Maria Rapala-Kozik

Mariusz Olczak

Katarzyna Ostrowska

Agata Starosta

Andrzej Kozik

Molecular characterization of thi3 gene involved in thiamine biosynthesis in Zea mays: cDNA sequence and enzymatic and structural properties of recombinant bifunctional protein with 4-amino-5-hydroxymethyl-2-methylpyrimidine (phosphate) kinase and thiamine monophosphate synthase activities

Keywords: cDNA cloning, enzyme kinetics, protein overexpression, thiamine phosphate diphosphorylase, site-directed mutagenesis, structural modelling

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INTRODUCTION

Thiamine diphosphate (TDP) plays an essential role in all organisms as a cofactor of several universal enzymes involved in carbohydrate and amino acid metabolism, including pyruvate decarboxylase, pyruvate-, α-ketoglutarate-, and branched fatty acid-dehydrogenases, transketolase, and α-acetolactate synthase [START_REF] Friedrich | Thiamin (Vitamin B1, aneurin)[END_REF]. TDP is synthesized from exogenous thiamine taken with food (as the vitamin B 1 ) by higher animals or taken up by microbial cells from the environment [START_REF] Yamada | Properties of the thiamine transport system in Escherichia coli[END_REF][START_REF] Iwashima | Thiamin transporters in yeast[END_REF], or from endogenous thiamine monophosphate (TMP) which is synthesized by many prokaryotic and eukaryotic microorganisms as well as higher plants. The last step of de novo TMP biosynthesis involves a condensation of two precursors, 4-amino-5hydroxymethyl-2-methylpyrimidne diphosphate (HMP-PP) and 4-methyl-5-(2hydroxyethyl)thiazole phosphate (HET-P) which are separately synthesized on the routes that significantly differ between various groups of thiamine-synthesizing organisms. Many microorganisms can additionally salvage HMP-PP and HET-P by phosphorylation of 4amino-5-hydroxymethyl-2-methylpyrimidne (HMP) and 4-methyl-5-(2-hydroxyethyl)thiazole (HET) taken up from the medium (Scheme 1).

The pathways of bacterial thiamine biosynthesis are well recognized (for review see: [START_REF] Begley | Thiamin biosynthesis in prokaryotes[END_REF]). Many of the enzymes which catalyze the specific steps of thiamine synthesis in model bacteria species such as Escherichia coli, Bacillus subtilis and Salmonella typhimurium have been obtained as recombinant proteins and their three-dimensional structures have recently been determined (for review see: [START_REF] Settembre | Structural biology of enzymes of the thiamin biosynthesis pathway[END_REF]). In a sharp contrast, our knowledge of this process in eukaryotic cells is at a very preliminary stage, in terms of both the identification of individual biosynthetic reactions and the structural biology of enzymes involved. In baker's yeasts Saccharomyces cerevisiae, at least fifteen TDP-biosynthesis genes were identified (for review see: [START_REF] Nosaka | Recent progress in understanding thiamin biosynthesis and its genetic regulation in Saccharomyces cerevisiae[END_REF]) but products of only four genes, THI80, THI6, THI4 and THI20, are known as purified, natural or recombinant proteins [START_REF] Baker | The crystal structure of yeast thiamin pyrophosphokinase[END_REF][START_REF] Kawasaki | Copurification of hydroxyethylthiazole kinase and thiaminephosphate pyrophosphorylase of Saccharomyces cerevisiae: characterization of hydroxyethylthiazole kinase as a bifunctional enzyme in the thiamine biosynthetic pathway[END_REF][START_REF] Jurgenson | Structural insights into the function of the thiamine biosynthetic enzyme Thi4 from Saccharomyces cerevisiae[END_REF][START_REF] Haas | Thi20, a remarkable enzyme from Saccharomyces cerevisiae with dual thiamin biosynthetic and degradation activities[END_REF]. Kinetic characteristics are available for THI80 protein with thiamine diphosphokinase (EC 2.7.6.2) activity [START_REF] Voskoboyev | Thiamin pyrophosphokinase: structure, properties and role in thiamin metabolism[END_REF] and for THI6 bifunctional enzyme with TMP synthase (TMP diphosphorylase, EC 2.5.1.3) and HET kinase (EC 2.7.1.50) activities [START_REF] Kawasaki | Copurification of hydroxyethylthiazole kinase and thiaminephosphate pyrophosphorylase of Saccharomyces cerevisiae: characterization of hydroxyethylthiazole kinase as a bifunctional enzyme in the thiamine biosynthetic pathway[END_REF]. Three-dimensional structures were reported for thiamine diphosphokinase [START_REF] Baker | The crystal structure of yeast thiamin pyrophosphokinase[END_REF] and THI4 protein [START_REF] Jurgenson | Structural insights into the function of the thiamine biosynthetic enzyme Thi4 from Saccharomyces cerevisiae[END_REF], involved in thiazole synthesis [START_REF] Dorrestein | The biosynthesis of the thiazole phosphate moiety of thiamin (vitamin B 1 ): the early steps catalyzed by thiazole synthase[END_REF] and possibly playing some role in yeast tolerance against DNA damage [START_REF] Machado | Dual role for the yeast THI4 gene in thiamine biosynthesis and DNA damage tolerance[END_REF]. In higher plants, a limited number of thiamine biosynthesis genes have been identified and sequenced, including several orthologues of yeast THI4 thiazole-synthesizing gene, found in Zea mays [START_REF] Belanger | Evidence for the thiamine biosynthetic pathway in higher-plant plastids and their developmental regulation[END_REF], Arabidopsis thaliana [START_REF] Machado | Thi1, a thiamine biosynthetic gene in Arabidopsis thaliana, complements bacterial defects in DNA repair[END_REF] and Alnus glutinosa [START_REF] Ribeiro | Identification of agthi1, whose product is involved in biosynthesis of the thiamine precursor thiazole, in actinorhizal nodules of Alnus glutinosa[END_REF], and a family of orthologues of Brassica napus bth1 gene [START_REF] Kim | A Brassica cDNA clone encoding a bifunctional hydroxymethylpyrimidine kinase/thiamin-phosphate pyrophosphorylase involved in thiamin biosynthesis[END_REF] which probably codes for a bifunctional enzyme with TMP synthase and Biochemical Journal Immediate Publication. Published on 14 Aug 2007 as manuscript BJ20070677 [START_REF] Begley | Thiamin biosynthesis in prokaryotes[END_REF] HMP/HMP-P kinase (EC 2.7.1.49 / EC 2.7.4.7) activities. Any protein product of bth1 gene was not isolated although its function in thiamine biosynthesis was drawn from the functional complementation of thiamine-requiring mutants of some bacteria [START_REF] Kim | A Brassica cDNA clone encoding a bifunctional hydroxymethylpyrimidine kinase/thiamin-phosphate pyrophosphorylase involved in thiamin biosynthesis[END_REF]. Recently, a threedimensional structure of A. thaliana orthologue of yeast THI4 protein was reported [START_REF] Godoi | Structure of the thiazole biosynthetic enzyme THI1 from Arabidopsis thaliana[END_REF] but its enzymatic activity remains to be defined.

In this work we cloned and sequenced cDNA of a Z. mays gene designated thi3 which is orthologous to B. napus bthi1 gene. A product of this gene was isolated as a recombinant active enzyme and its dual TMP synthase and HMP/HMP-P kinase activity was directly presented and kinetically characterized. Models for the structure of enzyme active centres are presented. This is the second plant thiamine-synthesizing enzyme which has been characterized on the purified protein level and the first one whose enzymatic properties were quantitatively analyzed.

EXPERIMENTAL

Cloning and sequencing of thi3 cDNA from Zea mays leaves

The 10-day old leaves were grinded in liquid nitrogen to a fine powder using mortar and pestle followed by NucleoSpin filter homogenization (Macherey-Nagel, Germany). Total RNA from 100 mg of disrupted tissue was purified using NucleoSpin RNA II Kit (Macherey-Nagel). The quantity and integrity of the total RNA was checked by formaldehyde agarose gel electrophoresis.

To identify the thi3 cDNA, a set of six degenerate primers based on the known eukaryotic and prokaryotic conservative sequences of BTH1-related proteins (sequences derived from EMBL database) were designed. The cDNA was synthesized using Superscript III reverse transcriptase (Invitrogen, USA) with poly-dT(20) primer. From nine reactions performed in the experiment, the pair of MO1-F primer and MO2-R primer (Table 1) gave a dominant PCR product of about 1000 bp. The product was purified from the agarose gel, cloned into the pCR4-TOPO vector, and sequenced. All PCR reactions were performed with Perpetual Opti Taq Polymerase (Eurx, Gdansk, Poland). The 5' and 3' RACE (rapid amplification of cDNA ends) was performed according to Frohman [START_REF] Frohman | Rapid amplification of complementary DNA ends for generation of full-length complementary DNAs: Thermal RACE[END_REF]. Based on the partial sequence of thi3, a set of 5'RACE reverse primers and 3'RACE forward primers was designed (Table 1). The synthesis of cDNA from 4 µg of the total RNA was performed using the ZTH51 primer in 5'RACE and adaptor-oligo-dT [START_REF] Kim | A Brassica cDNA clone encoding a bifunctional hydroxymethylpyrimidine kinase/thiamin-phosphate pyrophosphorylase involved in thiamin biosynthesis[END_REF] primers in 3'RACE. After 5'RACE RT reaction, primers, nucleotides and salts were removed by ultrafiltration on Microcon-100 columns (Millipore, USA), using sterile water as dilution liquid. The purified cDNA was tailed using terminal transferase (Promega, USA) in the presence of 0.25 mM dATP at 37°C for 10 min. The second strand of cDNA was synthesized using adaptor-dT [START_REF] Kim | A Brassica cDNA clone encoding a bifunctional hydroxymethylpyrimidine kinase/thiamin-phosphate pyrophosphorylase involved in thiamin biosynthesis[END_REF] primer. After adding of adaptor and ZTH52 primers, the first 5'RACE reaction was performed, followed by "semi-nested" PCR using ZTH53 primer and adaptor primer. The discrete product was purified and cloned into the pCR4-TOPO sequencing vector. The 3'RACE product was amplified after adding of ZTH31 primer and adaptor primer to 3'RT cDNA. Then ZTH32 primer and adaptor primer were used in "semi-nested" PCR. The specific product was purified and cloned into the pCR4-TOPO vector. All DNAs were analyzed by sequencing (Sequencing Laboratory, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland).

