Tatu J K Haataja

M Kristian Koski

J Kalervo Hiltunen

Tuomo Glumoff
email: tuomo.glumoff@oulu.fi

Peroxisomal multifunctional enzyme type 2 from the fruit fly: dehydrogenase and hydratase act as separate entities as revealed by structure and kinetics

Keywords: CoA, coenzyme A, DmMFE-2, Drosophila melanogaster multifunctional enzyme type 2, multifunctional enzyme type 2 (MFE-2), hydroxysteroid (17-beta) dehydrogenase 4 homolog, crystal structure, peroxisomes, fatty acid metabolism, substrate channelling

Abbreviations used: CoA, coenzyme A; DmMFE-2, Drosophila melanogaster multifunctional enzyme type 2; DH, 3R-hydroxyacyl-CoA dehydrogenase; H2, 2E-Enoyl-CoA hydratase 2; SCP-2L, sterol carrier protein-2-like; NAD + , nicotinamide

INTRODUCTION

Acyl-CoA esters are oxidized in the β-oxidation spiral that is the major pathway for the breakdown of fatty acids and is found, depending on the species, in peroxisomes and mitochondria. The mitochondrial system seems to be a property of mammals only, whereas peroxisomal β-oxidation is found in all eukaryotes studied this far [START_REF] Poirier | Peroxisomal betaoxidation-a metabolic pathway with multiple functions[END_REF].

The second and third reactions in the peroxisomal β-oxidation cycle (Figure 1) are performed by a multifunctional enzyme that consists of one single polypeptide chain harbouring both dehydrogenase and hydratase activities. In the mammalian peroxisomes, there are two structurally nonrelated multifunctional enzymes involved in the fatty acid breakdown with different stereo specificities towards the 3-hydroxy intermediate. The subject of the current study, multifunctional enzyme type 2 (MFE-2), is active towards the R-isomers of the substrate intermediates [START_REF] Hiltunen | Peroxisomal multifunctional beta-oxidation protein of Saccharomyces cerevisiae. Molecular analysis of the fox2 gene and gene product[END_REF][START_REF] Adamski | Molecular cloning of a novel widely expressed human 80 kDa 17 beta-hydroxysteroid dehydrogenase IV[END_REF]. Both peroxisomal MFE type 1 and the membrane-associated mitochondrial trifunctional enzyme complex house 3S-hydroxy specific dehydrogenase [START_REF] Osumi | Peroxisomal beta oxidation system of rat liver. Copurification of enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase[END_REF][START_REF] Uchida | Novel fatty acid beta-oxidation enzymes in rat liver mitochondria. II. Purification and properties of enoyl-coenzyme A (CoA) hydratase/3-hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase trifunctional protein[END_REF]. MFE-2 has been identified from various species, first in fungi [START_REF] Hiltunen | Peroxisomal multifunctional beta-oxidation protein of Saccharomyces cerevisiae. Molecular analysis of the fox2 gene and gene product[END_REF] and later on in mammals [START_REF] Adamski | Molecular cloning of a novel widely expressed human 80 kDa 17 beta-hydroxysteroid dehydrogenase IV[END_REF][START_REF] Corton | Rat 17 beta-hydroxysteroid dehydrogenase type IV is a novel peroxisome proliferator-inducible gene[END_REF][START_REF] Qin | Peroxisomal multifunctional enzyme of beta-oxidation metabolizing D-3-hydroxyacyl-CoA esters in rat liver: Molecular cloning, expression and characterization[END_REF][START_REF] Dieuaide-Noubhani | Identification and characterization of the 2-enoyl-CoA hydratases involved in peroxisomal beta-oxidation in rat liver[END_REF][START_REF] Jiang | Physiological role of D-3-hydroxyacyl-CoA dehydratase/D-3-hydroxyacyl-CoA dehydrogenase bifunctional protein[END_REF]. Curiously, MFE-2 deficiency forms the larger subgroup among peroxisomal disorders in humans [START_REF] Van Grunsven | Peroxisomal Dhydroxyacyl-CoA dehydrogenase deficiency: Resolution of the enzyme defect and its molecular basis in bifunctional protein deficiency[END_REF]. The protein has variable domain organizations depending on the source as summarized in figure 2.

The dehydrogenase domain of MFE-2 belongs to the short chain dehydrogenase/reductase (SDR) family [START_REF] Jörnvall | Short-chain dehydrogenases/reductases (SDR)[END_REF]. The structure from the rat domain [START_REF] Haapalainen | Binary structure of the two-domain (3R)-hydroxyacyl-CoA dehydrogenase from rat peroxisomal multifunctional enzyme type 2 at 2.38 A resolution[END_REF] includes the ligand NAD + , and in addition to the typical ´Rossman fold´ nucleotide binding core it also contains an extended C-terminal subdomain. This 60-residue part is unique: other proteins in the SDR family do not have it. In the dehydrogenase homodimer the C-terminal subdomain from one monomer folds on top of the substrate binding tunnel of the other monomer, and vice versa [START_REF] Haapalainen | Binary structure of the two-domain (3R)-hydroxyacyl-CoA dehydrogenase from rat peroxisomal multifunctional enzyme type 2 at 2.38 A resolution[END_REF]. In this way the monomers contribute to the active site architectures of each other in a cooperative manner, and monomer-monomer interactions are strengthened. The dehydrogenases in yeast MFE-2 form a structural dimer within the polypeptide (Figure 2) with the two domains preferring different chain length fatty acyl-CoA substrates [START_REF] Qin | Yeast peroxisomal multifunctional enzyme: (3R)-hydroxyacyl-CoA dehydrogenase domains A and B are required for optimal growth on oleic acid[END_REF][START_REF] Ylianttila | Abbreviations used: CoA, coenzyme A; DmMFE-2, Drosophila melanogaster multifunctional enzyme type 2; DH, 3R-hydroxyacyl-CoA dehydrogenase; H2, 2E-Enoyl-CoA hydratase 2; SCP-2L, sterol carrier protein-2-like; NAD + , nicotinamide adenine dinucleotide; IPTG, isopropyl β-D-1thiogalactopyranoside; C4, 2E-butenoyl-CoA; C6, 2E-hexenoyl-CoA[END_REF].

The hydratase domain structures of MFE-2 (which is R-specific) from human and C. tropicalis [START_REF] Koski | A two-domain structure of one subunit explains unique features of eukaryotic hydratase 2[END_REF][START_REF] Koski | Crystal structure of 2-enoyl-CoA hydratase 2 from human peroxisomal multifunctional enzyme type 2[END_REF] are structurally different from the S-specific hydratases (hydratase 1, crotonase) from mitochondria [START_REF] Engel | The crystal structure of enoyl-CoA hydratase complexed with octanoyl-CoA reveals the structural adaptations required for binding of a long chain fatty acid-CoA molecule[END_REF] and peroxisomal MFE-1 [18]. Hydratase 2 from MFE-2 folds into a hot-dog fold with a long D-helix wrapped inside a 4-stranded E-sheet. The bacterial R-hydratase [START_REF] Hisano | Crystal structure of the (R)-specific enoyl-CoA hydratase from Aeromonas caviae involved in polyhydroxyalkanoate biosynthesis[END_REF], however, has common structural features with the MFE-2 hydratase. Comparison of the two has revealed the structural basis for the adaptation of the eukaryotic enzyme to accept long chain substrates: the absence of the D-helix from one of the hot-dog fold subdomains creates space for bulky substrates [START_REF] Koski | A two-domain structure of one subunit explains unique features of eukaryotic hydratase 2[END_REF].

Mammalian species and such as Danio rerio MFE-2s have a compact 13 kDa domain in their C-terminus [START_REF] Haapalainen | Crystal structure of the liganded SCP-2-like domain of human peroxisomal multifunctional enzyme type 2 at 1[END_REF], which is structurally homologous to sterol carrier protein 2 (Figure 2). It is a nonspecific lipid-binding protein displaying broad ligand binding properties. Drosophila, however, is one of the species lacking this domain from its MFE-2 protein (Figure 2). Despite the structurally well-characterized domains of MFE-2, it is not known how the fulllength protein is assembled and how the enzymatically active domains possibly interact in catalysis. In the case of multifunctional proteins, possessing more than one enzymatic activity responsible for consecutive steps in a pathway, a substrate channelling is always worth considering. There are wellcharacterized substrate channelling enzymes (review by Weeks et al.) [START_REF] Weeks | Tunneling of intermediates in enzyme-catalyzed reactions[END_REF]; however, an emerging picture of substrate channeling is that there may be real tunnels for small molecules inside proteins, but for bulkier substrates, like fatty acyl-CoAs, the molecular mechanisms are not as obvious, as exemplified by studies on the mitochondrial trifunctional enzyme [START_REF] Ishikawa | Structural basis for channelling mechanism of a fatty acid beta-oxidation multienzyme complex[END_REF] and peroxisomal MFE-1 [18]. Abbreviations used: CoA, coenzyme A; DmMFE-2, Drosophila melanogaster multifunctional enzyme type 2; DH, 3R-hydroxyacyl-CoA dehydrogenase; H2, 2E-Enoyl-CoA hydratase 2; SCP-2L, sterol carrier protein-2-like; NAD + , nicotinamide adenine dinucleotide; IPTG, isopropyl β-D-1thiogalactopyranoside; C4, 2E-butenoyl-CoA; C6, 2E-hexenoyl-CoA; C10, 2E-decenoyl-CoA.

Here we characterize the Drosophila MFE-2 (DmMFE-2) as an enzyme both in vitro and in vivo. We show that DmMFE-2 complements a defect of peroxisomal MFE-2 in yeast. We also present the first crystal structure of a full-length MFE-2 protein revealing the assembly of domains and dimerization. Both the structure and the kinetic properties do not support a substrate channeling mechanism in DmMFE-2.

Biochemical

EXPERIMENTAL Construction of the bacterial over-expression plasmids

The D. melanogaster cDNA clone (GH14720) encoding a 598 amino acid polypeptide that possesses a stretch of amino acid sequence similar to short chain dehydrogenase/reductase (SDR) and maoC-like dehydratase enzymes (accession code CG3415) was purchased from Drosophila Genomics Resource Center, Bloomington, IN, USA. The 64.1 kDa expression product is here referred to as the peroxisomal multifunctional enzyme type 2 (DmMFE-2). The 1797 bp DNA fragment was amplified using the primers 5' ttttaaaaacatATGTCCTCATCCGATGGAAAACTTCGTTACGAT 3' and 3' tttaaactcgagCAGCTTGGCCTGTGAGCTCTTCAGGTCGACATA 5' with the restriction enzyme recognition sites underlined and the template specific sequence in capitals. The fragment was cloned into modified pET23b(+) expression vector [START_REF] Alanen | Functional characterization of ERp18, a new endoplasmic reticulum-located thioredoxin superfamily member[END_REF] resulting in plasmid pET23b::DmMFE-2. The same plasmid construction strategy was used when preparing the expression plasmids of the separate dehydrogenase (DmDH) and hydratase 2 (DmH2) domains of the DmMFE-2, resulting in plasmids pPAL7::DmDH and pET15b::DmH2 covering amino acids 1-309 and 310-598, respectively.

Over-expression and purification

The plasmid pET23b::DmMFE-2 was transformed into E. coli BL21(DE3) pLysS. A preculture for over-expression was cultured over night at 37 o C in Luria Bertani broth supplemented with 50 μg ml -1 carbenisillin and 34 μg ml -1 chloramphenicol. 20 ml of the pre-culture was transferred in 1.0 l of M9ZB medium and cultivated at 37 o C under aerobic conditions. The production of recombinant DmMFE-2 was induced by the addition of isopropyl-β-D-thiogalactopyranoside (IPTG) to 0.4 mM at OD 600 of 0.8 and the cells were harvested by centrifugation after 4 hours of induction. The cells were washed with PBS (137 mM NaCl, 10 mM Na 2 HPO 4 , 2.7 mM KCl, 1,8 mM KH 2 PO 4 pH 7.4), 5 g (wet weight) suspended in 20 ml of Ni-NTA Lysis buffer (50 mM Tris pH 8.0, 300 mM NaCl, 1.0 M Urea) and frozen using liquid nitrogen and stored at -70 o C until used.

