Wenjing Jia

Nicholas Tovell

Stephanie Clegg

Mark Trimmer

Jeffrey Cole
email: j.a.cole@bham.ac.uk

A single channel for nitrate uptake, nitrite export and nitrite uptake by Escherichia coli NarU and a role for NirC in nitrite export and uptake

Keywords: nitrate transport, E. coli NarU, mutagenesis of NarU, NarU topology, 15 N-nitrate experiments, nitrite uptake and export by NirC

Abbreviations used: Nap, periplasmic nitrate reductase; NarG and NarZ, the major and minor nitrate reductases with active sites located in the cytoplasm; Nrf, nitrite reduction by formate catalysed by the periplasmic nitrite reductase; Nir, NADH-dependent nitrite reductase; GlpT,

INTRODUCTION

During anaerobic growth, enteric bacteria reduce nitrate first to nitrite and then to ammonia. In the presence of mM concentrations of nitrate, synthesis of the periplasmic nitrate and nitrite reductases, Nap and Nrf, is substantially repressed, but a membrane-associated, cytoplasmic pathway is induced [START_REF] Cole | Physiology, biochemistry and genetics of nitrate dissimilation to ammonia[END_REF][START_REF] Cole | Nitrate reduction to ammonia by enteric bacteria: redundancy, or a strategy for survival during oxygen starvation?[END_REF][START_REF] Potter | Competition between Escherichia coli strains expressing either a periplasmic or a membrane-bound nitrate reductase: does Nap confer a selective advantage during nitrate-limited growth?[END_REF][START_REF] Rabin | Dual response regulators (NarL and NarP) interact with dual sensors (NarX and NarQ) to control nitrate-and nitrite-regulated gene expression in Escherichia coli K-12[END_REF][START_REF] Tyson | Definition of the nitrite and nitrate response elements at the anaerobically inducible Escherichia coli nirB promoter: interactions between FNR and NarL[END_REF][START_REF] Wang | The napF and narG nitrate reductase operons in Escherichia coli are differentially expressed in response to submicromolar concentrations of nitrate but not nitrite[END_REF]. This requires the energy-conserving NarGHI nitrate reductase complex, which is coordinately regulated during anaerobic growth in the presence of nitrate with an energy dissipating NADH-dependent nitrite reductase, NirBD, that reduces nitrite in the cytoplasm to ammonia [START_REF] Rabin | Dual response regulators (NarL and NarP) interact with dual sensors (NarX and NarQ) to control nitrate-and nitrite-regulated gene expression in Escherichia coli K-12[END_REF][START_REF] Tyson | Definition of the nitrite and nitrate response elements at the anaerobically inducible Escherichia coli nirB promoter: interactions between FNR and NarL[END_REF]. As the active site for nitrate reduction is located in the cytoplasm, nitrate must first be transported across the cytoplasmic membrane against a -180 mV proton electrochemical gradient for NarG to function [START_REF] Moir | Nitrate and nitrite transport in bacteria[END_REF].

Two polytopic membrane proteins of the major facilitator family, NarK and NarU, catalyse nitrate uptake into the Escherichia coli cytoplasm [START_REF] Clegg | The roles of the polytopic membrane proteins, NarK, NarU and NirC, in Escherichia coli K-12: two nitrate and three nitrite transporters[END_REF]. Both proteins also catalyse nitrite extrusion and nitrite uptake, but the transport mechanisms are unknown. A third protein, NirC, catalyses nitrite uptake, but it is unknown whether NirC also facilitates nitrite export [START_REF] Clegg | The roles of the polytopic membrane proteins, NarK, NarU and NirC, in Escherichia coli K-12: two nitrate and three nitrite transporters[END_REF][START_REF] Clegg | Role of the Escherichia coli nitrate transport protein, NarU, in survival during severe nutrient starvation and slow growth[END_REF]. Based upon the accumulation of nitrite as the product of nitrate reduction, it was initially proposed that NarK is a nitrate-nitrite anti-porter [START_REF] Demoss | NarK enhances nitrate uptake and nitrite excretion in Escherichia coli[END_REF][START_REF] Noji | The narK gene product participates in nitrate transport induced in Escherichia coli nitrate-respiring cells[END_REF]. An alternative suggestion that NarK is primarily a nitrite extrusion protein [START_REF] Rowe | NarK is a nitrite extrusion system involved in anaerobic nitrate respiration by Escherichia coli[END_REF] is now known to be incorrect [START_REF] Clegg | The roles of the polytopic membrane proteins, NarK, NarU and NirC, in Escherichia coli K-12: two nitrate and three nitrite transporters[END_REF][START_REF] Clegg | Role of the Escherichia coli nitrate transport protein, NarU, in survival during severe nutrient starvation and slow growth[END_REF]. In E. coli, the 12 membrane helices of GlpT form a single substrate translocation pore that mediates the exchange of glycerol-3-phosphate for inorganic phosphate [START_REF] Gott | The transmembrane topology of the sn-glycerol-3-phosphate permease of Escherichia coli analysed by phoA and lacZ protein fusions[END_REF][START_REF] Huang | Structure and mechanism of the glycerol-3-phosphate transporter from Escherichia coli[END_REF]. Also, the fourteen-helix TetL and TetK tetracycline efflux proteins from Staphylococcus aureus and Bacillus subtilis are believed to catalyse Na + , K + and tetracycline antiport via a single channel that alternately imports one substrate and expels another [START_REF] Jin | Tet(L) and Tet(K) tetracycline-divalent metal/H + antiporters: characterization of multiple catalytic modes and a mutagenesis approach to differences in their efflux substrate and coupling ion preferences[END_REF]. It is therefore possible that NarK and NarU each constitutes a single channel for nitrate uptake, nitrite export, and nitrite uptake. If so, any site-directed mutation that decreases the rate of nitrate uptake should also decrease the rates of nitrite uptake and extrusion. However, this would not resolve whether nitrate uptake and nitrite export are stoichiometrically linked, for example, by an alternating access mechanism. Alternative possibilities are that NarK and NarU form a single channel for both nitrate / H + symport and nitrite / H + export [START_REF] Wood | Two domains of a dual-function NarK protein are required for nitrate uptake, the first step of denitrification in Paracoccus pantotrophus[END_REF], a process that does not require strict stoichiometry between the two processes. Also possible, but less likely, is that NarK and NarU constitute two channels, one for nitrate uptake, the other for nitrite export: if so, it should be possible to separate the two functions by site-directed mutagenesis.

In preliminary experiments, we substantiated the 12-helix topology of NarK, and hence of NarU. This provided a model that predicted the location of highly conserved residues in the NarK protein family, and directed the design of site-directed mutagenesis experiments to reveal whether all known functions of NarU depend on a single transport channel. We demonstrate that NirC enhances nitrite export during nitrate-dependent growth of a strain expressing NarU, and that NirBD-dependent nitrite reduction to ammonia is enhanced by the nitrite uptake capacity of NirC. Finally 15 N isotope studies were designed to determine whether nitrate-nitrogen is exported into the periplasm as nitrite before it is re-imported for reduction to ammonia by the cytoplasmic nitrite reductase, NirBD.

EXPERIMENTAL PROCEDURES

Strains and growth conditions

Strains and plasmids used during this study are listed in Table 1. Bacteria were grown aerobically at 37˚C in LB (10 g . l -1 tryptone, 5 g . l -1 yeast-extract and 5 g . l -1 NaCl) or anaerobically in MS medium (4.5 g . l -1 KH 2 PO 4 , 10.5 g . l -1 K 2 HPO 4 , 1 g . l -1 NH 4 SO 4 , 0.05 g . l -1 MgCl 2 , 2.5 g . l -1 nutrient broth, 10 μM Na + -molybdate, 10 μM Na + selenate and 1 ml . l -1 of E. coli sulphur-free salts [START_REF] Pope | Pyruvate and ethanol as electron donors for nitrite reduction by Escherichia coli[END_REF]) supplemented as appropriate with 100 µg . ml -1 ampicillin and/or 50 µg . ml -1 kanamycin. For anaerobic growth experiments, the electron acceptor was 20 mM potassium nitrate and the primary carbon source was either 0.4% glycerol or 0.4% glucose (glucose was added as a sterile 40% w/v solution after the bulk medium had been autoclaved). The inoculum was 2% (v/v) of an exponential phase culture growing aerobically in LB. For experiments to determine the extent of nitratedependent growth and nitrite accumulation, bacteria were grown in 100 ml of medium in unshaken 100 ml conical flasks. Samples were removed at intervals, the optical density of the culture at 650 nm was determined, and bacteria were sedimented by centrifugation. The concentration of nitrite that had accumulated in the culture medium was then determined [START_REF] Clegg | The roles of the polytopic membrane proteins, NarK, NarU and NirC, in Escherichia coli K-12: two nitrate and three nitrite transporters[END_REF].

Mutagenesis of narU

Various codons in narU were changed using the Quikchange Site-Directed Mutagenesis kit (Stratagene) following the manufacturer's instructions and plasmids pSJC901 and pJCB901 as the template. Specific primers were used in some of these experiments, for example, to substitute the conserved glycine residues with alanine. In many of the preliminary experiments, degenerate primers were used to substitute R87, R303 and other conserved residues by a variety of amino acids. Sequences of the primers used are available on request.

Localization of Myc-tagged proteins and Western blotting procedures

Cultures were grown anaerobically to an OD 650 of 0.5 in 1 l of MS-glucose/nitrate medium supplemented with 10% (v/v) LB. Bacteria were harvested, washed with 50 mM Tris-HCl, pH 8.0 and resuspended in 15 ml of 0.5 M sucrose, 5 mM EDTA, 50 mM Tris-HCl, pH 8.0. Lysozyme was added to 600 µg . ml -1 and the suspension was incubated for 1 h at 30°C to release the periplasmic proteins [START_REF] Mcewan | The periplasmic nitrate reductase of Rhodobacter capsulatus; purification, characterization and distinction from a single reductase for trimethylamine-N-oxide, dimethylsulphoxide and chlorate[END_REF]. The sphaeroplasts were pelleted by centrifugation for 1 h at 12,000 g, resuspended in 5 ml of 5 mM MgSO 4, 50 mM Tris-HCl, pH 8.0, and passed through a French pressure cell at 70 MPa. Unbroken bacteria and any protein in inclusion bodies were removed by centrifugation at 5,000 g for 5 min. and the cytoplasmic and membrane fractions were separated by centrifugation at 170,000 g for 1 h. Note that the membrane fraction includes inner and outer membranes and insoluble cell wall components. Proteins were separated by SDS-PAGE, transferred to a PVDF membrane, and probed with anti-myc antibody (Invitrogen, Paisley, Scotland). Anti-mouse IgG was obtained from TROPIX (Bedford, USA). The antibodies were detected using a chemiluminescent detection system (TROPIX Inc, Bedford, USA).

Construction of plasmids and detection of PhoA and LacZ fusion proteins

Fragments of the 5' end of the narK gene, including its promoter, up to the desired fusion point were amplified using PCR. The primers introduced a PstI or EcoRI site upstream of the narK promoter and a BamHI site at the fusion point. The PstI-BamHI fragments were cloned into pSK4158 [START_REF] Paulsen | Molecular characterization of the staphylococcal multidrug resistance export protein QacC[END_REF] to create in-frame phoA fusions. The EcoRI-BamHI fragments were cloned into pNM480 [START_REF] Minton | Improved plasmid vectors for the isolation of translational lac gene fusions[END_REF] to create in-frame lacZ fusions. Transformants were plated onto selective LB-agar with 40 µg . ml -1 bromo-4-chloro indolyl phosphate (X-P) or 40 µg . ml -1 bromo-4-chloro indolyl-β-Dgalactoside (X-gal). Alkaline phosphatase and β-galactosidase activities were measured as described previously [START_REF] Jayaraman | Location and sequence of the promoter of the gene for the NADH-dependent nitrite reductase of Escherichia coli and its regulation by oxygen, the Fnr protein and nitrite[END_REF][START_REF] Manoil | Analysis of membrane protein topology using alkaline phosphatase and beta-galactosidase gene fusions[END_REF].