Expression and purification of THI3 protein in E. coli

The overexpression and purification of THI3 protein containing intein (with the chitinbinding domain, CBD) attached at either the N-or C-terminus of the protein was performed according to the published method [START_REF] Chong | Modulation of protein splicing of the Saccharomyces cerevisiae vacuolar membrane ATP-ase intein[END_REF]. For this purpose, the thi3 cDNA was cloned into the pTYB1 vector (New England Biolabs, USA) utilizing the NdeI and SapI restriction sites, and into pTYB11 vector utilizing the SapI and XhoI restriction sites, resulting in pMO1 and pMO2 plasmids, respectively. The N-tagged version of recombinant THI3 protein lacked Nterminal 21-amino acid sequence. Recombinant proteins containing the CBD tag were overexpressed after induction with 0.3 mM isopropyl-β-D-thiogalactopyranoside (IPTG) in ER2566 E. coli cells at 16°C for 16 h. The cells were suspended in 20 mM Tris/HCl buffer, pH 8.5, containing 500 mM NaCl and sonicated. After centrifugation at 40 000 x g for 20 min, the recombinant proteins containing the CBD tag were purified according to the IMPACT-CN expression system (New England Biolabs) from soluble fraction using 50 mM dithiothreitol to induce the tag cleavage.

The protein samples were dialyzed against 20 mM Tris/HCl buffer, pH 7.5, containing 150 mM NaCl and subjected to the fine purification on Superdex 200 HR 10/50 high performance size-exclusion column (Amersham, England) eluted with the same buffer. The purified protein samples were stored in aliquots at -20ºC. Under these storage conditions the enzyme was stable for at least 3 months.

Protein samples from various preparation steps were analyzed by SDS-PAGE in the Laemmli system [START_REF] Laemmli | Cleavage of structural proteins during assembly of the head of bacteriophage T4[END_REF] with 10% resolution gel. The protein bands were either visualized by Coomassie Brilliant Blue R-250 staining or subjected to Western blotting on nitrocellulose membranes, using primary polyclonal anti-CBD antibodies (New England Biolabs) and secondary anti-rabbit antibody-horse-radish peroxidase conjugate (Promega).

Site-directed mutagenesis

The QuickChange Multi Site-Directed Mutagenesis Kit (Stratagene, USA) and the pTyb11 bacterial expression plasmid with thi3 insert as a template was used for mutagenesis. Initially, two double mutants were produced: in the first mutant Gln-98 was replaced by Leu and Gln-373 by Leu, in the second Met-134 was replaced by Lys and Thr-472 by Asp. The single mutants Q98L, M134K, Q373L and T472D were constructed by digestion of the double mutants and the wild-type Tyb11-thi3 by SacI and XhoI restriction enzymes and appropriate ligation. Additionally, a single mutant in which Ser-444 was replaced by Ala was designed.

Synthesis of HMP and its phosphate-and diphosphate esters

HMP synthesis was performed through the intermediary syntheses of 4-amino-5aminomethyl-2-methylpyrimidine [START_REF] Dibella | Isomeric S-methylthiamins[END_REF] and 4-amino-5-bromomethyl-2-methylpyrimidine [START_REF] Morimoto | Bildungsmechanismus von 4-Amino-5-aminomethyl-2-methyl-pyrimidin aus 3-Athoxy-2-(diathoxymethyl)propionitril und Acetamidin[END_REF]. HMP-P and HMP-PP were obtained from HMP [START_REF] Brown | Preparation of the mono-and pyrophosphate esters of 2-methyl-4-amino-5-hydroxymethylpyrimidine for thiamine biosynthesis[END_REF].

Enzyme assays

For TMP synthase assay, the reaction mixture contained, unless otherwise specified, 15 µM HMP-PP, 15 µM HET-P and 10 mM MgCl 2 in 50 mM Tris-HCl buffer, pH 8.0 and 5 µg of the pure enzyme in the final volume of 100 µl. After incubation at 37ºC for 60 min, the reaction was stopped with 16 µl of 10% metaphosphoric acid and the samples were centrifuged at 10 000 x g for 10 min, diluted 3 times with 0.1 M potassium phosphate and analyzed for TMP content by reverse-phase HPLC (RP-HPLC) with postcolumn derivatization and fluorimetric detection [START_REF] Lee | Determination of thiamine and its phosphate esters by gradient-elution high-performance liquid chromatography[END_REF]. A Merck LiChrosphere 100 RP-18 (5 µm) column (250 mm × 4 mm) was used, with a two-solvent system including 0.15 M ammonium citrate, pH 4.2 (solvent A) and 0.1 M formic acid, containing 0.4% diethylamine, pH 3.2 (solvent B). A gradient elution (0-90% B in 16 min) at a flow rate of 1 ml min -1 was applied.

The fluorogenic derivatization was performed with 0.0025% sodium hexacyanoferrate (III) in 2.25% NaOH, pumped through a peristaltic pump with a flow rate of 0.8 ml min -1 . The fluorescence emission at 430 nm excited at 365 nm was monitored.

The production of TMP from HMP or HMP-P due to the consecutive action of HMP/HMP-P kinase and TMP synthase were determined in analogous samples, with HMP-PP replaced by these substrates (15 µM) and with the 10 mM ATP supplement. C). The following time program was applied: at t=0 min: 10%B, 30%C, at t=14 min: 50%B, 50%C, at t=16 min: 10%B, 80%C, at t=18 min: 10%B, 30%C, at t=35min: 10%B, 30%C. The absorbance of the eluate at 245 nm was monitored.

Kinetic data analysis

The plots of the initial reaction velocity versus substrate concentrations were analyzed for various kinetic parameters as outlined by Bisswanger [START_REF] Bisswanger | Enzyme kinetics. Principles and methods[END_REF]. For hyperbolic kinetics, the apparent K m and V max parameters were estimated by fitting the classic Michaelis-Menten equation (untransformed) with the use of GraphPad Prism software. In the cases where the reaction velocity reached a maximum and decreased thereafter, indicative of the inhibition by the excess of substrate, the following equation was fitted:

v = (V max [S])/(K m + [S] +[S] 2 /K iu )
where v is the reaction velocity, [S] -the substrate concentration, and K iu -the apparent substrate (uncompetitive) inhibition constant. K iu was first estimated from the abscissa intercept of the high-concentration part of the Dixon plot, 1/v versus [S]. The value obtained was used as a constant during the fitting of the above equation by the GraphPad Prism to estimate the remaining K m and V max parameters. The uncompetitive inhibition of TMP synthase by ATP was analyzed from the plots of the reaction velocities versus HET-P concentrations at various ATP concentrations which should follow the equation:

v = (V max [S])/(K m + [S] +[S][I]/K iu )
where [I] is the inhibitor concentration, and K iu -the apparent uncompetitive inhibition constant. The Hanes-Woolf transformations, [S]/v versus [S], were used to classify the type of inhibition, and their slopes, determined by linear regression, where re-plotted versus [I] to estimate K iu as the abscissa intercept of this secondary plot. In addition, all three parameters, K iu , K m and V max , of the above equation were also estimated by direct non-linear regression fits to the untransformed data.

Bioinformatic analyses

Searching for protein sequence similarities was performed with the BLAST (v. 2.2.16)

program [27] available on the server of National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov). The multiple sequence alignments were generated with the use of CLUSTAL W (v. 1.82) [START_REF] Thompson | CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice[END_REF] from the ExPASy Proteomics Server of Swiss Institute of Bioinformatics (http://www.expasy.org) and visualized with JalView 2.3 [START_REF] Clamp | The Jalview alignment editor[END_REF] multiple alignment editor (http://www.jalview.org). Putative chloroplast transit peptides were found by the ChloroP (v. 1.1) program [START_REF] Emanuelsson | ChloroP, a neutral networkbased method for predicting chloroplast transit peptides and their cleavage sites[END_REF] at the server of Center for Biological Sequence Analysis of the Technical University of Denmark (http://www.cbs.dtu.dk/services/ChloroP). The THI3 protein sequence was also analyzed using the Conserved Domains Search service [START_REF] Marchler-Bauer | CD-Search: protein domain annotations on the fly[END_REF] on the NCBI server. Models of three dimensional structures of THI3 protein domains, based on the known crystal structures of S. typhimurium HMP-P kinase (PDB code 1JXH, [START_REF] Cheng | Crystal structure of 4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate kinase from Salmonella typhimurium at 2.3 Å resolution[END_REF]) and B. subtilis TMP synthase (PDB code 2TPS, [START_REF] Chiu | Crystal structure of thiamin phosphate synthase from Bacillus subtilis at 1.25 Å resolution[END_REF]), were generated using the SWISS-MODEL automated homology modelling server [START_REF] Schwede | SWISS-MODEL: an automated protein homology-modeling server[END_REF] of Swiss Institute of Bioinformatics (http://swissmodel.expasy.org). The structures were visualized using the Swiss-PdbViewer and PyMOL programs available from the ExPASy Server.

RESULTS

Cloning and sequence analysis of maize thi3 cDNA

The amplification with a pair of degenerate MO1-F forward and MO2-R reverse primers (Table 1) resulted in a discrete, specific RT-PCR product, which was cloned into a sequencing vector. After E. coli transformation, 10 clones were randomly chosen for sequencing. All clones contained the same 997-nucleotide DNA fragment. Based on this sequence, 5'RACE and 3'RACE were performed. In both reactions, nested PCR gave dominant products, which were cloned into a sequencing vector. For each RACE, four randomly chosen colonies were picked up and a plasmid purified from each clone was sequenced.

Purification and basic molecular characterization of recombinant maize THI3 protein, overproduced in E. coli

Of several versions of THI3 recombinant protein tested, the protein lacking N-terminal 21amino acid fragment and expressed in E. coli with 55-kDa intein as a tag protein attached to the N-terminus gave a satisfactory level of expression, especially when bacterial cultures were propagated at temperature lowered to 16ºC. Under these conditions the majority of recombined fusion THI3 protein was found in the soluble fraction.

