same con-
centrations, it did not compete with phage 77-R2C16
for binding to DR5 (Fig. 2A). This indicates that the
peptide R2C16, in both monomeric and dimeric forms,
is capable of binding to DR5. Competitive ELISA was
also used to analyse the effect of an increasing concen-
tration of the monomeric and dimeric forms of R2C16
on TRAIL DR5-binding. In this competition ELISA,
the amount of bound sTRAIL was measured. The
results obtained show that both forms of the R2C16
peptide could compete with sTRAIL for binding to
DR5, not only confirming that the R2C16 peptide is
indeed a DR5-binding peptide, but also suggesting that
R2C16 and TRAIL bind to an overlapping area on
DR5 (Fig. 2B).
Binding studies with surface plasmon resonance
Binding of the monomeric and dimeric form of peptide
R2C16 to immobilized DR4-Fc and DR5-Fc receptor

was assessed in real time by using surface plasmon
resonance. Both forms of R2C16 bound in a dose-depen-
dent manner to DR5 (Fig. 3A, B). It was observed
that, after saturation was reached, injection of higher
concentrations of peptides resulted in increasing
response units (RUs), indicating the accumulation of
the peptide. Furthermore, at higher concentrations
(> 2000 nm monomer or > 120 nm dimer), some
binding to DR4 was observed (Fig. 3C, D). Because of
the hydrophobic nature of the peptide, we consider
that the accumulation on DR5 and binding to DR4 is
caused by aggregation of the peptides. Thus, for K
d
determination, we decided to use the lane coated with
DR4-Fc as a control lane instead of an empty lane.
The signal obtained at equilibrium (176 s after injec-
tion) was plotted against the concentration of the pep-
tide and apparent K
d
values were calculated from these
A
B
C
Fig. 1. ELISAs with phage 77-R2C16 and control phage. (A) Wells
are coated with DR5-Ig. Phage 77-R2C16 (•) bound to DR5 in a dose-
dependent manner; control phage (
) did not show any binding. This
indicates that binding to DR5 is not mediated by nonspecific binding
of the phage particle. (B) Wells are coated with different receptors of
the TNF-family. Phage 77-R2C16 showed specific binding to DR5,

and control phage showed no binding to any of the receptors. This
confirms that binding of the phage 77-R2C16 to DR5 is via the dis-
played peptide, and not via other parts of the phage particle. (C) Com-
petition ELISA of sTRAIL with phage 77-R2C16 (1 · 10
10
pfuÆmL
)1
)
for binding to DR5. Increasing amounts of sTRAIL decreased the
binding of phage 77-R2C16 to DR5, suggesting that phage 77-R2C16
and sTRAIL bind to an overlapping region on DR5.
Inhibition of DR5 signalling J. Vrielink et al.
1656 FEBS Journal 277 (2010) 1653–1665 ª 2010 The Authors Journal compilation ª 2010 FEBS
plots. The monomer has an apparent K
d
value of
272 nm (range 251–294 nm) and the dimer has a value
of 40 nm (37–44 nm) (Fig. 4A, B).
Binding studies towards Jurkat cells with
fluorescence-activated cell sorting (FACS) analysis
To further evaluate the binding of the monomeric and
dimeric peptides, we characterized their binding
towards Jurkat cells by flow cytometry. Jurkat cells are
a widely used model of DR5 only cells. The con-
strained peptide R2C16 was synthesized with a biotin
label at the C-terminus (YCKVILTHRCY-K[biotin]).
Again, both a monomeric and a dimeric form of the
peptide were formed and they were separated from
each other by HPLC. Both forms of biotin-R2C16
bound in a dose-dependent manner to DR5 (Fig. 5A1,

