The NMR structure of human obestatin in membrane-like environments: insights into the structure-bioactivity relationship of obestatin

The NMR Structure of Human Obestatin in MembraneLike Environments: Insights into the Structure-Bioactivity Relationship of Obestatin Begon ˜a O. Ale ´n 1,2,3. , Lidia Nieto 5.¤a , Uxı ´a Gurriara ´n-Rodrı ´guez 1,2,3 , Carlos S. Mosteiro 1,2 , Juan C. A ´lvarezPe ´rez 1,2,3¤b ,Marı ´a Otero-Ale ´n 4¤c , Jesu ´s P. Camin ˜a 1,2 , Rosalı ´a Gallego 3,4 , Toma ´s Garcı ´a-Caballero 3,4 , Manuel Martı ´n-Pastor 4,6 , Felipe F. Casanueva 1,2,3 , Jesu ´s Jime ´nez-Barbero 5 , Yolanda Pazos 1,2 * 1A ´rea de Endocrinologı ´a Molecular y Celular, Instituto de Investigacio ´n Sanitaria (IDIS), Complejo Hospitalario Universitario de Santiago de Compostela (CHUS), SERGAS, Santiago de Compostela, Spain, 2CIBER Fisiopatologı ´a de la Obesidad y Nutricio ´n (CB06/03), Instituto de Salud Carlos III, Santiago de Compostela, Spain, 3Universidad de Santiago de Compostela, Santiago de Compostela, Spain, 4Instituto de Investigacio ´n Sanitaria (IDIS), Complejo Hospitalario Universitario de Santiago de Compostela (CHUS), Santiago de Compostela, Spain, 5Centro de Investigaciones Biolo ´gicas, CIB-CSIC, Madrid, Spain, 6Unidad de Resonancia Magne ´tica, RIAIDT, Universidad de Santiago de Compostela, Campus Sur, Santiago de Compostela, Spain Abstract The quest for therapeutic applications of obestatin involves, as a first step, the determination of its 3D solution structure and the relationship between this structure and the biological activity of obestatin. On this basis, we have employed a combination of circular dichroism (CD), nuclear magnetic resonance (NMR) spectroscopy, and modeling techniques to determine the solution structure of human obestatin (1). Other analogues, including human non-amidated obestatin (2) and the fragment peptides (6–23)-obestatin (3), (11–23)-obestatin (4), and (16–23)-obestatin (5) have also been scrutinized. These studies have been performed in a micellar environment to mimic the cell membrane (sodium dodecyl sulfate, SDS). Furthermore, structural-activity relationship studies have been performed by assessing the in vitro proliferative capabilities of these peptides in the human retinal pigmented epithelial cell line ARPE-19 (ERK1/2 and Akt phosphorylation, Ki67 expression, and cellular proliferation). Our findings emphasize the importance of both the primary structure (composition and size) and particular segments of the obestatin molecule that posses significant a-helical characteristics. Additionally, details of a species-specific role for obestatin have also been hypothesized by comparing human and mouse obestatins (1 and 6, respectively) at both the structural and bioactivity levels. Citation: Ale ´n BO, Nieto L, Gurriara ´n-Rodrı ´guez U, Mosteiro CS, A ´lvarez-Pe ´rez JC, et al. (2012) The NMR Structure of Human Obestatin in Membrane-Like Environments: Insights into the Structure-Bioactivity Relationship of Obestatin. PLoS ONE 7(10): e45434. doi:10.1371/journal.pone.0045434 Editor: Giorgio Colombo, Consiglio Nazionale delle Ricerche, Italy Received June 27, 2012; Accepted August 17, 2012; Published October 4, 2012 Copyright: ß2012 Ale ´n et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by grants from Ministerio de Economı ´a y Competitividad (SAF2010-20451, CTQ2009-08536), Xunta de Galicia (INCITE09PXIB 918374PR) and Instituto de Salud Carlos III (Ministerio de Economı ´a y Competitividad, Spain; PS09/02202, PS09/02075). CIBER is an initiative of Instituto de Salud Carlos III (Ministerio de Ciencia e Innovacio ´n, Spain). Instituto de Salud Carlos III and SERGAS fund the work of J.P. Camina, and Y. Pazos through a research-staff stabilization contract. Xunta de Galicia funds U. Gurriara ´n-Rodriguez through a research-staff contract Isabel Barreto. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: yolanda.pazos.randul[email protected] ¤a Current address: Laboratory of Chemical Biology, Department of Biomedical Engineering, Eindhoven University of Technology, Eindhoven, The Netherlands ¤b Current address: Division of Endocrinology and Metabolism, Department of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America ¤c Current address: Nodo Central Biobanco de Tumores de Andalucı ´a, Fundacio ´n Progreso y Salud, Junta de Andalucı ´a, Granada, Spain .These authors contributed equally to this work. Introduction Obestatin, a 23-amino acid peptide derived from the ghrelin peptide precursor (preproghrelin), was identified in 2005 as a physiological opponent of ghrelin [1]. Obestatin was originally isolated from the stomach and has subsequently been shown to be a circulating peptide whose secretion