ERa17p, a peptide reproducing the hinge region of the estrogen receptor a associates 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, France b Fondation Pierre-Gilles de Gennes pour la Recherche, 29 rue d’Ulm, 75005 Paris, France c Laboratory of Pathology, University of Crete, School of Medicine, Greece d Laboratory of Experimental Endocrinology, University of Crete, School of Medicine, Greece e Laboratoire J.-C. Heuson de Cancérologie Mammaire, Institut Jules Bordet, Université Libre de Bruxelles, 1 rue Héger Bordet, 1000 Brussels, Belgium article info Article history: Received 10 October 2011 Received in revised form 22 December 2011 Accepted 29 February 2012 Available online 8 March 2012 Keywords: Peptide Estrogen receptor Membrane interaction Cellular internalization abstract Recently, we identified a peptide (ERa17p, P 295 LMIKRSKKNSLALSLT 311 ) that corresponds to the 295–311 sequence of the estrogen receptor a (ERa, hinge region) and which exerts a panel of pharmacological effects in breast cancer cells. Remarkably, these effects can result from the interaction of ERa17p with the 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 increases the fluidity of membrane mimics, it weakly internalizes in living cells. In light of the above, one may evoke one important role of the 295–311 region of the ERa: the corresponding peptide could be secreted/delivered to the extracellular medium to interact with neighboring cells, both intracellularly and 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/stabilization of the protein with the membrane. Ó 2012 Elsevier Inc. All rights reserved. 1. Introduction The fate of the estrogen receptor a (ERa) in the cell is highly complex: ERa dimerizes upon binding to estrogen, translocates to 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 cycles occur before receptor degradation through a proteasomal-driven process controlling transcription [1]. In addition, liganded or unli- ganded ERa may also act in the cytoplasm as a specific signaling initiator 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 of a number of various length peptides, some of them playing a biological function. In this context, we investigated the role of a 17 amino-acid peptide corresponding to a regulatory motif of the receptor located within its hinge region, i.e., P 295 LMIKRSKKN- SLALSLT 311 . This peptide was further detected in the extracellular medium of E 2 -treated cells (either the corresponding 17-mer or in the form of a longer N-extended peptide of 44 amino-acids; unpublished observations). Interestingly, the lysine/arginine-rich region K 299 RSKK 303 of this amino-acids sequence corresponds to the third nuclear localization signal (NLS) of ERa [5], a motif that overlaps a type II b-turn (residues 362–370) [6]. This KRSKK basic cluster is a component of the autonomous transcription activation function 2 (AF2a) of the ERa and more specifically of a platform that is in charge of the recruitment of calmodulin [7,8], Hsp70 [9], the ubiquitin ligase E6AP [10], as well as ERa itself [11]. The sequence also contains specific motifs for phosphorylation, acety- lation, SUMOylation and poly- mono-ubiquitination (reviewed in Ref. [12]). Moreover, it associates with ERa in vitro at a site distinct from 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 298–308 sequence by inhibiting the activation of IGF-1R/IRS-1/Akt and by restoring aromatase inhibitor-sensitivity in cells [13]. For aforementioned reasons, we were interested in exploring the biological action of the peptide corresponding to this 295–311 0039-128X/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.steroids.2012.02.022 ⇑ Corresponding author at: Department of Chemistry, Ecole Normale Supérieure, 24 rue Lhomond, 75231 Paris Cedex 05, France. Tel.: +33 (0) 1 44 32 24 48; fax: +33 (0) 1 44 32 24 02. E-mail address: yves.jacquot@upmc.fr (Y. Jacquot). 1 Equal contribution. Steroids 77 (2012) 979–987 Contents lists available at SciVerse ScienceDirect Steroids journal homepage: www.elsevier.com/locate/steroids