Rates of nitrate and nitrite uptake and reduction by washed bacterial suspensions

Bacteria from 1 or 2 l of an anaerobic culture were harvested by centrifugation, washed in 20 ml of Na + /K + phosphate pH 7.3, resuspended in 50 mM phosphate buffer, pH 7.3, and assayed for nitrate reduction by formate using a nitrate electrode [START_REF] Potter | Competition between Escherichia coli strains expressing either a periplasmic or a membrane-bound nitrate reductase: does Nap confer a selective advantage during nitrate-limited growth?[END_REF][START_REF] Clegg | The roles of the polytopic membrane proteins, NarK, NarU and NirC, in Escherichia coli K-12: two nitrate and three nitrite transporters[END_REF].

Rates of nitrite uptake and reduction by the cytoplasmic NirBD nitrite reductase were also determined [START_REF] Clegg | The roles of the polytopic membrane proteins, NarK, NarU and NirC, in Escherichia coli K-12: two nitrate and three nitrite transporters[END_REF][START_REF] Jia | Nitrate and nitrite transport in Escherichia coli[END_REF]. For these assays, diffusion of nitrous acid across the membrane was minimised by incubating washed suspensions of bacteria with an electron donor (glucose or formate) and 0.1 mM nitrite in 50 mM MOPS buffer, pH 8.5.

Concentrations of protein and nitrite were determined using the Folin and Griess methods described previously [START_REF] Jia | Nitrate and nitrite transport in Escherichia coli[END_REF].

N-nitrate reduction and recovery of N-nitrite in cultures expressing narU alone, or both narU and nirC

The bacteria from 1 l of culture were harvested by centrifugation, washed and resuspended in MOPS buffer (pH 8.5). MOPS buffer (pH 8.5) was added to a 50 ml conical flask containing 2 to 5 ml of cell suspension and 1 ml of 40 % (w/v) glucose to make a final volume of 50 ml. Oxygen dissolved in this mixture was removed by sparging with nitrogen for 15 minutes. The flask was then sealed with a stopper and incubated at 30°C for 5 min. To start the assay, 250 μl of 100 mM 15 Nnitrate (99.5 % 15 N atom, Sigma-Aldrich, Poole, UK) and 250 μl of 100 mM 14 N-nitrite were added and the contents of the flask were mixed thoroughly. Approximately 1.5 ml samples were removed from the culture at 2 min intervals and immediately passed through a 0.2 μm Acrodisc syringe filter (Pall corporation, UK) to remove the bacteria and to stop the reduction. Approximately 1 ml of filtered medium was collected in a 1.5 ml microfuge tube and stored at -20°C.

Any nitrite in the filtrate was converted to Sudan-1, recovered by solid phase extraction and its 15 N content was measured by combustion and continuous flow/isotope ratio mass spectrometry (CF/IRMS) at Queen Mary, University of London [START_REF] Preston | Isotope dilution analysis of combined nitrogen in natural waters. III. Nitrate and nitrite[END_REF][START_REF] Johnston | Isolation of NO 3 --N as 1-phenylazo-2-naphthol (Sudan-1) for measurement of δ 15 N[END_REF]. Enrichment with 15 N was calculated above the background control containing MOPS buffer, 0.5 mM 15 N-nitrate, 0.5 mM 14 N-nitrite and 0.8 % (w/v) glucose. A sample or control (200 μl) was diluted with ultra-high-purity water (18 MΩ, Ultra Pure, Elga, UK) to a final volume of 50 ml and the pH was adjusted to 8.2 with 1 ml of 25 % (w/v) ammonium chloride. Any nitrite in the diluted sample was diazotized with 400 μl of 0.4% (w/v) aniline chloride (diazotization reagent) in 3 M HCl (final pH 2.0). After 5 min., 400 μl of 0.21 % (w/v) 2-naphthol (coupling reagent) in 3 M NaOH (final pH 8.0) was added. The azo dye, Sudan-1 (1-phenylazo-2-napthol), was developed at 20˚C for 10 min. The pH of this mixture was then adjusted to between 5.0 and 6.0 using approximately 75 μl of 1M citric acid. C18 Extract-Clean columns (6 ml, 500 mg; Alltech Associates Applied Science Ltd., Carnforth, UK) were placed on a 20 position solid phase extraction manifold (Alltech, UK) and conditioned with 3 ml of ethyl acetate (analytical grade) for 1 min. The ethyl acetate was then removed under vacuum (0.35 bar) and each column was then washed (3 ml of 0.9 % w/v NaCl). Samples containing Sudan-1 were loaded onto a column and pulled through under vacuum (0.35 bar). Sudan-1 formed a discrete orange band at the top of the silica column which was then washed (50 ml of UHP water under vacuum) and eluted with 1 ml of ethyl acetate and collected in a 5 ml glass centrifuge tube. The Sudan-1 solution was dried overnight at 40-50 ˚C and resuspended in 200 μl of acetone, transferred to a 300 μl derivatization vial insert (2-DV; Chromacol, UK) and evaporated until approximately 20 μl of solution remained. This solution was then pipetted onto an ashed 4-mm GF/F filter disk and air-dried.

The filter disks were then placed into tin capsules for combustion and subsequent isotopic analysis by continuopus flow isotope ratio mass spectrometry (Flash EA 1112 coupled to Finnigan MAT Delta Plus , Thermo-Finnigan, Germany). Signals for m/z (mass charge ratio) were determined for mass 28, 29 and 30 and calibration was performed against known amounts of urea. The total accumulation of 15 N in nitrite was calculated as . These experiments allowed the quantities of nitrite expelled or further reduced to ammonia to be calculated.

N Δ 2 N Δ N Δ × + =

RESULTS

Strategies for identifying residues essential for transport functions of NarU

Five computer programmes, SPLIT, DAS, TMPRED, HMMTOP and TMHMM, all predict both NarK and NarU to be membrane proteins with 12 helices. The programme PREDTMR, however, failed to predict trans-membrane helix II within both NarK and NarU, possibly because of its lower hydrophobicity due to the presence within this helix of the arginine residue, R87 in E. coli NarU (Supplementary figure 1A). In preliminary experiments, we exploited the high level of expression of narK during anaerobic growth in the presence of nitrate to define a 12-helix model for NarU by constructing six in-frame fusions to both the β-galactosidase gene, lacZ, and to the alkaline phosphatase gene, phoA, using a plasmid-encoded narK gene as the template (Supplementary figure 1B). The ratio of β-galactosidase to alkaline phosphatase activities accumulated in E. coli strain ET8000 transformed with these plasmids was determined (Supplementary table S1). The combined data, especially the high alkaline phosphatase but low β-galactosidase activity of the fusions to P284, were consistent with the 12-helix model for NarK and NarU, but were inconsistent with models that predict fewer helices. Based on these results, a model of the topology of NarU was proposed and used to design site-directed mutagenesis experiments for functional analysis (Figure 1).

The strain used for most of the experiments in this study, JCB4520, lacks all three nitrate and nitrite transport proteins, NarK, NarU and NirC. It also lacks both Nap and NarZ, as well as the periplasmic nitrite reductase, Nrf. Consequently, this strain is unable to grow anaerobically in the presence of nitrate on a non-fermentable carbon source such as glycerol unless it is transformed with a plasmid encoding a functional nitrate transport protein. When glycerol is replaced by glucose, the strain grows as well as the parent in the absence of nitrate, but cannot augment glucose fermentation by nitrate respiration. Multicopy plasmids are available encoding NarU with or without a Myc tag (pJCB901 and pSJC901, respectively). The presence of the Myc tag has no affect on the ability of both plasmids to fully complement the defective nitrate transport phenotype of a narK narU double mutant [START_REF] Clegg | The roles of the polytopic membrane proteins, NarK, NarU and NirC, in Escherichia coli K-12: two nitrate and three nitrite transporters[END_REF] (reproduced in supplementary figure S2 E andF). The tagged proteins can be detected in Western blotting experiments using a commercial antibody [START_REF] Clegg | Role of the Escherichia coli nitrate transport protein, NarU, in survival during severe nutrient starvation and slow growth[END_REF]. The starting point for this study was therefore strain JCB4520 transformed with either of these plasmids (positive controls): the untransformed host provided a negative control.

NarU residues essential for membrane accumulation and all known transport functions

Moir and Wood [START_REF] Moir | Nitrate and nitrite transport in bacteria[END_REF] noted that few residues are totally conserved throughout cluster 6 of the major facilitator family of transport proteins that includes NarK and NarU. Site-directed mutagenesis was used to replace these residues by a range of other residues. Many of the substitutions were duplicated using two plasmid templates, plasmid pSJC901 that encodes unmodified NarU, but for which no antibody is available; and pJCB901 that encodes Myc-tagged NarU that can be detected by Western blotting using an anti-Myc antibody to detect whether the substituted protein has been incorporate into the membrane fraction. Modified plasmids were transformed into E. coli strain JCB4520, and the effects of each substitution on the ability of the modified NarU to support nitrate uptake required for growth on the non-fermentable carbon source, glycerol, and nitrite accumulation in the extra-cellular medium were determined. Each transformant was also grown in media supplemented with glucose and nitrate, bacteria were harvested, and rates of nitrate reduction were measured using a nitrate-specific electrode.

First we confirmed our previous report that both Myc-tagged NarU encoded by plasmid pJCB901 and NarU encoded by plasmid pSJC901 were equally able to complement all of these functions when transformed into E. coli strain JCB4520 (Table 2; [START_REF] Clegg | Role of the Escherichia coli nitrate transport protein, NarU, in survival during severe nutrient starvation and slow growth[END_REF]). No loss of function occurred when the conserved G414 in helix 11 was replaced by leucine, or when tyrosine 261 in helix 7 was replaced by glutamine (Table 2). Substitution of all of the 13 other conserved residues resulted in total loss of all three functions, with the following exceptions. The R87K substituted NarU supported slow nitrate-dependent growth and a slow rate of nitrite accumulation in the presence of glycerol and nitrate, although the rate of nitrate reduction by washed bacteria was insignificantly above the detection limit (Table 2; supplementary figures 2 and 3). All other substitutions in the totally conserved R87 and R303 were non-functional, consistent with the essential role of the conserved arginine residues in helices 2 and 8 of Aspergillus nidulans NrtA [START_REF] Unkles | Two perfectly conserved arginine residues are required for substrate binding in a high-affinity nitrate transporter[END_REF]. Proline, like glycine, is sometimes conserved in a membrane helix to introduce flexibility required for substrate transport through the membrane [START_REF] Yernool | Structure of a glutamate transporter homologue from Pyrococcus horikoshii[END_REF][START_REF] Trainer | Role of a conserved membrane glycine residue in a dicarboxylate transporter from Sinorhizobium meliloti[END_REF][START_REF] Javadpoor | Helix packing in polytopic membrane proteins: role of glycine in transmembrane helix association[END_REF]. Although substitution of P113 by cysteine or leucine resulted in partial loss of function, this proline is clearly not essential because all of the transport activities were retained by the P113A derivative (Table 2).

