Steroids 77 (2012) 979–987

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Steroids

journal homepage: www.elsevier .com/locate /s teroids

ERa17p, a peptide reproducing the hinge region of the estrogen receptor aassociates to biological membranes: A biophysical approach

Cillian Byrne a,b,1, Lucie Khemtémourian a,1, Vassiliki Pelekanou c, Marilena Kampa d, Guy Leclercq b,e,Sandrine Sagan a, Elias Castanas d, Fabienne Burlina a, Yves Jacquot a,b,⇑a Laboratoire des BioMolécules, CNRS-UMR 7203, 24 rue Lhomond, Ecole Normale Supérieure/UPMC Univ Paris 06, 75253 Paris Cedex 05, Franceb Fondation Pierre-Gilles de Gennes pour la Recherche, 29 rue d’Ulm, 75005 Paris, Francec Laboratory of Pathology, University of Crete, School of Medicine, Greeced Laboratory of Experimental Endocrinology, University of Crete, School of Medicine, Greecee Laboratoire J.-C. Heuson de Cancérologie Mammaire, Institut Jules Bordet, Université Libre de Bruxelles, 1 rue Héger Bordet, 1000 Brussels, Belgium

a r t i c l e i n f o

Article history:Received 10 October 2011Received in revised form 22 December 2011Accepted 29 February 2012Available online 8 March 2012

Keywords:PeptideEstrogen receptorMembrane interactionCellular internalization

0039-128X/$ - see front matter � 2012 Elsevier Inc. Ahttp://dx.doi.org/10.1016/j.steroids.2012.02.022

⇑ Corresponding author at: Department of Chemistr24 rue Lhomond, 75231 Paris Cedex 05, France. Tel.: +(0) 1 44 32 24 02.

E-mail address: yves.jacquot@upmc.fr (Y. Jacquot)1 Equal contribution.

a b s t r a c t

Recently, we identified a peptide (ERa17p, P295LMIKRSKKNSLALSLT311) that corresponds to the 295–311sequence of the estrogen receptor a (ERa, hinge region) and which exerts a panel of pharmacologicaleffects in breast cancer cells. Remarkably, these effects can result from the interaction of ERa17p withthe plasma membrane. Herein, we show that ERa17p adopts a b-sheet secondary structure when in con-tact with anionic phospholipids and that it is engulfed within the lipid bilayer. While ERa17p increasesthe fluidity of membrane mimics, it weakly internalizes in living cells. In light of the above, one mayevoke one important role of the 295–311 region of the ERa: the corresponding peptide could besecreted/delivered to the extracellular medium to interact with neighboring cells, both intracellularlyand at the membrane level. Finally, the 295–311 region of ERa being in proximity to the cystein-447,the palmitoylation site of the ERa raises the question of its involvement in the interaction/stabilizationof the protein with the membrane.

� 2012 Elsevier Inc. All rights reserved.

1. Introduction

The fate of the estrogen receptor a (ERa) in the cell is highlycomplex: ERa dimerizes upon binding to estrogen, translocatesto the nucleus and binds to DNA, either directly or through tether-ing other transcription factors, acting thereby as a proper tran-scription factor. Thereafter, multiple cytoplasmic–nuclear cyclesoccur before receptor degradation through a proteasomal-drivenprocess controlling transcription [1]. In addition, liganded or unli-ganded ERa may also act in the cytoplasm as a specific signalinginitiator after receptor post-translational modification (e.g., phos-phorylation) and growth factor receptor activation. In this regard,it should be stressed that cytoplasmic ERa may transiently associ-ate with the plasma membrane through specific post-transcrip-tional modifications (e.g., palmitoylation of cysteine 447 [2–4]),thereby inducing (some) membrane-initiated signaling events.

The proteasomal degradation of ERa results in the emergence ofa number of various length peptides, some of them playing a

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biological function. In this context, we investigated the role of a17 amino-acid peptide corresponding to a regulatory motif of thereceptor located within its hinge region, i.e., P295LMIKRSKKN-SLALSLT311. This peptide was further detected in the extracellularmedium of E2-treated cells (either the corresponding 17-mer orin the form of a longer N-extended peptide of 44 amino-acids;unpublished observations). Interestingly, the lysine/arginine-richregion K299RSKK303 of this amino-acids sequence corresponds tothe third nuclear localization signal (NLS) of ERa [5], a motif thatoverlaps a type II b-turn (residues 362–370) [6]. This KRSKK basiccluster is a component of the autonomous transcription activationfunction 2 (AF2a) of the ERa and more specifically of a platformthat is in charge of the recruitment of calmodulin [7,8], Hsp70[9], the ubiquitin ligase E6AP [10], as well as ERa itself [11]. Thesequence also contains specific motifs for phosphorylation, acety-lation, SUMOylation and poly- mono-ubiquitination (reviewed inRef. [12]). Moreover, it associates with ERa in vitro at a site distinctfrom the hormone-binding pocket [11], inhibits in vitro the recruit-ment of p160 co-activator LxxLL binding motifs [11] and may pre-vent the phosphorylation of the serine 305 through its 298–308sequence by inhibiting the activation of IGF-1R/IRS-1/Akt and byrestoring aromatase inhibitor-sensitivity in cells [13].