B2). Compared to the control, the increasing amounts
of biotinylated monomeric and dimeric peptide showed
an increased fluorescence signal. At higher concentra-
tions of peptide (> 23 nm biotinylated monomer or
>12nm biotinylated dimer), the fluorescence signal
suddenly and drastically dropped to almost control
levels (data not shown). Again, this suggests the for-
mation of aggregates as a result of the hydrophobic
nature of the peptides, which would only be increased
by the addition of the biotin label.
To determine the specificity of this binding interac-
tion, the Jurkat cells were co-incubated with the bioti-
nylated peptides (5.71 nm of monomer and 1.43 nm
of dimer) and increasing concentrations of sTRAIL
(0.21 pm to 2.1 nm). Compared to the monomeric or
dimeric biotinylated-R2C16 alone, analyses showed
that the fluorescence signal can be gradually reduced
by increasing the concentrations of sTRAIL
(Fig. 5A2, A3, B2, B3). This indicates that the bioti-
nylated peptides and sTRAIL bind at a similar posi-
tion on the cells, most likely at an overlapping patch
of DR5.
Constrained R2C16 peptide inhibits
TRAIL-induced apoptosis
To assess the effect of R2C16 on DR5 apoptosis
induction, Colo205 colon carcinoma cells were used.
We have previously reported that Colo205 cells were
sensitive to TRAIL-induced apoptosis and the
TRAIL-death signal was primarily transmitted by
DR5 in these cells [25]. Treatment of the cells with

increasing concentrations of the monomeric R2C16
peptide caused no cell death, as measured by annexin
V labelling of the dying cells (Fig. 6A). Treatment with
the dimeric form of R2C16 lead to similar results,
where only the highest concentration (3.6 lm) caused a
small (7.1 ± 3.2%) increase of cell death (Fig. 6A).
Next, the possibility of antagonistic action of R2C16
was tested. Colo205 cells were treated with increasing
concentration of monomeric or dimeric R2C16 for 1 h
before treatment with 20 ngÆmL
)1
TRAIL for 2 h and
annexin V staining was used to quantify cell death.
Both forms of R2C16 were able to inhibit TRAIL-
induced cell death, with the dimeric form being more
efficient than the monomer. In addition, the dimeric
A
B
Fig. 2. (A) Competition ELISAs of peptide R2C16, in both mono-
meric and dimeric forms, and control peptide WP9QY with phage
77-R2C16 (1 · 10
10
pfuÆmL
)1
). Increasing amounts of monomer (•)
and dimer (
) competed with phage 77-R2C16 for binding to
DR5-Ig, whereas peptide WP9QY (
) did not. This showed that the
monomeric form of the dimeric peptide can bind to DR5. (B)

Competition ELISA of peptide R2C16, in both monomeric and
dimeric forms, with sTRAIL (10 ngÆmL
)1
). The amount of sTRAIL
binding was measured. Increasing amounts of monomer (•) and
dimer (
) competed with sTRAIL for binding to DR5-Ig, not only
confirming that the R2C16 peptide is indeed a DR5-binding peptide,
but also suggesting that R2C16 and TRAIL bind in an overlapping
area on DR5.
J. Vrielink et al. Inhibition of DR5 signalling
FEBS Journal 277 (2010) 1653–1665 ª 2010 The Authors Journal compilation ª 2010 FEBS 1657
form of R2C16 acted as an antagonist best in the con-
centration range 1–5 lgÆmL
)1
(equal to 0.36–1.8 lm).
Above this concentration, the efficiency of the peptide
to inhibit TRAIL-induced cell death was reduced
(Fig. 6B). This reduction may be caused by aggrega-
tion of the peptide.
Discussion
TRAIL and its receptors (DR4, DR5, DcR1, DcR2
and OPG) are of high interest because of the potential
of TRAIL to specifically induce apoptosis in cancer
cells, as well as its involvement in many diseases with
dysregulated apoptosis. Using phage display, we identi-
fied peptides that share a homologous consensus,
C(K ⁄ I ⁄ L)V(Y ⁄ I ⁄ A)LT(Q ⁄ H ⁄ L)(K⁄ R)C, and, when dis-
played on a phage, bind to DR5. The phage displayed
peptide that showed the highest affinity for DR5 was

