Marcel Zamocky
email: marcel.zamocky@boku.ac.at

Paul G Furtmüller

Marzia Bellei

Gianantonio Battistuzzi

Johannes Stadlmann

Jutta Vlasits

Christian Obinger

Intracellular catalase-peroxidase from the phytopathogenic fungus Magnaporthe grisea: expression analysis and biochemical characterisation of the recombinant protein

Keywords: catalase-peroxidase, non-animal peroxidase superfamily, phytopathogenic fungi, Magnaporthe grisea, oxidative stress KatG, catalase-peroxidase, MagKatG1, intracellular catalase-peroxidase from Magnaporthe grisea, MagKatG2, secreted catalase-peroxidase from Magnaporthe grisea, katG1, gene encoding MagKatG1, katG2, gene encoding MagKatG2, NcKatG2, catalaseperoxidase from Neurospora grassa, SynKatG, catalase-peroxidase from Synechocystis PCC6803, ROS, reactive oxygen species, MCAC, metal chelate affinity chromatography, MCD, monochlorodimedone, ECD, electronic circular dichroism

Phytopathogenic fungi like Magnaporthe grisea are unique in having two catalase-peroxidase (KatG) paralogs located either intracellularly (KatG1) or extracellularly (KatG2). The coding genes have recently been shown to derive from a lateral gene transfer from a (proteo)bacterial genome followed by gene duplication and diversification. Here we demonstrate that Magnaporthe grisea KatG1 is expressed constitutively in rice blast fungus. It is the first eukaryotic catalase-peroxidase to be expressed heterologously in E. coli in high amount and purity with almost 100% haem occupancy. Recombinant MagKatG1 is an acidic, mainly homodimeric oxidoreductase with a predominant five-coordinated high-spin haem b. At 25 °C and pH 7.0, the reduction potential of the Fe(III)/Fe(II) couple is found to be (-186 ± 10) mV. It binds cyanide monophasically with an apparent bimolecular rate constant of (9.0 ± 0.4) × 10 5 M -1 s -1 (pH 7.0 and 25 °C) and a K D value of 1.5 µM. Its predominantly catalase activity is characterized by a pH optimum at pH 6.0 and k cat and K M values of 7010 s -1 and 4.8 mM, respectively. In addition it acts as versatile peroxidase with pH optimum in the range pH 5.0 -5.5 using both one-(ABTS, o-dianisidine, pyrogallol, guaiacol) and two-electron donors (Br -, I -, ethanol). Structure-function relationships are discussed with respect to data reported for prokaryotic KatGs as is the physiological role of MagKatG1. Phylogenetic analysis suggests that (intracellular) MagKatG1 can be regarded as typical representative for catalaseperoxidase of both phytopathogenic and saprotrophic fungi.

Introduction

Catalase-peroxidases (KatGs) are unique bifunctional oxidoreductases accomplishing efficiently both peroxidatic and catalatic activity within a single active site [START_REF] Smulevich | Probing the structure and bifunctionality of catalase-peroxidase (KatG)[END_REF][START_REF] Zamocky | The evolution of catalases from bacteria to man[END_REF]. Despite their striking sequence similarities and close phylogenetic relationship with ascorbate peroxidases and cytochrome c peroxidases [START_REF] Zamocky | Phylogenetic relationships in class I of the superfamily of bacterial, fungal, and plant peroxidases[END_REF] they are the sole representatives within the whole superfamily of non-animal haem (i.e. plant, fungal and bacterial) peroxidases [START_REF] Passardi | Prokaryotic origins of the non-animal peroxidase superfamily and organelle mediated transmission to eukaryotes[END_REF] capable of both the reduction and efficient oxidation of hydrogen peroxide [START_REF] Zamocky | The molecular peculiarities of catalase-peroxidases[END_REF] -in this way similar to typical catalases that represent a quite different evolutionary line. A recent phylogenetic analysis [START_REF] Passardi | Phylogenetic distribution of catalase-peroxidases: Are there patches of order in chaos?[END_REF] has demonstrated that most of the 347 so far found katG sequences were encoded within eubacterial and archaebacterial genomes. About 40% of all sequenced prokaryotes possess katG gene(s) and so far comprehensive functional and structural investigations were only performed with eubacterial and archaebacterial catalase-peroxidases [START_REF] Singh | Comparative study of catalase-peroxidases (KatGs)[END_REF], [START_REF] Yamada | The 2.0 A crystal structure of catalase-peroxidase from Haloarcula marismortui[END_REF], [START_REF] Bertrand | Crystal structure of Mycobacterium tuberculosis catalase-peroxidase[END_REF], [START_REF] Kumasaka | Expression, purification, crystallization and preliminary X-ray analysis of the Met244Ala variant of catalase-peroxidase (KatG) from the haloarchaeon Haloarcula marismortui[END_REF]. However, KatGs can be found also in Ascomycota and Basidiomycota as well as in a few protists [START_REF] Zamocky | Phylogenetic relationships in class I of the superfamily of bacterial, fungal, and plant peroxidases[END_REF], [START_REF] Passardi | Phylogenetic distribution of catalase-peroxidases: Are there patches of order in chaos?[END_REF]. In contrast to prokaryotic KatGs, the corresponding eukaryotic proteins are scarcely described and only a few scattered data on gene localisation and expression and enzyme activities have been reported [11], [START_REF] Peraza | Neurospora crassa catalases, singlet oxygen and cell differentiation[END_REF], [START_REF] Paris | Catalases of Aspergillus fumigatus[END_REF], [START_REF] Scherer | Aspergillus nidulans catalaseperoxidase gene (cpeA) is transcriptionally induced during sexual development through the transcription factor StuA[END_REF] mainly due to lack of successfully produced recombinant protein.

Most interestingly, the plant pathogenic fungus Magnaporthe grisea (anamorph Pyricularia grisea) has two katG genes. This ascomycete, known as rice blast fungus, is classified among the most dangerous phytopathogens worldwide possibly attacking not only most rice variants but also numerous other grass species including economically important crops such as wheat, barley, and millet [15]. Besides KatGs its genome [START_REF] Dean | The genome sequence of the rice blast fungus Magnaporthe grisea[END_REF] revealed numerous virulence-associated genes and enzymes involved in secondary metabolism. From the two distinct KatG paralogs of Magnaporthe grisea one seems to be located intracellularly (KatG1) and one might be secreted from the fungal cells (KatG2) since it contains a (predicted) signal sequence. Localisation could be related with distinct functions, i.e. intracellular degradation of hydrogen peroxide deriving from fungal catabolism [17] and/or contribution to the enzymatic armour of phytopathogenic fungi against ROS produced during plant -pathogen interaction (oxidative burst) [18], [START_REF] Xue | Fungal pathogenesis in the rice blast fungus Magnaporthe grisea[END_REF]. Even a possible signaling function of KatGs is under discussion [START_REF] Zamocky | The evolution of catalases from bacteria to man[END_REF]. As obvious from the inspection of corresponding sequences and phylogenetic analyses [START_REF] Passardi | Phylogenetic distribution of catalase-peroxidases: Are there patches of order in chaos?[END_REF] both KatG1 and KatG2 seem to represent two distinct and abundant fungal KatG groups with different structure-function pattern.

Here, we focus on the expression analysis, recombinant production and biochemical characterisation of intracellular catalase-peroxidase of Magnaporthe grisea (MagKatG1). The complete ORF of katG1 (2217 bp) has been successfully cloned and heterologously expressed enabling a comprehensive investigation of its biophysical and biochemical properties. Presented spectral, redox and kinetic data are compared with those reported for the corresponding prokaryotic enzymes and are discussed with respect to structure-function relationships and physiological role(s) of eukaryotic catalase-peroxidases.

Materials and methods

Magnaporthe grisea. The ascomycete Magnaporthe grisea, strain MA 829 (Cooke) was obtained from the internal collection of BOKU -University of Natural Resources and Applied Life Sciences, Vienna. It was first grown on MPG-agar plates (containing 20 g malt extract, 1 g peptone, 20 g glucose and 20 g agar in 1 L) for 4-6 days at room temperature. For the isolation of RNA and activity monitoring it was grown in liquid medium with the same composition (without agar) at 25 °C in a shaking incubator at 130 rpm for 3 days.

Sequence similarity searches -PeroxiBase. All available sequences of peroxidase genes and proteins have been systematically annotated, compared and analysed in PeroxiBase (http://peroxibase.isb-sib.ch/, see [20] for details). All sequence names used in this contribution correspond to the nomenclature of PeroxiBase where also links to the corresponding genes and genomes can be found. Name and identification number of the intracellular catalase-peroxidase from Magnaporthe grisea are MagKatG1 and 2288, respectively, the corresponding UniProt accession is A4R5S9.

Multiple sequence alignment. Multiple sequence alignment of fungal and bacterial KatG proteins was performed with ClustalX Version 2.0.5 [START_REF] Larkin | Clustal W and Clustal X version 2.0[END_REF]. Following optimised parameters were applied: Gonet protein weight matrix with gap opening penalty 8, gap extension penalty 0.2 and gap separation distance of 4. The aligned output was presented with Genedoc [START_REF] Nicholas | Genedoc: a tool for editing and annotating multiple sequence alignments[END_REF].

Promoter & sub-cellular targeting prediction. Prediction of intron and native promoter location within the genomic DNA of Magnaporthe grisea strain 70-15 was performed in SoftBerry suite (http://www.softberry.com) using the ascomycete-specific parameters.

Cloning, heterologous expression and purification of recombinant MagKatG1. The cloning strategy of cDNA and complete katG1 gene is described in Supplemental material 1. Briefly, the complete gene was first maintained in TOPO vector (Invitrogen) and after modification it was expressed within pET21d vector (Novagen) allowing a His-tag fusion. Heterologous expression was achieved from E. coli BL21 DE3 Star cells (Invitrogen) at 16 °C and addition of hemin (10 µM final) concomitantly with IPTG (1 mM final) in the cultivation medium. Cells of 16 h old culture were centrifuged for 15 min at 6000 rpm and pellets were resuspended in homogenisation buffer containing 50 mM sodium phosphate, pH 8.0, and protease inhibitors (1 mM PMSF, 1 µg/mL leupeptin and 1 µg/mL pepstatin). Homogenisation was performed by two ultra-sonication cycles on Vibra-Cell Model CV17 (Sonics Materials Inc.): length of each cycle: 45 s; pulser: 50% ; intensive cooling between the cycles. The crude homogenate was centrifuged for 20 min at 20,000 × g and the cleared supernatant was applied onto MCAC chromatography (Chelating Sepharose Fast Flow, GE Healthcare) loaded with Ni(II) ions (volume: 30 mL). After loading the column was washed with 150 mL buffer A (50 mM sodium phosphate, pH 8.0, containing 500 mM NaCl and protease inhibitors). Bound His-tagged protein was eluted with a linear gradient of buffer A to 100% buffer B (50 mM sodium phosphate, pH 6.5, containing 500 mM NaCl and 500 mM imidazole). For final purification and determination of putative oligomeric structure of MagKatG1 gel permeation chromatography (Superdex 200 prep grade, GE Healthcare) was performed: volume: 250 mL; sample volume: 1 mL; 5 mM sodium phosphate, pH 7.0, containing 0.15 M NaCl; flow rate: 0.5 mL min -1 ; maximal pressure: 0.3 MPa.