The frozen cell suspension was thawed out at 30 o C and the cell lysis was completed by the addition of 500 μg ml -1 lysozyme, 50 μg ml -1 Dnase I and 50 μg ml -1 Rnase A. After 30 min of incubation at 30 o C, the lysate was cleared by centrifugation (30 000g, 45 min, 4 o C). The soluble fraction was applied at 1 ml min -1 to a 2.5 ml Ni-NTA affinity column (Qiagen) equilibrated in the lysis buffer prior to use. After flushing with Ni-NTA wash buffer (50 mM Tris pH 8.0, 500 mM NaCl, 20 mM imidazol), the target protein was released from the column using an elution buffer (50 mM Tris pH 8.0, 500 mM NaCl, 500 mM imidazol). A 14 ml Red Sepharose CL-6B (GE Healthcare) dye affinity column was equilibrated in 50 mM potassium phosphate buffer pH 7.0. The protein sample was diluted by 10 volumes with distilled water and was applied to the column at 0.5 ml min -1 . The bound protein was eluted with a linear gradient of KCl (0-2.0 M in 140 ml). Fractions containing DmMFE-2 were pooled, stabilized by 10 % (v/v) glycerol and concentrated. The sample was then applied to a Superdex 200 10/300 GL size exclusion column (GE Healthcare) equilibrated in 50 mM sodium phosphate pH 7.5, 200 mM NaCl, 1 mM Na 2 EDTA, 5 % glycerol, 1 mM NaN 3 . Major peaks were fractionated, DmMFE-2 containing fractions pooled and finally concentrated to 2.9 mg ml -1 .

The separate domains were expressed using E. coli BL21(DE3) and EnBase Flo medium (BioSilta, Oulu, Finland). The high-yield recombinant protein expression was carried out in 500 ml volume according to the instructions provided with the kit. The recombinant protein was induced for 24 hours at 30 o C by adding of 0.4 mM or 1 mM IPTG to the culture medium. In a separate study, the use of EnBase Flo significantly improved the expression of the full-length enzyme [START_REF] Krause | A novel fed-batch based cultivation method provides high cell-density and improves yield of soluble recombinant proteins in shaken cultures[END_REF]. Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Abbreviations used: CoA, coenzyme A; DmMFE-2, Drosophila melanogaster multifunctional enzyme type 2; DH, 3R-hydroxyacyl-CoA dehydrogenase; H2, 2E-Enoyl-CoA hydratase 2; SCP-2L, sterol carrier protein-2-like; NAD + , nicotinamide adenine dinucleotide; IPTG, isopropyl β-D-1thiogalactopyranoside; C4, 2E-butenoyl-CoA; C6, 2E-hexenoyl-CoA; C10, 2E-decenoyl-CoA.

The purification of the 2E-enoyl-CoA hydratase 2 from DmMFE-2 followed the protocol for purifying the full-length DmMFE-2, except that the step with Red Sepharose CL-6B was omitted. The final yield of recombinant DmH2 from single 500 ml expression was 113 mg of purified protein. The recombinant 3R-hydroxyacyl-CoA dehydrogenase of DmMFE-2 was initially purified using a 5 ml Profinity eXact affinity column (Bio-Rad). After the expression, the cells were suspended in Profinity eXact lysis buffer (0.1 M sodium phosphate; pH 7.2, 0.3 M sodium acetate) and treated as in the purification of the full-length DmMFE-2 to obtain the cleared lysate for the first affinity purification step. After binding the dehydrogenase in the Profinity eXact column, the weakly bound proteins were flushed out using a wash buffer (0.1 M sodium phosphate; pH 7.2, 0.15 M sodium acetate). The elution of the tag-free recombinant protein was triggered using the elution buffer (0.1 M sodium phosphate; pH 7.2, 0.2 M NaF). After the elution, the purification of the Drosophila dehydrogenase proceeded as in the case of the DmH2. 5 mg of purified protein was obtained from the 500 ml expression.

Static light scattering (SLS) experiments

Superdex 200 HR 10/300 GL (GE Healthcare) size exclusion column connected to ÄKTA purifier (GE Healthcare) and multi-angle light scattering device, miniDAWN TREOS (Wyatt Technology Corp.), was used for analysing four different samples of DmMFE-2 by SLS-technique. The column was equilibrated in 50 mM sodium phosphate pH 7.5, 200 mM NaCl, 1 mM Na 2 EDTA, 5 % glycerol, 1 mM NaN 3 prior to use. Sample concentrations were DmMFE-2 7.4 mg/ml, DmDH 5.7 mg/ml, DmH2 6.1 mg/ml and DmDH + DmH2 (in stoichiometric ratio) 5.7 mg/ml. The samples were prepared in the same phosphate buffer in which the column was equilibrated. 50μl of protein was used for one injection and during the separation the system flow was 250 μl/min at 17 o C. The results were analysed using Astra V software (Wyatt Technology Corp.).

In vivo studies, enzyme kinetics and substrate synthesis

The DNA fragment encoding DmMFE-2 was cloned into pYE352::CTA1 Saccharomyces cerevisiae expression vector [25], replacing the CTA1-gene encoding the yeast peroxisomal catalase and resulting in pYE352::DmMFE-2. The plasmid was transformed [START_REF] Chen | One-step transformation of yeast in stationary phase[END_REF] into S. cerevisiae deletion strain BY4741 Δfox2 (YKR009c, Euroscarf, accession code Y0508) for the complementation studies. In addition, the Δfox2 deletion strain was also transformed with pYE352::CTA1 [25] and pYE352::ScMFE-2 [START_REF] Qin | Yeast peroxisomal multifunctional enzyme: (3R)-hydroxyacyl-CoA dehydrogenase domains A and B are required for optimal growth on oleic acid[END_REF].

For the yeast complementation study, the BY4741 Δfox2 + pYE352::DmMFE-2 dilution series (10 -1 -10 -4 ) was pipetted on thin YPD-plates supplemented with oleic acid (0.67 % yeast nitrogen base w/o amino acids, 0.1 % yeast extract, 0.5 % potassium dihydrogen phosphate; pH 6.0, 0.5 % (v/v) Tween 80, 0.14 % oleic acid, 0.2 % dextrose, ampicillin 50 μg/ml and 2.0 % Agar). BY4741 Δfox2 and BY4741 Δfox2 + pYE352::CTA1 served as the negative controls and BY4741 wt and BY4741 Δfox2 + pYE352::ScMFE-2 as positive controls on the examined plates. The cells for the dilution series were obtained from fresh saturated liquid cultures. The cells were washed with sterile water prior to use. The OD 600 of the cultures was evened out to 1.0 for preparing the dilution series in sterile water. The plates were incubated first at 30 o C for a week to force the yeast to utilize the oleic acid as the sole carbon source for growth and an additional week at 4 o C for optimizing the visualization of the obtained clearing zones around the yeast growth.

The enzyme activity assays were carried out according to Hiltunen et al. [27] using 2Ebutenoyl-CoA, 2E-hexenoyl-CoA and 2E-decenoyl-CoA as substrates. Substrates were synthesized as described by Qin et al. [START_REF] Qin | Yeast peroxisomal multifunctional enzyme: (3R)-hydroxyacyl-CoA dehydrogenase domains A and B are required for optimal growth on oleic acid[END_REF]. All the measurements were carried out as a double determination using Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Abbreviations used: CoA, coenzyme A; DmMFE-2, Drosophila melanogaster multifunctional enzyme type 2; DH, 3R-hydroxyacyl-CoA dehydrogenase; H2, 2E-Enoyl-CoA hydratase 2; SCP-2L, sterol carrier protein-2-like; NAD + , nicotinamide adenine dinucleotide; IPTG, isopropyl β-D-1thiogalactopyranoside; C4, 2E-butenoyl-CoA; C6, 2E-hexenoyl-CoA; C10, 2E-decenoyl-CoA. 0.5 ml quartz cuvettes at 22 o C, and K M , V max and k cat values were calculated with GraFit 5 (Erithacus Software).

Crystallization

The method by Jancarik and Kim [START_REF] Jancarik | Sparse matrix sampling: A screening method for crystallization of proteins[END_REF] was used for the initial crystallization screening. Sitting and hanging drop vapour-diffusion methods were used at 21 o C and set up by a Tecan Freedom Evo liquid handling robot operated via the Gemini software. The crystallization drops contained equal volumes of protein solution and mother liquor (1 μl). A tetragonal shaped crystal grown in 100 mM Tris pH 8.0, 1.0 M NaCl, 20 % (w/v) PEG 5000 MME, 5 mM NAD + was the most suitable for X-ray analysis. The crystal was approximately 0.23 mm x 0.21 mm x 0.05 mm in dimensions. The crystal was transferred into a cryo-protecting mother liquor containing 20 % (v/v) glycerol in addition to the original mother liquor, incubated for one minute and flash frozen at 100 K using liquid nitrogen.

Data collection and structure determination

A dataset (90 o of diffraction data in 180 frames) was collected at 100 K using synchrotron radiation (wavelength 0.9310 Å) at the ID29 beamline, ESRF, Grenoble. The images were processed using XDS [29] with the high resolution limit set to 2.14 Å. The data fitted best to the space group P4 3 2 1 2. The structure was solved by molecular replacement using Phaser [START_REF] Mccoy | Phaser crystallographic software[END_REF]. Molecule A of the 3Rhydroxyacyl-CoA dehydrogenase fragment of rat peroxisomal multifunctional enzyme type 2 (PDB ID 1GZ6) and molecule A of the 2E-enoyl-CoA hydratase 2 domain of C. tropicalis multifunctional enzyme type 2 complexed with 3R-hydroxydecanoyl-CoA (PDB ID 1PN4) were used as models in the rotation and translation search. The first search cycle was done using the hydratase 2 molecule and the dehydrogenase molecule was used in the second cycle. After solving the structure, the amino acid sequences of the homology models were modified by CHAINSAW [START_REF] Stein | CHAINSAW: A program for mutating pdb files used as templates in molecular replacement[END_REF] to match the DmMFE-2 amino acid sequence. Iterative cycles were used for building the structure manually using coot [START_REF] Emsley | Abbreviations used: CoA, coenzyme A; DmMFE-2, Drosophila melanogaster multifunctional enzyme type 2; DH, 3R-hydroxyacyl-CoA dehydrogenase; H2, 2E-Enoyl-CoA hydratase 2; SCP-2L, sterol carrier protein-2-like; NAD + , nicotinamide adenine dinucleotide; IPTG, isopropyl β-D-1thiogalactopyranoside; C4, 2E-butenoyl-CoA; C6, 2E-hexenoyl-CoA[END_REF]. PHENIX [START_REF] Adams | PHENIX: A comprehensive python-based system for macromolecular structure solution[END_REF] and Refmac [START_REF] Murshudov | Refinement of macromolecular structures by the maximum-likelihood method[END_REF][START_REF] Winn | Use of TLS parameters to model anisotropic displacements in macromolecular refinement[END_REF] Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Abbreviations used: CoA, coenzyme A; DmMFE-2, Drosophila melanogaster multifunctional enzyme type 2; DH, 3R-hydroxyacyl-CoA dehydrogenase; H2, 2E-Enoyl-CoA hydratase 2; SCP-2L, sterol carrier protein-2-like; NAD + , nicotinamide adenine dinucleotide; IPTG, isopropyl β-D-1thiogalactopyranoside; C4, 2E-butenoyl-CoA; C6, 2E-hexenoyl-CoA; C10, 2E-decenoyl-CoA.

RESULTS

Identification of D. melanogaster peroxisomal MFE-2

The NCBI BLASTP was used to identify amino acid sequences from D. melanogaster in the RefSeq protein database that show high similarity to the human peroxisomal MFE-2 (NCBI protein accession code NP_000405). Among the 43 sequences, CG3415 clone, encoding a polypeptide with a predicted molecular mass of 64073 Daltons, possessed the highest score in the blast results with a 632 bits score and 0.0 e-value. The obtained amino residues showed similarity to the Rossmann fold that is common among the short chain alcohol dehydrogenase/reductase (SDR) family [START_REF] Jörnvall | Short-chain dehydrogenases/reductases (SDR)[END_REF]. The C-terminus displayed a hydratase 2 motif [START_REF] Qin | Human peroxisomal multifunctional enzyme type 2. Site-directed mutagenesis studies show the importance of two protic residues for 2-enoyl-CoA hydratase 2 activity[END_REF] and similarities to amino acid sequences in the R-hydratase-like hot-dog fold [START_REF] Leesong | Structure of a dehydratase-isomerase from the bacterial pathway for biosynthesis of unsaturated fatty acids: Two catalytic activities in one active site[END_REF]. Sequence alignments (Supplementary Figure S1) to the CG3415 clone with the separate domains of MFE-2 from human, rat (Rattus norvegicus) and yeast (Candida tropicalis) demonstrate high sequence identities between the various species at the amino acid level. The Nterminus of the CG3415 clone (residues 1-316) shares 55 % identity at the amino acid level with the 3R-hydroxyacyl-CoA dehydrogenase from the human and rat. The C-terminal part (residues 317-598) shares 50 % identity with the human enoyl-CoA hydratase 2 and 38 % identity with the yeast enoyl-CoA hydratase 2. Similar to the C. tropicalis MFE-2, the DmMFE-2 also lacks the C-terminal SCP-2L domain. Another interesting feature is the linker between the dehydrogenase and hydratase domains, which is 22 amino acid residues shorter in the DmMFE-2 than in human MFE-2 (Supplementary Figure S1).