For aforementioned reasons, we were interested in exploring thebiological action of the peptide corresponding to this 295–311

980 C. Byrne et al. / Steroids 77 (2012) 979–987

region (ERa17p). Strikingly, we found an extended spectrum ofremarkable biological actions. When added to cell culture media,ERa17p was found (i) to elicit (pseudo)-estrogenic responsesthrough intracellular interactions in ERa-expressing breast carci-noma cells under estrogen deprivation condition, giving rise to bothcell proliferation and ERE-dependent transcription as well as prote-asomal down-regulation of ERa and decrease of its mRNA level [9],(ii) to induce the apoptosis of breast cancer cells (by acting on themembrane independently of the presence of ERa) in vitro and theregression of human breast cancer xenografts in experimental ani-mals [14]; (iii) to modify the migration of breast cancer cells [15]and, therefore, to be implicated in the metastatic potential of breasttumors. Importantly, these effects occur independently from thepresence of ERa suggesting alternative mode(s) of action. In this re-spect, ERa17p was found to associate with cell membranes,enhancing the binding of the impermeable E2-BSA conjugate, butnon-directly competing with their membrane binding [14].

In parallel, X-ray crystallographic data show that the 295–311region of ERa, from which ERa17p is issued, is in close proximity(�15 Å) to the palmitoylated cysteine 447 (Fig. 1), which is impor-tant for ERa membrane targeting [2–4]. This structural feature sug-gests strongly that electrostatic interactions involving thenegatively charged headgroups of lipids constituting the inner faceof the phospholipid bilayer of the plasma membrane could partic-ipate in the targeting and in the stabilization of the receptor.

In view of these results and observations, we conducted a thor-ough investigation of ERa17p association with cells and membranemodels. At first using CHO cells, we examined by MALDI TOF massspectrometry the ability of the peptide ERa17p to bind cell mem-branes and to penetrate cells. In the second part of this study andby means of cell membrane models composed of the zwitterioniclipid phosphatidylcholine (PC) or the anionic lipid phosphatidyl-glycerol (PG), we have explored the conformation of ERa17p, itscapacity to reorganize the lipidic membrane and its effect on mem-brane integrity.

Fig. 1. Representation of the ERa (ribbons) with the palmitoylated cysteine 447(Connolly surface) and the KRSKK motif (Connolly surface). PDB code: 2YAT [35].

2. Experimental

2.1. Reagents

Protected Fmoc amino acids were purchased from Novabio-chem (Merck Chemicals Ltd.). Fmoc-Thr(tBu)-Novasyn TGA resinwas from Merck Chemicals Ltd. MBHA resin was from Iris Biotech.All reagents for peptide synthesis (chloroform, methanol, acetoni-trile, trifluoroacetic acid) were obtained from SDS. Tris–HCl, calce-in, Triton X-100, trypsin inhibitor and bovine serum albumin (BSA)were obtained from Sigma–Aldrich. Lipids DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) and DMPC (1,2-dimyristoyl-sn-gly-cero-3-phosphocholine) were purchased from Genzyme. DOPG(1,2-dioleoyl-sn-glycero-3-phospho-10-rac-glycerol) and DMPG(1,2-dimyristoyl-sn-glycero-3-phospho-10-rac-glycerol) were pur-chased from Avantipolar lipids. Dulbecco’s Modified Eagle Medium(DMEM), fetal bovine serum (FBS), trypsin–EDTA (0.05% trypsin,0.02% EDTA) and Hank’s BSS were purchased from PAA, the cellcounting kit (CCK8) was from Dojindo Laboratories. Streptavidin-coated magnetic beads (MyOne™ Streptavidin C1) were purchasedfrom Invitrogen. The complete mini cocktail of protease inhibitortablets (Pronase) were supplied from Roche.

2.2. Peptide synthesis

ERa17p and ERa17pAA were synthesized via standard Fmocsolid phase peptide synthesis on an Applied Biosystems 433a auto-mated peptide synthesizer, using preloaded Fmoc-Thr-NovasynTGA resin (0.2 mmol g�1). Acylation with activated Fmoc-aminoacids was typically accomplished with a 10-fold excess of Fmoc-amino acid, using HBTU and DIEA as coupling and activating agentsrespectively. The Fmoc-deprotection was carried out using 20%piperidine in NMP and was monitored using an in-line UV detectorat 290 nm. Cleavage and deprotection of the protected peptide-resin were achieved using the cocktail TFA – iPr3SiH (TIPS) – H2O(95:2.5:2.5), approximately 10 mL per gram of peptide resin, for2 h. The suspension was filtered, the filtrate was evaporated todryness in vacuo, and the residual material was triturated withice-cold ether (�3) to furnish a white solid. The peptide productwas dissolved in Milli-Q water and lyophilized before analysisand purification by semi-preparative RP-HPLC.

RP-HPLC was performed using a Waters setup, comprised of aWaters 1525 binary pump system with a Waters 2487 dual wave-length absorbance detector and using Breeze software. AnalyticalRP-HPLC was performed using an ACE reversed-phase C8 column(4.6 � 100 mm), using Solvent A: 0.06% TFA in water and solventB: 0.06% TFA in CH3CN:H2O (90:10), at a flow rate of 1.0 mL min�1

and UV detection at 220 nm with a gradient of 5–60% B in 20 min.Semi-preparative RP-HPLC was performed using an ACE 5 Å C8 col-umn (250 � 100 mm) with a flow rate of 5 mL min�1 using theappropriate gradient. ERa17p: Semi-preparative (20–40% B in20 min) RT = 11.02 min; Analytical (5–60% B in 20 min) RT =11.08 min; MALDI-TOF: [M + H]+ calcd 1899.1, found 1899.7.ERa17pAA: Semi-preparative (30–70% B in 30 min) RT = 14.74 min,MALDI-TOF (M + H+) calcd 1785.03, found 1786.10.