further characterized and shown to bind exclusively to
DR5, with no affinity towards any of the other four
TRAIL receptors, receptor activator of nuclear factor-
jB or TNF-receptor I. The synthetic constrained
peptide, YCKVILTHRCY, in both monomeric and
dimeric forms, competed with the phage 77-R2C16
and with sTRAIL for binding to DR5 in a concentra-
tion-dependent manner and retained DR5 selectivity.
The dimeric form of the peptide displayed higher affin-
ity for DR5 compared to the monomeric form with an
apparent K
d
value of 40 nm versus 272 nm, respec-
tively. This is in accordance with the reported observa-
tions in the literature of higher affinities of dimeric
and even multimeric peptides compared to their mono-
meric form as a result of an avidity effect [30,37–40].
A similar phenomenon was seen for the biotin-labelled
synthetic constrained peptide. Both the monomeric
and the dimeric form were able to bind to Jurkat cells
in a concentration-dependent manner. The dimeric
form of the biotin-labelled peptide appeared to have a
higher affinity for the Jurkat cells, although part of
this effect can be attributed the double biotin label.
The higher avidity for the dimeric peptide, which was
observed in all binding studies, might also be a conse-
quence of the reduced rigidity compared to the mono-
meric peptide. This would allow the dimeric peptides
to adapt an improved confirmation for DR5-binding.
Co-incubation of the Jurkat cells with biotinylated

AB
CD
Fig. 3. BIAcore curves of peptide R2C16, in both monomeric and dimeric forms, binding to immobilized DR5-Ig (A, B) or DR4-Ig (C, D).
(A) Increasing concentrations of the monomeric peptide (4086, 2043, 1021, 511, 255, 128, 63.8, 31.9, 16.0 and 8.0 n
M) were injected,
demonstrating an increased signal. The curves shown are corrected for the signal obtained in the lane coated with DR4-Ig. (B) Increasing
concentrations of dimeric peptide (179, 119, 89.5, 59.7, 44.8, 29.8, 22.4, 14.9, 11.2 and 7.5 n
M) were injected, demonstrating an increased
signal. The curves shown are corrected for the signal obtained in the lane coated with DR4-Ig. (C, D) Increasing concentrations of
monomeric (C) or dimeric peptide (D) demonstrated some binding to DR4-Ig at higher concentrations. This binding is most likely caused by
aggregation of the peptide as a result of its hydrophobic nature.
Inhibition of DR5 signalling J. Vrielink et al.
1658 FEBS Journal 277 (2010) 1653–1665 ª 2010 The Authors Journal compilation ª 2010 FEBS
peptide and sTRAIL showed a decrease in peptide
binding with increasing sTRAIL concentrations. This
suggests that the peptides bind at a similar patch on
DR5. Furthermore, both the monomeric and the
dimeric peptide acted as DR5 antagonists because they
were able to inhibit TRAIL-mediated apoptosis in
Colo205 cells, with the dimeric form of R2C16 demon-
strating the most efficient antagonistic effect. This is
the first DR5-specific antagonistic peptide described.
In 2004, Kajiwara et al. [41] described synthetic pep-
tides inhibiting TRAIL-induced cell death, although
these peptides bound to TRAIL instead of DR5. More
recently, Li et al. [32] described peptides binding to
DR5, although none of these were antagonistic. We
hypothesize that, at higher concentrations, the peptide
might aggregate in an aqueous environment based on
the fact that the amino acids in the sequences of pep-