Many of these and other substitutions were then introduced into NarU::Myc encoded by plasmid pJCB901 (Table 2). Without exception, similar concentrations of all of the Myc-tagged NarU proteins with substitutions at R87 or R303 were shown to accumulate in the membrane fraction at concentrations similar to unsubstituted NarU::Myc: representative data are shown in Supplementary figure 2A and2B. Glycines G172 and G175 are part of the extended nitrate signature motif in trans-membrane helix 5 [START_REF] Kinghorn | Missense mutations that inactivate the Aspergillus nidulans nrtA gene encoding a highaffinity nitrate transporter[END_REF]. Replacement of G172 by the bulkier valine resulted in failure to accumulate and hence, loss of function (Table 2). However, nitrate uptake and nitrite export functions were retained when glycine was replaced by alanine (Table 2 and supplementary figures 4A and 4C), indicating that a small residue was essential at this point of helix 5 for the correct folding and accumulation of NarU in the membrane. By contrast, substitution of G175 by either serine or alanine resulted in loss of function (supplementary Figures 4B and4D), despite the fact that the G175A protein was readily detected within the membrane by Western analysis (Supplementary figure 5). Replacement of G139 in helix 4 by arginine or glutamate also resulted in complete loss of function: incorporation of the G139I protein into the membrane was confirmed (Supplementary figure 5). G266 in helix 7 and G405 in helix 11 were totally essential for function even when these glycines were replaced by alanine (Table 2), and none of the variants G266T, P or A, or G405 L or A, were detected in membrane preparations by Western analysis (Supplementary Figure 5). Replacement of G162 by either serine or alanine or G307 by leucine resulted in complete loss of function due to failure to be incorporated into the membrane (Table 2 & supplementary Figure 5), suggesting that these residues might be conserved to introduce tight turns in short cytoplasmic loops between helices 4 and 5 and 8 and 9, respectively. In contrast, substitution of G99 by threonine resulted in only partial loss of function, but no loss of transport activity resulted from the G99A substitution.

In summary, results of the site-directed mutagenesis experiments were readily interpreted on the basis of the predicted topology of NarU (Fig. 1), and they revealed for the first time that the majority, but not all, of the highly conserved residues of NarU are for various reasons important or essential for all four known functions of NarU. This indicated that a single transport channel is likely to be used for nitrate uptake and nitrite extrusion.

NirC stimulates growth rate, nitrite extrusion, and nitrite uptake during anaerobic growth of a NarU + strain in the presence of nitrate

Having demonstrated that NarU can catalyse nitrate uptake, nitrite export, and nitrite uptake, we next investigated the physiological role of NirC during anaerobic growth in the presence of nitrate. During nitrate-dependent growth on the non-fermentable carbon source, glycerol, the NarU + NirC + strain, JCB4014, grew more rapidly and accumulated nitrite more rapidly than the isogenic NarU + NirC -strain, JCB4514 (Figure 2A,B). By the stationary phase of growth, more than 70% of the nitrate added to the medium had been accumulated as extracellular nitrite. The rate of growth of the NarU + NirC + strain in the presence of glucose was also more rapid than that of the NarU + NirC - strain, JCB4514, (Fig. 2C), and the maximum concentration of nitrite accumulated in the extracellular medium was again 70% of the initial concentration of nitrate (Fig. 2D). However, only the NirC + strain consumed nitrite rapidly during the later stages of growth (Fig. 2D). These results indicated that NirC can facilitate both extrusion of nitrite generated during nitrate reduction, and uptake of nitrite.

To demonstrate directly that NirC can catalyse more rapid nitrite uptake than NarU, strains expressing various combinations of narU and nirC were harvested after growth in the presence of glucose and nitrate. Rates of nitrite uptake and reduction were determined under conditions in which rates of nitrous acid diffusion into the bacteria were minimized, but the rate of nitrite reduction by the cytoplasmic nitrite reductase, NirB, was limited by the rate of nitrite uptake (Fig. 3; [START_REF] Clegg | Role of the Escherichia coli nitrate transport protein, NarU, in survival during severe nutrient starvation and slow growth[END_REF]). Rates of nitrite reduction by the NarU + NirC + and NarU -NirC + strains, JCB4014 and JCB4018 were in the range 45 to 65 nmol of nitrite reduced (mg dry bacteria) -1 . min -1 (Fig. 3). Nitrite was reduced at only a very low rate (<2 nmol of nitrite reduced (mg dry bacteria) -1 . min -1 ) by strain JCB4520 that lacks both NarU and NirC, but more rapidly by the NarU + NirC -strain, JCB4514 (6 nmol of nitrite reduced (mg dry bacteria) -1 . min -1 ). All of the substituted NarU proteins encoded by plasmid pSJC901 and many with substitutions in NarU::Myc encoded by plasmid pJCB901 were also checked for ability to restore nitrite uptake and reduction to strain JCB4520. There was complete correlation between ability to restore nitrite uptake and reduction, nitrate-dependent growth and nitrite export in the presence of glycerol, and rates of nitrate reduction after growth in the presence of glucose (data not shown).

These experiments established that both nitrite efflux and nitrite uptake are largely dependent on NirC. Conversely, in the absence of NirC, the ability of NarU to catalyse nitrite export rather than nitrate uptake limits the rate of nitrate uptake and growth. This raised the question whether nitrite formed during nitrate reduction is exported by NarU and NirC before it is re-imported for reduction in the cytoplasm by NirBD.

Direct evidence from 15 N isotope experiments that the nitrate-N is exported as nitrite before it is re-imported for reduction to ammonia

The combined data from the site-directed mutagenesis experiments and biochemical experiments with strains expressing different combinations of NarU and NirC indicated that NarU is primarily a nitrate uptake protein that can also catalyse a much slower rate of nitrite transport (Figure 3 and reference [START_REF] Clegg | The roles of the polytopic membrane proteins, NarK, NarU and NirC, in Escherichia coli K-12: two nitrate and three nitrite transporters[END_REF]). Nitrate labelled with 15 N was used to demonstrate that the nitrogen atom of nitrate is first expelled as nitrite before it is re-imported for reduction to ammonia.

Bacteria that had been grown in minimal medium supplemented with glucose and nitrate were harvested, washed, and incubated with 0.5 mM 15 N-nitrate, 0.5 mM 14 N-nitrite and 0.8% glucose. To minimise the diffusion of 14 N-HNO 2 across the membrane, MOPS buffer pH 8.5 was used for these experiments [START_REF] Jia | Nitrate and nitrite transport in Escherichia coli[END_REF]. The extra-cellular concentrations of nitrate, 15 A c c e p t e d M a n u s c r i p t nitrite concentration ( 14 N-nitrite plus 15 N-nitrite determined by direct chemical assay) during incubation at 37˚C were measured, and the concentration of 14 N-nitrite was calculated by difference (Figure 4).

There was an immediate decrease in total nitrite concentration when the NarU + NirC + strain JCB4014 was incubated with the equimolar mixture of nitrate and nitrite (Figure 4A). Despite this net decrease, 15 N-nitrite accumulated in the buffer at a rate of 19.6 nM . min -1. (mg of dry bacteria) -1 . This result suggested that 37% of the 15 N-nitrite produced was expelled from the cytoplasm, while 63% was reduced to ammonia at a rate of 33.4 nM. . min -1. (mg of dry bacteria) -1 . The total rate of nitrite reduction by the NarU + NirC + strain was 66.4 nM . min -1. (mg dry bacteria) -1 , which was calculated as 63% of the rate of nitrate reduction plus the net rate of decrease in extracellular nitrite concentration (33 nM . min -1. (mg dry bacteria) -1 ).

In contrast, during the initial 10 minutes of incubation of the NarU + NirC -strain JCB4514, the concentration of 15 N nitrate decreased at a rate of 17.6 nM min .-1. (mg dry bacteria) -1 , but extracellular 15 N nitrite accumulated in the external medium at a rate of 15.0 nM . min -1. (mg of dry bacteria) -1 (Fig. 4B). Thus, 87% of the nitrate reduced had accumulated extracellularly as 15 NO 2 -. Therefore, despite the presence of a highly active NirBD nitrite reductase, only 13% of the nitrate-N had been reduced to ammonia at a rate of 2.2 nM . min -1. (mg of dry bacteria) -1 . There was also a net increase in the total extracellular nitrite concentration during this period. Results from the two groups of experiments clearly showed that most of the nitrate-nitrogen was expelled as 15 N-nitrite before it could be re-imported via NirC for reduction in the cytoplasm to ammonia.

DISCUSSION

Surprisingly little is known about how nitrate is transported across bacterial membranes. Moir and Wood [START_REF] Moir | Nitrate and nitrite transport in bacteria[END_REF] noted that multi-component, ATP-dependent transport complexes are common in organisms that assimilate nitrate. In contrast, the NarK homologues of the major facilitator family of transport proteins are more common in denitrifying bacteria, or in bacteria that reduce nitrate to ammonia during anaerobic nitrate respiration. They also noted that the NarK family fall into three distinct groups, two sub-clusters of bacterial nitrate transporters, and the more distantly related fungal nitrate transporters. In preliminary experiments, the high level of expression of E. coli narK was exploited to confirm that NarK, and therefore also NarU, are similar to A. nidulans NrtA, with 12 membrane-spanning helices and N-and C-termini located in the cytoplasm. We also showed that R89 is essential for nitrate transport by NarK [START_REF] Feng | Transport of nitrate and nitrite across the Escherichia coli cytoplasmic membrane[END_REF]. However, during growth in the presence of nitrate, so much NarK accumulates that any residual transport activity following substitution of other amino acids was sufficient to support nitrate-dependent anaerobic growth [START_REF] Clegg | Role of the Escherichia coli nitrate transport protein, NarU, in survival during severe nutrient starvation and slow growth[END_REF]. Substitutions engineered into a plasmid-encoded narU gene therefore provided a more sensitive and reliable experimental system to investigate the effects of substituting highly-conserved residues on nitrate and nitrite transport. Despite the very wide range of substitutions studied, in no case did mutation result in loss of one transport function without simultaneous loss of all other functions.

Irrespective whether the functional nitrate transporter available was NarK or NarU, in this and previous work, nitrite accumulated quantitatively in the medium when washed bacterial suspensions were incubated in the presence of nitrate and formate, which cannot donate electrons to the cytoplasmic NirBD nitrite reductase [START_REF] Clegg | The roles of the polytopic membrane proteins, NarK, NarU and NirC, in Escherichia coli K-12: two nitrate and three nitrite transporters[END_REF][START_REF] Clegg | Role of the Escherichia coli nitrate transport protein, NarU, in survival during severe nutrient starvation and slow growth[END_REF]. However, during growth in the presence of glucose and nitrate, at least some of the nitrate imported was reduced to ammonia rather then accumulated in the surrounding medium. A critical question is therefore whether nitrite generated during nitrate reduction is immediately reduced in the cytoplasm by NirBD to ammonia. Alternatively, if NarK and NarU are simply nitrate / nitrite antiporters, this would require nitrite to be exported at least into the periplasm before it can subsequently be re-imported via NarK, NarU or NirC for reduction by NirBD. That the latter possibility is correct was shown both by physiological experiments in which the effects of NirC on nitrite accumulation and reduction were investigated, and from the results of experiments in which 15 N-nitrate and 14 N-nitrite were reduced simultaneously in the presence or absence of a functional NirC. The combined data from these experiments revealed that NirC is up to 10-fold more active than NarK or NarU in nitrite uptake for subsequent reduction in the cytoplasm by NirBD. Despite a net rate of nitrite uptake and reduction by the NirC + strain, 15 N-nitrite still accumulated in the extra-cellular medium. We conclude that nitrate uptake and reduction is mechanistically linked to nitrite expulsion. Some evidence that nitrate uptake by E. coli strains expressing either only narK or only narU is sensitive to inhibition by chemicals that collapse the membrane potential was obtained previously using a proteoliposome system [START_REF] Rowe | NarK is a nitrite extrusion system involved in anaerobic nitrate respiration by Escherichia coli[END_REF]. Furthermore, some bacteria synthesize two types of nitrate transport protein that fall into the two different sub-clusters of the NarK protein family [START_REF] Moir | Nitrate and nitrite transport in bacteria[END_REF][START_REF] Sharma | Involvement of NarK1 and NarK2 proteins in transport of nitrate and nitrite in the denitrifying bacterium Pseudomonas aeruginosa PAO1[END_REF]. Wood et al [START_REF] Wood | Two domains of a dual-function NarK protein are required for nitrate uptake, the first step of denitrification in Paracoccus pantotrophus[END_REF] proposed that one sub-cluster catalyses nitrate-nitrite antiport, the other nitrate / H + symport. We suggest that NarU constitutes a channel for both nitrate / H + symport and nitrite / H + export, processes that do not require strict stoichiometry between uptake and export, and accounts for how nitrate uptake can be initiated in the absence of pre-formed nitrite.

