FEBS Letters 583 (2009) 3405–3411 journal homepage: www.FEBSLetters.org Short peptides derived from the BAG-1 C-terminus inhibit the interaction between BAG-1 and HSC70 and decrease breast cancer cell growth Adam Sharp a, Ramsey I. Cutress a, Peter W.M. Johnson a, Graham Packham a, Paul A. Townsend b,* a Cancer Research UK Centre, Cancer Sciences Division, University of Southampton, School of Medicine, Southampton General Hospital, Southampton S016 6YD, UK b Human Genetics Division, University of Southampton, School of Medicine, Southampton General Hospital, Southampton S016 6YD, UK article info abstract Article history: BAG-1, a multifunctional protein, interacts with a plethora of cellular targets where the interaction Received 8 July 2009 with HSC70 and HSP70, is considered vital. Structural studies have demonstrated the C-terminal of Revised 21 September 2009 BAG-1 forms a bundle of three alpha-helices of which helices 2 and 3 are directly involved in binding Accepted 24 September 2009 to the chaperones. Here we found peptides derived from helices 2 and 3 of BAG-1 interfered with Available online 1 October 2009 BAG-1:HSC70 binding. We confirmed that a 12 amino-acid peptide from helix 2 directly interacted Edited by Lukas Huber with HSC70 and when introduced into MCF-7 and ZR-75-1 cells, these peptides inhibited their growth. In conclusion, we have identified a small domain within BAG-1 which appears to play a crit- ical role in the interaction with HSC70. Keywords: BAG-1 HSC70 Structured summary: HSP70 MINT-7265269, MINT-7265296, MINT-7265324, MINT-7265339, MINT-7265351, MINT-7265364, MINT- Interaction 7265483, MINT-7265464, MINT-7265310: HSC70 (uniprotkb:P11142) binds (MI:0407) to BAG1 (uni- Binding protkb:Q99933) by peptide array (MI:0081) MINT-7265281: peptide 15L (uniprotkb:Q99933) binds (MI:0407) to HSC70 (uniprotkb:P11142) by surface plasmon resonance (MI:0107) Ó 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. 1. Introduction and HSP70, is considered key for many of BAG-1 functions [5,14,28,30–33]. For example, mutations which prevent binding BAG-1 is a multifunctional protein that can modulate a wide to HSC70/HSP70 interfere with the ability of BAG-1 to promote cell range of cellular processes [1]. For example, overexpression of survival and to modulate NHR function [2,6,21,22,29,34–36].In BAG-1 protects cells from a wide range of apoptotic stimuli, acti- addition, BAG-1 has been shown to modulate proteasomal degra- vates RAF-1 kinase-dependent signalling, enhances proliferation dation of Tau proteins, a process dependent on the interaction with and metastasis, regulates proteasomal degradation, and modulates HSC70/HSP70 [5]. Moreover, BAG-1 has cochaperone activity and the transcriptional activity of a variety of nuclear hormone recep- stimulates nucleotide exchange of HSC70/HSP70 [27,37–40] tors (NHR) [2–16]. Functional and expression studies suggest that HSC70 and HSP70 play important roles in multiple cell processes, overexpression of BAG-1 may play an important role in diverse for example, via effects on protein (re)folding and activation of cancer types [1,2,4,15,17–29]. NHR. Thus, binding to these multifunctional proteins may explain The mechanism(s) by which BAG-1 exerts its pleiotropic effects at least in part the multiple effects associated with BAG-1 are not fully understood. Although BAG-1 has many protein bind- overexpression. ing partners (including BCL-2, RAF-1, pRb, CHIP and the protea- The interaction between BAG-1 and HSC70/HSP70 has been some), interaction with the 70 kDa heat shock proteins, HSC70 studied by NMR and X-ray crystallography [34,41]. The 70 kDa heat shock proteins comprise an amino terminal ATPase domain, a central peptide binding domain and a carboxyl terminal region Abbreviations: BAG, bcl2-associated athanogene-1; HSC70, heat shock cognate that can form a ‘‘lid” over the peptide binding domain [42,43]. 70; HSP70, heat shock protein 70; SPR, surface plasmon resonance The interaction of BAG-1 with the ATPase domain of HSC70/ * Corresponding author. Address: Human Genetics Division, Duthie Building, HSP70 is mediated by the C-terminal BAG domain of BAG-1. This Southampton General Hospital (MP808), Southampton SO16 6YD, UK. Fax: +44 (0)23 8079 4264. evolutionarily conserved ‘‘BAG domain” [44] is formed of a bundle E-mail address: [email protected] (P.A. Townsend). of three alpha-helices (Fig. 1). Helices 2 and 3 are involved in elec- 0014-5793/$36.00 Ó 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2009.09.047 3406 A. Sharp et al. / FEBS Letters 583 (2009) 3405–3411 UK) for 1 h at room temperature. The wells were washed as before, prior to addition of GST-BAG-1S or GST (each 500 ng) in