tide are rather hydrophobic. In addition, we observed
that biotinylated was more prone to aggregation than
the label-free peptide, which is in accordance with
biotin being well known for its hydrophobic character-
istics.
One mechanism of DR5 that is not yet fully under-
stood is the ability to mediate distinct cell signals
when interacting with different receptor agonists. An
agonistic DR5 monoclonal antibody could induce
both caspase-dependent and caspase-independent cell
death in Jurkat cells, whereas TRAIL could only trig-
ger the caspase-dependent cell death [20]. Thomas
et al. [19] found that TRAIL and some agonistic anti-
bodies required the C-terminal tail of DR5 for
recruitment of Fas-associated death domain, whereas
other agonistic antibodies could function in the
absence of this C-terminal tail. Thus, different recep-
tor agonists can use distinct molecular mechanisms to
activate signalling from the same receptor. It is postu-
lated that the binding of different agonists to the
extracellular domain causes different conformational
changes in the intracellular domain, which may inter-
act with different cytoplasmic adaptor proteins and
trigger different cell signals [20]. To address these
questions, the R2C16 antagonistic peptide described
in the presrent study comprises a useful tool for eluci-
dating the mechanisms of binding and signalling initi-
ation of DR5. Accordingly, the manner in which the
peptide is able to antagonize TRAIL should be fur-
ther elucidated by studying the trimerization of DR5.

In addition, the effects of the peptide on the different
cell signals that it can trigger or block should be
investigated.
Up to now, the focus of TRAIL research has been
mainly on its therapeutic value in cancer, as a result of
the quality of TRAIL that leads to the induction of
apoptosis in a broad range of cancer cells but not in
most untransformed cells [1]. However, the involve-
ment of TRAIL in neurodegenerative diseases has not
received much attention, despite the mounting evidence
emphasizing the role of TRAIL in these disorders.
Recent data show that, although TRAIL is absent in
normal brain, it is upregulated under pathological con-
ditions such as Alzheimer’s disease. Human brain cells
express all four TRAIL receptors and are sensitive to
TRAIL-induced apoptosis. TRAIL is also suggested to
be involved in the pathogenesis of HIV-1-associated
dementia [2]. HIV infection triggers TRAIL expression
in macrophages and these TRAIL expressing macro-
phages can initiate neuronal injury. The involvement
of TRAIL in Alzheimer’s disease was shown by
A
B
Fig. 4. Dose–response curves of increasing concentration of pep-
tide R2C16, (A) monomeric (•) or (B) dimeric (
), binding to DR5-Ig
measured with surface plasmon resonance. The amount of binding
is depicted as the RU measured at 176 s. Graphs were fit with
four-parameter sigmoid curves. Apparent K
d

values were calculated
as the concentration of peptide that gives a signal of 50% of the
maximum RU.
J. Vrielink et al. Inhibition of DR5 signalling
FEBS Journal 277 (2010) 1653–1665 ª 2010 The Authors Journal compilation ª 2010 FEBS 1659
neutralization of the TRAIL death pathway, which
protected a human neuronal cell line from b-amyloid
toxicity [2]. TRAIL plays a dual role in T cell-induced
experimental autoimmune encephalomyelitis (EAE), an
animal model of multiple sclerosis. Blockade of
TRAIL within the periphery exacerbates EAE, whereas
blockade of TRAIL in the central nervous system sup-
presses EAE by inhibiting brain cell apoptosis [42].
Inhibition of TRAIL-induced apoptosis within the cen-
tral nervous system may represent a possible therapeu-
tic strategy for preventing neuronal damage in patients
with neurodegenerative diseases. The R2C16 peptide,
with its DR5 antagonistic activity and lipophilic prop-
erties, has the potential to act as a lead peptide in
studies aiming to block TRAIL and reducing its toxic-
ity in neurodegenerative diseases.
Overall, the peptides described in the present study,
or their derivatives, may have various applications in
the field of TRAIL-mediated signalling and diseases
caused by dysregulated TRAIL signalling. The small
size of this peptide offers the possibility of designing
structurally mimetic nonpeptidic molecules.
A1 B1
A2 B2
A3 B3