We are grateful to Dr Tim Overton for providing the myc-tagged vector for the construction of the C-terminal NarU fusion proteins; to Drs D. J. Slotboom and J. S. Lolkema for help predicting membrane spanning helices of the NarK-NarU protein family; to the Darwin Trust of Edinburgh for a research studentship for WJ; and to the UK Biotechnology and Biological Sciences Research Council for both a studentship for NT and project grant P20180 to fund this research.

Figure 3 Rates of nitrite uptake and reduction dependent upon NarU and NirC

The isogenic set of strains expressing all combinations of narU and nirC were grown in the presence of glucose and nitrate, harvested in the late exponential phase of growth, washed with MOPS buffer (pH 8.5) and resuspended in the same buffer. Low rates of nitrite reduction at pH 8.5 determined in the presence of formate were deducted from the rate in the presence of glucose to give the NirBD-dependent rate of nitrite uptake and reduction: these rates were typically 1 -2 nmol of nitrite reduced (mg dry bacteria) -1 . min -1 . Note that under the conditions used for these experiments, the rate of nitrite reduction is determined by the rate of nitrite uptake via NarU or NirC [START_REF] Clegg | The roles of the polytopic membrane proteins, NarK, NarU and NirC, in Escherichia coli K-12: two nitrate and three nitrite transporters[END_REF][START_REF] Jia | Nitrate and nitrite transport in Escherichia coli[END_REF].

Figure 4 15 N isotope experiments to demonstrate NarU-dependent export of nitrate-N as nitrite before it is re-imported for reduction to ammonia

The NarU + NirC + E. coli strain JCB4014 and the isogenic NarU + NirC -strain JCB4514 were grown in MS medium supplemented with glucose and nitrate, harvested, washed and resuspended in MOPS buffer, pH 8.5. These suspensions were incubated with 0.5 mM 15 N-nitrate, 0.5 mM 14 Nnitrite and 0.8% glucose. The concentrations of nitrate, 15 N-nitrite and total nitrite ( 14 N-nitrite plus 15 N-nitrite measured by direct chemical assay) were determined at intervals. The concentration of 14 N-nitrite was calculated as the difference between the concentrations of total nitrite and 15 N-nitrite. (A) The NarU + NirC + strain JCB4014 at a concentration of 0.96 mg dry mass . ml -1 . (B) The NarU + NirC -strain, JCB4514 at a concentration of 1.9 mg dry mass . ml -1 . Order of conserved residues: R87, G99, P113, G139, F145, G162, G172, G175, Y261, G266, R303, G307, D311, G405 and G414. Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

  N-nitrite and total 7 Biochemical Journal Immediate Publication. Published on 11 Aug 2008 as manuscript BJ20080746 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20080746

Figure 1

 1 Figure 1Location of conserved residues in E. coli NarU

Figure

  Figure 2Physiological role of NirC in nitrate-dependent growth and nitrite accumulation

Biochemical

  Journal Immediate Publication. Published on 11 Aug 2008 as manuscript BJ20080746 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20080746 A c c e p t e d M a n u s c r i p t

  Figure 1

Biochemical

  Journal Immediate Publication. Published on 11 Aug 2008 as manuscript BJ20080746 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20080746 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

Figure 2 0

 2 Figure 2

  Figure 3

2 Physiological role of NirC in nitrate-dependent growth and nitrite accumulation

  An isogenic set of strains expressing all combinations of narU and nirC were grown anaerobically in the presence of 20 mM nitrate, and the optical density and concentration of nitrite in the culture medium were determined at intervals. The strains used in these experiments and their relevant genotypes are JCB4014 narK narU + nirC + ; JCB4018 narK narU nirC + ; JCB4514 narK narU + nirC; and JCB4518 narK narU nirC. (A) and (B). The non-fermentable carbon source, glycerol was used for these experiments. As nitrate was the only available terminal electron acceptor, strains lacking both NarK and NarU are unable to grow in this medium. (C) and (D). Growth and nitrite accumulation of strains able to reduce nitrate in the presence of glucose and 20 mM nitrate. Note that, irrespective of the carbon source, both growth and nitrite export into the medium were stimulated by the presence of a functional NirC.

Table 1 Strains and plasmids used in this work

 1 Biochemical Journal Immediate Publication. Published on 11 Aug 2008 as manuscript BJ20080746 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20080746 A c c e p t e d M a n u s c r i p t

	Name	Genotype	Source
	Strains
	JCB4514		[9]
	JCB4518		[9]
	JCB4014		[9]
	JCB4018		[9]
	JCB4520		[9]
		Δ nrfA::chl
	ET8000	rbs lacZ::IS1 gyrA hutC K	[33]
	Plasmids
	pSJC901	3.5 kb PCR fragment containing the narU gene, ligated into pGEM®	[9]
		T Easy
	pJCB901	2.5 kb PCR fragment containing the narU gene and a myc tag,	[9]
		ligated into pGEM ® T Easy
	pSK4158	Chl R PhoA fusion vector	[19]
	pNM480	Carb R LacZ fusion vector	[20]

RK4353 Δ napA-B Δ narZ::Ω Δ narK::tet Δ nirC::cam RK4353 Δ napA-B Δ narZ::Ω Δ narK::tet Δ narU::kan Δ nirC::cam RK4353 Δ napA-B Δ narZ::Ω Δ narK::tet RK4353 Δ napA-B Δ narZ::Ω Δ narK::tet Δ narU::kan RK4353 Δ napA-B Δ narZ::Ω Δ narK::tet Δ narU::kan Δ nirC::cam Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

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

© 2008 The Authors Journal compilation © 2008 Biochemical Society

a. Strain JCB4520, deleted in narK, narU and nirC, was transformed with plasmids expressing mutated derivatives of NarU. Nitrate-dependent anaerobic growth with glycerol as carbon source and extrusion of nitrite were dependent upon nitrate and nitrite transport by the plasmid-encoded NarU protein.

b. Predicted locations are based on the proposed topology of NarU shown in Figure 1. TM3, transmembrane region 3; CL2/3, cytoplasmic loop between TM 2 and 3. c. pGEM-TEasy was the vector used to clone narU. d. NA: not applicable because no Myc-tag was attached. e. NA: not assayed because the substituted protein was functional, and therefore must have been incorporated into the membrane. f. Strain JCB4520 transformed with plasmid pSJC901 encoding wild-type NarU. g. Strain JCB4520 transformed with plasmid pJCB901 encoding wild-type NarU with a Myc tag.


Ph.D Sven Horke
email: horke@uni-mainz.de

Ines Witte

Petra Wilgenbus

Sebastian Altenhöfer

Maximilian Krüger

Huige Li

Ulrich Förstermann

Protective effect of paraoxonase-2 against ER stressinduced apoptosis is lost upon disturbance of calciumhomeostasis

Keywords: gene regulation / calcium / paraoxonase / apoptosis / calpain / endoplasmic reticulum , dithiotreitol, ER, endoplasmic reticulum, GRP78, glucose regulated protein of 78kDa, PON2, paraoxonase-2, ROS, reactive oxygen species, UPR, unfolded protein response pathway, UTR, untranslated region

Paraoxonase-2 (PON2) is a ubiquitously expressed anti-oxidative protein, which is largely found in the endoplasmic reticulum (ER). Addressing the cytoprotective functions of PON2, we observed that PON2 overexpression provided significant resistance to ER stress-induced caspase-3 activation when the ER stress was induced by interference with protein modification (by tunicamycin or dithiotreitol), but not when ER stress was induced by disturbance of Ca 2+ homeostasis (by thapsigargin or A23187). When analyzing the underlying molecular events, we found an activation of the PON2 promoter in response to all tested ER stress-inducing stimuli. However, only tunicamycin and dithiotreitol resulted in increased PON2 mRNA and protein levels. In contrast, when ER stress was caused by thapsigargin or A23187, we observed a Ca 2+ -dependent active degradation of PON2 mRNA, elicited by its 5'untranslated region. In addition, thapsigargin and A23187 also induced PON2 protein degradation by a Ca 2+ -dependent calpain-mediated mechanism. Thus, we provide evidence that independent mechanisms mediate the degradation of PON2 mRNA and protein after disturbance of Ca 2+ homeostasis. Further, because Ca 2+ -disturbance induces ER stress, but abrogates the otherwise protective function of PON2 against ER stress-induced apoptosis, we propose that the underlying cause of ER stress determines the efficacy of putative cellular defence mechanisms.

