Mohamed El Khattabi

Maarten L Van Roosmalen

Dennis Jager

Heidi Metselaar

Hjalmar Permentier #

Kees Leenhouts
email: leenhouts@biomade.nl

Jaap Broos

Lactococcus lactis as expression host for the biosynthetic incorporation of tryptophan analogues in recombinant proteins

Keywords: Lactococcus lactis, tryptophan auxotroph, 5-methyltryptophan, tryptophan analogues, IAA, glyphosate IAA, 3 -indoleacrilic acid, CDM, chemically defined medium, glyphosate, N-phosphonomethylglycine, 5-FTrp, 5-fluorotryptophan, 5-OHTrp, 5hydroxytryptophan, 7-AzaTrp, 7-azatryptophan, 5-methylTrp, 5-methyltryptophan, PA, protein anchor

Incorporation of tryptophan (Trp) analogues into a protein may facilitate its structural analysis by spectroscopic techniques. Development of a biological system for the biosynthetic incorporation of such analogues into proteins is of considerable importance.

The Gram-negative Escherichia coli is the only prokaryotic expression host regularly used for the incorporation of Trp analogues into recombinant proteins. Here we present the use of the versatile Gram-positive expression host Lactococcus lactis for the incorporation of Trp analogues. The availability of a tightly regulated expression system for this organism, the possibility to secrete modified proteins into the growth medium and the construction of the trp-synthetase deletion strain PA1002 of L. lactis rendered this organism potentially an efficient tool for the incorporation of Trp analogues in recombinant proteins. The Trp analogues 7-azatryptophan, 5-fluorotryptophan and 5hydroxytryptophan were incorporated with efficiencies of >97%, >97% and 89%, respectively. Interestingly, 5-methyltryptophan could be incorporated with 92% efficiency. Successful biosynthetical incorporation of 5-methylTrp in recombinant proteins has not been reported before.

INTRODUCTION

Tryptophan (Trp) is an attractive intrinsic fluorescence probe in protein chemistry [1;2]. Its emission maximum (λ max ) and the quantum yield (Q) are highly sensitive parameters for monitoring changes in structure and dynamics of a protein. In numerous studies the intrinsic protein fluorescence was used to monitor processes like enzyme kinetics, protein-ligand interactions, or protein (un)folding. Modern molecular biology techniques allow easy introduction of a Trp codon at a position sensitive to monitor the desired process while native Trp residues can be replaced by a tyrosine or phenylalanine residue, often without a noticeable effect on protein function. The use of Trp analogues with altered optical properties can further broaden the intrinsic fluorescence approach. A number of Trp analogues like 7-azatryptophan (7-AzaTrp), 5-hydroxytryptophan (5-OHTrp) and fluorotryptophans can be efficiently biosynthetically incorporated in recombinant proteins using an Escherichia coli Trp auxotroph [3;4] The Gram-positive bacterium Lactococcus lactis has been shown to be an excellent host for the expression of different proteins. Both homologous and heterologous proteins are well expressed [START_REF] Arnau | Heterologous gene expression of bovine plasmin in Lactococcus lactis[END_REF][START_REF] Miyoshi | Controlled production of stable heterologous proteins in Lactococcus lactis[END_REF][START_REF] Nouaille | Heterologous protein production and delivery systems for Lactococcus lactis[END_REF][START_REF] Kunji | Lactococcus lactis as host for overproduction of functional membrane proteins[END_REF]. This high-level production of proteins has been obtained using constitutive as well as inducible promoters [9;10]. Here we explored the possibility to use L. lactis as a production organism for the incorporation of Trp analogues. A suitable L. lactis Trp auxotroph was not available. Efficient incorporation of Trp analogues with Trp prototrophic E. coli strains has been achieved in some cases by adding inhibitors that block the biosynthesis of Trp. We have investigated this approach for L. lactis and we also report the construction of a Trp auxotroph of L. lactis by deleting the tryptophan synthase genes. Best results were obtained using the Trp auxotrophic mutant strain and conditions are described that result in the efficient replacement (89% ->97%) of Trp for the Trp analogues 5-fluoroTrp (5-FTrp), 5-OHTrp, 7-AzaTrp and 5-methyltryptophan (5-methylTrp) in a recombinant model protein. This is the first report for the efficient incorporation of 5-methylTrp in a recombinant protein.

EXPERIMENTAL Bacterial strains, plasmids, media and growth conditions

L. lactis strains and plasmids used in this study are listed in Table 1. Plasmids pPA6 and pPA90 used to express the studied proteins PA6 and PA90, respectively, are based on the nisin inducible expression vector pNZ8048 [START_REF] Kuipers | Controlled overproduction of proteins by lactic acid bacteria[END_REF]. The relevant parts of the plasmids are described in Figure 1. L. lactis strains were grown at 30 o C in M17 medium, supplemented with 0.5% glucose (GM17) [START_REF] Terzaghi | Improved medium for Lactic streptococci and their bacteriophages[END_REF] or in CDM [START_REF] Poolman | Relation of growth of Streptococcus lactis and Streptococcus cremoris to amino-acid transport[END_REF], supplemented with 0.5% glucose and 1 mM of all amino acids, except Trp. Antibiotics (chloramphenicol or erythromycin) were added at a concentration of 5 µg/ml for the maintenance of the plasmids. Media were solidified by 1.5% agar and β-galactosidase activity of LacZ was detected by supplementing the media with 80 µg/ml 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal). L-Trp, DL-5-FTrp, L-5-OHTrp, DL-7-AzaTrp and DL-5-methylTrp were from Sigma-Aldrich. Fresh stock solutions were prepared in dilute NaOH.

Conditions for the inhibition of tryptophan biosynthesis were as follows. L. lactis strain PA1001 harboring the plasmid pPA90 was grown at 30 o C for 16 h in GM17 medium.

The cells were harvested by centrifugation, washed with saline and diluted into CDM with or without 1 mM Trp, containing different concentrations of IAA or glyphosate (both from Sigma-Aldrich). The growth of the cultures was followed by optical density measurement at 600 nm using a Bio-Rad SmartSpec TM 3000 UV-VIS spectrophotometer (Bio-Rad Laboratories, Richmond, Calif.).

Expression conditions

Expression of PA6 and PA90 was induced by the addition of filtered sterilised culture supernatant of the nisin-producing strain NZ9700 [START_REF] Kuipers | Controlled overproduction of proteins by lactic acid bacteria[END_REF] to the strains harboring plasmid pPA6 and pPA90. Nisin containing medium was added to the cultures at 1000fold dilution (end concentration approximately 10 ng/ml). For efficient incorporation of Trp analogues, L. lactis strains harboring plasmids pPA6 or pPA90 were grown overnight in GM17 at 30 o C. Subsequently, the cultures were diluted 50-fold in 400 ml fresh GM17 and grown for approximately 4 h at 30 o C (OD 600 : 0.8). The cells were harvested by centrifugation at 7,000 g for 10 min, washed with saline (0.9% NaCl) and

General DNA techniques and transformation

Restrictions, ligations and PCRs were performed according to the enzyme suppliers instructions and essentially as described by Sambrook et al. [START_REF] Sambrook | Molecular Cloning, a Laboratory Manual[END_REF]. Genomic DNA was isolated as described earlier [14]. Plasmids were transferred to L. lactis strains by electroporation using a Gene Pulser (Bio-Rad Laboratories, Richmond, Calif.) [15].

Generation of trpBA deletion strain

Oligonucleotide primers were designed, based on the trp operon sequence of L. lactis strain MG1363 (G.