Localization and enzymatic properties of DmMFE-2

Kinetic properties of DmMFE-2 were determined (Table 1). K M and k cat values show that DmMFE-2 prefers the medium chain C10 substrate over the short chain C4 and C6 substrates. DmMFE-2 is also active in vivo since it rescues the growth of yeast S. cerevisiae 'fox2 strain devoid of the endogenous peroxisomal MFE-2 on oleic acid as the carbon source (Figure 3A). Catalytic domains of MFE-2s are known to be active also if expressed and purified as stand-alone proteins. Our data from static light scattering (SLS) experiments indicate that the catalytic domains do not assembly into heteromeric complexes in vitro (Supplementary Figure S2). We wanted to find support for a possible substrate channelling phenomenon being present in DmMFE-2 and therefore repeated the kinetic measurements by using the dehydrogenase and the hydratase 2 domains in 1:1 mixture instead of the full-length protein (Table 1 and Figure 3B and3C). Most interestingly, it was found that the fulllength DmMFE-2 is not more efficient in catalysis compared to individual enzymes. Furthermore, this applies in a similar fashion to all the short and medium chain substrates accessible to us.

The structure determination of DmMFE-2

Molecular replacement was done using the C. tropicalis 2E-enoyl-CoA hydratase 2 domain of MFE-2 (1PN4) and the rat 3R-hydroxyacyl-CoA dehydrogenase domain of MFE-2 (1GZ6) as the starting models for the initial phase determination using a native dataset from DmMFE-2 crystals. A successful molecular replacement solution was obtained using the space group P4 3 2 1 2 in the rotation and translation search. The asymmetric unit consists of one DmMFE-2 monomer in a unit cell with dimensions of 114.48 x 114.48 x 89.11 Å with corresponding 90 o angles. After solving the phases, the amino acid side chains were mutated to match the DmMFE-2 sequence and the missing residues were built in the positive electron density. Clear electron density was discovered from the hydratase 2 domain as well as from the C-terminal part of the dehydrogenase domain. 80 % of the total sequence Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Abbreviations used: CoA, coenzyme A; DmMFE-2, Drosophila melanogaster multifunctional enzyme type 2; DH, 3R-hydroxyacyl-CoA dehydrogenase; H2, 2E-Enoyl-CoA hydratase 2; SCP-2L, sterol carrier protein-2-like; NAD + , nicotinamide adenine dinucleotide; IPTG, isopropyl β-D-1thiogalactopyranoside; C4, 2E-butenoyl-CoA; C6, 2E-hexenoyl-CoA; C10, 2E-decenoyl-CoA.

was comprised of the clear density that initially enabled the tracking of the correct position of the atoms in the main chain. However, the density was poor in some areas of the map that are known to contain flexible regions in the homologous structures. The missing polypeptide fragments include regions 1 -6, 44 -55, 88 -98 and 200 -204 in the dehydrogenase domain and 373 -387, 451 -457 and 593 -598 in the hydratase domain. Despite the fact that the protein was crystallized in the presence of cofactor NAD + , no positive density was detected at the known cofactor binding region of the dehydrogenase domain. Structural refinement statistics are summarized in table 2.

The overall structure of DmMFE-2 monomer and dimer

The current structure described here is the first crystal structure of a full-length MFE-2 protein thus extending the understanding of MFE-2 proteins from a domain level to domain assembly level. According to the calculated Matthew's coefficient (2.25), the asymmetric unit is occupied by a single DmMFE-2 monomer. The monomer consists of one N-terminal 3R-hydroxyacyl-CoA dehydrogenase domain, a C-terminal 2E-enoyl-CoA hydratase 2 domain and a short loop that connects the two catalytic domains. The monomer is roughly L-shaped and it is hourglass-shaped in the side view. The constituent domain structures bear high similarity to the previously published MFE-2 single domain structures from the rat and yeast.

The monomer of DmMFE-2 reveals the contacts between the two enzymatic domains within the polypeptide. As detailed in figure 4, three salt bridges (Lys285 to Asp316, and Lys256 and Lys305 to Glu315) are found between the dehydrogenase and the hydratase 2 domains, and one between the connecting loop and the hydratase 2 (Asp312 to Lys412). These contacts reside at the narrowest section of the molecule. From the mentioned residues, only Lys305 is conserved among the MFE-2 family.

It is known, however, that MFE-2 is active as a dimer and many features in the new structure also support this. The DmMFE-2 homodimer is formed at the 2-fold symmetry axis in a way that the two polypeptides align each other crosswise creating a complex interaction network between the two dehydrogenase and hydratase monomers as previously shown [START_REF] Haapalainen | Binary structure of the two-domain (3R)-hydroxyacyl-CoA dehydrogenase from rat peroxisomal multifunctional enzyme type 2 at 2.38 A resolution[END_REF][START_REF] Koski | A two-domain structure of one subunit explains unique features of eukaryotic hydratase 2[END_REF]. Dimerization could also be understood such that both identical catalytic domains are forming their own dimers which are connected via short polypeptide linkers in the centre of the molecule. Sequence alignment using ClustalX [START_REF] Thompson | The CLUSTAL_X windows interface: Flexible strategies for multiple sequence alignment aided by quality analysis tools[END_REF] revealed that the linker region in the fruit fly MFE-2 appears to be shorter than in human MFE-2. In DmMFE-2 the unfolded linker region consists of 6 residues (determined using ClustalX and PROCHECK [START_REF] Thompson | The CLUSTAL_X windows interface: Flexible strategies for multiple sequence alignment aided by quality analysis tools[END_REF][START_REF] Laskowski | PROCHECK: A program to check the stereochemical quality of protein structures[END_REF]), but in human MFE-2 there are additional 22 amino acids forming a gap between the dehydrogenase dimer and the hydratase dimer that, in practice, are absent in the DmMFE-2 sequence (Supplementary figure S1). The overall shape of the DmMFE-2 dimer is rather rectangular when viewing the molecule from the front (Figure 5A and5B). When viewing the molecule from the side, the monomer in front covers the other monomer and the silhouette resembles the silhouette of the monomer (Figure 5C). Surprisingly, the centre of the molecule is not filled, and there exists a clear physical hole (Figure 5B). The active sites of one polypeptide are facing to the same sides (Figure 5D).

The two DmMFE-2 monomers in the dimer are kept together by several hydrogen bonds as well as hydrophobic and aromatic interactions as shown before [START_REF] Haapalainen | Binary structure of the two-domain (3R)-hydroxyacyl-CoA dehydrogenase from rat peroxisomal multifunctional enzyme type 2 at 2.38 A resolution[END_REF][START_REF] Koski | A two-domain structure of one subunit explains unique features of eukaryotic hydratase 2[END_REF][START_REF] Koski | Crystal structure of 2-enoyl-CoA hydratase 2 from human peroxisomal multifunctional enzyme type 2[END_REF]. The structure is further stabilized by salt bridges between the two polypeptides. In the dehydogenase dimer, the interactions are mainly of a hydrophobic nature between the long helixes α5 and α5' in addition to hydrogen bonds. However, a few salt bridges are formed that will give the dehydrogenase dimer additional stability. The first bonds are formed between Glu215 and Arg261' and between Glu215' and Arg261. Glu241 and Arg254' are also forming bonds as well as Glu241' and Arg254. In the hydratase domain, a small hydrophobic patch is formed by residues Ile326, Leu330, Leu490 and Leu493. This patch defines the bottom area of a local groove that is filled with several water molecules. This patch is also responsible for the hydrophobic interaction between α10 to α13' and α10' to α13. Two stabilizing salt bridges are formed between Arg492 and Glu347' and between Arg492' and Glu347. Monomers are possibly further cross-linked via Ser335 and Asp486' and between Ser335' and Asp486 since the side chains are only 2.6 Å apart.

When

examining the central opening that is formed between the dehydrogenase and hydratase subunits more carefully, one further observation is worth mentioning. The opening is bordered with lysine and histidine residues such that they create a positively charged patch in the middle of the dimer. The residues involved are His256, Lys285, Lys287, Lys305 and Lys372 from both of the polypeptides. The locations of positively charged residues in the DmMFE-2 dimer are shown in figure 5D.

3R-hydroxyacyl-CoA dehydrogenase domain of DmMFE-2

The 3R-hydroxyacyl-CoA dehydrogenase domain of DmMFE-2 consists of amino acids 1-307 such that residues 1-244 display a classic Rossmann fold with an α/β doubly-wound structure and residues 245-307 constitute the extension specific to MFE-2 proteins (Figure 5A). Five β-sheets (β1 -β5) align in a row in the core of the catalytic domain and α-helices (2 or 3 depending on the side) border the β-sheets in the middle of the core. Presumably, the missing loop regions and the partially unfolded part of the N-terminus both contribute to the binding of the cofactor NAD + . The phosphate binding consensus sequence GXXXGXG itself is ordered in the structure, but the non-conserved loop (between β2 and α2) also participating in the NAD + binding is completely absent. In addition, the polypeptide after the helix α2 is not forming a clear secondary structure as it is in the previously published MFE-2 dehydrogenase domain structures (1GZ6, 2ET6). The NAD + binding region is open in the structure, which would permit the cofactor binding. However, due to molecular packing within the crystal lattice the binding of NAD + probably becomes inhibited by a loop between sheets β12 and β13 from a symmetry-related hydratase molecule. Especially Gln474 is only 3.62 Å apart from the location of the superimposed NAD + molecule of the rat dehydrogenase. The crystal packing may also partially explain the weaker electron density in the N-terminal part of the structure.

The overall structural topology of the DmMFE-2 dehydrogenase is very similar to the previously published rat dehydrogenase domain (1GZ6) [START_REF] Haapalainen | Binary structure of the two-domain (3R)-hydroxyacyl-CoA dehydrogenase from rat peroxisomal multifunctional enzyme type 2 at 2.38 A resolution[END_REF]. The C-terminal extension of the rat dehydrogenase domain also exists well-ordered in the DmMFE-2. The extension is not common in the SDR superfamily [START_REF] Jörnvall | Short-chain dehydrogenases/reductases (SDR)[END_REF], but the feature is shared between the different MFE-2 homologues. According to the sequence alignments, the human and rat homologues share the highest similarity in the Cterminal region of the domain. When examining the rat C-terminal extension, the β-sheets from two monomers clearly intersect at Trp246. The corresponding region in the current structure shows intersection between Met252 and Met252' (Supplementary figure S1). Amino acids Ser154, Tyr167 and Lys171 form the widely conserved SDR family catalytic triad in DmMFE-2 (Supplementary figure S1). This triad, needed in the hydride transfer reaction, is well-ordered in the current structure. The dehydrogenase dimer of DmMFE-2 superimposed with the rat MFE-2 dehydrogenase is presented in figure 6A.

2E-Enoyl-CoA hydratase 2 domain of DmMFE-2

2E-Enoyl-CoA hydratase 2 domain is built from amino acids 314-598 in DmMFE-2. The Drosophila hydratase 2 subunit consists of 7 α-helices (α10-α16) and 10 β-sheets (β8-β17) that are well-defined (Figure 6B). The subunit can further be divided in two separate subdomains that are connected via a long loop region between sheets β12 and β13 (Figure 5A). A typical hot-dog fold [START_REF] Leesong | Structure of a dehydratase-isomerase from the bacterial pathway for biosynthesis of unsaturated fatty acids: Two catalytic activities in one active site[END_REF] Biochemical Journal Immediate Publication. Published on 14 Feb 2011 as manuscript BJ20101661 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101661 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.

