Damon A Lowes

Helen F Galley
email: h.f.galley@abdn.ac.uk

Mitochondrial protection by the thioredoxin-2 and glutathione systems in an in vitro endothelial model of sepsis

Keywords: Sepsis, oxidative stress, endothelium, inflammation, peroxiredoxin, thioredoxin

Mitochondrial protection by the thioredoxin-2 and glutathione systems in an in vitro endothelial model of sepsis

Damon A. Lowes, Helen F

INTRODUCTION

Sepsis is a severe infection causing dysregulation of inflammatory responses and may result in multiple organ dysfunction syndrome (MODS), which has a mortality rate of around 70% [START_REF] Porter | Current status of clinical trials with anti-TNF[END_REF][START_REF] Shapiro | The association of sepsis syndrome and organ dysfunction with mortality in emergency department patients with suspected infection[END_REF]. Oxidative stress and mitochondrial dysfunction are associated with sepsis and organ failure in animal models [START_REF] Supinski | Hemin prevents cardiac and diaphragm mitochondrial dysfunction in sepsis[END_REF][START_REF] Zapelini | Antioxidant treatment reverses mitochondrial dysfunction in a sepsis animal model[END_REF][START_REF] Crouser | Abnormal permeability of inner and outer mitochondrial membranes contributes independently to mitochondrial dysfunction in the liver during acute endotoxemia[END_REF][START_REF] Gellerich | Impaired energy metabolism in hearts of septic baboons: Diminished activities of Complex I and Complex II of the mitochondrial respiratory chain[END_REF][START_REF] Simonson | Altered mitochondrial redox responses in gram negative septic shock[END_REF] and with poorer outcome in patients with sepsis [START_REF] Fredriksson | Derangements in mitochondrial metabolism in intercostal and leg muscle of critically ill patients with sepsis-induced multiple organ failure[END_REF][START_REF] Fink | Reactive oxygen species as mediators of organ dysfunction caused by sepsis, acute respiratory distress syndrome, or hemorrhagic shock: potential benefits of resuscitation with Ringer's ethyl pyruvate solution[END_REF][START_REF] Singer | Mitochondrial function in sepsis: acute phase versus multiple organ failure[END_REF][START_REF] Wallace | Mitochondrial diseases in man and mouse[END_REF].

The impact of mitochondrial functional capacity and the mechanisms related to mitochondrial dysfunction during sepsis and MODS are still not fully understood. During sepsis, leucocytes and mitochondria produce elevated amounts of reactive oxygen species (ROS), reviewed in [START_REF] Bernal | Oxidative stress in critical care medicine[END_REF]. Enhanced mitochondrial generation of the superoxide anion radical is observed in non-surviving groups in various models of sepsis and may be a starting point of mitochondrial oxidative stress [START_REF] Zapelini | Antioxidant treatment reverses mitochondrial dysfunction in a sepsis animal model[END_REF][START_REF] Rhee | Controlled elimination of intracellular H 2 O 2 : Regulation of peroxiredoxin, catalase, and glutathione peroxidase via post-translational modification[END_REF][START_REF] Ritter | Treatment with N-acetylcysteine plus deferoxamine protects rats against oxidative stress and improves survival in sepsis[END_REF]. Improved outcomes in animal models of sepsis have been achieved by us and others utilising mitochondrial targeted antioxidants (extensively reviewed [START_REF] Galley | Bench-to-bedside review: Targeting antioxidants to mitochondria in sepsis[END_REF][START_REF] Lowes | The mitochondria-targeted antioxidant MitoQ protects against organ damage in a lipopolysaccharide-peptidoglycan model of sepsis[END_REF][START_REF] Dare | A systematic review of experimental treatments for mitochondrial dysfunction in sepsis and multiple organ dysfunction syndrome[END_REF]). Superoxide does not readily cross the mitochondrial membrane and is catalysed to hydrogen peroxide within the mitochondria by manganese-containing superoxide dismutase (MnSOD). Hydrogen peroxide can diffuse out of the mitochondrion and is metabolised by catalase in the peroxisome, but is primarily removed within the mitochondrion by oxidation of reduced mitochondrial glutathione (mGSH) catalysed by mitochondrial glutathione peroxidase-1 (mGPx-1) with recycling back to reduced glutathione catalysed by glutathione reductase (GRD) [START_REF] Watabe | SP-22 is a thioredoxindependent peroxide reductase in mitochondria[END_REF]. It is now known that oxidation of mitochondrial thioredoxin-2 (TRX-2) in the presence of peroxiredoxin-3 (PRX-3) with subsequent recycling via thioredoxin reductase-2 (TRX-2R) is also important [START_REF] Patenaude | Mitochondrial thioredoxin system -Effects of TrxR2 overexpression on redox balance, cell growth, and apoptosis[END_REF]. This TRX-2 system has been reported to be more efficient at maintaining mitochondrial proteins in a reduced state compared to the mGSH system [START_REF] Zhang | Mitochondrial thioredoxin-2/peroxiredoxin-3 system functions in parallel with mitochondrial GSH system in protection against oxidative stress[END_REF], although under conditions of low level oxidative stress, both systems keep hydrogen peroxide levels in check and have definitive roles in cell signalling [START_REF] Zhang | Mitochondrial thioredoxin-2/peroxiredoxin-3 system functions in parallel with mitochondrial GSH system in protection against oxidative stress[END_REF]. However under conditions of more severe oxidative stress, the TRX-2 system may become more important [START_REF] Galley | Bench-to-bedside review: Targeting antioxidants to mitochondria in sepsis[END_REF].

The relative importance of the mGSH and TRX-2 systems and mitochondrial dysfunction during the cellular events of sepsis are not known. Endothelial cells recognise and respond to invading pathogens [START_REF] Aird | The role of the endothelium in severe sepsis and multiple organ dysfunction syndrome[END_REF] and loss of endothelial integrity contributes to the morbidity and mortality associated with sepsis. Increased mitochondrial generation of hydrogen peroxide and pro-inflammatory cytokine production in response to bacterial cell wall components and inflammatory mediators by endothelial cells, has been well documented [START_REF] Zhang | Mitochondrial reactive oxygen species-mediated signaling in endothelial cells[END_REF][START_REF] Corda | Rapid reactive oxygen species production by mitochondria in endothelial cells exposed to tumor necrosis factor-α is mediated by ceramide[END_REF]. Therefore, the aim of our study was to examine the consequences of inhibiting either mGPX-1 with thiomalic acid or TRX-2R with the gold compound auranofin, on oxidative stress and relative mitochondrial function in human endothelial cells cultured under conditions which mimic a mixed Gram negative/Gram positive septic insult.

EXPERIMENTAL Materials

Auranofin was obtained from Alexis Biochemicals, AXXORA Ltd, Nottingham, UK; 5-(6)carboxy-2,7'-dichlorodihydro-fluorescein-diacetate and the Molecular Probes™ ATP determination kit were from Invitrogen, Paisley, UK. Caspase-Glo® was from Promega, Southampton, UK; protease /phosphatase inhibitor cocktail was from Roche, Hertfordshire, UK; Bradford reagent was from Bio-Rad, Hertfordshire, UK and antibodies for western blotting were from Abcam, Cambridge, UK. All other reagents were from Sigma-Aldrich, Poole, Dorset, UK.

3

Cell culture and treatment

The human umbilical vein endothelial cell line (HUVEC C) was obtained from ATCC.org and were used at passages 5-14. This in vitro model of sepsis has been described previously in detail [START_REF] Lowes | The mitochondria-targeted antioxidant MitoQ protects against organ damage in a lipopolysaccharide-peptidoglycan model of sepsis[END_REF]. For experimentation, cells were grown in 96 well plates or on coverslips (see experimental detail below) in the presence of 2µg/ml lipopolysaccharide (LPS, from E. coli strain 0111:134) plus 20µg/ml peptidoglycan G (PepG, from S. Aureus strain 6571), prepared as described [START_REF] Kumar | Comparative analysis of antibody repertoire against Staphylococcus aureus antigens in patients with deep-seated versus superficial staphylococcal infections[END_REF], plus either thiomalic acid or auranofin for up to 7d to allow mitochondrial dysfunction to develop. LPS/PepG are toll like receptor (TLR) 4 and TLR2 agonists respectively that play a key role in the innate immune system. We have shown previously that HUVEC increase their mitochondrial generation of hydrogen peroxide and pro-inflammatory cytokine production maximally in response to these concentrations of LPS/PepG [START_REF] Lowes | The mitochondria-targeted antioxidant MitoQ protects against organ damage in a lipopolysaccharide-peptidoglycan model of sepsis[END_REF]. Auranofin is a co-ordinated gold(I) compound that can react with selenol-containing residues and is a potent inhibitor of purified thioredoxin reductase protein. Thiomalic acid is a glutathione mimetic that can bind to active site -SH groups. Evidence suggests that the mitochondrial isoforms if TRX-R and GPx are more sensitive to inhibition than the cytosolic isoforms. To determine the selective inhibition of mitochondrial isoforms of the enzymes by auranofin and thiomalic acid we exposed cells to the inhibitors at a range of concentrations in preliminary experiments, and measured enzyme activities of mitochondrial and cytosolic isoforms of TRX-R and GPx (see below.) As a result of these experiements, 4mM thiomalic acid or 2µM auranofin were used in subsequent experiments. Some cells were treated with solvent only (untreated) or solvent plus inhibitor only, or solvent plus 50µM hydrogen peroxide or 25M t-butyl-hydroperoxide as positive controls. Cell viability was assessed using acid phosphatase activity [START_REF] Yang | An acid phosphatase assay for quantifying the growth of adherent and nonadherent cells[END_REF].