RESULTS

L. lactis PA1001 is not auxotrophic in Trp

L. lactis PA1001 is a strain that has been optimized for the inducible expression of proteins that contain the peptidoglycan binding domain of the lactococcal cell wall hydrolyzing enzyme AcmA [16;17]). The entire peptidoglycan binding domain is called PA90 and contains 5 Trp residues. PA6 is part of PA90 and contains 1 Trp residue. In the plasmids containing the genes that encode for PA90 or PA6, the genes are located behind a tightly regulated nisin promoter (Figure 1). Upon expression by the addition of nisin to the growth medium, PA90 and PA6 are secreted into the culture supernatant by means of a signal peptide present in the N-terminus of the recombinant proteins.

Secretion into the growth medium simplifies the isolation of the recombinant proteins.

L. lactis strain PA1001 was grown in CDM containing no Trp or 1 mM L-Trp to investigate its growth characteristics. Growth rates were similar in the presence or absence of Trp, showing that PA1001 is not auxotrophic in Trp and that the biosynthetic route for Trp is efficient enough to sustain similar growth rates as with Trp added to the medium (Figure 2).

Inhibition of the Trp biosynthesis pathway

In E. coli, inhibition of the Trp biosynthesis pathway by inhibitors like IAA or glyphosate has been used in prototroph E. coli strains to stimulate the incorporation of Trp analogues [START_REF] Leone | Selective incorporation of 19F-labeled Trp side chains for NMR-spectroscopy-based ligandprotein interaction studies[END_REF]. This approach was explored for L. lactis PA1001 in CDM. A concentration of 0.2 mg/ml of IAA was completely inhibiting the growth of L. lactis PA1001 (Figure 3A). However, addition of Trp did not elevate the IAA-inhibitory effect, suggesting that the growth inhibitory effect caused by IAA in L. lactis differed from that in E. coli.

Trp biosynthesis was also inhibited by glyphosate (Figure 3B). A glyphosate concentration of 10 mM had no effect on the growth, but 30 mM inhibited the growth of L. lactis strain PA1001 in CDM. In this case the growth was partially restored after the addition of Trp. Glyphosate inhibits the function of the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSP) [START_REF] Steinrucken | The herbicide glyphosate is a potent inhibitor of 5-enolpyruvyl-shikimic-acid 3-phosphate synthase[END_REF], which acts upstream in the formation of chorismate, a precursor of all aromatic amino acids. Growth inhibition may result from the limitation An increase in growth rate in the presence of glyphosate was only achieved by the addition of Trp. This result indicates that the Trp biosynthesis pathway is inhibited by glyphosate and that the concentrations of the aromatic amino acids phenylalanine and tyrosine in CDM were not limiting for the growth. Apparently, the biosynthesis routes for phenylalanine and tyrosine under the tested conditions are more efficient than for Trp.

Deletion of the trpBA genes from the chromosome of L. lactis strain PA1001

Since growth was only partially recovered in the case of inhibition with glyphosate, it is likely that incorporation of Trp analogues will not be very efficient. For this reason a Trp auxotroph L. lactis strain was generated by deleting the genes coding for the enzyme tryptophan synthase. Tryptophan synthase is the enzyme that converts indole-3-glycerolphosphate and L-serine into L-tryptophan, which is the last step in the biosynthesis of Trp. Its presence is wide spread among microorganisms and plants. The functional enzyme is a tetramer, formed by the two subunits α and β (2 α and 2 β subunits) [START_REF] Miles | Tryptophan synthase: a multienzyme complex with an intramolecular tunnel[END_REF],

which are encoded by trpA and trpB genes, respectively. In L. lactis strain IL1403, tryptophan synthase is part of an operon containing seven structural genes in the order trpEGDCFBA [21;22]. An operon with similar organisation is also present in the genome of L. lactis strain MG1363 (G. Buist, personal communication), which is the parent strain of PA1001.

The trpB and trpA genes are located at the end of the trp operon. Therefore, both genes can be deleted at the same time without disturbing the organisation of the rest of the operon. The DNA sequences upstream of trpB and downstream of trpA, amplified by using PCR, were inserted into the conditionally replicating plasmid pORI280 [14].

Deletion of the trpBA genes from the chromosome of strain PA1001 by the use of the pORI280-derivative pPA189, was confirmed by PCR and Trp auxotrophy was confirmed by the absence of growth on CDM lacking Trp (Figure 2). Growth of the generated L. lactis strain PA1002 in CDM + Trp was similar to that of the parent strain PA1001 (Figure 2). more difficult to incorporate biosynthetically using E. coli. Only the successful incorporation of 4-methyltryptophan has been reported using this host [START_REF] Budisa | Probing the role of tryptophans in Aequorea victoria green fluorescent proteins with an expanded genetic code[END_REF]. Surprisingly, when 5-methylTrp was used with PA1002 as expression host a good yield of PA6 was obtained, comparable to the yield of the other Trp analogues tested (Figure 4B). This suggests that 5-methylTrp can be biosynthetically incorporated using L. lactis. Replacing Trp for a Trp analogue in a protein can facilitate its characterization using spectroscopy techniques like absorbance, fluorescence or NMR. For many applications, a high to complete incorporation efficiency is desired. Most Trp analogue containing proteins have been produced using E. coli (reviewed in [3;24]). Good results have been obtained by using one of the available Trp auxotrophs and the incorporation efficiency was found to be dependent on the promotor used to express the recombinant proteins. A tightly controlled promotor system is needed, otherwise the recombinant protein is expressed during the biomass generation phase, in medium containing Trp. Best results have been obtained with the T5, tac, and λP L promotors [START_REF] Ross | Spectral enhancement of proteins by in vivo incorporation of tryptophan analogues[END_REF]. Starvation of the washed cells after growth in Trp containing medium to deplete the level of Trp was also found to be critical for high incorporation efficiency. Recently, efficient incorporation of 5-FTrp was reported with a non-Trp auxotropic strain of E. coli using IAA as inhibitor of the biosynthesis of Trp [START_REF] Leone | Selective incorporation of 19F-labeled Trp side chains for NMR-spectroscopy-based ligandprotein interaction studies[END_REF]. A similar approach using glyphosate has been reported [25].

Mass spectrometry

MALDI

This approach can be useful for expression organisms for which no Trp auxotroph is available like L. lactis. We have explored the potential of this approach but found that the growth inhibitory effect of IAA on L. lactis in CDM without Trp is not related with inhibition of the Trp biosynthesis, since addition of Trp did not alleviate the inhibition of the cell growth rate (Figure 3). Similarly, an inhibitory effect on the growth of L. lactis was found at 30 mM glyphosate, but addition of 1 mM Trp did only partly restore the growth rate. At this moment, the biological activity of these 2 compounds towards this lactis expression systems [START_REF] Arnau | Heterologous gene expression of bovine plasmin in Lactococcus lactis[END_REF][START_REF] Miyoshi | Controlled production of stable heterologous proteins in Lactococcus lactis[END_REF][START_REF] Nouaille | Heterologous protein production and delivery systems for Lactococcus lactis[END_REF][START_REF] Kunji | Lactococcus lactis as host for overproduction of functional membrane proteins[END_REF]. Therefore, it is likely that Trp analogs can be efficiently incorporated into many other proteins expressed in L. lactis PA1002.

In several studies with E. coli, 5-OHTrp turned out to be less efficiently incorporated than 7-AzaTrp and 5-FTrp using the same expression system [START_REF] Ross | Spectral enhancement of proteins by in vivo incorporation of tryptophan analogues[END_REF]. L. lactis PA1002 also shows a lower preference for 5-OHTrp than the other 2 Trp analogues. However, this

Trp auxotroph is the first organism allowing the biosynthetic incorporation of 5-methylTrp. Although closely resembling the structure of allowed substituents at the 5position (F, OH, NH 2 ), this analogue seems not to be accepted by the E. coli translation machinery. Early work on Trp analogue incorporation reported both chromosome-based expression of proteins in Trp starved E. coli cells upon addition of 5-methylTrp [26;27] and no expression under these conditions [START_REF] Pardee | Incorporation of azatryptophan into proteins of bacteria and bacteriophage[END_REF]. Recently, it was reported that 5-methylTrp can not be incorporated in a recombinant protein using an E. coli auxotroph [START_REF] Budisa | Probing the role of tryptophans in Aequorea victoria green fluorescent proteins with an expanded genetic code[END_REF].