The non deuterated (H-ERa17p) and deuterated (D-ERa17p)biotin sulfone-apa-GGGG-ERa17p peptides (apa = aminopentanoicacid) were synthesized via standard Boc solid phase peptidesynthesis, also on an Applied Biosystems 433a automated peptidesynthesizer using MBHA resin (0.51 mmol g�1). Acylation wasaccomplished with a 10-fold excess of Boc-amino acid, usingDCC/HOBt coupling in DMSO/NMP. Boc-deprotection was carriedout using multiple TFA in DCM washes. Note: Boc-D,DGlycine waspurchased from Cambridge isotope laboratories and coupling wascarried out manually using 2 � 2 equivalents Boc-D,DGlycine,2 � 1.95 equivalents HATU and 2 � 4 equivalents DIEA in NMP.

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Biotin sulfone was coupled manually using 5 equivalents biotinsulfone, 4.95 equivalents HATU, and 10 equivalents DIEA in DMSO: NMP (50:50) overnight. Cleavage and deprotection of the pro-tected peptide-resin was achieved using a solution of Me2S(250 lL g�1) and anisole (1.5 mL g�1) in HF (�5 mL) for 2 h at0 �C. The solution was partially dried in vacuo and the crude pep-tide was precipitated in ice-cold ether and washed a further fourtimes with cold ether. The precipitated peptide was dissolved inH2O:AcOH (50:50) and separated from the resin via filtration.The filtrate was diluted with water and lyophilized to furnish awhite solid, which was analyzed and purified by semi-preparativeRP-HPLC. RP-HPLC was performed using a Waters setup, comprisedof a Waters 1525 binary pump system with a Waters 2487 dualwavelength absorbance detector and using Breeze software. Ana-lytical RP-HPLC was performed using an ACE reversed-phase C8column (4.6 � 100 mm), using Solvent A: 0.06% TFA in water andsolvent B: 0.06% TFA in CH3CN:H2O (90:10), at flow rate of1.0 mL min�1 and UV detection at 220 nm with a gradient of5–60% B in 20 min. Semi-preparative RP-HPLC was performedusing an ACE 5 Å C8 column (250 � 100 mm) with a flow rate of5 mL min�1 using the gradient indicated. H-ERa17p: Biotin sul-fone-apa-HGHGHGHG-PLMIKRSKKNSLALSLT-OH. Semi-preparative(20–40% B in 20 min) RT = 11.25 min; Analytical (5–60% B in20 min) RT = 13.56 min; MALDI-TOF: [M + H]+ calcd 2484.38,found, 2484.58. D-ERa17p Biotin sulfone-apa-DGDGDGDG-PLMIKRSKKNSLALSLT-OH. Semi-preparative (20–40% B in 20 min)RT = 11.75 min. Analytical (5–60% B in 20 min) RT = 13.81 min;MALDI-TOF: [M + H]+ calcd 2492.63, found 2492.43.

2.3. Cell culture and cell viability assays

CHO-K1 cells were cultured in sterile conditions in DMEM sup-plemented with 10% heat-inactivated fetal bovine serum (FBS) in ahumidified atmosphere of 5% CO2, at 37 �C. Cell viability wasassayed using the Cell Counting Kit-8 (CCK-8). Cells were seededin 96-well microliter plates and after 24 h, the supernatant wasremoved and 100 lL of ERa17p solution (10 lM) in DMEM (withor without 10% FBS) was added to the cells. Cells were also incu-bated with DMEM (with or without FBS) and no peptide as a refer-ence. After 1 h 15 min or 24 h incubation at 37 �C, the supernatantwas removed and 100 lL of CCK8 (10% in DMEM) was added. After2 h incubation at 37 �C, the absorbance was measured at 450 nmusing a microplate reader (FLUOstar OPTIMA, BMG LABTECH) witha reference wavelength at 620 nm. The experiments were per-formed in triplicates and repeated twice independently.

2.4. In vitro degradation of ERa17p by trypsin or pronase

Fifty pmol of H-ERa17p were mixed with 50 lL of the commer-cial trypsin–EDTA solution (or 50 lL pronase, 0.5 mgmL�1 in50 mM Tris–HCl buffer pH 7.4). The mixture was incubated for3 min at 37 �C then transferred to ice. 50 lL of trypsin inhibitor(5 mg mL�1) (or complete mini for pronase inhibition) and 50 pmolof D-ERa17p were added. This was followed by the addition of900 lL of 50 mM Tris–HCl buffer pH 7.4 containing 0.1 mg mL�1

BSA (buffer A) and 5 lL of the commercial solution of streptavi-din-coated magnetic beads. The mixture was incubated for 1 h atroom temperature. After bead immobilization with a magnet (Dy-nal magnetic particle concentrator), the supernatant was removed.Beads were washed twice with buffer A (200 lL), twice with bufferA containing 0.1% sodium dodecylsulfate (200 lL), twice with buf-fer A containing 1 M NaCl (200 lL), then with H2O (2 � 200,2 � 50 lL) and finally with 50 lL of H2O:CH3CN (1:1). Beads weremixed with 3 lL of a saturated solution of CHCA matrix (containing0.1% TFA) and incubated at room temperature for 10 min to elutethe biotinylated peptides. Beads were immobilized again on the

magnet and 1 lL of the supernatant was deposited on the MAL-DI-TOF sample holder. The samples were analyzed by MALDI-TOFmass spectrometry.

2.5. Treatment of ERa17p by diazotized 2-nitroaniline

Diazotized 2-nitroaniline was prepared as described previously[16]. Briefly, 50 lL of an aqueous solution of NaNO2 (0.6 M) wereadded to 400 lL of a solution of 2-nitroaniline (0.06 M) and HCl(0.125 M) in EtOH:H2O (1:1). The mixture was kept 5 min at roomtemperature. Ten microliters of the diazotized 2-nitroaniline solu-tion was then added to 50 pmol of H-ERa17p in 50 lL PBS. Themixture was kept 10 min at 0 �C then the reaction was quenchedby addition of 1 lL 500 mM Tris–HCl buffer pH 7.4. 50 pmol ofD-ERa17p were added. The mixture was diluted by addition of940 lL buffer A. A commercial solution (5 lL) of streptavidin-coated magnetic beads was added. The biotinylated species werepurified and analyzed as described above.