TRX-R and GPx activity

TRX-R and GPx activity was determined in cytosolic and mitochondrial fractions using Sigma® thioredoxin reductase assay and glutathione peroxidase activity assay kits respectively. Briefly, cytosolic or mitochondrial lysates were added to the appropriate assay buffers containing NADPH and enzyme mixes in a 1ml cuvette. Reactions were started by the addition of 5,5'dithiobis(2-nitrobenzoic) acid or t-butyl-hydroperoxide for TRX-R or GPx activity respectively. TRX-R activity was determined by following the increase in absorbance at 412nm after a 2min delay and GPx activity by following the increase in absorbance at 430nm after a 15s delay, using a Helios beta single beam spectrophotometer.

Cytokine production

Cells were grown to confluence in 96 well plates and treated as above for 24h. Levels of interleukin (IL) -6 and IL-8 were measured in culture medium using a commercially available enzyme immunoassay kit.

Intracellular total radical production

Briefly, cells were grown to confluence in 96 well plates and loaded with 50µM oxidation sensitive 5-(6)-carboxy-2,7'-dichlorodihydrofluorescein diacetate (CDHFD) or 50M oxidationinsensitive 5,(6)-carboxy-2,7-dichlorofluorescein (CDF) for 60min in the dark. Cells were then washed twice with phosphate buffered saline (PBS) before being treated immediately as described above. The rate (positive) of total radical formation was determined fluorimetrically as a result of oxidation of 5-(6)-carboxy-2,7'-dichlorofluorescein (CDHF) as a continuous A c c e p t e d M a n u s c r i p t recording until saturation of CDHF was reached, at a temperature of 37C at an excitation wavelength of 485 nm and emission wavelength 530nm. MitoQ (1M) was used as a ROS scavenger to demonstrate the selectivity and reproducibility of CDHF.

Mitochondria isolation

Mitochondria were isolated following treatment, for analysis of GSH concentration and TRX-2, PRX-3 and mGPX-1 protein expression. Cells were typsinised, pelleted and washed with icecold PBS before being resuspended in 5vol homogenization buffer containing 0.25M sucrose, 20 mM HEPES-KOH pH7.5, 10mM KCl, 1.5mM MgCl 2 , 1mM EDTA, 1mM EGTA, 1mM dithiotreitol and protease/phosphatase inhibitor cocktail for 10min on ice. The cells were then homogenised with a Dounce borosilicate glass homogeniser before being centrifuged at 300g to remove cell debris and nuclei. Cytosolic and mitochondrial fractions were obtained by centrifugation of the cleared lysate for 10min at 13,000g. Protein concentrations of the cytosolic and mitochondrial fractions were determined by the Bradford protein assay procedure.

Mitochondrial and total GSH: GSSH ratio

The ratio of reduced:oxidized glutathione (GSH:GSSG) in isolated mitochondria and whole cell lysates was used to indicate mitochondrial glutathione system activity (mitochondrial oxidative stress) and total glutathione metabolism. Isolated mitochondria or cell lysate was added to reaction buffer containing 0.1%v/v Triton-X 100 and 0.1M potassium phosphate, pH 6.5. GSH was measured by adding 20μM mono-bromobimane and 0.3mg/ml glutathione-S-transferase and incubated at 37 o C in the dark for 15min. To measure oxidised glutathione, GSSG was reduced to GSH in separate lysed mitochondria or whole cell lysate samples with 2.22mM diethylenetriamine-penta-acetic acid and 2mM dithiothreitol in 1M HEPES buffer, pH8.5 for 30min at 37°C and measuring GSH as before. Fluorescence was determined at room temperature (excitation 355nm, emission 520nm) and normalized to g mitochondrial protein.

Mitochondrial TRx-2, PRX-3 and mGPX-1 protein expression

Mitochondrial expression of TRX-2 protein and the enzyme PRX-3 were measured using redox western blotting techniques to determine the redox (oxidation) states of TRX-2 and PRX-3 [START_REF] Damdimopoulos | Human mitochondrial thioredoxin involvement in mitochondrial membrane potential and cell death[END_REF][START_REF] Cox | Redox potential and peroxide reactivity of human peroxiredoxin 3[END_REF]. This was achieved by modifying any reduced protein with small molecules that are capable of covalently modifying reduced active sites. For redox western blotting of TRX-2, mitochondria were resuspended in 20mM Tris buffer, pH8.0, and incubated with 15mM 4acetoamido-4'-maleimidylstilbene-2,2'-disulphonic acid (AMS) for 3h at 37 o C. For redox determination of PRX-3, mitochondria were incubated with 100mM N-ethylmaleimide, 40mM HEPES pH7.4, 50mM NaCl, 1mM EDTA, 1mM EGTA and 10μg/ml catalase for 15min at room temperature before addition of 5%w/v 3-(3-cholamidopropyl) dimethylammonio)-1-propanesulphonate hydrate (CHAPS). mGPX-1 protein expression was measured using standard western blotting techniques without redox modification. All proteins were separated by 12-15% polyacrylamide gel electrophoresis and transferred to Immobilon-P™ transfer membrane using standard techniques. Blocked membranes were probed with primary polyclonal antibodies using rabbit anti-human PRX-3, sheep anti-human TRX-2 and rabbit anti-human GPX-1 (with appropriate horseradish peroxidase-linked secondary antibodies). Hypoxanthine-guanine phosphoribosyltransferase (HPRT) was used as loading control. Bands were visualized by A c c e p t e d M a n u s c r i p t enhanced chemiluminescence and quantified using GeneTools™ (SynGene, Synoptics Ltd, Cambridge, UK).

Measurement of caspase 3/7 activity

Apoptosis was assessed by measuring the activation of caspase-3 and caspase-7 using the Caspase-Glo® 3/7 assay. Briefly, cells were grown to confluence in 96 well plates and treated as described above for 7d, then an equal volume of Caspase-Glo® 3/7 substrate solution was added to the culture medium and mixed gently for 30s on a vibrating platform. Cells were then incubated for 1h at room temperature in the dark. Luminescence was measured immediately using a fluorimeter set to detect luminescence.

Mitochondrial membrane potential

Mitochondrial membrane potential was analysed in intact cells using the fluorescent probe 5,5,6,6-tetrachloro-1,1,3,3-tetraethylbenzimidazol-carbo-cyanine iodide (JC-1). Briefly, following cell treatments in 96 well plates for 7d, cells were washed twice with PBS and then incubated for 30min with 10µg/ml JC-1 at 37 o C in the dark. Following incubation, cells were washed twice with PBS and the orange/red and green fluorescence was measured immediately at 37 o C (excitation 490 nm, emission 590/520 nm). As a positive control, cells were incubated with the uncoupling agent, rotenone (1mM) for 4h prior to JC-1 analysis. In intact mitochondria J aggregates form and JC-1 fluoresces red. When the mitochondrial membrane potential drops the JC-1 assumes a monomeric form and fluoresces green (see online supplementary figure).

Metabolic activity

Metabolic activity was analysed by measuring the rate of reduction of AlamarBlue™ in intact cells after treatment for 7d. Alamar Blue is a novel redox indicator that exhibits both fluorescent and colourimetric changes in response to changes in metabolic activity (oxidative metabolism) [START_REF] Pagé | A new fluorometric assay for cytotoxicity measurements in vitro[END_REF]. Briefly, following cell treatments, 10µl AlamarBlue™ in 100µl culture medium was added to wells. Fluorescence was measured every 15min for 2h at 37 o C at an excitation wavelength of 530nm and emission wavelength 590nm.