Introduction

Upon synthesis, all proteins destined for transmembrane insertion or secretion translocate to the endoplasmic reticulum (ER). The ER is the site of protein amino-glycosylation, disulfide bond formation and proper folding of nascent proteins. In order to assist these processes, cells express molecular chaperones such as calnexin, glucose-regulated protein of 78 kDa (GRP78; BiP) and protein disulfide isomerase (PDI). Whereas formation and isomerisation of disulfide bonds is catalyzed by PDI [START_REF] Ellgaard | The human protein disulphide isomerase family: substrate interactions and functional properties[END_REF], calnexin governs proper protein structure by retaining incorrectly folded proteins in the ER [START_REF] Bergeron | Calnexin: a membrane-bound chaperone of the endoplasmic reticulum[END_REF]. Ample evidence indicates that various stimuli, e.g. deprivation of amino acids, disturbance of glucose-/ Ca 2+ -homeostasis or redox potential may interfere with protein maturation, which induces ER stress via the accumulation of unfolded proteins. This activates a conserved intrinsic stress response known as the unfolded protein response pathway (UPR). The UPR consists of three different orchestrated branches initiated by activation of ER stress transducers inositol-requiring protein 1 (IRE1), protein kinase RNA (PKR)-like ER kinase (PERK) and activating transcription factor-6 (ATF6); these enable cellular responses ranging from stress relieve to ER remodelling, protein degradation, autophagy and apoptosis [START_REF] Walter | Signal integration in the endoplasmic reticulum unfolded protein response[END_REF]. A key mediator of the UPR is GRP78. Although still under debate, one model of how ER stress is sensed suggests that under unstressed conditions, GRP78 binds to IRE1, PERK and ATF6 to keep them in an inactive state. Upon stress, however, the ER chaperone GRP78 is attracted by and bound to unfolded proteins, which results in a loss of interaction of GRP78 with IRE1, PERK and ATF6 [START_REF] Shen | ER stress regulation of ATF6 localization by dissociation of BiP/GRP78 binding and unmasking of Golgi localization signals[END_REF][START_REF] Bertolotti | Dynamic interaction of BiP and ER stress transducers in the unfolded-protein response[END_REF]. This activates these integral membrane proteins that thereby sense the folding status in the ER lumen and link this information to cytosolic events. As such, IRE1 performs unusual splicing of X-box-binding-protein-1 (XBP1) mRNA, PERK phosphorylates eIF2a protein and ATF6 is proteolytically processed in order to release a soluble cytosolic portion that acts as transcription factor [START_REF] Schroder | The mammalian unfolded protein response[END_REF]. As a consequence, general translation is diminished and gene expression is rapidly altered in order to alleviate stress or undergo apoptosis. In vivo relevance of the UPR is highlighted by the fact that this pathway is critically involved in several diseases [START_REF] Zhang | The unfolded protein response: a stress signaling pathway critical for health and disease[END_REF], such as Alzheimer [START_REF] Kudo | The unfolded protein response is involved in the pathology of Alzheimer's disease[END_REF], apoptosis of pancreatic β-cells [START_REF] Kharroubi | Free fatty acids and cytokines induce pancreatic beta-cell apoptosis by different mechanisms: role of nuclear factor-kappaB and endoplasmic reticulum stress[END_REF], diabetes mellitus and atherosclerosis [10,11]. The latter is characterized by an enhanced production of reactive oxygen species (ROS) and inflammatory responses of vascular cells and macrophages, both of which contributes to the pathophysiology of atherosclerosis [12][13][14]. For vascular endothelial cells it is known that oxidized low density lipoprotein particles (oxLDLs) or bioactive components thereof can induce oxidative stress [15], ER stress and inflammatory cytokine production [16]. Similarly, it was shown that accumulation of free cholesterol, which may represent a likely cause of cell death in later stages of atherosclerotic lesions, causes macrophage apoptosis via ER stress [17]. Thus, an understanding of underlying molecular mechanisms is mandatory, as it determines UPR induced gene expression, regulation of molecular stabilities as well as pro-and anti-apoptotic factors. We recently described that another cellular enzyme, paraoxonase-2 (PON2) is involved in ER stress [18]. The paraoxonase family of proteins consists of three highly similar enzymes, PON1, PON2 and PON3 [19] with characterized enzymatic activities [20,21]. Although it is known that PON2 plays a dominant role in the inactivation of certain lactone(s) involved in quorum sensing of Pseudomonas aeruginosa [22,[START_REF] Stoltz | Paraoxonase-2 deficiency enhances Pseudomonas aeruginosa quorum sensing in murine tracheal epithelia[END_REF], natural endogenous substrates remain relatively elusive. In the case of PON2, we reported the existence of two distinct, glycosylated splice-variants [18]. In vivo studies using transgenic and / or deficient mice revealed anti-atherogenic properties of all three PONs [24][25][START_REF] Ng | Adenovirus mediated expression of human paraoxonase 2 protects against the development of atherosclerosis in apolipoprotein E-deficient mice[END_REF][START_REF] Ng | Adenovirus-mediated expression of human paraoxonase 3 protects against the progression of atherosclerosis in apolipoprotein Edeficient mice[END_REF]. In accordance, we and others reported that PON2 significantly reduced intracellular oxidative stress [18,[START_REF] Ng | Paraoxonase-2 is a ubiquitously expressed protein with antioxidant properties and is capable of preventing cell-mediated oxidative modification of low density lipoprotein[END_REF]. Although regulation of PON2 expression remained largely enigmatic, it is known that PON2 expression is induced by both ER stress [18] and oxidative stress [29] and appears to be regulated, at least in part, by PPARγ and AP1 pathway activation [START_REF] Shiner | Macrophage paraoxonase 2 (PON2) expression is up-regulated by pomegranate juice phenolic anti-oxidants via PPARgamma and AP-1 pathway activation[END_REF]. Prompted by the observation that PON2 overexpression rendered endothelial cells significantly more tolerant to ER stress-induced apoptosis (i.e. caspase-3 activation), which was opposed after down-regulation of PON2 [18], we asked as to whether PON2 was generally protective against ER stress-induced apoptosis. We here report that the protective and anti-apoptotic effect of PON2 was not a general phenomenon; it was absent, when the ER stress was induced by Ca 2+ -deregulation. The current study was designed to uncover the underlying molecular events leading to a loss of protection by PON2 under specific ER stress conditions.

Biochemical

hy 926 cells and UPR induction

Human endothelial EA.hy 926 cells were cultured in Dulbeccos modified eagle medium (without phenol red; Sigma) containing Na-pyruvate (PAA), antibiotics penicillin / streptomycin, HAT supplement, Glutamax (Invitrogen) and 10% fetal calf serum (Perbio). Cells were cultured at 37°C in a humidified atmosphere at 10% CO 2 . Medium for cultivation of EA.hy 926 cells stably overexpressing PON2-isoform1-green-fluorescent-protein (PON2-iso1-GFP) or PON2-isoform1-hemagglutinin (PON2-iso1-HA) [18] contained geneticin (300 µg/ml; PAA) in addition. Cells were confluently seeded in 96-well plates (2.0 x 10 4 /well) or 24-well plates (1 x 10 5 /well). The next day, DTT (solved in water), tunicamycin (Sigma), thapsigargin (Invitrogen), A23187 (Sigma) or solvent control dimethylsulfoxid (DMSO) was added to fresh culture medium. Maximum DMSO concentration did not exceed 0.5%. Efficient UPR induction was verified in every experiment by confirming enhanced expression of GRP78 mRNA and / or protein.

Inhibitors, cell lysis and Western blotting

Cells were seeded and treated as outlined above. Unless otherwise indicated, the following inhibitors (or solvent control) were supplemented for 1 h in complete growth medium before addition of UPR inducing compounds: proteasome-inhibitors lactacystin (10 -50 µM; Cayman Chemicals) or MG132 (10 µM); autophagy-inhibitor 3-methyladenine (3-MA; 1 -5 µM); calpain inhibitor-I N-acetyl-Leu-Leu-norleucinal (ALLN; 1 -5 µM); cycloheximide (5 µg/ml); actinomycin D (5 µg/ml)(all from Sigma); BAPTA/AM (10µM; Calbiochem). Inhibitors remained present throughout subsequent addition of UPR inducing compounds. At the indicated time points, medium was aspirated, cells were washed with phosphate-buffered saline and lysed by addition of ice-cold lysis buffer (100 mM Tris/HCl pH 7.4, 200 mM NaCl, 2% Triton X-100, both protease and phosphatase inhibitor cocktails Complete and PhosphoStop (Roche)), followed by 20 min rocking at 4°C and mechanical disruption. Finally, lysates were cleared by centrifugation (30 min, 12,000 x g, 4°C). Protein content was determined using the BCA protein assay reagent (Pierce). Western blotting was performed as described earlier [18]. The following primary antibodies (all anti human) were used: mouse monoclonal-anti-α-tubulin (Ab-2 DM1a; Dianova), goat anti-GRP78 (C-20; Santa Cruz) and rabbitanti-PON2 [18].

Cloning of the PON2 5'untranslated region (UTR)

Earlier we described the generation of a pGL4.10 plasmid (Promega) containing genomic DNA of the PON2 gene ranging from 2249bp upstream of the 5'end to position +112 [18]. This plasmid was digested with restriction enzyme Nco-I, which cuts out a 147bp fragment (containing parts of Exon-1, Intron-1/2 and some vector backbone); religation of the larger fragment (6444bps) results in a plasmid harbouring the PON2 promoter fragment (-2249 to -1), directly followed by the putative PON2 5'UTR and the firefly luciferase gene. Thus, the putative PON2 5'UTR was cloned based on the Genatlas / Gene database; its sequence consists of 32bps 5'-GGAGCGAGGCAGCGCGCCCGGCTCCCGCGCC-3'. Because this sequence was cloned according to public database entries, it may not represent the complete PON2 5'UTR.

Cloning of the PON2 3'UTR by rapid amplification of cDNA ends (3'RACE)

Using EA.hy 926 total RNA, PON2 mRNA 3'UTR was amplified by 3'RACE with primers GAGAGATCTAGACTCGTCAGAGGTTCTCCGCATCCAG and CATCCTGGTCGAGCTGGGGCCGGCCTTTTTTTTTTTTTTTTTTTTTTTTTG with the Superscript-II RT-PCR system (Invitrogen) according to the supplier's instructions. Amplified DNA was gel-purified, blunt ends generated using Klenow-fragment, the DNA inserted into zero-blunt pCR4-TOPO plasmid (Invitrogen) and sequenced. The amplified sequence contains the 3'UTR and a limited base pair number of the coding sequence. From this plasmid, the 3'UTR was amplified by PCR using the same reverse primer as before together with the sense-primer GACAGACATCTAGAATTGTACTTTTGGCATGAAAGTGCG. The amplified 3'UTR was inserted into the plasmid pGL4-PON2-promoter [18] C-terminal to the luciferase gene by standard cloning procedures using restriction enzymes FseI and XbaI followed by seqencing.

RNA extraction, quantitative real-time RT-PCR (qRT-PCR) and mRNA decay analysis

To analyze mRNA decay, cells were solvent-treated (control) or were pre-treated for 4h with the UPR inducing compound as indicated in the figure legends. Then, 5,6-dichlorobenzimidazole 1-β-Dribofuranoside (DRB; 60 µM; Sigma) or actinomycin D (5 µg/ml) was added to inhibit RNA synthesis. UPR inducing compounds remained present throughout this treatment. At the indicated time points, total RNA was extracted from EA.hy 926 cells cultured in 24-well plates using E.Z.N.A. total RNA kit (Omega Bio-tek Inc) according to the suppliers instructions. Then, 80 ng RNA was used for cDNA generation using High Capacity cDNA Reverse Transcription kit (Applied Biosystems) according to the suppliers instructions. Finally, RNA expression levels were quantitatively analyzed using 2 µl cDNA, 1 U Taq-polymerase (PeqLab), 0.4 µl 10 mM dNTPs and 2 µl per primer per 20 µl reaction in a Bio-Rad iCycler (Bio-Rad). Pre-designed, validated Taqman primers for detection of PON2 or GRP78 were purchased from Applied Biosystems. Additionally, the following Taqman primers (synthesized by MWG) were used: for spliced XBP1 GGAGTTAAGACAGCGCTTG (sense); GCACCTGCTGCGGACTC (antisense); GAAGCCAAGGGGAATGAAGT (probe); for glycerinealdehyde-3-phosphate-dehydrogenase (GAPDH) CAACAGCCTCAAGATCATCAGC (sense); TGGCATGGACTGTGGTCATGAG (antisense); CCTGGCCAAGGTCATCCATGACAAC (probe); for EGFP CTGACCCTGAAGTTCATCTGC (sense); AAGTCGTGCTGCTTCATGTG (antisense); TCGTGACCACCCTGACCTAC (probe); for firefly luciferase TGCAAAAGATCCTCAACGTG (sense); AATGGGAAGTCACGAAGGTG (antisense); AAGACCGACTACCAGGGCTT (probe); for renilla luciferase ATTGGTATGGGCAAATCAGG (sense); GGCCGACAAAAATGATCTTC (antisense); TCTTACTGCATGGTTTGAACTTCT (probe). qRT-PCR was performed as described earlier [18]. All mRNA levels were normalized to GAPDH mRNA (or renilla luciferase mRNA where indicated) from the very same samples and calculated by the 2 [-∆∆C(T)] method. When analyzing the function of PON2 mRNA UTRs, cells were co-transfected with a plasmid allowing for constitutive renilla luciferase expression and the indicated pGL4-PON2-promoter plasmids (including the 3'-or 5' UTR). After the specific treatment, RNA was prepared as above, but additional extensive DNase-I oncolumn digestion (Omega Bio-tek Inc.) was performed. Principally, this method is somewhat susceptible to unwanted amplification of remaining plasmid DNA in RNA preparations. Therefore, we controlled effective plasmid digestion by including RNA preparations without DNase-I digestion in every experiment. In subsequent qRT-PCR analyses, these differed by a factor of >20 (data not shown).