The SDS-PAGE record of a typical THI3 purification run is presented in Figure 1.

After the affinity chromatography on a chitin-containing column followed by the Superdex-200 high performance gel filtration, a homogeneous protein was obtained in a typical yield of about 3 mg from 4 l of bacterial culture. The protein apparent monomer molecular mass was 55 kDa as determined by SDS-PAGE. Upon the Superdex-gel filtration the protein eluted in a volume corresponding to an apparent molecular mass of 95 kDa (results not shown), suggesting that under non-denaturing conditions the protein existed as a dimer.

Characterization of enzymatic activities of THI3 protein

As shown in Table 2, the recombinant maize THI3 protein could synthesize TMP from HMP-PP and HET-P substrates. This TMP synthase activity required the presence of magnesium ions in the reaction mixture. For this reaction, HMP-PP could not be substituted by HMP or HMP-P, nor HET-P by HET. The TMP synthase activity was inhibited by ATP, but in the presence of ATP TMP could be formed from HET-P plus HMP or HMP-P, clearly indicating that the enzyme possessed additional HMP kinase and HMP-P kinase activities. At the equivalent ATP concentration, the rate of TMP formation from HMP/HMP-P plus HET -P substrates, i.e. due to the sequential action of HMP/HMP-P kinase and TMP synthase, was ca.

30% of that from HMP-PP plus HET-P. On the other hand, no HET kinase activity could be assigned to THI3 protein as TMP was not formed from unphosphorylated thiazole in combination with any pyrimidine in the presence of ATP.

The conventional kinetic characteristics of maize TMP synthase is illustrated in Figure 2. A dependence of initial reaction rate on HET-P concentration was hyperbolic but the dependence on HMP-PP concentration showed a pattern indicating for a strong inhibition by excess substrate (Figure 2A). The rate was maximal at 15-20 µM HMP-PP and then gradually dropped. The inhibition of TMP synthase by ATP was analyzed against varying HET-P concentration at a constant HMP-PP concentration. An example analysis using Hanes-Woolf plots is shown in Figure 2B. These plots, prepared for various ATP concentrations, had a common intercept with the rate axis, suggesting that the inhibition is "uncompetitive" [START_REF] Bisswanger | Enzyme kinetics. Principles and methods[END_REF].

The kinetic characteristics of HMP kinase activity was obtained through direct determinations of HMP phosphorylation. The dependences on HMP or ATP concentration at the fixed concentration of the second substrate were hyperbolic (Figure 3). The values of estimated kinetic constants of maize HMP kinase and TMP synthase are collected in Table 3.

Site-directed mutagenesis of THI3 protein at the TMP synthase and HMP/HMP-P kinase active centres

The active sites of THI3 protein were probed by substitution of several amino acid residues which were selected based on the literature data on bacterial TMP synthases and HMP kinases, as well as on the model of THI3 structure (see Discussion). The rates of TMP synthase and HMP kinase reactions in the extracts from cells transformed with mutant plasmids are presented in Table 4. Substitutions of Leu for Gln-98 and Lys for Met-134 were detrimental to HMP kinase activity while TMP synthase activity was fully preserved. A substitution of Ala for Ser-444 represents an opposite situation of a complete elimination of TMP synthase activity without any significant effect on the second activity. Two further single mutants, Q373L and T472D, exhibited full TMP synthase activity and moderately reduced (by 30% and 50%, respectively) HMP kinase activity. A double mutant Q98L+Q373L differed from the Q98L mutant only in a detectable but still small (ca. 10%) HMP kinase activity. In contrast, another mutant with double amino acid substitution both in the TMP synthase and HMP/HMP-P kinase active centres, M134K+T472D, showed anomalous behaviour, presenting too high HMP kinase activity, ca. 25% of that of the wildtype protein.

DISCUSSION

The Z. mays gene which was identified in this work through cloning of its cDNA and characterization of its product in the form of pure, heterologously expressed recombinant protein is hereby designated thi3 as the third discovered gene of this species involved in thiamine biosynthesis. Searching through available protein databases revealed several orthologues of Z. mays THI3 in four higher plant species, including Oryza sativa, Medicago truncatula, Arabidodpis thaliana and Brassica napus. A multiple sequence alignment of plant THI3 orthologues (one from each species) is shown in Figure 4. Amino acid identity with Z. mays THI3 varies from 77% for rice ABA96049 protein to 63% for Medicago ABE87727 protein. In the Arabidopsis genome, three homologous sequences, AAM 91567 (Figure 4), BT020417 and AF000657 were identified. The first two differ by only one amino acid substitution; the third shows one short insertion and one deletion and may be the product of alternative splicing of the same gene. This gene exists in a single copy and maps to the locus AGT1G22950 on chromosome 1. Similarly, four homologous sequences, ABA96049 (Figure 4), EAZ19897, BAF29362 and ABG21903, were found in the rice genome. The first two probably originate by alternative splicing from one gene at the locus Os12g0192500 on chromosome 12, and two others are their truncated versions. Like all other members of this plant protein family, THI3 contains a putative Nterminal chloroplast transit peptide [START_REF] Wallace | Plant organellar targeting sequences In Plant Molecular Biology Labfax[END_REF]. All THI3 orthologues were described as having two putative conserved domains: an N-terminal domain with high sequence similarity to many bacterial HMP/HMP-P kinases, and a C-terminal domain homologous to bacterial TMP synthases. As analyzed with the use of NCBI CDD Search Server, THI3 shows an analogous two-domain structure, with the N-terminal kinase domain comprising residues 59-302 and the C-terminal synthase domain including residues 335-546.

In spite of the presence of putative HMP/HMP-P kinase and TMP synthase-encoding domains in cDNAs of several plant species, their protein products have not yet been obtained in isolated, pure form for which the suggested enzymatic activities could be directly demonstrated. However, the B. napus bthi1 cDNA was isolated based on its ability to complement an E. coli mutant deficient in TMP synthase and was additionally shown to complement some HMP kinase-deficient mutants [START_REF] Kim | A Brassica cDNA clone encoding a bifunctional hydroxymethylpyrimidine kinase/thiamin-phosphate pyrophosphorylase involved in thiamin biosynthesis[END_REF]. Our maize THI3 protein, obtained by heterologous expression in E. coli, is the first pure plant bifunctional enzyme with confirmed HMP/HMP-P kinase and TMP synthase activity.

The recombinant THI3 was homogeneous as determined by SDS-PAGE and apparently existed in solution as a dimer, similarly to bacterial HMP kinase and TMP synthase which form dimers in crystals [START_REF] Cheng | Crystal structure of 4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate kinase from Salmonella typhimurium at 2.3 Å resolution[END_REF][START_REF] Chiu | Crystal structure of thiamin phosphate synthase from Bacillus subtilis at 1.25 Å resolution[END_REF]. The enzymatic properties of recombinant THI3 were readily detectable, although the determined specific activities (Table 3) were much lower than those reported for corresponding bacterial enzymes. For example, the specific activity of THI3 HMP kinase for HMP as the substrate (20 nmol•min -1 •mg -1 ) was one-eighth of the lowest value reported for a bacterial HMP kinase, ThiD from E. coli [START_REF] Reddick | Overexpression, purification and characterization of two pyrimidine kinases involved in the biosynthesis of thiamin: 4-amino-5hydroxymethyl-2-methylpyrimidine kinase and 4-amino-5-hydroxymethyl-2methylpyrimidine phosphate kinase[END_REF]. Similarly, the specific activities of a bacterial TMP synthase, ranging from 65 nmol•min -1 •mg -1 for the enzyme isolated from E. coli [START_REF] Kawasaki | Thiamine phosphate pyrophosphorylase[END_REF] to 700 nmol•min -1 •mg -1 for ThiC from B. subtilis [START_REF] Zhang | Characterization of the Bacillus subtilis thisC operon involved in thiamine biosynthesis[END_REF], exceed at least eight times the value for the plant bifunctional enzyme (8.5 nmol•min -1 •mg -1 ).

Whether these comparisons indicate for an intrinsically lower turnover of the bifunctional plant enzyme or for some conformational imperfections of its recombinant form, remains to be established. The kinetic characterization of maize THI3 protein revealed two interesting properties of the TMP synthase activity of this enzyme: the substrate inhibition by excess of HMP-PP and uncompetitive inhibition by ATP. The uncompetitive inhibition by ATP, ADP and inorganic diphosphate was also reported for E. coli TMP synthase [START_REF] Kawasaki | Thiamine phosphate pyrophosphorylase[END_REF].