ATP: ADP ratio

Cellular ATP: ADP ratio was determined after 7d treatment using the Molecular Probes™ ATP determination kit. Briefly, following cell treatments in 96 well plates, 1x10 6 cells were washed with ice cold PBS before the addition of 50µl ice cold lysis buffer (25mM HEPES pH7.8, 5mM MgCl 2 , 140mM NaCl and 1x detergent (somatic cell ATP-releasing reagent). Immediately following lysis, 10µl cell lysate was added to 100µl ATP determination buffer containing 1mM dithiothreitol, 0.5mM D-luciferin and 12pg firefly luciferase. To determine ADP levels, samples were treated with ADP converting solution, which contains ATP sulphurylase to remove endogenous ATP, and pyruvate kinase and phosphoenolpyruvate to convert ADP to ATP. ATP was then measured as above. Luminescence was read immediately.

Oxygen consumption

Cellular oxygen consumption was measured following 7d treatments, using a Clark-type oxygen electrode. Briefly, cells were grown on poly-L-lysine coated coverslips and, following cell 6 treatments, were added to a 7ml oxygen electrode containing normal growth media maintained at 37 o C. Oxygen consumption was measured for 15min.

Lactate production

Lactate was measured in culture medium after 7d treatment in 96 well plates, using the Abcam® lactate fluorometric assay kit. Briefly, conditioned culture media was removed and diluted in lactate assay buffer before being added to 96 well plates containing the lactate enzyme mix and probe. Appropriate calibration standards were also made and added to the plate. The reaction was allowed to proceed for 30min in the dark before fluorescence was measured at an excitation wavelength of 535nm and emission wavelength 590nm.

Statistical analysis

Six separate independent experiments were undertaken using 6 different cultures on different days including western blotting (n=6). No assumptions were made about the distribution of the data and non-parametric testing was performed using Kruskal Wallis analysis of variance and Mann Whitney post hoc testing as appropriate. A p value of <0.05 was taken to be significant.

RESULTS

TRX-R and GPx activity

The concentrations of the inhibitors chosen for this study were determined from initial experiments where maximal inhibition of the mitochondrial isoforms with no significant loss in cytosolic enzyme activity was seen. Our results showed that mitochondrial TRX-R and GPx enzyme activity decreased maximally at 2M and 4mM respectively (p=0.004 and 0.024 respectively, Fig. 1A and1C), with no change in cytosolic enzyme activity (Fig. 1B and1D). Higher concentrations reduced mitochondrial isoform enzyme activities a little further, but with loss of mitochondrial selectivity such that cytosolic enzyme activities were also decreased.

Cell viability

Cell viability was 100% for all cell treatments, measured at 24 hours or 7 days, with the exception of cells exposed to LPS-PepG plus auranofin for 7 days treatment only, where the viability decreased to (median [interquartile range] 69.6 [63-79]% (p=0.0022).

Cytokine production

As expected, IL-6 and IL-8 concentrations were significantly increased in cells exposed to LPS/PepG compared to untreated cells (p<0.0001, Fig. 2). There was no effect of auronofin or thiomalic acid on LPS/PepG induced cytokine production (Fig. 2)

Intracellular total radical production

To assess whether inhibition of either PRX-3 or mGPX-1 under conditions of sepsis would induce a higher rate of intracellular oxidative stress, we incubated cells with LPS/PepG and with auranofin or thiomalic acid. Results showed that the rate of total intracellular radical formation in cells cultured under our conditions of sepsis was significantly greater than in untreated cells (p=0.03, Fig. 3). The rate was higher still in cells exposed to the inhibitors in addition to LPS and A c c e p t e d M a n u s c r i p t 7 PepG (p=0.03, Fig. 3). Radical production was also slightly higher in cells treated with the inhibitors alone (p=0.041, Fig. 3).

Mitochondrial thioredoxin and glutathione

To evaluate the inhibition of PRX-3 or mGPX with auranofin or thiomalic acid respectively, on overall glutathione and thioredoxin-2 systems, we measured intra-mitochondrial expression of TRX-2 protein and GSH concentrations following 7d septic insult. Mitochondrial TRX-2 protein levels were higher in all LPS and PepG treated cells than in untreated cells (p<0.001, Fig. 4A). However, cells treated with thiomalic acid in addition to LPS and PepG had significantly more TRX-2 expression than other cell treatments (p=0.01, Fig. 4A). In comparison, the intramitochondrial GSH:GSSG ratio was lower in cells treated with LPS/PepG (p<0.001, Table 1) and lower still in cells also treated with auranofin (p <0.001). Cells treated with thiomalic acid, with or without LPS-PepG, had similar mitochondrial GSH:GSSG ratios to untreated cells. In addition, all cell treatments resulted in a significant reduction in the total GSH:GSSG ratio (p=0.0159, Table 1).

Expression of PRX-3 and mGPX-1

We determined the effect of inhibition of PRX-3 and mGPX-1 activity on subsequent mitochondrial protein expression of these enzymes after 7d treatment with the inhibitors plus LPS/PepG. In cells treated with either LPS or PepG, or with LPS/PepG and the inhibitor, there was higher PRX-3 expression, which was greatest in cells treated with auranofin (p<0.001, Fig. 4C). Expression of mGPX-1 was higher in all cell treatments compared to untreated cells and was highest in cells treated with auranofin plus LPS/PepG (p<0.05, Fig. 4B).

Caspase 3/7 activation

The effect of inhibition of PRX-3 and mGPX on apoptosis was determined by measuring the activation of caspase 3/caspase 7 following 7d culture. There was no effect of LPS and PepG exposure when compared to untreated cells (p=0.3, Fig. 5A). However, when cells were treated with auranofin, which inhibits TRX2-R, both with and without LPS-PepG, caspase activation was higher than in cells without auranofin (p<0.03, Fig. 5A).

Mitochondrial membrane potential

Mitochondrial membrane potential was determined by the ratio of red/green JC-1 fluorescence after 7d treatments (Fig. 5B). Cells exposed to LPS and PepG had a significantly lower JC-1 fluorescence ratio than untreated cells, indicating loss of mitochondrial membrane potential (p<0.001, Fig. 5B). In those cells treated with LPS-PepG plus auranofin, there was an even greater loss of mitochondrial membrane potential (p=0.01 and <0.001 respectively, Fig. 5B). This was not seen with thiomalic acid treated cells.

Metabolic activity, oxygen consumption, ATP: ADP ratio and lactate production

Investigation of the functional capacity of mitochondria after 7d septic insult was achieved by measuring the metabolic activity as determined by the rate of reduction of Alamar blue™ by mitochondrial respiratory complex activity, total oxygen consumption, ATP:ADP ratios and lactic acid production (Table 2). The metabolic activity in cells treated with auranofin plus LPS/PepG was significantly higher than all other cell treatments (p<0.001). Cells treated with 8 LPS/PepG had significantly reduced oxygen consumption rates (p=0.028) and ATP: ADP ratios (p<0.001) and increased lactate formation (p<0.001). However, cells treated with LPS/PepG plus auranofin had a considerably lower oxygen consumption rate (p=0.02) and ATP: ADP ratio (p<001) and a large increase in lactate formation (p<0.001).

DISCUSSION

We have shown that inhibition of the mitochondrial thioredoxin-2 or glutathione systems in our endothelial cell model of sepsis resulted in higher total intracellular radical production and loss of mitochondrial membrane potential, with altered protein expression of various components related to these systems. Inhibition of the TRX-2 system resulted in higher mitochondrial metabolic activity, lower ATP/ADP ratios, lower oxygen consumption, increased lactate formation and evidence of caspase 3/7 activation. Inhibition of the mGSH system had little effect on mitochondrial function. This suggests that the TRX-2 system may be more important preserving mitochondrial function in endothelial cells under conditions of sepsis.

