Interaction of the Anti-Proliferative GPER Inverse Agonist ERα17p with the Breast Cancer Cell Plasma Membrane: From Biophysics to Biology.

Michaël Trichet,Lucie Khemtemourian,Delphine Ravault,Yves Jacquot,Isabel Alves,Rosamaria Lappano,Lilian Salazar Vazquez,Sandrine Sagan,Marcello Maggiolini,Mathilde Belnou

doi:10.3390/cells9020447

Abstract

The peptide ERα17p, which corresponds to the 295-311 fragment of the hinge/AF2 domains of the human estrogen receptor α (ERα), exerts apoptosis in breast cancer cells through a mechanism involving the G protein-coupled estrogen-dependent receptor GPER. Besides this receptor-mediated mechanism, we have detected a direct interaction (Kd value in the micromolar range) of this peptide with lipid vesicles mimicking the plasma membrane of eukaryotes. The reversible and not reversible pools of interacting peptide may correspond to soluble and aggregated membrane-interacting peptide populations, respectively. By using circular dichroism (CD) spectroscopy, we have shown that the interaction of the peptide with this membrane model was associated with its folding into β sheet. A slight leakage of the 5(6)-fluorescein was also observed, indicating lipid bilayer permeability. When the peptide was incubated with living breast cancer cells at the active concentration of 10 μM, aggregates were detected at the plasma membrane under the form of spheres. This insoluble pool of peptide, which seems to result from a fibrillation process, is internalized in micrometric vacuoles under the form of fibrils, without evidence of cytotoxicity, at least at the microscopic level. This study provides new information on the interaction of ERα17p with breast cancer cell membranes as well as on its mechanism of action, with respect to direct membrane effects.

Highlights

The Pro295 –Thr311 sequence of the human estrogen receptor α (ERα) encompasses residues that are located in the C-terminal part of the hinge region (D domain) and in the N-terminus of the AF2 transactivation function (E/F domains)
By using circular dichroism (CD) spectroscopy and differential scanning calorimetry (DSC), we have previously shown that the peptide ERα17p was able to interact with membrane models (LUVs) that are composed of one species of anionic lipids such as 1,2-dimyristoyl-sn-glycero-3phosphoglycerol (DMPG) [25]
Far-UV circular dichroism (CD) spectroscopy, plasmon waveguide resonance (PWR) and fluorescein leakage experiments were carried out to explore the secondary structure of the peptide under membrane interaction, the characteristics of this interaction, and the effects of ERα17p on the membrane integrity, respectively

Summary

Introduction

The Pro295 –Thr311 sequence of the human estrogen receptor α (ERα) encompasses residues that are located in the C-terminal part of the hinge region (D domain) and in the N-terminus of the AF2 transactivation function (E/F domains). This short sequence corresponds to an interaction platform. It is subjected to trypsin- and chymiotrypsin-dependent proteolysis at the K303 N304 and at the K302 K303 sites [6] In this regard, it should be noted that the K299 RSKK303 motif corresponds to the third nuclear localization signal (NLS) of the ERα [7]. The ERα mutation K303R, which is found in several invasive breast tumors [9], is associated with methylation modifications by the SET7 methyltransferase and, with transcription profile changes [10]

Methods

Results

Conclusion