Abbreviations used: CoA, coenzyme A; DmMFE-2, Drosophila melanogaster multifunctional enzyme type 2; DH, 3R-hydroxyacyl-CoA dehydrogenase; H2, 2E-Enoyl-CoA hydratase 2; SCP-2L, sterol carrier protein-2-like; NAD + , nicotinamide adenine dinucleotide; IPTG, isopropyl β-D-1thiogalactopyranoside; C4, 2E-butenoyl-CoA; C6, 2E-hexenoyl-CoA; C10, 2E-decenoyl-CoA. is formed in the cores of the subdomains: in the N-terminal subdomain helix α12 is covered by five βsheets (β8-β12) and the C-terminal hot-dog fold is formed by α-helix 16 and β-sheets β13-β17.

The DmMFE-2 hydratase domain possesses an interesting structural difference in comparison to the known yeast and human hydratases and the Aeromonas caviae R-hydratase [START_REF] Hisano | Crystal structure of the (R)-specific enoyl-CoA hydratase from Aeromonas caviae involved in polyhydroxyalkanoate biosynthesis[END_REF]. We have shown earlier that the ability of the eukaryotic hydratase 2´s to accept long chain substrates is due to extra space in the core of the domain created by the lack of the central α12-helix. This helix is present in the bacterial counterpart and it fills the whole interior space of the β-sheet structure, thus rendering the protein active for short chain substrates only. In Drosophila hydratase the α12-helix is of intermediate length (Figure 7B). In this respect the current structure resembles more the bacterial protein, but on the other hand retains the ability to utilize a broader spectrum of substrates as in eukaryotic MFE-2s. In fact, when the liganded yeast structure (1PN4) is superimposed on top of the fruit fly hydratase 2, it can immediately be seen that the fatty acid tail is in close physical contact to the helix α12. Despite this the DmMFE-2 prefers the C10 substrate over shorter ones (see above). The structural comparison of known R-hydratase structures is presented in figure 7. Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Abbreviations used: CoA, coenzyme A; DmMFE-2, Drosophila melanogaster multifunctional enzyme type 2; DH, 3R-hydroxyacyl-CoA dehydrogenase; H2, 2E-Enoyl-CoA hydratase 2; SCP-2L, sterol carrier protein-2-like; NAD + , nicotinamide adenine dinucleotide; IPTG, isopropyl β-D-1thiogalactopyranoside; C4, 2E-butenoyl-CoA; C6, 2E-hexenoyl-CoA; C10, 2E-decenoyl-CoA.

DISCUSSION

The assembly of a full-length MFE-2 has been unknown. The DmMFE-2 dimer is dominated by dehydrogenase and hydratase subdimers that are connected via a short loop (residues 308-313, Supplementary Figure S1). The loop, which is short enough to limit large domain movements, is involved in domain-domain contacts within each MFE-2 monomer. The monomers in turn contribute to the rigid assembly with the extensive dimerization contacts between monomers. The centre of the dimeric molecule contains a hole. Both dehydrogenase and hydratase catalytic sites within one polypeptide are accessible on the same side: the CoA binding region of the dehydrogenases is facing the centre of the dimer while the hydratase active site is pointing out from the centre.

Due to of the structural similarity of the enzymatic domains of all known MFE-2 proteins, and due to the extensive dimerization present in DmMFE-2, it is very likely that assembly of the enzymatic domains in mammalian and yeast counterparts will be similar despite variation in the connecting linkers and the fact that Drosophila MFE-2 lacks the SCP-2L domain.

The hydratase subunit of DmMFE-2 shares only a 17 % sequence identity with the A. caviae Rspecific hydratase and 38 % with the yeast C. tropicalis hydratase 2 from MFE-2. It is staggering to discover that all these proteins have such a similar structure, even though the sequence similarity shows such variation. The most visible difference between the three structures is the hot-dog fold αhelix (in the fruit fly hydratase labelled as α12) that is varying in length. The helix is virtually absent in both the liganded and unliganded forms of the C. tropicalis hydratase 2 domain [START_REF] Koski | A two-domain structure of one subunit explains unique features of eukaryotic hydratase 2[END_REF]. In the bacterial hydratase homodimer the α-helix extends up to the upper part of the β-sheet bundle in the N-terminal end of a monomer. The α-helix in the D. melanogaster hydratase 2 domain is not as long as in the A. caviae R-hydratase monomer and it is also slightly leaning away from the active site. It is also clear from the kinetic data in table 1 that DmMFE-2 uses the C10 substrate efficiently.

The structure of the A. caviae R-hydratase suggests the existence of two active sites and activity against short-chain enoyl-CoAs that are up to C6 in length. The substrate binding pocket in C. tropicalis hydratase is much more capacious due to replacement of the long hot-dog α-helix (corresponding α12 in DmMFE-2) with a highly mobile polypeptide and a short α-helix. This has resulted in the broader substrate utilization range in the yeast counterpart. Similarly to the yeast, in the current structure there exists only one active site in a hydratase 2 monomer. The size of the substrate binding pocket is restricted by the hot-dog helix α12. When superimposing the hydratase domain with the liganded yeast hydratase domain, the end of the fatty acid tail of the substrate is in close contact to the hot-dog α-helix. This could indicate narrower substrate specificity for the fruit fly hydratase domain, unless the hot-dog α-helix is exceptionally flexible.

In the catalytic reactions by MFE-2, the 2E-enoyl-CoA substrate is first hydrated by the hydratase 2 domain and the 3R-hydroxy intermediate then serves as the substrate for the dehydrogenase domain for the subsequent oxidation. However, the assembly does not indicate whether the substrate leaving from a hydratase is further catalyzed in the dehydrogenase of the same MFE-2 polypeptide or whether it proceeds to the dehydrogenase of the other polypeptide. The latter possibility, together with the subdimer assembly mode, well explains the necessity of dimerization for activity found for MFE-2 proteins.

Enzyme kinetic data does not support efficiency-enhancing substrate channelling between the enzymatic domains. The structure, however, has some features that can be discussed relative to a channelling mechanism being present. The surface of the central opening of the dimer has a number of positively charged residues that could bind and route the phosphate-containing substrates. However, the hole appears too narrow (ca. 9 Å) to let a bulky (and possibly non-extended [START_REF] Koski | A two-domain structure of one subunit explains unique features of eukaryotic hydratase 2[END_REF]) fatty acyl-CoA substrate pass through. In addition, there is also a track of positively charged residues leading from the hydratase active site towards the dimer centre. One pathway towards the centre opening could be the residues which lie at the hydratase dimer interface. A small opening from the hydratase substrate binding pocket exists in the subunit that has access to the positively charged residues in the dimer interface. However, an alternate pathway should be considered since the CoA molecule is too monolithic to transfer through the opening. The amount of charged residues on the surface of the hydratase 2 domain on the way from hydratase active site to the dehydrogenase active site via the outer edge of the hydratase appear to form just a narrow path. It is tempting to see these residues providing access between the active sites and thus contributing to the enzymatic efficiency. We attempted to test this by mutating two to five of these residues to glutamates, but the mutant proteins were insoluble. Dihydrofolate reductase-thymidylate synthase [START_REF] Knighton | Abbreviations used: CoA, coenzyme A; DmMFE-2, Drosophila melanogaster multifunctional enzyme type 2; DH, 3R-hydroxyacyl-CoA dehydrogenase; H2, 2E-Enoyl-CoA hydratase 2; SCP-2L, sterol carrier protein-2-like; NAD + , nicotinamide adenine dinucleotide; IPTG, isopropyl β-D-1thiogalactopyranoside; C4, 2E-butenoyl-CoA; C6, 2E-hexenoyl-CoA; C10, 2E-decenoyl-CoA. Abbreviations used: CoA, coenzyme A; DmMFE-2, Drosophila melanogaster multifunctional enzyme type 2; DH, 3R-hydroxyacyl-CoA dehydrogenase; H2, 2E-Enoyl-CoA hydratase 2; SCP-2L, sterol carrier protein-2-like; NAD + , nicotinamide adenine dinucleotide; IPTG, isopropyl β-D-1thiogalactopyranoside; C4, 2E-butenoyl-CoA; C6, 2E-hexenoyl-CoA; C10, 2E-decenoyl-CoA. Abbreviations used: CoA, coenzyme A; DmMFE-2, Drosophila melanogaster multifunctional enzyme type 2; DH, 3R-hydroxyacyl-CoA dehydrogenase; H2, 2E-Enoyl-CoA hydratase 2; SCP-2L, sterol carrier protein-2-like; NAD + , nicotinamide adenine dinucleotide; IPTG, isopropyl β-D-1thiogalactopyranoside; C4, 2E-butenoyl-CoA; C6, 2E-hexenoyl-CoA; C10, 2E-decenoyl-CoA. Abbreviations used: CoA, coenzyme A; DmMFE-2, Drosophila melanogaster multifunctional enzyme type 2; DH, 3R-hydroxyacyl-CoA dehydrogenase; H2, 2E-Enoyl-CoA hydratase 2; SCP-2L, sterol carrier protein-2-like; NAD + , nicotinamide adenine dinucleotide; IPTG, isopropyl β-D-1thiogalactopyranoside; C4, 2E-butenoyl-CoA; C6, 2E-hexenoyl-CoA[END_REF] is proposed to contain such an "electrostatic highway", but it consists of a larger, generally negative surface instead of a few residues.

CONCLUDING REMARKS

The D. melanogaster MFE-2 is a peroxisomal protein active in the E-oxidation of fatty acyl-CoAs; (i) its domains show amino acid sequence as well as (ii) the fold similarities to those of other MFE-2s, (iii) DmMFE-2 complements functionally the S. cerevisiae peroxisomal MFE-2 in vivo, and (iv) the recombinant domains display the predicted catalytic activities in vitro. The assembly of domains renders DmMFE-2 a "double-dimeric" protein, where dimerization can equally well be seen as the dimer of polypeptides or the dimer of like enzymes coming from different polypeptides. There is a physical hole in the middle of the protein, but it is not large enough to allow fatty acyl-CoA molecules from using it as a channel between active sites. Negative surface charge distribution is also not extensive enough to convincingly support the hypothesis of an electrostatic substrate channelling mechanism. On the contrary, forceful evidence against the presence of substrate channelling in DmMFE-2 comes from enzyme kinetic data. The turnover number and the catalytic efficiency for separate hydratase and dehydrogenase enzymes are of the same order of magnitude as for the native, full-length protein. Thus, the question remains why the two enzymatic activities reside together in a multifunctional protein. Possibly the answer is simply the need for stability and dimerization for efficient catalysis. Should, however, a substrate channelling phenomenon be present, it could serve the purpose of achieving something else than efficiency, such as the protection of an intermediate. Abbreviations used: CoA, coenzyme A; DmMFE-2, Drosophila melanogaster multifunctional enzyme type 2; DH, 3R-hydroxyacyl-CoA dehydrogenase; H2, 2E-Enoyl-CoA hydratase 2; SCP-2L, sterol carrier protein-2-like; NAD + , nicotinamide adenine dinucleotide; IPTG, isopropyl β-D-1thiogalactopyranoside; C4, 2E-butenoyl-CoA; C6, 2E-hexenoyl-CoA; C10, 2E-decenoyl-CoA.

Table 1

Kinetic parameters for the full-length DmMFE-2 protein in comparison with the values obtained using the dehydrogenase (DH) and the hydratase 2 (H2) domains of DmMFE-2 as separate monofunctional enzymes in a 1:1 ratio C4, C6 and C10 stand for 2E-butenoyl-CoA, 2E-hexenoyl-CoA and 2E-decenoyl-CoA, respectively.