2.6. Determination of membrane-bound and internalized peptide byMALDI-TOF mass spectrometry

CHO-K1 cells were seeded in sterile conditions in 12 well plates24 h before starting the internalization experiment. Cells werethen incubated with H-ERa17p (10 lM in DMEM with 10% FBSor without FBS) for 1 h 15 min. The incubation experiments wereperformed on sub-confluent cells (106 cells/well).

For internalization experiments, at the end of the incubation,cells were washed three times with 1 mL Hank’s BSS, treated for3 min at 37 �C with 500 lL trypsin–EDTA and transferred at 4 �C.Trypsin inhibitor (100 lL, 5 mg mL�1) and BSA (100 lL, 1 mg/mL)were added. The cell suspension was transferred to a 1.5 mL conictube and the well was washed with 500 lL of 50 mM Tris–HCl buf-fer (pH 7.4). Both suspensions were pooled and centrifuged for2 min at 640g. The pellet was washed with 1 mL of 50 mM Tris–HCl buffer (pH 7.4), 0.1% BSA (buffer A) and centrifuged again.The pellet was mixed with the adequate amount of deuteratedinternal standard (D-ERa17p) and 150 lL of a solution containing0.3% Triton X-100, 1 M NaCl. The mixture was then heated for15 min at 100 �C. The cell lysate was centrifuged for 5 min at7080g. The supernatant was mixed with 850 lL of buffer A. Fivemicroliters of the commercial solution of streptavidin-coated mag-netic beads were added to the sample and the mixture was incu-bated for 1 h at room temperature to capture all biotinylatedpeptides. After bead immobilization with the magnet, the superna-tant was removed and beads were washed. The samples were ana-lyzed as described above. MALDI-TOF mass spectrometry analyseswere performed in the ion positive reflector mode on an ABIVoyager DE-Pro MALDI-TOF mass spectrometer (Applied Biosys-tems) using as matrix a saturated solution of a-cyano-4-hydroxy-cinnamic acid (CHCA) in CH3CN:H2O:CF3COOH (50:50:0.1). On theMALDI-TOF mass spectra corresponding to the average of severalhundreds laser shots, the area of the [M + H]+ signals (includingall isotopes) of the H- and D-ERa17p signals were measured andthe amount of intact internalized H-ERa17p was calculated fromthe area ratio. All internalization experiments were performed intriplicates and repeated at least twice independently.

The same procedure was used to measure membrane boundpeptide, with the omission of trypsin treatment. After incubationof the cells with peptide, cells were washed three times with Hanksbuffer and treated directly at 100 �C for 15 min with lysis solutionin the presence of D-peptide in the plate. The cell lysate was addedto a 1.5 mL conic tube and 850 lL of buffer A was used to removethe remaining lysate. The combined suspension was centrifugedfor 5 min at 7080g and the supernatant was transferred to a fresh

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conic tube. Abstraction of the peptides and analysis was carried outas described above.

2.7. Preparation of multilamellar vesicles (MLVs)

Lipid films were made by dissolving the appropriate amount oflipid into chloroform or a mixture of chloroform and methanol(2:1). The solvent was evaporated with dry nitrogen gas yieldinga lipid film that was further dried under vacuum for at least30 min. Lipid films were hydrated in Tris–HCl, 100 mM NaCl buffer(pH 7.4). The films were then shaken extensively with a vortex at atemperature superior to the phase transition temperature of thelipid to obtain MLVs and were then subjected to one freeze-thawcycle, at temperatures of approximately �80 and 40 �C, to homog-enize the size of the multilamellar vesicles.

2.8. Differential scanning calorimetry (DSC)

DSC experiments were performed on a high-sensitivity calorim-eter (TA Instruments). All experiments were preceded by a heatingand a cooling cycle. The temperature range was 0–40 �C with ascan rate of 1 �C min�1 and a delay of 10 min between sequentialscans in a series to allow thermal equilibration. Data analysiswas performed by the fitting software NanoAnalyze provided byTA Instruments. The lipid concentration was 1 mg mL�1. ERa17pwas gradually added to the lipid MLVs to obtain peptide:lipidmolar ratios of 1:100, 1:50, 1:25 and 1:10. A minimum of fourheating and cooling scans was performed for each analysis.

2.9. Preparation of large unilamelar vesicles (LUVs)

Lipid films were made by dissolving the appropriate amount oflipid into chloroform or a mixture of chloroform and methanol(2:1). The solvent was evaporated with dry nitrogen gas yieldinga lipid film that was further dried under vacuum for at least30 min. Lipid films were hydrated with a buffer containing70 mM calcein and 10 mM Tris–HCl, 100 mM NaCl (pH 7.4) for atleast 30 min, at a lipid concentration of 10 mM. The lipid suspen-sions were subjected to 10 freeze-thaw cycles, at temperatures ofapproximately �80 and 40 �C, and subsequently extruded 19 timesthrough 0.2 lm pore size filters. Free calcein was separated fromthe calcein-filled LUVs using size-exclusion column chromatogra-phy (Sephadex G-50 fine) and elution with 10 mM Tris–HCl,100 mM NaCl (pH 7.4). The phospholipid content of lipid stocksolutions and vesicle preparations was determined as inorganicphosphate according to Rouser [17].