In an attempt to identify specific amino acid residues which form the active centres on THI3 molecule we took advantage of a significant sequence similarity of THI3 with S. typhimurium HMP/HMP-P kinase (Code P55882) and B. subtilis TMP synthase (P39594) whose crystal structures have been reported [START_REF] Cheng | Crystal structure of 4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate kinase from Salmonella typhimurium at 2.3 Å resolution[END_REF][START_REF] Chiu | Crystal structure of thiamin phosphate synthase from Bacillus subtilis at 1.25 Å resolution[END_REF]. As presented in Table 5, most of aminoacid residues that occur in the active sites of bacterial enzymes are conserved in the maize enzyme except that Thr-96 and Gln-98 replaced Val and Glu, respectively, in HMP/HMP-P kinase domain and Val-523 replaced Ile in TMP synthase domain. The sequence identity of S. typhimurium HMP/HMP-P kinase and B. subtilis TMP synthase with the maize THI3 protein, ca. 39% as estimated by BLAST, was sufficient to use the three-dimensional structures of those bacterial enzymes as templates for modelling the THI3 structure. For this purpose, we used the SWISS-MODEL automatic homology-based modelling server of the Swiss Institute of Bioinformatics. The predicted overall structures of THI3 domains were very similar to those of the reference enzymes (Figure 5). Thus, the model of HMP kinase domain revealed a ribokinase-like fold with a core structure composed of a central eight strand β-sheet covered by two layers of five and three α-helices [START_REF] Cheng | Crystal structure of 4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate kinase from Salmonella typhimurium at 2.3 Å resolution[END_REF]. The modelled TMP synthase domain had the triosephosphate isomerase fold, containing a core barrel composed of eight α-helices and eight parallel β-strands [START_REF] Chiu | Crystal structure of thiamin phosphate synthase from Bacillus subtilis at 1.25 Å resolution[END_REF][START_REF] Peapus | Structural characterization of the enzyme-substrate, enzyme-intermediate, and enzyme-product complexes of thiamin phosphate synthase[END_REF]. More importantly, the predicted arrangements of active-site residues within the three-dimensional protein structure basically match those in the reference bacterial proteins. In the HMP kinase domain of THI3 protein (Figure 5A), eight residues: Gly-65, Ala-72, Asp-77, Thr-96, Gln-98, Met-134, Val-161 and Cys-268, may be involved in HMP binding. Gln-98 probably forms a direct hydrogen bond to 4-amino group of HMP and the other residues may interact with the pyrimidine ring indirectly, through fixed water molecules, or by making some hydrophobic contacts [START_REF] Cheng | Crystal structure of 4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate kinase from Salmonella typhimurium at 2.3 Å resolution[END_REF]. Three residues, Lys-230, Thr-266 and Lys-292 may contribute to a putative ATP-biding site. At least three residues in TMP synthase domain of THI3 (Figure 5B), Thr-470, Thr-472 and Lys-473, are shifted from the locations which the corresponding residues of B. subtilis protein occupy to make direct hydrogen bonds with thiazole phosphate group (Thr-156 and Thr-158) and van der Waals contacts with thiazole ethyl chain as well as electrostatic interactions with HMP-PP diphosphate group (Lys-159) [START_REF] Peapus | Structural characterization of the enzyme-substrate, enzyme-intermediate, and enzyme-product complexes of thiamin phosphate synthase[END_REF]. A new location of Lys-473 may provide favourable interactions with thiazole phosphate group. Positions of other active centre residues of plant and bacterial proteins nearly match. Residues Ser-524, Gly-500 and Val-523 may constitute the phosphate-binding site, Arg-375, Lys-377, Asn-405, Asp-406, Asp-425 and Ser-444 may fix the diphosphate group and Gln-373 may interact with pyrimidine moiety, possibly via two hydrogen bonds to the ring N-3 and the 4-amino group [START_REF] Peapus | Structural characterization of the enzyme-substrate, enzyme-intermediate, and enzyme-product complexes of thiamin phosphate synthase[END_REF].

We performed a preliminary experimental verification of the above models of the active centres of Z. mays HMP kinase/TMP synthase by mutations at selected residues. We exchanged two residues, Gln-98 and Met-134, at the HMP kinase site and three residues, Gln-373, Ser-444 and Thr-472 at the TMP synthase site. The enzymatic properties of Q98L, M134K and S444A mutants exactly followed the above structural predictions. Two first exclusively express full TMP synthase activity, and the last has only the HMP kinase activity.

The last type mutation (S130A) had been extensively exploited during the structural studies of the B. subtilis TMP synthase to prove the essential role of Ser-130 residue in the catalytic mechanism [START_REF] Chiu | Crystal structure of thiamin phosphate synthase from Bacillus subtilis at 1.25 Å resolution[END_REF][START_REF] Peapus | Structural characterization of the enzyme-substrate, enzyme-intermediate, and enzyme-product complexes of thiamin phosphate synthase[END_REF]. A full TMP synthase activity of T472D mutant is consistent with the predicted escape of Thr-472 from the bound ligand. A lack of effect of Q373L mutation is more difficult to explain, although the model of TMP synthase active site shows a possible rotation of amide group which may prevent the formation of hydrogen bonds to aminopyrimidine moiety. Unfortunately, there is no data available on the effects of similar mutations in the bacterial enzyme. Two double-mutants of THI3 protein, Q98L+Q373D and M134K+T472D, were additionally studied, with a single residue substituted at each active centre. As expected, both exhibited full TMP synthase activity and decreased HMP kinase activities, although the HMP kinase of M134K+T472D mutant exceeds the predictions from the single mutant data. A systematic study of the mutations of all residues which were Biochemical Journal Immediate Publication. Published on 14 Aug 2007 as manuscript BJ20070677 preliminarily classified in this work as occurring at active centres of THI3 protein is currently carried out in our laboratory on the level of purified mutant proteins.

Several features of recombinant bifunctional enzyme of Zea mays described in this paper implicate putative biological functions of the natural product of thi3 gene. In vivo, the full-length THI3 polypeptide would be targeted to chloroplasts, within which the entire thiamine biosynthesis pathway is known to occur [START_REF] Belanger | Evidence for the thiamine biosynthetic pathway in higher-plant plastids and their developmental regulation[END_REF]. The matured two-domain enzyme with HMP/HMP-P and TMP synthase activities could catalyze two consecutive steps of thiamine biosynthesis that lead from HMP-P to TMP. An additional ability of THI3 to phosphorylate HMP in the presence of ATP does not appear to be important in vivo as no mechanisms by which HMP could enter chloroplasts have ever been discussed. This reaction is well known in various microorganisms as a salvage pathway. Bacterial TMP synthases were often suspected to play a regulatory function, primarily because of their susceptibility to inhibition by ATP, ADP and inorganic diphosphate [START_REF] Kawasaki | Thiamine phosphate pyrophosphorylase[END_REF]. The unique fusion of HMP-P kinase and TMP synthase activities into one molecule of higher plant protein such as THI3 may result in a more selective response to ATP as the TMP synthesis is quenched by HMP-PP (which is formed in the presence of ATP on the same enzyme and is immediately consumed for TMP formation).

It may be that ATP levels regulate the yield of entire thiamine synthesis in higher plants through the inhibitory action on the bifunctional enzyme HMP/HMP-P kinase / TMP synthase. The THI3 orthologues occur in several higher plant species, strongly suggesting a specific role of the combination of two activities into one protein for the thiamine biosynthesis in higher plants.

TABLES

  For a direct HMP kinase assay, the reaction mixtures contained 20 µM HMP, 1 mM ATP, 10 mM MgCl 2 , 50 mM Tris-HCl buffer pH 8.0 and 5 µg enzyme in the final volume of 100 µl. After 60 min-incubation at 37ºC, the reaction was stopped and the sample, diluted 3 times with 0.1 M potassium phosphate, was analyzed for HMP-P content by RP-HPLC on a Supelcosil LC-18 column (250 mm x 4.6 mm), eluted with a ternary solvent system composed Biochemical Journal Immediate Publication. Published on 14 Aug 2007 as manuscript BJ20070677 ' 2007 The Authors Journal compilation ' 2007 Biochemical Society of 4 mM tetrabutylammonium hydrogensulphate (TBA) in water (solvent A), 4 mM TBA in methanol (solvent B) and 4 mM TBA in 50 mM potassium phosphate buffer pH 6.6 (solvent

Biochemical

  Journal Immediate Publication. Published on 14 Aug 2007 as manuscript BJ20070677 ' 2007 The Authors Journal compilation ' 2007 Biochemical Society 10

  All 3'RACE sequences were identical. From clones received after 5'RACE we choose one with the longest region upstream of the putative ATG start codon. This thi3 cDNA clone (Accession No. AM16973) had a total length of 1947 bp and contained a 48 bp untranslated 5'region, a 1653 bp-long open reading frame and a 246 bp untranslated 3' region with the stop codon and a polyA tail. A putative open reading frame-encoded protein (THI3, Accession No. CAJ45026) consisted of 551 amino acid residues and its predicted molecular mass and isoelectric point were 57.8 kDa and 8.51, respectively.

Biochemical

  Journal Immediate Publication. Published on 14 Aug 2007 as manuscript BJ20070677 ' 2007 The Authors Journal compilation ' 2007 Biochemical Society

Figure 4

 4 Figure 4 Multiple alignment of the amino acid sequences of THI3 and its orthologues occurring in various plant speciesThe accession codes of the sequences compared were emb/CAJ45026 (Zea mays, this work), gb/AAM91567 (Arabidopsis thaliana), gb/AAC31298 (Brassica napus, BTH1), gb/ABE87727 (Medicago truncatula) and gb/ABA96049 (Oryza sativa). The alignment was performed using the CLUSTALW program and the result visualized in JalView. Black, dark grey and light grey highlights mark the positions in the primary structures where all 5, 4 of 5 and 3 of 5 residues are identical, respectively. The box at the N-terminus of Zea mays THI3 protein includes the putative 36-amino acid residue chloroplast transit peptide predicted by ChloroP program[START_REF] Lee | Determination of thiamine and its phosphate esters by gradient-elution high-performance liquid chromatography[END_REF].

Figure 5 33 ]

 533 Figure 5 The models of overall structures and arrangements of putative active centre residues of THI3 HMP/HMP-P kinase (A) and TMP synthase (B) domains, overlaid on the corresponding features of three-dimensional structures of S. typhimurium HMP/HMP-P kinase [32] and B. subtilis TMP synthase [33] The models of THI3 domains were obtained with the SWISS-MODEL server and all structures were visualized using the Swiss-PdbViewer and PyMOL programs. The ribbon diagrams of the overall architectures of THI3 domains (in violet) and the corresponding bacterial enzymes (in orange) are shown in background. Stick representations of the active site residues in the bacterial proteins and corresponding putative active site residues of THI3 domains are marked with similar colours, lighter for THI3. Additionally, the THI3 residues are indicated by solid arrows and labelled with normal font, and the bacterial protein residues with dashed arrows and italic font. Dark grey stick models of substrates, HMP in A, and HMP-PP and HET-P in B, are shown in the positions they occupy in the crystal structures of bacterial enzymes.

Biochemical Journal Immediate Publication. Published on 14 Aug 2007 as manuscript BJ20070677

Biochemical Journal Immediate Publication. Published on 14 Aug 2007 as manuscript BJ20070677

Biochemical Journal Immediate Publication. Published on 14 Aug 2007 as manuscript BJ20070677

Table 1

 1 contains primers used in site-directed mutagenesis. All seven mutagenesis constructs were sequenced.