5-methylTrp is an interesting analogue since its electron-donating methyl group lowers the ionization potential of the indole moiety by ~0.12 eV [START_REF] Liu | Ionization potentials of fluoroindoles and the origin of nonexponential tryptophan fluorescence decay in proteins[END_REF]. A lowering of the ionization potential will stimulate photo-induced electron transfer from the excited probe to nearby acceptors like peptide bonds [START_REF] Liu | Ionization potentials of fluoroindoles and the origin of nonexponential tryptophan fluorescence decay in proteins[END_REF]. For Trp embedded in a protein, this process is regarded the dominant non-radiative depopulation process, and the photophysical properties of Trp (quantum yield, fluorescence decay kinetics) are strongly dependent on this process. The ability of changing the ionization potential of a Trp position to a higher value, via 5-fluoro substitution [START_REF] Liu | Ionization potentials of fluoroindoles and the origin of nonexponential tryptophan fluorescence decay in proteins[END_REF], or a lower value, via 5-methyl substitution, can become an interesting experimental approach to study this quenching process in more

detail.

An anisotropy study using 5-methylindole revealed that it shows mixed emission from the 1 L a and 1 L b states while indole was found to emit only from 1 L a [START_REF] Eftink | Photophysics of indole-derivatives -experimental resolution of L a and L b transitions and comparison with theory[END_REF]. Trp embedded in a protein emits from 1 L a , only for one protein showing an extremely blue-shifted λ max , 1 L b emission has been reported [START_REF] Broos | The emitting state of tryptophan in proteins with highly blue-shifted fluorescence[END_REF]. Likely, biosynthetic incorporation of 5-methylTrp will result more often in 1 L b emitting proteins. The impact of the protein matrix on the spectroscopic parameters of 5-methylTrp can now be experimentally explored using L. lactis PA1002 as expression host. [32] NZ9000 MG1363 containing an insertion of the nisRK genes into the pepN gene.

[10] NZ9700 Nisin producing strain.

[10]

PA1001

A derivative of strain NZ9000 lacking the entire acmA and htrA genes. Plasmid pPA90 contains the entire sequence of L. lactis AcmA peptidoglycan binding domain (from spacer 1 to LysM3, aa sequence GNTN….YLRIK as in [START_REF] Buist | Molecular-cloning and nucleotide-sequence of the gene encoding the major peptidoglycan hydrolase of Lactococcus lactis, a muramidase needed for cell-separation[END_REF]).

The position of the Trp codons in the LysM domains is indicated. The growth was followed by measuring the absorbance at 600 nm at the indicated times.

  S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 2 Oct 2007 as manuscript BJ20070909 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20070909 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society finally, resuspended into 100 ml CDM supplemented with 0.5% glucose. After a starvation period of 45 min at 30 o C, nisin and 1 mM (L-enantiomer) of either Trp or Trp analogue were added. The cells were subsequently incubated for approximately 24 h at 30 o C. After spinning down the cells, PA90 or PA6 were precipitated from the culture using 5% trichloro acetic acid (TCA) during for 1 h at 0 o C, followed by 20 min centrifugation at 13,000 g and 0 o C. Samples were analyzed using SDS-PAA gel electrophoresis. Per lane TCA precipitate originating from 0.4 ml culture was loaded.

  the chromosome (from nt 1002272 to 1004262 according to EMBL/GenBank databases with accession number AM406671) without leaving the Em r and LacZ genes and any other plasmid sequences in the chromosome. The deletion in the chromosome was confirmed by PCR using primers flanking the deleted gene region, Del.fw (5'-CCCTTCATGGATGGAAGAAC-3') and Del.rev (5'-GTTCTACGATTCCACTTGG-3'). A PCR reaction on the chromosomal DNA of the deletion mutant results in that case in a DNA fragment of 613 basepair (bp), whereas the chromosomal DNA of the wildtype as a template generates in such a PCR assay a DNA fragment of 2603 bp. A strain with the trpBA deletion was designated PA1002.Mass spectrometryCoomassie-stained bands of PA6 protein were excised from the gel and washed several times with water and acetonitrile. After the last wash step, the bands were dried completely and resuspended in 20 l of 2.5 ng/ l porcine trypsin (Promega, Leiden, The Netherlands) dissolved in 100 mM ammonium bicarbonate and incubated for 1-2 h at 37 ºC. Of the overlaying liquid, 0.75 l was spotted on a MALDI target and mixed immediately with an equal volume of 10 mg/ml -cyano-hydroxycinammic acid (LaserBio Labs, Sophia-Antipolis Cedex, France) in 50% acetonitrile with 0.1% (v/v) trifluoroacetic acid. Spots were air dried and positive mode reflectron mass spectra were recorded on a Voyager DE-PRO MALDI-TOF instrument (Applied Biosystems, Foster City, CA, USA) and a 4700 Proteomics Analyzer MALDI-TOF/TOF instrument (Applied Biosystems). Spectra were calibrated externally with standard peptides. S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 2 Oct 2007 as manuscript BJ20070909 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20070909 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society Revised manuscript ref.no BJ2007/0909 7

  S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 2 Oct 2007 as manuscript BJ20070909 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20070909 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Societyof the other aromatic amino acids phenylalanine and tyrosine. This was tested by the addition of 1 mM extra of these two amino acids to CDM (final concentrations 2.7 and 2.1 mM, respectively), but no stimulation in the growth rates were observed (not shown).

S t a g e

  2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 2 Oct 2007 as manuscript BJ20070909 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20070909 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society Production of recombinant proteins in the presence of Trp analogues using Trp auxotroph PA1002L. lactis strain PA1002 carrying plasmids pPA90 or pPA6 was used to express PA90 or PA6, respectively. L. lactis strain PA1002 did not grow when inoculated in CDM that was supplemented with the Trp analogues 5-FTrp, 5-OHTrp or 7-AzaTrp. However, proteins could be produced after the formation of biomass in the absence of nisin in rich growth medium (GM17) followed by washing of the cells and resuspending in CDM supplemented with Trp or a Trp analogue and nisin (Figure4A). Compared to expression in the presence of Trp, production levels of PA6 and PA90 were only minimally affected by the presence of the Trp analogues.The range of Trp analogues which can be biosynthetically incorporated is mainly determined by the substrate specificity of Trp-tRNA synthetase. This enzyme charges tRNA trp with Trp and the protein from E. coli only accepts minor changes in the indole moiety of the Trp structure like small substituents (F, OH, NH 2 ) or introduction of a nitrogen or sulphur atom in the indole nucleus[3;23]. Methylated Trp analogues are

  -TOF was used to establish the incorporation efficiency of the Trp analogues into PA6. PA6 bands were cut out from the SDS-PAA gel and the proteins were digested with trypsin yielding a fragment of 11 amino acids containing the single Trp residue in PA6. In Figure5, MALDI-TOF spectra are shown for this tryptic fragment of PA6 containing 5-OHTrp, 5-methylTrp, 5-FTrp, and 7-AzaTrp respectively. In the case of 7-AzaTrp and 5-FTrp, no significant peak of the peptide containing Trp is visible, indicating that the incorporation efficiency is >97%. The incorporation efficiencies were 89% and 92% for 5-OHTrp and 5-methylTrp, respectively. S t a g e 2 ( a ) P O S T -P R I N T DISCUSSION In this manuscript the construction of a Trp auxotroph of L. lactis is presented and we demonstrate the quantitative biosynthetic incorporation (>97%) of the Trp analogues 7-AzaTrp and 5-FTrp into a secreted recombinant model protein. An 89% efficiency for the incorporation of 5-OHTrp into the recombinant PA6 protein using this Trp auxotroph was found. The biosynthetic incorporation of 5-methylTrp in a recombinant protein is reported for the first time. The incorporation efficiency for this Trp analogue was 92% using the L. lactis PA1002 Trp auxotroph.