Fig. 5. Dose– response histograms of
biotinylated peptide R2C16, (A1) monomeric
or (B1) dimeric, on Jurkat cells. Increasing
amounts of both monomeric (m1 = 0.57 n
M,
m2 = 2.86 n
M, m3 = 11.42 nM) and dimeric
(d1 = 0.14 n
M, d2 = 1.43 nM, d3 = 5.71 nM)
biotinylated peptide give rise to a right shift
in the fluorescent PE signal compared to
the control (C, filled grey). Competition his-
tograms of 5.71 n
M monomeric biotinylated
peptide (m, tinted) (A2) and 1.43 n
M dimeric
biotinylated peptide (d, tinted) (B2) show a
strong right shift compared to control
(C, filled grey). Co-incubation of the biotiny-
lated peptides with 2.1 n
M sTRAIL [m + T
(A2) and d + T (B2), respectively] show a
decrease in the right shift that they were
initially able to cause. The curves represent
the relative binding signal binding of the
(A3) monomer and (B3) dimer upon co-
incubation with sTRAIL compared to the
signal obtained when no sTRAIL was used.
Inhibition of DR5 signalling J. Vrielink et al.
1660 FEBS Journal 277 (2010) 1653–1665 ª 2010 The Authors Journal compilation ª 2010 FEBS

Experimental procedures
TRAIL purification
cDNA corresponding to soluble human TRAIL (C-terminal
amino acids 114–281) was cloned into the NcoI and BamHI
sites of a pET15b vector and transformed to and expressed
in Escherichia coli BL21 (DE3). The trimeric soluble protein
was purified as described previously [43].
Biopanning
The Ph.D.–C7C phage display peptide library (New Eng-
land Biolabs, Hitchin, UK), consisting of randomized hept-
amer constrained peptides (1.2 · 10
9
individual clones), was
used to identify peptides binding to DR5. The disulfide-
constrained heptapeptides are expressed at the N-terminus
of g3p, with the first cysteine preceded by an alanine
residue, and the second cysteine followed by a short spacer
(Gly-Gly-Gly-Ser). DR5-Fc, fusion of the ecto-domain of
the receptor to the Fc-portion of human IgG
1
(R&D Sys-
tems, Minneapolis, MN, USA), was used to coat Protein A
magnetic dynabeads (Dynal, Hammerfest, Norway). To
6 lL of beads, 1 lg of DR5-Fc was added in 0.1 m NaH-
CO
3
(pH 8.6) and incubated overnight at 4 °C. The beads
were then blocked using 0.1 m NaHCO
3
(pH 8.6),

5mgÆmL
)1
BSA and 0.02% NaN
3
. Phage library was
added to the beads at 2 · 10
11
pfu and incubated for
45 min. Unbound phages were removed by washing ten
times with washing buffer (NaCl ⁄ Tris containing 0.1%
Tween 20). Bound phages were eluted with 1 mL of 0.2 m
glycine ⁄ HCl (pH 2.2), 1 mgÆmL
)1
BSA for no more then
10 min, and immediately neutralized with 150 l Lof1m
Tris-HCl (pH 9.1). The eluted phages were amplified in
E. coli ER2738 and titred according to the manufacturer’s
instructions (New England Biolabs). Another two rounds
of biopanning were then performed. The incubation time
was decreased to 30 min in the second round and to 15 min
in the third round. In both rounds, the concentration of
Tween 20 in the washing buffer was increased to 0.5%, and
1 lm sTRAIL in NaCl ⁄ Tris was used for competitive elu-
tion. Before the third round, a subtractive round was per-
formed, by incubating the amplified phage of round two
with human IgG Fc fragment (Rockland Immunochemi-
cals, Inc., Gilbertsville, PA, USA) bound to protein A
beads. To 20 lL of beads, 10 lg of Fc fragment was added,
incubated and blocked as described above. Phages were
incubated for 30 min with the beads, and unbound phages

