Jörg Andrä

Igor Jakovkin

Joachim Grötzinger

Oliver Hecht

Anna D Krasnosdembskaya

Torsten Goldmann

Thomas Gutsmann

Matthias Leippe

Structure and mode of action of the antimicrobial peptide arenicin

Keywords: AMP, antimicrobial peptide, CFU, colony forming unit, FRET, Förster resonance energy transfer, LPS, lipopolysaccharide, MIC, minimal inhibitory concentration, MBC, minimal bactericidal concentration, PC, phosphatidylcholine, DPhyPC, Diphytanoyl-PC, PE, phosphatidylethanolamine, PG, phosphatidylglycerol antimicrobial peptide, atomic force microscopy, epithelial defence, lipopolysaccharide, membrane permeabilisation, planar lipid bilayer

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Two isoforms of a new class of antibacterial cationic peptides, termed arenicins, have been recently isolated from the coelomocytes of the marine polychaeta lugworm, Arenicola marina [START_REF] Ovchinnikova | Purification and primary structure of two isoforms of arenicin, a novel antimicrobial peptide from marine polychaeta Arenicola marina[END_REF]. The mature peptides consist of 21 amino-acid residues comprising a disulfide bond, and a unique 18 amino-acid residue loop structure. They are rich in arginine and hydrophobic amino-acid residues (Tyr and Trp) and differ only by a single, conservative amino acid substitution (V10I). Both isoforms exhibit indentical potent antimicrobial activity against Escherichia coli, Listeria monocytogenes, and Candida albicans [START_REF] Ovchinnikova | Purification and primary structure of two isoforms of arenicin, a novel antimicrobial peptide from marine polychaeta Arenicola marina[END_REF]. Due to their molecular size, net charge and origin, arenicins are classified as natural antimicrobial peptides (AMPs), also referred to as host defence peptides [START_REF] Zasloff | Antimicrobial peptides of multicellular organisms[END_REF][START_REF]Mammalian host defense peptides[END_REF]. This expanding group of peptides forms a part of the innate immune system of all animals. They are secreted by specific immune cells, such as neutrophils [START_REF] Ganz | Defensins. Natural peptide antibiotics of human neutrophils[END_REF], NK-cells, or coelomocytes [START_REF] Bruhn | Dissection of the mechanisms of cytolytic and antibacterial activity of lysenin, a defence protein of the annelid Eisenia fetida[END_REF], and are also found on exposed body surfaces, e.g. skin and lung, where they provide a first line of defence against invading pathogens [START_REF] Harder | A peptide antibiotic from human skin[END_REF][START_REF] Harder | Isolation and characterization of human beta-defensin-3, a novel human inducible peptide antibiotic[END_REF][START_REF] Laube | Antimicrobial peptides in the airway[END_REF]. These peptides display a highly diverse tertiary structure.

Prominent examples are linear α-helical peptides such as magainins [START_REF] Zasloff | Magainins, a class of antimicrobial peptides from Xenopus skin: Isolation, characterization of two active forms, and partial cDNA sequence of a precursor[END_REF] and cecropins [START_REF] Boman | Antibacterial peptides: key components needed in immunity[END_REF], or β-sheet structures with a complex disulfide pattern, e.g. the defensins [START_REF] Ganz | Defensins. Natural peptide antibiotics of human neutrophils[END_REF][START_REF] Lehrer | Defensins: endogenous antibiotic peptides of animal cells[END_REF]. The arenicin structure, however, appears to be unique. As arenicins bear a net positive charge (+6) in combination with a substantial number of hydrophobic amino-acid residues, a membranolytic step involved in the killing of microorganism may be assumed. However, information on the the arenicin-membrane interaction is incomplete and the mechanism of the antibiotic action remains elusive.

Here, we present a comprehensive study on the mode of action of this extraordinary peptide with a focus on Gram-negative bacteria. Using NMR spectroscopy, we elucidated the solution structure of arenicin isoform 1 (RWCVYAYVRVRGVLVRYRRCW-COOH), and investigated and visualized its interaction with viable bacteria possessing defined lipopolysaccharide structures and with the corresponding reconstituted membrane mimetics.

This study included determination of the biological activity of arenicin, an ultrastructural analysis of arenicin-treated bacteria by atomic force microscopy and electron microscopy, permeabilisation of bacterial membranes, elucidation of the different steps of arenicin´s membrane interaction, i.e. binding, insertion and permeabilisation using FRET spectroscopy, arenicin-mediated dissipation of the membrane potential of liposomes, and electrical measurements of arenicin-induced lesions in planar lipid bilayers. Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

EXPERIMENTAL Peptides, phospholipids, and reagents

Chemically synthesized arenicin (RWCVYAYVRVRGVLVRYRRCW-COOH) with a non-amidated C-terminus was purchased from Biosyntan GmbH (Berlin, Germany). The sequence is identical to that of arenicin isoform-1, which differs from isoform 2 by a single amino acid substitution (V10I), as previously identified [START_REF] Ovchinnikova | Purification and primary structure of two isoforms of arenicin, a novel antimicrobial peptide from marine polychaeta Arenicola marina[END_REF] and is referred to throughout this manuscript as arenicin. The correct formation of the disulfide linkage (Cys3-Cys20) of the synthetic peptide was confirmed by mass spectrometry (kindly performed by C. Gelhaus).

Melittin was synthesized by solid phase synthesis by Fmoc chemistry and purified by HPLC as described previously [12]. Asolectin and polymyxin B, in its sulfate salt form, were purchased from Sigma (Deisenhofen, Germany). Phospholipids, cardiolipin, 1,2-diphytanoylsn-glycero-3-phosphocholine (DPhyPC, synthetic), L-α-phosphatidylcholine (PC, chicken egg), L-α-phosphatidyl-DL-ethanolamine (PE, E. coli), L-α-phosphatidyl-DL-glycerol (PG, chicken egg) were purchased from Avanti Polar Lipids (Alabaster, AL, USA). Lipids (purity >99%) were used without further purification. N-(7-nitrobenz-2-oxa-1,3-diazol-4yl)phosphatidyl-ethanolamine (NBD-PE) and N-(lissamine rhodamine B sulfonyl)-phosphatidylethanolamine (Rh-PE) were from Molecular Probes (Eugene, OR, USA). All other chemicals were analytical grade and purchased from Merck (Darmstadt, Germany).

Bacteria and bacterial culture

The bacterial strains used were six Gram-negative and one Gram-positive isolate: i) Deep rough mutant strains with defined LPS Re structures (Fig. 1): Escherichia coli WBB01, Salmonella enterica Sv Minnesota R595, Proteus mirabilis R 45, ii) strains with the complete Lipopolysaccharide Deep-rough type lipopolysaccharide (LPS) was extracted from E. coli strain WBB01 grown at 37 °C by the phenol/chloroform/petrol ether method, purified, and lyophilized [START_REF] Galanos | A new method for the extraction of R lipopolysaccharides[END_REF].

NMR experiments and structure calculation

Arenicin (1.8 mg) was dissolved in 0.7 ml of 88% H 2 O/10% D 2 O/2% (v/v) deuterated acetic acid. Two-dimensional TOCSY (τ m =75ms) and NOESY (τ m =150ms) spectra with WATERGATE solvent suppression were recorded on a Varian Inova 600 MHz Spectrometer at 283.15 K. Hydrogen bonding was evaluated using H-D exchange experiments. Hydrogen bonds were detected by recording one-dimensional 1 H-spectra after dissolving 1.8 mg arenicin in D 2 O; hydrogen bonds were assigned only to amide protons that still were detectable after 16 h. The assignment of proton resonances was carried out using TOCSY and NOESY data. Interproton distance restraints were derived from the cross-peak intensities in the NOESY spectrum by 1/r 6 -calibration. Additional distance restraints were added to represent the Cys3-Cys20 disulfide bond and hydrogen bonds in the peptide backbone.

Structure calculations were performed with the CYANA 2.1 program [START_REF] Guntert | Torsion angle dynamics for NMR structure calculation with the new program DYANA[END_REF] applying a simulated annealing-molecular dynamics protocol. Five hundred structures were calculated and the 10 structures with the lowest target function were chosen to represent the ensemble and to calculate the average structure. For graphical representation the RIBBONS and Grasp software was used [START_REF] Kraulis | MOLSCRIPT: a program to produce both detailed and schematic plots of protein structures[END_REF]17].

Assay for hemolytic activity

Twenty µl of washed human erythrocytes (5 x 10 8 cells/ml) in 10 mM phosphatebuffered saline, pH 7.4, were incubated with 80 µl of a peptide sample in the same buffer for 30 min at 37°C or 4°C in a round bottom microtiter plate (Nunclon Surface, Nunc, Roskilde, Denmark). After the incubation period the plate was centrifuged at 1000 x g for 10 min to remove intact erythrocytes, and the concentration of released hemoglobin was measured in a microtiter plate reader at 405 nm (Rainbow, Tecan, Grödig/Salzburg, Austria) after ten-fold dilution of the supernatant. Hemolytic activity was expressed as percent hemolysis (% hemolysis = ((OD Sample -OD buffer )/(OD max -OD buffer )*100). Maximal lysis (OD max ) was achieved by adding distilled water instead of the peptide sample to the cells. Data shown represent the mean of at least two experiments each performed in duplicate. Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Assay for cytotoxic activity

The influence of peptides on the viability of human cells was determined by monitoring the increase of the fluorescence of alamarBlue (BioScource) caused by metabolically active cells only. Jurkat cells were grown in RPMI 1640 medium (GIBCO-BRL) supplemented with 10 % fetal bovine serum (PAA-laboratories, Parkerford, USA) at 37°C and 5% CO 2 , washed twice in 20 mM MES, 150 mM NaCl, pH 5.5 and resuspended in the same buffer. Five x 10 5 cells (40 µl) were added to two-fold serial dilutions of the proteins in the same buffer (50 µl) in a precoated microtiter plate (0.1 % bovine serum albumin (Sigma-Aldrich) at 20°C for 10 min) and incubated at 37°C and 5% CO 2 . After 30 min, 10 µl of alamarBlue (final concentration: 10 %) were added and the cells were incubated for another 30 min. The increase of fluorescence was measured in the microtiter plate fluorescence spectrophotometer (Fluoroskan II, Labsystems) using an excitation wavelength of 538 nm and an emission wavelength of 590 nm. The fluorescence difference between Jurkat cells treated with 0.1 % Triton-X100 and cells treated with buffer only under the same conditions was taken as the value for 100 % cytotoxic activity. Peptide concentrations at which 50 % of the cells were killed (LD 50 ) were derived from dose-response curves. Each curve represents the mean of two independent experiments, each performed in duplicate.