Gram-positive organism

  is unclear. These results prompted us to make a Trp auxotroph of L. lactis and this was achieved via deletion of the trpBA genes. Growth conditions were found for the L. lactis Trp auxotrophic strain PA1002 that result in high to very high incorporation efficiencies of the 4 Trp analogues tested into the secreted model recombinant protein PA6. The expression of protein anchor (PA) proteins was under S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 2 Oct 2007 as manuscript BJ20070909 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20070909 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society control of the nisin promotor, a promotor system known to be tightly regulated. The PA proteins used in this study are essentially homologous L. lactis proteins, only PA6 contains the heterologous c-myc sequence. However, a broad range other proteins of pro-and eukaryotic origin, including membrane proteins, have been expressed in L.

S t a g e 1 Table 1 .

 11 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 2 Oct 2007 as manuscript BJ20070909 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20070909 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society Strains and plasmids used in this study

1 a 3 S

 13 nisin inducible expression-secretion vector derived from pNZ8048, containing the 1 st peptidoglycan binding motif (LysM domain) of acmA fused C-terminally to c-LacZ, conditionally replicating cloning and reporter plasmid suitable for use in gene-replacement strategies.[14] S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 2 Oct 2007 as manuscript BJ20070909 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20070909 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society pPA189 Em r , LacZ, gene-replacement vector. Derivative of pORI280 containing DNA sequences 5' and 3' of the trpB and the trpA genes, respectively. This study Cm r : chloramphenicol resistance. Em r : erythromycin resistance. LacZ: -galactosidase. 2 t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 2 Oct 2007 as manuscript BJ20070909 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20070909 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

Figure legends Figure 1 .

 legends1 Figure legends

Figure 2 .

 2 Figure 2. Growth of L. lactis strains PA1001 and PA1002 in CDM in absence or presence of Trp. Cells were grown in CDM containing 1 mM Trp overnight at 30 o C. Subsequently, the cells were washed with saline, then inoculated into fresh CDM without Trp during 45 min, followed by the addition Trp sources or no Trp at all (T=0).

Figure 3 .

 3 Figure 3. Growth-inhibition of L. lactis strain PA1001 by Trp biosynthesis pathway inhibitors. Cells were grown in CDM containing 1 mM Trp overnight at 30 o C. Subsequently, the cells were washed with saline, then inoculated into fresh CDM containing either IAA (A) or glyphosate (B). The cultures contained the inhibitor without any Trp (solid line) or in combination with 1 mM Trp (dashed line). The concentrations of IAA tested are indicated in (A). Two concentrations of glyphosate were used, a permissive concentration of 10 mM (circles) and an inhibitory concentration of 30 mM (squares).

Figure 4 . 18 (B) 1mM 5 -

 4185 Figure 4. Coomassie stained SDS-PAA gels showing the proteins produced by L.lactis strain PA1002. The equivalent of 0.4 ml of the cell-free supernatants (TCA precipitated) of strain PA1002 producing either secreted PA6 or PA90 were loaded into each lane. Biomass of L. lactis strain PA1002 containing the plasmids pPA6 or pPA90 was formed in GM17 for approximately 4 h. Subsequently, the cells were cultivated for 24 h in CDM supplemented with 0.5% glucose and nisin, and containing (A) 1 mM Trp ("WT"); 1 mM 5-FTrp ("5F"); 1 mM 5-OHTrp ("5OH"); 1 mM 7-AzaTrp ("7aza") or S t a g e 2 ( a ) P O S T -P R I N T

Figure 5 . 1 Time 9 S

 519 Figure 5. MALDI-TOF mass spectra of PA6 with different tryptophan analogues digested with trypsin. The mass region of the SGDTLWGISQR peptide is shown: the monoisotopic mass of the protonated peptide with 5-OHTrp, 5-mehylTrp, 5-FTrp, and 7-AzaTrp incorporated is 1235.6, 1233.6, 1237.6, and 1220.6 Da, respectively. The monoisotopic peak of the peptide with unmodified tryptophan is indicated with an asterisk at m/z 1219.6. S t a g e 2 ( a ) P O S T -P R I N T

  ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 2 Oct 2007 as manuscript BJ20070909 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20070909 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society B.

Figure 4

 4 Figure 4.

  ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 2 Oct 2007 as manuscript BJ20070909 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20070909 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

Kim, H. W., Perez, J. A., Ferguson, S. J. and Campbell, I. D. (1990) The specific incorporation of labeled aromatic-amino-acids into proteins through growth of bacteria in the presence of glyphosate -Application to fluorotryptophan labeling to the H+-ATPase of Escherichia coli and NMR-studies. FEBS Lett. 272, 34-36

ACKNOWLEDGEMENTS

This work was supported by NanoNed grant 7919 from the Technology Foundation STW in the Netherlands.


Charles Affourtit

Martin D Brand

Uncoupling protein-2 contributes significantly to high mitochondrial proton leak in INS-1E insulinoma cells and attenuates glucose-stimulated insulin secretion

Keywords: pancreatic beta cells, efficiency of oxidative phosphorylation, mitochondrial uncoupling, UCP2, glucose-stimulated insulin secretion, type 2 diabetes FCS, foetal calf serum, GSIS, glucose-stimulated insulin secretion, ∆ψ, mitochondrial membrane

Uncoupling protein-2 contributes significantly to high mitochondrial proton leak in INS-1E insulinoma cells and attenuates glucose-stimulated insulin secretion

Charles Affourtit, Martin D Brand

INTRODUCTION

Type 2 diabetes is rapidly becoming a global pandemic. Although the development of this metabolic disorder is understood incompletely, it is evident that pancreatic beta cell dysfunction is responsible for its final progression to hyperglycemia. To appreciate fully how type 2 diabetes arises, it is essential to obtain a detailed understanding of beta cell function and regulation. When blood glucose levels rise, beta cells increase their oxidative glucose catabolism, leading to increased mitochondrial protonmotive force and an increased cytoplasmic ATP/ADP ratio. This closes plasma membrane K ATP channels, causing plasma membrane depolarisation, opening of voltage-sensitive calcium channels, calcium influx, and insulin secretion [START_REF] Rutter | Nutrient-secretion coupling in the pancreatic islet beta-cell: recent advances[END_REF]. This canonical model of glucose-stimulated insulin secretion (GSIS 1 ) relies heavily on efficient mitochondrial energy transduction, since it appears essential that glucose oxidation is coupled tightly to ATP synthesis.

In general, oxidative phosphorylation is coupled incompletely due to proton leakage across the mitochondrial inner membrane. Proton leak in isolated mitochondria is mainly constitutive and depends on the amount of the adenine nucleotide translocase [START_REF] Brand | The basal proton conductance of mitochondria depends on adenine nucleotide translocase content[END_REF]. Importantly, certain proton leak activity is inducible, which provides a mechanism to regulate the efficiency of energy transduction.

Inducible proton leak is mostly accounted for by uncoupling proteins (UCPs). The archetypal uncoupling protein, UCP1, catalyses mitochondrial proton conductance in mammalian brown adipose tissue, where it plays the central role in adaptive thermogenesis. The UCP1 orthologues UCP2 and UCP3 mediate mitochondrial proton leak too, but only when activated by (e.g.) superoxide or reactive oxygen species derivatives [START_REF] Esteves | The reactions catalysed by the mitochondrial uncoupling proteins UCP2 and UCP3[END_REF]. The physiological functions of UCP2 and UCP3 remain uncertain [START_REF] Brand | Physiological functions of the mitochondrial uncoupling proteins UCP2 and UCP3[END_REF] and are subject to ongoing debate [5].

