Perturbation of estrogen receptor α localization with synthetic nona-arginine lxxll- peptide coactivator binding inhibitors
Chemistry & Biology
Perturbation of Estrogen Receptor a Localizationwith Synthetic Nona-Arginine LXXLL-Peptide Coactivator Binding Inhibitors
Mae¨lle Carraz,Wilbert Zwart,Trang Rob Michalides,and Luc 1Chemical Genomics Centre of the Max Planck Society, 44227 Dortmund, Germany2Laboratory of Chemical Biology, Department of Biomedical Engineering, Eindhoven University of Technology, 5600MB Eindhoven,The Netherlands3Division of Cell Biology, The Netherlands Cancer Institute, 1066CX Amsterdam, The Netherlands*Correspondence: DOI 10.1016/j.chembiol.2009.06.009
coactivators includes the p160 protein family, consisting of threemembers: SRC-1, Tif-2/GRIP1, and SRC-3/AIB1. The impor-
The interaction of estrogen receptor a (ERa) with the
tance of these coactivators in ERa signaling has been high-
consensus LXXLL motifs of transcriptional coactiva-
lighted by knockout studies and by the discovery of p160 gene
tors provides an entry for functional ERa inhibi-
amplification in ERa-positive breast cancer
tion. Here, synthetic cell-permeable LXXLL peptide
These p160 coactivators
probes are brought forward that allow evaluation of
have the ability to interact with ERa and other nuclear receptors
the interaction of specific recognition motifs with
in a hormone-dependent manner via small hydrophobic andamphiphatic a-helical peptide sequences with the common
ERa in the context of the cell. The probes feature a
signature motif, LXXLL (L = leucine, and X = any amino acid).
nona-arginine tag that facilitates cellular entry and
Crystallographic studies showed that this motif, also called
induces probe localization in nucleoli. The nucleoli
nuclear receptor box (NR box), binds to a hydrophobic cleft on
localization provides an explicit tool for evaluating
the ERa ligand binding domain (LBD) surface, under control of
the LXXLL motif interaction with ERa. The probes
activating ligand (p160 coactivators contain
compete with coactivators, bind ERa, and recruit it
three NR box copies within their central nuclear receptor interac-
into the nucleoli. The physical inhibition of the ERa-
tion domain. Although the three leucines in the LXXLL motif are
coactivator interaction by the probes is shown to
conserved, the amino acid residues flanking this sequence are
be correlated with the inhibition of ERa-mediated
not and specify NR box recognition for a particular nuclear
gene transcription. This chemical biology approach
receptor in cooperation with specificities of the ligand. These
allows evaluating the ER
characteristic features have raised the idea that the ERa-coacti-
vator interaction is a specific amendable interface for LXXLL
and inhibitor binding directly in cells.
motif–like inhibitors, preventing transactivation by ERa The formation of
the coactivator binding surface on ERa requires a hormone-dependent conformational change. Inhibitors of coactivator
Estrogen receptor a (ERa) is a member of the nuclear receptor
binding (ICBs) may only bind to
superfamily and is an estrogen-inducible transcription factor. In
hormone-occupied ERa, thus featuring a different mechanism
the normal physiology of the female reproductive system, ERa
than the classic anti-estrogen drugs. These ICBs might thus be
regulates the growth and function of such tissues as breast,
useful in anti-estrogen therapy for patients with tamoxifen-resis-
uterus, and ovaries. In addition, ERa has been identified as play-
tant breast cancers or could provide a possibility to target
ing a role in many pathological processes, such as breast cancer
orphan nuclear receptors for which antagonists are difficult to
(). Because 70% of breast tumors depend on
the stimulatory effect of estrogens, anti-estrogen therapies
Several types of compounds blocking ERa-cofactor interac-
have been developed that significantly reduce breast cancer
tions have already been developed. The first proof that the
recurrence ). However, the long-term effects
concept of inhibiting ERa-cofactor interactions results in the inhi-
of tamoxifen, one of the most successful anti-estrogen drugs,
bition of ERa-mediated transcription has been obtained with
in metastatic disease are limited to an average of 15 months
LXXLL peptides overexpressed in cells from transfected DNA
(), after which tamoxifen resistance develops
in more than 80% of treated women In the
induction of gene transcription, ERa acts in complex association
has been enforced by the identification and the use of synthetic
with other cellular factors, including coactivators, which are
ICBs, either peptide based or small molecule
required for the transcriptional activation process to occur
(The most widely studied group of ERa
702 Chemistry & Biology 16
, 702–711, July 31, 2009 ª2009 Elsevier Ltd All rights reserved
Chemistry & Biology
Cellular Modulation of ERa with Synthetic Peptides
Most of these compounds inhibit
Table 1. Sequences of the Selected LXXLL-Motif Peptides
ERa-coactivator binding in biochemical assays, and a selected
set of compounds was also able to inhibit ERa gene transactiva-
tion in cellular assays ). The translation from biochemical studies
on the ERa LBD to cellular activity on the full-length protein is,
however, not trivial. For example, it has been reported that the
conformation of the coactivator binding groove in the full-length
receptor tends to be different from the isolated LBD (
addition, direct binding of inhibitors to ERa in cells and their
capacity to displace coactivators are two processes that aredifficult to observe directly. The process of inhibiting the ERa-co-activator interaction thus requires more molecular insight. There-
complete peptide. In addition to the LXXLL molecular probes,
fore, methods need to be developed that directly visualize the
two AXXAA-molecular probes, based on SRC-1 Box2 and Tif-2
interaction of inhibitors with ERa and provide information on
Box2 and mutated for the recognition motif by alanines, were
the resulting ERa inhibition. Here, we show that synthetic cell-
also generated as negative control peptides.