Sepsis is associated with increased pro-inflammatory cytokine production, oxidative stress, consumption of endogenous antioxidants and mitochondrial damage [START_REF] Supinski | Hemin prevents cardiac and diaphragm mitochondrial dysfunction in sepsis[END_REF][START_REF] Zapelini | Antioxidant treatment reverses mitochondrial dysfunction in a sepsis animal model[END_REF][START_REF] Crouser | Abnormal permeability of inner and outer mitochondrial membranes contributes independently to mitochondrial dysfunction in the liver during acute endotoxemia[END_REF][START_REF] Gellerich | Impaired energy metabolism in hearts of septic baboons: Diminished activities of Complex I and Complex II of the mitochondrial respiratory chain[END_REF][START_REF] Simonson | Altered mitochondrial redox responses in gram negative septic shock[END_REF][START_REF] Fredriksson | Derangements in mitochondrial metabolism in intercostal and leg muscle of critically ill patients with sepsis-induced multiple organ failure[END_REF]. Tight control of cellular redox homeostasis is essential for protection against oxidative damage and for maintenance of normal metabolism. Therefore, mitochondrial generation of ROS is strictly regulated by mitochondrial antioxidant enzymes including MnSOD, mGSH/GPX-1 and the TRX-2/PRX-3 systems [START_REF] Patenaude | Mitochondrial thioredoxin system -Effects of TrxR2 overexpression on redox balance, cell growth, and apoptosis[END_REF][START_REF] Pedrajas | Mitochondria of Saccharomyces cerevisiae contain one-conserved cysteine type peroxiredoxin with thioredoxin peroxidase activity[END_REF]. Although both systems remove mitochondrial hydrogen peroxide, redox potentials differ substantially between the two, to facilitate the complex multiple signalling roles of ROS [START_REF] Hansen | Nuclear and mitochondrial compartmentation of oxidative stress and redox signaling[END_REF]. Auranofin is a co-ordinated gold(I) compound that reacts with selenol-containing residues and is a potent inhibitor of purified thioredoxin reductase protein. At the concentrations used in our in vitro study, we showed inhibition of mitochondrial TRX-R only, which resulted in no loss in cell viability in unstimulated cells (higher concentrations were required to inhibit the cytosolic TRX-R isoform and caused 50% reduction in cell viability). This concentration effect could be due to auranofin's characteristics as it belongs to the phosphine class of compounds that can be selectively concentrated within mitochondria at low concentrations'. In addition, the concentrations required to inhibit the isoforms of THX-R is cell type specific indicating the complex nature of the thioredoxin system, extensively reviewed in [START_REF] Arnér | Focus on mammalian thioredoxin reductases -important selenoproteins with versatile functions[END_REF][START_REF] Gromer | Human placenta thioredoxin reductase -Isolation of the selenoenzyme, steady state kinetics, and inhibition by therapeutic gold compounds[END_REF]. Thiomalic acid is a glutathione mimetic that can bind to active site -SH groups. We inhibited mGPX-1 exclusively at the concentrations used here. It been shown previously that mitochondrial GPx is much more sensitive to thiomalic acid than the cytosolic isoform [START_REF] Mercurio | Mercaptans decrease selenium-dependent glutathione-peroxidase activity in the chick[END_REF].

During sepsis, the basal rate of ROS production increases, specifically mitochondrial superoxide formation [START_REF] Zapelini | Antioxidant treatment reverses mitochondrial dysfunction in a sepsis animal model[END_REF][START_REF] Rhee | Controlled elimination of intracellular H 2 O 2 : Regulation of peroxiredoxin, catalase, and glutathione peroxidase via post-translational modification[END_REF][START_REF] Ritter | Treatment with N-acetylcysteine plus deferoxamine protects rats against oxidative stress and improves survival in sepsis[END_REF]. This is presumed to deplete antioxidants, increase free radical formation, and is associated with pro-inflammatory cytokine production and ultimately damage to mitochondria [START_REF] Supinski | Hemin prevents cardiac and diaphragm mitochondrial dysfunction in sepsis[END_REF][START_REF] Zapelini | Antioxidant treatment reverses mitochondrial dysfunction in a sepsis animal model[END_REF][START_REF] Crouser | Abnormal permeability of inner and outer mitochondrial membranes contributes independently to mitochondrial dysfunction in the liver during acute endotoxemia[END_REF][START_REF] Gellerich | Impaired energy metabolism in hearts of septic baboons: Diminished activities of Complex I and Complex II of the mitochondrial respiratory chain[END_REF][START_REF] Simonson | Altered mitochondrial redox responses in gram negative septic shock[END_REF][START_REF] Fredriksson | Derangements in mitochondrial metabolism in intercostal and leg muscle of critically ill patients with sepsis-induced multiple organ failure[END_REF][START_REF] Fink | Reactive oxygen species as mediators of organ dysfunction caused by sepsis, acute respiratory distress syndrome, or hemorrhagic shock: potential benefits of resuscitation with Ringer's ethyl pyruvate solution[END_REF][START_REF] Singer | Mitochondrial function in sepsis: acute phase versus multiple organ failure[END_REF][START_REF] Wallace | Mitochondrial diseases in man and mouse[END_REF][START_REF] Lowes | The mitochondria-targeted antioxidant MitoQ protects against organ damage in a lipopolysaccharide-peptidoglycan model of sepsis[END_REF]. Antioxidant therapies directed at mitochondria are now being studied, and we and others have demonstrated beneficial effects on mitochondrial function both in vitro and in animal models of sepsis [START_REF] Lowes | The mitochondria-targeted antioxidant MitoQ protects against organ damage in a lipopolysaccharide-peptidoglycan model of sepsis[END_REF][START_REF] Dare | A systematic review of experimental treatments for mitochondrial dysfunction in sepsis and multiple organ dysfunction syndrome[END_REF]. In endothelial cells treated with LPS/PepG plus inhibitors, the rate of total radical formation were enhanced above that of cells treated with LPS/PepG alone. However, this increase did not augment IL-6 or IL-8 production when compared to LPS-/PepG stimulated cells.

Mitochondrial oxidative stress was assessed by measuring intra-mitochondrial TRX-2 expression and intra-mitochondrial GSH:GSSG ratio compared to total GSH:GSSG ratio. After 24h (data not shown) and at 7d, we found that LPS/PepG treatments increased TRX-2 expression. A higher level of expression was seen in LPS/PepG treated cells treated with thiomalic acid and is likely to be compensatory for mGPx-1 inhibition. In addition, LPS/PepG treatment resulted in smaller decrease in both intra-mitochondrial and total GSH:GSSG ratios. However, auranofin caused an even lower intra-mitochondrial GSH:GSSG ratio without affecting total GSH:GSSG ratios, suggesting mitochondrial oxidative stress. We performed redox western blotting by covalent modification of any remaining reduced (after experimental treatment) TRX-2 in the mitochondrial lysates to assess the redox form of TRX-2. However, we found only one strong band indicating no oxidised TRX-2 in any treatment group. This result is consistent with a previous study using bovine aortic endothelial cells where TRX-2 was present only in the reduced form even when these cells were exposed to extreme oxidative stress (12mM hydrogen peroxide) [START_REF] Winterbourn | Thiol chemistry and specificity in redox signaling[END_REF].

Peroxiredoxins may be damaged by excessive ROS production such that a condition termed hyper-oxidation and inactivation occurs [START_REF] Zhang | Mitochondrial thioredoxin-2/peroxiredoxin-3 system functions in parallel with mitochondrial GSH system in protection against oxidative stress[END_REF]. Previous work has shown that endothelial mitochondrial PRX3 is much less sensitive to oxidation and inactivation than cytosolic PRX isoforms [START_REF] Damdimopoulos | Human mitochondrial thioredoxin involvement in mitochondrial membrane potential and cell death[END_REF]. We found only reduced PRX-3 in all cell treatments using redox western blotting. This may be due to the effects of the enzyme sulfiredoxin, which is able to reactivate peroxiredoxins following oxidation. This enzyme is up-regulated during oxidative stress and sepsis and can migrate into mitochondria, reactivating oxidised PRX-3 [START_REF] Noh | Sulfiredoxin translocation into mitochondria plays a crucial role in reducing hyperoxidized peroxiredoxin III[END_REF]. We propose that sulfiredoxin expression may be higher in endothelial cells than other cell types, explaining the increased resistance of PRX-3 to oxidation.

ROS removal in oxidative stress and sepsis is controlled in part by the level of endogenous antioxidant defences. The rate of removal of hydrogen peroxide in vivo by GPX-1 has been presumed to be independent of GSH concentration. However, kinetic studies have now revealed that this is not the case [START_REF] Ng | The rate of cellular hydrogen peroxide removal shows dependency on GSH: Mathematical insight into in vivo H 2 O 2 and GPx concentrations[END_REF]. It has been shown in vitro using purified enzyme that the total upper limit of hydrogen peroxide removal by GPX-1 is approximately 700nM, although under conditions of oxidative stress and sepsis, levels of intracellular hydrogen peroxide may reach 5µM [START_REF] Stone | An assessment of proposed mechanisms for sensing hydrogen peroxide in mammalian systems[END_REF]. This would cause the pool of mGSH to fall rapidly, which is replaced very slowly by diffusion of cytosolic GSH into the mitochondrion, reviewed in [START_REF] Kalinsky | Mitochondrial GSH[END_REF]. The relative reactivity of PRX-3 with hydrogen peroxide is approximately the same as mGPX-1. However, peroxiredoxins have been shown to be expressed at higher levels than glutathione peroxidases/glutaredoxins [START_REF] Hansen | Nuclear and mitochondrial compartmentation of oxidative stress and redox signaling[END_REF][START_REF] Noh | Sulfiredoxin translocation into mitochondria plays a crucial role in reducing hyperoxidized peroxiredoxin III[END_REF] and TRX-2 contains a mitochondrial targeting sequence and is actively transported into the mitochondrion as our results suggest.