Assays for antibacterial activity a) Determination of minimal inhibitory and bactericidal concentrations

Peptides were dissolved in the indicated buffer (Table 1). Each solution (180 µl) was pipetted into the first well of a microtiter plate. For a two-fold serial dilution, 90 µl of each solution was transferred to the next well filled with the same volume of buffer. Subsequently, a suspension of log-phase bacteria in LB medium was added (10 µl, containing 10 4 colony forming units, CFU) to each peptide solution (90 µl). The plates were incubated overnight in a wet chamber at 37°C (or 4°C when indicated) with constant shaking and bacterial growth was monitored by measuring the absorbance at 620 nm in a microtiter plate reader (Rainbow, Tecan, Crailsham, Germany). The minimal inhibitory concentration (MIC) was defined as the lowest peptide concentration, at which no bacterial growth was measurable. Portions of each well (10 µl) were diluted with buffer, plated out in duplicate on LB-agar plates, incubated overnight at 37°C, and bacterial colonies were counted. The minimal bactericidal concentration (MBC) was defined as the peptide concentration where no colony growth was observed. The values were expressed as the mean of at least two independent experiments, each performed in duplicate, with a divergence of not more than one dilution step. NaCl, pH 7.4) at 37 °C for 1 h. Permeabilisation of the bacterial outer and /or cytoplasmic membrane allows the dye to cross the membranes and to intercalate into the DNA. When excited at 495 nm, the binding of the dye to DNA resulted in an increase of emitted fluorescence at 538 nm which was measured in a microtiter plate reader (Fluoroskan II; Labsystems, Milford, MA, USA). Membrane-permeabilizing activity of the peptides was expressed as percentage of permeabilized bacteria. For maximum permeabilisation of the bacteria (100% value), cells were incubated with 70% ethanol for 5 min. The values were expressed as the mean of two independent experiments, each performed in duplicate.

Circular dichroism (CD) spectroscopy

CD measurements were carried out on a Jasco J-720 spectropolarimeter (Japan Spectroscopic Co., Ltd., Tokyo, Japan), calibrated according to [18]. CD spectrum measurement represents the average of at least three scans obtained by collecting data at 1 nm intervals with a bandwidth of 2 nm. The measurements were performed in 50 mM sodium phosphate, pH 5.8, at 10°C in a 1.0 cm tandem quartz cuvette. The two chambers of the tandem cuvette were filled with either the peptide solution or the liposome suspension, respectively. After recording the CD-spectrum the cuvette was shaken for 20 min in order to mix the peptide solution and liposome suspension and a second CD-spectrum was recorded.

The ratio of arenicin and PG was 0.85:1 (w/w).

Förster resonance energy transfer (FRET) spectroscopy

Intercalation of arenicin into phospholipid liposomes and LPS aggregates was determined at indicated temperatures by FRET spectroscopy applied as a probe-dilution assay as described earlier [START_REF] Schromm | Lipopolysaccharide-binding protein mediates CD14-independent intercalation of lipopolysaccharide into phospholipid membranes[END_REF]. The peptide was added to the liposomes (suspended in 20 mM Hepes, 150 mM NaCl, pH 7.0), and intercalation was monitored as the increase of the quotient between the donor fluorescence intensity I D at 531 nm and the acceptor intensity I A at 593 nm (FRET signal) in dependence of time. All measurements were performed at least twice with no major discrepancies. Representative curves are shown.

Preparation of planar bilayers and electrical measurements

Planar lipid bilayers were prepared according to the Montal-Mueller technique [START_REF] Montal | Formation of bimolecular membranes from lipid monolayers and a study of their electrical properties[END_REF] as described earlier in detail [START_REF] Wiese | Electrophysiological measurements on reconstituted outer membranes[END_REF][START_REF] Böhling | Lipid-specific membrane activity of human beta-defensin-3[END_REF]. For the reconstitution of the cytoplasmic membrane of human cells, instead of natural PC, DPhyPC was used to obtain a higher stability of the membranes. The inner leaflet of the outer membrane of Gram-negative bacteria was reconstituted by a phospholipid mixture (PL) consisting of PE, PG, and cardiolipin (molar ratio 81:17:2). In all experiments, the compartment to which peptide was added is named first (cis), and the compartment opposite to the addition (trans) was grounded. To be consistent with the literature, we used a negative sign for voltages if the potential applied to the ciscompartment is positive (i.e. reflecting a cell inside negative membrane potential). All

RESULTS

Solution structure of arenicin

In total, 194 distance restraints obtained by NMR spectroscopy were used to calculate the structure of arenicin. Table 2 summarizes the analysis of the ensemble representing the solution structure of arenicin. Arenicin is a two-stranded anti-parallel β-sheet (Cys3-Val10 and Val13-Cys20) stabilized by nine intra-backbone hydrogen bonds and a disulfide bond between Cys3 and Cys20 (Fig. 1A). The strands of the β-sheet are connected by a type I' βturn. Arenicin shows an amphiphilic surface with distinct hydrophobic and hydrophilic areas.

The electrostatic potential surface of the averaged arenicin structure reveals large hydrophobic areas separated by the positively charged arginine side chains (Fig 1B). Since the charged arginine side chains are highly mobile, this picture reflects only a static image of the charge distribution. However, this characteristic distribution of charged and hydrophobic areas is achieved by inserting a significant right-handed twist into the β-sheet-structure, thus preventing an even charge distribution on the molecular surface. A comparison with the previously published structure of arenicin isoform 2 [START_REF] Ovchinnikova | Recombinant expression, synthesis, purification, and solution structure of arenicin[END_REF] revealed that, despite the righthanded twist, the β-sheet of the arenicin isoform 2 is almost planar whereas in case of the isoform 1 the β-sheet is bent. As a consequence, the overall shape of arenicin isoform 1 resembles a globular protein, thus contrasting with the elongated shape of the isoform 2.

Cytotoxic activity

The cytotoxicity of arenicin and two reference peptides, i.e. bee venom melittin and polymyxin B, was monitored by measuring the release of hemoglobin from freshly isolated human erythrocytes and by monitoring their effect on the metabolic activity of human Jurkat T-cells. Arenicin, in sharp contrast to the highly hemolytic melittin (LD 50 = 3-4 µM), appeared to be only moderately lytic with an LD 50 above 40 µM (35.5 % lysis at 30 µM).

Notably, polymyxin B, despite its considerable hydrophobicity due to its alkyl chain, was completely non-hemolytic up to a concentration of 30 µM. Reduction of the incubation temperature from 37°C to 4°C did not abolish hemolysis, but led to a significantly reduced hemolytic effect of arenicin (9 % at 30 µM). By contrast, arenicin was substantially toxic to Jurkat cells. Killing was time-and concentration-dependent with an LD 50 of 4, 3.5, and 2.7 µM at 37°C after 1, 2, and 3 h of incubation, respectively. For comparison, melittin was effective against Jurkat cells with an LD 50 between 0.15 and 0.3 µM under the same conditions.

Antibacterial activity

We tested the antibacterial activities of the peptides under various buffer conditions using three deep-rough mutant (LPS Re chemotype) Gram-negative bacterial strains with well defined LPS structures (Fig. 1C,D), one of which we used in parallel for the preparation of model membranes to correlate biological with biophysical data. The bacterial panel includes human pathogens and laboratory strains. From the perspective of drug development, it was chosen to reflect strains which are in general more sensitive to the action of cationic peptides, i.e. E. coli WBB01, and strains which appear more resistant, in particular to the action of polymyxin B, the prototype of a cationic peptidic antibiotic with potent activity against Gramnegative bacteria, i.e. Proteus mirabilis R45. In addition, we tested the antibacterial activity of the peptides against two rough-mutant (LPS Ra chemotype) strains with LPS having complete core oligosaccharides.

Arenicin was active against all bacterial strains at similar low concentration and, among the peptides tested, it was the most potent one against P. mirabilis (Tab. 1).

Its activity was lower at a physiological salt concentration (150 mM) compared to buffer without any added salt. However, in buffers with varying ionic strength (150-500 mM), the activity of the peptide remained virtually constant. In particular the MIC of arenicin against P. mirabilis was very low, even under conditions where polymyxin B was completely inactive. The further addition of a bivalent cation (i.e. Mg 2+ ) was without any measurable effect. Notably, the arenicin concentration necessary to kill E. coli WBB01 and P. mirabilis R45 was the same at 37°C and at 4°C.

The kinetics of bacterial killing by arenicin is rapid, as assessed by plating out bacterial suspensions and colony counting after incubation with the peptide (Fig. 2). At 5 µM arenicin, complete killing of E. coli WBB01 was observed even after 5 min. It is worth mentioning that inhibitory and bactericidal concentrations which can be derived from these curves may differ from those shown in Table 1, as here bacteria were incubated in buffer alone to avoid growth during the incubation period. However, a substantial replication of bacteria was observed overnight even in buffer alone. This is in particular reflected in the case of P. mirabilis in the presence of 1 µM peptide (Fig. 2).

Ultrastucture of E. coli WBB01

The effect of arenicin (10 µM) on bacteria (exemplified here for E. coli WBB01) was visualized by atomic force and electron microscopy. For AFM measurements, bacteria were incubated with the peptide in LB medium. As depicted in Fig. 3 impact on the structural integrity of bacteria when compared to an untreated control. For electron microscopy, bacteria were incubated alone and in the presence of the peptide for 30 min at 37°C in Hepes buffer supplemented with 10% LB medium (Fig. 4). Under these conditions, 79% of bacteria were killed. Apparent steps in arenicin-mediated killing of E. coli became visible at higher magnification (Fig. 4B), beginning with the formation of membrane blebs, release and condensation of cytoplasmic material, detachment of the outer membrane from the plasma membrane and formation of electron-dense spots on the outer membrane.

Finally, the complete cytoplasm disappeared and the bacteria were covered with the spots which may represent aggregated lipopolysaccharide from the outer membrane. Interestingly, bacteria undergoing cell division always exhibited two cytoplasm clearing sites and the electron-dense spot formation was concentrated at the septum.

Permeabilisation of bacterial membranes

As the observed ultrastructure of arenicin-treated bacteria strongly suggested a membranolytic step in the mode of action of this peptide, we investigated the uptake of a DNA-intercalating dye, SYTOX green, by a suitable bacterial strain, i.e. E. coli D31, during incubation with arenicin. Dye uptake directly reflects membrane permeabilisation, as SYTOX green itself is not permeable through an intact lipid bilayer. In the case of Gram-negative bacteria, inner and outer membrane permeabilisation is apparently necessary to accomplish dye uptake. Considerable dye uptake was measurable even after 5 min of incubation of bacteria with the peptide (Fig. 5). The minimal inhibitory concentration of arenicin for E. coli D31, measured in parallel under these conditions, was 0.125 µM. Arenicin was also capable of permeabilizing the plasma membrane of a Gram-positive bacterial strain, B. megaterium (data not shown).

Circular dichroism (CD)

CD spectroscopy was used to study the conformation of arenicin bound to liposomes.