permeable LXXLL-peptide probes with specific localization and
The effect of the biotin and nona-arginine tags, incorporated in
visualization tags are ideal molecular probes to inhibit and eval-
the peptides, on the binding to the ERa coactivator binding
uate ERa-coactivator interactions in living cells and to address
surface was evaluated in a biochemical fluorescent polarization
the above-mentioned issues. By use of confocal laser scanning
assay with the recombinant LBD of ERa The assay
microscopy (CLSM), these probes can be used to monitor the
measures the displacement of a fluorescein-labeled reference
association of specific peptide sequences with the full-length
peptide (SRC-1 box2) by the nonfluorescent LXXLL peptides
ERa and to evaluate their effects on the assembly of ERa
under study. The R9 tag alone did not bind to ERa LBD, as
signaling protein complex within the organized microenviron-
expected. The dose-dependent fluorescence depolarization
ment of the living cell. The peptide probes feature a nona-argi-
curves obtained with peptides SRC-1 Box2 and SRC-1 Box2-
nine tag that ensures efficient cellular entry of the peptides and
PEG-biotin were overlapping A), showing that the
induces a specific localization of the peptides into the nucleoli.
PEG-biotin spacer does not influence ERa binding. For both
This specific property of the peptides is used to evaluate the
the SRC-1 Box2 and the SRC-1 Box2-R9 peptides and the Tif-
affinity of different LXXLL-motif peptides for the ERa. The
2 Box2 and Tif-2 Box2-R9 peptides, the same binding affinities
peptides bind and translocate ERa into the nucleoli, resulting
were found B), showing that the nona-arginine tag
in the displacement of ERa from the DNA and coactivators.
also does not disturb the capacity of the peptides to bind to ERa.
Overall, the peptides function as functional inhibitors as theyeffectively lower ERa-mediated gene transcription.
Cellular Toxicity and Intracellular Distributionof the Synthetic LXXLL Molecular Probes
To study and evaluate any toxic effects of the synthetic peptideprobes, the viability of cells after an overnight incubation with
Selection and Synthesis of LXXLL-Motif Molecular
increasing concentrations of peptides was tested using the
WST-1 assay (see available online) (
For the generation of molecular probes inhibiting the ERa-coac-
). Peptides conjugated to PEG-biotin but lacking the
tivator interaction, we selected a set of natural LXXLL-motif
nona-arginine tag were found to be toxic on the human cancer
sequences reported to have a high affinity for ERa
cell line U2OS at concentrations of 1 mM, whereas all of their
and capable of blocking ERa transcriptional activity
nona-arginine counterparts showed toxicity at around 0.1 mM.
when overexpressed in cells—boxes 1 and 2 of the SRC-1 coac-
The nona-arginine tag alone was found to be less toxic to the
tivator protein and boxes 1, 2, and 3 of the Tif-2 coactivator
cells. Interestingly, the two peptides SRC-1 Box2-Ala-R9 and
protein (). Phage display peptides D2 and D11, which
Tif-2 Box2-Ala-R9 mutated for the recognition motif by alanines
were previously reported to possess a high affinity for full-length
were not lethal to cells at concentrations up to 0.6 mM. This
ERa and to bind at the same coactivator binding surface (
difference in minimal toxic concentration may be explained
), were also included in the selection The
by a different sequence-dependent cellular uptake capacity of
plasma membrane of mammalian cells is generally impermeable
these peptides, but may also be related to their intracellular phar-
to the vast majority of peptides and proteins. We therefore
macological effects. From the dose-dependent toxicity curves
decided to take advantage of the cell-permeable properties of
obtained, we determined the concentrations of peptides that
the nona-arginine (R9) peptide sequence
gave less than 20% of cell death, compared with nontreated
) and use it as a carrier for the selected
cells (). These peptide concentrations (0.02–0.07 mM),
LXXLL peptides. The arginine tags were attached to the peptides
were also tested by fluorescent assisted cell sorting (FACS) for
at their C terminus by means of peptide synthesis. To monitor the
apoptosis induction using the specific fluorescently labeled
localization of the LXXLL peptides within the cells via immuno-
apoptotic marker Annexin-V (). Also in this
labeling, they were provided with a biotin epitope connected
assay (data not shown), the nona-arginine peptides induced
via an ethylene glycol linker to the C-terminal part of the
less than 20% apoptosis at these concentrations.
Chemistry & Biology 16
, 702–711, July 31, 2009 ª2009 Elsevier Ltd All rights reserved 703
Chemistry & Biology
Cellular Modulation of ERa with Synthetic Peptides
Figure 1. Biochemical Fluorescence Polari-zation Studies of the ERa-LBD BindingCapacity of the Synthetic Peptides(A) Representative dose-dependent inhibitioncurves of selected peptides for the binding ofa fluorescein-labeled SRC-1 Box2 peptide to theERa-LBD.
(B) IC50 values of all tested peptides. Error barsrepresent mean ± standard error.
The cell-entry and intracellular distributions of the synthetic
zinc-finger motifs present in its DNA binding domain. Inhibiting
peptides were assessed by immunolabeling of cells treated
peptides expressed from DNA constructs fused to a fluorescent
with the peptides using a fluorescent Cy5-conjugated anti-biotin
protein are evenly distributed throughout the nucleus, but also in
antibody. CLSM imaging of these cells revealed that the PEG-
the cytoplasm, and are colocalized with ERa upon hormone
biotin spacer and the LXXLL peptides lacking a nona-arginine
addition We investigated here whether
tag did cross the plasma membrane to some extent but clus-
peptide probes of a synthetic nature and featuring a specific
tered into vesicular structures in the cytoplasm ).