Purity of obtained fractions was analysed with SDS PAGE under standard conditions (BioRad Mini-Protean, 12 % separating gel) with gels being either stained with Coomassie Brilliant Blue or blotted onto nitrocellulose membrane (Amersham Biosciences) for detection of MagKatG1 by immunoblotting using a polyclonal antibody raised against catalaseperoxidase from Neurospora crassa [23].

To reveal the expression pattern of MagKatG1 native polyacrylamide gel electrophoresis with a linear gradient of 4-18% acrylamide was used. Native electrophoresis was run with 10 mM Tris/HCl buffer, pH 8.3, containing 14.4 g/L glycine (Mini-Protean apparatus, BioRad) at 150 V for 30 min, followed by 65 V for 3 h. Catalase staining was performed according to [24] with catalatically active bands ocurring as white bands on a dark-green background. Peroxidase activity staining was performed by using o-dianisidine as electron donor.

Mass spectrometric analysis. In order to analyse proteolytic degradation of MagKatG1 bands in SDS-PAGE were excised, destained, carbamidomethylated and digested with sequencing-grade bovine trypsin (Sigma-Aldrich). The extraction from gel pieces was performed in the same mode as described recently [START_REF] Kolarich | Structural analysis of the glycoprotein allergen Hev b 4 from natural rubber latex by mass spectrometry[END_REF]. Extracts were taken to dryness in a Speed Vac concentrator and reconstituted with water containing 0.1 % formic acid. Subsequent MALDI-TOF-MS analysis was performed on a Voyager-DE STR (Applied Biosystems) with α-cyano-4-hydroxycinnamic acid (ACH) as matrix.

Electronic circular dichroism spectrometry and structure prediction. ECD spectra were recorded on a PiStar-180 Spectrometer equipped with a thermostatic cell holder (Applied Photophysics; Leatherhead, U.K.). For recording far-UV spectra (260-190 nm) the quartz cuvette had a path length of 1 mm. Spectral bandwidth: 5 nm; step size: 1 nm; scan time: 624 s; protein concentration: 4 µM MagKatG1. Protein concentration was calculated from the known amino acid composition and absorption at 280 nm according to [START_REF] Gill | Calculation of protein extinction coefficients from amino acid sequence data[END_REF]. All ECD measurements were performed in 5 mM phosphate buffer, pH 7.0, at 25 °C. Each spectrum was automatically corrected with the baseline to remove birefringence of the cell. The instrument was flushed with nitrogen with a flow rate of 5 L min -1 .

In addition the secondary structure of MagKatG1 was predicted with Psipred [START_REF] Mcguffin | The PSIPRED protein structure prediction server[END_REF]. The 3D structure of MagKatG1 was modeled using EsyPred 3D [START_REF] Lambert | EsyPred3D: prediction of proteins 3D structures[END_REF] and KatG from Burkholderia pseudomallei (1MWV). The reliability of the obtained model was verified using What IF (http://swift.cmbi.ru.nl/servers/htmL/index.htmL).

Electronic UV-VIS spectroscopy. The UV-VIS spectrum of purified recombinant ferric MagKatG1 in 5 mM phosphate buffer, pH 6.5, was routinely recorded on Zeiss Specord 10 diode array photometer in the range between 200 -800 nm at 25 °C. We have determined the extinction coefficient at Soret band (ε 408 nm = 110,650 M -1 cm -1 ) for highly purified MagKatG1 from the slope of the linear correlation between A 408 nm and seven distinct protein concentrations (not shown). Ferrous MagKatG1 was produced by addition of sodium dithionite either directly or from a freshly prepared anaerobic stock solution in a glove box (Meca-Plex, Neugebauer) with a positive pressure of nitrogen (25 millibars). All solutions were made anaerobic by flushing with nitrogen gas (oxygen < 3 ppm) and stored in the glove box. The spectrum of the low-spin cyanide complex of MagKatG1 was recorded after mixing 7 µM protein with 800 µM cyanide in 5 mM phosphate buffer, pH 6.5.

Spectroelectrochemistry.

Determination of the standard reduction potential, E°, of the Fe(III)/Fe(II) couple of MagKatG1 was carried out using a homemade OTTLE cell [START_REF] Battistuzzi | Redox thermodynamics of low-potential iron-sulfur proteins[END_REF]. The three-electrode configuration consisted of a gold mini-grid working electrode (Buckbee-Mears, Chicago, IL), a homemade Ag/AgCl/KCl sat microreference electrode, separated from the working solution by a Vycor set, and a platinum wire as counter electrode. The reference electrode was calibrated against a saturated calomel electrode before each set of measurements. All potentials are referenced to the SHE. Potentials were applied across the OTTLE cell with an Amel model 2053 potentiostat/galvanostat. The constant temperature was maintained by a circulating water bath, and the OTTLE cell temperature was monitored with a Cu-costan microthermocouple. UV-VIS spectra were recorded using a Varian Cary C50 spectrophotometer. Spectra of 28 µM MagKatG1 equilibrated in 50 mM sodium phosphate buffer, pH 7.0, containing 10 mM NaCl were recorded at various applied potentials. The following electron mediators were used: 850 µM methyl viologen, 3 µM lumiflavine-3acetate, indigo disulfonate, phenazine methosulfate and methylene blue. Results were Kinetics and thermodynamics of formation of the MagKatG1-cyanide complex. In order to probe haem accessibility apparent bimolecular rate constants of complex formation between cyanide and ferric MagKatG1 were determined by convential stopped-flow spectroscopy (model SX-18MV Applied Photophysics, U.K.). For a total of 100 µL/shot into the optical observation cell with 1 cm light path, the fastest time for mixing two solutions and recording the first data point was of the order of 1.3 ms. Formation of low-spin cyanide complex was followed at 426 nm, the Soret maximum difference between high-spin MagKatG1 and the cyanide complex. In a typical experiment, one syringe contained 2 µM MagKatG1 in 5 mM phosphate buffer, pH 7.0, and the second syringe contained at least a 2.5fold excess of cyanide in 100 mM phosphate buffer, pH 7.0. At high molar excess of cyanide over catalase-peroxidase, the formation of cyanide complex was too fast, and the majority of absorbance change occurred within the dead time of the instrument. As a consequence, pseudo-first order conditions could not be maintained over the whole concentration range. Three determinations were performed for each ligand concentration. The mean of the pseudofirst-order rate constants, k obs , was used in the calculation of the second-order rate constant obtained from the slope of a plot of k obs versus cyanide concentration. All measurements were performed at 25 °C.

Equilibrium binding of cyanide to MagKatG1 was studied by spectroscopic titration of 1 µM enzyme in 100 mM phosphate buffer, pH 7.0, with increasing concentrations of cyanide (1 µM -800 µM). Enzyme spectrum was recorded before and after addition of constant volumes of cyanide solution and the free enzyme spectrum was substracted from the complex spectrum after correction for volume dilution. Plotting the ratio of cyanide concentration to absorbance difference between HS and LS MagKatG1 (peak maximum minus valley) against the cyanide concentration allowed determination of K D .

Overall kinetic parameters. Catalase activity was determined both polarographically using a Clark-type electrode (YSI 5331 Oxygen Probe) inserted into a stirred water bath (YSI 5301B) or spectrophotometrically by measuring the decomposition of hydrogen peroxide at 240 nm according to [30]. For spectrophotometrical measurement one unit was defined as the amount of enzyme catalyzing the conversion of 1 µmol H 2 O 2 min -1 at an initial concentration of 15 mM H 2 O 2 at pH optimum and 25 °C.

Peroxidase activity was monitored spectrophotometrically using 1 mM H 2 O 2 and 5 mM ABTS (ε 414 = 31.1 mM -1 cm -1 ) [31] or 5 mM guaiacol (ε 470 = 26.6 mM -1 cm -1 ) or 1 mM odianisidine (ε 460 = 11.3 mM -1 cm -1 ) or 1 mM pyrogallol (ε 430 = 2.5 mM -1 cm -1 ). One unit of peroxidase was defined as the amount of enzyme that decomposes 1 µmol of electron donor min -1 at pH optimum and 25 °C.

Additionally, peroxidase activity with ethanol as 2-electron donor was determined at 340 nm in a coupled reaction using aldehyde dehydrogenase as described previously [START_REF] Zamocky | Site-directed mutagenesis of the lower parts of the major substrate channel of yeast catalase A leads to highly increased peroxidatic activity[END_REF]. Chlorination and bromination activity of recombinant MagKatG1 was followed by halogenation of monochlorodimedon MCD (100 µM) dissolved in 100 mM phosphate buffer, pH 7.0, containing 10 mM H 2 O 2 and either 10 mM bromide or 100 mM chloride. Rates of halogenation were determined from the initial linear part of the time traces using an extinction coefficient for MCD at 290 nm of 19.9 mM -1 cm -1 [START_REF] Kettle | Assays for the chlorination activity of myeloperoxidase[END_REF]. One unit was defined as the amount that decomposes 1 µmol of MCD min -1 at pH 7.0 and 25 °C. Iodide oxidation was followed at 353 nm, i.e. absorbance maximum of triiodide (ε = 25.5 mM -1 cm -1 ) in 100 mM phosphate buffer, pH 7.0, containing 10 mM H 2 O 2 and 10 mM iodide at pH 7.0 and 25 °C [START_REF] Jakopitsch | Catalase-Peroxidase from Synechocystis is capable of chlorination and bromination reactions[END_REF]. One unit was defined as the amount that produces 1 µmol of I 3 -min -1 at pH 7.0 and 25 °C. In all steady-state measurements at least three determinations for each electron donor concentration were performed and reactions were started by addition of MagKatG1.

Results and Discussion

Statistics from PeroxiBase clearly suggests that in eukaryotic organisms KatGs are predominantly spread among the fungal kingdom. A recent phylogenetic analysis [START_REF] Zamocky | The evolution of catalases from bacteria to man[END_REF] demonstrated the existence of two distinct types of fungal katG genes. Mainly phytopathogenic fungi (e.g. Magnaporthe grisea or Gibberella zeae) contain both paralogs, with katG1 most probably encoding an intracellular and katG2 an extracellular catalaseperoxidase.