Substrate

C4 C6 C10

Full-length DmMFE-2

V max (Pmol min -1 mg - In C. elegans the enzymes of MFE-2 have been separated from each other such that hydratase 2 is a separate protein, while dehydrogenase and SCP-2L -domains form a united polypeptide. The yeast MFE-2 is also missing the SCP-2L -domain and has duplicated the dehydrogenase domain that has evolved altered substrate specificities in the two domains [START_REF] Qin | Yeast peroxisomal multifunctional enzyme: (3R)-hydroxyacyl-CoA dehydrogenase domains A and B are required for optimal growth on oleic acid[END_REF]. The Nterminal dehydrogenase domain displays the highest activity towards medium and long chain 3Rhydroxyacyl-CoAs and the middle dehydrogenase domain shows the preference towards the short chain (C4) substrates [START_REF] Qin | Yeast peroxisomal multifunctional enzyme: (3R)-hydroxyacyl-CoA dehydrogenase domains A and B are required for optimal growth on oleic acid[END_REF]. The two polypeptides forming the dimer have been coloured in pale cyan and gray, and the dimeric dehydrogenase and hydratase 2 enzymes denoted by DH and H2, respectively. Of the positively charged residues (Lys, Arg and His), the conserved residues among MFE-2 family have been coloured in marine blue and the rest have been coloured in dark grey. The 3R-hydroxydecanoyl-CoA ligands have been positioned to the active sites by superimposing the liganded hydratase 2 domain structure of MFE-2 [START_REF] Qin | Yeast peroxisomal multifunctional enzyme: (3R)-hydroxyacyl-CoA dehydrogenase domains A and B are required for optimal growth on oleic acid[END_REF] and the proposed ligand-containing structure of dehydrogenase of yeast MFE-2 [START_REF] Ylianttila | Abbreviations used: CoA, coenzyme A; DmMFE-2, Drosophila melanogaster multifunctional enzyme type 2; DH, 3R-hydroxyacyl-CoA dehydrogenase; H2, 2E-Enoyl-CoA hydratase 2; SCP-2L, sterol carrier protein-2-like; NAD + , nicotinamide adenine dinucleotide; IPTG, isopropyl β-D-1thiogalactopyranoside; C4, 2E-butenoyl-CoA; C6, 2E-hexenoyl-CoA[END_REF]. When viewing the molecule from the front a positively charged surface patch could be delineated leading from the hydratase active site (lower right corner, on the back of the enzyme) directly to the dehydrogenase CoA-binding site on the right side upwards from the central opening.

Figure S1 Amino acid sequence alignment of MFE-2s from various species

The Drosophila melanogaster MFE-2 has been aligned using ClustalX [START_REF] Thompson | The CLUSTAL_X windows interface: Flexible strategies for multiple sequence alignment aided by quality analysis tools[END_REF] together with human (HsMFE-2), rat (RnMFE-2), zebra fish (DrMFE-2) and yeast C. tropicalis 3R-hydroxyacyl-CoA dehydrogenase domain B and 2E-Enoyl-CoA hydratase 2 domain fragment (CtDhB+H2). The Nterminal 3R-hydroxyacyl-CoA dehydrogenase domain A of C. tropicalis MFE-2 has been left out from the comparison for clarity. The UniProtKB identifiers of the selected sequences are Q9VXJ0, P51659, P97852, Q8AYH1 and P22414, respectively. The secondary structure elements have been highlighted above the sequences with bars and arrows. The rectangle box is pointing out the linker region between the catalytic domains. The black vertical arrows indicate the location of residues that are essential for the enzyme catalysis [START_REF] Haapalainen | Binary structure of the two-domain (3R)-hydroxyacyl-CoA dehydrogenase from rat peroxisomal multifunctional enzyme type 2 at 2.38 A resolution[END_REF][START_REF] Koski | A two-domain structure of one subunit explains unique features of eukaryotic hydratase 2[END_REF]. Complete identity or similarity of residues between the sequences has been indicated with black shade, while decreasing greyness is indicating regions with four or three matching residues.

Figure S2 Investigation of heterogenic protein complex formation in vitro using static light scattering (SLS) technique

The formation of protein complex between the dimers of catalytic domains of DmMFE-2 was studied using multi-angle light scattering device, miniDAWN TREOS (Wyatt Technology Corp.). The curves in the chromatogram represent single injection each. Red colour is indicating the full-length DmMFE-2, blue colour 3R-hydroxyacyl-CoA dehydrogenase domain of DmMFE-2, green colour 2E-Enoyl-CoA hydratase 2 domain of DmMFE-2 and black colour the both catalytic domains as separate polypeptides in the same injection. The data demonstrate that the DmMFE-2 dehydrogenase and hydratase domains do not form a complex in vitro when expressed as separate polypeptides. According to the numeric data provided by the experiment, in the provided conditions the DmMFE-2 dimer has a molecular mass of 122 kDa, the dehydrogenase dimer 62 kDa, the hydratase 2 dimer 69 kDa and the mixture of the two dimers 65 kDa. With the exception of the mixture sample, this is in agreement with the theoretical values when taking into account the monodispersity of each of the samples.

Biochemical

  Journal Immediate Publication. Published on 14 Feb 2011 as manuscript BJ20101661 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101661 A c c e p t e d M a n u s c r i p t

  Abbreviations used: CoA, coenzyme A; DmMFE-2, Drosophila melanogaster multifunctional enzyme type 2; DH, 3R-hydroxyacyl-CoA dehydrogenase; H2, 2E-Enoyl-CoA hydratase 2; SCP-2L, sterol carrier protein-2-like; NAD + , nicotinamide adenine dinucleotide; IPTG, isopropyl β-D-1thiogalactopyranoside; C4, 2E-butenoyl-CoA; C6, 2E-hexenoyl-CoA; C10, 2E-decenoyl-CoA.

Biochemical

  Journal Immediate Publication. Published on 14 Feb 2011 as manuscript BJ20101661 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101661 A c c e p t e d M a n u s c r i p t

Biochemical

  Journal Immediate Publication. Published on 14 Feb 2011 as manuscript BJ20101661 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101661 A c c e p t e d M a n u s c r i p t

  was used for TLS refinement cycles. The final structure factors were validated using MolProbity [36]. Biochemical Journal Immediate Publication. Published on 14 Feb 2011 as manuscript BJ20101661 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101661 A c c e p t e d M a n u s c r i p t

  Biochemical Journal Immediate Publication. Published on 14 Feb 2011 as manuscript BJ20101661 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101661 A c c e p t e d M a n u s c r i p t

Biochemical

  Abbreviations used: CoA, coenzyme A; DmMFE-2, Drosophila melanogaster multifunctional enzyme type 2; DH, 3R-hydroxyacyl-CoA dehydrogenase; H2, 2E-Enoyl-CoA hydratase 2; SCP-2L, sterol carrier protein-2-like; NAD + , nicotinamide adenine dinucleotide; IPTG, isopropyl β-D-1thiogalactopyranoside; C4, 2E-butenoyl-CoA; C6, 2E-hexenoyl-CoA; C10, 2E-decenoyl-CoA.

Biochemical

  Journal Immediate Publication. Published on 14 Feb 2011 as manuscript BJ20101661 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101661 A c c e p t e d M a n u s c r i p t

Biochemical

  Abbreviations used: CoA, coenzyme A; DmMFE-2, Drosophila melanogaster multifunctional enzyme type 2; DH, 3R-hydroxyacyl-CoA dehydrogenase; H2, 2E-Enoyl-CoA hydratase 2; SCP-2L, sterol carrier protein-2-like; NAD + , nicotinamide adenine dinucleotide; IPTG, isopropyl β-D-1thiogalactopyranoside; C4, 2E-butenoyl-CoA; C6, 2E-hexenoyl-CoA; C10, 2E-decenoyl-CoA.

Biochemical

  Abbreviations used: CoA, coenzyme A; DmMFE-2, Drosophila melanogaster multifunctional enzyme type 2; DH, 3R-hydroxyacyl-CoA dehydrogenase; H2, 2E-Enoyl-CoA hydratase 2; SCP-2L, sterol carrier protein-2-like; NAD + , nicotinamide adenine dinucleotide; IPTG, isopropyl β-D-1thiogalactopyranoside; C4, 2E-butenoyl-CoA; C6, 2E-hexenoyl-CoA; C10, 2E-decenoyl-CoA.

Biochemical

  Journal Immediate Publication. Published on 14 Feb 2011 as manuscript BJ20101661 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101661 A c c e p t e d M a n u s c r i p tLicenced copy. Copying is not permitted, except with prior permission and as allowed by law.

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.

  Abbreviations used: CoA, coenzyme A; DmMFE-2, Drosophila melanogaster multifunctional enzyme type 2; DH, 3R-hydroxyacyl-CoA dehydrogenase; H2, 2E-Enoyl-CoA hydratase 2; SCP-2L, sterol carrier protein-2-like; NAD + , nicotinamide adenine dinucleotide; IPTG, isopropyl β-D-1thiogalactopyranoside; C4, 2E-butenoyl-CoA; C6, 2E-hexenoyl-CoA; C10, 2E-decenoyl-CoA.

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.

A c c e

  p t e d M a n u s c r i p t Biochemical Journal Immediate Publication. Published on 14 Feb 2011 as manuscript BJ20101661 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101661 A c c e p t e d M a n u s c r i p t

Figure 1

 1 Figure 1 Schematic view of β-oxidation cycle of an acyl-CoA molecule in the peroxisomesThe first step is FAD-dependent dehydrogenation that results to a carbon double bond at the Δ 2position. The formed 2E-enoyl-CoA intermediate becomes hydrated in the second step which is followed by nicotinamide adenine dinucleotide (NAD + ) -dependent dehydrogenation that converts the intermediate to 3-ketoacyl-CoA. In the final step, the original carbon chain becomes shortened by two carbon atoms and acetyl-CoA is formed. The shortened substrate enters another degradative cycle. MFE-2 is responsible for the second and third steps of the cycle.

Figure 2

 2 Figure 2Comparison of peroxisomal multifunctional enzyme type 2s from various speciesThe figure demonstrates the versatility of the domain organization between MFE-2s from the selected species. Black colour corresponds to the 3R-hydroxyacyl-CoA dehydrogenase domain, vertical black stripes 2E-enoyl-CoA hydratase 2 domain and the sterol carrier protein 2-like domain (SCP-2L) is coloured in white. Human, rat and zebra fish MFE-2 share the same domain composition and organization. Drosophila MFE-2 is lacking the SCP-2L -domain. Nematodes and yeasts have yet different domain compositions. In C. elegans the enzymes of MFE-2 have been separated from each other such that hydratase 2 is a separate protein, while dehydrogenase and SCP-2L -domains form a united polypeptide. The yeast MFE-2 is also missing the SCP-2L -domain and has duplicated the dehydrogenase domain that has evolved altered substrate specificities in the two domains[START_REF] Qin | Yeast peroxisomal multifunctional enzyme: (3R)-hydroxyacyl-CoA dehydrogenase domains A and B are required for optimal growth on oleic acid[END_REF]. The Nterminal dehydrogenase domain displays the highest activity towards medium and long chain 3Rhydroxyacyl-CoAs and the middle dehydrogenase domain shows the preference towards the short chain (C4) substrates[START_REF] Qin | Yeast peroxisomal multifunctional enzyme: (3R)-hydroxyacyl-CoA dehydrogenase domains A and B are required for optimal growth on oleic acid[END_REF].

Figure 3

 3 Figure 3Activity of DmMFE-2 in vivo and in vitro (A) Rescue experiments with DmMFE-2 in yeast mutant devoid of endogenous MFE-2. Yeast dilution series were prepared on YP-oleic acid plates (0.125 % oleic acid, 0.2 % D+ Glucose). 1. Δfox2 (halo-), 2. Δfox2 + pYE352::CTA1 (halo-), 3. wt (halo+), 4. Δfox2 + pYE352::ScFOX2 (halo+), 5. Δfox2 + pYE352::DmMFE-2 (halo+). Samples 1. and 2. do not display development of halo due to the absence of a functional fox2 gene. Samples 3. and 4. serve as positive controls in the experiment and demonstrate clearance of the oleic acid around the yeast cells. The DmMFE-2 (Sample 5.) complements well the yeast null mutant strain. (B and C) Michaelis-Menten saturation curves and Lineweaver-Burk plots (inserts) of kinetic data for the full-length DmMFE-2 (B) and for 1:1 mixture of the dehydrogenase domain and the hydratase 2 domain of DmMFE-2 in stoichiometric ratio (C). 2Edecenoyl-CoA was used as the substrate, while the formation of the magnesium complex of 3ketodecanyl-CoA was monitored spectrophometrically at 303 nm. GraFit 5 was used for fitting the data.

Figure 4

 4 Figure 4Theoverall fold of DmMFE-2 monomer offering a close up view into the dehydrogenase and hydratase 2 domain borderThe C-terminal subdomain of the dehydrogenase (A; in the middle) and the hydratase 2 domain (A; on the bottom) share only a few contacts as indicated in (B) in stereo, and in the text.