Figure 6 shows the CD spectra of arenicin in the absence as well in the presence of PG liposomes. The CD spectrum of arenicin in solution shows the typical shape of a β-sheet peptide with a positive absorption band at 230 and a negative absorption band at 216 nm. This shape is dramatically changed in the presence of PG liposomes. The overall shape of the spectrum is indicative of a helical conformation of the peptide, although the two minima (219 and 231 nm) of the spectra are shifted to a higher wavelength compared to a CD spectrum of a helical protein in solution (208 and 222 nm). This shift indicates that arenicin inserts into the hydrophobic core of the PG liposomes and thereby undergoes a conformational change.

Insertion into model membranes

We used FRET spectroscopy as a sensitive tool to detect the specific interaction of arenicin with pure phospholipid and LPS bilayers. Binding and subsequent insertion of peptide into the lipid bilayer is assumed to result in an increase in membrane area and with that in fluorescent probe dilution and in an increase of the observed FRET signal. Liposomes composed of zwitterionic phosphatidylcholine (PC) and of negatively charged phosphatidylglycerol (PG) were chosen to mimic the plasma membranes of human cells and of bacteria, respectively. Aggregates made of LPS Re purified from E. coli strain WBB01 were used as a mimetic of the Gram-negative outer membrane. At 37 °C, rapid intercalation of arenicin was observed in all types of model membranes (Fig. 7). In the different lipid systems the changes in the FRET signal cannot be compared directly due to different aggregate structures. However, different intercalation characteristics were observed at lower temperatures as compared to 37 °C for the various lipids. An enhanced intercalation of peptide was observed particularly into the LPS membrane but also into the negatively charged PG membrane when compared to PC. By contrast, arenicin intercalation into zwitterionic PC liposomes was decreased at lower temperature.

Permeabilisation of model membranes

Addition of arenicin to asolectin liposomes, loaded with potassium and a fluorescent dye, led to the immediate dissipation of a valinomycin-induced membrane potential (Fig. 8), and was the first direct evidence for a pore-forming activity of the peptide. To further characterize the electrical and geometrical properties of the arenicin-induced membrane lesions or pores and to investigate the lipid specificity of the peptide-membrane interaction, we employed the Montal-Mueller planar lipid bilayer technique. This system allows the reconstitution of membranes with virtually any lipid composition, including symmetrical membranes of DPhyPC (Fig. 9A) and a defined phospholipid mixture (PL) mimicking roughly the plasma membrane of human cells and the cytoplasmic membrane of bacteria (Fig. 9B). Moreover, the asymmetrical outer membrane of the envelope of Gram-negative, consisting of LPS on its outer leaflet and PL on its inner leaflet could also be reconstituted (Fig. 9C). Upon addition of arenicin to the cis side of a planar lipid bilayer and setting of a

DISCUSSION

Arenicins are remarkably active effector molecules of the lugworm´s immune system and exhibit a broad spectrum antimicrobial activity [START_REF] Ovchinnikova | Purification and primary structure of two isoforms of arenicin, a novel antimicrobial peptide from marine polychaeta Arenicola marina[END_REF]. These particular antimicrobial peptides (AMPs) caught our attention because of their unique cysteine-bridged large loop structure. Mechanistic information about how this new class of AMPs interacts with and kills bacteria has not been published so far. Arenicin isoform 1, which we have studied here in detail, forms a stable tertiary structure in solution, which resembles that of isoform 2 [START_REF] Ovchinnikova | Recombinant expression, synthesis, purification, and solution structure of arenicin[END_REF], though isoform 1 exhibits a more globular form when compared with the elongated structure of arenicin isoform 2. Detailed analyses of the structures revealed that the two isoforms do not differ significantly in their backbone-dihedral angles and that these are not responsible for the overall structural difference. The right handed twist in one of the β-strands in arenicin isoform 1 is due to experimentally derived 1 H-1 H distances that have not been observed in isoform 2 [START_REF] Ovchinnikova | Recombinant expression, synthesis, purification, and solution structure of arenicin[END_REF]. In addition, the bend of the arenicin isoform 1 β-sheet originates from observed longrange side chain contacts that also have not been described for the isoform 2. The anti-parallel β-sheet is twisted to expose an amphipathic surface. Other β-sheet AMPs, such as protegrin-1 and tachyplesin-1, adopt similar solution structures [START_REF] Fahrner | Solution structure of protegrin-1, a broad-spectrum antimicrobial peptide from porcine leukocytes[END_REF][START_REF] Laederach | Solution and micelle-bound structures of tachyplesin I and its active aromatic linear derivatives[END_REF] though the β-sheet of arenicin is much more extended and is not caged between two disulfide bonds. This is in contrast to most α-helical AMPs, which only adopt a defined secondary structure upon interaction with membranes or membrane-mimetic environments [12,[START_REF] Degrado | Induction of peptide conformation at apolar/water interfaces. 1. A study with model peptides of defined hydrophobic periodicity[END_REF]. As arenicin is amphipathic and rich in arginine and hydrophobic amino-acid residues, it is reasonable to suppose that disruption of the membrane is involved in killing of the bacteria. To address this hypothesis, we investigated its interactions with Gram-negative bacteria and model membranes. In our comprehensive studies on the antibacterial properties of arenicin, unequivocal evidence was found for the peptide-induced permeabilisation of the LPS-containing outer membrane and the phospholipid-containing cytoplasmic membrane of bacteria. Killing of E. coli and of polymyxin B-resistant P. mirabilis occurred within minutes and the ultrastructural analyses of arenicin-treated E. coli revealed signs of cell envelope destruction. Accordingly, the permeabilisation of both membranes of viable E. coli also occurred within minutes. No pronounced lipid specificity for intercalation and pore formation of arenicin in model membranes composed to mimic the respective biological targets (i.e. zwitterionic PC for the erythrocyte plasma membrane, negatively charged PG or a phospholipid mixture of PG, cardiolipin, and of zwitterionic phosphatidylethanolamine for the bacterial cytoplasmic enterica, and P. mirabilis) was observed. This is in contrast to the situation with other AMPs, such as polymyxin B and human β-defensin 3 for which the LPS structure, in particular the distribution of negatively charged groups, was decisive for the antibacterial activity [START_REF] Böhling | Lipid-specific membrane activity of human beta-defensin-3[END_REF][START_REF] Wiese | Molecular mechanisms of polymyxin B-membrane interactions: direct correlation between surface charge density and self-promoted transport[END_REF].

Arenicin-induced permeabilisation of model membranes is accompanied by peptide intercalation into the bilayer and a transmembrane current flow characteristic for structurally heterogeneous lesions. At a first glance, the characteristics of the obtained current fluctuations in planar lipid bilayers may fit into the carpet model [START_REF] Oren | Mode of action of linear amphipathic α-helical antimicrobial peptides[END_REF] rather than to a barrel-stave pore with defined geometry [START_REF] Boheim | Statistical analysis of alamethicin channels black lipid membranes[END_REF]. However, in general this view may be oversimplified [START_REF] Bechinger | Detergent-like actions of linear amphipathic cationic antimicrobial peptides[END_REF].

How does the structure of arenicin fit into the experimentally observed characteristics of the membrane lesions? Oligomerization of β-sheet domains is well docomented for bacterial pore-forming toxins, such as α-toxin from Staphylococcus aureus. However, membrane pores formed by these toxins have a highly ordered cylindrical geometry [START_REF] Song | Structure of staphylococcal alpha-hemolysin, a heptameric transmembrane pore[END_REF].

Apparently, arenicin undergoes a pronounced conformational change after insertion into a membrane. Since it is too short to span an entire bilayer it may be speculated that membrane defects were only elicited in the cis monolayer which could explain the heterogenicity of the observed lesions. Further studies will shed more light on this issue.

The concentration of arenicin effective in killing bacteria was almost identical to the concentration that induced measurable effects on the respective model membranes. Although this does not rule out potential intracellular targets, it implies that membrane interaction, if it is not the lethal event itself, is at least an essential step in bacterial killing. This is in concordance with recent data, where we have shown that the interaction of various AMPs with model membranes of lipid compositions resembling those of different target cells, reflected their biological activity [12,[START_REF] Böhling | Lipid-specific membrane activity of human beta-defensin-3[END_REF][START_REF] Schröder-Borm | Molecular basis for membrane selectivity of NK-2, a potent peptide antibiotic derived from NKlysin[END_REF].

The main focus of this study is the interaction of arenicin with Gram-negative bacteria and respective biomembrane mimetics. However, we would expect comparable results with Gram-positive bacteria and this has been proven exemplarily for membrane permeabilisation of B. megaterium. The broader target cell selectivity of arenicin compared to other AMPs, reflected in particular in a considerable cytotoxicity against Jurkat cells, may be attributed to the high salt concentration of the natural habitat of the lugworm. As the selectivity of peptides is mainly based on electrostatic interactions of the cationic peptide with the negatively charged bacterial surface [START_REF] Schröder-Borm | Molecular basis for membrane selectivity of NK-2, a potent peptide antibiotic derived from NKlysin[END_REF][START_REF] Matsuzaki | Molecular basis for membrane selectivity of an antimicrobial peptide, magainin 2[END_REF], this interaction is impaired at the high salt concentration found in sea water and thus a more pronounced hydrophobic interaction, which is presumably responsible for cytotoxicity, is necessary to guarantee a potent antimicrobial activity. The In summary, we have elucidated the structure and mode of action of arenicin isoform 1, a representative of a new class of AMPs. The peptide folds into a defined β-sheet tertiary structure and permeabilizes bacterial membranes which is a key step in bacterial killing. Its potent activity against polymyxin B resistant P. mirabilis, even at high ionic strength, its short length, and disulfide-bond stabilized tertiary structure make arenicin a particularly interesting antimicrobial compound and a lead structure for antibiotic drug development. Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

  S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 15 Oct 2007 as manuscript BJ20071051 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071051

  S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 15 Oct 2007 as manuscript BJ20071051 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071051

  S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 15 Oct 2007 as manuscript BJ20071051 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071051 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society b) Time course of bacterial killing Bacteria were grown to log-phase, washed with 2 8021 se (25 µl) and 2 µM of the fluorescent dye SYTOX Green (25 µl; in 10 mM Hepes, 25 mM

  S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 15 Oct 2007 as manuscript BJ20071051 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071051 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society Assay for pore-forming activity Pore-forming activity of arenicin was determined by measuring fluorimetrically the dissipation of a valinomycin-induced membrane potential in asolectin liposomes [20]. The measurements were repeated eight times and a representative curve is shown.

  measurements were performed in 5 mM Hepes, 100 mM KCl, 5 mM MgCl 2 , pH 7.0 (specific electrical conductivity 17.2 mS/cm) at 37 °C. Representative current traces of at least five independent measurements are shown. S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 15 Oct 2007 as manuscript BJ20071051 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071051 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

  S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 15 Oct 2007 as manuscript BJ20071051 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071051 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

  , arenicin had a dramatic S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 15 Oct 2007 as manuscript BJ20071051 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071051 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

  S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 15 Oct 2007 as manuscript BJ20071051 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071051 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

  negative trans-membrane voltage, we observed spontaneous current fluctuations after a certain lag period. The fluctuations appeared and vanished in a cooperative manner and reached conductivity levels of maximal 4 nS and a life time of several ms. The accumulation S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 15 Oct 2007 as manuscript BJ20071051 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071051Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

  of a number of lesions eventually led to a dramatic increase in current flow and the disruption of the membrane (arrow). Both conductivity and lifetime are indistinguishable in all three types of membranes suggesting in all cases the formation of structurally heterogeneous lesions rather than defined pores. Representative current traces are shown in Fig.9A-C. S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 15 Oct 2007 as manuscript BJ20071051 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071051 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

  S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 15 Oct 2007 as manuscript BJ20071051 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071051 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society membrane, and of LPS for the outer membrane of Gram-negative bacteria, such as E. coli, S.