Biochemical Journal Immediate Publication. Published on 14 Aug 2007 as manuscript BJ20070677 ' 2007 The Authors Journal compilation ' 2007 Biochemical Society

Biochemical Journal Immediate Publication. Published on 14 Aug 2007 as manuscript BJ20070677 ' 2007 The Authors Journal compilation ' 2007 Biochemical Society

Biochemical Journal Immediate Publication. Published on 14 Aug 2007 as manuscript BJ20070677 ' 2007 The Authors Journal compilation ' 2007 Biochemical Society

Biochemical Journal Immediate Publication. Published on 14 Aug 2007 as manuscript BJ20070677 ' 2007 The Authors Journal compilation ' 2007 Biochemical Society

Table 1 Primers used in this study

 1

	__________________________________________________________________________________________
	Name of	Sequence	Description
	primer
	__________________________________________________________________________________________
	MO1-F	TCGTGAAGACTGGGATGCT(TC)CC	Degenerate, forward
			primer
	MO2-R	GTAGTC(CT)GCACCATCTTTCCA	Degenerate, reverse
			primer
	ZTH51	GCTTCTTTCACATTTGGAGTGAC	5'RACE RT primer
	ZTH52	GTAGCGAGAGTAGATGGTCC	First 5'RACE primer
	ZTH53	GCTGGTGGACACCATGACC	"Nested" 5'RACE
			primer
	ZTH31	GCGTCCATGTTGGCCAATC	First 3'RACE primer
	ZTH32	CATACCAGCATGGGAAGTAC	"Nested" 3'RACE
			primer
	Adaptor	GACTCGAGTCGACATCG	Adaptor 3' and 5'RACE
			primer
	ZTH1-F	GGTGGTCATATGACGTCCGTACCGCTAC	Forward primer for PCR
			of thi3 cloned into
			pTYB1 vector
	ZTH1-R	GGTGGTTGCTCTTCCGCAAGACCTTGAGTGAGCATTCTTC Reverse primer for PCR
			of thi3 cloned into
			pTYB1 vector
	ZTH11-F	GGTGGTTGCTCTTCCAACGCCACCCTTGGCTTCCGCA	Forward primer for PCR
			of thi3 cloned into
			pTYB11 vector
	ZTH11-R	GGTGGTCTCGAGCTAAGACCTTGAGTGAGCATTC	Reverse primer for PCR
			of thi3 cloned into
			pTYB11 vector
	MutQ98L	GCTGTCACCGCGCTGAACACCGTCGG	Mutagenetic primer,
			replaces Gln-98 to
			Leu-98
	MutQ373L	GGCGCTACCATTGTCCTACTGAGAGAAAAAGACGCC	Mutagenetic primer,
			replaces Gln-373 to
			Leu-373
	MutM134K	GGTGAAAACAGGGAAGCTCCCTTCAGCTGG	Mutagenetic primer,
			replaces Met-134 to
			Lys-134
	MutT472D	GTGTCTTCCCGACCACGGACAAGGCAAACAATCCTACC	Mutagenetic primer,
			replaces Thr-472 to
			Asp-472
	MutS444A	GGAAAAATCATCGGCGTCGCATGTAAGACCCTGG	Mutagenetic primer,
			replaces Ser-444 to
			Ala-444
	__________________________________________________________________________________________

Biochemical Journal Immediate Publication. Published on 14 Aug 2007 as manuscript BJ20070677 ' 2007 The Authors Journal compilation ' 2007 Biochemical Society

Table 2 Enzymatic activity of maize THI3 protein: the rates of TMP formation from various combination of substrates and cofactors

 2 Concentrations in the reaction mixture were: 15 µM HMP, HMP-P, HMP-PP, HET-P or HET, 10 mM ATP and 10 mM MgCl 2 . Samples containing 5 µg protein/100 µl in 50 mM Tris-HCl buffer pH 8.0 were incubated for 60 min at 37 o C and then analyzed for TMP content by RP-

	HPLC with fluorogenic postcolumn derivatization. Values are means ± standard deviations
	from at least 3 independent kinetic experiments.
	___________________________________________________________________________
	Activity tested	Substrates and cofactors	Specific activity
			(nmol TMP/min•mg protein)
	___________________________________________________________________________
	TMP synthase	HMP-PP, HET-P	0.52 ± 0.05
		HMP-PP, HET-P, Mg 2+	2.46 ± 0.12
		HMP-PP, HET-P, ATP	0.05 ± 0.02
		HMP-PP, HET-P, Mg 2+ , ATP	0.83 ± 0.11
	HMP kinase /TMP synthase	HMP, HET-P, ATP, Mg 2+	0.22 ± 0.03
	HMP-P kinase /TMP synthase HMP-P, HET-P, ATP, Mg 2+	0.24 ± 0.02
	HET kinase /TMP synthase	HMP-PP, HET, ATP, Mg 2+	0

___________________________________________________________________________ Biochemical Journal Immediate Publication. Published on 14 Aug 2007 as manuscript BJ20070677 ' 2007 The Authors Journal compilation ' 2007 Biochemical Society

Table 3 Kinetic constants of TMP synthase and HMP kinase activities of THI3 protein

 3 The apparent kinetic parameters were determined at the second substrate concentration of 15 µM (HMP-PP), 1.5 µM (HET-P), 0.5 mM (HMP) or 5 mM (ATP). K m -Michaelis constant;

	K iu	HMP-PP -substrate (HMP-PP) uncompetitive inhibition constants for TMP synthase, K iu	ATP
	-the constant for uncompetitive inhibition of TMP-synthase by ATP (determined against
	variable concentration of HET-P as the substrate), V max -maximal velocity recalculated per 1
	mg protein. The applied kinetic equations with these parameters are specified under Methods.
	Values are means ± standard deviations; the number of independent kinetic experiments are
	given in parentheses.
	___________________________________________________________________________
	Activity	Parameter	Parameter value
	___________________________________________________________________________
	TMP synthase	K m HMP-PP (µM)	12.8 ± 6.9 (4)
			K iu	HMP-PP (µM)	14.8 ± 6.3 (4)
			V max HMP-PP (nmol TP/min•mg protein)	8.5 ± 3.9 (4)
			K m HET-P (µM)	1.79 ± 0.63 (8)
			V max HET-P (nmol TP/min•mg protein)	2.66 ± 0.22 (8)
			K iu	ATP (mM)	3.4 ± 0.9 (3)
	HMP kinase	K m HMP (µM)	6.58 ± 1.55 (4)
			V max HMP (nmol HMP-P/min•mg protein)	20.1 ± 1.90 (4)
			K m ATP (µM)	7.24 ± 1.80 (7)
			V max ATP (nmol HMP-P/min•mg protein)	17.7 ± 0.34 (7)
	___________________________________________________________________________

Biochemical Journal Immediate Publication. Published on 14 Aug 2007 as manuscript BJ20070677 ' 2007 The Authors Journal compilation ' 2007 Biochemical Society

Table 4 Enzymatic activity of THI3 mutants

 4 Activity was determined in the lysates from E. coli cells transformed with plasmids containing THI3 sequence with indicated amino acid substitutions. The expression levels of all mutants were similar to that of wild-type protein as judged from immunochemical analysis using CBD-specific antibodies (results not shown). The activity were expressed relatively to that determined in the wild-type protein samples. Values given are ranges from at least three independent determinations.

	___________________________________________________________________________
	Mutant	Relative activity (%)
		TMP synthase	HMP kinase
	___________________________________________________________________________
	Q98L	104-124	0-2
	M134K	97-111	0-5
	Q373L	96-113	64-75
	S444A	0-1	80-91
	T472D	101-126	46-54
	Q98L+Q373L	93-105	7-13
	M134K+T472D	83-97	19-32
	___________________________________________________________________________

Biochemical Journal Immediate Publication. Published on 14 Aug 2007 as manuscript BJ20070677 ' 2007 The Authors Journal compilation ' 2007 Biochemical Society detection

  . Ordinary hyperbolic fits are shown with K m and V max values of 7.46 µM and 20.5 nmol HMP-P per min per mg protein, respectively, for HMP concentration dependence, and of 6.27 µM and 17.4 nmol HMP-P per min per mg protein, respectively, for the dependence on ATP concentration.

' 2007 The Authors Journal compilation ' 2007 Biochemical Society

Biochemical Journal Immediate Publication. Published on 14 Aug 2007 as manuscript BJ20070677 ' 2007 The Authors Journal compilation ' 2007 Biochemical Society

Acknowledgements

This work was partly supported by the grant No. 2P04C 017 27 from the Ministry of Science and Higher Education, Poland (to M.R.-K.).

Table 5 Active centre residues of S. typhimurium HMP kinase, B. subtilis TMP synthase and the corresponding domains of Z. mays THI3 bifunctional enzyme

For bacterial enzymes, the residues found in the closest neighbourhood of the bound ligands in the crystal structures [START_REF] Cheng | Crystal structure of 4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate kinase from Salmonella typhimurium at 2.3 Å resolution[END_REF][START_REF] Chiu | Crystal structure of thiamin phosphate synthase from Bacillus subtilis at 1.25 Å resolution[END_REF][START_REF] Peapus | Structural characterization of the enzyme-substrate, enzyme-intermediate, and enzyme-product complexes of thiamin phosphate synthase[END_REF] are listed, except the residues of putative ATP-binding site of S. typhimurium HMP kinase, modelled through the homology to other members of ribokinase family [START_REF] Cheng | Crystal structure of 4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate kinase from Salmonella typhimurium at 2.3 Å resolution[END_REF]. The corresponding putative active centre residues of Zea mays THI3 domains were identified by sequence alignment with CLUSTAL W program and structural modelling with the use of the SWISS-MODEL server.

LEGENDS TO SCHEMES AND FIGURES Scheme 1 Proposed scheme for thiamine and TDP biosynthesis in Eukaryota

The biosynthesis pathways may differ in some details between yeasts and higher plants, as exemplified by different substrates (underlined) used by plants for the synthesis of thiazole moiety (HET-P). The sequenced genes and purified proteins, unequivocally assigned to thiamine biosynthesis in baker's yeast and higher plants (underlined) as specified. The symbols of proteins whose three-dimensional structures were reported are bolded. A -Arabidopsis thaliana, Z -maize, b -Brassica napus, ag -Alnus glutinosa.