is pulsatile. Obestatin displays an ultradian rhythmicity similar to that of ghrelin and GH secretion [2]. Moreover, obestatin was shown to bind selectively to the orphan receptor GPR39, which belongs to the same family as the ghrelin receptor GHS-R1a and the motilin receptor [2], [3]. Although obestatin has been known as a controversial ghrelinassociated peptide due to the lack of reproducible biological actions on feeding, additional activities for this molecule have been reported [4]. In particular, the proliferative abilities of obestatin were first demonstrated in human retinal pigmented epithelial cells [5], and the intracellular mechanisms responsible of this action were later elucidated in human gastric cancer cell lines [6], [7], thus defining the functionality to this biologically active peptide. The ERK1/2 molecules control a large number of different or even opposed cellular processes such as proliferation, survival, development, stress response, and apoptosis [8]. Akt is a serine/ threonine kinase that acts as central player in the regulation of several cell functions: protein synthesis, cell survival (one of the PLOS ONE | www.plosone.org 1 October 2012 | Volume 7 | Issue 10 | e45434 main factors in many cancer types) and metabolism (glucose uptake, lipid homeostasis and protein synthesis) [9]. Deregulation of these signals usually provokes diseases, such as cancer or diabetes. Once obestatin activates GPR39, two routes are triggered in parallel: i) sequential activation of Gi, PI3K, novel PKCeand Src and the subsequent ERK1/2 activation; and ii) a barrestin 1-mediated signaling pathway that involves the recruitment of Src to the b-arrestin 1 scaffolding complex, thus causing Akt phosphorylation. In this way, Src acts as a switch that activates matrix metalloproteinases (MMPs) to initiate the proteolytic release of the EGF-like ligands onto the cell surface, which later bind to EGFR. The binding of the ligands leads to receptor dimerization, which activates the intrinsic kinase and the specific binding sites of phosphorylation, including PI3K. This kinase activation allows for Akt phosphorylation in the A-loop (T308) and the HM (S473) by PDK1 and mTORC2, respectively. The activated Akt then inactivates the TSC1/TSC2 heterodimer to activate mTORC1 and phosphorylate downstream targets, including p70S6K1 [7]. The fact that obestatin modulates cell proliferation, especially that of gastric cancer cells, suggests the involvement of this peptide in diverse processes, such as the repair of gastric mucosal damage or as a fuel for gastric cancer cell proliferation. Subsequently, obestatin displayed a novel role as an autocrine/paracrine regulator of adipogenesis and adipocyte metabolism [10], [11]. All of the effects observed for obestatin indicate that this molecule is a biologically relevant peptide and not only a non-functional connective peptide. Regarding the structure of obestatin, this peptide contains 23 amino acids with a post-translational amide modification on the C-terminus. It is believed that this modification is essential for the bioactivity of obestatin [1]. Recently, the relationship between the primary structure and the bioactivity of mouse obestatin has been studied, and it was concluded that the N-terminal 13 residues of obestatin exhibit structural behavior most similar to that of the full peptide [12]. Additionally, CD and NMR studies have been performed to determine the possible secondary structure of mouse obestatin (6) and its (11–23)-obestatin truncated isoform in the presence of DPC/SDS micelles. The results showed that both peptides assume regular secondary structures at the C-terminus and that carboxy-amidation is a prerequisite for the biological activity because it is necessary to induce and to stabilize the regular conformations [13]. More recently, Subasinghage et al. have performed a structure-bioactivity study using human obestatin. These authors described the effects of human obestatin and its (11–23)-obestatin isoform in rats [14]. In addition to these studies, the availability of new data regarding the signaling mechanisms of obestatin in human cell lines [5–7] has prompted us to develop further studies on this topic. In this context, we have used CD, NMR spectroscopy, and modeling techniques to determine the solution structure of human obestatin (1), human non-amidated obestatin (2) and the fragment peptides (6–23)-obestatin (3), (11– 23)-obestatin (4), and (16–23)-obestatin (5) in SDS micelles (Figure 1). Furthermore, comparisons have been made between the structural characteristics and the biological activities observed for these peptides, as determined by their capabilities to stimulate Akt and