  S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 15 Oct 2007 as manuscript BJ20071051 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071051 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society evolutionary selection of arginines instead of lysines may also be an adaptation to high salt environment. It has been recently published that a lysine derivative of human α-defensin-1 is much less active and has a higher sensitivity to increasing salt concentration than the argininecontaining wild-type molecule [35].

35

  Zou, G., de Leeuw, E., Li, C., Pazgier, M., Zeng, P., Lu, W. Y., Lubkowski, J. and Lu, W. (2007) Toward understanding the cationicity of defensins. Arg and Lys versus their noncoded analogs. J. Biol. Chem. 282, 19653-19665 S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 15 Oct 2007 as manuscript BJ20071051 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071051Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Figure legends Fig. 1 Ribbon

 legends1 Figure legends

Fig. 2 Time

 2 Fig. 2 Time course of bacterial killing by arenicin. Bacteria (E. coli WBB01 and P. mirabilis R45) were incubated alone (control) and in the presence of three different concentrations of arenicin in buffer (20 mM Hepes, 150 mM NaCl, pH 7.0) at 37°C. Viability of bacteria was assessed by plating out the bacterial suspensions at various time points and is presented as CFU (% of control) = (CFU peptide / CFU control ) * 100.

Fig. 3 Effect

 3 Fig. 3 Effect of arenicin treatment on the morphology of bacteria (I). AFM images of an untreated E. coli WBB01 (A) and of one incubated with 10 µM arenicin (B) in LB medium at 37°C for 30 min. Images were taken in air in AC (tapping) mode. Length (bar) and height (colour code) scales are indicated.

Fig. 4 Effect

 4 Fig. 4 Effect of arenicin treatment on the morphology of bacteria (II). Transmission electron microscopy of untreated E . coli WBB01 (A, control) and of those incubated with 10 µM arenicin (A, + Arenicin) at 37°C for 30 min. Selected higher magnification pictures of bacteria incubated with the peptide are shown in B. Length scale: each bar represents 1 µm. S t a g e 2 ( a ) P O S T -P R I N T

Fig. 5 PermeabilisationFig. 6 CD

 56 Fig. 5 Permeabilisation of bacterial membranes by arenicin. Damage of the outer and inner membrane of E. coli D31 induced by arenicin incubated for 5, 30, and 60 min was monitored via the uptake of the DNA-intercalating fluorescent dye SYTOX green.

Fig. 7 Intercalation

 7 Fig. 7 Intercalation of arenicin into liposome membranes. Peptide intercalation into PC (A), PG (B) liposomes, and E. coli WBB01 LPS aggregates (C) measured at three different temperatures by FRET spectroscopy. An increase of the FRET-signal (I Donor /I Acceptor ) corresponds to a reduced FRET efficiency by dye dilution and is indicative for membrane insertion of the peptide.

Fig. 8 Permeabilisation

 8 Fig. 8 Permeabilisation of liposomes by arenicin. Addition of peptide (0.1 nmol) led to the immediate dissipation of a valinomycin-induced membrane potential in phospholipid vesicles (1 ml cuvette buffer, pH 7.5) and concomitantly to dequenching of the fluorescent dye. Grey areas indicate time periods at which the spectrofluorometer shutter was temporarily closed to enable compound addition (arrows).

Fig. 9 Permeabilisation

 9 Fig. 9 Permeabilisation of planar lipid membranes by arenicin. Time courses of peptide-induced current traces are shown for symmetrical DPhyPC (A), for PL membranes (B), and for an asymmetrical E. coli WBB01 LPS Re / PL membrane (C). The final peptide concentrations and the applied voltages were 0.7 µM / -100 mV (A), 0.5 µM / -20 mV (B), and 0.8 µM / -52 mV (C), respectively. An arrow indicates disruption of the respective membrane. Bar represents 100 ms, all traces are displayed using the same time scale.

Figure 2 SFigure 3 S 4 S 5 S

 2345 Figure 2

Figure 6 SFigure 7 S 8 SFigure 9 S

 6789 Figure 6

Table 1 -

 1 Antibacterial activity of arenicin compared to that of melittin and polymyxin B against three deep rough (LPS Re) and two rough (LPS Ra) mutant bacterial strains at various buffer conditions: (1) 20 mM Hepes, pH 7.0; (2) 20 mM Hepes, 150 mM NaCl, pH 7.0; (3) 20

	mM Hepes, 300 mM NaCl, pH 7.0; (4) 20 mM Hepes, 500 mM NaCl, pH 7.0; (5) 5 mM
	Hepes, 100 mM KCl, pH 7.0; (6) 5 mM Hepes, 100 mM KCl, 5 mM MgCl 2 , pH 7.0; (7) PBS,
	pH 7.4. The minimal inhibitory concentration (MIC) and the minimal bactericidal
	concentration (MBC, in brackets) are given in µM. The assay was performed in 10 % LB / 90
	% buffer at 37°C and 4°C (when indicated). *, bacterial killing is not complete but >90 % at
	indicated concentration; n.a., not accomplishable; n.d., not done.
	Strain	LPS	Buffer / T	Salt conc. (M)	Arenicin	Melittin	Polymyxin B
	E. coli WBB01	Re	1	0	0.3 (0.6)	0.6 (2.5)	0.15 (0.6)
			1 / 4°C	0	n.a. (0.6)	n.d.	n.d.
			2	0.15	1.25 (2.5)	2.5 (5)	1.25 (1.25)
			3	0.3	2.5 (5)	40 (40)	1.25 (2.5)
			4	0.5	2.5 (5)	40 (>40)	1.25 (5)
			5	0.1	1.25 (2.5)	1.25 (1.25)	0.6 (2.5)
			6	0.1	1.25 (5)	5 (10)	1.25 (2.5)
			7	0.15	1.25 (5)	1.25 (1.25)	1.25 (1.25)
	P. mirabilis R45	Re	1	0	0.6 (2.5)	2.5 (10)	0.6 (5)
			1 / 4°C	0	n.a. (2.5)	n.d.	n.d.
			2	0.15	5 (20*)	20 (>40*)	>40 (>40)
			3	0.3	5 (20*)	>40 (>40)	>40 (>40)
			4	0.5	5 (20*)	>40 (>40)	>40 (>40)
			5	0.1	5 (20)	20 (40*)	> 40 (>40)
			6	0.1	2.5 (>10)	40 (40)	> 40 (>40)
			7	0.15	5 (20*)	20 (40)	> 40 (>40)
	S. enterica R595	Re	1	0	0.6 (1.25)	0.6 (1.25)	0.3 (0.3)
			2	0.15	1.25 (2.5)	2.5 (5)	0.3 (0.3)
	S. enterica R60	Ra	1	0	1.25 (2.5)	2.5 (5)	0.3 (0.3)
	E. coli ATCC 23716	Ra	1	0	0.6 (0.6)	5 (10)	1.25 (1.25)

S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 15 Oct 2007 as manuscript BJ20071051 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071051

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Table 2

 2 Structural statistics for the 20 conformers of arenicin 1. No NOE distance restraint was violated by more than 0.5 Å in any of the structures.

	Distance restraints a
	Intraresidue (i-j = 0)	111
	Sequential (|i-j| = 1)	41
	Medium range (2 ≤ |i-j| ≤ 4)	2
	Long range (|i-j| ≤ 5)	16
	Hydrogen bonds	9*2
	Disulfide bonds	2*3
	All	194
	Average r.m.s.d. b to mean in Å
	Average backbone r.m.s.d. to mean	0.66 ± 0.27
	Average heavy atom r.m.s.d. to mean	1.51± 0.26
	Average r.m.s.d. to mean for secondary structures c in Å
	Average backbone r.m.s.d. to mean	0.45 ± 0.16
	Average heavy atom r.m.s.d. to mean	1.34 ± 0.22

a Only the number of upper distance restraints is given; to each upper distance restraint corresponds one lower distance restraint. b r.m.s.d. root mean square deviation.

© 2007 The Authors Journal compilation © 2007 Biochemical Society

Biochemical Journal Immediate Publication. Published on 15 Oct 2007 as manuscript BJ20071051 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071051 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

Acknowledgements

We thank Annemarie Brauser for taken AFM images, Dr. Christoph Gelhaus for performing mass spectrometry, Christine Hamann for FRET measurements, Heike Kühl for electron microscopy, Christoph Lemke for measurements on planar lipid bilayers, Kerstin

Stephan for antibacterial testings, and Dr. Lee Shaw for critical reading of the manuscript.

This study has been carried out with financial support from the Deutsche Forschungsgemeinschaft (SFB 617 ´Molecular mechanisms of epithelial defense´, projects A9, A17, and A18). A.K. was supported by a short-term fellowship of the SFB 617 during her stay in Kiel, Germany. S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 15 Oct 2007 as manuscript BJ20071051 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071051

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Valerie Petegnief

Miriam Font-Nieves

M Elena Martin

Matilde Salinas

Anna M Planas

Valérie Petegnief

Míriam Font-Nieves

M Elena Martín

Nitric oxide mediates NMDA-induced persistent inhibition of protein synthesis through dephosphorylation of eukaryotic initiation factor 4E-binding protein 1 and eukaryotic initiation factor 4G proteolysis

Keywords: 4E-BP1, eIF4G, calpain, excitotoxicity, ischemia, NO eIF2 , eukaryotic initiation factor 2, eIF4E, eukaryotic initiation factor 4E, eIF4G, eukaryotic initiation factor 4G, 4E-BP1: eukaryotic initiation factor 4E-binding protein 1, mTOR, mammalian target

Nitric oxide mediates NMDA-induced persistent inhibition of protein synthesis through dephosphorylation of eukaryotic initiation factor 4E-binding protein 1 and eukaryotic initiation factor 4G proteolysis