2.10. Circular dichroïsm

Circular dichroism spectroscopy was carried out on a Jasco 815Circular Dichroism Spectropolarimeter using a Peltier to controlthe temperature. Spectra were recorded as an average of fourscans. Scans were acquired over the range of 190–270 nm, in0.2 nm increments, at a scan rate of 20 nm per minute and witha 1 nm bandpass. Samples were run at 20 �C in a cell of 0.1 cmpathlength. For each spectrum, the background was subtractedprior to smoothing and processing. Smoothing was carried outusing second order polynomial smoothing on Prism software. Mo-lar Residual Ellipticity [h]MRE was calculated using the Eq. (1):

½h�MRE ¼ h� 100=ðc � n� lÞ ð1Þ

h is ellipticity in mDeg, c is the concentration in M of peptide, n isthe number of backbone amide bonds and l is the pathlength ofthe sample.

2.11. Calcein leakage assay

A fluorescence plate reader (Fluostar Optima, BmgLabtech) wasused to perform leakage experiments in standard 96-wells trans-parent microtiter plates. The leakage assay was started by adding10 ll of a 0.2 mM ERa17p stock solution to 190 lL of a mixtureof calcein containing LUVs (100 and 200 lM lipids) and 10 mMTris–HCl, 100 mM NaCl (pH 7.4). Directly after addition of the pep-tide, the microtiter plate was shaken for 10 s using the shakingfunction of the plate reader. The plate was not shaken during themeasurement. Fluorescence was measured from the top, every5 min, using a 485 nm excitation filter and a 535 nm emissionfilter. The maximum leakage at the end of each measurementwas determined by adding 1 lL of 10% Triton X-100 to a final con-centration of 0.05% (v/v). The release of fluorescent dye was calcu-lated according to Eq. (2):

LðtÞ ¼ ðFt � F0Þ=ðF100 � F0Þ ð2Þ

L(t) is the fraction of dye released (normalized membrane leakage),Ft is the measured fluorescence intensity, and F0 and F100 are thefluorescence intensities at times t = 0, and after addition of TritonX-100, respectively.

3. Results

3.1. Cellular internalization and membrane binding of ERa17p

The membrane binding and cellular internalization of ERa17pin living CHO cells were examined using a method based on MAL-DI-TOF mass spectrometry (MS), relying on the use of labeled(polydeuteriated) peptide as an internal standard. This method,developed in our laboratory to study cell-penetrating peptides(CPPs), can be used to quantify the amount of membrane-boundand intact (not degraded) internalized peptides, and finally theanalysis of their intracellular degradation [18,19]. For the presentinvestigation, ERa17p was functionalized with an N-terminalisotope/affinity tag consisting of a biotin sulfone and four non-deu-terated Gly residues (H-ERa17p, non labeled species incubatedwith cells) or four bi-deuterated Gly residues (D-ERa17p, internalstandard for MALDI-TOF MS quantification).

CHO-K1 cells were chosen to run our experiments because theydo not express ERa [20]. ERa17p extracellular concentration was inthe same range as that used to observe pharmacological effects inbreast cancer cell lines (10 lM) [7–9,11,14,15]. Incubation timewas 1 h 15 min, both in the absence or the presence of FBS. Nocytotoxic effect was observed even after 24 h incubation in thesame conditions with CHO-K1 cells (data not shown), permittingthe quantification of internalization.

To quantify the net amount of internalized peptide, the potentialmembrane-bound peptide was removed with trypsin. After enzymeinhibition and addition of the internal standard (D-ERa17p), cell ly-sis was performed, as previously described [21], under conditionsthat release the content of all cellular compartments and com-pletely block the action of intracellular proteases. The lysate wasincubated with streptavidin-coated magnetic beads to recover allbiotinylated species (i.e., the internalized intact or degraded pep-tide and the internal standard) and samples were analyzed by MAL-DI-TOF mass spectrometry. The amount of intact internalizedpeptide (0.3 ± 0.1 pmol/106 cells) was calculated from the areas ofthe H- and D-ERa17p signals (Fig. 2A). This amount correspondsto peptides that were totally protected from trypsin, includingpeptides localized inside cell and possibly embedded in the plasmamembrane. Noteworthy, the amount of internalized ERa17p wasmuch lower than that observed for CPPs, ranging from 1–10pmol [18]. However, the concentration of intact ERa17p was not

Fig. 2. (A) MALDI-TOF mass spectrum obtained for a cellular internalization experiment. 106 CHO cells were incubated for 75 min at 37 �C with 10 lM H-ERa17p, 1 pmolinternal standard (D-ERa17p) was added before cell lysis. Inset zoom : signals of intact H-ERa17p and D-ERa17p. (B) Amounts of internalized and membrane-bound H-ERa17p obtained with one million CHO-K1 cells in presence (right) or absence (left) of fetal bovine serum (FBS).

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negligible since it would correspond to a calculated concentrationof peptide associated with cells (intracellular and possibly embed-ded) of approximately 200 nM, assuming a CHO volume of 1.5 pL. Itshould be stressed that this concentration was not influenced bythe presence of FBS (Fig. 2B). Neither modification of ERa17p (suchas phosphorylation, acetylation, methylation) nor peptide degrada-tion was detected by MALDI-TOF mass spectrometry.

The amount of peptide bound to the cell surface was then mea-sured by omitting the trypsin treatment after peptide incubationwith CHO-K1 cells (Fig. 2B). It was almost identical in the presenceor absence of serum and was found to be 9.7 ± 2.0 pmol/106 cells.The quantity of ERa17p linked to the cell membrane is thusmuch higher (about 30 times) than the amount of internalized/membrane-embedded peptide.

We therefore concluded that ERa17p strongly associates withcell membranes and, to a lesser extent, internalizes in CHO cells.In an attempt to further decipher the mechanism of this associa-tion/penetration, we analyzed the physiochemical properties ofERa17p in membrane models, composed of lipids that comprisecell membranes and more specifically with the membrane surface.