Figure 1 SDS-PAGE patterns from a representative purification of maize THI3 protein overproduced in E. coli

The electrophoresis was carried out in the Laemmli system [START_REF] Laemmli | Cleavage of structural proteins during assembly of the head of bacteriophage T4[END_REF] with 10% resolution gel. plots of the dependences of TMP-formation rates on HET-P concentration in the presence of ATP. In A, the line drawn through data points for the HMP-PP concentration dependence was generated with K iu =24 µM found from Dixon plot, and K m =6.35 µM and V max =5.05 nmol TMP per min per mg protein, estimated by fitting the equation for the uncompetitive inhibition by excess substrate. For the HET-P concentration dependence, the classic hyperbola with K m =1.74 µM and V max =2.52 nmol TMP per min per mg protein, was fitted. In B, from the Hanes-Woolf plot obtained in the absence of ATP, the K m and V max values for HET-P were estimated as 2.55 µM and 2.88 nmol TMP per min per mg protein, respectively, and the K iu for ATP (4.3 mM in this example) was obtained from the abscissa intercept of the secondary plot of the slope versus the ATP concentration.


Isabel Cardoso

Maria Rosário Almeida

Nelson Ferreira

Gemma Arsequell

Gregorio Valencia

Maria João Saraiva
email: mjsaraiv@ibmc.up.pt

Ferreira

Comparative in vitro and ex vivo activities of selected inhibitors of Transthyretin aggregation -relevance in drug design

Keywords: Transthyretin, amyloid, iododiflunisal, aggregation, anti-amyloidogenic drugs

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

Introduction

Familial amyloidotic polyneuropathy (FAP) is an autosomal dominant disorder characterized by the extracellular deposition of Transthyretin (TTR) amyloid fibrils. This systemic disease has a special involvement of the peripheral nerve. Over 80 TTR mutations have been related to amyloid deposition and disease [START_REF] Saraiva | Familial amyloidotic polyneuropathy: protein aggregation in the peripheral nervous system[END_REF], being the most frequent a substitution of a methionine for valine at position 30 -V30M. TTR is a homotetrameric protein produced in the liver and in the choroid plexus of the brain and functions as a transporter of T 4 and vitamin A.

The knowledge acquired in the last years on structural and biochemical features characteristic of amyloidogenic intermediates, has paved the way for the investigation of drugs capable of interfering early on the amyloidogenic pathway. For TTR related amyloidosis, the first step of a rational intervention is to obstruct tetramer dissociation and thus, prevent potential aggregation prone species from being generated. A second target is to inhibit the association between conformational intermediate species that lead to fibril elongation. Following these principles, several authors have reported the identification of compounds capable of inhibiting TTR fibril formation [2][START_REF] Oza | Synthesis and evaluation of anthranilic acid-based transthyretin amyloid fibril inhibitors[END_REF][START_REF] Baures | Discovering transthyretin amyloid fibril inhibitors by limited screening[END_REF][5][START_REF] Razavi | Benzoxazoles as transthyretin amyloid fibril inhibitors: synthesis, evaluation, and mechanism of action[END_REF]. However, most of these fibril inhibition studies were performed under non-physiological acidic conditions believed to induce rapid conformational changes and no specific distinction was made regarding the molecular species affected by those drugs. Furthermore, these in vitro tests were performed in the test tube using prokaryotic rather than eukaryotic systems for the synthesis of human TTR. Cellular systems represent a more physiological in vitro approach to study inhibitors of amyloidogenesis which avoids isolation of the target protein. Here, we describe, for the first time, a TTR based cellular system for the screening of amyloid inhibitors. Using this system we also report comparative results of activity of a series of drugs referred to in the literature as inhibitors of TTR fibril formation.

Materials and methods

Vector constructions

Vectors p169ZT carrying human wt TTR (p169ZT-hTTRwt) and p169ZT carrying human TTR L55P (p169ZT-hTTR55) were generated as previously described [START_REF] Sousa | Evidence for early cytotoxic aggregates in transgenic mice for human transthyretin[END_REF]. We further produced a construct carrying the V30M TTR mutation by site directed mutagenesis using the Quickchange mutagenesis kit (Stratagene) following the manufacturer's instructions and the appropriate primers: 5'-AATGTGGCCATGCATGTGTTC-3' and 5'-GAACACATGCATGGCCACATT-3'.

Transfection and TTR expression in cell culture

Rat Schwanomas (RN22) (American Type Cell Collection) were stably co-transfected with each of the TTR cDNA constructs (WT, V30M or L55P) plus a plasmid with resistance to neomycin (pLNeo, kindly provided by Dr. Paulo Vieira, Pasteur Institute) following the CaPO 4 -DNA precipitate method [START_REF] Sambrook | Molecular cloning: a laboratorial manual[END_REF]. Briefly, cells were grown to approximately 30-50% confluence in 10 cm dishes (Costar). 25 µg of the appropriate purified cDNA TTR plasmid plus 5 µg of purified pLNeo plasmid were resuspended in 500 µl of 250 mM CaCl 2 and diluted with 500 µl 2X HEPES-buffered saline (2X HEBS). The CaPO 4 -DNA precipitate was allowed to form by standing the mixture 20 min at room temperature and was subsequently added to the cells. After 5 hours incubation, cells were washed twice with phosphate-buffered saline (PBS) and fed with 10 mL of DMEM (Gibco BRL) supplemented with 10% FCS and 1% pen/strep (complete medium). 24 hours later, transfection medium was replaced by complete medium supplemented with G418 (1 mg/mL). Resistant colonies arose approximately after 7 days of selection and were isolated and separated over the course of the following weeks.

TTR secretion was tested by quantitative enzyme-linked immunosorbent assay (ELISA) [START_REF] Sousa | Transthyretin in high density lipoproteins: association with apolipoprotein A-I[END_REF]: 96 well plates (Maxisorp-Nunc) were coated overnight at 4ºC with polyclonal rabbit antihuman TTR (1:500 dilution, DAKO) and blocked with 5% skim milk in PBS; conditioned media was applied to the wells for 1 h at room temperature; as secondary antibody peroxidase conjugated anti-human TTR (The Binding Site; 1:500 dilution in PBS-0.05% Tween 20 (PBS-T)) was used. Development was performed with ABTS (Sigma) / H 2 O 2 (Merck) . TTR concentration in cell media was calculated from a standard curve ranging from 5 to 340 ng/mL.

Since the vector used is under the control of the metallothionein promoter, experiments were performed in presence of 100 µM ZnSO 4 in the cell culture media in order to induce TTR synthesis.

Immunoprecipitation and Mass spectrometry analysis

Zinc treated cells expressing TTR were grown to approximately 100% confluence in complete medium supplemented with G418 which was then replaced by 14 C-Leucine containing medium, and collected 4 hours later. Cells were harvested and then lysed with 1 ml of lysis solution (30 mM Tris pH 7.5, 300 mM NaCl, 1% Triton x-100, 10 µM PMSF), centrifuged 30 minutes at 16,000 g, the pellet discarded and the supernatant collected.

Both medium and cell supernatant were incubated with rabbit anti-human TTR (1:20 times with washing solution (30 mM Tris pH7.5, 300 mM NaCl, 1% Triton x-100) and the complex eluted with SDS loading buffer (62.5 mM Tris, pH 6.8; 2% SDS; 10% glycerol; 5% 2-mercaptoethanol). Finally, samples were fractioned on a 15% polyacrylamide gel under denaturing conditions (SDS-PAGE) and the gel was processed for autoradiography.

In some experiments, cells were grown in medium supplemented with 1% serum and G418, for 24 hours; the medium was collected and processed for immunoprecipitation as above.

After separation in a SDS-PAGE gel, proteins were stained following a silver stain protocol.

The The compounds were weighed and dissolved at approximately 10 mg/ml in DMSO and used at a final concentration of 1 µM.

Recombinant protein

Recombinant TTR Y78F were produced in a bacterial expression system using Escherichia coli BL21 [11] and purified as previously described [12]. Protein concentration was determined using the Lowry method [13].

Western blotting

In some experiments, after application of the medium on the membrane filter, each dot was cut and boiled in SDS loading buffer. The removed material was run on a 15% SDS denaturing acrylamide gel and then transferred to a nitrocellulose membrane (Amersham).

The membrane was blocked with 5% skim milk in PBS for 1 hour at room temperature and then incubated with 1:500 dilution of rabbit anti-human TTR anti-serum (DAKO) for another hour at room temperature; as secondary antibody (1:5000 dilution) peroxidaseconjugated anti-rabbit IgG (Amersham) was used. Development was performed with 3', 3'diaminobenzidine (DAB) (Sigma).

Transmission Electron Microscopy

Inhibition of fibrillogenesis by selected drugs was evaluated using TTR Y78F which aggregates under physiological conditions (PBS, pH 7.4) after 2 days at 37ºC. For negative staining, 5 µl of these preparations were adsorbed to glow-discharged carbon collodion film on 200-square mesh copper grids for 1 min. The grids were washed with deionized water and stained with 1% uranyl acetate (Fluka). Specimens were visualized with a Zeiss electron microscope operating at 60 kV and grids were exhaustively examined.

Assessment of tetrameric TTR stability by isoelectric focusing (IEF) in semi-denaturing conditions

The conditions used for the IEF of plasma TTR were described previously by Altland et al 1981 and Almeida et al 2004 [14, 15]. Briefly, 30 µl of plasma from a TTR V30M heterozygotic carrier were incubated at 37ºC for 1 h, with 5 µL of a 10 mM solution of the compound to be tested. The preparations were then subjected to native polyacrylamide gel electrophoresis to isolate TTR. The gel band containing TTR was excised and subjected to IEF in a gel with 4 M urea and 5% ampholytes pH 4-6.5, at 1200 V for 6 hours. Proteins in the gel were stained with Coomassie blue. These semi-denaturing conditions allow the visualization of bands corresponding to the TTR monomer and tetramer and also to an oxidized form of the monomer. Gels were scanned (hp Scanjet 4470c; Hewlett Packard) and subjected to densitometry using the ImageQuant program. Experiments were repeated at least three times and data shown is representative of the obtained results.

Statistical analyses

All data examined were expressed as mean ±S.E. Comparison between groups was made using the Student's t test. A p value of less than 0.05 was considered statistically significant.