INTRODUCTION

Brain ischemia induces early and deep inhibition of protein synthesis that is persistent in vulnerable neurons such as CA1 hippocampal and striatal neurons [START_REF] Hossmann | Disturbances of cerebral protein synthesis and ischemic cell death[END_REF][START_REF] Dienel | Regional protein synthesis in rat brain following acute hemispheric ischemia[END_REF][START_REF] Hata | Evolution of brain infarction after transient cerebral ischemia in mice[END_REF]. In this regard, longlasting inhibition of protein synthesis is one of the alterations that best correlates with neuronal injury after ischemia. Indeed, translation recovery is associated with survival, whereas neurons unable to overcome protein synthesis blockade are destined to die [START_REF] Hermann | Relationship between metabolic dysfunctions, gene responses and delayed cell death after focal cerebral ischemia in mice[END_REF]. Translation is mainly inhibited via regulation of the initiation and elongation steps, by impairing the activity of certain eukaryotic initiation or elongation factors (eIFs or eEFs). Modulation of the phosphorylation status and/or degradation of these proteins account for their inactivation. For instance, changes in the phosphorylation status of eIF2 , 4E-BP1 or eEF2 are known to modulate the rate of protein synthesis [START_REF] Rhoads | Signal transduction pathways that regulate eukaryotic protein synthesis[END_REF]. Phosphorylation of eIF2 by the double-stranded RNA-activated protein kinase-like endoplasmic reticulum (ER) kinase (PERK) is sufficient to abrogate protein synthesis soon after reperfusion following an ischemic episode [START_REF] Burda | Phosphorylation of the subunit of initiation factor 2 correlates with the inhibition of translation following transient cerebral ischaemia in the rat[END_REF][START_REF] Degracia | Eukaryotic initiation factor 2 kinase and phosphatase activity during postischemic brain reperfusion[END_REF][START_REF] Kumar | Brain ischemia and reperfusion activates the eukaryotic initiation factor 2 kinase[END_REF][START_REF] Paschen | Transient cerebral ischemia activates processing of xbp1 messenger RNA indicative of endoplasmic reticulum stress[END_REF]. In addition, a number of other factors are affected. For instance, depletion of ATP activates eEF2 kinase through AMP kinase in cortical neurons and slows elongation [START_REF] Maus | 2-Deoxyglucose and NMDA inhibit protein synthesis in neurons and regulate phosphorylation of elongation factor-2 by distinct mechanisms[END_REF]. Furthermore, global cerebral ischemia leads to eIF4G and eIF4E breakdown [START_REF] Degracia | Global brain ischemia and reperfusion: modifications in eukaryotic initiations factors associated with inhibition of translation initiation[END_REF][START_REF] Neumar | Eukaryotic initiation factor 4E degradation during brain ischemia[END_REF][13]. Production of NO bursts after ischemia and NO is involved in inflammation, cerebral damage [14] and protein synthesis reduction through eIF2 phosphorylation [15][16][17]. Likewise, excitotoxicity is one of the main promoters of neuronal loss after an ischemic episode, and is also known to inhibit protein synthesis [18][19][20][21]; however the underlying mechanisms are not fully elucidated. The rise in intracellular calcium induced by glutamate activates eEF2 kinase, which in turn phosphorylates eEF2 and results in the transient blockade of elongation [18]. The increase in cytosolic calcium concentration following NMDA activates the cysteine protease calpain [22]. eIF4G, the scaffolding protein that enables the assembly of eIF4E and eIF4A with the mRNA and the ribosomal 40S subunit is a substrate of calpain [13], and is thus a possible target of NMDA. However, most studies of eIF4G cleavage have reported that eIF4G is the target of caspase-3 in apoptotic conditions (reviewed in [23]). Caspase-3 cuts eIF4GI at two sites to generate three fragments, as referred to as: Fragments of Apoptotic cleavage of eIF4G (FAG): N-FAG, M-FAG and C-FAG, corresponding to the N-, middle-and C-terminal parts of the protein, respectively (reviewed in [23]). M-FAG 533-1176 is the only fragment that remains able to promote some cap-dependent translation. Therefore, proteolysis of eIF4G is presumably associated with the switch from cap-dependent to cap-independent translation and favours, for example, the translation of specific internal ribosome entry site (IRES)-containing mRNAs [23]. Unlike with FAG, the role of eIF4G fragments resulting from calpain activity has not been explored. eIF4E binding protein 1 (4E-BP1) is another key regulator in protein synthesis because it associates to eIF4E [24]. In cell cultures, insulin, amino acids and rapamycin modulate the phosphorylation status of 4E-BP1 through mammalian target of rapamycin (mTOR) signalling [25,26]. In the hypophosphorylated form, 4E-BP1 competes with the binding site of eIF4G and sequesters eIF4E, whereas the hyperphosphorylated form dissociates from eIF4E, enabling it to engage with other components of the eIF4F complex [24,[27][28][29][30][31]. Therefore, proteolysis of eIF4G together with dephosphorylation of 4E-BP1 precludes the formation of eIF4F complexes [32] that may reduce the cap-mRNA translation. There is compelling evidence of crosstalk between NO and NMDA. In pure neuron cultures, uncoupling between the NMDA receptor and nNOS signaling pathways [33] and pharmacological inhibition of nNOS [34] strongly prevent neuronal death after an excitotoxic challenge. Reciprocally, production of NO by iNOS in activated glia leads to neuronal death, which is prevented by , thus suggesting that NO leads to glutamate release [38] in a positive feed-back loop. Furthermore, NO challenge triggers excitotoxicity in cerebellar neurons [39]. The mechanisms responsible for NO toxicity are not completely elucidated but energy depletion is likely involved. NO induces a fast depolarisation of the mitochondria, which leads to reduced ATP production [40]. In addition, NO exposure triggers the activation of PARP that leads to ATP consumption and may therefore account for energy depletion [41].

Here we addressed the molecular mechanisms underlying NMDA-mediated long-term inhibition of protein synthesis in neuronal cultures, and considered whether NO is involved in this process.

MATERIALS AND METHODS

Materials

All products for culture, except MEM, were purchased from Invitrogen (Paisley, Scotland, UK). ATP bioluminescent assay kit, somatic cell ATP releasing agent, chemicals and reagents were obtained from Sigma-Aldrich (Alcobendas, Spain), unless otherwise stated. Diethylamine/Nitric oxide sodium complex (DEA/NO) and N -propyl-L-arginine were purchased from Tocris (Avonmouth, UK). The calpain inhibitors calpeptin and MDL28170, as well as rapamycin were from Calbiochem (LaJolla, CA, USA). The non-specific nitric oxide synthase (NOS) inhibitor N -nitro-L-arginine methyl ester (L-NAME) and the neuronal specific NOS (nNOS) inhibitor N -propyl-L-arginine (NPLA) were purchased from Sigma-Aldrich. The mouse antibodies against eIF4E and STAT3 were from BD Transduction Laboratories (Heidelberg, Germany). The goat antibodies against eIF4G (N-20) and anti-eIF2 were purchased from Santa Cruz Biotechnology (Heidelberg, Germany). Rabbit antiphospho-eIF2 (Ser 51 ) was from Epitomics (Burlingame, CA, USA) and mouse anti-spectrin was from Chemicon (Temecula, CA, USA). Anti-4E-BP1 antibody was obtained from Cell Signaling Technology (Beverly, MA, USA). Anti-mouse, anti-goat and anti-rabbit HRPconjugated antibodies were from Amersham (Buckinghamshire, England). Complete protease inhibitor cocktail and the antibody against -tubulin were from Boehringer Mannheim (Germany). L- [4,5-3 H]leucine (S.A. = 73 Ci/mmol) and 7-methyl GTP-Sepharose were purchased from Amersham (Buckinghamshire, England).

Cell cultures and treatments

Mixed primary cortical cultures of neurons and glia were prepared from 18-day-old Sprague-Dawley rat embryos (Charles River, France), as previously described [20]. Animals were anaesthetized and killed by cervical dislocation. All procedures were approved by the Ethical Committee for Animal Use (CEEA) at the University of Barcelona. Neuron-enriched cultures were prepared as mixed cultures, but ARA-C was added on day in vitro 4 and then in the subsequent partial medium changes on days in vitro 7 and 10 to limit glial proliferation. Cells were seeded on 24-well plates at a density of 3680 cells/mm 2 . For the ATP content measurement, cells were seeded on 6-well plates. All experiments were performed on day in vitro 11-13 cultures. DEA/NO was prepared as a 20 mM stock in NaOH pH10 to avoid NO release and stored at -20ºC for further use. A 2 mM DEA/NO solution in NaOH pH10 was prepared extemporaneously and added to the culture medium in order to achieve a final concentration of 4-40 µM. Cells were exposed for 30 min to DEA/NO and medium was thereafter replaced with MEM supplemented with B27 and gentamycin. Excitotoxic lesion was performed by treating cultures for 30 min with 35 µM NMDA. NOS inhibitors (L-NAME + NPLA) were added simultaneously to NMDA and also after medium change. Calpain inhibitors were added at 30 M, unless otherwise stated, 1 h before NMDA or DEA/NO and also after medium change. Thapsigargin at 1 or 10 M was added for 30 min and rapamycin at 1 or 2 M for 3 h with no previous medium change. Calpeptin, MDL28170, thapsigargin and rapamycin were dissolved in DMSO.

Measurement of LDH activity

Cell death was estimated 24 h after the lesion by measuring the activity of lactate dehydrogenase (LDH) released in the medium as previously described [20]. Briefly, the decrease in 0.75 mM NADH absorbance at 340 nm was followed in a phosphate buffer (50 mM, pH 7.4) in the presence of 4.2 mM pyruvic acid as substrate. The kinetic assay of NADH consumption was monitored for 4 min and the slope values of decreased absorbance at 340 nm (negative values) were converted to positive values and expressed in arbitrary units (AU).

Propidium iodide nuclear staining

Cells were treated for 30 min with 35 µM NMDA and then fixed and stained with propidium iodide (PI) 24 h later as previously described [20]. PI-positive nuclei were counted in three fields per well and the sum of the three, corresponding to a total area of 0.4416 mm 2 was calculated. The result was expressed as PI positive cells/mm 2 .

to NMDA or DEA/NO, cells were incubated with NMDA or DEA/NO for 30 min, medium was changed to MEM/B27 and, 90 min later, cells were incubated with [ 3 H]leucine for 30 min as described above. Medium was removed, proteins were precipitated with trichloroacetic acid and lysates were processed as previously described [20] for the determination of [ 3 H]leucine incorporated into proteins. Results were expressed as dpm in the TCA fraction/mg protein/min.

Measurement of ATP levels

After drug treatments, cells were washed with cold PBS and collected in a 5 mM EDTA, 0.1 M Na 2 HPO 4 , pH 7.5 buffer. After a 5 min spin at 590 X g at 4ºC, pellets were resuspended in 200 µl of the same buffer, sonicated and centrifuged again for 15 min at 10 000 X g at 4ºC. ATP content was determined in the fresh supernatants using an ATP bioluminescent assay kit and following manufacturer's instructions. Samples were run together with a 0.05-8 µM ATP standard curve and luminescence was monitored in an Orion microplate luminometer (Berthold detection system). Relative light units (RLU) were converted to pmols of ATP and results were expressed as nmol ATP/mg protein.