3.2. Far-UV circular dichroism study

Using far-UV CD spectroscopy, we have assayed whetherERa17p adopts a well-defined conformation, in the presence ofphospholipidic vesicles, relevant to a physical contact betweenthe peptide and lipids. The CD spectrum of ERa17p alone in solu-tion is characterized by a strong minimum at 197 nm, a spectralsignature that is typical of a random state [11]. The same signature

was observed when ERa17 was incubated at a range of 50–100 lMin the presence of large unilamellar vesicles (LUV) composed of thezwitterionic lipid DMPC up to a lipid:peptide ratio of 25:1, suggest-ing an absence of peptide–lipid interaction (Fig. 3A). The spectrumwas significantly different at the 10:1 lipid:peptide ratio and wascharacterized by two weak minima at �199 and �218 nm. Thisobservation is most likely due to independent aggregation ofERa17p observed at high peptide concentration (between 50 and100 lM) and a peptide:lipid ratio of 1:10 (data not shown).

In contrast, when membrane mimics are composed of glycerol-derived negatively charged lipids (DMPG and DOPG), a positiveband at 198 nm and a weak negative band at 219 nm characteristicof a b-sheet secondary structure were observed, suggesting aninteraction between the peptide and the negatively charged lipids(Fig. 3B). It should be stressed that the intensity of the absorptionbands were relatively consistent for lipid:peptide ratios rangingfrom 100:1 to 10:1.

Strikingly, the mutated peptide ERa17pAA, i.e., PLMIKRSAAN-SLALSLT, in which basic amino-acids represent only �11% of theresidues, shares the same tendency to adopt a b-sheet secondarystructure although the intensity of the absorption bands was sig-nificantly lower than observed with ERa17p (about 10% of itsintensity) and the signal more noisy (Fig. 3C).

The above data suggest that ERa17p interacts directly with lip-ids in unilamellar vesicles, depending on the charge state of thelipid headgroups. It adopts a b-sheet structure in the presence ofanionic lipids (DMPG and DOPG) whereas it remains random inthe presence of zwitterionic lipids (DMPC and DOPC). Because ofthe similar spectral signature of the peptide in the presence or

Fig. 3. CD spectra of ERa17p at 20 �C in the presence of (A) zwitterionic DMPC and(B) anionic DMPG vesicles. The spectra have been recorded with differentpeptide:lipid ratios of 1:100, 1:50, 1:25 and 1:10. Molar Residual Ellipticity[h]MRE was calculated using the formula [h]MRE = h � 100/(c � n � l) where h isellipticity in mDeg, c is the concentration of peptide, n is the number of backboneamide bonds and l is the pathlength of the sample. (C) CD spectrum of the peptideERa17pAA at 20 �C in the presence of anionic DMPG vesicles.

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absence of zwitterionic vesicles, an interaction with anionic lipids,only, seems likely. In addition, as cells are normally negativelycharged, this finding provides an additional clue about a possibleinteraction of the peptide with cell membranes, explaining in partour aforementioned data of the peptide association with cells [14].

3.3. Interaction of ERa17p with cell membrane mimics

To confirm a link between the conformational changes ofERa17p and its interaction with anionic phospholipid bilayers, dif-ferential canning calorimetry (DSC) assays were performed withvesicles composed of DMPC (a zwitterionic phospholipid, Fig. 4A)or DMPG (an anionic phospholipid, Fig. 4B). This approach is par-ticularly adapted to this aim, the phase behavior of lipid membranesystems being highly affected by the presence of guest moleculessuch as peptides [22,23]. Briefly, the thermally induced gel to

liquid–crystalline phase transition and the related thermodynamicvariables (melting temperature, enthalpy and entropy change)depend on the nature of the interactions between the peptideand the membrane as well as on the topology of the peptide withrespect to the phospholipid bilayer [24].

In the pure lipid scans (bottom of each figure), there is a tall nar-row transition temperature (Tm) near 23 �C and a large enthalpy.This corresponds to the chain melting transition (rippled gel tofluid lamellar) with high cooperativity. A smaller and broader peaknear 13 �C corresponding to the pre-transition (lamellar gel to rip-pled gel) was also observed. This pre-transition has a lower enthal-py, is less cooperative, and corresponds to melting or untilting ofthe lipid headgroups.

The addition of ERa17p to the zwitterionic DMPC vesicles at alipid:peptide ratio from 100:1 to 10:1 had a modest effect on thepre-transition and no effect on the main transition (Fig. 4A). Thisindicates that intact lipid–lipid interactions occur and that thepeptide does not bind consequently to the membrane. Thus,the random state observed by far UV–CD is relevant to an absenceof interaction between the peptide and the zwitterionicphospholipids.

In contrast, the addition of ERa17p to the anionic DMPG vesi-cles leads to large changes in the thermotropic behavior of the lipidbilayer (Fig. 4B). We observed a decrease followed by the disap-pearance of the pre-transition at a lipid:peptide ratio 50:1, indicat-ing that the peptide interacts with the negatively chargedheadgroups of DMPG. At low ERa17p concentration (lipid:peptideratio 100:1), the shape of the main transition peak appears verysimilar to that of the pure lipid, while at higher concentrations(lipid:peptide ratio 50:1 and 25:1), the shape of the main transitionpeak becomes asymmetric and shifts to a higher temperature. Thisasymmetric shape suggests that the peptide interacts strongly withthe acyl chains of the lipids, with a possible coexistence of the twophases. We assume that one phase might represent peptide-richregions and the other peptide-poor regions, a feature related tothe propensity of ERa17p for aggregation (data not shown). Inaddition, the increase in melting temperature implies that the pep-tide might induce membrane rigid domains. At a lipid:peptide ratioof 10:1, we observed a broad phase transition indicating a loss oflipid cooperativity, suggestive of membrane damage.