Mitochondria from cultured beta cells exhibit GDP-sensitive proton conductance that is stimulated by superoxide [START_REF] Echtay | Superoxide activates mitochondrial uncoupling proteins[END_REF]. Since UCP2 mRNA is expressed in pancreatic islets, this suggests strongly that beta cells contain a functional UCP2 that is activated by reactive oxygen species [START_REF] Brand | Physiological functions of the mitochondrial uncoupling proteins UCP2 and UCP3[END_REF].

Given its proton-conducting function, it is thought that UCP2 activity attenuates the response of the protonmotive force and ATP/ADP to glucose, and thus impairs GSIS. The most persuasive support for this notion comes from genetic knock-out studies, which reveal that pancreatic islets from Ucp2ablated mice exhibit higher GSIS rates than wild-type controls [START_REF] Zhang | Uncoupling protein-2 negatively regulates insulin secretion and is a major link between obesity, beta cell dysfunction, and type 2 diabetes[END_REF]. Moreover, insulin secretion is improved when endogenous superoxide is lowered using a superoxide dismutase mimetic.

Importantly, this effect is observed exclusively in wild-type animals, which demonstrates that UCP2 diminishes GSIS when activated by superoxide [START_REF] Krauss | Superoxide-mediated activation of uncoupling protein 2 causes pancreatic beta cell dysfunction[END_REF]. The ability of UCP2 to modulate insulin release is not undisputed: beta cell-specific over-expression of human UCP2 in mice and doxycycline-inducible over-expression of the protein in cultured beta cells did not appear to affect GSIS [START_REF] Produit-Zengaffinen | Increasing uncoupling protein-2 in pancreatic beta cells does not alter glucose-induced insulin secretion but decreases production of reactive oxygen species[END_REF].

In many cell types, e.g. hepatocytes [10], thymocytes [START_REF] Buttgereit | ConA induced changes in energy metabolism of rat thymocytes[END_REF] and neuronal cells [START_REF] Jekabsons | In situ respiration and bioenergetic status of mitochondria in primary cerebellar granule neuronal cultures exposed continuously to glutamate[END_REF], a significant proportion (20-25%) of cellular respiratory activity is used to drive 'futile' mitochondrial proton leak. Despite the potentially pathological effect of UCP2 activity and uncoupling in pancreatic beta cells, little is known about the magnitude and importance of proton leak in this cell type. We recently predicted that proton leak activity may be relatively high in beta cells, because proton conductance is considerably higher in mitochondria isolated from a well-established clonal beta cell line (INS-1E, see reference [START_REF] Merglen | Glucose sensitivity and metabolism-secretion coupling studied during two-year continuous culture in INS-1E insulinoma cells[END_REF]) than in those isolated from rat skeletal muscle. As a consequence of its high rate, proton leak exerts a relatively high control over the membrane potential (∆ψ) and ATP/ADP ratio in INS-1E mitochondria [START_REF] Affourtit | Stronger control of ATP/ADP by proton leak in pancreatic beta-cells than skeletal muscle mitochondria[END_REF]. The nature of this relatively high proton conductance is unclear, however, and it is uncertain whether the high leak in INS-1E mitochondria is of physiological relevance or merely represents a mitochondrial isolation artefact.

In the present study we show that, similar to the situation in isolated mitochondria, proton leak rates are significantly higher in INS-1E cells than in myoblasts. Myxothiazol-sensitive, oligomycin-resistant respiratory activity (a good approximation of leak) is roughly 4-fold higher in INS-1E than in muscle cells. RNA interference (RNAi) experiments demonstrate that over 30% of the leak activity in INS-1E cells is accounted for by UCP2. We estimate that roughly 20% of INS-1E respiration is caused by proton cycling through UCP2. In agreement with the loss of function studies in mice, GSIS is enhanced upon UCP2 knock-down in INS-1E cells.

EXPERIMENTAL

Cell culture and mitochondrial isolation INS-1E cells were grown as described in [START_REF] Merglen | Glucose sensitivity and metabolism-secretion coupling studied during two-year continuous culture in INS-1E insulinoma cells[END_REF] in medium containing 11 mM glucose; antibiotics were omitted in RNAi experiments. C2C12 myoblasts were grown at 37˚C under 5% CO 2 in Dulbecco's Modified Eagle's medium (Sigma-Aldrich, Gillingham, UK) supplemented with 10% (v/v) foetal calf serum (FCS) and 4 mM glutamine. Mitochondria were prepared as described in [START_REF] Affourtit | Stronger control of ATP/ADP by proton leak in pancreatic beta-cells than skeletal muscle mitochondria[END_REF]. INS-1E cells and myoblasts harvested from 500 cm 2 trays (Nunclon; 80-90% confluence) yielded typically ~10 and 3 mg mitochondrial protein per tray, respectively. The comparatively low yield from myoblasts is due to the relatively high mechanical force required to rupture cells of this type. Importantly, mitochondria from INS-1E cells and myoblasts were well coupled, with respiratory control ratios during succinate oxidation of ~3 and 4, respectively.

UCP2 knock-down and detection siRNA oligos (Ambion, Huntingdon, UK) were targeted at rat Ucp2 exons 3, 5 and 8 (Table 1). Their ability to lower UCP2 levels was assessed in cells, seeded in 175 cm 2 flasks (BD Falcon™), grown to ~50% confluence. Following complex-formation, siRNA and the transfection agent Lipofectamine™ 2000 (Invitrogen, Paisley, UK) were added at final concentrations of 50 nM and 1.7 µg/mL, respectively. To identify potential non-specific effects, cells were transfected in parallel with scrambled siRNA (Ambion, Silencer® Negative Control 1). After 3 days' further growth, transfected cells were trypsinized; mitochondria were then isolated, resuspended and stored at -80˚C in gel- Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. loading buffer (10% (w/v) SDS, 250 mM Tris-HCl (pH 6.8), 5 mM EDTA, 50% (v/v) glycerol, 5% (v/v) ß-mercaptoethanol, 0.05% (w/v) bromophenol blue). To detect UCP2, 20 µg mitochondrial protein was separated on a 12% SDS polyacrylamide gel and transferred to nitrocellulose. The membrane was blocked for 2 h at room temperature in Tris-buffered saline containing 0.1% (v/v) Tween 20 and 5% (w/v) dried skimmed milk (Marvel), then probed overnight at 4˚C with goat anti-UCP2 1˚ antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA) diluted to 0.2 µg/mL in this blocking buffer. Subsequently, the membrane was incubated for 1 h at room temperature with peroxidase-conjugated 2˚ antibodies (Pierce Biotechnology, Rockford, IL, USA) diluted to 0.1 µg/mL in blocking buffer, then cross-reacted proteins were visualised by enhanced chemiluminescence (Amersham Biosciences, Little Chalfont, UK). Finally, the membrane was stained with GelCode® Blue reagent (Pierce Biotechnology, Rockford, IL, USA) to confirm equal protein loading. Protein was quantified by densitometry using ImageJ software (http://rsb.info.nih.gov/ij/).

Mitochondrial proton leak

O 2 -uptake rates and ∆ψ were measured simultaneously as described in [START_REF] Affourtit | Stronger control of ATP/ADP by proton leak in pancreatic beta-cells than skeletal muscle mitochondria[END_REF]. Mitochondria were incubated at ~0.9 mg/mL with 350 ng/mL nigericin to collapse ∆pH, 4 µM rotenone to inhibit complex I, and 0.9 nmol oligomycin/mg protein to inhibit phosphorylation. Succinate (4 mM) was added to initiate state 4 respiration, which was titrated with malonate (up to 0.4 mM) causing stepwise inhibition of respiratory chain activity and decrease of ∆ψ. Expression of the successive O 2 -uptake rates as a function of the concomitant ∆ψ values revealed the kinetic response of proton leak to ∆ψ.