Analysis and comparison of the whole genome of Magnaporthe grisea [START_REF] Dean | The genome sequence of the rice blast fungus Magnaporthe grisea[END_REF] with katG1 and katG2 showed a significant difference in the G+C contents (for katG1 59.2% versus 51.6%) suggesting a lateral gene transfer (LGT) of katG genes from bacteria to a fungal progenitor as has been demonstrated for other fungi [START_REF] Zamocky | Phylogenetic relationships in class I of the superfamily of bacterial, fungal, and plant peroxidases[END_REF][START_REF] Passardi | Phylogenetic distribution of catalase-peroxidases: Are there patches of order in chaos?[END_REF]. Interestingly, the rather high G+C contents of MagkatG1 resembled that of various Burkholderia species (58-68 %) thereby indicating a putative proteobacterial ancestor. In order to answer the question whether only the katG1 gene or a longer genome portion was transferred by LGT, we have also analysed the putative promoter region of MagkatG1 with GENSCANW (http://genes.mit.edu/GENSCAN.htmL). A conserved 40 bp long promoter motif was identified at positions -1838 to -1799 (see PeroxiBase ID=2288 for further details). Moreover, a typical CGGAGT-box was found in positions -657 to -652 similar to a promoter region in the catB gene of Aspergillus oryzae [35]. The G+C contents of this 1838 bp long region is only 49.73%, thus even lower than the average value for the entire Magnaporthe genome. This suggests that only the coding region was transferred via LGT from a proteobacterial into the fungal genome.

In order to probe whether expression of MagKatG1 is induced by oxidative stress, cultures of Magnaporthe grisea were grown in MPG in the absence and presence of hydrogen peroxide or peroxyacetic acid or paraquat (final concentrations: 0.5 -2 mM) added in the middle exponential or early stationary growth phase, respectively. RT-PCR analysis of total RNA isolated from filtered mycelia allowed quantification of katG1-specific mRNA under various conditions. The RT-PCR signal intensities obtained from various cDNA samples (Figure 1) clearly demonstrated that katG1 is expressed constitutively. Only with paraquat the expression level was enhanced to 120% compared to standard conditions. This might suggest a role of intracellular MagKatG1 in continuous degradation of H 2 O 2 deriving from fungal metabolism and is in contrast to MagKatG2 that -due to the presence of a (predicted) signal sequence -is located extracellularly and is produced on demand, i.e. its expression is significantly enhanced under oxidative stress conditions that could occur during attacking of plants [17]. Subsequently MagKatG1 was heterologously expressed and purified. The entire ORF (2217 bp) was cloned in TOPO vector (Invitrogen) using outer primers and modified by introducing of NcoI, AgeI and NotI restriction sites without affecting the coding sequence (Supplemental material 1). This allowed cloning in pET21d vector (Novagen) in frame with a C-terminal hexa-His-tag fusion. Heterologous expression of the recombinant plasmid pMZM1 was performed in E. coli strain BL21 DE3 Star (Invitrogen) at 16 °C and in the presence of hemin avoided formation of inclusion bodies and produced recombinant protein with almost 100% haem occupancy. In average about 30 mg of soluble KatG1 could be obtained by lysis activity of the eukaryotic enzyme was very small (Table 1), namely ~0.4% and ~15% of the NADH oxidase activity reported for Burkholderia pseudomallei [START_REF] Singh | Catalase-peroxidases (KatG) exhibit NADH oxidase activity[END_REF] and Escherichia coli KatG [START_REF] Singh | Comparative study of catalase-peroxidases (KatGs)[END_REF].

Thermostability tests of MagKatG1 were performed in the range from 25 °C to 65 °C by incubating the enzyme at the corresponding temperature for defined time intervals followed by measuring the catalase and peroxidase activities at 25 °C (Table 2). Comparison with equivalent data of bacterial counterparts [START_REF] Singh | Comparative study of catalase-peroxidases (KatGs)[END_REF] revealed that MagKatG1 is a mesophilic enzyme that is more thermostable than Synechocystis KatG or Burkholderia pseudomallei KatG [START_REF] Singh | Comparative study of catalase-peroxidases (KatGs)[END_REF]. Generally, the catalase activity is more susceptible to temperature-induced inactivation than the peroxidase activity. It has to be investigated whether the observed (limited) thermostability of the peroxidatic activity may be related to the fact that Magnaporthe grisea attacks predominantly various cultivars of rice which grow optimally only in subtropical climate.

Summing up, phytopathogenic fungi are unique in having two catalase-peroxidase (KatG) paralogs located either intracellularly (KatG1) or extracellularly (KatG2). The coding genes have been shown to derive from a lateral gene transfer from a (proteo)bacterial genome followed by gene duplication and diversification. MagKatG1 is the first eukaryotic catalaseperoxidase to be heterologously expressed in E. coli in high amount and purity with almost 100% haem occupancy. The acidic, mainly homodimeric oxidoreductase contains a predominantly five-coordinated high-spin haem b with spectral and redox properties comparable to prokaryotic enzymes. Its overwhelming catalase activity is characterized by a pH optimum at pH 6.0 and k cat and K M values of 7010 s -1 and 4.8 mM, respectively. The intracellular metalloenzyme is expressed constitutively and might contribute to continuous degradation of H 2 O 2 deriving from fungal metabolism in the cytosol. In addition the bifunctional enzyme acts as versatile peroxidase with pH optimum in the range pH 5.0 -5.5 oxidising both one-(ABTS, o-dianisidine, pyrogallol, guaiacol) and two-electron donors (Br -, I -) at reasonable rates. However, similar to prokaryotic KatGs, the biochemical and physiological role of the peroxidase activity of fungal KatGs is unknown as is the nature of the (putative) endogenous electron donor.

Table 1 Selected kinetic parameters of recombinant MagKatG1. Presented are average values of typical clones. Kinetic traces are presented in Supplemental material 6.

Sample (clone) Activity parameter Data (average values)

M133 K M (H 2 O 2 ) (4.8 ± 0.4) mM M133 k cat (H 2 O 2 ) (7010 ± 230) s -1 M133 k cat / K M (H 2 O 2 ) (1.46 ± 0.06) × 10 6 M -1 s -1 M133+M134+M166+M167 catalatic activity (3430 ± 280) U mg -1 M133+M134+M166+M167 peroxidatic activity (o-dianisidine) (14.2 ± 4.6) U mg -1 M133+M134+M166+M167 peroxidatic activity (ABTS) (32.3 ± 2.1) U mg -1 M133+M134+M166+M167 peroxidatic activity (guaiacol) (2.7 ± 0.4) U mg -1 M133+M134+M166+M167 peroxidatic activity (pyrogallol) (3.3 ± 0.3) U mg -1 M133+M134+M166+M167 chlorination of MCD* (0.36 ± 0.06) U mg -1 M133+M134+M166+M167 bromination of MCD* (2.56 ± 0.11) U mg -1 M133+M134+M166+M167 iodination of MCD* (0.07 ± 0.02) U mg -1 M133+M166 (per)oxidation of ethanol (0.3 ± 0.1) U mg -1 M133+M166 NADH oxidase (2.2 ± 0.5) U µmol haem -1 M133 K D cyanide (1.4 ± 0.3) µM M133 k on cyanide (9.0 ± 0.4) × 10 5 M -1 s -1 M133 k off cyanide (1.4 ± 0.4) s -1
* MCD has been demonstrated not to function as electron donor for recombinant MagKatG1. A c c e p t e d M a n u s c r i p t

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LEGENDS TO FIGURES

Figure 1 DNA electrophoresis (0.85% agarose) of RT-PCR products isolated from Magnaporthe grisea grown under standard conditions (lane 1) or oxidative stress induced by addition of 0.1 mM paraquat (lane 2) or 0.3 mM peroxyacetic acid (lane 3). Lane 4: amplification of whole gene after induction of oxidative stress with paraquat; lane 5: molar mass standards with size given in base pairs (bp). In lanes 1-3 the same amount of RT-PCR product was loaded.

  Biochemical Journal Immediate Publication. Published on 10 Nov 2008 as manuscript BJ20081478 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081478A 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.

  presented as Nernst plot that allowed calculation of the standard reduction potential, E°, of the Fe(III)/Fe(II) couple MagKatG1.

Biochemical

  Journal Immediate Publication. Published on 10 Nov 2008 as manuscript BJ20081478 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081478 A c c e p t e d M a n u s c r i p t

Biochemical

  Journal Immediate Publication. Published on 10 Nov 2008 as manuscript BJ20081478 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081478 A c c e p t e d M a n u s c r i p t

Figure 2 (

 2 Figure 2 (A) SDS PAGE of recombinant MagKatG1 purified from E. coli BL21 DE3 Star homogenate. Lane 1: Coomassie Brilliant Blue stained crude extract; lane 2: pool from MCAC affinity column fractions 4 -6; lane 3: pool from MCAC affinity column fractions 7 -10; lane 4: protein from main elution peak of Superdex 200 chromatography (i.e. sample from lane 3 purified over a second column); lanes 5 & 6: Western blot analysis using a polyclonal antibody raised against NcKatG1, lane 5: MCAC fractions 4 -6 (corresponding to CBB -lane 2), lane 6: MCAC fractions 7 -10 (corresponding to CBB -lane 3). (B) Active staining on catalase (lanes 1 & 2) and peroxidase activity (lanes 3 & 4): lane 1: pool from MCAC fractions 7 -10; lane 2: pool from main peak of Superdex 200 chromatography (sample from lane 1 further purified); lane 3: pool from MCAC fractions 7 -10; lane 4: pool from main peak of Superdex 200 (sample from lane 3 further purified). Bovine liver catalase (Sigma) and Comamonas testosteroni KatG (PeroxiBase ID 5351) were used as native standards. Molar mass of standard proteins is given in kDa.

Figure 3 (

 3 Figure 3 (A) Electronic UV-VIS and far-UV ECD spectrum (inset) of purified recombinant MagKatG1 (black line, 4.2 µM) in 5 mM phosphate buffer, pH 7.0. For comparison the corresponding spectra of SynKatG (grey line) recorded under identical conditions are depicted. (B) Homology model of the 3D structure of MagKatG1 monomer revealed by EsyPred [28]. An overlay with the known structure of KatG from Burkholderia pseudomallei (PDB code 1MWV) is presented. The figure was rendered by Swiss PDB Viewer.