Mitochondrial dysfunction is thought to be linked to the development of sepsis-induced organ dysfunction [START_REF] Supinski | Hemin prevents cardiac and diaphragm mitochondrial dysfunction in sepsis[END_REF][START_REF] Zapelini | Antioxidant treatment reverses mitochondrial dysfunction in a sepsis animal model[END_REF][START_REF] Crouser | Abnormal permeability of inner and outer mitochondrial membranes contributes independently to mitochondrial dysfunction in the liver during acute endotoxemia[END_REF][START_REF] Gellerich | Impaired energy metabolism in hearts of septic baboons: Diminished activities of Complex I and Complex II of the mitochondrial respiratory chain[END_REF][START_REF] Simonson | Altered mitochondrial redox responses in gram negative septic shock[END_REF][START_REF] Fredriksson | Derangements in mitochondrial metabolism in intercostal and leg muscle of critically ill patients with sepsis-induced multiple organ failure[END_REF][START_REF] Fink | Reactive oxygen species as mediators of organ dysfunction caused by sepsis, acute respiratory distress syndrome, or hemorrhagic shock: potential benefits of resuscitation with Ringer's ethyl pyruvate solution[END_REF][START_REF] Singer | Mitochondrial function in sepsis: acute phase versus multiple organ failure[END_REF][START_REF] Wallace | Mitochondrial diseases in man and mouse[END_REF][START_REF] Dare | A systematic review of experimental treatments for mitochondrial dysfunction in sepsis and multiple organ dysfunction syndrome[END_REF][START_REF] Singer | Mitochondrial dysfunction in sepsis[END_REF]. Many studies have shown that derangements in cellular oxygen utilization result from damage to the electron transport chain and damage to the inner mitochondrial membrane that can result in impairment of ATP generation, increased lactic acid production and mitochondrial swelling. We assessed the relative roles of the TRX-2 and mGSH systems on mitochondrial dysfunction in LPS/PepG treated cells. Collapse of the mitochondrial membrane potential, which may further impact on the oxidative phosphorylation pathway for ATP generation, may trigger apoptotic cell death pathways. The loss of mitochondrial membrane potential with a low ATP:ADP ratio, reduced oxygen consumption and increased lactate production, but no change in metabolic activity was seen in LPS/PepG treated cells and was similar to cells where mGPX was inhibited. However, inhibiting TRX-2R caused further loss of membrane potential, substantially reduced oxygen consumption, a greater increase in lactate production, and a large reduction in the ATP:ADP ratio, accompanied by a subsequent 5 fold 10 increase in metabolic activity. The increase in metabolic activity is most likely due to the low ATP concentration acting as a switch to produce more ATP. AlamarBlue™ is an accurate and sensitive indicator of mitochondrial function as it is an exceptional detector of reduction of all the elements of the electron transport chain that are not affected by oxidants [START_REF] Takato | Reduction of Alamar Blue, a novel redox indicator, is dependent on both the glycolytic and oxidative metabolism of glucose in rat cultured neurons[END_REF].

Changes to intracellular redox state (oxidative stress) have important implications for cell survival. Oxidation of TRX-2 and/or mGSH increases the susceptibility of the cell to oxidants and promotes apoptosis through the intrinsic pathway [START_REF] Chen | Mitochondrial thioredoxin in regulation of oxidant induced cell death[END_REF][START_REF] Circu | Glutathione and apoptosis[END_REF]. The low ATP: ADP ratio and mitochondrial dysfunction seen upon inhibition of TRX-R2 in LPS/PepG treated cells was accompanied by increased caspase 3/7 activation and a 30% reduction in cell viability. This shows that the TRX-2 system in endothelial cells is important to prevent cell death via adequate LPS/PepG-induced hydrogen peroxide removal.

In conclusion we have shown that in our in vitro endothelial cell model of sepsis, the proteins of the TRX-2 system are better resistant to the effects of oxidative stress and appear to have a larger protective role against mitochondrial dysfunction induced by LPS/PepG exposure than the mGSH system. Endothelial cells have an important role in host defence and inflammation during sepsis. However, further studies will be necessary to evaluate the relative importance of the mitochondrial hydrogen peroxide removal systems of other cell types during LPS/PepG insult.

Biochemical

  Journal Immediate Publication. Published on 28 Feb 2011 as manuscript BJ20102135 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20102135

Biochemical

  Journal Immediate Publication. Published on 28 Feb 2011 as manuscript BJ20102135 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20102135

Biochemical

  Journal Immediate Publication. Published on 28 Feb 2011 as manuscript BJ20102135 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20102135

Fig. 1 .

 1 Fig. 1. Inhibition of TRX-R and GPx in mitochondrial (panels A and C) and cytosolic (panels B and D) fractions from endothelial cells treated with solvent control (untreated), auranofin or thiomalic acid. Box and whisker plots show median, interquartile and full range (n=6). P value shown is Kruskal Wallis. * = significantly different from untreated cells (Mann Whitney p<0.05).

Fig. 2

 2 Fig.2Interleukin (IL)-6 and IL-8 concentratrions in cell culture supernatants from endothelial cells treated with solvent control (untreated), 2g/ml LPS plus 20g/ml PepG with 2M auranofin or 4mM thiomalic acid for 24h. Box and whisker plots show median, interquartile and full range (n=6). P value shown is Kruskal Wallis. * = significantly different from untreated cells and (Mann Whitney p<0.05).

Fig. 3 .

 3 Fig. 3.Oxidative stress (total radical production), measured as the rate of change in fluorescence in endothelial cells treated with solvent control (untreated), 2g/ml LPS plus 20g/ml PepG with and without 2M auranofin or 4mM thiomalic acid or 50M hydrogen peroxide. Box and whisker plots show median, interquartile and full range (n=6). P value shown is Kruskal Wallis. * = significantly different from untreated cells and + = significantly different from LPS-PepG alone (Mann Whitney p<0.05).

Fig. 4 .

 4 Fig. 4.Expression of key proteins in mitochondria. Mitochondria were extracted from endothelial cells treated with solvent control (untreated), 2g/ml LPS plus 20g/ml PepG with and without 2M auranofin or 4mM thiomalic acid, or 50M hydrogen peroxide. HPRT was used a loading control. Densitometry analysis and representative western blot of protein expression of A. TRX-2. B. mGPx-1. C. PRX-3. Box and whisker plots show median, interquartile and full range from six separate cultures (n=6). P value shown is Kruskal Wallis. * = significantly different from untreated cells and + = significantly different from LPS-PepG alone (Mann Whitney p<0.05).

Fig. 5 .

 5 Fig. 5. Caspase 3/7 activation and mitochondrial membrane potential. Endothelial cells were treated with solvent control (untreated), 2g/ml LPS plus 20g/ml PepG with and without 2M auranofin or 4mM thiomalic acid, 50M hydrogen peroxide (or 1mM rotenone as control, JC-1 only). A. Caspase 3/7 activation measured by luminescence. C. Mitochondrial membrane potential measured using JC-1. Box and whisker plots show median, interquartile and full range (n=6). P value shown is Kruskal Wallis. * = significantly different from untreated cells and + = significantly different from LPS-PepG alone (Mann Whitney p<0.05).