localization tag are able to interact with ERa as well, when
However, the cellular distribution of the nona-arginine-tagged
applied externally to cells, and whether the introduced localiza-
molecular probes was completely different, localizing in both
tion tag could be used to evaluate this interaction in a novel
the cytoplasm and the nucleus. In the nucleus, these peptides
manner. Therefore, U2OS cells transiently transfected with full-
were mainly found located at the nucleoli, as anticipated
length ERa fused to cyan fluorescent protein (CFP) were incu-
() because the nucleolar accumulation of such positively
bated with the LXXLL-nona-arginine molecular probes in the
charged peptides has been described elsewhere
presence of estradiol. The ERa-binding capacity of these
Because this particular localization at the nucleoli
peptides was assessed by colocalization with ERa by CLSM.
was not found for the non-R9-tagged peptides, we conclude
When cells were not incubated with the molecular probes, ERa
that this feature can be exclusively ascribed to the R9 tag.
localized as expected in the nucleoplasm, in punctuated foci
Also, in accordance with the results of a recent study
(data not shown). The same ERa distribution was observed in
), we observed that the concentration of serum
presence of the nona-arginine tag alone, whereas this peptide
used in the culture medium had a significant impact on the distri-
itself was clearly observed into the nucleoli (indicating
bution of these molecular probes inside cells (data not shown). In
a lack of interaction between the receptor and this peptide.
addition, we noticed that supplementing the medium with 10%
However, incubating the cells with the peptides SRC-1 Box2-
of non-heat-inactivated serum could abrogate the cellular
R9, Tif-2 Box1-R9, Tif-2 Box2-R9, and D2-R9 and, to a lesser
uptake of R9 peptides, which is most likely due to unspecific
extent, with the peptide Tif-2 Box3-R9 resulted in a clear nuclear
binding between the peptides and serum components.
colocalization of these peptides with the estradiol-liganded
However, this did not occur when the medium was beforehand
ERa into the nucleoli. The affinity of these peptides for ERa is
heat inactivated, resulting in an efficient cellular uptake.
apparently strong enough to bind to ERa and subsequently totranslocate it to the alternative location in the nucleoli. This effect
Binding Capacity of LXXLL Molecular Probes
occurs in the presence of a concentration of estradiol (106 M)
that normally would result in complete recruitment of ERa to
ERa is typically localized in the nucleus, excluded from the
DNA templates. The incorporated nona-arginine tag thus serves
nucleoli, and its uniform distribution in the nucleoplasm rapidly
both to facilitate cellular uptake of the peptide probes and as
changes into a hyperspeckled foci pattern upon the addition of
a specific localization tag, generating a spacial resolution
either agonistic or antagonistic ligands ).
within the nucleus that enables visualization of peptides/ERa
When bound to estradiol, ERa associates with specific regions
complexes. Using the statistic tools of the CLSM Leica software,
in the DNA, called estrogen responsive elements (EREs), via
we quantified the ERa-nucleoli recruitment by the peptides,
Figure 2. CLSM Imaging of U2OS Cells afterIncubation with Biotinylated Peptide ProbesCells were overnight incubated with peptidesfeaturing different tags and probe elements, fol-lowed by immunolabeling with a Cy5-conjugatedanti-biotin antibody. PB = PEG-biotin; whitearrows indicate nucleoli, and scale bars represent10 mm.
704 Chemistry & Biology 16, 702–711, July 31, 2009 ª2009 Elsevier Ltd All rights reserved
Chemistry & Biology
Cellular Modulation of ERa with Synthetic Peptides
Figure 3. ERa-Binding Capacity of Syn-thetic Peptides in U2OS Cells(A) CLSM imaging of cells (positive events) aftertransient transfection with ERa-CFP and incuba-tion with nona-arginine peptides and estradiol(106 M). White arrows show ERa sequestrationinto the nucleoli, and scale bars represent 10 mm.
(B) Quantification of ERa-nucleoli recruitmentexpressed as the ratio of the ERa-CFP fluores-cence intensity per pixel into nucleoli versus thenucleoplasm. Circles represent ratio values percell (6 % n % 19); medians are indicated by theblack line in the middle of the box plots and whosesizes represent ± 25% of the medians (programProfit); asterisks (*) indicate results statisticallydifferent from the control (p < 0.01).
expressed as the ratio of the CFP-ERa fluorescence intensity per
SRC-1 coactivator but has a distinct preference for the second
pixel at the nucleoli versus the nucleoplasm Interest-
motif (Although no binding was observed
ingly, significant ERa-nucleoli recruitment was found when cells
in our biochemical assay between the ERa LBD and the peptides
were incubated with all the synthetic R9 peptides, except for the
SRC-1 Box1-R9, Tif-2 Box3-R9, and D11-R9 (IC50 >300 mM),
peptides mutated in their recognition motif by alanines (i.e.,
these peptides did induce a weak but significant ERa-nucleoli
SRC-1 box2-Ala-R9 and Tif-2 Box2-Ala-R9 and the R9 alone;
recruitment (B). This finding underlines the value of
B and ), showing that the nucleoli recruitment
assessing ERa-peptide binding directly in cells, where ERa is
is specifically due to the selective interaction of the peptides
expressed as full length and reveals ERa-LXXLL-peptide interac-
with the ERa coactivator binding surface via their LXXLL motifs.
tion surfaces beyond the isolated and not posttranslational
The ERa-nucleoli recruitment is concentration dependent
modified LBD alone.