Results

TTR Expression

To set up a cellular system for the production of TTR aggregates, cells were transfected with different human TTR constructs (p169ZT-hTTRWT, p169ZT-hTTRV30M, and p169ZT-hTTRL55P). Demonstration of TTR secretion and quantification of TTR levels in conditioned media was performed by quantitative ELISA after 24 hours in serum-free conditions. Cells were first treated with 100 µM ZnSO 4 for 3 days to produce TTR. The media was then replaced by serum-free media for 24 hours and TTR in the media was quantified by ELISA as detailed in materials and methods. The interval range of TTR expression differed; for TTR WT varied from 100-300, was 350-1000 for TTR V30M and 300-900 for TTR L55P (expressed in ng/ml)/10 6 cells/24 hours).

Protein expression and degradation was also assessed by immunoprecipitation on both media and cell lysates.

Production of amyloidogenic TTR species

We next investigated the ability of the different constructs to induce the production of amyloidogenic species. Cells were grown in serum free medium for 24, 48 or 72 hours and 10 then the media was analyzed for the presence of aggregated material retained by a 0.2 µm filter in a dot blot assay. While TTR aggregates were not detected in media from cells transfected with constructs carrying the TTRWT or TTR V30M cDNA even after 72 hours of cell culture, TTR L55P protein material was partially retained by the membrane filter as early as 24 hours of culture (figure 2A), suggesting the formation of oligomers or aggregates.

The doted area was analyzed by SDS-PAGE gel; the proteins were then transferred to a nitrocellulose membrane followed by immunodetection against TTR (figure2B), revealing the intact TTR monomer; under these conditions we did not have evidence for aggregate formation since SDS disrupted those aggregates. TTR, even under denaturing conditions does not completely dissociate into monomers, originating a second band corresponding to the dimer with approximately 32 kDa of apparent mass.

Comparative effects of Diflunisal and Iododiflunisal on TTR L55P aggregation

We next evaluated this system in the screening of drugs capable of interfering with TTR fibrillogenesis. Very recently, it has been reported that an iodinated diflunisal (Dif) derivative, iododiflunisal (I-Dif), was very potent as a TTR fibrillogenesis inhibitor, and as a stabilizer of the TTR tetrameric fold when compared with diflunisal [15] as assessed by in vitro and ex vivo approaches, respectively. In this study, we started by studying in our cellular system the effect of different I-Dif concentrations (0.01-10 µM) on TTR aggregation. As depicted in figure 3A, I-Dif inhibited L55P aggregate formation at concentrations between 0.1-10 µM.

We next compared the inhibitory activity on TTR L55P fibrilogenesis of diflunisal and iododiflunisal at three different concentrations : 0.1, 1 and 10 µM. Results are displayed in figure 3B and showed that iododiflunisal was able to inhibit aggregate formation at 0.1 µM whereas for diflunisal, the same effect was only observed at 10 µM concentration. Thus, I-Dif presented a 100 fold increase in inhibitory activity when compared to diflunisal. These observations confirmed the above referred data on the much higher capacity of iododiflunisal over diflunisal to prevent TTR fibrillogenesis It is noteworthy that some variation occurred in these assays. However, the relative intraassay activities between different drugs and between drugs and control maintained significance, despite inter-assay fluctuations. For example, 0.1µM of I-Dif inhibited L55P fibril formation by 25% as displayed in figure 3A, but by 90% as shown in figure 3B. Yet, statistical significance is observed with the referred concentration of I-Dif in both experiments, when compared with the respective control.

Comparative aggregate inhibitory activities of selected compounds Following the results described above and to further assess the usefulness of the cellular model for screening purposes, we continued our studies on this cellular system with different drugs. We evaluated a series of compounds selected based on their ability to inhibit amyloid fibril formation according to literature data (for references see Table 1), which in most cases used isolated recombinant TTR upon acidification to pH 4. Other small organic compounds also referred to as good amyloid inhibitors such as plant compounds (genistein and resveratrol), DES, benzoxazole and triiodophenol were also selected for testing. Thus, compounds listed in table 1 TIP inhibited aggregate and fibril formation whereas the addition of genistein and DES resulted in no visible effect by TEM (not shown).

In vitro and ex vivo activities of selected compounds

The same panel of compounds was further tested ex-vivo for their effect on TTR stability by IEF analysis under semi-denaturing conditions, namely in the presence of 4M urea. To perform the assays we used basically the same conditions as in previous studies [15]. Plasma from a heterozygotic carrier of TTR V30M was incubated with each of the compounds referred to in the legend of figure 6 and, following IEF, the bands corresponding to the TTR tetramer and monomer were visualized, and the tetramer/monomer ratio measured. Under the conditions used, TTR presents a characteristic pattern of two main bands, representing monomers (normal and oxidized form) and several bands of lower pI, representing different forms of tetramers.

As compared to iododiflunisal, a similar stabilization effect was obtained with DCPA and with TIP, not evident for DFPB and benzoxazole under the conditions used. All the other compounds without inhibitory TTR fibrillogenesis activity in the cellular assay failed to stabilize V30M ex vivo.

Discussion

The process that converts a soluble protein into amyloid is complex and not fully understood. TTR amyloid deposits have been suggested as the causative agent of FAP.

However, the finding that asymptomatic individuals carrying the TTR V30M mutation show a positive reaction for inflammatory markers and present non fibrillar TTR deposits in the nerve raised the possibility that species besides mature fibrils could be involved in the disease [16]. In fact, in vitro studies showed that TTR L55P aggregates are toxic to a Schwanoma cell line, inducing caspase-3 activation, while long fibrils do not produce significant caspase-3 activation. These observations suggest that a major target on FAP therapy is to inhibit the formation of intermediate species and that comprehension of how native TTR converts into toxic species is of major importance.

Initially, most in vitro studies on TTR amyloidogenesis were performed by acidifying the protein [2,[START_REF] Oza | Synthesis and evaluation of anthranilic acid-based transthyretin amyloid fibril inhibitors[END_REF]5,[START_REF] Razavi | Benzoxazoles as transthyretin amyloid fibril inhibitors: synthesis, evaluation, and mechanism of action[END_REF], using an environment certainly different from in vivo conditions. More recently, TTR variants such as Y78F, which are able to produce aggregates and fibrils at physiological pH have benefited research on anti-amyloidogenic drugs and disease mechanisms by avoiding pH related artifacts. In both cases (with and without acidification), most studies are still conducted with purified recombinant proteins [2][START_REF] Oza | Synthesis and evaluation of anthranilic acid-based transthyretin amyloid fibril inhibitors[END_REF][START_REF] Baures | Discovering transthyretin amyloid fibril inhibitors by limited screening[END_REF][5][START_REF] Razavi | Benzoxazoles as transthyretin amyloid fibril inhibitors: synthesis, evaluation, and mechanism of action[END_REF] and thus, such approaches lack factors that occur in vivo. Thus, a cellular system producing the amyloidogenic protein would represent an improved approach to study effects of modulating factors and drugs on fibrillogenesis. In fact, different cellular models have been generated in order to study amyloidogenesis. A cell culture system for generation of . Furthermore, different cellular models have been created in order to study many different aspects of Alzheimer's disease [START_REF] Matsumoto | Overexpression of amyloid precursor protein induces susceptibility to oxidative stress in human neuroblastoma SH-SY5Y cells[END_REF][START_REF] Iuvone | The spice sage and its active ingredient rosmarinic acid protect PC12 cells from amyloid-{beta} peptideinduced neurotoxicity[END_REF][START_REF] Yang | Intracellular A beta 1-42 aggregates stimulate the accumulation of stable, insoluble amyloidogenic fragments of the amyloid precursor protein in transfected cells[END_REF]]. Here we report on the establishment of a cellular system of transfected RN22 (rat Schwanoma) with WT-TTR, V30M and L55P mutants. The latter was shown to form species that were retained by a filter, in contrast to the WT and V30M proteins.

14 Although the most prevalent mutation responsible for the pathology, V30M does not show a very high amyloidogenic potential in vitro, since amyloid fibrils are only obtained after acidification. Furthermore, crystal X-ray studies [22] revealed a structure similar to the WT counterpart and the differences found could not explain a significant instability of the protein. Thus, in vivo, this mutation plays a very important role in the pathology of FAP but in vitro, the protein does not lend itself to study by the assays described here.

One of the advantages of this in vitro cellular system is that it measures aggregation under good physiological conditions because: 1) uses an eukaryotic system for the production of TTR; 2) the secreted protein is not isolated avoiding modifications, such as oxidations that might influence aggregation; 3) aggregation occurs in the cell media, under physiological pH and in the presence of serum proteins. None of the previous "in vitro" available methodologies to screen for TTR inhibitors take into consideration these aspects, as TTR aggregation is measured on isolated recombinant TTR isolated from bacteria and in the absence of serum proteins.

When the 12 compounds classified in the literature as good TTR fibrillogenesis inhibitors were tested on the cellular system, having iododiflunisal as the reference compound, only DCPA, benzoxazole, DFPB, and TIP, were able to prevent L55P aggregate formation in the conditioned media, which reflects the above mentioned limiting conditions, such as the aggressive environment imposed by acidification, the exclusive use in most instances of WT rather than mutant TTR and the absence of serum proteins [23,[START_REF] Greeen | Synthesis and characterization of potent bivalent inhibitors that bind prior to Transthyretin tetramerization[END_REF][START_REF] Baures | Discovering transthyretin amyloid fibril inhibitors by limited screening[END_REF]. Regarding the ultrastructural analysis, DCPA did not show an inhibitory effect as high as DFPB and benzoxazole, indicating that the Y78F mutant may not be as sensitive to this drug as TTR L55P and V30M are.

Stabilization of tetrameric V30M TTR by iododiflunisal was previously shown in ex vivo tests using plasma from hetero and homozygous carriers. The sample was directly analyzed without previous treatment with cross-linkers and did not suffer further processing such as immunoprecipitation. 4 M urea allowed discrimination of susceptibility to tetramer dissociation into monomeric species, directly visualized on the IEF gel without the use of TTR antibodies for quantification of the tetramer/monomer ratio. We also did not observe a stabilizing effect of benzoxazole and DFPB by ex vivo testing under the conditions used;

this fact is suggestive that these 2 drugs either inhibit TTR fibrillogenesis by mechanisms other than tetramer stabilization, such as inhibition of polymerization of soluble intermediary species generated after tetramer dissociation; alternatively they might have a more effective anti-fibrillogenic effect in L55P and in Y78F.