  LPS= lipopolysaccharide, PepG = peptidoglycan G * = significantly different from untreated cells (p<0.05) + = significantly different from LPS/PepG alone (p<0.05) Biochemical Journal Immediate Publication. Published on 28 Feb 2011 as manuscript BJ20102135 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20102135 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. © 2011 The Authors Journal compilation © 2011 Portland Press Limited Publication. Published on 28 Feb 2011 as manuscript BJ20102135 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20102135 A c c e p t e d M a n u s c r i p t

Table 1 Ratio of reduced/oxidised glutathione (GSH/GSSG)

 1 Biochemical Journal Immediate Publication. Published on 28 Feb 2011 as manuscript BJ20102135 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20102135 A c c e p t e d M a n u s c r i p t

	Treatment	Total GSH/GSSH	Mitochondrial
			GSH/GSSH
	Untreated	43.7 [41.1 -51.9]	49. 5 [46.8 -51.9]
	LPS/PepG	35.8 [34.6 -36.0]*	14.3 [13.9 -15.6]*
	Auronofin	36.0 [34.6 -39.4]*	30.6 [29.8 -32.3]*+
	Auronofin + LPS/PepG	34.4 [33.2 -35.4]*	6.5 [5.6 -7.5]*+
	Thiomalic acid	37.3 [32.5 -39.6]*	36.2 [34.9 -39.0]*
	Thiomalic acid + LPS/PepG 35.9 [35.6 -36.5]*	45.7 [42.0 -52.3]
	Hydrogen peroxide	41.1 [40.5 -41.7]*	16.2 [16.0 -17.1]*
	Median [interquartile range], n=6. P=0.0089 for total GSH/GSSG and p=<0.0001 for
	mitochondrial GSH/GSSH (Kruskal Wallis).
	LPS= lipopolysaccharide, PepG = peptidoglycan G
	*= significantly different from untreated cells (p<0.05)
	+=significantly different from LPS/PepG alone (p<0.05)

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Table 2 Mitochondrial metabolic activity, oxygen consumption, ADP/ATP and lactate

 2

	Treatment	Metabolic activity	Oxygen consumption	ADP/ATP ratio	Lactate
		Units/min/10 6 cells	nMol/min/10 6 cells	10 6 cells	nmol/10 6 cells
	Untreated	365 [272 -450]	6.5 [6.1-7.8]	6.3 [6.0 -6.6]	2.3 [2.0 -2.5]
	LPS/PepG	340 [238 -352]	4.9 [4.7 -5.3]*	4.7 [4.0 -5.0]*	3.0 [2.9 -3.6]*
	Auronofin	343 [323 -468]	5.3 [4.7 -6.0]*	5.3 [4.7 -5.4]	2.3 [2.0 -3.1]
	Auronofin + LPS/PepG	1250 [1050 -1416]*	2.9 [2.7 -3.3]*+	2.3 [2.0 -2.7]*	4.1 [3.8 -5.4]*+
	Thiomalic acid	346 [227 -467]	5.0 [4.7 -5.4]*	5.6 [5.1 -6.0]	2.0 [1.8 -2.2]
	Thiomalic acid + LPS/PepG 341 [252 -668]	5.3 [4.6 -5.9]*	4.4 [4.2 -4.4]*+	2.6 [2.4 -2.9]*
	Hydrogen peroxide	378 [221 -418]	4.5 [4.2 -4.6]*	6.1 [5.3 -6.6]	2.2 [1.8 -3.3]

Median [interquartile range], n=6. For Kruskal Wallis analysis of variance p values, see text.

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

© 2011 The Authors Journal compilation © 2011 Portland Press Limited

"#1) 3


Laura R Pearce

Eeva M Sommer

Kei Sakamoto

Stephan Wullschleger

Dario R Alessi
email: d.r.alessi@dundee.ac.uk

Protor-1 is required for efficient mTORC2mediated activation of SGK1 in the kidney

Keywords: mTOR, mTORC2, NDRG1, PI3K, Protor/PRR5, SGK1

The mTOR (mammalian target of rapamycin) protein kinase is an important regulator of cell growth and is a key target for therapeutic intervention in cancer. Two complexes of mTOR have been identified: complex 1 (mTORC1), consisting of mTOR, Raptor and mLST8 and complex 2 (mTORC2) consisting of mTOR, Rictor, Sin1, mLST8 and Protor-1 or Protor-2. Both complexes phosphorylate the hydrophobic motifs of AGC kinase family members: mTORC1 phosphorylates S6K, while mTORC2 regulates phosphorylation of Akt, PKC and SGK1. To investigate the roles of the Protor isoforms, we generated single as well as double Protor-1 and Protor-2 knockout mice and studied how activation of known mTORC2 substrates was affected. We observed that loss of Protor-1 and/or Protor-2 did not affect the expression of the other mTORC2 components, nor their ability to assemble into an active complex. Moreover, Protor knockout mice display no defects in the phosphorylation of Akt and PKC at their hydrophobic or turn motifs. Strikingly, we observed that Protor-1 knockout mice displayed markedly reduced hydrophobic motif phosphorylation of SGK1 and its physiological substrate NDRG1 in the kidney. These data suggest that Protor-1 may play a role in enabling mTORC2 to efficiently activate SGK1, at least in the kidney.

3

Introduction. mTOR protein kinase plays a key role in controlling cell growth and is likely to be overactivated in the majority of cancers. mTOR exists in two evolutionarily conserved complexes, termed mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2) (reviewed in [START_REF] Guertin | Defining the role of mTOR in cancer[END_REF]). mTORC1 consists of mTOR bound to Raptor (Regulatory associated protein of mTOR) and mLST8 (mammalian lethal with SEC13 protein 8) and is inhibited by rapamycin [START_REF] Loewith | Two TOR complexes, only one of which is rapamycin sensitive, have distinct roles in cell growth control[END_REF][START_REF] Hara | Raptor, a binding partner of target of rapamycin (TOR), mediates TOR action[END_REF][START_REF] Kim | mTOR interacts with raptor to form a nutrient-sensitive complex that signals to the cell growth machinery[END_REF]. mTORC1 is activated downstream of growth factors via the PI3K (phosphoinositide 3-kinase) pathway, involving Akt-mediated phosphorylation of PRAS40 (Proline-rich Akt substrate of 40kDa) and TSC2, leading to activation of the Rheb GTPase [START_REF] Sancak | PRAS40 is an insulin-regulated inhibitor of the mTORC1 protein kinase[END_REF][START_REF] Huang | The TSC1-TSC2 complex: a molecular switchboard controlling cell growth[END_REF]. In addition, mTORC1 activity is stimulated by amino acids through a pathway including Rag GTPases and MAP4K3 [START_REF] Findlay | A MAP4 kinase related to Ste20 is a nutrient-sensitive regulator of mTOR signalling[END_REF][START_REF] Sancak | The Rag GTPases bind raptor and mediate amino acid signaling to mTORC1[END_REF][START_REF] Kim | Regulation of TORC1 by Rag GTPases in nutrient response[END_REF]. While mTORC2 also contains mLST8, Raptor is replaced by Rictor (Rapamycin-insensitive companion of mTOR), Sin1 (SAPK-interacting protein 1) and one of the closely related isoforms of Protor-1 or Protor-2 [START_REF] Jacinto | Mammalian TOR complex 2 controls the actin cytoskeleton and is rapamycin insensitive[END_REF][START_REF] Sarbassov | Rictor, a novel binding partner of mTOR, defines a rapamycin-insensitive and raptor-independent pathway that regulates the cytoskeleton[END_REF][START_REF] Yang | Identification of Sin1 as an essential TORC2 component required for complex formation and kinase activity[END_REF][START_REF] Frias | mSin1 is necessary for Akt/PKB phosphorylation, and its isoforms define three distinct mTORC2s[END_REF][START_REF] Jacinto | SIN1/MIP1 maintains rictor-mTOR complex integrity and regulates Akt phosphorylation and substrate specificity[END_REF][START_REF] Pearce | Identification of 13 Protor as a novel Rictor-binding component of mTOR complex-2[END_REF][START_REF] Woo | PRR5, a novel component of mTOR complex 2, regulates platelet-derived growth factor receptor beta expression and signaling[END_REF][START_REF] Thedieck | PRAS40 and PRR5-like protein are new mTOR interactors that regulate apoptosis[END_REF]. mTORC2 is insensitive to acute rapamycin treatment and although it is also activated by growth factors through an ill-defined PI3K-dependent mechanism. Some of the best-characterised substrates of mTOR complexes are the Akt, PKC, SGK and S6K protein kinases. These belong to the AGC kinase family and typically members of this family are activated by phosphorylation of two key sites: the T-loop and the hydrophobic motif [18]. mTORC1 controls the hydrophobic motif of p70 ribosomal S6 kinase (S6K) [START_REF] Hara | Raptor, a binding partner of target of rapamycin (TOR), mediates TOR action[END_REF][START_REF] Kim | mTOR interacts with raptor to form a nutrient-sensitive complex that signals to the cell growth machinery[END_REF], and mTORC2 regulates the equivalent site in Akt [19], protein kinase C (PKC ) [START_REF] Sarbassov | Rictor, a novel binding partner of mTOR, defines a rapamycin-insensitive and raptor-independent pathway that regulates the cytoskeleton[END_REF] and serum-and glucocorticoid-induced protein kinase (SGK1) [START_REF] Garcia-Martinez | mTOR complex 2 (mTORC2) controls hydrophobic motif phosphorylation and activation of serum-and glucocorticoidinduced protein kinase 1 (SGK1)[END_REF], accounting for the ability of mTOR to control protein synthesis, cell proliferation, survival, metabolism and reorganisation of the actin cytoskeleton. Akt (Thr450) and PKC (Thr638) are also stabilised by phosphorylation of their turn-motif, an event which is mediated by mTORC2 and that probably occurs during translation [START_REF] Ikenoue | Essential function of TORC2 in PKC and Akt turn motif phosphorylation, maturation and signalling[END_REF][START_REF] Facchinetti | The mammalian target of rapamycin complex 2 controls folding and stability of Akt and protein kinase C[END_REF][START_REF] Oh | mTORC2 can associate with ribosomes to promote cotranslational phosphorylation and stability of nascent Akt polypeptide[END_REF]. SGK1 was originally identified as a serum and glucocorticoid-inducible gene and it is activated downstream of insulin and growth factors (reviewed in [START_REF] Tessier | Serum and glucocorticoid-regulated protein kinases: variations on a theme[END_REF]). The generation of PtdIns [START_REF] Hara | Raptor, a binding partner of target of rapamycin (TOR), mediates TOR action[END_REF][START_REF] Kim | mTOR interacts with raptor to form a nutrient-sensitive complex that signals to the cell growth machinery[END_REF][START_REF] Sancak | PRAS40 is an insulin-regulated inhibitor of the mTORC1 protein kinase[END_REF])P 3 by PI3-kinase triggers a signalling cascade that leads to the phosphorylation of the hydrophobic motif (Ser422 in SGK1) by mTORC2 [START_REF] Garcia-Martinez | mTOR complex 2 (mTORC2) controls hydrophobic motif phosphorylation and activation of serum-and glucocorticoidinduced protein kinase 1 (SGK1)[END_REF]. This does not directly activate SGK1, but instead creates a docking site for PDK1, which can subsequently phosphorylate SGK1 at the T-loop residue (Thr256), resulting in its activation [START_REF] Biondi | The PIF-binding pocket in PDK1 is essential for activation of S6K and SGK, but not PKB[END_REF][START_REF] Collins | In vivo role of the PIF-binding docking site of PDK1 defined by knock-in mutation[END_REF]. One of the best-characterised substrates of SGK1 is NDRG1 and the phosphorylation of this protein is frequently used as a marker for SGK1 activity [START_REF] Garcia-Martinez | mTOR complex 2 (mTORC2) controls hydrophobic motif phosphorylation and activation of serum-and glucocorticoidinduced protein kinase 1 (SGK1)[END_REF][START_REF] Murray | Exploitation of KESTREL to identify NDRG family members as physiological substrates for SGK1 and GSK3[END_REF].