ERK1/2, the expression of the proliferation marker Ki67 and the cellular proliferative capabilities [15] in the human retinal pigmented epithelial cell line ARPE-19. Additionally, the details of the species-specific role of obestatin have also been deduced by comparing human and mouse obestatin (1and 6, respectively). Materials and Methods Materials Human obestatin (1) and mouse obestatin (6) were obtained from California Peptide Research Inc. (Napa, CA, US). Human non-amidated obestatin and human (16–23)-obestatin were purchased from Bachem Ltd. (St. Helens, UK). Human (6–23)- obestatin was obtained from Biomedal (Sevilla, ES). Human (11– 23)-obestatin was obtained from Peptides International (Louisville, KY, US). The rabbit polyclonal IgG antibodies against phospho-p44/42 mitogen-activated protein kinase (MAPK), p44/42 MAPK, phospho-Akt HM(S473), and Akt HM(S473) were purchased from Cell Signaling Technology (Beverly, MA, US). The rabbit polyclonal IgG antibodies against GPR39 and actin were obtained from Abcam (Cambridge, UK). The anti-rabbit IgG horseradish peroxidase was purchased from Jackson ImmunoResearch Europe Ltd. (Suffolk, UK). Cell culture The retinal pigmented epithelium cell line ARPE-19 was cultured as described by the supplier (ATCC, Manassas, VA, US). Briefly, ARPE-19 cells were seeded in 100-mm dishes and cultured in DMEM:F12 medium supplemented with 10% (v/v) fetal bovine serum (FBS), 100 U/mL penicillin G, 100 mg/mL streptomycin sulfate and 2.5 mM L-glutamine with 5% CO 2 at 37uC. Immunoblotting analysis Serum-starved cells were stimulated with the peptides for the indicated time period and doses at 37uC. The medium was then aspirated, and the cells were lysed in ice-cold lysis buffer [RIPA buffer: 50 mM Tris-HCl (pH 7.2), 150 mM NaCl, 1 mM EDTA, 1% (v/v) NP-40, 0.25% (w/v) Na-deoxycholate, protease inhibitor cocktail (1:100, Sigma Chemical Co., St. Louis, MO, US), phosphatase inhibitor cocktail (1:100, Sigma Chemical Co., St. Louis, MO, US)]. The soluble cell lysates were pre-cleared by centrifuging at 13,0006g for 15 min. The protein concentration was evaluated using the QuantiProTM BCA Assay kit (Sigma Figure 1. Primary structure of the obestatins used in this work: human obestatin (1), human non-amidated obestatin (2), human (6– 23)-obestatin (3), human (11–23)-obestatin (4), human (16–23)-obestatin (5) and mouse obestatin (6). The red-labeled residues in 6are different from those of 1. doi:10.1371/journal.pone.0045434.g001 Structure-Bioactivity Analysis of Human Obestatin PLOS ONE | www.plosone.org 2 October 2012 | Volume 7 | Issue 10 | e45434 Chemical Co., St. Louis, MO, US). The same amount of protein for each sample was separated on 10% sodium dodecyl sulfate (SDS)/polyacrylamide gels and transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA, US). The blots were incubated with 5% non-fat milk in a Tris buffer solution containing Tween-20 (TBST) [20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.1% (v/v) Tween-20, used for all incubation and washing steps] for 1 h. The blots were then incubated for 1 h with the corresponding antibodies, according to the manufacturer’s instructions, and were subsequently incubated with the peroxidase-conjugated IgG antibody. After washing, the signals were visualized using an ECL plus Western Blotting Detection System (GE-Amersham, Buckinghamshire, UK). The blots shown are representative of three experiments. The image processing was performed using the NIH Image Software ImageJ 1.38x. Small Interfering RNA (siRNA) silencing of gene expression The following double-stranded siRNA duplexes of GPR39 were used (Thermo Fisher Scientific, Dharmacon, Lafayette, CO, US; ON-TARGETplus SMART pool L-005569-00-0005, Human GPR39, NM_001508): 39-UCCAAUAUGUCCAUCUGUA-59, 39-GCGCGAAACCAGCCAAUUC-59,39-GAGGCUGAUUGUUGUGACA-59, and 39-AACCAGAUUCGGAGGAUCA-59. A non-silencing RNA duplex was used as a control for all siRNA experiments. The ARPE cells were transfected using Lipofectamine 2000 (Invitrogen; CA, US). Immunocytochemistry detection of Ki67 The ARPE-19 cells were cultured at a density of 4610 3 cells per well in the culture medium described above on 8-well Lab-Tek II chamber slides covered with CC2 glass slide coverslips. After 2 days, the medium was renewed, and the cells were cultured in a serum-free medium (300 mL) for 24 h. The cells were then treated with FBS (10% v/v), human obestatin (1; 100 and 200 nM), human non-amidated obestatin (2; 100 and 200 nM) and the fragment peptides (6–23)-obestatin (3; 100 and 200 nM), (11–23)-obestatin (4; 100 and 200 nM), (16–23)-obestatin (5; 100 and 200 nM) and mouse obestatin (6; 100 and 200 nM) in fresh DMEM:F12. After 24 h, the intact cells were