(A), in line with the expected molecular mechanism ofthe interaction. Additionally, a Tif-2 Box2 peptide not featur-
Displacement of SRC1 and Tif-2 Coactivators
ing the R9-motif does not show ERa-nucleoli recruitment
by LXXLL Molecular Probes in ERa Binding
B), as expected on the basis of the nonnucleoli localization
To investigate whether the LXXLL-peptide-ERa binding can
of this peptide.
displace overexpressed full-length coactivators in living cells,
Among the peptides tested, the highest ERa relocation score
we tested the ERa binding of the synthetic molecular LXXLL
was found for the SRC-1 Box2-R9, Tif-2 Box1-R9, and D2-R9
probes again, but now in the presence of the transfected SRC-1
peptides, with median ratios of ERa signal into the nucleoli
and Tif-2 coactivators, tagged with yellow fluorescent protein
versus the nucleoplasm of 1.2, 0.9, and 0.8, respectively
(YFP). ERa indeed still colocalized with the nona-arginine
(B). These cellular observations are in line with our
peptides into the nucleoli except for the Tif-2 Box3-
biochemical binding results B) and with previous reports
R9, R9, and the alanine mutant peptides (We also
indicating that ERa interacts with all three LXXLL motifs of the
observed that the full-length SRC-1 coactivator could at least
Figure 4. ERa-Binding Capacity of Syn-thetic Peptides in U2OS Cells in the Pres-ence of Overexpressed Coactivators(A) CLSM imaging of cells (positive events) trans-fected with ERa-CFP and SRC-1-YFP, in the pres-ence of estradiol (106 M). Scale bars represent10 mm.
(B) Quantification of ERa-nucleoli recruitment incell cotransfected with SRC-1-YFP expressed asthe ratio of the ERa-CFP fluorescence intensityper pixel into nucleoli versus the nucleoplasm.
Circles represent ratio values per cell (5 % n % 43).
Medians are indicated by the black line in themiddle of the box plots, whose sizes represent ±25% of the medians (program Profit). Asterisks (*)indicate results statistically different from thecontrol (*p < 0.01).
Chemistry & Biology 16, 702–711, July 31, 2009 ª2009 Elsevier Ltd All rights reserved 705
Chemistry & Biology
Cellular Modulation of ERa with Synthetic Peptides
Figure 5. Inhibition of ERa-Gene-MediatedTransactivation by Synthetic Nona-ArginineLXXLL Peptides(A) U2OS cells were transfected with ERa and theERa-DNA binding sequence ERE-tk-Luc. Lucif-erase activities are expressed as the ratio of Fireflyluciferase signal to Renilla luciferase signal and arenormalized to the ratio obtained when cells weresolely treated with the R9-tag peptide.
(B) Inhibition by R9 peptides of an endogenous ER-mediated pS2 gene in MCF-7 cells. MCF-7 cellswere cultured with 70 mM of the indicated peptidesin the presence of 10 nM estradiol (E2) (unless indi-cated). Cells were lysed after 3 hr of estradiol treat-ment, after which RNA was isolated and preparedfor QPCR analysis of endogenous ER-mediatedpS2 mRNA levels (normalized). Error bars repre-sent mean ± standard error.
partially be recruited to the nucleoli together with ERa in cells
transcription (The largest inhibition, compared with
treated with the most potent ERa-binding peptides (data not
untreated cells, was obtained with the Tif-2 Box2-R9 peptide
shown). This could be due to the recruitment of ERa as a dimer
(61.0% of inhibition), the Tif-2 Box1-R9 peptide (49.8% of inhibi-
to the nucleoli in which one monomer is bound to an arginine-
tion), and the SRC-1 Box2-R9 peptide (48.2% of inhibition). For
tagged peptide and the other monomer is still bound to SRC-1,
comparison, when overexpressed in U2OS cells from a trans-
or to the ligand-independent interaction between SRC-1 and
fected DNA construct, an SRC-1 Box2 peptide exhibited a
the AF-1 domain of ERa (
degree of inhibition (51.5%; similar to that of the
Among the set of peptides tested, the strongest nucleoli
synthetic SRC-1 Box2-R9 peptide on ERa-mediated gene tran-
recruitment effect for ERa was observed with the Tif-2 Box2-R9
scription. The concentration dependence of the inhibition of
peptide (median ratios, 2) and the SRC-1 Box2-R9 peptide
the ERa transcriptional activity by the peptides was verified
(median ratios, 0.7) with 95% and 88% of cells,
with the Tif-2 peptide constructs (A), and no inhibition
respectively, exhibiting a ratio higher than that found for cells
was observed with external applied Tif-2 Box2 peptide without
treated with the control peptide R9 (
an arginine tag B).
Similar experiments performed on cells cotransfected with
We also examined the effect of the most efficacious peptide
CFP-ERa and full-length YFP-Tif-2 coactivator followed by treat-
from the luciferase reporter assay (Tif-2 Box2-R9) on the expres-
ment with the synthetic SRC-1 Box2-R9 or Tif-2 Box2-R9
sion of an endogenous ERa target gene in the breast cancer cell
peptides showed that these peptides could also efficiently
line MCF-7 through quantitative polymerase chain reaction
recruit ERa into the nucleoli in the presence of Tif-2 coactivator
(QPCR) analysis MCF-7 cells treated with estradiol
(It is interesting to note that ERa nucleoli-recruitment
or with estradiol and the R9 peptide as reference showed the
upon the Tif-2 Box2-R9 peptide treatment was found to be less
expected up-regulation of ERa-mediated pS2 mRNA levels.
efficient when cells expressed the corresponding Tif-2 coactiva-
MCF-7 cells treated with estradiol and the Tif-2 Box2-R9 peptide
tor (median ratios, 0.6) than the SRC-1 coactivator (median
featured pS2 mRNA levels analogous to cells not treated with
ratios, 2), possibly indicating a higher affinity of the Tif-2 coacti-
estradiol, showing the effective down-regulation of the pS2
vator or a different binding mechanism.