Software, statistics and image acquisition

Device specific software provided by the suppliers was used. In addition, GraphPad Prism software was used for any calculations of collected data, for non-linear regression curve fitting or statistical evaluation using 1-way ANOVA with Bonferronis multiple comparisons post-test (see figure legends). A probability level of 0.05 was considered significant. Adobe Photoshop software was used for image acquisition of scanned Western blots. If at all, solely brightness and / or contrast were changed and adjusted simultaneously for all lanes and areas of any blot.

All other relevant materials such as plasmids, promoter reporter analyses and caspase-3/7 activity determination have been described recently [18].

Results

PON2 protects against ER stress-induced apoptosis unless ER stress is caused by disturbance of Ca 2+ -homeostasis

Prolonged ER stress can induce apoptosis, which can be monitored by the activity of executioner caspase-3. Here we determined the effect of PON2 overexpression on caspase-3/7 activation in response to a 16h-treatment with varying concentrations of different ER stress inducing compounds, namely tunicamycin, DTT or thapsigargin. These induce ER stress via distinct mechanisms: DTT obliterates protein structures, tunicamycin interferes with protein amino-glycosylation and thapsigargin disturbs cellular Ca 2+ homeostasis. As control, we first determined the maximum caspase-3 activation by treating naïve EA.hy 926 cells with pro-apoptotic staurosporin (Figure 1A). Within the concentrations tested, 0.3 µM staurosporin resulted in the highest (approximately 5-fold) stimulation. We then treated EA.hy 926 cells with increasing concentrations of DTT and observed concentration-dependent caspase-3 activation (Figure 1B). In cells stably overexpressing PON2-iso1-GFP or PON2-iso1-HA, maximum DTT-induced caspase-3 activation was markedly reduced. Similar results were obtained with tunicamycin (Figure 1C). Here, PON2 overexpressing cells showed only very limited caspase-3 activation. However, different results were obtained for thapsigargin-induced caspase-3 activation. PON2 overexpression somewhat decreased the potency of thapsigargin to induce caspase-3 activation, but the maximum effect was not significantly reduced (Figure 1D).

Stimulators of ER stress enhance PON2 promoter activity

Addressing underlying molecular events causing loss of protection of PON2 against thapsigargin-, but not tunicamycin-or DTT-induced caspase-3/7 activation, we analyzed the relative alterations in PON2 gene expression caused by the tested compounds. We recently described the generation of a plasmid harbouring a firefly luciferase gene under the expressional control of a PON2 promoter fragment (pGL4-PON2-promoter; corresponding to region -2249 to -1), which was used for reporter gene analyses [18]. Within this promoter fragment, sequences with high homology to ER stress-responsive cis-acting elements can be found, such as ER stress element (ERSE)-like sequence [START_REF] Yoshida | Identification of the cis-acting endoplasmic reticulum stress response element responsible for transcriptional induction of mammalian glucose-regulated proteins. Involvement of basic leucine zipper transcription factors[END_REF] and the UPR element [START_REF] Wang | Activation of ATF6 and an ATF6 DNA binding site by the endoplasmic reticulum stress response[END_REF]. This plasmid was used to evaluate the effect of different UPR inducing compounds on PON2 promoter activation. Transiently transfected human endothelial EA.hy 926 cells were treated for 16h with varying concentrations of DTT, tunicamycin or thapsigargin. A concentration dependent increase in promoter activity was observed with all three substances (Figure 2). With the highest concentrations tested, a 5-fold (tunicamycin; Figure 2B) to 9-fold (DTT, thapsigargin; Figures 2A,C) enhancement was observed. Interestingly, there was no decline in promoter activity even with concentrations that evidently induced pro-apoptotic executioner caspase-3 (see Figure 1). Non-specific induction of the PON2 promoter by serum components was excluded by the similar induction levels observed in the absence or presence of serum (Figure 2D).

Tunicamycin and DTT induce whereas thapsigargin declines PON2 mRNA expression

We next determined the effect of the UPR inducers on endogenous PON2 mRNA levels. EA.hy 926 cells were treated with constant concentrations of DTT (2.5 mM) or tunicamycin (1 µg/ml) and samples were taken at different time points for PON2 mRNA quantification by real-time qRT-PCR. UPR induction was verified by significantly increased levels of endogenous GRP78 mRNA (not shown), whose induction is considered characteristic of ER stress. Figure 3A shows that DTT led to a moderate increase (~180%) of endogenous PON2 mRNA in a time-dependent fashion. Similarly, tunicamycin treatment caused enhanced PON2 mRNA expression (Figure 3B). Here, PON2 mRNA levels increased approximately 3-fold after six hours. Upon exposure to thapsigargin, however, the reverse result was obtained. Specifically, we observed both a time-dependent and concentrationdependent decline in PON2 mRNA levels after thapsigargin treatment (Figures 3C+D,respectively). Hence, although DTT, tunicamycin and thapsigargin equally induce both the UPR pathway and PON2 promoter, they show opposed effects on endogenous PON2 mRNA levels. Principally, a thapsigargin-induced decrease in PON2 mRNA levels may result from both a discontinued transcription and an active degradation. The latter was tested by analyzing PON2 mRNA stability. To this end, cells were pre-treated with lowered, non-cytotoxic concentrations of DTT (2.5 mM), tunicamycin (0.5 µg/ml) or thapsigargin (1 nM) for 4 hours to induce the UPR followed by inhibition of RNA synthesis by addition of DRB. In parallel, basal PON2 mRNA decay in indigenous EA.hy 926 cells was monitored. These analyses revealed a surprisingly long half-life of PON2 mRNA. Even 24 hours after inhibition of RNA synthesis by DRB, on average 70% of PON2 mRNA remained present (Figure 4). Similar results were obtained using actinomycin D (see below). This is in agreement with our observation of a relatively long half-life (approx. 2-3 days) of PON2 protein, which was seen in experiments based on treatment with RNA interference (not shown) or cycloheximide (see below). UPR induction in EA.hy 926 cells either by DTT or tunicamycin (Figures 4A+B,respectively) did not significantly change decay of endogenous PON2 mRNA. UPR induction by thapsigargin, however, resulted in a significantly faster decay of PON2 mRNA (Figure 4C). Already at the first time point analyzed (4 hours), PON2 mRNA was degraded by approx. 25%, whereas no significant decay was observed at this time point with the other conditions tested. Thus, in contrast to DTT or tunicamycin, thapsigargin treatment of EA.hy 926 cells induced an active degradation of PON2 mRNA.

A disturbed Ca 2+ -homeostasis, but not the secondary activation of the UPR pathway causes PON2 mRNA decay Thapsigargin blocks the sarco-/endoplasmic reticulum calcium-ATPase SERCA, which depletes the ER calcium storage thereby causing ER stress. We next investigated whether the observed PON2 mRNA decay upon thapsigargin treatment results from the primary stimulus, namely a deregulated intracellular Ca 2+ -signalling, or from a secondary thapsigargin-specific UPR induction. For this purpose, naïve EA.hy 926 cells were treated for 16h with thapsigargin (10 nM) or the potent cytosolic Ca 2+ -chelating compound BAPTA/AM (10 µM), or combinations thereof. While BAPTA/AM treatment by itself resulted in somewhat reduced PON2 mRNA levels, it clearly inhibited the thapsigargin-induced degradation of PON2 mRNA (Figure 5A). The UPR pathway was still activated in the thapsigargin+BAPTA/AM treated samples as evidenced by more than 15-fold and 11-fold increases in GRP78 mRNA and spliced X-box binding protein-1 (sXBP1) mRNA, respectively (Figures 5B+C). This suggests that PON2 mRNA degradation results from deregulated Ca 2+ -signalling rather than the UPR. Therefore we explored if a mechanistically different disturbance of intracellular Ca 2+ -homeostasis results in similar effects. Indeed, EA.hy 926 cells treated with the calcium ionophore A23187 showed significantly reduced PON2 mRNA levels, which could be inhibited by BAPTA/AM (Figure 5D). Similar to thapsigargin, also A23187 produced a marked enhancement of GRP78 and sXBP1 mRNA levels, even in the presence of BAPTA/AM (more than 16-fold and 18-fold, respectively; Figures 5E+F).

Next we examined if the PON2 mRNA Ca 2+ -responsive element is located in the untranslated region(s) (UTR) or e.g. the coding sequence. To this end, we performed 3'RACE experiments and cloned the 3'UTR of PON2, as well as the 32bps-long 5'UTR (according to public database entries) into the above mentioned pGL4-PON2-promoter plasmid. As a result, this plasmid was modified such that the encoded firefly luciferase mRNA was N-or C-terminally fused to the 5'UTR or 3'UTR of endogenous PON2 mRNA, respectively. Basal expression was comparable for all constructs and was independent from the addition of 5' or 3'UTRs (not shown). As shown in Figure 5G, thapsigargin treatment enhanced PON2 promoter activity and resulted in a marked increase of firefly luciferase mRNA (in agreement with firefly luciferase activity, Figure 2C). However, after PON2 5'UTR was fused to firefly luciferase mRNA, thapsigargin caused pronounced destabilization of this unrelated mRNA (Figure 5G). The 3'UTR of PON2 showed no such effect (Figure 5G). Also, the Ca 2+independent stressor tunicamycin did not induce decay of the firefly luciferase mRNA independent of the addition of the PON2 5' or 3'UTR (Figure 5H). Thus, we presumed that the Ca 2+ -responsive element of endogenous PON2 mRNA is located in its 5'UTR. In agreement with this assumption, firefly luciferase mRNA levels in thapsigargin-treated cells overexpressing the 5'UTR construct were reduced to less than control levels (~80% in thapsigargin treated relative to untreated cells; Figure 5G). As further evidence for the occurrence of an Ca 2+ -responsive element in the 5'UTR of PON2 mRNA we show that thapsigargin treatment did not cause degradation of overexpressed PON2-GFP mRNA (Figure 5J), which solely contains the PON2 coding sequence without UTRs.