Western blot

Neuron-enriched cultures were treated with NMDA, NMDA+NOS inhibitors, or DEA/NO for 30 min. Ninety minutes later, cultures were washed with cold 10 mM phosphate-buffered saline (PBS) and harvested in radio-immunoprecipitation (RIPA) lysis buffer (10 mM PBS, 1% Igepal AC-630, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulphate) supplemented with a protease inhibitor cocktail (Complete) and 1 mM sodium orthovanadate. Protein content was determined by the Bradford assay (Bio-Rad Laboratories, München, Germany). Thirty µg of proteins was separated by electrophoresis on 7%, 12% or 17% polyacrylamide gels (for spectrin-eIF4G, eIF2 and 4E-BP1, respectively) in denaturing conditions and transferred to 0.2 µm polyvinylidene difluoride Immun-Blot membranes (Bio-Rad Laboratories, Hercules, CA, USA). Membranes were incubated overnight at 4ºC with the following primary antibodies: goat anti-eIF4G diluted 1:500, goat anti-eIF2 , rabbit antiphospho-eIF2 (Ser 51 ), rabbit anti-4E-BP1 diluted 1:2000, mouse anti-spectrin diluted 1:4000, mouse anti-STAT3 and mouse anti-eIF4E diluted 1:1000. After two washes in Tris buffer containing Tween-20 (T-TBS), membranes were then incubated for 1 h at room temperature with either anti-rabbit, anti-mouse or anti-goat horseradish peroxidase-conjugated antibody. The reaction was visualized using a chemiluminescence detection system based on the luminol reaction. Autoradiograms were scanned with a GS-800 Densitometer scanner (Bio-Rad) and band density was quantified with the Quantity One image analysis software (Bio-Rad). Molecular weight markers are indicated in kDa on the left side of some of the blots shown in the Figures. For the analysis of eIF2 phosphorylation the phosphorylated/total protein ratio was calculated for each sample. Since the molecular weights of spectrin and eIF4G are 240 and 220 kDa, respectively, we decided to use the 90 kDa protein STAT3 of higher MW than tubulin, to check for equal loading, since none of the treatments affected STAT3 expression (not shown). The densities of the 150-145 kDa bands corresponding to cleaved spectrin were quantified together. The spectrin/STAT3 and eIF4G/STAT3 ratios were calculated for each sample. Data from all quantifications are expressed as a percentage of controls. In the case of 4E-BP1, the proportion of each phosphorylated form ( , and ) was calculated as a percentage of total 4E-BP1 as in [32].

Determination of the formation of eIF4E/eIF4G and eIF4E/eIF4E-B1 complexes

After the different treatments, cells were washed with buffer A containing 20 mM Tris-HCl, pH 7.6, 1 mM dithiothreitol, 1 mM EDTA, 1 mM PMSF, 1 mM benzamidine, 120 mM KCl, 10 g/ml leupeptin, 1 g/ml pepstatin A, 10 g/ml antipain, 50 mM sodium fluoride, 10 mM glycerophosphate and 10 mM sodium molybdate; they were then lysed with buffer A containing 0.5% Igepal AC-630 and 0.1% triton X-100. Cell extracts were spun at 12000 X g for 10 min at 4ºC and the supernatants were collected and frozen at -80ºC for further use. Two hundred g of proteins was incubated with 7-methylguanosine triphosphate (m 7 GTP)-Sepharose for 45 min at 4ºC in buffer A containing 0.1 mM GTP. Proteins were eluted from m 7 GTP-Sepharose with SDS loading buffer and subjected to 7.5%-17% SDS-PAGE and Western blot. Membranes were incubated separately with antibodies against eIF4G, eIF4E and 4E-BP1.

Statistical analysis

Results are expressed as mean SEM and the number of replicates (n) is indicated in the legend of each figure. Unless indicated, one-way analysis of variance (ANOVA) with the Bonferroni post-hoc test was performed to evaluate significant differences between groups. Kruskal-Wallis analysis followed by Dunn's test for multiple comparisons was used to compare groups with non-homogenous variance. One symbol indicates P<0.05, two P<0.01 and three P<0.001.

RESULTS

NOS inhibitors protected against NMDA toxicity in mixed and neuron-enriched cultures.

One of our objectives was to determine whether NO participates in NMDA-induced inhibition of protein synthesis in our primary cultures. First, we confirmed that NMDA toxicity was dependent on NO. When testing the effect of several NOS inhibitors we found that the combination of the non-specific NOS inhibitor L-NAME with the specific nNOS inhibitor NPLA at 2 mM and 100 M, respectively provided almost complete protection against transient (30 min) exposure to 35 M NMDA in mixed neuron/glia and neuron-enriched cultures, as assessed by lactate dehydrogenase activity and propidium iodide staining assays (Figs. 1A and1B). Further experiments were therefore performed with the NAME+NPLA combination of NOS inhibitors (referred to as NOS inhibitors in the text). Treatment with the above concentrations of NOS inhibitors did not affect neuronal viability (Fig. 1A). To check for direct NO toxicity, i.e. not induced by NMDA, we performed a dose-response study with DEA/NO, an NO-donor. DEA/NO was applied for 30 min and toxicity was assessed at 24 h. At 40 µM DEA/NO induced similar toxicity to treatment with 35 µM NMDA (Fig. 1C) and this concentration of DEA/NO was therefore selected to perform further experiments.

NMDA and NO induced protein synthesis failure

We have previously demonstrated that NMDA decreases the protein synthesis rate after 1 h exposure [20]. Here we explored whether transient exposure to NMDA caused long-lasting inhibition of protein synthesis. A time-course study showed that NMDA inhibited by 52 and 43% the incorporation of [ 3 H]leucine into proteins after 30-min exposure in mixed (Fig. 2A) and neuron-enriched cultures (Fig. 2B), respectively. After removal of NMDA, protein synthesis partially recovered but remained significantly lower than in controls 2 h later (i.e. 90 min after NMDA removal) in both mixed (Fig. 2A) and neuron-enriched cultures (Fig. 2B). NOS inhibitors were ineffective at 30 min (during NMDA exposure) but did promote complete recovery of protein synthesis at 2 h. Treatment with NOS inhibitors alone had no effect on protein synthesis (Fig. 2B). DEA/NO also led to a slight reduction of protein synthesis (less than 20%) at the end of a 30-min exposure and the effect was more pronounced 2 h later (33% decrease) in neuron-enriched cultures (Fig. 2C). Altogether these data suggest that NO impairs translation and contributes to the late phase of protein synthesis inhibition induced by NMDA.

NMDA and NO induced a moderate decrease in ATP content and did not regulate the phosphorylation of eIF2 ATP depletion can induce endoplasmic reticulum stress, which leads to phosphorylation of eIF2 and subsequent inhibition of protein synthesis [15,42]. Here we evaluated ATP content after the treatments to determine whether ATP depletion was involved in the observed inhibition of protein synthesis. NMDA caused a 26% reduction of ATP levels at the end of the 30-min exposure, and this effect was maintained 2 h later (Fig. 3A). Although DEA/NO reduced ATP content to 53% after 30-min exposure, this effect was not significantly different from controls 2 h later (Fig. 3B), thus indicating that in contrast to NMDA the action of NO on ATP is not long-lasting. NOS inhibitors did not prevent the ATP decrease 2 h after NMDA exposure (Fig. 3C), and this illustrates that NO was not involved in the persistent ATP reduction after NMDA. Our data suggest that the late protein synthesis inhibition caused by NMDA is not mediated by the corresponding ATP reduction. Phosphorylated eIF2 binds to the GDP/GTP exchanger eIF2B and inhibits its function ([reviewed in [30]). The inability to restore the binding of GTP to eIF2 prevents the formation of new eIF2•GTP•Met-tRNAi ternary complexes and consequently protein synthesis is inhibited. NMDA and NO did not induce eIF2 phosphorylation on Ser 51 (Figs. 3D and3E), indicating that eIF2 activity was not inhibited. In order to determine whether our cultures might show increased phosphorylation of eIF2 , neurons were treated for 30 min with thapsigargin. This endoplasmic reticulum stress inducer is known to cause eIF2 phosphorylation through PERK activation. Figure 3F shows that thapsigargin triggers eIF2 phosphorylation when compared to control cells.

NMDA and NO led to calpain activation and eIF4G proteolysis

NMDA is known to induce cytosolic calcium increase [START_REF] Maus | 2-Deoxyglucose and NMDA inhibit protein synthesis in neurons and regulate phosphorylation of elongation factor-2 by distinct mechanisms[END_REF]20] during the period of exposure. As expected, NMDA activated calpain, a calcium-dependent cysteine protease, which was assessed by the proteolysis of -spectrin (Figs. 4A and4C). The 240 kDa full-length protein is processed into 150 and 145 kDa fragments by calpain and to 150 kDa and 120 kDa fragments by caspase-3 [43]. NMDA generated the formation of the 145-150 kDa fragments only (Fig. 4A), this being indicative of calpain-dependent hydrolysis. Furthermore, eIF4G was cleaved 2 h after NMDA exposure (39% decrease, P<0.05; Figs. 4A and4C). Proteolysis of both spectrin and eIF4G was completely blocked in the presence of NOS inhibitors (Figs. 4A and4C), thus indicating an NO-dependent mechanism. In agreement with a previous report [39], NO dramatically stimulated calpain and reduced eIF4G levels by 52% (Figs. 4B and4D). Several calpain inhibitors (not shown) were then used to determine the contribution of calpain to eIF4G processing, and we found that two of them, calpeptin and MDL28170, completely inhibited calpain activation and blocked the eIF4G degradation induced by NMDA (Figs. 5A and5C) and DEA/NO (Figs. 5B and5D). Therefore, eIF4G degradation was totally dependent on calpain activation.