were subsequently used in the third positive panning round.
After round three, 25 individual phage clones were picked
from agar plates and amplified. The single-stranded DNA
was isolated and sequenced according to the manufacturer’s
instructions. Supernatants were screened using ELISA, as
described below. Samples that showed a high binding signal
were further purified using poly(ethylene glycol) precipita-
tion according to the manufacturer’s instructions (New
England Biolabs).
ELISA with phage
Maxisorp 96-wells plates (Nunc, Roskilde, Denmark) were
coated for 1–2 h with 100 lLof1ngÆlL
)1
receptor-Fc
A
B
Fig. 6. Annexin V cell assays with peptide R2C16 using Colo205
cells. (A) Treatment of cells with increasing concentrations of the
monomeric R2C16 peptide (•) caused no cell death. Treatment with
the dimeric form of R2C16 (
) gave similar results. Only the high-
est concentration (8 l
M) caused a small (7.1 ± 3.2%) increase of
cell death, suggesting that the R2C16 peptide is not a DR5 agonist.
(B) Reduction of TRAIL-induced cell death in Colo205 cells by pep-
tide R2C16. Colo205 cells were treated with increasing concentra-
tion of monomeric (•) or dimeric (
) R2C16 before treatment with
20 ngÆmL
)1

TRAIL and annexin V staining was used to quantify the
dying cells. Treatment of Colo205 cells with only 20 ngÆmL
)1
TRAIL
induced 80% cell death. Both forms of R2C16 were able to reduce
TRAIL-induced cell death, with the dimeric form being more
efficient than the monomer. The dimeric form of R2C16 acted as
an antagonist best in the concentration range 1–5 lgÆmL
)1
(equal
to 0.76–3.8 l
M). Above this concentration, the efficiency of the
peptide to inhibit TRAIL-induced cell death was reduced, most likely
as a result of aggregation of the peptide.
J. Vrielink et al. Inhibition of DR5 signalling
FEBS Journal 277 (2010) 1653–1665 ª 2010 The Authors Journal compilation ª 2010 FEBS 1661
(R&D Systems) at 4 °C in 0.1 m NaHCO
3
(pH 8.6). The
wells were blocked for 1–2 h with 200 lL of 2% BSA in
0.1 m NaHCO
3
and washed three times with NaCl ⁄ Tris,
0.5% Tween 20 (TBST). Phage supernatant, purified phage
diluted in TBST with 0.5% BSA or a premix of different
concentration of synthesized peptides with 1 · 10
10
pfuÆmL
)1
of phage was added at 100 lL per well and incu-

bated for 30 min. After washing the wells six times with
TBST, 200 lL of 1 : 5000 diluted HRP ⁄ anti-M13 monoclo-
nal conjugate (Amersham Pharmacia Biotech, Little Chal-
font, UK) in TBST was added for 1 h. Wells were washed
six times with TBST and bound phages were detected with
100 lL of tetramethylbenzidine (TMB; one-step Turbo TMB-
ELISA) (Pierce Biotechnology, Rockford, IL, USA). The
reaction was stopped with 100 lL of approximately 1.8 m
H
2
SO
4
. The signal was read at 450 nm in a multiscan Ascent
plate reader (Thermo Labsystems, Helsinki, Finland).
Curves were fitted with four-parameter sigmoid curves.
Competition ELISA with peptides and sTRAIL
Maxisorp plates were coated for 1–2 h with 100 lLof
1ngÆlL
)1
DR5-Fc at 4 °C in 0.1 m NaHCO
3
(pH 8.6). The
wells were blocked for 1–2 h with 200 lL of 2% BSA in
0.1 m NaHCO
3
(pH 8.6) and washed three times with
TBST. Different concentrations of synthesized peptides
were premixed with 10 ngÆmL
)1
sTRAIL in TBST and