gene expression by the inhibitor peptide. Together, the resultsshow that inhibition of the ERa-coactivator interactions in cells
Inhibition of ERa Gene Transactivation
by exogenous synthetic peptides resulted in an inhibition of
by Synthetic LXXLL Motif Molecular Probes
ERa-mediated gene transcription, the extent of which depends
The synthetic nona-arginine peptides induced sequestration of
on the amino acid sequences of the peptides. There is thus
ERa into the nucleoli. We therefore presumed a concomitant
a clear relationship between the potential of the LXXLL molecular
decrease of ERa transcriptional activity when perturbed with
probes to bind and recruit ERa to the nucleoli and their capacity
these peptides. Moreover, because these peptides are in fact
to antagonize ERa-mediated gene transcription. The results
mimicking the interactions between ERa and endogenous
show that a cell-based study of the ERa binding and antago-
cofactors, we expected that they might function in a dominant
nizing activity of inhibitors directly evaluates their physiological
negative manner when added to cells, disrupting these interac-
tions and blocking the ERa transcriptional activity. To studysuch an activity, a firefly luciferase reporter gene controlled by
EREs was cotransfected with an expression vector for ERa. Cellswere incubated in the presence or absence of estradiol, and the
We have designed a set of synthetic peptidic LXXLL molecular
reporter gene product was quantified in the resulting cell lysates
probes bearing both a nona-arginine tag, promoting cell-entry
by measuring the luminescence generated from the oxyluciferin
and specific nucleoli localization, and a biotin tag, allowing the
formed ). All the peptides, except for the control
visualization of their intracellular localization. Neither the arginine
R9 and alanine mutant peptides, decreased ERa gene-mediated
tag nor the biotin epitope changed the binding affinity of the
706 Chemistry & Biology 16, 702–711, July 31, 2009 ª2009 Elsevier Ltd All rights reserved
Chemistry & Biology
Cellular Modulation of ERa with Synthetic Peptides
peptides for the ERa LBD, showing that these tags are located at
Tif-2 coactivators, normally found bound to ERa in the nucleo-
sufficient distance from the binding pharmacophore. Even
plasm. The probes thus can compete with full-length coactiva-
though all the LXXLL-peptide sequences under study are
tors in living cells. Quantification of ERa-nucleoli recruitment
reported to have an affinity for the full-length ERa
upon LXXLL-peptide treatment therefore provides, to our knowl-
not all of the sequences were found
edge, a new, simple, and efficient method to assess the potency
to bind the ERa LBD strongly. The fact that the full-length
of molecular probes to act as ERa-coactivator inhibitors under
receptor can have a different surface topology as the isolated
physiological conditions. Among the panel of peptides tested,
the Tif-2 Box2-R9 and SRC-1 Box2-R9 peptides were found as
) is probably reflected in these observations,
the most potent inhibitors of SRC-1 and Tif-2 coactivator binding.
especially since a cellular activity of all these LXXLL-peptide
The specific physical inhibition of the ERa-coactivator interac-
sequences could be shown in our studies.
tion could be correlated with a decrease in ERa transcriptional
In order to perform subsequent cellular studies without the
activity when perturbed with these peptides, both in a luciferase
occurrence of any toxicity side effects, the concentrations at
reporter assay and for activity of the endogenous pS2 gene
which these probes showed cellular toxicity were determined.
in MCF-7 cells. Again, all LXXLL probes were functional and
Interestingly, the peptides that featured both a nona-arginine
decreased ERa gene-mediated luciferase transcription, whereas
tag and an LXXLL-motif showed higher toxicity to the cells
the control nona-arginine peptide and the AXXAA probes did not.
than either the nona-arginine tag alone or the peptide featuring
In line with the nucleoli recruitment results, the Tif-2 Box2 and
an arginine tag but with the LXXLL motif mutated to an AXXAA
SRC-1 Box2 showed the strongest inhibition. The luciferase
motif. Since no strong differences in cellular uptake are to be
gene transcription is inhibited to around 50% This
expected between the LXXLL and AXXAA peptides, the different
value is also obtained when a similar peptide is overexpressed
pharmacological behavior is more likely related to their intracel-
in U2OS cells from a transfected DNA construct and in accor-
lular effects. This furthermore indicates that the effects observed
dance with literature data on overexpressed peptides (
for the molecular probes are highly sequence specific.
The Tif-2 Box2-R9 showed a
CLSM imaging of cells incubated with these molecular probes
full inhibition of the pS2 gene transcription in MCF-7 cells. The
showed that they successfully entered the cells and adopted
extent of the transcriptional inhibition may depend on the cell
a particular localization into the nucleoli. This particular nucleoli
type, assay format, and ligand-exposure time.
localization was not found for the non-R9-tagged peptides and
It was previously shown that interaction of Tif-2 with ERa is
is therefore exclusively due to the R9 tag. The efficient cellular
strongly and selectively affected by mutations in NR-Box 2
uptake and specific nucleoli localization provide a strong meth-
(). In a similar manner, it was shown that estra-
odological tool to study any interactions of these molecular
diol-bound full-length ERa displays unambiguous SRC-1 box
probes with proteins, as it can be expected that either the local-
preferences for NR-Box 2, while the recruitment of boxes 1
ization of the probes or that of the proteins will be perturbed upon
and 3 of SRC-1 was not detected in vitro ().
binding. Transient transfection of cells with ERa together with
The 75% homology found between the SRC-1 Box2 and Tif-2
their incubation with the LXXLL probes showed that these
Box2 sequences provides a good explanation for the common
peptides indeed resulted in perturbation of the ERa through
features, both in terms of ERa-binding capacity and ERa-medi-
recruitment to the nucleoli. This specific localization of ERa
ated transcription inhibition that were observed in cells for these
was not observed for a peptide consisting of the nona-arginine
two peptides. Overall, there is a clear relationship between the
tag alone, nor was it observed for the AXXAA-motif peptides or
inhibitory effect on ERa-mediated gene transcription and ERa
peptides not featuring the nona-arginine tag. The ERa recruit-
translocation into the nucleoli by the molecular probes.