NMDA and DEA/NO induced the dephosphorylation of 4E-BP1 and abrogated the formation of eIF4F complexes

The formation of eIF4F complexes plays a key role in the regulation of translation initiation. Since eIF4E is a component of the eIF4F complex and it is a rate-limiting translation factor for cap-mRNA, we measured its level of expression, observing that none of the treatments altered its cell content (Fig. 6A). The phosphorylation status of 4E-BP1 also governs the formation of eIF4F complexes. 4E-BP1 resolves into three electrophoretic forms , and of increasing migration velocity. Only the hyperphosphorylated form of 4E-BP1 does not bind eIF4E [31] and therefore it increases the availability of eIF4E to interact with eIF4G. We studied the phosphorylation of 4E-BP1 and performed a semi-quantitative analysis of the proportion of , and forms with respect to total 4E-BP1 (Fig. 6B and Table 1). NMDA was observed to promote a shift from the form to the form (Table 1), an effect that was abrogated by NOS inhibitors (Fig. 6B and Table 1). Furthermore, DEA/NO induced the dephosphorylation of 4E-BP1, illustrated by a decrease in the form and an increase in the form, which is the band of lowest mobility (Fig. 6B and Table 1). In contrast, none of the treatments affected the form. Since eIF4G and 4E-BP1 compete for the same binding site in eIF4E, we evaluated the proteins bound to eIF4E after its purification by m 7 GTP-sepharose affinity chromatography. DEA/NO and NMDA almost completely abolished the interaction between eIF4G and eIF4E, and the NMDA effect was prevented by NOS inhibitors (Fig. 6C). We also studied whether calpain inhibitors influenced the phosphorylation status of 4E-BP1. Calpeptin and MDL28170 did not reverse the dephosphorylation of 4E-BP1 induced by NMDA and DEA/NO, respectively (Fig. 6B and Table 1). Moreover, calpeptin decreased the form and enhanced the formation of the form compared to NMDA alone (Fig. 6B and Table 1). When the protein synthesis rate was measured, we observed that none of the calpain inhibitors prevented the blockade of protein synthesis induced by NMDA or DEA/NO (Fig. 6D), thus suggesting that the prevention of eIF4G cleavage alone is not sufficient to abrogate the inhibition of protein synthesis. In contrast, NOS inhibitors that blocked eIF4G processing and dephosphorylation of 4E-BP1 did promote the complete recovery of translation (Figs. 2B, 4C and 6B). In addition, we observed that none of the calpain inhibitors rescued neurons from death (Fig. 6E), suggesting that survival is compromised when the normal rate of protein synthesis is not achieved.

Rapamycin reduced the formation of eIF4F complexes and inhibited protein synthesis in a similar way to NMDA

In order to determine whether hypophosphorylation of 4E-BP1 was by itself sufficient to reduce the formation of eIF4F complexes in the absence of eIF4G degradation, we incubated neurons with rapamycin, an inhibitor of the 4E-BP1 kinase mTOR (reviewed in [30]). Three hours after addition of rapamycin we observed a dephosphorylation of 4E-BP1 but no loss of eIF4G (Fig. 7A). When we looked at the amount of eIF4G bound to eIF4E after m 7 GTPsepharose affinity chromatography, we found that rapamycin induced a dose-dependent inhibition of eIF4G binding to eIF4E (Fig. 7B) that correlated with a decrease in protein synthesis (Fig. 7C) of similar proportions to that achieved with NMDA (Fig. 6D).

DISCUSSION

The present study investigated the mechanisms underlying excitotoxicity-mediated translation repression. In particular, we explored the contribution of eIF4G proteolysis and 4E-BP1 dephosphorylation to NMDA-and DEA/NO-induced protein synthesis inhibition. To our knowledge, this is the first study to report molecular changes in translation factors that might be involved in the long-lasting inhibition of protein synthesis after transient exposure to NMDA. We showed that neurons can withstand a transient blockade of translation with limited neuronal loss, and also provided evidence that protein synthesis recovery closely correlates with neuronal survival, as is observed in animal models of stroke [START_REF] Hossmann | Disturbances of cerebral protein synthesis and ischemic cell death[END_REF]. Indeed, the inactivation of NOS enabled full recovery of global protein synthesis, along with an almost complete rescue from NMDA-induced neurotoxicity; this latter finding has been previously reported by other groups [44,45]. Interestingly, NOS inhibitors were able to prevent the delayed but not the initial inhibition of protein synthesis following NMDA exposure. Similarly, ischemic preconditioning suppresses persistent but not acute inhibition of translation (reviewed in [46]).

NOS inhibitors did not impede NMDA-induced ATP reduction, despite the fact that they promoted a complete recovery of protein synthesis. Therefore, the moderate decrease in ATP after NMDA and DEA/NO treatments is unlikely to be the cause of protein synthesis inhibition. This result is in agreement with previously published data showing that severe protein synthesis inhibition precedes ATP reduction after treatment with an NO-donor [17]. Furthermore, suppression of protein synthesis occurs in the penumbra where ATP levels are maintained in transient focal ischemia [START_REF] Hata | Evolution of brain infarction after transient cerebral ischemia in mice[END_REF]. Interestingly, our data are in accordance with studies suggesting that transient inhibition of protein synthesis is not lethal, whereas the persistent phase of translation suppression is related to neurodegeneration [18,[46][47][48].

Release of calcium from ER stores through blockade of Ca 2+ /ATPase with thapsigargin [17] was shown to induce protein synthesis alterations due to phosphorylation of eIF2 . Glutamate also mobilizes Ca 2+ from ER reservoirs but depresses protein synthesis in an eIF2 phosphorylation-independent manner [19], in agreement with our data. We did not detect phosphorylation of eIF2 after exposure to DEA/NO, a fast NO-releasing complex, in contrast to previous observations in neuroepithelial cells [16]. S-nitroso-Nacetylpenicillamine (SNAP), a slow NO-releasing agent, has been shown to mobilize thapsigargin-sensitive calcium ER stores in neurons [17] and to phosphorylate eIF2 similarly to thapsigargin in neuroblastoma [15]. The shorter exposure time, faster NO release and lower concentration of NO-donor used in our study may explain the discrepancies with those studies. Therefore, under our experimental conditions DEA/NO inhibited translation by a mechanism that was independent of eIF2 phosphorylation, at least in the late phase. Our data suggest that NO does not contribute to the initial inhibition of protein synthesis by NMDA, but rather takes part in delayed translation deterioration.

Calpain activation and subsequent proteolysis of its substrates is known to occur after excitotoxicity in neurons [22,39,43]. While some reports show an attenuation of NMDA toxicity with pharmacological inhibitors of calpain [22], others have found no protection [43], as we did in the present study. Nevertheless, we found that calpain was responsible for eIF4G breakdown after exposure to NMDA or DEA/NO, in accordance with previous observations in the ischemic brain [13]. Proteolysis of several translation factors has been reported, mainly in apoptotic conditions. Research has shown that eIF2 and 4E-BP1 are the substrate for caspase-3 (reviewed in [23]), while eIF4E and 4E-BP1 are the substrate for calpain in vitro [START_REF] Neumar | Eukaryotic initiation factor 4E degradation during brain ischemia[END_REF]49]. However, to the best of our knowledge there is no evidence that eIF4E and 4E-BP1 are cleaved by calpain in vivo [START_REF] Neumar | Eukaryotic initiation factor 4E degradation during brain ischemia[END_REF]49]. Here, we did not observe any degradation of eIF2 , eIF4E or 4E-BP1, suggesting that calpain specifically processed eIF4G in our settings. The eIF4G fragment generated by calpain cleavage does not bind to eIF4E, thus precluding the formation of eIF4F complexes, as shown in both the present study and a model of global cerebral ischemia [32]. Therefore, it is likely that the strong proteolysis of eIF4G contributed to impaired translation in our experimental conditions.

The binding of 4E-BP1 to eIF4E also compromises the association of eIF4F components [24]. We observed that NMDA and DEA/NO insults led to the dephosphorylation of 4E-BP1, which is expected to facilitate its binding to eIF4E. A similar process has been described after global ischemia and was shown to cause a deficit in eIF4E/eIF4G complexes and, therefore, to contribute to translation inhibition [32]. NOS inhibitors enabled the complete recovery of protein synthesis by preventing eIF4G proteolysis and by precluding the dephosphorylation of 4E-BP1. The lack of protein synthesis recovery in the presence of a normal amount of eIF4G (in the presence of calpain inhibitors) suggests that the hypophosphorylation of 4E-BP1 is sufficient to reduce the protein synthesis rate. This result is supported by the data obtained with rapamycin. Rapamycin that dephosphorylated 4E-BP1 but maintained eIF4G integrity reduced the rate of protein synthesis. The rapamycininduced inhibition of translation correlated with the reduced formation of eIF4E/eIF4G complexes. eIF4E is crucial for cap-dependent translation initiation and is a mandatory element of the eIF4F complex since it functions as the cap-binding protein. Reduced availability of eIF4E resulting from protease-mediated degradation or trapping by 4E-BP1 would severely compromise cap-mRNA translation. Therefore, eIF4E is a rate-limiting factor and its sequestration by 4E-BP1 might slowdown the process of translation. The lower amount of full-length eIF4G may worsen the process by decreasing the probability of eIF4E-eIF4G binding. Our data suggest that preventing eIF4G degradation and 4E-BP1 dephosphorylation are necessary to unlock translation repression. The maintenance of eIF4G levels while 4E-BP1 is hypophosphorylated is not sufficient to prevent protein synthesis inhibition. Our results are in agreement with research showing that ischemic tolerance, which promotes translation recovery, tends to prevent eIF4G processing and to favour the phosphorylation of 4E-BP1 [47,[START_REF] García-Bonilla | .6 ** Neuron-enriched cultures were treated with NMDA, NMDA+NOS inhibitors or DEA/NO for 30 min. Calpain inhibitors were added as described in Figure 5. Ninety minutes after NMDA or DEA/NO removal, cells were collected and processed for 4E-BP1 Western blot. For each sample, the proportion of each phosphorylated form of 4E-BP1 ( , and ) was calculated as the percentage of each band over the total as explained in Methods. Data are mean ± SEM of 5-10 replicates[END_REF].

Since eIF4G and eIF4E play a key role in the translation of cap-mRNAs, we propose that limited availability of both proteins accounts for long-lasting translation failure (summarized in Figure 8) and contributes to neuronal loss after NMDA and NO challenges. Further studies are required to unravel the mechanisms controlling 4E-BP1 dephosphorylation.

Figure legends

Figure 3 Effect of NMDA and NO on ATP content and phosphorylation status of eIF2

A. Neuron-enriched cultures were incubated for 30 min with NMDA. Cells were scrapped either at the end of the 30-min incubation (0.5 h) or 90 min after medium change (2 h) and processed for ATP determination as detailed in Methods. Results were calculated as nmol/mg protein and expressed as % control. Data are mean ± SEM of 8-18 replicates. B. Neuronenriched cultures were treated for 30 min with DEA/NO. ATP content was determined at 0.5 h or 2 h after DEA/NO as in A. Data are mean ± SEM of 10-20 replicates from three different experiments. C. Neuron-enriched cultures were co-incubated for 30 min with NMDA and NOS inhibitors. Cells were scraped 90 min after medium change and processed for ATP determination as detailed in A. Data are mean ± SEM of 9 replicates for each condition from two different experiments. Neuron-enriched cultures were treated with NMDA or NMDA+NOSI (D) or with DEA/NO (E) for 30 min. Ninety minutes later, cells were collected, proteins were separated on 12% SDS-PAGE and processed for Western blot as indicated in Methods. Membranes were incubated first with an eIF2 -P antibody and then reprobed with the antibody recognizing eIF2 independently of its phosphorylation state.