Hence, these DSC data demonstrate that ERa17p interacts andinserts exclusively into membranes containing anionic lipids con-firming that the b-sheet conformation adopted by ERa17p is linkedto an interaction with anionic lipids, compatible with physiologicalcellular changes. Interestingly, high peptide:lipid concentrationsmay also induce lipid layer disruption, leading (in a cellular sys-tem) to cellular loss.

3.4. Membrane disruption induced by ERa17p in DOPC and DOPGvesicles

In order to further verify our hypothesis of a membrane damageinduced by ERa17p, we analyzed the leakage of a fluorescent dye(calcein) from large unilamellar vesicles, under peptide treatment[25]. When the vesicles are intact, calcein remains at a high con-centration in the interior and is self-quenched while when theirmembrane is disrupted, calcein may escape, eliminating the self-quenching effect and increasing thereby fluorescence.

When large unilamellar anionic DOPG vesicles loaded withcalcein were treated with 10 lM ERa17p for 1 h, about 45% ofmembrane leakage at a lipid:peptide ratio 10:1 and around 20%at a lipid:peptide ratio 20:1 was observed (Fig. 5). In contrast, inzwitterionic DOPC vesicles, ERa17p failed to affect the barrierproperties of the membrane even after several hours of incubationat the same concentrations, verifying our calorimetry results.Hence, our data show that the b-sheet secondary structure adopted

Fig. 4. DSC thermograms illustrating the effect of the addition of ERa17p to (A) zwitterionic DMPC and (B) anionic DMPG vesicles. The curves correspond to pure lipid andpeptide:lipid ratio of 1:100, 1:50, 1:25 and 1:10.

Fig. 5. Kinetics of membrane permeabilization by 10 lM ERa17p. The peptide wasadded to the calcein containing DOPC at a lipid:peptide ratio of 20:1 (emptytriangles) and 10:1 (empty circles) LUVs at t = 0; and DOPG at a lipid:peptide ratioof 20:1 (full diamonds) and 10:1 (full squares) LUVs at t = 0. The maximum leakage,after complete disruption of all vesicles by Triton X-100, was normalized at 1.

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by ERa17p in the presence of anionic vesicles is correlated not onlyto membrane association but also to membrane damage. Interest-ingly, this result might explain our previous data that (in ERa-negative breast cancer cells) long incubation of cells with 10�5 MERa17p results in massive necrosis [14].

4. Discussion

Endocrine resistance of breast cancer remains a major and per-sisting problem in oncology. Thus, in the context of new therapeu-tic approaches, a better understanding of the mechanism of actionof ERa appears necessary. In addition to the design and synthesis ofnovel SERMs targeting the hormone-binding pocket of the recep-tor, attempts are in progress to identify molecules targeting alter-native interaction sites and the synthesis of novel specificendocrine disruptors [26,27]. A new category of ERa modulatorshas therefore emerged, called peptide estrogen receptor modula-

tors (PERMS). ERa17p has been proposed as a prototype of sucha category. Indeed, the peptide mimics a part of the receptor pro-tein which is an important platform for various post-translationalmodifications and association with peptidic activity modulators[12] regulating its interaction with the type II b-turn/H4 ERa sur-face region (residues 362–370) of the AF-2 [6,12]. The topologyof this site is, indeed, significantly modified by the nature of the li-gand, a feature that could be related to the recruitment of specificco-activators and to the regulation of transcription [6,11]. Accord-ingly, ERa mutants devoid of this site exhibit a constitutive tran-scriptional activity, which is relevant to a more aggressivephenotype as well as to resistance phenomena towards aromataseinhibitors [7,11,13].

A natural peptide including the 295–311 sequence, issued mostprobably at least in part from the proteasomal degradation of ERa,has been detected in the culture medium of breast cancer cells(unpublished) providing biological relevance to reported proper-ties of ERa17p. This peptide elicits (pseudo)estrogenic effects inbreast cancer cells (ERa-positive cells under steroid starvation[7]) as well as migration modifications and apoptosis in the lattersthrough an ERa-independent mechanism [14,15]. In the presentwork, we extend these previous investigations by showing thatthe peptide can associate to cell membranes independently fromthe presence of ERa, penetrates weakly into the cell, and, at highconcentrations, induces membrane damage.

According to our data in ERa-negative CHO cells, ERa17p isinternalized, when incubated at the concentration of 10 lM inthe cell culture medium, and attains an intracellular concentration(200 nM) compatible with its previously described binding to thenative form of ERa in breast cancer cells [28]. Interestingly, a pre-liminary analysis of transcriptome data, seems to verify this effect(unpublished observations). In the present study, we have used anenzymatic treatment together with a MALDI-TOF mass spectrome-try-based method to quantify and analyze intracellular and mem-brane-associated peptide. The main difficulty when quantifyingpeptide cellular uptake is to accurately distinguish the internalizedspecies from those bound to the cell surface. Cell washes (even un-der acidic conditions and/or at high salt concentration) are oftennot sufficient to completely remove the membrane-bound peptide.Thus, proteases or chemical modification (such as diazotization)are often used [16]. In all cases, the treatment must be non-toxicand must induce total and fast modification/degradation of theextracellular peptide to avoid exchange between the intracellularand extracellular milieu. In the case of ERa17p, trypsin was foundto be more efficient than pronase or diazotized 2-nitroanilinetreatment, allowing total degradation of the peptide within 3 min.