ment to the nucleoli by the nona-arginine LXXLL probes was
The mechanism of ERa inhibition by ICBs typically is via
furthermore found to be concentration dependent. These results
a direct inhibition of the ERa-coativator interaction
show that the observed effects are due to a specific interaction
The nona-arginine-based inhibitors
of the LXXLL motif with the ERa in combination with the localiza-
reported here feature the same mechanism, but additionally
tion properties of the nona-arginine tag. The fact that the nucleoli
displace the ERa after inhibition of the ERa-coactivator interac-
recruitment of ERa was observed for all the nona-arginine LXXLL
tion. The mechanism of action is thus most likely a combination
probes underlines the affinity of all these peptides for the full-
of inhibition and sequestration of the receptor. The method of
length ERa in a cellular context. The absence of this interaction
cellular evaluation of inhibitory capacity on the full-length ERa
in a biochemical context for some of these peptides on the iso-
as presented here could therefore be applied to evaluate coac-
lated ERa LBD shows that cellular evaluation, as presented
tivator binding inhibitors in general. These inhibitors could be
here, provides a more physiologically relevant view on the
either provided themselves with localization and visualization
binding interaction. The binding affinity of the peptides for the
tags or be used in competition assays with the molecular peptide
ERa can be evaluated by quantifying the ERa-nucleoli recruit-
probes presented here. Also, this method can provide a basis to
ment. This shows that certain peptide sequences, such as
address NR selectivity for LXXLL-based inhibitors in a cellular
SRC-1 Box2, Tif-2 Box1, and Tif-2 Box2, show a higher affinity
context, when applied to a panel of different NRs.
than others. This is in line with observations that the ERa has
The receptor's conformation is postulated to be a better indi-
distinct preferences for the different NR boxes of coactivators
cator of the antagonist activity of different ERa ligands than
cell-based reporter gene assays (
The LXXLL probes maintain the property to recruit the ERa into
). The molecular probes presented
the nucleoli even in the presence of overexpressed SRC-1 and
here and derivatives thereof could provide a valuable entry to
Chemistry & Biology 16, 702–711, July 31, 2009 ª2009 Elsevier Ltd All rights reserved 707
Chemistry & Biology
Cellular Modulation of ERa with Synthetic Peptides
diagnose the antagonist capacity of specific ligands in different
The resin was washed 5 times with dichloromethane and ether successively
cell types. In the presence of specific ERa antagonists, the
and dried under vacuum for at least 1 hr. Cleavage of side chain protecting
receptor adopts a conformation that prevents coactivators from
groups and of the peptides from the resin was achieved by shaking the resinin a mixture of trifluoroacetic acid (TFA), triisopropyl silane, ethane-1,2-dithiol,
binding ). It can be speculated that the molec-
and H2O (95/1/2.5/1.5) for 2 hr. Then, the resin was removed by filtration and
ular probes presented here will not interact with this antagonist-
washed twice with TFA. Most of the TFA was evaporated from the combined
liganded ERa. However, in the case of antagonist resistance,
filtrates, and a 10-fold volume excess of cold diethyl ether was added drop-
the ligand frequently acquires an agonistic function
wise to achieve peptide precipitation. The crude peptides were isolated by
and the molecular probes might
centrifugation (3500 rpm, 10 min), washed with cold ether, taken up in water
interact again with the ERa and thus induce nucleoli recruitment.
and lyophilized, and finally purified by reverse-phase high-performance liquidchromatography on a nucleodur C
The method presented here will thus provide ways to probe the
18 Gravity column (125 3 21 mm, Macherey-
Nagel) with a linear 60 min gradient of A (0.1% TFA in H2O) and B (0.1% TFA in
surface of the ERa and determine its conformation in different
MeCN) from 10% B to 100% B and a flow rate of 20 mL/min1. After purifica-
cell-types, dependent on ligand, or with specific posttranslational
tion, all the synthesized peptides were lyophilized and stored at 80C. The
modifications or point mutations.
purity of the peptides was assessed by analytical liquid chromatography–mass spectrometry at 210 nm, 254 nm, and 280 nm using a diode array ultra-violet/visible detector. All the nona-arginine peptides were obtained pure with
yields ranging from 13% to 41%.
The ERa-coactivator interaction, which is based on the
In Vitro Competitive Fluorescence Polarization Assay Expression
molecular recognition between consensus helical LXXLL
and Purification of ERa-LBD Protein
motifs of the coactivator and a hydrophobic pocket at the
The pET15b-ERa-LBD plasmid (gift from Bayer-Schering Pharma AG) encod-ing the LBD of the human ERa was used as template for PCR amplification
surface of the receptor, is an essential step for the ERa tran-
using the primers 50-CCGGATGGATCCATGGGCAGCAGCCATCATCAT-30
scriptional activation process to take place. This interaction
and 50-CCGGATCTCGAGTTAAGTGGGCGCATGTAGGCGG-30. The PCR
is an important target for the development of anti–breast
product (ERa-LBD residues 302–553) was digested with BamHI and XhoI
cancers drugs. Here, a new, to our knowledge, chemical
enzymes (Fermentas) and subcloned into a pHT401 plasmid to introduce a
biology approach is reported in which synthetic LXXLL-
Strep-tag and His-tag at the N terminus of the ERa-LBD construct. This
peptide probes provided with specific visualization and
plasmid (pHT503) was transformed into Escherichia coli Rosetta 2-DE3-placI
localization tags interact directly with ERa in cells and per-
(Novagen) cells that were then grown in Luria Bertani medium with 100 mg/Lampicilline and 34 mg/L chloramphenicol at 37C. At OD
turb its localization and transcriptional activity. These
600nm 0.8, protein
expression was induced with 0.5 mM isopropyl-beta-D-thiogalactopyranoside