Cell respiration

Non-transfected INS-1E cells and myoblasts, seeded in 175 cm 2 flasks, were grown to ~80% confluence. In RNAi experiments, cells were grown to roughly 50% confluence, transfected as described above, and then grown another 3 days. Cells were then trypsinized, resuspended in their respective growth media at roughly 10 7 cells/mL, and stored on ice. Cell aliquots (600 µL) were put in glass vessels, flushed with 5% CO 2 -95% air, sealed, incubated for 30 min in a shaking (100 cycles/min) water bath at 37˚C, and then transferred to an oxygraph. Respiratory rates were calculated from the initial steady parts of the O 2 -uptake traces and expressed per 10 6 viable cells. Experiments were also performed with 7 µM oligomycin to approximate proton leak, and 9 µM myxothiazol to

Insulin secretion

Cells were seeded in 24-well plates (BD Falcon™) at 3x10 5 cells/well, grown overnight, and then transfected with UCP2-targeted (exon 8) or scrambled siRNA. After 3 days' further growth, transfected cells were starved for 2 h in growth medium that lacked glucose and pyruvate, and contained only 1% (v/v) FCS. Cells were washed 2x with a glucose-free Krebs-Ringer bicarbonate HEPES buffer (KRBH: 135 mM NaCl, 3.6 mM KCl, 10 mM Hepes (pH 7.4), 0.5 mM MgCl 2 , 1.5 mM CaCl 2 , 5 mM NaHCO 3 , 0.5 mM NaH 2 PO 4 and 0.1% (w/v) bovine serum albumin) and were then incubated in this buffer for 30 min in a shaking (30 cycles/min) water bath at 37˚C. Next, medium was replaced with KRBH containing 2, 5 or 30 mM glucose, or 2 mM glucose + 30 mM KCl. After a further 30 min, medium was collected and centrifuged to pellet any detached cells. Supernatants were assayed for insulin by ELISA (Mercodia, Uppsala, Sweden) using mouse insulin as a standard.

Statistics

Mean differences between experimental systems and conditions were tested for statistical significance by Student's t-tests.

RESULTS

Higher proton leak in INS-1E cells than myoblasts

INS-1E mitochondria exhibit proton conductance that is significantly higher than that observed in mitochondria from rat skeletal muscle [START_REF] Affourtit | Stronger control of ATP/ADP by proton leak in pancreatic beta-cells than skeletal muscle mitochondria[END_REF]. However, it may be that this difference solely indicates a discrepancy between cell and tissue mitochondria, or merely reflects a mitochondrial isolation artefact. As evidenced by the data shown in Fig. 1, both these concerns are ungrounded. Fig. 1A demonstrates that proton leak rate in myoblast mitochondria is substantially lower at any measured ∆ψ than the equivalent rate in INS-1E mitochondria. The considerable leak difference between mitochondria also manifests itself when leak is measured in intact cells. The proton leak rate in cells was estimated as the intracellular mitochondrial respiration rate (defined as respiration sensitive to the electron transport chain inhibitor myxothiazol) that was not sensitive to the ATP synthase inhibitor oligomycin. This method overestimates the leak rate a little because oligomycin causes a small increase in ∆ψ that slightly increases leak, but based on experiments with hepatocytes [10] the overestimate is not expected to be large. Although total INS-1E O 2 -uptake is only about half of that observed in myoblasts, the absolute oligomycin-resistant rate is nearly 4x higher in INS-1E cells, and relative to the rate without oligomycin, the rate with oligomycin is 7x higher in INS-1E cells (Fig. 1B). These cellular differences are higher than predicted from the mitochondrial data, which illustrates the relative importance of respiratory experiments in intact cells.

From Fig. 1B, the ratio of O 2 -uptake rates with and without oligomycin can be calculated.

This ratio provides an estimation of the proportion of respiratory activity that is used to drive 'futile' Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. mitochondrial proton leak in INS-1E (0.76 ± 0.06) and muscle cells (0.11 ± 0.02). Thus 75% of respiration would appear to drive proton leak in INS-1E cells, whilst only 10% of respiration in myoblasts is used for this purpose. Keeping in mind that the in vitro total respiratory rates used to calculate these proportions are not necessarily representative of those prevalent in vivo, the obtained numbers may be compared with similar ratios reported in the literature. The equivalent values are ~25% in hepatocytes [10], and ~20% in thymocytes [START_REF] Buttgereit | ConA induced changes in energy metabolism of rat thymocytes[END_REF] and neuronal cells [START_REF] Jekabsons | In situ respiration and bioenergetic status of mitochondria in primary cerebellar granule neuronal cultures exposed continuously to glutamate[END_REF]. The high INS-1E value disagrees with a value of 10% in INS-1 cells [START_REF] Produit-Zengaffinen | Increasing uncoupling protein-2 in pancreatic beta cells does not alter glucose-induced insulin secretion but decreases production of reactive oxygen species[END_REF], perhaps due to extensive differences in experimental conditions. Alternatively, this discrepancy could be related to the different origins of the respective cell lines. INS-1 is an insulin-secreting line that was isolated from a radiation-induced rat insulinoma [START_REF] Asfari | Establishment of 2-mercaptoethanol-dependent differentiated insulin-secreting cell lines[END_REF]. Because of their non-clonal nature, INS-1 cells exhibit a limited stability over passages, which has been suggested as a possible explanation for the large variability in published results [START_REF] Merglen | Glucose sensitivity and metabolism-secretion coupling studied during two-year continuous culture in INS-1E insulinoma cells[END_REF]. INS-1E on the other hand, is a clonal line derived from the parental INS-1 cells [START_REF] Janjic | Free radical modulation of insulin release in INS-1 cells exposed to alloxan[END_REF], has been characterised in detail, and represents a significant improvement in terms of beta cell differentiation and stability [START_REF] Merglen | Glucose sensitivity and metabolism-secretion coupling studied during two-year continuous culture in INS-1E insulinoma cells[END_REF].

UCP2 contributes to high INS-1E proton leak

Given the pronounced effect of UCP2-ablation on GSIS in pancreatic islets [START_REF] Zhang | Uncoupling protein-2 negatively regulates insulin secretion and is a major link between obesity, beta cell dysfunction, and type 2 diabetes[END_REF], we reasoned that the relatively high proton leak activity observed in INS-1E cells (Fig. 1B) could be due to UCP2 activity.

We used RNAi to reduce UCP2 levels, and thus test this hypothesis. We routinely use a polyclonal antibody to detect UCP2 in mouse tissues, and find no UCP2 band in samples from UCP2 knockout mice (not shown). Fig. 2A shows that in INS-1E cells this polyclonal antibody cross-reacts with a single protein of ~32 kD in samples obtained from both non-transfected cells (lane 1) and those transfected with scrambled siRNA (lane 5). The intensity of this band is reduced dramatically in samples from cells that have been transfected, separately, with three different siRNA oligonucleotides targeted at independent Ucp2 exons (lanes 2-4). Densitometric comparison confirms equal loading of total mitochondrial protein (Figs 2B and2D), and reveals that UCP2 levels are reduced substantially within 72 h after transfection (Fig. 2C). The observed densities upon UCP2 knock-down are roughly 15% (exons 5 and 3) and 10% (exon 8) of the scrambled control.

O 2 -consumption rates of cells transfected with scrambled siRNA were 10.6 ± 2.1 in the absence and 7.7 ± 1.5 nmol O/min/10 6 cells in the presence of oligomycin. These activities are comparable with those observed in non-transfected INS-1E cells (Fig. 1A). The overall respiratory activity of cells transfected with Ucp2-targeted siRNA is on average nearly 13% lower than that of cells transfected with scrambled siRNA (Fig. 3A). This difference is statistically significant (p = 0.02) and, importantly, it arises irrespective of the particular Ucp2 exon targeted. Similarly, oligomycininsensitive respiratory activity is lowered, on average by more than 30% (p = 1 x 10 -5 ), when UCP2 levels are diminished by siRNA targeted at 3 separate exons (Fig. 3B). As a result, the fraction of overall respiration that is oligomycin-resistant is reduced significantly (p = 0.01) upon UCP2 knock-S t a g e 2 ( a ) P O S T -P R I N T down (Fig. 3C). This suggests strongly that UCP2 contributes substantially to the proton leak rate of non-transfected INS-1E cells.