Fig. 6. Interaction of the PLMIKRSKKNSLALSLT sequence. (A) With respect to the action displayed by ERa17p the peptide is recruited from the culture medium to the plasmamembrane through its basic block KRSKK. Thereafter, the peptide adopts a b-sheet conformation and is embedded in the phospholipid bilayer. A weak internalization(0.3 ± 0.1 pmol/106 cells) occurs whereas the majority of the peptide (10.0 ± 2.0 pmol/106 cells) remains at the membrane; (B) In the context of the entire estrogen receptorand in concert with the palmitoylated Cyst-447, we hypothesize that this sequence may interact with negatively charged residues present in the inner face of plasmamembrane and participates in the stabilization of the protein in the membrane.

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Our data show that, in addition to the internalized peptide, amuch higher quantity (30-fold) is linked to the cell membrane incells not bearing ERa. Noteworthy, this is consistent with previousconfocal microscopy data in which the main bulk of a FITC-labeledERa17p was found on the cell membrane, while only a weak fluo-rescence signal was detected intracellularly [14], suggesting acommon mechanism in CHO cells and breast cancer cells express-ing a different spectrum of ERa and b, a hypothesis actually underinvestigation. This may explain how ERa17p exerts its apoptoticand migration-modulatory actions when introduced in the culturemedium, again independently from the presence of ER isoforms[14,15].

Given the ERa17p’s propensity to bind cell membranes, we con-centrated our efforts in deciphering the interaction of the peptidewith lipids using model synthetic membranes composed of phos-phatidylcholine (PC) or phosphatidylglycerol (PG) and devoid ofother potential membrane targets such as glycoproteins, glycolip-ids or proteins eventually associated with lipids. This is done inview of our previous findings that ERa17p does not compete forbinding with protein-conjugated steroids at the membrane level,although it enhances their binding [14]. Accordingly, we clearlyidentified here a physical interaction of the peptide with mem-branes. Indeed, ERa17p (i) adopts a b-sheet conformation in thepresence of anionic lipids and (ii) may not be adsorbed solely atthe membrane surface but could be embedded within. The fact thatERa17p interacts with anionic lipids (DMPG and DOPG) but notwith neutral lipids (DMPC and DOPC) raises the question, in a bio-logical context, about the ability of the latter to recruit anionic lip-ids at the outer face of the cell membrane. In this regard, a key roleof the positive charges in ERa17p (+5 net charge) must be evoked[29,30]. In this context, it is noteworthy that the partial insertion

into negative model membranes of peptides adopting a b-sheetconformation has been previously described, as exemplified withprotegrin [31] and vinculin [32].

Lastly, we show that ERa17p disrupts lipid vesicles at high con-centrations and high peptide:lipid ratios. Whether this latter effectis of physiological value is questionable in the absence of concretedata concerning the concentrations of the endogenously producedpeptide in the extracellular milieu. Nevertheless, we can reason-ably hypothesize that under an intense estrogen stimulation andERa-turnover, sufficient proteasomal degradation may occur, lead-ing to a sufficient ERa17p production and secretion, and, thereby, amembrane disruption. Further experiments are required to explorethis possibility. However, a hint about a possible physiological rel-evance of this finding derives from the fact that a long incubationtime (24–48 h) of the peptide (in addition to apoptosis) induces amassive necrosis of breast cancer cells independently of the pres-ence of ERa [14].

Hence, our data outline that ERa17p interacts with anionicmembrane lipids, leading to its association with lipid bilayers. Infact, the contact between ERa17p and anionic lipids may operateas follows: (i) first an electrostatic interaction involving the poly-basic motif KRSKK and negatively charged phospholipids (drivingforce of the interaction) and (ii) a folding of the flanked regionsleading to a b-sheet conformation of the peptide (Fig. 6A). Indeed,using a mutated peptide lacking the basic KK residues (ERa17pAA),the peptide–lipid interaction was decreased by 90%.

Our previous work has clearly revealed that ERa17p not onlyassociates with membranes of breast cancer cells but also triggersspecific rapid signaling cascades important for cell survival orapoptosis, as already observed with other estrogens [33,34]. It isnot clear, until now, which is the protein counterpart for such an

C. Byrne et al. / Steroids 77 (2012) 979–987 987

action. Indeed, our preliminary data suggest a potential interactionof ERa17p with the membrane associated E2-binding proteinGPR30/GPER1 [15], although not all ERa17p effects can be ex-plained by such an interaction. The results presented here solelysuggest that an association/integration/penetration mechanismmay be taken into account.

An arising question deriving from our data is whether theERa17p sequence, in the context of ERa molecule might conferto the receptor a specific membrane-associated label. Our dataclearly indicate that the full-length peptide associates withphospholipid bilayers that are composed of the anionic lipids(which are present in normal and cancer cells). Even if a link be-tween our observations and a participation of this motif in theassociation of the ERa in the membrane seems likely (see Fig. 6B,hypothetical model), additional mutagenesis experiments are re-quired to validate this hypothesis.

5. Conclusion

In conclusion, our data suggest that the ERa17p peptide corre-sponding to the ERa hinge region can penetrate, even modestly,into cells and attains concentrations compatible with an ER-relatedaction, explaining previous findings of a (pseudo)estrogenic effectof the peptide. However, the major finding of this work is the asso-ciation of ERa17p with polar lipids in membrane models and thepenetration of the peptide into the membrane, explaining previousdata in breast cancer cells. Even if the polar headgroups of negativelipids are required for this interaction, a role of their hydrophobicpart is likely, a partial engulfing of the latter within the membraneand a resulting increase of its fluidity, leading to a membrane dis-ruption and ultimately to cell necrosis, a result previously reportedin breast cancer cells. Finally, the protein surface exposure of the295–311 region of ERa and its proximity from the cystein-447raises the question of its involvement in the interaction/stabiliza-tion of the protein with the membrane. Even if it seems likely,further mutation studies targeting the KRSKK region will be neces-sary to explore this hypothesis.

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