DTT and tunicamycin enhance PON2 protein levels, whereas disturbed Ca 2+ -homeostasis causes its degradation

To test if disturbed Ca 2+ -homeostasis also results in PON2 protein degradation, similar experiments as above were performed and protein levels were controlled by Western blotting. As shown recently [18] and in Figure 6A, DTT (2.5 mM) treatment of EA.hy 926 cells resulted in a moderate, time-dependent increase in PON2 protein expression. UPR activation was verified by probing for GRP78 (not shown). Similarly, tunicamycin treatment caused enhanced PON2 protein expression in a concentration-and time-dependent manner (Figures 6B+C,respectively). Since both isoforms of PON2 are glycosylatedwhich is inhibited by tunicamycin -newly synthesized PON2 isoforms 1 + 2 appear with lower apparent molecular weights. De novo synthesis of these isoforms is indicated by the fact their emergence is obliterated by cycloheximide (Figure 6C). Importantly, no degradation of PON2 protein A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

was observed even with concentrations of tunicamycin that strongly activate pro-apoptotic caspase-3 (Figure 1C). By contrast, thapsigargin treatment resulted in a concentration-dependent degradation of PON2 protein (Figure 7A). This was partially inhibited by pre-treatment with BAPTA/AM (Figure 7B). Similar to the results obtained for PON2 mRNA, A23187 mimicked the thapsigargin-mediated effects on PON2 protein; A23187 treatment also caused PON2 degradation, which was mostly prevented by BAPTA/AM (Figures 7C+D). Finally, we investigated the underlying mechanism of this active PON2 degradation. Thapsigargin-or A23187-mediated increases in cytosolic Ca 2+ -concentrations may give rise e.g. to proteasomal degradation of proteins, calpain protease activation or autophagy. In order to distinguish between these possibilities, EA.hy 926 cells were treated for 16h with solely 10 nM thapsigargin or together with inhibitors of proteasome activity (MG132, 10 µM, not shown) and lactacystin (50 µM), calpain inhibitor-I (N-acetyl-Leu-Leu-norleucinal, ALLN, 5 µM) or an inhibitor of autophagy (3-methyladenine, 3-MA, 5 µM). As shown in Figure 7E, thapsigargin-induced decay of PON2 did not result from proteasomal activity or autophagy, because the well-known and potent inhibitors lactacystin and 3-MA did not prevent PON2 degradation. By contrast, we show that PON2 protein is subject to Ca 2+ -activated calpain protease activity, because the calpain inhibitor-I ALLN inhibited thapsigargin-induced PON2 decay. In summary, PON2 protein stability negatively correlates with increased cytosolic Ca 2+ -concentrations and calpain activation. Similar to endogenous PON2, this also applies to stably overexpressed PON2-GFP, which is degraded upon thapsigargin exposure in a time-dependent manner (Figures 7F+G,respectively). Thus, the degradation of both endogenous and overexpressed PON2 in response to Ca 2+ -disturbances elucidates the cause of lack of protection by PON2 overexpression against thapsigargin-induced apoptosis. To demonstrate the close correlation between thapsigargin-induced PON2 mRNA-and protein degradation on the one hand, and caspase-3 activation on the other hand, these parameters were plotted in one graph in Figure 7H.

Discussion

ER stress is critically involved in the pathogenesis of diseases such as diabetes [10] or neurodegenerative disorders [START_REF] Zhang | The unfolded protein response: a stress signaling pathway critical for health and disease[END_REF]. In atherogenesis, enhanced uptake of free cholesterol by macrophages has been shown to induce ER stress [START_REF] Feng | The endoplasmic reticulum is the site of cholesterol-induced cytotoxicity in macrophages[END_REF]. Due to the central position of the ER and the UPR, there is significant interest in factors modulating the cellular pro-and anti-apoptotic response to ER stress. Since the underlying cause of ER stress appears critical for the cellular response, we investigated whether the recently discovered anti-apoptotic function of PON2 was applicable for several forms of ER stress or if it was dependent on the inducing stimulus and if different causes of the stress similarly affected PON2 gene expression. Stable overexpression of PON2 decreased caspase-3 activation stimulated by DTT or tunicamycin, but not that stimulated by thapsigargin. Most probably, this is due to the fact that deregulation of cytosolic Ca 2+ -levels in response to thapsigargin or A23187 caused a rapid degradation of both endogenous and even the overexpressed PON2. Because even strongly pro-apoptotic concentrations of tunicamycin did not result in PON2 degradation, this implies that tunicamycin-induced apoptosis does not elicit a secondary rise in cytosolic Ca 2+ , at least not to the extent caused by yet low concentrations of thapsigargin or A23187. Alternatively, our data could suggest that the protective effect of PON2 lies upstream of caspase-3 and that PON2 does not e.g. interfere with caspase-3 activation directly. In addition, it appears unlikely that UPR-accompanying ROS production is the underlying common proapoptotic mechanism, as this would probably be mostly reduced by overexpression of anti-oxidative PON2. Moreover, it may suggest that PON2 has different effects on the pro-apoptotic pathways activated by the tested compounds. Thapsigargin mediates apoptosis by mitochondrial caspasedependent death cascades, Ca 2+ -dependent anti-apoptotic phosphatidylinositol 3-kinase / MAPK activation [34] and depends on enhanced nitric oxide synthase activity [35]. Tunicamycin-induced cell death, in contrast, is facilitated rather by ER specific apoptosis pathways, i.e. by ER-resident protein calnexin [36] or caspase-2 /-9 activation [START_REF] Cheung | Involvement of caspase-2 and caspase-9 in endoplasmic reticulum stress-induced apoptosis: a role for the IAPs[END_REF]. Due to its ER localization [18,38], PON2 may primarily interfere with ER derived apoptotic pathways. In line with this, neither PON2 overexpression nor knock-down altered nitric oxide production in EA.hy 926 cells (M.K. & S.H.; unpublished observations), which is considered essential for thapsigargin-induced apoptosis [35]. When addressing ER stress-induced alterations in PON2 gene expression, we found that a heterogeneous group of compounds whose common denominator is the induction of ER stress all stimulated PON2 promoter activity. Provided the PON2 promoter 2249-bp fragment contains all relevant regulatory sites, our data indicate that transcriptional activation of UPR-responsive genes is the consequence of a secondary UPR pathway activation and is independent of the UPR-inducing stimulus. ER stress responsive promoter elements such as ERSE, ERSE2, UPRE or others are not assigned to specific forms of stress (e.g. disturbed Ca 2+ -homeostasis vs. hindered protein modification). PON2 induction thus differs from the UPR-mediated induction of upstream, ER stressresponsive transcriptional activators such as ATF6 and XBP1, which appears stimulus-specific [39]. Additionally, ER stress transducers such as IRE1, PERK, ATF6 and XBP1 are activated with different time kinetics in order to meet cellular requirements for protein refolding / degradation, or apoptosis [40,41]. At the level of mRNA turn-over and / or translation, however, mechanism(s) evidently exist that can distinguish between DTT-, tunicamycin-, thapsigargin-or A23187-induced ER stress. These mechanism(s) may sense whether the increased expression of a specific protein is beneficial for stress relieve and may regulate the mRNA / protein stability accordingly. This is in line with a global profiling of ER stress-regulated mRNAs, where clear differences were found between thapsigargin or tunicamycin treatment of the same cells [START_REF] Kawai | Global mRNA stabilization preferentially linked to translational repression during the endoplasmic reticulum stress response[END_REF]. In the current study, all ER stress-inducing compounds tested resulted in a transcriptional activation of the PON2 gene. As expected, DTT and tunicamycin moderately increased PON2 mRNA and protein levels. In contrast, thapsigargin and A23187 produced a pronounced degradation of endogenous PON2 mRNA and protein. It is currently unclear which RNase activity (or other destabilizing mechanism) mediates the decay of PON2 mRNA. One candidate protein could be IRE1, which possesses RNase activity and was recently found to mediate the ER stress-induced degradation of specific mRNAs in Drosophila [43]. IRE1 activation in our experimental setup was evidenced by markedly increased levels of spliced XBP1 mRNA. However, because BAPTA/AM prevented PON2 mRNA degradation, but not UPR induction or IRE1 activation, it appears unlikely that IRE1 fulfils this function in our model. Rather, increased cytosolic Ca 2+ -levels may activate mechanisms destabilizing PON2 mRNA. Ca 2+ is known to regulate mRNA stability through stabilizing or destabilizing proteins such as HuR or AUF-1 [START_REF] Misquitta | Control of protein expression through mRNA stability in calcium signalling[END_REF]. Interestingly, these proteins mostly act on UTRs, which is in line of our finding that Ca 2+ -induced PON2 mRNA decay depends on the 5'UTR. Alternatively, Ca 2+ -stimulated RNase L [45] may represent a mechanism for PON2 mRNA decay. In contrast to PON2, it has recently been shown that ER stress rather than cytosolic Ca 2+ upregulated HIF1-α mRNA [46]. In this context, it is to be noted that the PON2 mRNA 5'UTR mediated the degradation of an unrelated (firefly luciferase) mRNA upon cytosolic Ca 2+ -increase; however, this effect was less pronounced when compared to results obtained for the Ca 2+ -induced degradation of endogenous PON2 mRNA. Thus, one may speculate that additional sequences and / or structures found in the native PON2 mRNA may augment the destabilizing effect of the 5'UTR upon cytosolic Ca 2+ -increase. PON2 is a long-lived and stable protein (see Figure 6C). Nevertheless, thapsigargin (and also Ca 2+- ionophore A23187) stimulated an active, rapid and calpain-mediated degradation of this protein (Figure 7). This is in agreement with the finding that an increase in intracellular Ca 2+ can be a potent activator of cytoplasmic proteases, thereby contributing to cell death [47]. To our knowledge, it is unknown whether (patho)-physiological settings exist, in which Ca 2+ -stimulated down-regulation of PON2 is observed. Given that PON2 protects cells from apoptosis (at least under some ER stress conditions), its decay upon Ca 2+ -disturbance may contribute to the deleterious effects associated with various pathophysiological situations. Examples could include ischemia, which perturbs Ca 2+homeostasis and activates calpains [48], or the effects of oxysterols, which lead to increased cytosolic Ca 2+ -levels and endothelial cell death [49]. Therefore, it is mandatory to unravel the anti-apoptotic mechanism of PON2 and to explore if this is a general phenomenon or restricted to the UPR. We present several pieces of evidence indicating that two independent mechanisms mediate the degradation of PON2 mRNA and the destabilization of PON2 protein following disturbance of Ca 2+ homeostasis: (1) PON2 protein per se is very stable; protein levels were unchanged 24h after actinomycin D or 32h after cycloheximide (Figures 7F and 6C,respectively). Given this protein stability, an accelerated decay of PON2 mRNA in response to thapsigargin or A23187 cannot explain the parallel decay of PON2 protein (down to 42% after 16 h, Figure 7F); (2) PON2-specific siRNA results in a 90% reduction of PON2 mRNA after 1 day, but PON2 protein is not reduced before 3 days (data not shown); (3) PON2 protein degradation occurs independent of the mRNA's 5'UTR, because PON2-GFP protein is degraded after Ca 2+ -disturbance although the PON2-GFP mRNA lacks any UTR; [START_REF] Shen | ER stress regulation of ATF6 localization by dissociation of BiP/GRP78 binding and unmasking of Golgi localization signals[END_REF] We show that calpain proteases mediate Ca 2+ -stimulated PON2 protein decay. Taken together, our findings suggest that UPR-induced activation of the PON2 promoter is a relatively non-specific effect independent of the inducing stimulus. In contrast, the subsequent regulation of PON2 mRNA and protein stability seems to be specific for the stress-inducing mechanism(s). Increases in cytosolic Ca 2+ -concentrations lead to rapid degradation of both PON2 mRNA and protein by separate, unrelated mechanisms. We thus demonstrate that the mechanisms causing ER stress are of major importance for PON2 regulation and its protective function against ER stress-induced caspase-3 activation. Financial support came from the Deutsche Forschungsgemeinschaft Collaborative Research Center 553 (project A1 to U.F.), and by intra-mural funds of the Faculty of Medicine, Gutenberg University Mainz (to S.H.). Disclosures: none. EA.hy 926 cells were co-transfected with plasmids encoding for a firefly luciferase gene under the expressional control of the PON2 promoter fragment and with a plasmid for constitutive expression of renilla luciferase (normalization). Four hours after transfection, cells were treated for 16h with increasing concentrations of DTT (A), tunicamycin (B) or thapsigargin (C). Finally, firefly luciferase activity reflecting PON2 promoter induction was recorded, normalized to renilla luciferase activity and expressed as fold induction. (D) Addressing if serum components influence PON2 promoter activity, EA.hy 926 cells were seeded, transiently transfected with the PON2 promoter construct, serum deprived for 16h, and finally either left untreated or stimulated by adding medium with 10% fetal calf serum for 6h and analyzed as above. Symbols represent means ± SEM; n=3. (A-C), Ca-ionophore A23187 (10 µM) (D-F), the intracellular Ca 2+ -chelator BAPTA/AM (BAPTA; 10 µM; 1h pre-treatment where indicated) or combinations thereof. Then, cells were lysed, RNA was prepared and mRNA levels of PON2 (A+D), GRP78 (B+E) or spliced XBP1 (C+F) were monitored by qRT-PCR. (G+H) EA.hy 926 cells were transfected with plasmids pGL4-PON2-promoter, pGL4-PON2-promoter+5'UTR or pGL4-PON2-promoter+3'UTR. A plasmid for constitutive expression of renilla luciferase was co-transfected. Cells were treated with DMSO (0.1%; solvent control), thapsigargin (10 nM; panel G) or tunicamycin (10 µg/ml; panel H) for 16h. Then, total RNA was prepared with concomitant DNase I digestion (see Methods) and firefly luciferase mRNA levels were determined by qRT-PCR. These were normalized to renilla luciferase mRNA levels to control for transfection efficiencies. Basal expression was comparable for all constructs and was independent from the addition of 5' or 3'UTRs (not shown). (J) EA.hy 926 cells stably overexpressing PON2-GFP were left untreated or were treated with actinomycin D (5 µg/ml; Actino) alone or in combination with thapsigargin (10 nM) for 16h. Subsequently, total RNA was prepared and PON2-GFP mRNA levels were determined by qRT-PCR. Symbols represent means ± SEM; n=4-9. Differences were tested for statistical significance using 1way ANOVA and Bonferronis multiple comparisons post-test; n.s. = not significant; *P<0.05; **P<0.01; ***P<0.001. Lysates (50 µg of protein) were analyzed by Western blotting using anti-PON2 and anti-α-tubulin antibodies. Results of densitometric quantification of relative signal intensities (after normalization to α-tubulin) are given below the lanes. (B) EA.hy 926 cells were left untreated or were treated with the indicated concentrations of tunicamycin for 8 hours and analyzed as described for panel A. Arrowhead indicates tunicamycin-induced, newly synthesized PON2 isoform-1; newly synthesized PON2 isoform-2 is not detectable after 8h treatment (see next panel). (C) EA.hy 926 cells were untreated (lane 1) or were treated with tunicamycin (tunica; 0.5 µg/ml) or cycloheximide (cyclo; 5 µg/ml) or both. After 8, 16, 24 or 32 h cells were harvested and lysates (50 µg) were analyzed by Western blotting using anti-PON2, anti-GRP78 and anti-α-tubulin antibodies. Arrows indicate tunicamycin-induced, newly synthesized PON2 isoforms1+2. Each blot is representative of two to four others showing similar results.