fixed in 96% ethanol for 1 h. The immunocytochemical technique was automatically performed using an AutostainerLink 48 instrument (Dako, Glostrup, DK). The FLEX primary antibody to Ki-67 (clone MIB 1; Dako) was used. En Vision FLEX/HRP (Dako) was employed as a detection system. Briefly, the procedure comprised the following steps: 1) epitope retrieval in 10 mM citrate buffer (pH 6.0) using a microwave (750 W, 10 min); 2) incubation with peroxidase-blocking agent (5 min); 3) incubation with primary antibody (30 min); 4) incubation with labeled polymer-horseradish peroxidase (HRP, dextran polymer conjugated with HRP and affinity-isolated goat anti-mouse immunoglobulins; 30 min); 5) incubation with a diaminobenzidine (DAB) chromogen substrate solution (10 min); and 6) counterstaining with Harris hematoxylin (9 min). In all cases, triplicate dishes were used for each experimental point. Cell proliferation assay The cell proliferation was measured using a BrdU cell proliferation enzyme-linked immunosorbent assay (ELISA) kit (Roche Diagnostics, Mannheim, DE). The BrdU assay was performed according to the manufacturer’s protocol. ARPE-19 cells were cultured in a 96-well multiplate at a density of 2610 3 cells per well in the culture medium described above for 24 h. The Figure 2. Secondary structure analyses performed using the DICHROWEB web server with the CONTINLL algorithm and reference data set 4. The relative amounts of a-helix and b-sheet were determined by adding together the contributions from helix 1 plus helix 2 and strand 1 plus strand 2, respectively, whereas the amounts of b-turn and random structure were read directly from the output. The peptides clearly showed greater helicity in SDS than in PBS. doi:10.1371/journal.pone.0045434.g002 Structure-Bioactivity Analysis of Human Obestatin PLOS ONE | www.plosone.org 3 October 2012 | Volume 7 | Issue 10 | e45434 procedure comprised the following steps: 1) 0% FBS for 24 h; 2) stimulation with FBS (10% v/v), human obestatin (1, 100 nM), human non-amidated obestatin (2, 100 nM) and the fragment peptides (6–23)-obestatin (3, 100 nM), (11–23)-obestatin (4, 100 nM), (16–23)-obestatin (5, 100 nM) and mouse obestatin (6, 100 nM) in serum-deprived medium for 48 h; 3) incubation with BrdU-labeling solution (10 mL, 3 h, 37uC); 4) removal of the labeling solution and fixing with FixDenat solution (200 mL, 30 min, 25uC); 5) incubation with an anti-BrdU-peroxidase (POD) antibody solution (100 mL, 90 min, 25uC); and 6) washing followed by the addition of the substrate solution (100 mL, 30 min). The BrdU incorporation was quantified using the spectrophotometric absorbance (370 nm) measured with a Reader VersaMaxPLUS. The mean absorbance of the control cells represented 100% cell proliferation, and the mean absorbance of the treated cells was related to the control values to determine the sensitivity. In all cases, each experimental point was replicated eight times. Immunocytochemistry detection of GPR39 The ARPE-19 cell samples were processed using standard procedures [10]. The slides were consecutively incubated with 1) anti-GPR39 rabbit polyclonal antibody (1:500) in Dako ChemMate antibody diluent (Dako, Glostrup, DK); 2) EnVision TM peroxidase rabbit (Dako, Carpinteria, CA, US) as the detection system; and 3) 3,39-diaminobenzidine-tetrahydrochloride (Dako Figure 3. Plot of the chemical shifts indices (CSIs) for residues at the C-termini of the studied peptides. (A) CSIs of the backbone amide protons (H N ) and (B) CSIs of the alpha protons (H alpha ). The peptide sequence (6) is aligned with that of the other peptides. The CSI is defined as d obs – d random-coil . doi:10.1371/journal.pone.0045434.g003 Figure 4. Secondary helical structure based on the H N and H alpha chemical shifts indices determined using the RCI server. The underlined residues were predicted to have secondary helical structure based on the H N and H alpha chemical shifts indices that were determined using the RCI server (http://wishart.biology.ualberta.ca/rci). The residues within helical structures that extended over more than two residues are represented in bold; these residues are located primarily at the C-terminus. doi:10.1371/journal.pone.0045434.g004 Structure-Bioactivity Analysis of Human Obestatin PLOS ONE | www.plosone.org 4 October 2012 | Volume 7 | Issue 10 | e45434 Liquid DAB +Substrate-chromogen system). The cell and tissue sections were faintly counterstained with Harris hematoxylin. NMR spectroscopy Samples for the NMR experiments were prepared by dissolving peptides in 500 mL of a d25-SDS aqueous solution (9:1 H 2 O:D 2 O, PBS buffer at pH 6.5) to make a final concentration of approximately 0.8–1.0 mM and a peptide/SDS ratio