In this context, it is anticipated that ∆ψ increases upon UCP2 knock-down. This will not only lead to an underestimation of UCP2's contribution to proton leak (see Discussion), but may also result in increased reactive oxygen species levels. It is possible that this in turn activates additional uncoupling mechanisms. However, the nature of UCP2-independent proton leak is unclear at present.

UCP2 knock-down enhances insulin secretion

In the current experiments, insulin secretion rates in INS-1E cells are relatively high at 2 mM glucose. However, these rates are increased significantly when the glucose level is raised to 30 mM, or cells are incubated in the additional presence of 30 mM KCl (Fig. 4A). Since INS-1E cells are expected to respond to such stimuli in this manner [START_REF] Merglen | Glucose sensitivity and metabolism-secretion coupling studied during two-year continuous culture in INS-1E insulinoma cells[END_REF], we deemed our assay suitable to assess the effect of UCP2 knock-down on GSIS. In cells transfected with scrambled siRNA, insulin secretion is similar at 2 and 5 mM glucose (Fig. 4A), but whilst UCP2 knock-down does not affect secretion at 2 mM glucose, it enhances insulin release at 5 mM glucose. This indicates GSIS is improved, which agrees with observations made in islets from Ucp2 knock-out mice [START_REF] Zhang | Uncoupling protein-2 negatively regulates insulin secretion and is a major link between obesity, beta cell dysfunction, and type 2 diabetes[END_REF]. Upon UCP2 knock-down, insulin secretion is also increased at 30 mM glucose but, importantly, KCl-induced secretion, which does not rely on mitochondrial energy metabolism, is not affected (Fig. 4A). From Fig. 4B it can be seen that cell number and cellular insulin content are not altered significantly after UCP2 down-regulation, although the average total insulin level appears somewhat increased.

DISCUSSION

Because the cytoplasmic ATP/ADP of pancreatic cells is governed predominantly by ATP supply [START_REF] Rutter | Nutrient-secretion coupling in the pancreatic islet beta-cell: recent advances[END_REF], these cells are interesting from a bioenergetic perspective. Glucose-induced increases in ATP/ADP observed in cells [START_REF] Rutter | Nutrient-secretion coupling in the pancreatic islet beta-cell: recent advances[END_REF] are not seen in most other cell types, where this ratio is mainly controlled by ATP demand [17]. Based on a metabolic control analysis of oxidative phosphorylation in muscle and INS-1E mitochondria, we suggested recently that this difference in control is partly due to mitochondrial peculiarities [START_REF] Affourtit | Stronger control of ATP/ADP by proton leak in pancreatic beta-cells than skeletal muscle mitochondria[END_REF]. In particular, proton leak rate was relatively high in INS-1E mitochondria and, consequently, exerted relatively high control over ∆ψ and the external ATP/ADP ratio. The data reported here demonstrate that a relatively high proton conductance does not merely represent a mitochondrial isolation artefact, but is a characteristic feature of intact INS-1E cells (Fig. 1B). The estimated proportion of overall respiratory activity used to drive 'futile' proton leak in INS-1E cells is much larger than that used in hepatocytes [10], thymocytes [START_REF] Buttgereit | ConA induced changes in energy metabolism of rat thymocytes[END_REF] or neuronal cells [START_REF] Jekabsons | In situ respiration and bioenergetic status of mitochondria in primary cerebellar granule neuronal cultures exposed continuously to glutamate[END_REF].

Our results establish that a large part of the high leak rate in INS-1E cells is accounted for by UCP2, as oligomycin-resistant O 2 -consumption is decreased by ~30% when the protein is knocked down using RNAi (Fig. 3B). Fig. 3A indicates that UCP2 also contributes to the total respiratory Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. activity. A contribution of 13% is an underestimate, however, because UCP2 knock-down will raise ∆ψ and cause a compensatory increase in proton leak. A better estimate comes from multiplying the slightly underestimated contribution of UCP2 to the oligomycin-insensitive respiration (30%) by the slightly overestimated proportion of overall respiration that drives proton leak (75%) to give a value of about 20% of the cell respiration rate caused by proton cycling through UCP2 in cells. This value is calculated assuming RNAi removes all UCP2, which will cause a further small underestimation of its contribution. Nonetheless, 20% is high compared to the calculated 10% in thymocytes: ~20% of thymocyte respiration is caused by proton cycling [START_REF] Buttgereit | ConA induced changes in energy metabolism of rat thymocytes[END_REF] and ~50% of this is lost in thymocytes from UCP2 knock-out mice [18].

The relatively high contribution of UCP2 to INS-1E respiration (Fig. 3), its attenuating effect on GSIS (Fig. 4A), and the significant control of proton leak over ATP/ADP [START_REF] Affourtit | Stronger control of ATP/ADP by proton leak in pancreatic beta-cells than skeletal muscle mitochondria[END_REF] support the view that UCP2 may play an important regulatory role in cell physiology. Our data indicate that UCP2 is most likely active under pathological and physiological conditions with its activation state being dependent on fluctuating levels of effectors such as superoxide [START_REF] Echtay | Superoxide activates mitochondrial uncoupling proteins[END_REF]. In other words, UCP2 activation is unlikely just a pathological side effect of a damage limitation mechanism that is induced during highfat feeding or hyperglycemia and that causes cell dysfunction [START_REF] Zhang | Uncoupling protein-2 negatively regulates insulin secretion and is a major link between obesity, beta cell dysfunction, and type 2 diabetes[END_REF][START_REF] Krauss | Superoxide-mediated activation of uncoupling protein 2 causes pancreatic beta cell dysfunction[END_REF][START_REF] Joseph | Free fatty acid-induced beta-cell defects are dependent on uncoupling protein 2 expression[END_REF]. We suggest that the relatively high mitochondrial proton leak in cells is a mechanism that amplifies the effect of physiological regulators of UCP2 proton conductance on the cytoplasmic ATP/ADP ratio and hence on GSIS.