  Journal Immediate Publication. Published on 11 Aug 2008 as manuscript BJ20080775 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20080775 A c c e p t e d M a n u s c r i p t Experimental Cultivation of EA.

Biochemical

  Journal Immediate Publication. Published on 11 Aug 2008 as manuscript BJ20080775 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20080775 A c c e p t e d M a n u s c r i p t

  Biochemical Journal Immediate Publication. Published on 11 Aug 2008 as manuscript BJ20080775 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20080775 A c c e p t e d M a n u s c r i p t

Biochemical

  Journal Immediate Publication. Published on 11 Aug 2008 as manuscript BJ20080775 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20080775

  Biochemical Journal Immediate Publication. Published on 11 Aug 2008 as manuscript BJ20080775 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20080775 A c c e p t e d M a n u s c r i p t

  Biochemical Journal Immediate Publication. Published on 11 Aug 2008 as manuscript BJ20080775 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20080775A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Figure 1 :

 1 Figure 1: PON2-mediated tolerance to ER stress-induced apoptosis is lost after disturbance of Ca 2+ -homeostasis. (A)To test maximum caspase-3/7 activation, naïve EA.hy 926 cells were treated for 16h with the indicated concentrations of apoptosis-inducing compound staurosporin with subsequent caspase-3/7 activity determination. Alternatively, naïve or PON2-iso1-HA or PON2-iso1-GFP overexpressing EA.hy 926 cells were treated for 16h with various concentrations of DTT (B), tunicamycin (C) or thapsigargin (D). Then caspase 3/7 activity was determined. Symbols represent mean ± SEM; n=6-14. Caspase-3/7 activation in PON2-GFP / PON2-HA overexpressing cells was significantly lower than in naïve EA.hy 926 cells with concentrations equal to or higher than 10 mM DTT or 1 µg/ml tunicamycin (P<0.05; P<0.001, respectively).

Figure 2 :

 2 Figure 2: UPR stimulates PON2 promoter activity independent from the nature of ER stress.EA.hy 926 cells were co-transfected with plasmids encoding for a firefly luciferase gene under the expressional control of the PON2 promoter fragment and with a plasmid for constitutive expression of renilla luciferase (normalization). Four hours after transfection, cells were treated for 16h with increasing concentrations of DTT (A), tunicamycin (B) or thapsigargin (C). Finally, firefly luciferase activity reflecting PON2 promoter induction was recorded, normalized to renilla luciferase activity and expressed as fold induction. (D) Addressing if serum components influence PON2 promoter activity, EA.hy 926 cells were seeded, transiently transfected with the PON2 promoter construct, serum deprived for 16h, and finally either left untreated or stimulated by adding medium with 10% fetal calf serum for 6h and analyzed as above. Symbols represent means ± SEM; n=3.

Figure 3 :

 3 Figure 3: PON2 mRNA levels are increased by DTT or tunicamycin treatment, but decreased after thapsigargin treatment. EA.hy 926 cells were left untreated or were treated with DTT (2.5 mM) (A), tunicamycin (1µg/ml) (B), or thapsigargin (10 nM) (C) for the indicated time points; in panel (D), varying concentrations of thapsigargin (0.1 -10 nM) were applied for 16h. Afterwards, total RNA was prepared and PON2 mRNA levels were determined by qRT-PCR. Symbols represent means ± SEM; n=3-4. In panels C and D, nonlinear regression was performed using an equation of one phase exponential decay.

Figure 4 :

 4 Figure 4: Analysis of PON2 mRNA stability upon ER stress induction. EA.hy 926 cells were left untreated or were pre-treated for 4 hours with the UPR-inducing compounds DTT (2.5 mM) (A), tunicamycin (0.5 µg/ml) (B) or thapsigargin (1 nM) (C). Then, RNA synthesis was inhibited by addition of 5,6-dichlorobenzimidazole 1-β-D-ribofuranoside (DRB; 60 µM) with prolonged presence of the UPR inducing compounds. At the indicated time points, RNA was prepared and PON2 mRNA

Figure 5 :

 5 Figure5: PON2 mRNA degradation is instigated by disturbed Ca 2+ -homeostasis, but not by general activation of the UPR pathway. EA.hy 926 cells were left untreated (control) or were treated for 16h with thapsigargin (thapsi; 10 nM) (A-C), Ca-ionophore A23187 (10 µM) (D-F), the intracellular Ca 2+ -chelator BAPTA/AM (BAPTA; 10 µM; 1h pre-treatment where indicated) or combinations thereof. Then, cells were lysed, RNA was prepared and mRNA levels of PON2 (A+D), GRP78 (B+E) or spliced XBP1 (C+F) were monitored by qRT-PCR. (G+H) EA.hy 926 cells were transfected with plasmids pGL4-PON2-promoter, pGL4-PON2-promoter+5'UTR or pGL4-PON2-promoter+3'UTR. A plasmid for constitutive expression of renilla luciferase was co-transfected. Cells were treated with DMSO (0.1%; solvent control), thapsigargin (10 nM; panel G) or tunicamycin (10 µg/ml; panel H) for 16h. Then, total RNA was prepared with concomitant DNase I digestion (see Methods) and firefly luciferase mRNA levels were determined by qRT-PCR. These were normalized to renilla luciferase mRNA levels to control for transfection efficiencies. Basal expression was comparable for all constructs and was independent from the addition of 5' or 3'UTRs (not shown). (J) EA.hy 926 cells stably overexpressing PON2-GFP were left untreated or were treated with actinomycin D (5 µg/ml; Actino) alone or in combination with thapsigargin (10 nM) for 16h. Subsequently, total RNA was prepared and PON2-GFP mRNA levels were determined by qRT-PCR. Symbols represent means ± SEM; n=4-9. Differences were tested for statistical significance using 1way ANOVA and Bonferronis multiple comparisons post-test; n.s. = not significant; *P<0.05; **P<0.01; ***P<0.001.

Figure 6 :

 6 Figure 6: UPR activation by DTT or tunicamycin induces PON2 protein expression. (A) EA.hy 926 cells were left untreated or were treated with DTT (2.5 mM) for 4, 8 or 16 hours.Lysates (50 µg of protein) were analyzed by Western blotting using anti-PON2 and anti-α-tubulin antibodies. Results of densitometric quantification of relative signal intensities (after normalization to α-tubulin) are given below the lanes. (B) EA.hy 926 cells were left untreated or were treated with the indicated concentrations of tunicamycin for 8 hours and analyzed as described for panel A. Arrowhead indicates tunicamycin-induced, newly synthesized PON2 isoform-1; newly synthesized PON2 isoform-2 is not detectable after 8h treatment (see next panel). (C) EA.hy 926 cells were untreated (lane 1) or were treated with tunicamycin (tunica; 0.5 µg/ml) or cycloheximide (cyclo; 5 µg/ml) or both. After 8, 16, 24 or 32 h cells were harvested and lysates (50 µg) were analyzed by Western blotting using anti-PON2, anti-GRP78 and anti-α-tubulin antibodies. Arrows indicate tunicamycin-induced, newly synthesized PON2 isoforms1+2. Each blot is representative of two to four others showing similar results.

Figure 7 :

 7 Figure 7: PON2 protein is degraded by a Ca 2+ -dependent, calpain-mediated mechanism. (A)EA.hy 926 cells were solvent treated or were treated for 16h with the indicated concentrations of thapsigargin. Lysates (100 µg of protein) were analyzed by Western blotting using anti-PON2, anti-αtubulin and anti-GRP78 antibodies. (B) EA.hy 926 cells were solvent treated or pre-treated with the intracellular Ca 2+ -chelating compound BAPTA/AM (BAPTA; 20 µM) for 1h. Then, thapsigargin (10 nM) was added as indicated. After 16h, lysates were prepared and analyzed as in panel A. (C) Similar experiment as in panel A, but using Ca-ionophore A23187 for 16h at the indicated concentrations. (D) Similar experiment as in panel B, but replacing thapsigargin with A23187 at the indicated concentrations. (E) EA.hy 926 cells were solvent treated (DMSO) or pre-treated with the inhibitor of proteasome activity lactacystin (Lacta; 50 µM), calpain inhibitor-I N-acetyl-Leu-Leu-norleucinal (ALLN; 5 µM) or the autophagy inhibitor 3-methyladenin (3-MA, 5 mM) for 1 hour before thapsigargin (Thapsi; 10 nM) was added for another 16h. Lysates were analyzed as stated in panel A. (F) Naïve EA.hy 926 cells or (G) EA.hy 926 cells stably overexpressing PON2-iso1-GFP were left untreated or were treated with actinomycin D (5 µg/ml; Actino) alone or in combination with thapsigargin (10 nM) for the indicated time. Lysates were analyzed as stated in panel A. Results of semi-quantitative densitometric quantification for relative expression levels of endogenous PON2 (F) or PON2-GFP (G) are indicated below the lanes. Each blot is representative of 2 -5 others with similar results. Comparable results were obtained using MG132 instead of lactacystin and cycloheximide instead of actinomycin D (panels E and F, respectively; data not shown). In panel (H),

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

© 2008 The Authors Journal compilation © 2008 Biochemical Society

Acknowledgement