PBS. Fol- lowing incubation at room temperature for 1.5 h, unbound protein was removed by washing in PBS. Bound BAG-1 was detected using the BAG-1-specific monoclonal antibody 3.10 G3E2 [46] at a 1/ 1000 dilution of ascities in PBS (200 ll). Following incubation at room temperature for 1 h, wells were washed and then incubated for 1 h with 200 ll of horseradish peroxidase conjugated sheep anti-mouse Ig antibody (GE/Amersham Biosciences, UK) diluted in PBS (200 ll). Wells were washed and developed by addition of 200 ll of o-Phenylenediamine Dihydrochloride (Sigma, UK) and incubation for 10 min at 37 °C. Absorbance at 450 nm was deter- mined on a Dynatech MR5000 plate reader (Dynatech, Germany). The absorbance of wells containing HSC70 and GST was used to determine the 0% interaction and the absorbance of wells contain- ing HSC70 and GST-BAG-1S was used to determine the 100% inter- action. Peptides were added to the interaction assay at various concentrations prior to addition of GST-BAG-S. Fig. 1. The BAG-1:HSC70 interaction. The figure shows the interaction between the 2.3. Surface plasmon resonance (SPR) three helices comprising the BAG domain of BAG-1 (red) and the ATPase domain of HSC70 (blue). The 8 amino-acid binding core of helix 2 (pink) and the 16 amino-acid binding core of helix 3 (light blue) identified in this study are indicated. Figure was SPR analysis was performed using BIAcore 2000 biosensor and drawn using PyMOLÓ and structural coordinates from Protein Data Bank id 1hx1. BIA evaluation 2.0 software (BIAcore, Sweden). CM5 carboxy- methyl dextran sensor chips were activated with injection of 100 ll of a 1:1 mixture of N-ethyl-N0-[(dimethylamino)propyl]car- trostatic interactions with subdomains IB and IIB of the ATPase do- bodiimide and N-hydroxysuccinimide. Immediately after activa- main of HSC70 [41]. Although helix 1 is not directly involved in tion, 30 ll of peptide 15L (100 lM) in HBS buffer (10 nM HEPES binding, it may contribute to intramolecular interactions which with 0.15 M NaCl, 3.4 mM EDTA, and 0.005% surfactant P20; BIA- stabilize the overall structure of the BAG domain. In this paper, core, Sweden) was injected for covalent coupling to the surface we have identified small peptides derived from the BAG domain of the sensor chip. Unreacted sites were blocked by injection of that were sufficient to inhibit the interaction of BAG-1 and 70 ll of 1 M ethanolamine pH 8.5. Recombinant HSC70 was diluted HSC70 and investigated their effect on breast cancer cell growth. to the desired concentrations in HBS buffer and injected separately over the chip-bound peptide 15L. Sensograms were collected at 2. Materials and methods 25 °C, with a flow rate of 10 ll/min and data collection rate of 1 Hz. Sensograms were normalised to a base line of 0 resonance 2.1. BAG-1 peptides and GST-BAG-1 units (RU). Equivalent concentrations of each protein were injected over an untreated surface to serve as blank sensograms for subtrac- For in vitro studies, peptides were generated by solid phase syn- tion of bulk refractive index background. The sensor chip surface thesis (Mimotopes Pty Ltd., Australia), dissolved in DMSO and was regenerated between runs with a 1 min injection of 100 lM 10 mM stock solutions stored at À80 °C. For cell studies, Penetratin HCl, at 10 ll/min. [45] and Penetratin-coupled peptides were obtained from the Can- cer Research UK Research Services (London, UK), dissolved in water 2.4. Cell culture and 1 mM stocks stored at À80 °C. The sequence of the Penetratin- coupled peptides are shown below (Penetratin sequences are ZR-75-1 and MCF-7 human breast cancer cell lines were ob- underlined). tained from ATCC (American Type Culture Collection, USA) and H2-Pen CKLDRRVKATIERQIKIWFQNRRMKWKK maintained in Dulbecco’s modified Eagle’s media (Life Technolo- H2mut-Pen CKLDAAVKATIERQIKIWFQNRRMKWKK gies Inc., UK) supplemented with 10% (v/v) fetal calf serum (PAA H3-Pen LKRKRGLVKKVQAFLAECDTVERQIKIWFQNRRMKWKK Laboratories, UK) and 1% (v/v) penicillin/streptomycin/glutamine. Con-Pen RQIKIWFQNRRMKWKKC-CVARTEPLQT To measure growth inhibition, cells were plated at a density of Penetratin RQIKIWFQNRRMKWKK 5000 cells per well of a 96 well plate. The following day, cells were treated with various concentrations of BAG-1 or control peptides. BAG-1 peptides were synthesised with Penetratin at their C-ter- After 6 days, media was removed from each well and replaced with minus coupled via a peptide bond. The control peptide was an 200 ll RPMI-1640 media (Life Technologies Inc., UK) supple- unrelated BCL-2 derived sequence coupled to Penetratin by a disul- mented with 10% (v/v) fetal calf serum and 1% (v/v) penicillin/ Ò phide bond at its N-terminus.