Figure 5

 5 Figure 5 Domain organization in the DmMFE-2 dimer (A) Standard view of the enzyme revealing the dehydrogenase and hydratase subdimers that are connected via short linkers (black arrows). The Rossmann fold region has been coloured in marine blue and C-terminal subdomain of the dehydrogenase subunit is in aquamarine. Lime green and dark gray colours represent the N-and C-terminal hot dog fold in the hydratase dimer. The central α-helix bundle in the hydratase dimer interface and the remaining loop regions are coloured in pale gray. (B) Surface presentation of the MFE-2 dimer in the same orientation as in (A). Gray and aquamarine are highlighting the crosswise dimerization of the two monomers. (C) Side view of the hourglass-shaped DmMFE-2 dimer. The colouring corresponds to the colouring in (B) and the molecule has been turned 90 o clockwise from the standard view of the protein. (D) Surface presentation of DmMFE-2 dimer and the positively charged surface residues.The two polypeptides forming the dimer have been coloured in pale cyan and gray, and the dimeric dehydrogenase and hydratase 2 enzymes denoted by DH and H2, respectively. Of the positively charged residues (Lys, Arg and His), the conserved residues among MFE-2 family have been coloured in marine blue and the rest have been coloured in dark grey. The 3R-hydroxydecanoyl-CoA ligands have been positioned to the active sites by superimposing the liganded hydratase 2 domain structure of MFE-2[START_REF] Qin | Yeast peroxisomal multifunctional enzyme: (3R)-hydroxyacyl-CoA dehydrogenase domains A and B are required for optimal growth on oleic acid[END_REF] and the proposed ligand-containing structure of dehydrogenase of yeast MFE-2[START_REF] Ylianttila | Abbreviations used: CoA, coenzyme A; DmMFE-2, Drosophila melanogaster multifunctional enzyme type 2; DH, 3R-hydroxyacyl-CoA dehydrogenase; H2, 2E-Enoyl-CoA hydratase 2; SCP-2L, sterol carrier protein-2-like; NAD + , nicotinamide adenine dinucleotide; IPTG, isopropyl β-D-1thiogalactopyranoside; C4, 2E-butenoyl-CoA; C6, 2E-hexenoyl-CoA[END_REF]. When viewing the molecule from the front a positively charged surface patch could be delineated leading from the hydratase active site (lower right corner, on the back of the enzyme) directly to the dehydrogenase CoA-binding site on the right side upwards from the central opening.

Figure 6

 6 Figure 6Comparison of the dehydrogenase and hydratase domains with known structures of separate MFE-2 domains (A) Stereo view of the Drosophila dehydrogenase monomer of MFE-2 superimposed with rat dehydrogenase monomer of MFE-2 (1GZ6) that is in complex with NAD + . The Drosophila dehydrogenase monomer is coloured in marine blue and the rat dehydrogenase monomer in silver. Black arrows point out locations of the flexible loops, which are absent in the DmMFE-2 structure. The N-and C-termini have been indicated in the figure as well. (B) Stereo view of the Drosophila 2Eenoyl-CoA hydratase 2 dimer of MFE-2 superimposed with the homologous hydratase 2-dimer from the yeast C. tropicalis (1PN4) and hydratase 2 from human MFE-2 (1S9C). Substrate binding site in each hydratase monomer is evident since the C. tropicalis protein is a complex with the reaction product 3R-hydroxydecanoyl-CoA. The Drosophila hydratase is coloured in marine blue, human and yeast counterparts are coloured in pale cyan and in silver, respectively.

  Abbreviations used: CoA, coenzyme A; DmMFE-2, Drosophila melanogaster multifunctional enzyme type 2; DH, 3R-hydroxyacyl-CoA dehydrogenase; H2, 2E-Enoyl-CoA hydratase 2; SCP-2L, sterol carrier protein-2-like; NAD + , nicotinamide adenine dinucleotide; IPTG, isopropyl β-D-1thiogalactopyranoside; C4, 2E-butenoyl-CoA; C6, 2E-hexenoyl-CoA; C10, 2E-decenoyl-CoA.

Figure 7

 7 Figure 7 Structural comparison of known R-hydratase structures with the hydratase domain of DmMFE-2 (A) The homodimeric R-hydratase from A. caviae (1IQ6) consists of two 14 kDa subunits. (B) Hydratase 2 domain of DmMFE-2. The black arrow points out the front loop. Helix α12 is also labelled. The monomeric domain has molecular mass of 30.8 kDa. (C) Yeast C. tropicalis hydratase 2 domain of MFE-2 (1PN4) in complex with 3R-hydroxydecanoyl-CoA. The hydratase monomer has a molecular mass of 31.5 kDa. While the interior of the bacterial hydratase is completely filled, there are different amount of free space left for differently-sized substrates in the insect and yeast hydratases. The eukaryotic hydratases (B and C) have only one catalytic site, while the bacterial hydratase possesses two catalytic sites.

Biochemical

  Journal Immediate Publication. Published on 14 Feb 2011 as manuscript BJ20101661 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101661 A c c e p t e d M a n u s c r i p tLicenced copy. Copying is not permitted, except with prior permission and as allowed by law.

  Abbreviations used: CoA, coenzyme A; DmMFE-2, Drosophila melanogaster multifunctional enzyme type 2; DH, 3R-hydroxyacyl-CoA dehydrogenase; H2, 2E-Enoyl-CoA hydratase 2; SCP-2L, sterol carrier protein-2-like; NAD + , nicotinamide adenine dinucleotide; IPTG, isopropyl β-D-1thiogalactopyranoside; C4, 2E-butenoyl-CoA; C6, 2E-hexenoyl-CoA; C10, 2E-decenoyl-CoA.

ARTWORK Figure 1

 1 Figure 1

  Abbreviations used: CoA, coenzyme A; DmMFE-2, Drosophila melanogaster multifunctional enzyme type 2; DH, 3R-hydroxyacyl-CoA dehydrogenase; H2, 2E-Enoyl-CoA hydratase 2; SCP-2L, sterol carrier protein-2-like; NAD + , nicotinamide adenine dinucleotide; IPTG, isopropyl β-D-1thiogalactopyranoside; C4, 2E-butenoyl-CoA; C6, 2E-hexenoyl-CoA; C10, 2E-decenoyl-CoA.

Figure 2 Biochemical

 2 Figure 2

Figure 3 Biochemical

 3 Figure 3

Figure 4 Biochemical

 4 Figure 4

  Abbreviations used: CoA, coenzyme A; DmMFE-2, Drosophila melanogaster multifunctional enzyme type 2; DH, 3R-hydroxyacyl-CoA dehydrogenase; H2, 2E-Enoyl-CoA hydratase 2; SCP-2L, sterol carrier protein-2-like; NAD + , nicotinamide adenine dinucleotide; IPTG, isopropyl β-D-1thiogalactopyranoside; C4, 2E-butenoyl-CoA; C6, 2E-hexenoyl-CoA; C10, 2E-decenoyl-CoA.

Figure 5 Biochemical

 5 Figure 5

Figure 6 Biochemical

 6 Figure 6

Figure 7 Biochemical

 7 Figure 7

Table 2 Data collection, refinement statistics and quality of the structure

 2 Values in parentheses refer to the highest resolution shell. b Value in parentheses refer to the percentage of reflections taken randomly for the test calculations. c The non-hydrogen atoms used in the refinement. d Full B-factors after applying TLS refinement in Refmac are given in accordance with the procedure required by the RCSB Protein Data Bank (PDB).

		Biochemical Journal Immediate Publication. Published on 14 Feb 2011 as manuscript BJ20101661
	THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101661	Beamline Data set Data collection statistics Wavelength (Å) Temperature (K) Rotation range ( o ) Space group a (Å) b (Å) c (Å) Resolution range (Å) a Crystal mosaicity ( o ) Hkl (observed/unique) Redundancy Completeness (%) a Overall I/σI a R merge a Refinement statistics R free b R work Number of residues c Number of atoms c Protein c Water c Geometry statistics Root mean square deviations Lengths (Å)/angles ( o ) Average B-factors d All atoms (Å 2 ) Main chain atoms (Å 2 ) Side chain atoms (Å 2 ) Water molecules (Å 2 ) Ramachandran plot Favored region (%) Allowed regions (%) Disallowed regions (%) A c c e p t e d ID29, ESRF, Grenoble, France DmMFE-2 0.9310 100 90 P4 3 2 1 2 114.48 114.48 89.11 28.5 -2.15 (2.21 -2.15) 0.61 230188/32494 7.1 99.4 (96.7) 22.9 (3.0) 4.7 (63.1) 28.5 (5%) 23.4 532 4173 4035 138 0.011/1.20 65.8 68.8 62.8 52.9 97.3 2.7 0.0 M a n u s c r i p t

a + , nicotinamide adenine dinucleotide; IPTG, isopropyl β-D-1thiogalactopyranoside; C4, 2E-butenoyl-CoA; C6, 2E-hexenoyl-CoA; C10, 2E-decenoyl-CoA.

© 2011 The Authors Journal compilation © 2011 Portland Press Limited

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.© 2011 The Authors Journal compilation © 2011 Portland Press Limited

ACKNOWLEDGEMENTS

We thank Päivi Pirilä, Tuula Kurvinen and Marja Lajunen for synthesizing the substrates. Biochemistry student Henri Urpilainen is acknowledged for preparing the plasmid constructs for the separate dehydrogenase and hydratase enzymes of DmMFE-2 during a laboratory rotation study. The European Synchrotron Radiation Facility in Grenoble is acknowledged for beamtime on the beamline ID29. This research was carried out on grants from the Academy of Finland to T.G. and Sigrid Jusélius Foundation to K.H.

ACCESSION NUMBERS

Atomic coordinates and structure factors for the crystal structure of DmMFE-2 have been deposited in the Protein Data Bank under the ID code 3OML.

Abbreviations used: CoA, coenzyme A; DmMFE-2, Drosophila melanogaster multifunctional enzyme type 2; DH, 3R-hydroxyacyl-CoA dehydrogenase; H2, 2E-Enoyl-CoA hydratase 2; SCP-2L, sterol carrier protein-2-like; NAD + , nicotinamide adenine dinucleotide; IPTG, isopropyl β-D-1thiogalactopyranoside; C4, 2E-butenoyl-CoA; C6, 2E-hexenoyl-CoA; C10, 2E-decenoyl-CoA.


Jelena Stanisavljevic

Josue Curto

Clara Francí

Víctor Díaz

Manuel

García De Herreros

Josep Baulida
email: jbaulida@imim.es

LSF/TFCP2c/LBP-1c is Required for Snail1-induced Fibronectin Gene Expression

Keywords: Snail1, TFCP2c, epithelial to mesenchymal transition, E-cadherin, Fibronectin

Fibronectins are cell-secreted glycoproteins that modulate cell attachment, spreading, migration, morphology, differentiation and oncogenic transformation. Fibronectin expression is activated during epithelial-mesenchymal transition (EMT) and is a hallmark of mesenchymal cells. Results presented in this article reveal that a transcription factor previously unrelated with EMT, LSF/TFCP2c/LBP-1c, was translocated to the nucleus and bound to the Fibronectin promoter upon EMT induction by Snail1. Consequently, the interference of TFCP2c/LSF/LBP-1c activity prevented Fibronectin expression. Moreover, TFCP2c/LSF/LBP-1c was detected in nuclei of embryonic dermal mesenchymal cells adjacent to the hair bud, a cell population that expresses endogenous nuclear Snail1 and Fibronectin. Therefore, our data indicate a new molecular role for TFCP2c/LSF/LBP-1c in Fibronectin expression.