100 lL of this premix was added to each well, and incu-
bated for 30 min. After washing the wells six times with
TBST, 200 lL of 1 : 200 diluted anti-human TRAIL sera
(AF375; R&D Systems) in TBST was added to the wells
for 1 h, washed six times with TBST and 200 lLof
1 : 25 000 diluted HRP conjugated swine anti-goat (Bio-
Source International, Camarillo, CA, USA) was added for
1 h. After washing wells six times with TBST, 100 lLof
TMB was added to measure the amount of bound sTRAIL.
The reaction was stopped with 100 lL of approximately
1.8 m H
2
SO
4
. The signal was read at 450 nm. Curves were
fitted with four-parameter sigmoid curves.
Synthetic peptides
The constrained peptide (YCKVILTHRCY), in both mono-
meric and dimeric forms, was synthesized by Pepscan (Lelys-
tad, the Netherlands). Both peptides had a free amine at the
N-terminal and a free acid at the C-terminal. The lyophilized
peptides were solubilized in acetonitril ⁄ water (1 : 1) to give a
stock concentration of 20 mgÆmL
)1
. A control peptide
(YCWSQYLCY) was purchased from Bachem AG (Buben-
dorf, Switzerland). The lyophilized peptide was solubilized in
50% acetic acid to give a stock concentration of 10 mgÆmL
)1
.

The stock of each peptide was stored at )20 °C.
The constrained peptide with biotin label (YCK-
VILTHRCY-K[biotin]), in both monomeric and dimeric
forms, was also synthesized by Pepscan Systems, again with
a free amine at the N-terminal and the biotin label at the
C-terminus. The biotin label was coupled to the C-terminal
tyrosine, resulting in two biotin labels for the dimeric
peptide. The lyophilized peptides were solubilized in
acetonitril ⁄ water (1 : 1) to give a stock concentration of
20 mgÆmL
)1
. The stock of each peptide was stored at
)20 °C.
Interaction studies by surface plasmon resonance
To evaluate the binding of the peptides to DR5, BIAcore
2000 (BIAcore AB, Uppsala, Sweden) was used. All
reagents used were also purchased from BIAcore AB.
Immobilization of the receptor DR4-Fc and DR5-Fc on
the sensor surface of a CM5-chip was performed in accor-
dance with a standard amine coupling procedure at a flow
rate of 10 lLÆmin
)1
. To activate carboxyl groups on the
sensor surface, 70 lL of a solution containing 0.2 m
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochlo-
ride and 0.05 m N-hydroxysuccinimide was injected. Recep-
tors (6.7 lgÆmL
)1
in 10 mm NaAc buffer, pH 5.0) were
flowed over the chip surface until a surface density of

approximately 3500 RU was reached. Remaining active
groups were blocked by injecting 70 lL of 1.0 m ethanol-
amine-HCl (pH 8.5). Assays were performed at 25 °C with
NaCl ⁄ Po containing 0.005% (v ⁄ v) P20 surfactant as
running buffer and a flow rate of 70 lLÆmin
)1
. Peptides
were injected at variable concentrations for 3 min followed
by 4 min of running buffer. The surface was regenerated
after each binding step by removing all bound peptide by
injecting 35 lLof10mm glycine (pH 2.0). Sensorgrams
were evaluated with BIAevaluation software, version 4.1
(Biacore, GE Healthcare, Chalfont St Giles, UK). The lane
coated with DR4-Fc was used as a reference. The signal
obtained at equilibrium (176 s after start injection) was
plotted against the concentration of the peptide and fitted
with four-parameter sigmoid curves. From these curves, the
apparent K
d
values were calculated.
Interaction studies on Jurkat cells by FACS
analysis
Jurkat cells were maintained in RPMI 1640
medium + GlutaMAX-I supplemented with 10% fetal
bovine serum, 100 UÆmL
)1
penicillin and 100 lgÆmL
)1
strep-
tomycin (all from Gibco, Gaithersburg, MD, USA) at 37 °C