At any rate, testing of the V30M variant is most pertinent in studies searching for TTR antifibrillogenic drugs as besides being the most common mutant found in FAP, further application of the drug under study encompasses in vivo testing in animal models which are available for this mutant [START_REF] Sousa | Evidence for early cytotoxic aggregates in transgenic mice for human transthyretin[END_REF].

The

Compound reference

Triiodophenol (TIP) [START_REF] Miroy | Inhibiting transthyretin amyloid fibril formation via protein stabilization[END_REF] Flufenamic acid (Fluf) [START_REF] Baures | Discovering transthyretin amyloid fibril inhibitors by limited screening[END_REF] Diflunisal (Dif)

Legends to figures

Figure 1 A

 1 shows TTR from media and cell lysates (pellet), separated under denaturing conditions (SDS-PAGE) after immunoprecipitation with antihuman TTR antibody and reveals that most of the produced protein is secreted into the medium, as soluble non-degraded material. To further confirm the identity of each TTR variant expressed and secreted by the cells, we performed mass spectrometry analysis of the protein in the conditioned media of transfected cells. The results are displayed in figure1B which highlights the particular peaks corresponding to each mutation, thus confirming the expected TTR variant in each transfection. As shown in the left panel of figure1-B2, a peptide of 2181 mass, representing residues 16-35 of TTR but with methionine replacing valine, was detected in the preparation deriving from TTR V30M transfected cells, not present in the preparation of the right panel (deriving from TTR L55P transfected cells), which has the corresponding non-mutated, normal 2149 mass peptide. Conversely, in the preparation deriving from TTR L55P transfected cells (right panel), a mutated peptide of 3126 mass, representing residues 49-76 of TTR but with proline replacing leucine, was detected, which was absent from the V30M preparation; instead, a normal, non-mutated peptide of 3142 is observed.

  4 and monitorization of turbidity at 400nm. A further criteria for lead compound selection was individual products representative of different therapeutic categories such as, for instance, non-steroid anti-inflammatory drugs (NSAIDs) (diflunisal, diclofenac, flufenamic acid) or compounds sharing molecular structural similarities with those (DCPA, DFPB, diclonisal).

were tested at 1 µM concentration and results are shown in figure 4 .

 4 Different levels of inhibition were evident when compared to the iododiflunisal control (Figure4, histogram). Among the compounds tested, triidophenol (TIP) showed an inhibitory capacity similar to that of I-Dif. (~57% inhibition).Three of the compounds analyzed revealed to be more efficient than iododiflunisal: 2-((3, 5-Dichlorophenyl) amino) benzoic acid (DCPA), 4-(3, 5-difluorophenyl) benzoic acid (DFPB) and Benzoxazole (Benzo), inhibiting aggregate formation in more than 80%. For the other assessed compounds including DES, resveratrol, diflunisal, DNP, genistein, diclofenac, diclonisal and flufenamic acid, no inhibitory effect was evident when compared to the control. Conditioned media incubated with compounds just prior application onto the membrane showed a signal equivalent to conditioned media alone (not shown) ruling out a possible effect of the compounds on the immunoassay.Transmission electron microscopyTo compare the results obtained in the cellular model, we analyzed by TEM the effect of six of the drugs under study: Genistein, TIP, DES, DCPA, DFPB and benzoxazole. This study was performed with a different TTR variant: TTR Y78F which is very prone to aggregation as compared with the L55P mutant. The soluble Y78F variant aggregates in about 48 hours 12 at pH 7.4 as documented byAlmeida et al., 2004 [15] who observed that upon incubation with I-Dif no aggregates were visible and only round particles resembling the native TTR were present; in contrast, incubation with diflunisal or diclofenac did not prevent aggregate formation[15].

Figure 5

 5 Figure 5 is representative of the results obtained in the present study and shows a complete inhibition of aggregate and fibril formation with DFPB and benzoxazole (lower panels); DCPA was only able to partially inhibit aggregate and fibril formation (upper right panel).

  amyloid A associated amyloidosis (AA) was established using murine peritoneal macrophages after repeated injections of Hammersten casein [17]. Staining with congo red, thioflavin T, and anti-AA revealed amyloid like structures associated with macrophage clusters. Such systems can be useful in studies on the influence of chaperon molecules and other factors on the formation and degradation of amyloid fibrils. Similarly, transfected cells with presenilin 2 (PS2) gene were used to examine the processing and degradation pathways of PS2 [18]

Figure 1 -A:

 1 Figure 1-A: Immunoprecipitation analysis of RN22 cells media and pellet from: non-transfected cells (NT); transfected with plasmids for TTR WT (WT), V30M (V30M) and L55P (L55P). B: Mass spectrometry analysis of immunoprecipitated TTR from conditioned media of TTR V30M and L55P transfected cells. After immunoprecipitation, proteins were separated in a SDS-PAGE gel which was silver stained (B1): molecular weights (MW); standard TTR (TTR std); non-transfected cells (NT); cells transfected with plasmids for TTR V30M (V30M) and L55P (L55P). The bands corresponding to the TTR protein (arrow) were cut, digested by trypsin and analyzed by mass spectrometry (B2), revealing specific peptides for TTR V30M (left panel) and for L55P (right panel).

Figure 2 -B:

 2 Figure 2-Aggregation in conditioned media of RN22 cells A: Dot blot filter assays of media from: non-transfected RN22 cells or transfected with plasmid for WT and TTR mutants (WT, V30M and L55P). TTR was quantified in the medium by ELISA and 500 ng were applied in the filter, followed by TTR immunodetection. B: Western blotting of the material retained on the membrane filter, revealing the presence of TTR (lane1); TTR standard (lane 2). In both lanes the TTR monomer and dimmer are observable; Standard molecular weights (MW).

Figure 3 -A-

 3 Figure 3-A-Effect of different concentrations of I-Dif in L55P aggregate formation. Conditioned media containing 500 ng of total TTR of cells expressing TTR L55P treated with different concentrations of I-Dif (0.01, 0.02, 0.1, 0.5, 1, 5 and 10 µM) or non-treated (o µM I-Dif) was applied onto an acetate cellulose membrane filter, followed by immunodetection of TTR aggregates with a specific anti-human TTR antibody, and quantified using ImageQuant program. * p< 0.02. B-Dot blot showing the effect of three different concentrations of I-Dif and Dif in L55P aggregate formation. The experiment was performed as in A and reveals that I-Dif has an ability of preventing L55P aggregate formation approximately 100X higher than Dif.

Figure 4 -

 4 Figure 4-Screening for TTR anti-amyloidogenic drugs.Filter assays of the conditioned media from TTR L55P cells incubated with different drugs at 1 µM. Conditioned media of cells incubated with I-Dif, TIP, showed significant less aggregates than control. DCPA, DFPB and benzoxazole showed the highest inhibitory effect whereas all the other 8 compounds tested produced no effect. Histogram: densitometry of dot blot showing % of inhibition of aggregate formation by each compound, compared to the control (0 % inhibition, no drug added). * p< 0.006.

Figure 5 -Figure 6 -

 56 Figure 5-Morphological analysis by TEM TTR Y78F incubated 48 hours at 37ºC evolved from round particles to fibrils (control, top left panel). When incubated with DFPB or benzoxazole fibril formation was completely inhibited (bottom panels) whereas incubation with DCPA produced only a partial inhibition (top right panel)

Figure 1 Biochemical 28 Figure 6 Biochemical

 1286 Figure 1

381:351-6. 16-

  main conclusions of this screening work indicate that: (i) DCPA and TIP are the best stabilizers of V30M tetramers in plasma from carriers of this mutant, and clearly inhibit aggregation in the cellular system; also, DFPB displayed anti-aggregation properties for L55P and Y78F but did not stabilize V30M tetramers, possibly by acting through mechanisms other than tetrameric stabilization. Therefore, iododiflunisal, DCPA and TIP are promising for the treatment of V30M associated FAP but need to undergo further drug development stages to overcome their toxicity. (ii) Compounds with virtually null stabilization of tetrameric structure (as assessed ex vivo), such as flufenamic acid, diclofenac, resveratrol, ginestein, diflunisal, and DES clearly had no effect on TTR aggregation in cell culture and thus further testing them may not be worthwhile. Sousa, M. M., Cardoso, I., Fernandes, R., Guimaraes, A. and Saraiva, M. J.(2001)

	10-Henzel WJ, Billeci TM, Stults JT, Wond SC, Grimley C and Watanabe C. Identifying
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	13-Lowry, O. H., Rosenbrough, N. J., Farr, A. L., and Randall, J. (1951) Protein
	The cellular model here described represents a step forward in the study of events occurring
	very early in TTR amyloidogenesis and of factors/drugs affecting such process; moreover,
	our cellular model supplies a unique tool to test drugs independently of the mechanism
	behind their action, i.e., tetramer stabilization, inhibition of aggregation and others. We
	believe this system will have important implications in future drug screening and design
	while constituting an important tool for the selection of compounds that merit preclinical
	testing.
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measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275. 14-Altland, K., Rauh, S. and Hacker, R. (1981) Demonstration of human prealbumin by double one-dimensional slab gel electrophoresis. Electrophoresis 2, 148-155 15-Almeida MR, Macedo B, Cardoso I, Alves I, Valencia G, Arsequell G, Planas A, Saraiva MJ. (2004). Selective binding to transthyretin and tetramer stabilization in serum from patients with familial amyloidotic polyneuropathy by an iodinated diflunisal derivative. Biochem J.

Table 1 -

 1 Compounds tested for the inhibition of aggregate formation by TTR L55P expressing cells.

Biochemical Journal Immediate Publication. Published on 8 Aug 2007 as manuscript BJ20070689 © 2007 The Authors Journal compilation © 2007 Biochemical Society

© 2007 The Authors Journal compilation © 2007 Biochemical Society

Acknowledgements

We thank Paul Moreira for his assistance in the production of recombinant TTRs. This work was supported by the Portuguese Foundation for Science and Technology (FCT) through a fellowship to Cardoso I (SFRH/BPD/9416/2002), POCTI and POCI grants, and by the Gulbenkian Foundation.