of approximately 1/70. The NMR spectra were recorded at 298 K using Bruker 600 and 700 MHz spectrometers equipped with a tripleresonance Z gradient probe and processed using XWIN-NMR software (Bruker Inc.; Billerica, MA, US). The resonance of 2,2,3,3-tetradeutero-3-trimethylsilylpropionic acid (TSP) was used as a chemical shift reference in the 1 H NMR experiments (d TSP = 0 ppm). The one-dimensional (1D) 1 H homonuclear spectra were recorded in the Fourier mode using quadrature detection. The two-dimensional (2D) 1 H homonuclear TOCSY (total correlation spectroscopy) and NOESY (nuclear Overhauser effect spectroscopy) spectra were collected in the phase-sensitive mode using time-proportional phase increments in t1. For each of these experiments, 512 t1 increments were used. The free induction decay in t2 consisted of 2048 data points over a spectral width of 6009.615 Hz. In general, 409661024 data points were collected for each block, and 96 transients were collected for the 2D experiments. The TOCSY spectra were recorded using the MLEV-17 pulse sequence with mixing times (spin-lock) of 60– 80 ms. The NOESY experiments were acquired with mixing times of 250–400 ms. The experimental data were acquired and processed using the TopSpin TM (Bruker Inc; Billerica, MA, US) program on a PC station. The data matrices were multiplied by a qsine function in both dimensions and then zero-filled to 1024 data points in F1 prior to the Fourier transformation. The peptide resonance assignments were obtained using standard strategies based on the 2D NMR experiments. Figure 5. Summary of sequential and medium-range NOE connectivities involving the NH, Haand Hbprotons of the peptides in SDS micelles, as derived from CYANA calculation. The thickness of the bar indicates the intensities of the NOEs for the following peptides: (A) human obestatin (1), (B) human non-amidated obestatin (2), (C) human (6–23)-obestatin (3), (D) human (11–23)-obestatin (4), (E) human (16–23)- obestatin (5) and (F) mouse obestatin (6). The asterisk (*) represents the C-terminal amidation of the molecule. The dagger (#) represents the differences between human obestatin (1) and mouse obestatin (6). doi:10.1371/journal.pone.0045434.g005 Structure-Bioactivity Analysis of Human Obestatin PLOS ONE | www.plosone.org 5 October 2012 | Volume 7 | Issue 10 | e45434 Structure calculation The peak lists for the NOESY spectra recorded with a 0.25 s mixing time were generated by interactive peak picking using the CARA software [16]. The NOESY cross-peak volumes were determined using the automated peak integration routine implemented in CARA. Conversion of NOE peak intensities to distance restraints was done using automatic calibration as implemented in CYANA 2.1 [17]. The three-dimensional (3D) structures of human obestatin (1) and its derivatives were determined using the standard protocol of combined automated NOE (nuclear Overhauser effect) assignment and the structure calculation implemented in the CYANA 2.1 program. Seven cycles of combined automated NOESY assignment and structure calculations were followed by a final structure calculation. The structure calculation started in each cycle from 100 randomized conformers, and the standard simulated annealing schedule was used. The 20 conformers with the lowest final CYANA target function values were retained for analysis and passed to the next cycle. Weak restraints on the phi/psi torsion-angle pairs and on the side-chain torsion angles between tetrahedral carbon atoms were applied temporarily during the high-temperature and cooling phases of the simulated annealing schedule to favor the permitted regions of the Ramachandran plot and the staggered rotamer positions, respectively. The list of upper-distance bonds for the final structural calculation consists of unambiguously assigned upper-distance bonds and does not require the possible swapping of diastereotopic pairs. The 20 conformers with the lowest final CYANA target function values were subjected to restrained energy-minimization in a water shell using the AMBER 8.0 program [18]. The resulting 20 energy-minimized CYANA conformers represent the solution structures of human obestatin (1) and its derivatives. The MOLMOL program was used to visualize the 3D structures [19]. CYANA was used to obtain statistics on the target function values, restraint violations and Ramachandran plots according to the PROCHECK-NMR conventions [20]. The RMSD (root mean square deviation) values were calculated using CYANA for the superpositions of the backbone N, Caand C’ atoms and the heavy atoms throughout the protein. To obtain the RMSD value of a structure represented by a bundle of conformers, all of the conformers