in 5% CO
2
in a humidified environment. Cells were
harvested at 10
6
cells per sample and washed in ice-cold
NaCl ⁄ Pi ⁄ 2% fetal bovine serum to prevent peptide reduc-
tion by the reducing agent glutathione present in the RPMI
medium. Increasing concentrations of biotin-labelled pep-
tides were added to the cells and incubated for 1 h on ice.
The cells were washed twice in ice-cold NaCl ⁄ Pi ⁄ 2% fetal
bovine serum. The cells were incubated for an additional
1 h with streptavidin-phycoerythrin (PE) (BD Pharmingen,
Inhibition of DR5 signalling J. Vrielink et al.
1662 FEBS Journal 277 (2010) 1653–1665 ª 2010 The Authors Journal compilation ª 2010 FEBS
San Diego, CA, USA) on ice, in the dark. The cells were
washed twice and then resuspended in 300 lL of ice-cold
NaCl ⁄ Pi ⁄ 2% fetal bovine serum. Finally, the samples were
measured on a FACSCalibur Flow cytometer (Becton-Dick-
inson, Franklin Lakes, NJ, USA) and analysed with FlowJo
(Tree Star Inc., Ashland, OR, USA). The PE signals
obtained are displayed as histograms.
For the competition assays with sTRAIL, a similar pro-
tocol was used. Cells were harvested 10
6
cells ⁄ sample and
washed in ice-cold NaCl ⁄ Pi ⁄ 2% fetal bovine serum. Cells
were co-incubated with 5.71 nm monomeric or 1.43 nm
dimeric peptide and increasing concentrations sTRAIL for
1 h on ice. The cells were washed twice in ice-cold

NaCl ⁄ Pi ⁄ 2% fetal bovine serum. The cells were incubated
for another hour with Streptavidin-PE (BD Pharmingen)
on ice, in the dark. The cells were washed twice and then
resuspended in 300 lL of ice-cold NaCl ⁄ Pi ⁄ 2% fetal bovine
serum. Finally, the samples were measured on a FACSCali-
bur Flow cytometer (Becton-Dickinson) and analysed with
Flowjo (Tree Star Inc.). The PE signals obtained are
displayed as histograms. In addition, the signals obtained
in triplo were expressed as a percentage of the PE signal
related to the signal obtained with no sTRAIL.
Apoptosis assay
Colo205 cells were maintained in RPMI 1640 medium
(Sigma, St Louis, MO, USA) supplemented with 10% fetal
bovine serum (Sigma), 100 UÆmL
)1
penicillin, 100 lgÆmL
)1
streptomycin (Sigma), 2 mml-glutamine (Sigma) and 1 mm
sodium pyruvate (Gibco) at 37 °Cin5%CO
2
in a humidi-
fied environment. One hour before treatment, the cells were
seeded in a 24-well plate at a concentration of 300 000 cell-
sÆmL
)1
in NaCl ⁄ Pi ⁄ 1% BSA to prevent peptide reduction
by the reducing agent glutathione present in the RPMI
medium. Varying concentrations of peptides were added to
the cells and incubated for 1 h before the addition of
20 ngÆmL

)1
sTRAIL for 2 h. After treatment, the cells were
transferred into Eppendorf tubes and collected by centrifu-
gation at 3000 g for 5 min. The cell pellets were resus-
pended in 50 lL of calcium buffer [10 mm Hepes, pH 7.5
(set with NaOH), 140 mm NaCl and 2.5 mm CaCl
2
] con-
taining 3 lL of annexin V (IQ Corporation, Groningen, the
Netherlands) and incubated for 15 min on ice. The staining
was terminated by diluting the annexin V solution to
300 lL with calcium buffer and the samples were analysed
immediately on a FACSCalibur Flow cytometer (Becton
Dickinson). The results were expressed as percentage of
annexin V positive cells.
Acknowledgements
We thank Almer van der Sloot for providing sTRAIL
and for his support with the BIAcore. We also thank
Geert Mesander and Henk Moes for their technical
assistance with the FACS analysis. Finally, we thank
the EU Sixth Framework Program LSH-2005-2.2.0-2
(TRIDENT) and TIPharma PROJECT T3-112 (TNF
ligands in cancer) for providing financial support.
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