Protor-1 and Protor-2 isoforms are encoded by different genes and interact with Rictor through a conserved N-terminal region [START_REF] Pearce | Identification of 13 Protor as a novel Rictor-binding component of mTOR complex-2[END_REF][START_REF] Woo | PRR5, a novel component of mTOR complex 2, regulates platelet-derived growth factor receptor beta expression and signaling[END_REF][START_REF] Thedieck | PRAS40 and PRR5-like protein are new mTOR interactors that regulate apoptosis[END_REF]. This may stabilise Protor, as total protein levels of Protor are vastly reduced in Rictor knockout fibroblasts [START_REF] Pearce | Identification of 13 Protor as a novel Rictor-binding component of mTOR complex-2[END_REF]. As Protor isoforms have no other obvious functional domains it is hard to predict their physiological function. To address how Protor isoforms influence mTORC2 we generated single Protor-1 -/-, Protor-2 -/-as well as Protor-1 -/-Protor-2 -/-double knockout mice. While phosphorylation of the hydrophobic and turn motifs of both Akt and PKC was unaffected in Protor-1 -/-mice, we observed a significant reduction in phosphorylation of the hydrophobic motif of SGK1 in kidney, a tissue in which SGK1 phosphorylation and activity is high. Protor-1 deficient mice also displayed a marked reduction in phosphorylation of the SGK1 T-loop and also that of NDRG1, a specific SGK1 substrate. Our results indicate that Protor-1 plays a critical role in mediating the phosphorylation of SGK1 by mTORC2 in the kidney. A c c e p t e d M a n u s c r i p t 5 TCAACATGACGAAGTCAAGTGTC-3') were used to detect the wild type, heterozygous and knockout alleles. For Protor-2 mice, Primer 1 (5'-AACCTGGGAAAGGAAGAAGC-3'), Primer 2 (5'-GCACTCAAAATCTCTGTGGC-3') and Primer 4 (5'-TCTGATTCTCCCACCTGAAGT-3') were utilized. The PCR programme consisted of 5min at 95°C, 35 cycles of 30sec at 95°C, 30sec at 60°C and 1min at 72°C, followed by 10min at 72°C on a PTC-200 Peltier Therma Cycler DNA engine. Mice were maintained under specific pathogen-free conditions and routine animal ear notching for genotyping was carried out by staff in the College of Life Science Transgenic Unit (University of Dundee). All procedures were carried out in accordance with the regulations set by the University of Dundee and the United Kingdom Home Office.

Blood glucose and plasma insulin measurement. Blood glucose levels were measured using the Ascensia Breeze 2 blood glucose monitoring system (Bayer) following tail incision. For plasma insulin measurements, blood was collected in haematocrit capillary tubes (Hawksley) from mice following tail incision and incubated on ice. Blood was centrifuged at 3,000xg for 15min to obtain plasma. Insulin levels were measured using a rat/mouse insulin enzyme-linked immunosorbent assay (ELISA) kit (Millipore, EZRMI-13K). Assays were carried out according to the manufacturer's instructions, typically this involved the use of 5 l of plasma per assay and mouse insulin standards from 0 to 10ng/ml were used to generate a standard curve. Samples were assayed in duplicate.

Glucose tolerance test.

Mice were deprived of food overnight (16hrs), weighed and basal blood glucose levels measured following tail incision. Mice were injected intraperitoneally with 2mg/g D-glucose solution and blood glucose levels measured at 15, 30, 45, 60 and 120min post-injection.

Insulin tolerance test.

Mice were allowed to feed overnight ad libitum and then food was removed for 3hrs before the experiment. Mice were weighed and the basal blood glucose level determined following tail incision. Mice were then injected intraperitoneally with 1mU/g insulin and blood glucose levels measured at 15, 30, 45, 60 and 120min post-injection.

Injection of mice with IGF1. Mice were starved for 3hrs before being anaesthetised using sodium pentobarbital (80-90 g/g, intraperitoneally injected) and placed on a heating pad to maintain body temperature. This was followed by an intravenous injection through the inferior vena cava of either PBS or 0.5 g/g IGF1 dissolved in PBS [START_REF] Shioi | Akt/protein kinase B promotes organ growth in transgenic mice[END_REF]. Tissues were rapidly excised 5min after injection and frozen in liquid nitrogen.

Buffers.

The following buffers were used: Tris-CHAPS lysis buffer [50mM Tris-HCl (pH 7.5), 1mM EGTA, 1mM EDTA, 0.3% (w/v) CHAPS, 1mM sodium orthovanadate, 10mM sodium-ß-glycerophosphate, 50mM sodium fluoride, 5mM sodium pyrophosphate, 0.27M sucrose, 0.15M NaCl, 0.1 % (v/v) 2-mercaptoethanol, 1mM benzamidine and 0.1mM phenylmethylsulphonylfluoride], Buffer A [50mM Tris-HCl (pH 7.5), 0.1mM EGTA and 0.1% (v/v) 2-mercaptoethanol], Hepes-CHAPS lysis buffer [40mM Hepes (pH 7.5), 120mM NaCl, 1mM EDTA, 0.3% (w/v) CHAPS, 10mM sodium pyrophosphate, 10mM sodium-ß-glycerophosphate, 50mM sodium fluoride, 0.5mM sodium orthovanadate, 1mM benzamidine and 0.1mM phenylmethylsulphonylfluoride], Hepes kinase buffer [25mM Hepes (pH 7.5), 50mM KCl], TBS-Tween Buffer [50mM Tris-HCl pH 7.5, 0.15M NaCl and 0.1% (v/v) Tween-20] and Sample Buffer [50mM Tris-HCl pH 6.8, 6.5% (v/v) Glycerol, 1% (w/v) SDS, and 1% (v/v) 2-mercaptoethanol]. Preparation of tissue lysates. Mouse tissues were rapidly excised, snap frozen in liquid nitrogen and stored at -80°C until use. Frozen tissues were weighed and homogenised in a 10-fold excess of ice-cold Tris-CHAPS or Hepes-CHAPS lysis buffer using a polytron. Lysates were centrifuged at 3,000rpm for 15min at 4°C and supernatants then further centrifuged at 12,000rpm for 20min at 4°C to remove all insoluble material. The protein concentration of each lysate was determined before being snap frozen in aliquots and stored at -80°C.