Introduction

Advanced tumour cells frequently show a down-regulation of epithelial markers accompanied by increased expression of Fibronectin (FN1) and other mesenchymal genes. This morphogenetic process, referred to as epithelial to mesenchymal transition (EMT), was first described to be essential during embryogenesis [START_REF] Nieto | The snail superfamily of zinc-finger transcription factors[END_REF] and it provides epithelial tumour cells with properties that support cancer progression, such as migration, invasion, extravasation [START_REF] Thiery | Epithelial-mesenchymal transitions in tumour progression[END_REF] as well as cell stemness associated to cancer [START_REF] Hollier | The Epithelial-to-Mesenchymal Transition and Cancer Stem Cells: A Coalition Against Cancer Therapies[END_REF]. Fibronectins are a class of high molecular weight cell-secreted glycoproteins that modulate cell-substrate adhesion as well as a set of cellular processes, among them cell attachment and spreading, migration, morphology, differentiation and oncogenic transformation [START_REF] Yamada | Cell surface interactions with extracellular materials[END_REF]. Fibronectin expression is a feature of mesenchymal cells and correlates with stimuli (such as TGF-that induce Snail1 expression and EMT [START_REF] Thuault | HMGA2 and Smads co-regulate SNAIL1 expression during induction of epithelial-to-mesenchymal transition[END_REF]. Snail1 is a member of SNAI genes that is expressed in the EMT processes analyzed [START_REF] Barrallo-Gimeno | The Snail genes as inducers of cell movement and survival: implications in development and cancer[END_REF]. This factor directly represses epithelial markers such as E-cadherin (the product of the CDH1gene) by binding to E-boxes in their promoters [START_REF] Batlle | The transcription factor snail is a repressor of E-cadherin gene expression in epithelial tumour cells[END_REF][START_REF] Cano | The transcription factor snail controls epithelial-mesenchymal transitions by repressing E-cadherin expression[END_REF].

This article provides evidence that Snail1-induced FN1 gene expression requires the transcription factor TFCP2c/LSF/LBP-1c [START_REF] Veljkovic | Lineage-specific and ubiquitous biological roles of the mammalian transcription factor LSF[END_REF] (TFCP2c from now on), and that SNAI1 and TFCP2c are coexpresed along with FN1 during embryogenesis. Although this transcription factor is known to interact with cellular and viral promoters, any interaction with FN1 or other mesenchymal genes has not been previously described. Therefore, our data indicate a new role for TFCP2c in Fibronectin regulation in Snail1induced EMT.

Materials and methods

Cell lines, transfection and infection

RWP1, HT29 M6, MEFs and NMuMG cells were grown in Dulbecco's modified Eagle's medium (DMEM, Invitrogen) plus 10% FBS under standard conditions. For NMuMG cells medium was supplemented with 10 g/mL of Insulin. DNA transfection was performed with LipofectAMINE-PLUS (Invitrogen) reagent according to manufacturer's instructions and DNA transduction with lentivirus. These experimental procedures are detailed in the supplementary data.

Transcription activity and DNA constructs

Luciferase-based reporter assays were performed as described elsewhere [10,[START_REF] Solanas | E-cadherin controls beta-catenin and NF-kappaB transcriptional activity in mesenchymal gene expression[END_REF]. DNA vectors used in the experiments are detailed in the supplementary data. A c c e p t e d M a n u s c r i p t RNA was extracted with Gene Elute TM Mammalian Total RNA Miniprep Kit (Sigma-Aldrich). For quantitative analysis, 1 g of RNA was retro-transcribed with Transcriptor First Strand cDNA Synthesis Kit (Roche) and 100 ng of the obtained cDNA was used as a template for quantitative SYBR-Green-based PCR. Correct product size was confirmed in agarose gels. The calculated RNA amount was systematically normalized by the Pumilio RNA levels. For the semi-quantitative analysis, 50 ng RNA was analysed with SuperScript One-Step RT-PCR with Platinum Taq (Invitrogen) and agarose gels. See the supplementary table S1 for specific oligonucleotides.

Analysis of RNA

Analysis of proteins

Immunofluorescence: A standard IF protocol on cells grown for at least 48 hours on ethanol sterilized glass coverslips was applied. All steps were done at room temperature. Cells were fixed with 4% paraformaldehyde (PFA) for ten minutes. PFA auto-fluorescence was quenched with 50 mM NH 4 Cl/PBS for five minutes and cells were permeabilised with 0.2% Triton X-100 for five minutes. Blocking was carried out for 1 h with PBS containing 3% bovine serum albumin (BSA). Coverslips were then incubated in humid atmosphere for 1 h with a primary antibody (20 g/ml for TFCP2c antibody) and, after extensive washing, for 45 min with the secondary antibody (in dark). Nuclear counterstaining was performed by incubating the coverslips for one minute with 5 g/ml propidium iodide and 100 g/ml RNAse. Coverslips were mounted with fluoromont G and fluorescence was viewed and captured through a Leica TCS-SP2 confocal microscope.

Analysis of proteins in the mouse embryo: The protocol was reported by Francí et al. [START_REF] Francí | Expression of Snail protein in tumor-stroma interface[END_REF]. CD-1 pregnant mice (Harlan) were killed by cervical dislocation; the embryos were removed by Caesarean section, fixed in formalin and embedded in paraffin. Sections (4 m) were dewaxed and rehydrated. Antigens were retrieved by boiling the samples in Tris-EDTA pH 9 for 15 min. Endogenous peroxidase activity was quenched with 4% hydrogen peroxide in PBS containing 0.1% sodium azide for 15 min. After several rinses with PBS, sections were incubated with PBS containing 1% BSA to block non-specific binding and were washed with PBS. Sections were incubated with either 10 g/ml of purified anti-Snail1 MAb EC3, 5g/ml anti-Fibronectin PAb or 20 g/ml anti-TFCP2 PAb overnight at 4ºC. After several further rinses with PBS, bound Ab was detected using anti-mouse or anti-rabbit Envision (Envision System Peroxidase, DAKO, Glostrup, Denmark). Sections were counterstained with hematoxylin.

Chromatin Immunoprecipitation (ChIP)

Cells seeded in 150 mm-diameter plates were washed twice with pre-warmed PBS (37ºC) and cross-linked with 1% formaldehyde for 10 minutes at 37ºC in DMEM. Reaction was stopped by adding 250 l of glycine 2.5 M (0.125 M final concentration) and incubating for 2 more minutes. Cells were washed twice with cold PBS and 1ml of Soft Lysis Buffer (50 mM Tris pH 8.0, 2 mM EDTA, 0.1% Nonidet P-40, 10% glycerol and protease and phosphatase inhibitors) was added to the plates on ice. After scrapping, lysates were transferred to Eppendorf tubes, incubated for 10 minutes on ice and centrifuged for 15 minutes at 3,000 rpm. Supernatant was discarded and pellet resuspended in SDS lysis buffer (1% SDS, 10 mM EDTA and 50 mM Tris pH 8.0) and sonicated to generate fragments of DNA from 200 to 1,000 bp (40% amplitude in Branson DIGITAL Sonifier® UNIT Model S-450D sonicator, 10 pulses of 10 seconds each). Lysates were incubated for 20 minutes on ice and subsequently centrifuged at maximum speed for 10 minutes.

Protein concentration in the supernatants was determined by Lowry and 500 g of protein per immunoprecipitation (IP) was diluted in Dilution Buffer (0.001% SDS, 1.1 Triton X-100, 16.7 mM Tris pH 8.0, 2 mM EDTA, 2 mM EGTA, 167 mM NaCl). Preclearing in order to reduce background was performed for 3 hours at 4ºC with agitation using mouse IgG (Dako) and salmon sperm-BSA (bovine serum albumin) blocked agarose-protein G (Upstate). Samples were then centrifuged at 2,000 rpm to remove the beads, 10% of the samples was stored as the input and the rest of the samples was divided for IP and incubated with either specific TFCP2c antibody (ab42973, Abcam) or an irrelevant antibody of the same species overnight at 4ºC with agitation.

Blocked beads were added to each sample and incubated for one more hour at 4ºC. Five washes were performed in MoBiTec columns with each of the buffers: Low Salt Buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris pH 8.0 and 150 mM NaCl), High Salt Buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris pH 8.0 and 500 mM NaCl), LiCl Buffer (250 mM liCl, 1% Nonidet P-40, 1% Sodium deoxycholate, 1 mM EDTA and 10 mM Tris pH 8.0) and TE Buffer. Columns were centrifuged to eliminate traces of buffer (2 minutes, 2,000 rpm) and the remaining beads were incubated with Elution buffer (100 mM Na 2 CO 3 and 1% SDS) at 37ºC for 30 minutes. DNA was recovered by centrifugation (5 minutes, 2,000 rpm). 4 l of 5 M NaCl (250 mM final concentration) was added to each eluate and decrosslinking was performed by incubating samples at 65°C overnight. After 2-4 hours digestion with proteinase K (55ºC), DNA was purified by the GFX PCR DNA and Gel Band Purification kit (Amersham Biosciences) and finally analyzed by quantitative SYBR-green PCR (Supplementary table S3).

For ChIP of exogenous promoters [START_REF] James | HPV16-E6 associated hTERT promoter acetylation is E6AP dependent, increased in later passage cells and enhanced by loss of p300[END_REF], 3.5x10 6 RWP1 cells were plated in 150 mm dishes and 5 g of indicated FN1 promoter was transfected with LipofectAMINE®-PLUS reagent (Invitrogen), according to manufacturer's instructions. 48 hours after transfection, cells were treated following the protocol previously described. The amount of protein used per IP was 100 g.

Results

TFCP2c binds to the FN1 promoter in Snail1 expressing cells

Upregulation of mesenchymal genes, such FN1, is a characteristic molecular event related with EMT [10,[START_REF] Solanas | E-cadherin controls beta-catenin and NF-kappaB transcriptional activity in mesenchymal gene expression[END_REF]. Accordingly, Fibronectin RNA and protein levels increased when RWP1 and HT29 M6 epithelial cells were forced to transit towards a mesenchymal phenotype by ectopic Snail1HA expression (Fig. 1a). In line with this increment, the transcriptional activity of the -341/+265 proximal FN1 promoter was higher in Snail1 than in control cells (Fig. 1b). Whereas Fibronectin basal levels in HT29 M6 were lower than in RWP1 cells, the increase induced by Snail1 was higher. As expected for EMT, we detected reduced expression of the epithelial gene E-cadherin in Snail1 expressing cells (Fig. 1a andb). To find out transcription factors mediating FN1 activation by Snail1, we looked for transcription factor binding sites in the FN1 proximal promoter. We narrowed our search to the initial 19 bp (-341/-323) sequence of the proximal promoter, since the exclusion of this region was sufficient to interfere FN1 transcription by Snail1, as observed through reporter assays of 5'-deleted proximal FN1 promoters (Fig. 1c). The transcription factor binding algorithm (TESS) detected three consensus binding motives for the transcription factor TFCP2c/LSF/LBP-1c, conserved among species (Fig. 1d); therefore, we decided to evaluate the possibility that TFCP2c was mediating Fibronectin activation in Snail1 cells.

We analyzed whether TFCP2c was binding to the FN1 promoter by means of ChIP assays. We detected binding of TFCP2c to the proximal sequence of the FN1 promoter in HT29 M6-Snail1HA but not in control cells. In contrast, Snail1 expression did not stimulate the binding of an irrelevant transcription factor (Fig. 2a). Taking advantage of the fact that the transfection efficiency of RWP1 is higher than that of HT29 M6, we tested whether the TFCP2c is binding to the exogenous proximal FN1 promoter transfected into RWP1 control and Snail1 cells. Similarly to the endogenous promoter, we found higher binding of TFCP2c to the transfected proximal promoter (-341/+265) in RWP1 Snail1HA than in control cells; however, this was not the case for a promoter missing the sequence (-341/-332) including the TFCP2c binding motives (Fig. 2b). Next, we analyzed by immunofluorescence whether the increased TFCP2c binding to the FN1 promoter in Snail1 cells was accompanied by an increment of the nuclear amount of TFCP2c. We detected higher nuclear TFCP2c staining in HT29 M6 Snail1 than in control cells (Fig. 2c). Since total levels of TFCP2c analysed by western blot were not increased in Snail1 cells, altogether, our data indicate that Snail1 expression triggers the nuclear localization of TFCP2c and its binding to the FN1 proximal promoter.