probes monitor the affinity of specific peptide sequences
for 16–18 hr at 16C. Bacteria were then concentrated by centrifugation for
for the full-length ERa in cells. This approach represents
10 min at 5,000 rpm and were kept at 80C until purification. Cells lysate
a significant advancement over biochemical interaction
chromatography was performed at 4C on an A¨KTA purifier using a HisTrap
studies, which frequently focus on the isolated ERa LBD
HP 5 ml column (GE Healthcare) pre-equilibrated with the protein extract
alone. The molecular probes feature a nona-arginine tag
buffer: 300 mM NaCl, 50 mM Na2HPO4, 2 mM NaH2PO4, and 20 mM imidazole(pH 8.0). Protein elution was obtained with 500 mM NaCl, 50 mM Na
that facilitates cellular entry and also accounts for specific
2 mM NaH2PO4, 500 mM imidazole, and 10% (v/v) glycerol (pH 8.0); fractions
subcompartmental localization at the nucleoli. This specific
containing the ERa-LBD protein were then desalted on a Sephadex G25
feature allows these probes to dislocate the ERa from the
PD-10 column (Amersham Biosciences) pre-equilibrated with 25 mM NaCl,
DNA to the nucleoli upon binding and thus perturb the
20 mM Tris-HCl (pH 8.0), 10% (v/v) glycerol, and 1 mM tris (2-carboxyethyl)
assembly of the receptor transcription complex. This molec-
ular displacement of the ERa is accompanied by an inhibi-tion of its transcriptional activity. Quantification of the
Fluorescence Polarization Measurements
A reaction mixture containing a fluorescein-labeled coactivator (SRC-1 Box2)
ERa-nucleoli recruitment is a simple and rapid method to
peptide FL-CQLLTERHKILHRLLQEGSPSD (108 M), the ERa-LBD protein
assess the affinity of peptide sequences for ERa in the phys-
(106 M), and estradiol (105 M) was prepared. Fluorescence polarization inhi-
iological context of a cell. Moreover, since the timing of addi-
bition experiments were performed in 384-well plates (Optiplate-384 F, Perkin
tion and concentration are easily controlled and their amino
Elmer) by adding 10 ml of the reaction mixture to 40 ml of inhibitor peptides at
acid sequences can be fully modified, these arginine-tagged
increasing concentrations. After 2 hr of incubation at 4C, the fluorescence
probes promise to be useful molecular tools to elucidate and
polarization of the labeled coactivator peptide was measured on a plate readerfluorometer (Safire2TM, Tecan) with excitation at 470 nm and emission at
modulate ERa functioning.
519 nm for fluorescein. The concentration of inhibitor peptide that resultedin a half-maximum decrease in the polarization value of the fluorescent coac-
tivator peptide displayed from the purified ERa was defined as IC50 and wasdetermined from three independent experiments with the ORIGIN 7 program
(Scientific Graphing and Analysis Software, OriginLab Corp).
The synthesis of all peptides was performed on solid support, using the Fmocstrategy. Biotin-PEG Nova Tag resin (0.48 mmol/g, Novabiochem) was
allowed to swell in dimethylforamide (DMF) at room temperature for 30 min.
U2OS cancer cells (human osteosarcoma; ATCC number HTB-96) were split at
After DMF was drained, the beads were incubated with 40% piperidine in
25% confluence in 96-well plates and maintained at 37C under 5% CO2 in
DMF for the deprotection of the Fmoc group, and subsequently the amino
Dulbecco's modified Eagle's medium (DMEM) supplemented with 1% (v/v)
acids (AA) were introduced using the following coupling conditions: AA
antibiotics penicillin/streptomycin (GIBCO) and 10% heat-inactivated fetal
(3 eq), 2-(6-chloro-1H-benzotriazole-yl)-1,1,3,3-tetramethylaminium hexa-
calf serum (GIBCO). After 1 day, cells were incubated with 100 ml of increasing
fluorophosphate (3 eq), and N-methylmorpholine (6 eq) in DMF for 40 min.
concentrations of peptides (range from 0.01 to 5 g/L) directly diluted in the
The Fmoc deprotection and introduction of amino acids was repeated until
complete medium, for 14 hr. Subsequently, 10 ml of the cell proliferation
the complete sequence was assembled.
reagent WST-1 was added to each well, and cells were incubated again in
708 Chemistry & Biology 16, 702–711, July 31, 2009 ª2009 Elsevier Ltd All rights reserved
Chemistry & Biology
Cellular Modulation of ERa with Synthetic Peptides
a humidified atmosphere for 2.5 hr before measurement. The absorbance of
cells were maintained at 37C under 5% CO2 for 4 hr before treatment with
the samples was then measured using a microplate reader (Safire2TM, Tecan)
synthetic peptides directly dissolved in the medium. For comparison, a
at 450 nm, and cell toxicity was expressed as the percentage of inhibition of
pcDNA3.1 plasmid encoding for the SRC-1 Box2 peptide sequence (pSF5
WST-1 incorporation, compared with control cells not treated with peptides.
vector) was also tested: cells were transfected with 20 ng of pSF5, 0.2 ng of
All cell experiments were further performed at peptide concentrations that
pCMV-HA-ERa, 200 ng of pERE-tK-firefly luciferase, and 2 ng of SV40 Renilla
did not induce more than 20% of cell toxicity.
luciferase plasmids. Four hours after adding the peptides, estradiol (108 M)was incubated with the cells for 16 hr before the reading of firefly luciferase
Cell Imaging by Confocal Laser Scanning Microscopy (CLSM):
expression by a dual-luciferase reporter assay system (Promega). Lumines-
Cellular Localization of the Synthetic Peptides
cent intensities were recorded on a Berthold Centro XS3 LB 960 (Berthold
U2OS cells were cultured on coverslips for 48 hr before imaging in the same
technologies). Firefly luciferase activity signal was first normalized with respect
complete medium as mentioned above. Fourteen hours before imaging, cells
to the constitutive R. reniformis luciferase signal and second normalized to the
were incubated with peptides at the nontoxic peptide concentrations
luciferase activity found in control cells incubated with the R9 tag alone.
described above and then were fixed with 4% paraformaldehyde in PBS for
Results shown are representative of three independent experiments.