were superimposed on the average structure, and the average of the RMSD values between the individual conformers and their average coordinates was calculated. CD spectroscopy CD experiments of the peptides were performed using a 720Jasco spectropolarimeter (Tokyo, JP) and a 1-mm-path-length quartz cuvette. The CD spectra of the peptides in the SDS micellar solution were recorded using a H 2 O solution containing 40 mM peptide and SDS micelle at a concentration of 2.8 mM (25uC). In all cases, 25 mM NaH 2 PO 4 /Na 2 HPO 4 buffer was used to maintain the pH at 6.5. The CD spectra presented are the Table 1. Structural statistics for the ensemble of the best 20 structures of human obestatin (1), its fragments and mouse obestatin (6). Human obestatin (1) Human non amidatedobestatin (2) Human (6–23)-obestatin (3) Human (11–23)-obestatin (4) Human (16–23)-obestatin (5) Mouse obestatin (6) Experimental restraints [a] Sequential distances 267 145 145 184 123 284 Medium-range distances (i–j) ,5 99 28 31 63 38 68 Long-range distances (i–j) $5 200 0 2 7 Total 368 173 176 247 163 359 Final CYANA target function value (A ˚ 2 ) [b] 0.18 0.22 0.02 0.11 0.27 0.04 RMS deviations from ideal geometry [c] Bond lengths (A ˚) 0.005 0.005 0.004 0.004 0.004 0.005 Bond angles (u) 0.7 0.7 0.6 0.5 0.5 0.6 RMSD to mean coordinates (A ˚) [d] (14–20) (14–20) (14–20) (14–20) (16–23) (14–20) Backbone N, C a , C’ 0.4260.21 1.4960.56 0.5560.23 0.0960.04 0.4960.24 0.4660.39 All heavy atoms 1.1960.38 2.6960.78 1.2460.27 0.4960.22 1.5060.40 0.9360.52 Ramachandran plot statistics [c] Most favorable regions (%) 71.4 72.3 69.6 70.5 60.0 77.4 Additional allowed regions (%) 26.4 27.2 30.4 29.5 38.7 22.6 Generously allowed regions (%) 0.0 0.1 0.0 0.0 1.3 0.0 Disallowed regions (%) 2.2 0.3 0.0 0.0 0.0 0.0 [a] The final CYANA target function value was computed for the structures calculated using CYANA. [b] Average values of the 20 final energy-minimized CYANA conformers. [c] Calculated using PROCHECK-NMR. [d] Atomic differences are given as the average RMS difference of the mean coordinate structure (mean). doi:10.1371/journal.pone.0045434.t001 Structure-Bioactivity Analysis of Human Obestatin PLOS ONE | www.plosone.org 6 October 2012 | Volume 7 | Issue 10 | e45434 average of 5 accumulations from 190 to 250 nm, which were recorded with a bandwidth of 1 nm and a scanning speed of 20 nm/min. During all of the measurements, the trace of the hightension voltage remained less than 700 V, which should ensure the reliability of the obtained data [21]. Baselines of either solvent or micellar solutions without peptide were subtracted from each respective sample to yield the contribution of the sample. The secondary structure composition was estimated using the DICHROWEB web server [22], [23] with the following algorithms: CONTINLL, involving 2 different reference data sets, and K2d [23]. Data analysis All of the data are reported as the mean 6SE. A statistical ANOVA analysis was performed using an analysis of variance with the Bonferroni post hoc test. Values of P,0.05 were considered to be statistically significant and are marked with an asterisk (*). Figure 6. Superimposition of the 20 best representative structures of peptides 1 to 6, as calculated from the NMR data for the peptides in SDS micelles. (A) Human obestatin (1), (B) human non-amidated obestatin (2), (C) human (6–23)-obestatin (3), (D) human (11–23)- obestatin (4), (E) human (16–23)-obestatin (5) and (F) mouse obestatin (6). The Tyr16 side chain is shown in blue. doi:10.1371/journal.pone.0045434.g006 Figure 7. Comparison between the secondary helical structure based on the H N and H alpha chemical shifts indices determined using the RCI server and the secondary helical structure obtained in our structures. The underlined residues were predicted to have secondary helical structure based on the H N and H alpha chemical shifts indices obtained using the RCI server (http://wishart.biology.ualberta.ca/rci). The residues within helical structures that extended over more than two residues are represented in bold; these residues are located primarily at the C-terminus. The secondary helical structure obtained in our structures, as calculated by CYANA, included a-helix formation (green labels) and 3 10 -helix formation (blue labels). doi:10.1371/journal.pone.0045434.g007 Structure-Bioactivity Analysis of Human Obestatin PLOS ONE | www.plosone.org 7 October 2012 | Volume 7 | Issue 10 | e45434 Results and Discussion 1. Circular dichroism Circular dichroism is a suitable and rapid approach to provide information about the secondary structural features of peptides such as obestatin in solution [24]. First, the influence of SDS micelles on the secondary