Figure 4

 4 Figure 4 Spectral characterisation of recombinant MagKatG1 (black line) in comparison with prokaryotic SynKatG (grey line). Arrows indicate peak maxima. (A) Ferric proteins in 5 mM sodium phosphate buffer, pH 7.0, and 25 °C. (B) Ferrous proteins in 5 mM sodium phosphate buffer, pH 7.0, and 25 °C. (C) Low-spin cyanide complexes obtained by mixing of ferric proteins with 800 µM NaCN in 5 mM sodium phosphate buffer, pH 7.0, and 25 °C.

Figure 5

 5 Figure[START_REF] Zamocky | The molecular peculiarities of catalase-peroxidases[END_REF] Electronic spectra of catalase-peroxidase from Magnaporthe grisea obtained at various applied potentials in spectroelectrochemical experiments. Spectra were recorded at 25 °C. The corresponding Nernst plot is reported in the inset, where X stands for: [(A λred Max -A λred )/(A λox Max -A λox )] , λ ox =406 nm and λ red =435 nm. Conditions: sample volume: 1 mL; 28 µM protein in 50 mM phosphate buffer, pH 7.0, containing 10 mM NaCl; 850 µM methyl viologen and 3 µM lumiflavine-3-acetate, indigo disulfonate, phenazine methosulfate and methylene blue.

Figure 6

 6 Figure 6 Kinetics and thermodynamics of cyanide binding to recombinant MagKatG1. (A) Spectral transition showing the formation of the low spin cyanide complex. The reaction of 2 µM ferric MagKatG1 with 30 µM cyanide was measured in the conventional stopped-flow mode. Arrows indicate changes of absorbance with time. Conditions: 50 mM phosphate buffer, pH 7.0, and 25 °C.; (B) Typical time trace at 423 nm with single-exponential fit; (C) Dependence of k obs -values from the cyanide concentration. The association rate constant k on was calculated from the slope, the dissociation rate constant k off from the intercept; (D) Hanes

  plot deduced from titration of MagKatG1 with increasing concentration of cyanide. Conditions: 7 µM MagKatG1 in 50 mM phosphate buffer, pH 7.0, and 25 °C.

Figure 7 B 3 BiochemicalC 5 0 6 -D 7

 73567 Figure 7 pH-profile of catalase and peroxidase activities of recombinant MagKatG1 (clone M133). Concentration of MagKatG1 was 0.2 µM and all measurements were performed at 25°C in 100 mM phosphate or citrate buffers in the range between pH 3.0 -9.0.

Table 2

 2 Thermostability of recombinant MagKatG1 (clones M133 and M166). n. d., not determined. Biochemical Journal Immediate Publication. Published on 10 Nov 2008 as manuscript BJ20081478 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081478

	Temperature	Loss of catalase activity	Loss of ABTS-peroxidase activity
	40 °C	95% after 22 h	stable, < 10% loss after 48 h
	45 °C	95% after 330 min	stable, < 10% loss after 28 h
	50 °C	95% after 48 min	stable, < 10% loss after 28 h
	55 °C	95% after 15 min	n. d.
	60 °C	95% after 8 min	50% after 7 h
	65 °C	95% after 1 min	50% after 4 h

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

© 2008 The Authors Journal compilation © 2008 Portland Press Limited

A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Portland Press Limited

Acknowledgements

This research was supported by the Austrian Science Foundation (FWF project P19793). We would like to thank Christian Würtz and Hanspeter Rottensteiner for providing us with KatGspecific antibody.


Moritz Hentschke
email: m.hentschke@uke.uni-hamburg.de

Ute Süsens
email: suesens@zmnh.uni-hamburg.deub

Uwe Borgmeyer
email: uwe.borgmeyer@zmnh.uni-hamburg.de

Transcriptional ERRγ2-mediated activation is regulated by Sentrin-specific proteases

Keywords: AF, activation function, DBD, DNA binding domain, EGFP, enhanced green fluorescent protein, ERR, estrogen receptor-related receptor, HEK, human embryonic kidney, N2A, neuroblastoma-2A, NDSM, negatively charged amino acid-dependent sumoylation motif, PDSM, phosphorylation-dependent sumoylation motif, PGC-1, peroxisome

Modification with small ubiquitin-like modifiers (SUMOs) has emerged as an important means of regulating the activity of transcription factors, often by repressing their activity. The estrogen receptor-related receptor γ (ERRγ, ERR3, NR3B3) is a constitutively active orphan nuclear receptor. A phosphorylation-dependent sumoylation motif (PDSM) is located in close vicinity of the amino-terminally located ERRγ2-specific activation function 1 (AF-1). Its function can be substituted by a negatively charged amino aciddependent sumoylation motif (NDSM). A mutational analysis reveals that ERRγ2-activity is modulated through sumoylation of a lysine residue at position 40 which in turn is regulated by phosphorylation. Phosphorylation in the +5-position relative to the sumoylation target is directly visualized by a high resolution electrophoretic mobility shift assay (EMSA). Sumoylation represses the activity of ERRγ both, with and without forced expression of the peroxisome proliferator-activated receptor gamma coactivator 1β (PGC-1β). Fusion proteins of a heterologous DNA binding domain with the ERRγ2 amino terminus demonstrate the function of the PDSM as the repression function 1 (RF-1) for the neighboring AF-1. Derepression is achieved by coexpression of Sentrin/SUMO-specific protease (SENP) family members. Together, our results demonstrate reversible phosphorylation-dependent sumoylation as a means to regulate the activity of an orphan nuclear receptor.

Orphan nuclear receptors are a class of endogenous receptors whose cognate ligands are unknown. Although they are often studied to ascertain the identification of physiological ligands, finding additional means to regulate their transcriptional activity is of great interest. Most transcription factors undergo post-translational modification like phosphorylation, acetylation and ubiquitination, which are known to modify their properties [START_REF] Faus | Post-translational modifications of steroid receptors[END_REF][START_REF] Fu | Acetylation of nuclear receptors in cellular growth and apoptosis[END_REF]. Recently, covalent modification by the small ubiquitinlike protein modifier (SUMO) has emerged as an important mechanism to regulate many protein activities [START_REF] Geiss-Friedlander | Concepts in sumoylation: a decade on[END_REF][START_REF] Meulmeester | Cell biology: SUMO[END_REF]. Similar to ubiquitin SUMO is activated by an E1 enzyme, conjugated by an E2 enzyme (UBC9) and bound to a substrate by a member of the E3 family. Sumoylation usually takes place on a lysine which is part of the core consensus motif ψKxE, where ψ represents an aliphatic branched amino acid residue and x is any amino acid [START_REF] Anckar | SUMO: getting it on[END_REF]. Sumoylation is thought to regulate protein functions by altering protein-interactions which in turn affects their subcellular localization and their activities. In case of transcription factors, sumoylation is generally accompanied by repression, suggestive of a promoted interaction with corepressors [START_REF] Lyst | A role for SUMO modification in transcriptional repression and activation[END_REF]. Sumoylation is a highly dynamic process regulated by the modulation of expression and activity of SUMO enzymes. In addition there is a crosstalk with other posttranslational modifications, including phosphorylation and acetylation [START_REF] Anckar | SUMO: getting it on[END_REF]. Phosphorylation-dependent sumoylation is mediated by a composite motif ψKxExxS that possesses a serine residue as a potential target for phosphorylation in the +5 position [7][START_REF] Stankovic-Valentin | An acetylation/deacetylation-SUMOylation switch through a phylogenetically conserved psiKXEP motif in the tumor suppressor HIC1 regulates transcriptional repression activity[END_REF][START_REF] Yang | A recurrent phospho-sumoyl switch in transcriptional repression and beyond[END_REF][START_REF] Gregoire | Control of MEF2 transcriptional activity by coordinated phosphorylation and sumoylation[END_REF][START_REF] Hietakangas | Phosphorylation of serine 303 is a prerequisite for the stress-inducible SUMO modification of heat shock factor 1[END_REF][START_REF] Shalizi | A calcium-regulated MEF2 sumoylation switch controls postsynaptic differentiation[END_REF]. In several SUMO substrates the phospho-sumoyl switch is mediated by the bipartite motif ψKxExxSP, termed phosphorylation-dependent sumoylation motif (PDSM) with an SP motif, a proline-directed phosphorylation site [7]. The SP motif corresponds to the ERK consensus site and thus may link the MAPK pathway and sumoylation [START_REF] Guo | Signalling pathways and the regulation of SUMO modification[END_REF]. A well studied example is the sequential phosphorylation and sumoylation of a PDSM motif in the amino terminus of the peroxisome proliferatoractivated receptor γ (PPARγ) turning off PPARγ-dependent transcription [START_REF] Floyd | Control of peroxisome proliferatoractivated receptor gamma2 stability and activity by SUMOylation[END_REF][START_REF] Ohshima | Transcriptional activity of peroxisome proliferator-activated receptor gamma is modulated by SUMO-1 modification[END_REF][START_REF] Pascual | A SUMOylationdependent pathway mediates transrepression of inflammatory response genes by PPAR-gamma[END_REF][START_REF] Yamashita | The transactivating function of peroxisome proliferator-activated receptor gamma is negatively regulated by SUMO conjugation in the amino-terminal domain[END_REF]. A PDSM can be viewed as a regulated version of the negatively charged amino aciddependent sumoylation motif, NDSM, characterized by acidic amino acids within the 10-amino acid downstream region [START_REF] Anckar | SUMO: getting it on[END_REF][START_REF] Yang | An extended consensus motif enhances the specificity of substrate modification by SUMO[END_REF]. SUMO-conjugation can be reversed by SUMO-specific proteases, also known as sentrin-specific proteases (SENPs) and as SMT3-specific isopeptidases [START_REF] Gong | Differential regulation of sentrinized proteins by a novel sentrin-specific protease[END_REF]. Apparently six mammalian SENPs regulate the pool of conjugatable SUMO and the half-life of conjugated proteins, albeit with different specificity towards SUMO paralogues and with different subcellular localization [START_REF] Mukhopadhyay | Modification in reverse: the SUMO proteases[END_REF]. During deconjugation SENPs can discriminate between SUMO-1 and SUMO-2/3 but the enzymatic efficiency can in addition be target protein-dependent [START_REF] Ihara | Noncovalent binding of small ubiquitin-related modifier (SUMO) protease to SUMO is necessary for enzymatic activities and cell growth[END_REF]. SENP3 and SENP5 are preferentially active in deconjugating SUMO-2/3-conjugated targets. The recent finding of an unexpected specificity of SENP6 and 7 towards synthetic substrates suggests that SUMOs may not be their physiological substrates at all [22]. SENP1 has been localized in nuclear pores and nucleoplasmic speckles, whereas SENP2 has been mainly detected in nuclear pores. SENP3 and SENP5 are nucleolar and SENP6 and SENP7 are nucleoplasmic family members. The capacity of SENPs to discriminate among substrates together with their distinct subcellular localization and expression pattern argues for non-redundant roles in vivo [START_REF] Mukhopadhyay | Modification in reverse: the SUMO proteases[END_REF]. Hietakangas et al. [7] identified a bipartite PDSM in the amino terminus of ERRγ2 and demonstrated that Lys 40 of ERRγ2 is the main SUMO acceptor. Resulting from different promoter usage the ERRγ gene encodes at least two isoforms with distinct amino termini, γ1 and γ2, the latter of which has a 23 residue extension [START_REF] Süsens | Alternative splicing and expression of the mouse estrogen receptor-related receptor gamma[END_REF]. This extension functions as an autonomous activation function, the AF-1, and thereby contributes to the higher transcriptional activity of ERRγ2 [START_REF] Hentschke | Identification of PNRC2 and TLE1 as activation function-1 cofactors of the orphan nuclear receptor ERRγ[END_REF]. Gal4 fusion proteins with the amino terminus of ERRγ1 repress, while those with the amino terminus of ERRγ2 activate appropriate reporter genes. Importantly, a GAL4 fusion protein containing only ERRγ2 residues 1-23 exhibits higher activity than the whole amino terminal domain [25]. These findings suggest that the repressive function coincides with the sumoylation motif. Here, we show that the AF-1 of ERRγ2 is regulated by the sumoylation site in its vicinity which can be defined as the repression function, RF-1, of ERRγ. Mutagenesis of the RF-1 in the context of a GAL4 fusion protein revealed that all mutants proposed to inhibit sumoylation led to a higher activity of the AF-1. Similarly, a Lys 40 to arginine mutant, as well as the mutation of the hydrophobic residue Ile 39 and of the acidic residue Glu 42 to alanine led to higher activity of full-length ERRγ2. Both, superactivation by replacing the potential phosphorylation acceptor Ser 45 with alanine and a phosphorylation-dependent mobility shift of protein DNA complexes during gel electrophoresis speak in favor of a functional PDSM. Generating an acidic moiety, by replacing both Ser 44 and Ser 45 with glutamic acid, results in down-regulation, supporting the view that the PDSM is the regulated form of an NDSM. This is further corroborated by the repression effect of phosphomimetic mutants in the downstream region. Furthermore, coexpression of SENPs suggest that regulation by phosphorylation is only one means to regulate the activity of ERRγ2. ERRγ2 is in addition derepressed by forced expression of desumoylation enzymes SENP1, SENP2, SENP3 and SENP5. Coimmunoprecipitation of ERRγ2 and SENP1, as well as the requirement of a functional sumoylation site for SENP-mediated activation speak in favor of a direct mechanism.