10 min at room temperature. A 10-min treatment of the fixed cells by PBS-NH4Cl 50 mM and then PBS-Glycine 20 mM was used to avoid self-fluores-
pS2 Gene Inhibition in MCF-7 Cells
cence bleaching. Cells were permeabilized with Triton X-100 0.1% for
For quantitative RT-PCR in MCF-7 cells, cells were cultured in phenol red–free
10 min, washed 3 times, and incubated for 30 min with a blocking solution
DMEM containing 5% charcoal-treated serum for 72 hr prior to the onset of the
of PBS-BSA 0.1%. The cellular distribution of peptides was revealed after
experiment in order to deplete activated ER in the cells. Subsequently, cells
immunolabeling of the cells using a Cy5-conjugated monoclonal mouse anti-
were cultured overnight in the presence of the indicated peptides at a final
biotin antibody (Jackson ImmunoResearch) diluted 1:400 in PBS and their
concentration of 70 mM. After overnight treatment, 10 nM estradiol was added
CLSM imaging (Leica TCS SP2), with the following settings: lex = 633 nm
for 3 hr or the cells were left untreated. After exposure to hormones, cells were
and lem = 650–700 nm.
lysed, and RNA was extracted using Trizol (Invitrogen), according to the manu-facturer's protocol. RNA was reverse-transcribed using SuperScript(tm) III
Cellular Assessment of ERa-LXXLL-Peptides Binding
Reverse Transcriptase (Invitrogen), on which QPCR was performed using
U2OS cells were cultured on coverslips in a 6-well plate for 48 hr before
CYBR Green (Applied Biosystems), according to the manufacturer's proto-
imaging in DMEM without phenol red supplemented with 1% penicillin/strep-
cols. The pS2 cDNA was amplified with the forward primer 50 CATCGAC
tomycin and 10% hormone-depleted (charcoal/dextran treated) and heat-
GTCCCTCCAGAAGA and the reverse primer 50 CTCTGGGACTAATCACCG
inactivated fetal calf serum (Thermo Scientific HyClone). At 50% of confluence,
TGCT. As a control for equal loading, the observed signals were related to
U2OS cells (neither expressing ERa nor ERb) were tran-
b-actin RNA levels, using a forward primer 50 CCTGGCACCCAGCACAAT
siently transfected by PEI (Polysciences) or Fugene 6 (Roche) with 1 mg of
and reverse primer 50 GGGCCGGACTCGTCATACT. Input levels were related
pcDNA3.1 plasmid containing the HA-ERa-CFP construct
to b-actin house keeping gene mRNA levels. Average values and SD were
Cells were incubated overnight with the nona-arginine peptides dissolved in
obtained for samples without peptide (duplicate) or with peptide (triplicate).
the culturing medium. Afterward, estradiol was added to the cultures at a finalconcentration of 106 M for 3 hr. Cells were then fixed and stained asdescribed above and imaged by CLSM (objective X63) with the followed
independent settings: lex = 458 nm and lem = 470–500 nm for CFP, andlex = 633 nm and lem = 650–700 nm for Cy5.
Supplemental data include two tables and five figures and can be found with
The quantification of ERa-CFP in the nucleoli was performed with the Quan-
this article online at
tify Histogram module within the CLSM Leica software. First, on the basis of
Cy5 fluorescence, for each cell, regions of interest (ROIs) were designed,encompassing the nucleoli containing peptides and the nucleoplasm. Then,
the average CFP fluorescence intensity per pixel in the ROIs were subtractedby the fluorescence intensity of the background to calculate the enrichment of
We thank Emmanuel Margeat and Julien Savatier (Centre de Biochimie
ERa-CFP in the nucleoli compared to the nucleoplasm as the mean ratio (CFP
Structurale, Montpellier, France) for advice on microscopy and for providing
intensity nucleoli/CFP intensity nucleoplasm). Results are presented as the
the pcDNA3-YFP-Tif-2 plasmid. We thank Sascha Fuchs (Chemical Genomic
ratios (nucleoli/nucleoplasm) found per peptide for two independent experi-
Centre, Dortmund, Germany) for providing the pSF5 plasmid. We thank Scher-
ments, for a 10–20 cell population.
ing Plough, Bayer-Schering Pharma, and Merck-Serono for financial support.
W. Zwart was supported by TI Pharma project T3-107.
Cellular Assessment of LXXLL Peptides/Coactivators
(SRC-1 and Tif-2) Competitive Binding for ERa
Received: February 6, 2009
U2OS cells were cultivated and treated in the same conditions as above and
Revised: May 21, 2009
transiently cotransfected with 1 mg of pcDNA3-ERa-CFP plasmid and 2 mg
Accepted: June 19, 2009
of pcDNA3-SRC-1-YFP plasmid or 2 mg of pcDNA3-YFP-Tif-2 plasmid. After
Published: July 30, 2009
cells were fixed and labeled with the Cy5-conjugated anti-biotin antibody,they were imaged by CLSM with the successive settings of CFP, Cy5, and
YFP (lex = 514 nm and lem = 525–550 nm). The same method as describedabove was used to quantify ERa-CFP fluorescence into the nucleoli and was
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BioFactors 25 (2005) 97–107 Coenzyme Q10 protects SHSY5Y neuronalcells from beta amyloid toxicity andoxygen-glucose deprivation by inhibiting theopening of the mitochondrial permeabilitytransition pore Geng Lia,∗, Liang-Yu Zoua,b, Chun-Mei Caoc and Edward S. Yanga aThe Jockey Club MRI Centre, The University of Hong Kong, Hong Kong, China bDepartment of Neurology, The First Affiliated Hospital, Harbin Medical University, China
Volume 11(Suppl 3) 2011 Official Journal of Malaysian P ublic Health Physicians'Association EDITORIAL BOARD Chief Editor Prof. Dato' Dr. Syed Mohamed Aljunid (Unit ed Nations University – International Institute for Global Health) Deputy Chief Editor Assc. Prof. Dr. Sharifa Ezat Wan Puteh