structure of the different peptides was studied. A preliminary scanning of the conformational preferences of the peptides was performed in 25 mM PBS and in an SDS micelle solution. SDS was adopted based on the works of Schwyzer, who hypothesized that previous contact with the membrane is essential for the peptide to adopt the proper conformation to further interact with its receptor [25]. As shown in Figure S1, the CD spectra of the peptides (40 mM) suggested that random coil conformations were prevalent in PBS, whereas the spectra recorded in the micelle solution indicated the presence of a certain amount of a-helical structure (Figure S2). Both human (1) and mouse obestatin (6) exhibited similar tendencies (Figure S3). The secondary structure analyses were performed using the DICHROWEB web server [22], [23] with the following algorithms: CONTINLL, which incorporated 2 different reference data sets, and K2d [23]. The conformational weights of the secondary structures obtained with CONTINLL were paired with 2 different data sets for the data ranging between 190 and 240 nm. Although an important caveat of this method is that the reference data sets are primarily appropriated for aqueous environments, it is nonetheless evident that the peptides in SDS have the greatest helicity (Table S1). An example of the obtained results is represented in Figure 2. 2. Structure determination using nuclear magnetic resonance (NMR) The NMR spectra of the peptides were collected in the presence of the SDS micelles. Peptides 1–6 provided well-dispersed 2D spectra. A small number of cross-peaks were observed for peptides 2and 3. Figure S4 and Figure S5 exemplify the quality of the spectra, of which the amide region of the 2D NOESY spectra of human obestatin (1) and its truncated analogue (4) are shown. The assignment process was straightforward because most of the proton resonances of these peptides at 298 K and pH 6.5 were well resolved and narrow. The chemical shift assignment and collection of the NOE data were performed by analyzing the 2D TOCSY and NOESY spectra using the CARA software and following standard procedures [16]. The proton chemical shifts of the peptides in SDS micelles are summarized in the Supporting Information: human obestatin (1), Table S2; human non-amidated obestatin (2), Table S3; human (6–23)-obestatin (3), Table S4; human (11–23)-obestatin (4), Table S5; human (16– 23)-obestatin (5), Table S6; and mouse obestatin (6), Table S7. The experimental NMR data were used to generate 3D models of peptides 1–6. The three-dimensional structures were calculated using the CYANA software based on the inter-proton distance restraints (sequential and medium-range NOE-derived restraints). The best 20 structures, out of 50 calculated, were chosen according to the lowest values of the penalty (f) for the target function. A subsequent energy refinement was performed in explicit solvent using the AMBER program. The corresponding results, including the further analysis of the resulting structures performed using MOLMOL and PROCHECK-NMR, are shown below. 2. 1. Backbone HN amide and H-alpha 1 H chemical shifts. First, the variation of the backbone amide H N and H alpha chemical shift values was monitored for significant changes that might have structural relevance. These chemical shifts in peptides 1–6 were, on average, shifted upfield in relation to the typical values observed for random coils, as might be expected for helical structures (Figure 3). Furthermore, the chemical shift analysis performed using the RCI server [26] predicted helical conformations for the residues located mainly at the C-termini of the peptides (Figure 4). Figure 3 shows the chemical shift indices (CSI) for the H N and H alpha protons of the residues at the C-termini of some of these peptides. The CSIs for the H N and H alpha protons of human obestatin (1) between residues Tyr16 and Leu23 are generally (with a few exceptions) negative and much stronger than those of the non-amidated obestatin (2), indicating that the latter has a smaller percentage of helical structure at the C-terminus. The CSIs for the H N and H alpha protons of human (6–23)-obestatin (3) generally indicate a higher percentage of helical structure relative to (1), suggesting that the lack of the first 5 amino acids does not have an important conformational impact on the structure. Figure 8. Analysis of the expression and functionality of GPR39 in ARPE-19 cells. (A) Immunocytochemical detection of GPR39 in ARPE-19 cells (objective magnification of 20x). (B) The effect of siRNA depletion of GPR39 on pAkt(S473) and pERK1/2(T202/Y204) in ARPE-19 cells after human obestatin treatment (1, 100 nM, 10 min). The ARPE-19 cells