EXPERIMENTAL

Plasmids

To express GAL4 fusion proteins Gal4-ERRγ2/1-38 and Gal4-ERRγ2/1-48, ERRγ fragments were generated with the polymerase chain reactions using γ2F (5'-GTCGACATGGATTCGGTAGAACTTTGCCT-3' with a SalI restriction site shown in italics and the start ATG underlined) and reverse primers ERRγ-48stop (5'-TCTAGACTAGGAGGCTGGGCTGGAGGGTT-3') and ERRγ-38stop (5'-TCTAGACTAGAAGGACGAACAGCTGGAATCA-3') with XbaI and a BseY1 restriction site in italics and translational stop codons underlined. Mutants of Gal4-ERRγ2/1-48 were introduced by generating amplicons with the respective mutated reverse primers. Products were inserted into pGem-Teasy (Promega) and following sequence verification the SalI/XbaI inserts were excised and inserted in-frame into pBind3∆ in which the cytomegalovirus immediate early promoter drives expression of the DBD of the yeast GAL4 protein. The pBind3∆ vector is a derivate of pBind (Promega) with an altered multiple cloning site and lacking the Renilla luciferase module [25]. To generate mutants of ERRγ2, the mutant fragments and the nonmutated 5'end were excised via SalI and BseY1 and inserted into pSPUTK-ERRγ, a plasmid with a newly introduced 5'-located SalI restriction site. The insert was then -4 -

Cell culture, transient transfections, and reporter gene analysis

Human embryonic kidney 293 cells (293) and mouse neuroblastoma Neuro-2a (N2a) cells were grown in Opti-MEM without phenol red (Invitrogen) supplemented with 5% fetal calf serum, 200 units/ml of penicillin G, and 200 µg/ml streptomycin at 37 °C under a 5% CO 2 atmosphere. For transient transfections cells were seeded in 96well tissue culture plates (Greiner) and transfected the next day at ~ 50% confluence. 293 were transfected with FuGENE 6 (Roche), using 3 µl transfection reagent per microgram of DNA, whereas N2a cells were transfected with 2.5 µl Lipofectamine 2000 (Invitrogen) per microgram of DNA. A CMX expression plasmid coding for ßgalactosidase was used as a substitute in the controls of Fig. 1C. EGFP fluorescence generated by pEGFP-C3 (Clontech) was determined the next day (N2a) and two days after transfection (293), respectively, with a Wallac 1420 Multilabel Counter (PerkinElmer). Then light emission generated by luciferase activity with the Bright-Glo Luciferase Assay System according to the manufacturer's instructions (Promega) was measured with the Multilabel Counter. The light emission generated by luciferase activity was normalized to the level of EGFP-fluorescence activity derived from the EGFP control plasmid. Transfections were generally performed in septuplicate. Average relative luciferase light units and standard deviations were calculated using the Prism-4 program (GraphPad.com).

Electrophoretic mobility shift assay

N2A cells (2 x 10 5 ) were seeded in 6 well plates and transfected the next day with 2 µg of expression plasmids as described above. Extracts were prepared after two days by 3 cycles of freeze-thaw in 40µl extraction buffer consisting of 20mM HEPES, pH 7.4, 80mM NaCl, 20mM KCl, 2 mM dithiothreitol, 6 % glycerol and 1x Complete protease inhibitor cocktail (Roche) and centrifugation at 16,000 g. The supernatants were stored at -80 °C. For DNA binding, 2.5 µl of the extracts were diluted in 10 µl extraction buffer containing 0.5 µg human Cot-1 DNA and incubated with an excess of radioactively labelled double stranded oligonucleotides. Sequence of the DR0 element, generation of the radioactive probe and electrophoresis have been described [START_REF] Hentschke | Domains of ERRgamma that mediate homodimerization and interaction with factors stimulating DNA binding[END_REF]. The Gal4 binding element was generated by annealing of the oligonucleotides 5'-AGCTCGGAGGACTGTCCTCCGA-3' and 5'-AGCTTCGGAGGACAGTCCTCCG-3'. To achieve protein charge-dependent resolution, protein DNA complexes were resolved on 5% PAGE in 0.5 x TBE (pH 8.3) at 20V/cm in a cold room at 4 ºC until complexes covered a distance of approximately 18 cm. Dephosphorylation was achieved by incubating 8 µl of the -5 - A c c e p t e d M a n u s c r i p t protein extracts with 1 unit calf intestine alkaline phosphatase (Roche) for 30' at room temperature.

RESULTS

A sumoylation target sequence represses the autonomous AF-1 of ERRγ2

The 126 residues long N-terminus of ERRγ2 preceding the DNA binding domain (DBD) functions as a transcriptional activator when fused to the heterologous DBD of the yeast transcriptional activator Gal4 (Gal4-ERRγ2/1-126). In contrast, an analog fusion with the ERRγ1 amino terminus starting with the 24 th amino acid of ERRγ2 leads to transcriptional repression [25]. These findings not only argue in favor of a 23 amino acid long AF-1 but also in favor of a repressive function in the remaining 103 amino acids. An amino acid sequence comprising a sumoylation consensus sequence, a PDSM (φKxExxS), lies in the vicinity of the activation domain (Fig. 1A). The lysine in position 40 (K40) of ERRγ2 has already been shown to be the major sumoylation target of ERRγ [7]. Therefore, we compared transcriptional activation of the Gal4 DBD with three fusion constructs of the ERRγ2 amino terminus in human embryonic kidney derived 293 cells. Whereas activation by Gal4-ERRγ2/1-48 and by Gal4-ERRγ2/1-126 was similar, a further truncation of ten amino acids caused a fivefold higher activation (Fig. 1B). This fusion protein, Gal4-ERRγ2/1-38, lacks the sumoylation site, suggesting that sumoylation represses the AF-1. To test whether this effect is specific for human embryonic kidney cells we included mouse neuroblastoma-derived N2a cells in this study. Although overall activation was higher, the effects of the truncations in 293 and N2A cells were similar (Fig. 1C). In summary, these results suggested that amino acids 39 to 48 of ERRγ2 constitute an autonomous repression function-1 (RF-1) which regulates the AF-1 of ERRγ2.

Mutation of the sumoylation target sequence derepresses the autonomous AF-1 of ERRγ2

To get additional support for a functional sumoylation site, we introduced single amino acid mutations in the Gal4-ERRγ2/1-48 fusion protein. Neither an F38A nor a T41A mutation in the vicinity of the putative target Lys 40 did affect the activity of the fusion protein in 293 cells (Fig. 1D). The same holds true for an F38E mutation (not shown). Both amino acids, Phe 38 and Thr 41 , are not critical residues of the sumoylation consensus sequence. In contrast, the I39A and the E42A mutation, representing the hydrophobic and the acidic residue of the ψKxE consensus sequence respectively, both caused a fourfold increase in reporter gene activity. A sevenfold increase was observed by the conservative K40R mutation of the putative sumoylation target supporting the hypothesis that a Lys 40 modification leads to a reduced activity and that the AF-1 is regulated by sumoylation at Lys 40 . Similar results were obtained in N2A cells (Fig. 1E) supporting the above findings and arguing for a general regulatory mechanism.

Mutations of a putative phosphorylation site affect transcriptional activity of the AF-1

Next, we elaborated whether modification of the neighboring phosphorylation site affects the function of the RF-1 as shown for other PDSMs. In contrast to Ser 44 A c c e p t e d M a n u s c r i p t is a predicted target of phosphorylation. The S44A and S44E mutations did not significantly affect the transcriptional activity of the fusion proteins in HEK cells (Fig. 1F) or N2A cells (Fig. 1G). However, expression of the S45A mutant enhanced luciferase activity more than twofold in both cell lines, suggesting that phosphorylation of this residue enhances sumoylation. Introducing a negative charge by an S45E mutation diminished the activation effect (Fig. 1F,G). A double mutation of both serines to glutamic acid (S44/45E) was neutral. These findings suggest that a negative charge introduced by phosphorylation of Ser 45 leads to increased sumoylation and consequently to a reduced transcriptional activity mediated by the AF-1. To analyze, whether mutations affect protein expression or DNA binding, we performed an EMSA with N2A extracts expressing Gal4-ERRγ2/1-48 as well as several of its mutants. DNA binding was tested with a radioactive labeled Gal4 DNA binding site. All bands showed similar intensity indicating that differences in activation were neither the result of altered expression nor of altered DNA binding.