Bottom panels are quantitative analysis of the blots. Band density was measured, the eIF2 -P/eIF2 total ratio was calculated for each sample and results are expressed as % control. Data are mean ± SEM of 11-13 replicates in D and 9-12 replicates in E. Significance for all graphs: *P<0.05, ***P<0.001 vs. control cultures; $$ P<0.01 vs. NMDA-treated cultures. Unpaired t-test was performed for statistical analysis in E. F. Neurons were treated with thapsigargin for 30 min and phosphorylation of eIF2 was examined by Western blot as described in D. C=control, N=NMDA, N+NOSI=NMDA+NOS inhibitors (NAME+NPLA), Thap=thapsigargin. Proteins were separated on 7% SDS-PAGE and processed for eIF4G immunoblotting. Membranes were then reprobed with an antibody recognizing -spectrin. NMDA treatment induced a loss of eIF4G (bottom blot) and of full-length -spectrin and the generation of the 150 kDa spectrin fragment but not of the 120 kDa product (lowest band, top blot). NOS inhibitors impeded spectrin and eIF4G proteolysis. B. Neuron cultures were exposed for 30 min to DEA/NO as in Figure 3E. Proteins were processed for eIF4G and -spectrin Western blot as in A. DEA/NO triggered calpain-mediated breakdown of spectrin (top blot) and eIF4G (bottom blot). Blots are representative of two different experiments. C. Quantitative analysis of cleaved spectrin (top graph) and eIF4G (bottom graph). Cells were treated as in A. Results were calculated as arbitrary units and expressed as % control. Data are mean ± SEM of 4-6 replicates for spectrin and 8-13 for eIF4G. D. Quantitative analysis of cleaved eIF4G after DEA/NO treatment. Cells were treated as in B. Data are mean ± SEM of 7 replicates. Significance for all graphs:*P<0.05, **P<0.01 vs. control cultures and $ P<0.05, $$$ P<0.001 vs. NMDA-treated cultures. C=control, N=NMDA, N+NOSI=NMDA+NOS inhibitors (NAME+NPLA).

Figure 5 Effect of calpain inhitors on -spectrin and eIF4G cleavage

A. Neuron-enriched cultures were pre-treated for 1 h with 30 µM calpeptin and exposed to NMDA for 30 min. B. Neuron-enriched cultures were pre-treated for 1 h with 30 µM MDL28170 and then exposed to DEA/NO. Sister cultures were treated with vehicle. Ninety minutes after medium change, cells were collected and processed for eIF4G and -spectrin Western blot as in Figure 4. The calpain inhibitors completely blocked spectrin and eIF4G proteolysis. C. Quantitative analysis of cleaved spectrin (top graph) and eIF4G (bottom graph). Cells were treated as in A. Data are mean ± SEM of 5 replicates for spectrin and 9-10 for eIF4G. D. Quantitative analysis of cleaved spectrin (top graph) and eIF4G (bottom graph). Cells were treated as in B. Data are mean ± SEM of 9-10 replicates. Significance for all graphs: *P<0.05, ***P<0.001 vs. control cultures and $ P<0.05, $$ P<0.01 or $$$ P<0.001 vs. NMDA-or DEA/NO-treated cultures. C=control, N=NMDA, N+NOSI=NMDA+NOS inhibitors (NAME+NPLA), D=DEA/NO, Cp= calpeptin and M=MDL28170.

Figure 8 NMDA targets responsible for delayed translation repression

Following the stimulation of NMDA receptors, NO is produced and induces the hypophosphorylation of 4E-BP1 and the subsequent binding to eIF4E, thus reducing its availability to interact with eIF4G. Increased intracellular calcium activates calpain that cleaves eIF4G into fragments unable to bind eIF4E. Lower amounts of full-length eIF4G together with the sequestration of eIF4E result in the formation of fewer eIF4E/eIF4G complexes and translation inhibition. Alterations of additional translation factors by regulation of phosphorylation status and/or degradation most likely contribute to achieve the full inhibition of translation.

  Biochemical Journal Immediate Publication. Published on 16 Jan 2008 as manuscript BJ20071060 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071060 S t a g e 2 ( a ) P O S T -P R I N T

  Biochemical Journal Immediate Publication. Published on 16 Jan 2008 as manuscript BJ20071060 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071060 S t a g e 2 ( a ) P O S T -P R I N T

Figure 1

 1 Figure 1 Neuronal death induced by NMDA and NO A. Neuron-enriched cortical cultures at 11-12 DIV were co-incubated with 2 mM L-NAME plus 100 µM NPLA (NOSI) and NMDA for 30 min. Medium was then removed and replaced with fresh medium supplemented with the same concentration of NOS inhibitors. Lactate dehydrogenase activity was determined 24 h later as indicated in Methods. Values in arbitrary units are mean ± SEM of 4-8 individual determinations. B. Mixed cultures were treated with NMDA and NOSI as indicated in A. At 24 h post-injury, cells were fixed and stained with PI as indicated in Methods. The number of PI-stained nuclei was counted under the fluorescence microscope (see Methods). Results are expressed as PI-positive neurons/mm 2 . Values are mean ± SEM of 6 replicates per condition from two different experiments. C. Dose-response study (4-40 M) for DEA/NO toxicity in mixed cultures. Lactate dehydrogenase activity was determined in culture media 24 h after a 30-min exposure to DEA/NO. Values are mean ± SEM of 7-8 replicates from two different experiments. Significance for all graphs:*P<0.05, ***P<0.001 vs. control cultures; $$$ P<0.001 vs. NMDA-treated cultures. C=control, N=NMDA, NOSI=NOS inhibitors (NAME+NPLA). N+NOSI=NMDA+NOS inhibitors.

Figure 2

 2 Figure 2 Long-lasting inhibition of protein synthesis induced by NMDA and NO in cortical cultures A. Protein synthesis was assessed by incorporation of [ 3 H]leucine into proteins in mixed cultures as detailed in Methods. [ 3 H]leucine was added simultaneously with NMDA (0.5 h), 30 min (1 h) or 90 min (2 h) after a 30-min incubation with NMDA. NOS inhibitors were added immediately before NMDA and after NMDA removal. Results were calculated as dpm/mg prot/min and expressed as % control (n=4-16). B. Incorporation of [ 3 H]leucine into proteins in neuron cultures was performed as for mixed cultures and detailed in Methods. Data are mean ± SEM (n=4-15). C. Protein synthesis was assessed by incorporation of [ 3 H]leucine into proteins in neuron cultures as detailed in Methods. [ 3 H]leucine was added simultaneously with DEA/NO (0.5 h) or 90 min (2 h) after a 30-min incubation with DEA/NO 40 M. Results were calculated as dpm/mg prot/min and expressed as % control. Data are mean ± SEM (n=9-17). Significance for all graphs: *P<0.05, **P<0.01, ***P<0.001 vs. control cultures; $$$ P<0.001 vs. NMDA-treated cultures. C=control, N=NMDA, NOSI=NOS inhibitors (NAME+NPLA), N+NOSI=NMDA+NOS inhibitors. Bars in the graphs represent the incubation duration with NMDA or DEA/NO.

Figure 4 -

 4 Figure 4 -spectrin and eIF4G cleavage after NMDA and NO A. NMDA leads to the proteolysis of -spectrin and eIF4G. Neuron cultures were exposed to NMDA or NMDA+NOS inhibitors for 30 min as in Fig 3D.Proteins were separated on 7% SDS-PAGE and processed for eIF4G immunoblotting. Membranes were then reprobed with an antibody recognizing -spectrin. NMDA treatment induced a loss of eIF4G (bottom blot) and of full-length -spectrin and the generation of the 150 kDa spectrin fragment but not of the 120 kDa product (lowest band, top blot). NOS inhibitors impeded spectrin and eIF4G proteolysis. B. Neuron cultures were exposed for 30 min to DEA/NO as in Figure3E. Proteins were processed for eIF4G and -spectrin Western blot as in A. DEA/NO triggered calpain-mediated breakdown of spectrin (top blot) and eIF4G (bottom blot). Blots are representative of two different experiments. C. Quantitative analysis of cleaved spectrin (top graph) and eIF4G (bottom graph). Cells were treated as in A. Results were calculated as arbitrary units and expressed as % control. Data are mean ± SEM of 4-6 replicates for spectrin and 8-13 for eIF4G. D. Quantitative analysis of cleaved eIF4G after DEA/NO treatment. Cells were treated as in B. Data are mean ± SEM of 7 replicates. Significance for all graphs:*P<0.05, **P<0.01 vs. control cultures and $ P<0.05, $$$ P<0.001 vs. NMDA-treated cultures. C=control, N=NMDA, N+NOSI=NMDA+NOS inhibitors (NAME+NPLA).

Figure 6

 6 Figure 6 Phosphorylation status of 4E-BP1 and eIF4E/eIF4G complexes formation after NMDA and NO. Effect of calpain inhibitors on 4E-BP1 dephosphorylation and protein synthesis inhibition induced by NMDA or DEA/NO.A. Neuron-enriched cultures were treated with NMDA or NMDA+NOS inhibtors for 30 min and 90 min after medium change, cells were collected and processed for eIF4E Western blot. B. Cultures were pretreated for 1 h with 30 µM calpeptin or MDL28170. Then NMDA or DEA/NO were added to the culture medium. Thirty minutes later, medium was changed and replaced with fresh medium containing the same concentration of calpain inhibitors. Sister cultures were treated with NMDA or NMDA+NOS inhibitors. Ninety minutes after medium

Figure 7

 7 Figure 7 Effect of rapamycin on phosphorylation status of 4E-BP1, eIF4E/eIF4G complexes formation and protein synthesisNeuron-enriched cultures were treated for 3 h with rapamycin (1 or 2 M) or vehicle and were then processed for eIF4G or 4E-BP1 Western blot (A) or for eIF4E purification and subsequent determination of the eIF4E/eIF4G and eIF4E/4E-BP1 complexes (B). C. Protein synthesis was assessed by incorporation of [ 3 H]leucine into proteins in neuron cultures 3 h after addition of rapamycin (1 or 2 M) or vehicle as detailed in Methods. Results were calculated as dpm/mg prot/min and expressed as % control (n=5-11). Significance: **P<0.01 and **P<0.001 vs. control cultures. Rap=rapamycin.

Biochemical

  Journal Immediate Publication. Published on 16 Jan 2008 as manuscript BJ20071060 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071060 S t a g e 2 ( a ) P O S T -P R I N T

Table 1 Phosphorylation status of 4E-BP1 after NMDA and NO. Effect of calpain or NOS inhibitors

 1

		Isoforms
	Gamma	Beta	Alpha

S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.© 2008 The Authors Journal compilation © 2008 Biochemical Society

Biochemical Journal Immediate Publication. Published on 16 Jan 2008 as manuscript BJ20071060 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071060 S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

Acknowledgements:

The authors would like to thank Araceli Salinas and Elisabet Gomez for their excellent technical assistance and the Language Advice Service from the University of Barcelona for revising the English. VP is a recipient of a Ramon y Cajal grant and MFN a recipient of a FPI grant from the Spanish Ministry of Education and Science (MEC). This study was supported by grants FIS05/0271 from the Spanish Ministry of Health and SAF2005-05793-C01 from the Ministry of Education and Science.