were transfected with GPR39 siRNA prior to obestatin 1treatment. Equal amounts of protein in each sample were used to assess the expression of GPR39 by western blotting. The GPR39 level was expressed as the fold change relative to the control siRNA-transfected cells (mean 6SE). The protein expression was normalized relative to actin. The data are expressed as the mean 6SE. The asterisk (*) denotes P,0.05 when comparing the treated control siRNA group with the control siRNA group; the dagger (#) denotes P,0.05 when comparing the GPR39 siRNA group with the control siRNA group. doi:10.1371/journal.pone.0045434.g008 Structure-Bioactivity Analysis of Human Obestatin PLOS ONE | www.plosone.org 8 October 2012 | Volume 7 | Issue 10 | e45434 The truncated peptide 4, which lacks the first 10 residues, has the strongest negative CSIs for both H N and H alpha protons, thus providing further support for the formation of an a-helix at the Cterminus of this peptide. In the case of mouse obestatin (6), the negative CSIs for H N and H alpha are indicative of an a-helix, although the comparison with the human obestatin (1) shows considerable variations along the sequence without a clear trend. 2. 2. NOE pattern. Figure 5 contains a summary of all (short, medium and long range) connectivities deduced from an analysis of the NOESY spectra of the peptides. It has been previously reported that poorly ordered structures of obestatin were observed in water solutions. These peptides generally exhibited bend structures in the central Lys10-Ala14 fragment [13], [14]. For peptides 1–6 in buffered SDS micelles, the backbone and sidechain proton NMR resonances could be completely assigned in a sequential manner with the aid of the TOCSY and NOESY spectra. A significant population of conformations containing an ‘‘ordered’’ a-helix is present, with characteristic short-range dNN(i, i+1) and medium-range daN(i, i+3) and dab (i, i+3) NOE connectivities [16]. Fairly strong dNN(i, i+1) and daN(i, i+1) NOEs were observed for most of the residues, although some gaps resulted from resonance overlap. The data that were obtained clearly suggest the presence of a significant population of ordered a-helical structures for these peptides in SDS micelle solutions. In fact, for human obestatin (1), clear mediumand long-range connectivities were observed including 1) medium-range Hai/HNi+3 (Gly8/Leu11, Lys10/ Gly13, Leu11/Val14) and Hai/Hbi+3 (Val7/Lys10, Gly8/Leu11, Gly13/Tyr16, Ser20/Leu23) connectivities and 2) a long-range Hai/HNi+4 (Lys10/Val14) connectivity. For the non-amidated peptide 2, fewer medium-range connectivities were observed relative to the amidated peptide 1, including 1) medium-range Hai/HNi+3 (Leu11/Val14, Gly13/Tyr16) connectivities but no Hai/Hbi+3 connectivities and 2) a long-range Hai/HNi+4 (Lys10/Val14) connectivity. For the truncated human (6–23)- obestatin (3), fewer medium-range connectivities were observed: 1) medium-range Hai/HNi+3 (Gln17/Ser20, His19/Ala22) connectivities but no Hai/Hbi+3 connectivities were observed, and 2) no long-range Hai/HNi+4 connectivities were observed. The presence of medium-range connectivities (Hai/HNi+3, Hai/Hbi+3) and long-range connectivities (Hai/HNi+4) from Gly13 to Leu23 confirmed the presence of a fairly well defined a-helical structure at the C-terminus of human (11–23)-obestatin (4): 1) mediumrange Hai/HNi+3 (Gly13/Tyr16, Gln17/Ser20, Gln18/Gln21, Ser20/Leu23) and Hai/Hbi+3 (Gly13/Tyr16, Gln15/Gln18, Gln17/Ser20, Ser20/Leu23) connectivities were observed, and 2) long-range Hai/HNi+4 (Gly13/Gln17, Glu17/Gln21) connectivities were observed. The observed structure of the human (16– 23)-obestatin (5) is not as well defined as that of 4. Only residues His19/Leu23 are involved in a-helix formation: 1) medium-range Hai/HNi+3 (His19/Ala22, Ser20/Leu23) connectivities and no Hai/Hbi+3 connectivities were observed, and 2) a long-range Hai/HNi+4 (His19/Leu23) connectivity was also observed. A fairly similar pattern of NOEs, especially at the C-terminal region Figure 9. Time-course of the effect of the different peptides on ERK1/2 [pERK 1/2 (T202/Y204)] and Akt [pAkt HM (S473)] phosphorylation in ARPE-19 cells. The serum-starved cells were treated with the peptides (200 nM) at 37uC for the indicated times. The cells were lysed and analyzed using SDS-PAGE with specific antibodies. ERK1/2 and Akt phosphorylation were quantified using densitometry and expressed as the fold change relative to the phosphorylation obtained for unstimulated cells (mean 6SE of five independent experiments). doi:10.1371/journal.pone.0045434.g009 Structure-Bioactivity Analysis of Human Obestatin PLOS ONE | www.plosone.org 9 October 2012 | Volume 7 | Issue 10 | e45434