An NDSM can substitute a mutated PDSM

The question arose whether an NDSM could substitute a mutated PDSM. To this end, the transcriptional activities of Gal4-ERRγ2/1-48(S45A) and of the phosphomimetic mutant S45D were compared with S45A mutants with additional single or combinatory P46D, A47D and S48D substitutions. As expected for an NDSM, all negative charges in the +6 to +8 position reduced the transcriptional activity of Gal4-ERRγ2/1-48(S45A) (Fig. 2). The PDSM was functionally replaced by acidic amino acids in all three positions, as well as only in the +7/+8 position. Together, these results showed that the AF-1 is regulated by a repressive function, the RF-1, located in its close vicinity and similar results in two distinct cell lines suggest a general regulatory mechanism. The repressive activity which overlaps with a sumoylation site was relieved by mutations predicted to reduce or to abolish sumoylation. The altered activity is not greatly regulated through DNA binding or stability of the proteins. Mutating the putative phosphorylation site suggests that sumoylation is regulated by kinases and phosphatases.

The PDSM is phosphorylated

Indirect evidence suggested that the serine in the +5 position of the sumoylation site is a target of phosphorylation. Since separation of protein-DNA complexes in EMSAs is dependent on the charge of the protein, we investigated protein-DNA complexes at high resolution. The non-mutated fusion protein (Gal4-ERRγ2(1-48)) has a pI of 6.45 and a net charge of -1. Phosphorylation changes the pI to 6.11 and, depending whether the phosphate group is single or doubly ionized, the net charge to -2 or to -3. In the non-mutated fusion protein as well as in all mutants with an unaffected Ser 45 , three complexes can be distinguished (Fig. 3A). Based on the fact that the Gal4-DBD binds as a dimer to DNA, these bands presumably reflect unmodified, single and double modified dimer-DNA complexes, respectively. The triplet of the E42A mutant ran slower, whereas the K40A mutant ran faster, supporting the interpretation that two charges significantly altered the velocity of the complex. In contrast, the Ser 45 mutants S45A, S44/45A, and S44/45E exhibited dominant single bands with the expected velocities. Therefore, these data support the assumption that Ser 45 is the major phosphorylation target. In addition, the analysis of mutants affecting sumoylation -7 - A c c e p t e d M a n u s c r i p t showed that the sumoylation site does not affect protein modification by phosphorylation.

To proof that the occurrence of triplet bands were indeed the result of partial phosphorylation, extracts were pre-incubated with alkaline phosphatase. Dephosphorylation turned the triplet to a single band, reflecting the non-modified protein (Fig. 3B). As expected, Ser 45 mutants were not affected by dephosphorylation.

Activation of full-length ERRγ2 by mutation of the amino-terminal sumoylation motif

Given that the sumoylation motif negatively influenced the AF-1 in the context of a fusion protein, we next asked whether this was true in the full-length protein. Therefore, we tested transcriptional activation by ERRγ2 in N2A cells with pBV-4xDR0, a well established ERRγ reporter with four DR0 elements previously shown to be bound by ERRγ2 dimers [START_REF] Hentschke | Domains of ERRgamma that mediate homodimerization and interaction with factors stimulating DNA binding[END_REF][START_REF] Huppunen | Dimerization modulates the activity of the orphan nuclear receptor ERRgamma[END_REF]. The I39A, the K40R, and the E42A mutation, all within the sumoylation motif resulted in an approximate four-to six-fold increase in transcription when compared to the wild-type (WT) protein (Fig. 4A). These results support the hypothesis that sumoylation represses the transcriptional activity of ERRγ2. To question whether sumoylation is regulated by phosphorylation, we tested the S44/45A mutation in the context of the full-length protein. The activity of the mutated protein was about threefold when compared to the wild-type protein (Fig. 4A). To verify that ERRγ and its mutants were properly expressed, cell extracts of transiently transfected N2A cells were tested in an EMSA (Fig. 4B). There were no gross differences in the intensity of protein complexes with the DR0 element. The specificity of the complexes was verified by generating supershift bands with αERRγ, an ERRγ-specific antiserum [START_REF] Hentschke | Domains of ERRgamma that mediate homodimerization and interaction with factors stimulating DNA binding[END_REF]. In summary, our results imply that the transcriptional activity of ERRγ2 is regulated by sumoylation of Lys 40 which in turn is regulated by phosphorylation.

Sumoylation mutants in the context of a coactivator

Transcriptional activation of ERRγ is greatly enhanced by coactivators, namely by PGC-1β [30,[START_REF] Kamei | PPARgamma coactivator 1beta/ERR ligand 1 is an ERR protein ligand, whose expression induces a high-energy expenditure and antagonizes obesity[END_REF]. Therefore, we tested whether coactivation in N2A cells is affected by the sumoylation site. The activities of the I39A mutant as well as those of the K40R mutant were clearly enhanced by PGC-1β, indicating that both, the wild-type and the mutants deficient in sumoylation are targets for the coactivator (Fig. 5). In addition, the results revealed that PGC-1β did not overcome the repressive effect of the RF-1.

AF-1-dependent transcription is enhanced by SENP1 expression

Transcriptional repression by sumoylation can best be verified by a partial relief mediated by SUMO-protease expression. Indeed, forced expression of mammalian sentrin-specific protease SENP1, a widely expressed enzyme clearly enhanced reporter gene activation by Gal4-ERRγ2/1-48 (Fig. 6A, lanes 1 and 2). To question whether sumoylation is a prerequisite for the SENP1 effect, various Gal4-ERRγ2/1-48 mutants were coexpressed with SENP1 in N2a cells (Fig. 6A). Clearly, the SENP1 effect is attenuated or abolished in mutants I39A, K40A and S45A all affecting amino acids of the PDSM consensus. Namely, the K40A mutant is not further activated. The reduced activation by SENP1 of the S45A mutant corroborates the claim that Ser 45 phosphorylation leads to enhanced sumoylation (Fig. 6A).

-8 - To verify that the activation depends on the enzyme activity, we introduced a C603S mutation in the catalytic center of SENP1. This mutation has previously been shown to abolish the activity of the cysteine protease and may function as a dominant negative enzyme [START_REF] Bailey | Characterization of the localization and proteolytic activity of the SUMO-specific protease, SENP1[END_REF]. Coexpression of the mutated SENP1 protein (mut-SENP1) did not affect reporter gene expression indicating a direct activation of the ERRγ2 fusion protein by desumoylation (Fig. 6B). As expected, the K40A mutant was not affected by coexpression of the mutated protein. Sumo proteases bind to the Sumo moiety through salt bridges [START_REF] Ihara | Noncovalent binding of small ubiquitin-related modifier (SUMO) protease to SUMO is necessary for enzymatic activities and cell growth[END_REF]. The notion that the enzymatic activity rather than binding alone activates ERRγ2 is underlined by coexpression of a SENP1 fusion protein with the viral VP16 activator domain. Activation by coexpression of SENP1 and SENP1-VP16 were indistinguishable (not shown).

SENP proteins regulate the activity of the AF-1

Mammalian sentrin-specific proteases (SENP1, 2, 3, 5, 6, and 7) differ in their expression pattern, their cellular localization as well as their substrate specificity. Therefore SENP1 to 7 were cotransfected with the AF-1 fusion construct Gal4-ERRγ2/1-48 and with the respective K40R mutant (Fig. 7A,B). Activation was limited to the non-mutated fusion protein and to SENP1, 2, 3 and 5. The highest activation was observed with SENP2, a protease with a substrate preference for SUMO-2/3. Activation by the SUMO-2/3-specific proteases SENP3 and 5 speaks in favor of ERRγ regulation by SUMO-2/3.

ERRγ2-mediated gene activation is regulated by SENP

To analyze whether overexpression of SENPs activate ERRγ2 driving DR0 elementmediated luciferase expression, the full-length protein was transfected along with SENP1 to 7 into N2a cells (Fig. 8A). The results were very similar to those with the AF-1 fusion protein. Activation was observed with SENP1, 2, 3 and 5. However the effects of SENP1 and 2 were indistinguishable. The negative control with a βgalactosidase plasmid as a substitute for the ERRγ plasmid proofed that SENPmediated activation of DR0 reporter gene expression was ERRγ-dependent (Fig. 8B). To elaborate whether activation by SENP was mediated by the RF-1, we tested the SENP effect with the K40R mutation. SENP-mediated activation was greatly diminished in the presence of the RF-1 mutant (Fig. 8C). This finding supports the biochemical analysis identifying K40 as the major sumoylation target of ERRγ [7]. A spurious activation of the K40R mutant suggests a minor involvement of a sumoylation site in the ligand binding domain. Next, we elaborated whether SENP activation depends on the presence of the AF-1. Compared to ERRγ2, coexpression of ERRγ1 with SENP1 and with SENP2 resulted in a weak activation emphasizing the importance of the AF-1 (Fig. 8D). Together these data reveal the regulation of ERRγ2 by phosphorylation-dependent sumoylation and underscore the importance of the γ2-specific AF-1.