TFCP2c is required for Snail1-induced Fibronectin expression

The binding of TFCP2c to the FN1 promoter in Snail1 cells suggests that TFCP2c activity is required for FN1 transcription in these cells. Therefore, we analyzed the effect of a dominant negative for TFCP2c (TFCP2c Q234L/K236E) on Fibronectin expression. This mutant interferes with TFCP2c transcriptional activity by preventing the binding of the endogenous wild-type TFCP2c to DNA [START_REF] Shirra | One exon of the human LSF gene includes conserved regions involved in novel DNA-binding and dimerization motifs[END_REF]. While expression of the dominant negative did not affect the low Fibronectin protein levels in RWP1 and HT29 M6 control cells, it clearly decreased the Fibronectin levels detected in Snail1 cells (Fig. 3a), especially in the case of HT29 M6 cells. Similar decrease was detected at the mRNA level, quantified as being of 50 or 60% in RWP1 Snail1HA or HT-29 M6 Snail1HA cells, respectively (Suppl. Fig. S1). In accordance with the effect of the dominant negative, we found that HT29 M6 Snail1 cells depleted of TFCP2c by infection of a shRNA specific for TFCP2c produced less Fibronectin than Snail1 cells infected with an irrelevant shRNA (Suppl. Fig. S1). The involvement of TFCP2c in

A c c e p t e d M a n u s c r i p t

Fibronectin activation was also tested in epithelial cells forced to transit to mesenchymal by TGF-, which induces endogenous Snail1 levels. As shown in Fig. 3b, Fibronectin mRNA was increased at 8 h of TGF- treatment in control NMuMG sensitive cells but remained at basal levels in cells transduced with the dominant negative of TFCP2c.

To further analyze the relevance of TFCP2 on cells expressing endogenous Snail1 we transduced the dominant negative form of TFCP2 into mouse embryonic fibroblasts (MEFs) in which Fibronectin RNA expression depends of endogenous Snail1 (Supplementary materials and methods). Like in epithelial cells expressing exogenous Snail1, the expression of TFCP2c Q234L/K236E also decreased FN1 expression (Fig. 3c). Other mesenchymal genes analyzed such as LEF1, Snail1 or ZEB1 were not altered by the dominant negative form. Since Fibronectin modulates the cell-substrate interaction we analyzed whether the dominant negative form also affected the migration of the MEFs. Wound healing (Fig. 3d) and single cell track assays (Fig. 3e) showed that the dominant negative form of TFCP2 reduced the migratory capacity of fibroblasts and the effect was rescued by providing exogenous fibronectin to the cells (Suppl. Fig. S2).

Nuclear TFCP2c is expressed in embryonic dermal mesenchymal cells adjacent to the hair bud together with Snail1 and Fibronectin

To verify that the effect of TFCP2c on FN1 expression was not restricted to cultured Snail1 expressing cells, we analyzed the subcellular localization of TFCP2c in vivo. Expression of endogenous Snail1 is higher in embryonic than in adult tissues, but it is not constitutive and has a limited distribution in a subset of mesenchymal cells; for instance, Snail1 is expressed in embryonic mesenchymal cells adjacent to hair bud cells [START_REF] Francí | Expression of Snail protein in tumor-stroma interface[END_REF][START_REF] Jamora | A Signaling Pathway Involving TGF-β2 and Snail in Hair Follicle Morphogenesis[END_REF]. In line with this data, by immunohistochemical analysis of whole mounted mice embryos at day E14.5, we found embryonic dermal mesenchymal cells with positive nuclear staining for Snail1 (Fig. 4a). Interestingly, we found a similar pattern of nuclear expression of TFCP2c (Fig. 4b) and Fibronectin was detected in the same areas (Fig. 4c). These data, therefore, suggest that the transcription factor TFCP2c is involved in the transcriptional activation of Fibronectin by Snail1 in vivo.

Discussion

Our results indicate for the first time a role for the transcription factor TFCP2/LSF/LBP-1c in FN1 transcription. LSF was known to regulate diverse cellular and viral promoters [START_REF] Veljkovic | Lineage-specific and ubiquitous biological roles of the mammalian transcription factor LSF[END_REF][START_REF] Hansen | Transcription factors LSF and E2Fs: Tandem cyclists driving G0 to S?[END_REF] but none of them have a direct role in EMT regulation. Indirect relationship between TFCP2c targets and EMT can be established considering the fact that EMT and Snail1 expression generate cells with stem properties that hold potential for tumour initiation, tumour recurrence and metastasis [START_REF] Hollier | The Epithelial-to-Mesenchymal Transition and Cancer Stem Cells: A Coalition Against Cancer Therapies[END_REF][START_REF] Moody | The transcriptional repressor Snail promotes mammary tumor recurrence[END_REF][START_REF] Mani | The epithelial-mesenchymal transition generates cells with properties of stem cells[END_REF]. Thus, it was described that TFCP2 activates the chicken Embryonic Stem 1 gene (cENS-1) expression early in embryonic development [19] and the osteopondin gene in the context of hepatocellular carcinoma [20]. We have observed that overexpression of 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.

TFCP2c in epithelial HT26 M6 or RWP1 cells did not increase Fibronectin RNA levels (Suppl. Fig. S3). Moreover, the epithelial phenotype was maintained and no changes in E-cadherin RNA levels (Suppl. Fig. S3) were observed in these cells. Although an increase of TFCP2c was not sufficient to induce a complete EMT in the epithelial cells tested, our results directly involve TFCP2c in the molecular events triggering the expression of the mesenchymal gene FN1. Thus, we unveil a new molecular mechanism which could be targeted to prevent cancer cell properties associated to Fibronectin expression. (a) TFCP2c binds to the FN1 promoter in HT29 M6 cells. ChIP assays were performed in HT29 M6 control and stably expressing Snail1HA cells. Endogenous FN1 promoter was amplified with specific primers covering the -375/-320 region. Levels of FN1 promoter precipitated with specific antibody against TFCP2c or WT1 were compared with those precipitated with non-specific antibody. Error bars show the standard deviation of four independent experiments. In Snail1 cells, the binding of TFCP2c to the proximal FN1 promoter was increased over 2.8 +/-0.5 fold when compared with that to a -2kb (-1958/-1870; supplementary table S3) distal sequence. (b) TFCP2c binds to the FN1 promoter in RWP1 Snail1HA cells. RWP1 control and stably expressing Snail1HA cells were transiently transfected with the -341/+265 or -322/+265 FN1 promoter and 48 hours later a ChIP assay was performed using a TFCP2c specific antibody. The amount of exogenous FN1 promoter and an irrelevant DNA were analyzed by qPCR. Bars represent the FN1 promoter/irrelevant DNA ratio in Snail1HA relative to control cells obtained from four independent experiments (mean +/-standard deviation). (c) Subcellular localization of TFCP2 in HT29 M6 control and Snail1HA cells. TFCP2c sublocalization was analyzed by immunofluorescence. Propidium iodide was used for nuclear staining. All mice involved in this study were maintained in a rodent barrier facility in order to guarantee the specific pathogen free health status of the animals and the protocol was previously approved by the Animal Research Ethical Committee from the PRBB.

Biochemical

  Journal Immediate Publication. Published on 02 Mar 2011 as manuscript BJ20102057 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20102057A 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.

Biochemical

  Journal Immediate Publication. Published on 02 Mar 2011 as manuscript BJ20102057 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20102057

Biochemical

  Journal Immediate Publication. Published on 02 Mar 2011 as manuscript BJ20102057 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20102057

Figure 1 .

 1 Figure legends Figure 1. The FN1 promoter contains a responsive element at -341/-323 including TFCP2c binding elements. (a) CDH1 and FN1 gene expression in HT29 M6 and RWP1 control and Snail1HA cells. The left panel shows the protein levels detected by Western blot using indicated antibodies from total cell extracts. The right panel shows the relative mRNA levels for FN1 and CDH1 genes. HPRT was used for normalization. Bars represent an x-fold RNA increase in Snail1HA relative to control cells. (b) CDH1 and FN1 promoter activity in HT29 M6 and RWP1 control and Snail1HA cells. Transcription activity was calculated by luciferase reporter assays in control and Snail1HA cells. Cells were transiently co-transfected with 150 ng of a firefly luciferase promoter reporter plasmid, including the -341/+265 FN1 promoter or the -178/+92 CDH1 promoter, and 10 ng of a renilla luciferase TK vector for normalization. Bars show the increase of normalized luciferase activity in Snail1HA in relation to control cells. Error bars show the standard deviation of at least three independent experiments. (c) Transcription activation of FN1 promoter fragments by Snail1. Reporter experiments were performed by transiently cotransfecting RWP1 parental cells with 100 ng of an indicated FN1 promoter fragment cloned into a firefly luciferase vector, either 5 ng of pcDNA3-Snail1HA or an empty vector, and 10 ng of renilla TK vector for normalization. Bars represent the activity of the promoters in presence of Snail1HA relative to pcDNA3. Error bars show the standard deviation of four independent experiments. (d) The -341/-314 sequence of the FN1 promoter includes a consensus site for TFCP2c binding. Putative TFCP2c binding motives detected by TESS are boxed. Consensus sequence is annotated on the top, where N = T, C, A or G; and R = A or G. The Homo sapiens -341/-314 FN1 promoter sequence was aligned with that of the indicated species.

Figure 2 .

 2 Figure 2. TFCP2c binds to the FN1 promoter in Snail1HA cells.(a) TFCP2c binds to the FN1 promoter in HT29 M6 cells. ChIP assays were performed in HT29 M6 control and stably expressing Snail1HA cells. Endogenous FN1 promoter was amplified with specific primers covering the -375/-320 region. Levels of FN1 promoter precipitated with specific antibody against TFCP2c or WT1 were compared with those precipitated with non-specific antibody. Error bars show the standard deviation of four independent experiments. In Snail1 cells, the binding of TFCP2c to the proximal FN1 promoter was increased over 2.8 +/-0.5 fold when compared with that to a -2kb (-1958/-1870; supplementary tableS3) distal sequence. (b) TFCP2c binds to the FN1 promoter in RWP1 Snail1HA cells. RWP1 control and stably expressing Snail1HA

Figure 3 .

 3 Figure 3. TFCP2c is required for Snail1-induced Fibronectin expression. (a) TFCP2c Q234L/K236E expression reduces Fibronectin protein levels in Snail1HA cells. RWP1 and HT29 M6 control and Snail1HA cells were infected with empty pBABE (-) or pBABE-TFCP2c Q234L/K236E-myc (DN), and protein levels were analyzed by Western blot from total protein extracts; Annexin II was used as loading control. (b) TFCP2c Q234L/K236E mutant reduces Fibronectin RNA levels in MEFs. RNA levels corresponding to the indicated genes were analyzed by RT-PCR from MEFs transduced with pBABE-TFCP2c Q234L/K236E-myc or with empty pBABE vector. (c) TFCP2c Q234L/K236E mutant reduces Fibronectin activation by TFG- in NMuMG cells. mRNA levels corresponding to the indicated genes were analyzed by RT-PCR or RT-qPCR from TGF- 1 -treated (8h, 50 ng/mL) NMuMG cells previously transduced (48 h) with pBABE-TFCP2c Q234L/K236E-myc or with empty pBABE vector. (d) TFCP2c Q234L/K236E mutant reduces migration of MEFs assayed by wound healing. Pictures were taken 48 hours after performing the scratch. Borders of the scratch at time zero are indicated with dashed lines. (e) TFCP2 Q234L/K236E mutant reduces migration of MEFs assayed by single cell track. Movement of three representative cells is plotted.

Figure 4 .

 4 Figure 4. Nuclear TFCP2c is expressed in embryonic dermal mesenchymal cells adjacent to the hair bud together with Snail1 and Fibronectin. (a) Snail1, (b) TFCP2c and (c) Fibronectin expression in dermal mesenchymal cells of E14.5 embryos. Protein expression was detected by immunohistochemistry, as described in Supplementary Data. Nuclei were counter-stained with hematoxylin. Red arrowheads indicate positive cells. Electronic amplification (2.5x) is shown in boxes.

Figure 1 Biochemical

 1 Figure 1

Figure 2 Biochemical

 2 Figure 2

Figure 3 Biochemical

 3 Figure 3

Figure 4 Biochemical

 4 Figure 4

  Biochemical Journal Immediate Publication. Published on 02 Mar 2011 as manuscript BJ20102057 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20102057

© 2011 The Authors Journal compilation © 2011 Portland Press Limited

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Acknowledgements

We are grateful for the technical assistance of Dr. Raúl Peña, Alicia Riquel and Marta Garrido. This study was supported by a grant (PI071054) from the Fondo de Investigaciones Sanitarias of the ISCIII to JB and by grants from the Ministerio de Ciencia y Tecnología (SAF2006-00339) and the Fundació La Marató of TV3 to A.G. H. Partial support from the Instituto Carlos III (RD06/0020/0040) and from the Generalitat de Catalunya (2005SGR00970) is also appreciated.