The notion that UCP2 regulates GSIS is corroborated by a steadily increasing body of evidence. Initial studies showed that adenoviral over-expression of UCP2 leads to attenuated GSIS in rats [START_REF] Chan | Increased uncoupling protein-2 levels in beta-cells are associated with impaired glucose-stimulated insulin secretion -Mechanism of action[END_REF] and insulinoma cells [START_REF] Hong | Effects of adenoviral overexpression of uncoupling protein-2 and -3 on mitochondrial respiration in insulinoma cells[END_REF]. Despite the fact that over-expression of UCPs tends to cause nonspecific proton conductance [START_REF] Esteves | The reactions catalysed by the mitochondrial uncoupling proteins UCP2 and UCP3[END_REF], such observations demonstrate that GSIS is impaired when oxidative phosphorylation becomes less efficient. More importantly, investigations involving Ucp2-ablated mice showed convincingly that UCP2 is a negative modulator of GSIS under conditions that are of (patho)physiological relevance [START_REF] Zhang | Uncoupling protein-2 negatively regulates insulin secretion and is a major link between obesity, beta cell dysfunction, and type 2 diabetes[END_REF][START_REF] Krauss | Superoxide-mediated activation of uncoupling protein 2 causes pancreatic beta cell dysfunction[END_REF][START_REF] Joseph | Free fatty acid-induced beta-cell defects are dependent on uncoupling protein 2 expression[END_REF][START_REF] Zhang | Genipin inhibits UCP2-mediated proton leak and acutely reverses obesity-and high glucose-induced beta cell dysfunction in isolated pancreatic islets[END_REF]. Our results (Fig. 4A) agree with these gene knockout studies: lowering UCP2 levels in INS-1E cells through RNAi results in increased GSIS. INS-1E data are inevitably of less physiological relevance than those obtained using islets. Arguably, however, the interpretation of experiments with Ucp2-knockout mice is relatively complicated, since the ab initio absence of the Ucp2 gene may have caused potentially complex developmental adaptations. Additionally, the observed altered islet behaviour may be an indirect result from the absence of Ucp2 in other tissues such as the brain. Because our experimental model does not suffer from such complications, the data reported in this paper demonstrate that GSIS can be modulated acutely by UCP2 activity. Such acute regulation is in line with observed immediate effects on GSIS of a superoxide dismutase mimetic [START_REF] Krauss | Superoxide-mediated activation of uncoupling protein 2 causes pancreatic beta cell dysfunction[END_REF] and a pharmacological UCP2 inhibitor [START_REF] Zhang | Genipin inhibits UCP2-mediated proton leak and acutely reverses obesity-and high glucose-induced beta cell dysfunction in isolated pancreatic islets[END_REF]. It is also worth notice that acute short-term inhibition of UCP2 expression by antisense oligonucleotides has recently been shown to ameliorate the hyperglycemic syndrome in two different animal models of type 2 diabetes [START_REF] De Souza | Inhibition of UCP2 expression reverses diet-induced diabetes mellitus by effects on both insulin secretion and action[END_REF]. Interestingly, this metabolic improvement appears to be due to a combined effect of UCP2 knock-down on insulin secretion by pancreatic cells and insulin action on peripheral tissues.

Despite the mounting evidence for a GSIS-modulatory role, it remains uncertain whether UCP2 indeed affects insulin secretion through uncoupling ATP synthesis from mitochondrial electron transfer. Previously reported effects of UCP2 on ATP/ADP (e.g. [START_REF] Zhang | Uncoupling protein-2 negatively regulates insulin secretion and is a major link between obesity, beta cell dysfunction, and type 2 diabetes[END_REF][START_REF] De Souza | Inhibition of UCP2 expression reverses diet-induced diabetes mellitus by effects on both insulin secretion and action[END_REF]), ∆ψ [START_REF] Krauss | Superoxide-mediated activation of uncoupling protein 2 causes pancreatic beta cell dysfunction[END_REF][START_REF] Zhang | Genipin inhibits UCP2-mediated proton leak and acutely reverses obesity-and high glucose-induced beta cell dysfunction in isolated pancreatic islets[END_REF], and O 2consumption [START_REF] Joseph | UCP2 knockout mouse islets have lower consumption and faster oscillations of beta cell oxygen[END_REF] favour such an uncoupling function, but are indirect measures providing circumstantial support only. In fact, the strongest direct indication that UCP2 uncouples oxidative phosphorylation in situ in any mammalian cell, comes from kinetic experiments with thymocytes [18]. At any given ∆ψ, thymocytes from Ucp2 knock-out mice exhibit lower non-phosphorylating (i.e. state 4) respiratory activity than wild-type cells. The effect of UCP2 knock-down on oligomycinresistant (i.e. state 4) O 2 -uptake in INS-1E cells (Fig. 3) is in close agreement with the kinetic measurements in thymocytes, and provides direct evidence for an uncoupling function of UCP2 in cells.

The results reported in this paper validate predictions of relatively high proton leak activity in INS-1E cells, and demonstrate that UCP2 contributes substantially to this activity. They show furthermore that UCP2 modulates insulin secretion in INS-1E cells in a fashion that is similar, qualitatively, to that observed in pancreatic islets. Our work thus highlights that INS-1E cells are a convenient and appropriate model to further clarify the role of UCP2 in islet physiology. Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

  S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 10 Sep 2007 as manuscript BJ20070954 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20070954

  determine non-mitochondrial O 2 -uptake. Inhibitors were dissolved in DMSO (final concentration ≤ 0.2%) and added just before flushing with CO 2 -air. Both concentrations gave maximum effects as checked by doubling concentrations. S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 10 Sep 2007 as manuscript BJ20070954 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20070954 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

  S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 10 Sep 2007 as manuscript BJ20070954 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20070954

  S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 10 Sep 2007 as manuscript BJ20070954 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20070954

  S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 10 Sep 2007 as manuscript BJ20070954 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20070954 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

[

Table 1 -

 1 siRNA oligonucleotide sequences (5'->3' sense) targeted at the rat Ucp2 g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 10 Sep 2007 as manuscript BJ20070954 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20070954 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

Figure 1 -

 1 Figure 1 -Higher proton leak in INS-1E cells than myoblasts A: Proton leak (J L ) dependence on ∆ψ was measured in INS-1E () and myoblast () mitochondria. Data and error bars represent mean ± SEM of 5 ( ) and 6 ( ) samples; data were fitted to exponentials. B: Whole-cell respiration (J O ) ± 7 µM oligomycin was measured in myoblasts (open) and INS-1E cells (grey); non-mitochondrial activity (3.3 ± 0.4 and 2.8 ± 0.3 nmol O/min/10 6 myoblasts and INS-1E cells, respectively) was subtracted. Data and error bars represent means ± SEM of 4 experiments.

Figure 2 -

 2 Figure 2 -UCP2 knock-down A: Western blot (UCP2) of mitochondrial protein from non-transfected INS-1E cells (1) and cells transfected with scrambled siRNA (5) or siRNA targeted at Ucp2 exons 5, 8, and 3 (2-4, respectively). B: nitrocellulose membrane stained with GelCode® Blue after Western analysis; contrast was enhanced in silico to aid visualisation. C: Densitometric profiles of lanes 2-5 of Western blot shown in panel A. D: Whole-lane relative densitometric quantification of protein stained with GelCode® Blue; quantification was performed using an image of which the contrast had not been enhanced. Numbers left to panel A represent molecular weight markers (kDa).

Figure 3 -

 3 Figure 3 -UCP2 knock-down lowers proton leak Respiration (J O ) was measured in the absence (A) or presence (B) of 7 µM oligomycin in INS-1E cells transfected with scrambled siRNA (black), or siRNA targeted at UCP2 exons 5 (dark grey), 8 (light grey) or 3 (open); non-mitochondrial activity (3.0 ± 0.7, 2.0 ± 0.5, 1.9 ± 0.4 and 1.7 ± 0.5 nmol O/min/10 6 cells, respectively) was subtracted. Data and error bars represent means ± SEM of 5 experiments and are expressed as a fraction of the scrambled control rates (A and B ), or as the oligomycin-insensitive respiratory fraction (C). Scrambled control rates ± oligomycin were 7.7 ± 1.5 and 10.6 ± 2.1 nmol O/min/10 6 cells, respectively.

Figure 4 - 12 S

 412 Figure 4 -UCP2 knock-down enhances GSIS INS-1E cells were transfected with scrambled (grey) or UCP2-exon 8 (open) siRNA. A: Insulin secretion was measured at 2, 5 and 30 mM glucose (G2, G5, G30), and at 2 mM glucose + 30 mM KCl (KCl). Data and error bars represent means ± SEM of 6 (G2, G5, G30) and 12 (KCl) experiments with each condition assayed 4-8x. B: Cells were counted on a haemocytometer after trypsinization from 24-well plates (3 wells were pooled for each count). Total cellular insulin content was measured in acidified-ethanol extracts. Data and error bars represent means ± SEM of 3 experiments performed in quadruplicate.S t a g e 2 ( a ) P O S T -P R I N T

ACKNOWLEDGEMENTS INS-1E cells were kindly donated by Drs P. Maechler and C. Wollheim (Department of Internal Medicine, University Medical Center, Geneva, Switzerland). This work was supported by the Medical Research Council, United Kingdom.