DISCUSSION

Small ubiquitin-like modifier conjugation has emerged as an important posttranslational control of protein functions [START_REF] Geiss-Friedlander | Concepts in sumoylation: a decade on[END_REF][START_REF] Meulmeester | Cell biology: SUMO[END_REF]. Here, we provided evidence for the regulation of ERRγ activity by dynamic SUMO conjugation of a lysine in the amino terminal domain. This sumoylation site, apparently regulated by a prolinedirected serine phosphorylation site defines an autonomous repression function, the -9 - In addition, the transcriptional activity of the closely related receptor ERRα1 (NR3B1) is regulated by phosphorylation-dependent sumoylation [START_REF] Vu | Phosphorylation-dependent sumoylation of estrogen-related receptor alpha1[END_REF]. While this study was in preparation, Tremblay et al. [34] reported functional sumoylation site in the amino termini of both, ERRα and ERRγ. Their data support the hypothesis that the SUMO moiety conveys a repressive effect. In addition Tremblay et al. showed that sumoylation site mutants display wild-type nuclear localization and DNA binding properties. Our analysis supports their main finding concerning ERRγ, an upregulation in transcriptional activity by the K40R mutation. In contrast to this study, in our experimental settings there was no necessity for a forced expression of coactivator proteins. In addition, we provided evidence that the RF-1 regulates the activity of the neighboring AF-1 independently of additional domains. This goal was achieved by analysis of GAL4-ERRγ2/1-48 mutants, constructs including the AF-1 (amino acids 1-23) as well as the sumoylation target sequence "IKTE" (amino acids 39-42). F38A and T41A mutations that do not alter the consensus sumoylation motif did not greatly alter the transcriptional activity of the fusion protein. In contrast, GAL4-ERRγ2/1-38 lacking the motif, as well as the I39A and E42A mutant were derepressed supporting the function of both, the hydrophobic and the acidic residue. In addition, both the K40A and the K40R mutant that preclude sumoylation showed a high constitutive activity. The mechanisms by which sumoylation interferes with the AF-1 remain unresolved. Based on the study of [START_REF] Tremblay | Phosphorylation-dependent sumoylation regulates estrogen-related receptor-alpha and -gamma transcriptional activity through a synergy control motif[END_REF] and our study, a dramatic effect on protein expression and DNA binding can be excluded. Sumoylation of the RF-1 may interfere with coactivator binding or interaction with the transcriptional initiation complex. On the other hand, there may be a gain of function by attracting corepressors. One possible candidate is the corepressor Daxx which has been shown to be actively recruited by sumoylated transcription factors, including the androgen receptor [START_REF] Kuo | SUMO modification negatively modulates the transcriptional activity of CREB-binding protein via the recruitment of Daxx[END_REF][START_REF] Lin | Role of SUMO-interacting motif in Daxx SUMO modification, subnuclear localization, and repression of sumoylated transcription factors[END_REF]. Alternatively, binding of coactivator and corepressor could be mutually exclusive. However, we can not exclude an additional regulation by acetylation of K40 as for example in the case of the transcription factor MEF2. In MEF2 a phosphorylation-regulated sumoylation-acetylation switch of a PDSM is essential for postsynaptic differentiation [START_REF] Gregoire | Control of MEF2 transcriptional activity by coordinated phosphorylation and sumoylation[END_REF][START_REF] Shalizi | A calcium-regulated MEF2 sumoylation switch controls postsynaptic differentiation[END_REF]. The Gal4 fusion proteins allowed us to explore the ERRγ PDSM with its putative Ser 45 phosphorylation site at the +5 position by a high resolution EMSA. The nonmutated protein which binds as a dimer gives rise to three complexes. The triplets of the K40A and E42A mutants are shifted up and down by one position, respectively. This suggests that the triplet represents the non-modified, the single and the double phosphorylated dimer, each differing by 2 charges. In vitro phosphatase treatment shifts the triplet to the respective slow migrating band, a direct evidence for the phosphorylation status. The high resolution EMSA may be of broader interest as a prototype for studying phosphorylation pathways. The functional PDSM analysis showed derepression of the S45A mutant and efficient repression of the phosphomimetic S45E and S45D mutants suggesting that the negative charge imparted by phosphorylation is the main trigger for ERRγ sumoylation. Therefore, this orphan receptor belongs to the large group of protein whose activity is regulated by kinase and phosphatase activities. A proline in position 46 points to proline-directed protein kinases as possible regulators of ERRγ. Possible candidates are extracellular signal-regulated kinase (Erk)-1 and -2, MAPKs whose optimum consensus sequence for phosphorylation was defined as "PxSP" where x is a neutral or basic amino acid but ideally not a proline [START_REF] Clark-Lewis | Definition of a consensus sequence for peptide substrate recognition by p44mpk, the meiosisactivated myelin basic protein kinase[END_REF][START_REF] Gonzalez | Identification of substrate recognition determinants for human ERK1 and ERK2 protein kinases[END_REF]. This motif is in -10 - A c c e p t e d M a n u s c r i p t accordance with the primary sequence "PSSP" directly following the sumoylation motif "IKTE" in ERRγ. The S44/45E mutant being less active than the S45E mutant is in agreement with the finding that acidic residues in NDSMs are more or less overrepresented within the 10-amino acid downstream region [START_REF] Anckar | SUMO: getting it on[END_REF][START_REF] Yang | An extended consensus motif enhances the specificity of substrate modification by SUMO[END_REF]. To directly approach the question whether an NDSM can substitute the PDSM, negative charged amino acids in the +5 to +8 position were introduced into the S45A mutant. The activity of Gal4-ERRγ2/1-48(S45A) was clearly repressed by additional aspartate residues proving that these negative charges functionally substitute serine phosphorylation. The ERRγ PDSM also modulated the activity of the full-length protein, both in the absence and in the presence of forced coactivator expression. This finding contradicts the recent report by Tremblay et al. [34] who reported the necessity for coactivator expression to achieve activation by a K40R mutation. One plausible cause for this discrepancy could be a higher level of coactivator proteins in our cell lines. This hypothesis is supported by the observation that the mere expression of ERRγ2 resulted in a more robust activation under our experimental settings. However, our experiments with a heterologous DBD support a direct repression of a construct without involvement of the AF-2. Therefore, it is worthwhile to clarify whether this discrepancy is due to the different cell lines, reporter constructs or other experimental settings.

In addition to a regulation by phosphorylation, our data is suggestive of an ERRγ regulation through control of the SUMO pathway itself. Accumulating evidence shows that target sumoylation is affected by regulated expression of various components of the sumoylation pathway [START_REF] Liu | Regulation of the sumoylation system in gene expression[END_REF]. Namely PIAS [protein inhibitor of activated STAT (signal transducer and activator of transcription)] proteins promote SUMOylation of transcription factors in a manner that resembles the action of RINGtype ubiquitin E3 ligases [START_REF] Palvimo | PIAS proteins as regulators of small ubiquitin-related modifier (SUMO) modifications and transcription[END_REF]. Since PIASxα/ARIP3 (androgen receptor-interacting protein) [START_REF] Kotaja | PIAS proteins modulate transcription factors by functioning as SUMO-1 ligases[END_REF] [START_REF] Janne | Androgen-receptor-interacting nuclear proteins[END_REF][START_REF] Kotaja | ARIP3 (androgen receptor-interacting protein 3) and other PIAS (protein inhibitor of activated STAT) proteins differ in their ability to modulate steroid receptor-dependent transcriptional activation[END_REF] is involved in nuclear receptor regulation we studied the effect of a forced expression on ERRγ regulation. Our data do not support a specific effect (data not shown). However, at this point we can not exclude that sumoylation proteins were not limited in the cell lines. Forced expression of PIASy has been shown to increase sumoylation of ERRγ [34]. However, pronounced reporter gene repression by forced expression PIASy and activation by PIASy-VP16 in the absence of ERRγ did not allow us to determine the ERRγ-specific effects (data not shown). In contrast, reporter gene activation by SENPs was strictly ERRγ-dependent. Therefore, we propose ERRγ regulation by the level of SUMO proteases, as well as by their localization and activity. Enzyme activity rather than stable binding as the basis of activation is supported by a fusion with the VP16 domain which did not act as a superactivator. The cysteine mutants of SENP1 and 2 still bind to SUMOs [START_REF] Ihara | Noncovalent binding of small ubiquitin-related modifier (SUMO) protease to SUMO is necessary for enzymatic activities and cell growth[END_REF][START_REF] Bailey | Characterization of the localization and proteolytic activity of the SUMO-specific protease, SENP1[END_REF] consistent with a weak activation of Gal4-ERRγ/1-48 by mut-SENP1-VP16 constructs (data not shown).

SENPs can be discriminated according to their substrate specificity, their subcellular distribution and their expression pattern. Coexpression of SENP1, 2, 3, and 5 clearly activated full-length ERRγ and fusion proteins in an RF-1-dependent manner. The regulated RF-1 depends on an intact PDSM. The observation of SENP2 being superior to SENP1 when coexpressed with Gal4-ERRγ/1-48 but not with full-length ERRγ may reflect substrate specificity. Coexpression of SENP6, SENP7 and of SENP8 (not shown) did not change ERRγ activity. Whereas SENP8/DEN1 -11 - A c c e p t e d M a n u s c r i p t demonstrates activity only against Nedd8-modified proteins [START_REF] Gan-Erdene | Identification and characterization of DEN1, a deneddylase of the ULP family[END_REF][START_REF] Mendoza | NEDP1, a highly conserved cysteine protease that deNEDDylates Cullins[END_REF][START_REF] Wu | DEN1 is a dual function protease capable of processing the C terminus of Nedd8 and deconjugating hyper-neddylated CUL1[END_REF], it is generally assumed that all other SENPs cleave SUMOs. However, this has only been investigated in detail with SENP1, 2, 3 and 5. Sparse data suggesting that SENP6 and 7 are desumoylating enzymes and their unexpected substrate specificity led to the speculation that they either recognize still uncharacterized natural substrates or have evolved a different mechanism of recognition of SUMO substrates from SENP [START_REF] Drag | DeSUMOylating enzymes--SENPs[END_REF]. Indeed, a recent biochemical analysis reveals specificity for SUMO2/3, especially when poly-SUMO2 or poly-SUMO3 chains are used as substrates [START_REF] Lima | Structure of the Human SENP7 Catalytic Domain and Poly-SUMO Deconjugation Activities for SENP6 and SENP7[END_REF]. Of the three mammalian SUMO paralogues SUMO-1 is ≈45% identical to SUMO-2 and SUMO-3 which are 96% identical to each other. Studies with SENPs reveal that SENP1 preferentially deconjugates SUMO-1, whereas SENP2 is less efficient at deconjugating SUMO-1 substrates [START_REF] Mukhopadhyay | Modification in reverse: the SUMO proteases[END_REF]. SENP3 and 5 are SUMO-2/3-specific with a high level of discrimination against SUMO-1. Although SUMO-2 and -3 are nearly identical, SUMO-3 is only inefficiently processed by SENP1 and SENP2. Therefore, activation by overexpression of SENP1 to 5 speaks in favor of ERRγ modification by SUMO-2. Experimental data show that dynamics is much slower for SUMO-1 than for SUMO-2, supporting a regulatory function of SUMO-2.

Studying reversible sumoylation of transcription factors like PPARs and ERRs offers fascinating opportunities to understand the cellular roles of SENPs. In addition, it will be important to decipher the signals and pathways for turning the phosphorylation switch. PPARs and ERRs together with their coregulators have been implicated in the regulation of energy the metabolism [49]. In addition accumulating evidence points the implication of ERRγ as a therapeutic for cancer [START_REF] Ariazi | Estrogen-related receptors as emerging targets in cancer and metabolic disorders[END_REF][START_REF] Yu | ERRgamma suppresses cell proliferation and tumor growth of androgen-sensitive and androgeninsensitive prostate cancer cells and its implication as a therapeutic target for prostate cancer[END_REF]. Therefore, these receptors attract attention as potential targets for pharmaceutical design. Understanding their coordinated regulation is of general interest and may lead to means of disease prevention or intervention in diseases related to the metabolic syndrome. C,E,G) were transfected with the GAL4 reporter pGL4/5-Luc and expression vectors coding for fusion proteins of the Gal4 DBD with the indicated elements of ERRγ2. The luciferase activity was normalized to the fluorescence generated by a cotransfected EGFP vector. Relative activity is given with the standard deviation based on a typical experiment performed in septuplicate. B, C) A repression function is located between residues 38 and 48 of ERRγ2. The activity generated by the Gal4 fusion with the amino terminal 126 amino acids of ERRγ2 was set as 1 and compared with fusions of the first 48 and of the first 38 amino acids, respectively, as well as with the empty Gal4 vector (-). D, E) Mutants of the sumoylation motif in the ERRγ2/1-48 fusion protein lead to an elevated transcriptional activity. Activation by non-mutated Gal4-ERRγ2/1-48 was arbitrarily set to 1 and compared to its mutants as indicated, as well as to the empty vector. F, G) Mutations of a putative phosphorylation site affect transcriptional activation. As in D and E activity of the mutants is shown in relation to the ERRγ2/1-48 fusion protein as indicated. H) Mutations do not affect expression and DNA binding of Gal4 fusion proteins. Extracts of N2A transfected with the Gal4 expression vector (Gal4) or constructs giving rise to the indicated Gal4-ERRγ2/1-48 fusion proteins were subjected to EMSA with a radioactive labeled Gal4 response element. Only the shifted bands are shown. Cell extracts transfected with a β-Galactosidase plasmid served as a negative control. Fig. 2 Functional substitution of a PDSM by NDSMs. Transcriptional activation of pGL4/5-Luc, a luciferase reporter with five Gal4 binding sites upstream of a TATA box, by the Gal4 fusion protein Gal4-ERRγ2/1-48 (nonmut.) was set as 1 and compared to activation by the phosphorylation-deficient mutant S45A and the phosphomimetic mutant S45D. To elaborate whether a negative charge in the downstream region is accompanied by down-regulation of the S45A mutant, the activity of mutants with aspartate residues substituting Pro 46 , Ala 47 , and Ser 48 was analyzed, as indicated. Fig. 4 Mutations of the sumoylation motif led to an enhanced transcriptional activity of ERRγ2 in N2A cells. A) Transcriptional ERRγ2 (WT) activation of a luciferase reporter with four DR0 response elements was set as 1 and compared to activation by I39A, K40R and E42A mutants, respectively. In addition, the S44/45A mutant affecting the phosphorylation site and a βGal expression plasmid as negative control (-) were tested. B) An EMSA was performed with cellular extracts of N2A cells transfected with expression plasmids coding for wild-type, mutant ERRγ2 and βGalactosidase (-) as indicated. The addition of αERRγ, a rabbit antiserum directed against ERRγ is indicated. Only the upper part of the gel is shown. Fig. 5 ERRγ2 and its sumoylation mutants are activated by the nuclear receptor coactivator PGC1β. ERRγ2-dependent activation of pBV-4xDR0-Luc in N2A cells was studied in the absence (-) and in the presence (+) of PGC1β as indicated. The activation by wild-type ERRγ2 (WT) was set as 1 and compared with the ERRγ2 mutants I39A and K40R. Fig. 6 Enhancement of AF-1 mediated gene activation by SENP1 in the presence of a functional PDSM. A) Activation by SENP1 in 293 cells is attenuated or abolished by mutations affecting the sumoylation consensus sequence. Gal4-ERRγ2/1-48 (nonmut.) and its mutants were coexpressed with either SENP1 (+) or with βGal expression plasmids (-) as negative controls. Activity in the presence of Gal4-ERRγ2/1-48 (lane 1) was set as 1. B) Activation of Gal4-ERRγ2/1-48 and of its Lys 40 to arginine mutant K40R in the presence of either a negative control plasmid (-/-), of a SENP1 expression plasmid (SENP1 +) or of a plasmid driving the expression of a desumoylation deficient SENP1 mutant (mut-SENP1 +). Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Figure legends

M D S V E L C L P E S F S L H Y E E E L L C R

Biochemical

  Journal Immediate Publication. Published on 10 Dec 2008 as manuscript BJ20081556 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081556 A c c e p t e d M a n u s c r i p t excised via flanking HindIII and EcoR5 restriction sites and inserted into pCMX. Due to the cloning strategy these constructs code for the full-length ERRγ2 with an amino terminal tag of three additional amino acids. The SENP1 expression plasmid pCDNA-FH-Senp1 was provided by Dr. O'Hare of the Marie Curie Research Institute, United Kingdom [26]. To generate non-functional mut-Senp1, Cys 603 was mutated to a serine by a PCR approach. Full-length pCMV-SPORT6 expression plasmids (Invitrogen) coding for SENP2 (IMAGE ID 3483046), SENP3 (IMAGE ID 5344274), SENP6 (IMAGE ID 5149995) and SENP7 (IMAGE ID 5364345) were obtained by the RZPD (Deutsches Ressourcenzentrum fur Genomforschung GmbH), Berlin, Germany [27]. The Senp5 cDNA obtained by the RZPD in the cloning vector pYX-Asc (IMAGE ID 6812169) was cloned via SalI and NotI restriction enzymes into pCMV-SPORT6. The other plasmids have been previously described.

Biochemical

  Journal Immediate Publication. Published on 10 Dec 2008 as manuscript BJ20081556 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081556

, Ser 45 - 6 -

 456 Biochemical Journal Immediate Publication. Published on 10 Dec 2008 as manuscript BJ20081556 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081556

  Biochemical Journal Immediate Publication. Published on 10 Dec 2008 as manuscript BJ20081556 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081556

Biochemical

  Journal Immediate Publication. Published on 10 Dec 2008 as manuscript BJ20081556 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081556 A c c e p t e d M a n u s c r i p t

Biochemical

  Journal Immediate Publication. Published on 10 Dec 2008 as manuscript BJ20081556 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081556 A c c e p t e d M a n u s c r i p t RF-1. Hietakangas et al. [7] already showed that Lys 40 of ERRγ2 is the major target of sumoylation.

Biochemical

  Journal Immediate Publication. Published on 10 Dec 2008 as manuscript BJ20081556 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081556

Biochemical

  Journal Immediate Publication. Published on 10 Dec 2008 as manuscript BJ20081556 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081556

Fig. 1 A

 1 Fig. 1 A sumoylation motif represses the amino terminal ERRγ2-specific AF-1. A) The amino terminus of ERRγ2 is shown in the one letter code. Residues 1 to 23 corresponding to the AF-1 are shown in italics, amino acid 24 is the putative start methionine of ERRγ1. Amino acids complying with the sumoylation consensus sequence are underlined, the putative sumoylation target Lys 40 (K40) and the putative phosphorylation target Ser 45 (S45) are highlighted. The carboxy termini of the Gal4 fusion proteins ERRγ2/1-38 and ERRγ2/1-48 are indicated. B-G) 293 cells (B, D, F) and N2A cells (C, E, G) were transfected with the GAL4 reporter pGL4/5-Luc and expression vectors coding for fusion proteins of the Gal4 DBD with the indicated elements of ERRγ2. The luciferase activity was normalized to the fluorescence generated by a cotransfected EGFP vector. Relative activity is given with the standard deviation based on a typical experiment performed in septuplicate. B, C) A repression function is located between residues 38 and 48 of ERRγ2. The activity generated by the Gal4 fusion with the amino terminal 126 amino acids of ERRγ2 was set as 1 and compared with fusions of the first 48 and of the first 38 amino acids, respectively, as well as with the empty Gal4 vector (-). D, E) Mutants of the sumoylation motif in the ERRγ2/1-48 fusion protein lead to an elevated transcriptional activity. Activation by non-mutated Gal4-ERRγ2/1-48 was arbitrarily set to 1 and compared to its mutants as indicated, as well as to the empty vector. F, G) Mutations of a putative phosphorylation site affect transcriptional activation. As in D and E activity of the mutants is shown in relation to the ERRγ2/1-48 fusion protein as indicated. H) Mutations do not affect expression and DNA binding of Gal4 fusion proteins. Extracts of N2A transfected with the Gal4 expression vector (Gal4) or constructs giving rise to the indicated Gal4-ERRγ2/1-48 fusion proteins were subjected to EMSA with a radioactive labeled Gal4 response element. Only the shifted bands are shown. Cell extracts transfected with a β-Galactosidase plasmid served as a negative control.

Fig. 3

 3 Fig. 3 Charge-dependent resolution of protein DNA complexes allows the direct visualization of Ser 45 -phosphorylation. Following electrophoretic separation, only the shifted bands detected by phosphor imaging of a 32 P-labeled Gal4 binding site are shown. A) Cellular extracts of N2A cells forced to express Gal4-ERRγ2/1-48 (nonmut.) and its mutant were analyzed by high-resolution EMSA as indicated. B) Before subjecting to an EMSA, cell extracts of transfected cells were preincubated in the absence (-) or in the presence (+) of alkaline phosphatase.

Fig. 7

 7 Fig.7AF-1 dependent gene activation in N2A cells is modified by coexpression of desumoylation enzymes. Activation of pGL4/5-Luc in the absence of SENP proteins (-) is set as 1. A) AF-1 dependent activation by Gal4-ERRγ2/1-48 was studied in the absence (-) and in the presence of SENP proteins as indicated. B) Activation by the Lys40 to arginine mutant K40R of Gal4-ERRγ2/1-48 in the absence (-) and in the presence of SENP proteins as indicated.

Fig. 8

 8 Fig. 8 ERRγ2 activity is mediated by coexpression of SENP proteins. Plasmids coding for ERRγ2 (A), β-Galactosidase as a negative control (B), the Lys 40 to arginine mutation of ERRγ2 (C) and for ERRγ1 (D) were transfected without (-) or along with one of six SENP family members in N2A cells, as indicated. Luciferase activities generated by pBV-4xDR0-Luc were measured and the activities in the absence of coexpressed proteases were set as 1.

1 2 3 MBiochemical

 3 Figure 1

Figure 2 nBiochemical

 2 Figure 2

Figure 3 F

 3 Figure 3

  Figure 4

  Figure 5

  Figure 7

Figure 8 -D

 8 Figure 8

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

© 2008 The Authors Journal compilation © 2008 Portland Press Limited

Acknowledgements

We are grateful to Peter O'Hare (Marie Curie Research Institute) and Jorma J. Palvimo (University of Helsinki) for supplying expression plasmids for SENP1 and ARIP3, respectively.

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.