Purification of inactive GST-Akt1 and GST-N-SGK1 [61-431] from HEK293 cells 10cm diameter dishes of HEK293 cells were cultured and each dish was transfected with 5 g of either GST-Akt1 or GST-N-SGK1 [61-431] using the polyethylenimine (PEI) method. 24hrs post-transfection, HEK293 cells that had been transfected with GST-Akt1 or GST-N-SGK1 [61-431] were serum starved for 16hrs. Cells were treated with 1 M PI-103 for 30min and harvested in Tris-CHAPS lysis buffer. 3mg Lysate was affinity purified on 10 l glutathione-Sepharose for 1hr at 4°C on a rotating wheel. The resulting precipitates were washed twice with Tris-CHAPS lysis buffer, twice with Buffer A and twice with Buffer A containing 0.27M sucrose. GST-tagged proteins were eluted from the resin by resuspension in an equal amount of Buffer A containing 0.27M sucrose and 10mM glutathione (pH7.5-8) for 1hr on ice. Supernatants were filtered through a 0.22 m-spin column and aliquots were snap-frozen and stored at -80°C. mTORC2 activity assays. Mouse tissues or MEFs were lysed in Hepes-CHAPS lysis buffer and 1-4mg of lysate was pre-cleared by incubation with 5 l of Protein G-Sepharose conjugated to pre-immune IgG. The lysates were then incubated with 5 l of Protein G-Sepharose covalently conjugated to either 5 g anti-Sin1 antibody, or 5 g preimmune IgG for 2hrs at 4ºC on a vibrating platform. The immunoprecipitates were washed four times with Hepes-CHAPS lysis buffer, followed by two washes with Hepes kinase buffer. Assays were carried out in a final volume of 40 l Hepes kinase buffer containing 1 g GST-Akt1 or 1 g GST-N-SGK1 [61-431] and kinase reactions initiated upon addition of 0.1mM ATP and 10mM MgCl 2 . Reactions were carried out for 30min at 30ºC on a vibrating platform and stopped by the addition of SDS sample buffer. Samples were heated to 70ºC for 5min, passed through a Spin-X column and subjected to electrophoresis and immunoblot analysis.

Immunoprecipitation of endogenous

Protor-1, Protor-2. 1mg (for Protor-1 immunoprecipitates) or 2mg (for Protor-2 immunoprecipitates) lysate from each of the indicated tissues was pre-cleared by incubation with 5 l of Protein G-Sepharose for 30min. The lysates were then incubated with 5 g of anti-Protor-1 or anti-Protor-2 covalently coupled to Protein G-Sepharose for 1.5hrs at 4°C. Immunoprecipitates were washed four times with Tris-CHAPS lysis buffer lacking 2-mercaptoethanol and twice with Buffer A in which reducing agent was omitted. Immunoprecipitates were resuspended in 20 l of SDS sample buffer (lacking reducing agent) and filtered through a Spin-X column to remove the Sepharose resin. 1X NuPAGE reducing agent (Invitrogen) was added to the eluted samples. These were then subjected to electrophoresis and immunoblot analysis as described below.

Kidney membrane preparations for ENaC immunoblots. This was undertaken as previously described [30]. Harvested mouse kidneys were snap frozen immediately after isolation and homogenized in detergent-free lysis buffer containing 50mM Tris pH 7.5, 1mM EDTA, 1mM EGTA, 1mM sodium orthovanadate, 5 mM sodium fluoride, 5 mM sodium pyrophosphate, 0.27M sucrose, 0.1% (v/v) 2-mercaptoethanol and protease inhibitors (1 tablet per 50ml) with a Polytron homogenizer. Nuclei and debris were pelleted by centrifugation at 1,500 g for 5min. The procedure was repeated at 3,000 g for 10min and the low speed supernatants were next centrifuged at 100,000 g for 1 hour. The A c c e p t e d M a n u s c r i p t 7 pellet was washed with 1 volume of lysis buffer without reducing agent and centrifuged again at 100,000g for 15 min to remove any remaining cytosolic particles. The pellet was then resuspended in lysis buffer including 1% (v/v) NP40 and centrifuged at 16,500g for 10min. The protein content of the membrane preparations was quantified and resuspended in SDS sample buffer.

Immunoblotting. Tissue or cell lysates (10-20 g) or immunoprecipitated samples were heated at 70°C for 5min in sample buffer, and subjected to polyacrylamide gel electrophoresis and electrotransfer to nitrocellulose membranes. Membranes were blocked for 1hr in TBS-Tween buffer containing 5% (w/v) skimmed milk. The membranes were probed with the indicated antibodies in TBS-Tween containing 5% (w/v) skimmed milk or 5% (w/v) BSA for 16hrs at 4°C. Detection was performed using horseradish peroxidase-conjugated secondary antibodies and enhanced chemiluminescence reagent.

Generation and stimulation of MEFs (mouse embryonic fibroblasts)

MEFs isolated from mouse embryos at E13.5 (embryonic day 13.5) were generated and cultured as described previously [31]. Cells were serum-starved in DMEM for 16h prior to stimulation with 50ng/ml IGF1 for 30min. Cells were subsequently lysed and the lysates centrifuged at 18000g for 15min at 4°C. Supernatants were snap-frozen and stored at 80 °C. Lysates (10=g) were analysed by immunoblotting using the antibodies indicated in the figures.

Results.

Generation of Protor-1 -/-, Protor-2 -/-and Protor-1 -/-Protor-2 -/-knockout mice. To study the physiological role of the Protor isoforms, we generated Protor-1 -/-and Protor-2 - /-knockout mice as described in the materials and methods and Figure 1. The breeding strategy for the single Protor-1 -/-and Protor-2 -/-, as well as the Protor-1 -/-Protor-2 -/-double knockout mice is summarised in Table 1. Mice were generated and maintained on a pure C57BL/6J background. The single and double knockout mice were viable, born at the expected Mendelian frequency (Table 1) and did not display any overt phenotype. The genotype of the Protor-1 -/-and Protor-2 -/-mice was verified by PCR (Figure 1C&G). In order to confirm the ablation of protein expression, Protor-1 was immunoprecipitated from the tissues of the Protor-1 -/-mice and as expected, there was a reduction in Protor-1 expression of approximately 50% in the Protor-1 +/-mice and Protor-1 could not be detected in the homozygous knockout mice (Figure 1D). A similar result was also obtained in the Protor-2 -/-mice (Figure 1H). There was no significant difference in the body weight of male or female single Protor-1 -/-or Protor-2 -/-or Protor-1 -/-Protor-2 -/- double knockout mice compared to wild type mice up to 4 months of age (data not shown). In addition, the Protor-1 -/-knockout animals displayed normal blood glucose and plasma insulin levels under both fasted and fed conditions and there was no detectable change in whole body glucose or insulin tolerance compared to wild type animals (Supplementary Figure 1).

NDRG1 phosphorylation is diminished in the kidney of Protor-1 -/-knockout mice. We initially examined the phosphorylation state of Akt, its substrate PRAS40 and FoxO1 as well as NDRG1, an SGK1 substrate, in various tissues from fed littermate Protor-1 +/+ , Protor-1 +/-and Protor-1 -/-mice. This revealed no significant difference in Akt, PRAS40 or FoxO1 phosphorylation in any of the tissues examined among the three genotypes (Figure 2). The only tissue in which significant phosphorylation of NDRG1 was observed in the kidney of Protor-1 +/+ animals (Figure 2A). Consistent with this the NDRG1 protein migrated as several electrophoretic species in kidney and only as a single species in other tissues. Interestingly, there was a partial reduction in the phosphorylation of NDRG1 in the kidney of Protor-1 +/-animals and this was more pronounced in the Protor-1 -/- knockouts (Figure 2A).

We next investigated NDRG1 phosphorylation in kidney extracts derived from single and double Protor-1 and Protor-2 knockout animals. Although there was no change in NDRG1 phosphorylation in the kidney of single Protor-2 -/-mice (Figure 3B), the Protor-1 -/-Protor-2 -/-double knockout mice displayed reduced NDRG1 phosphorylation, similar to that seen in the single Protor-1 -/-knockouts (Figure 3C). In all cases Akt (Thr308 and Ser473) and PRAS40 phosphorylation was unaffected by the loss of Protor-1 and/or Protor-2. In addition, the phosphorylation of Akt and PKC at the turn motif (Thr450 and Thr638 respectively) and the PKC hydrophobic motif (Ser657) remained unchanged in the kidney of the Protor isoform knockout mice (Figure 3A-C). We also investigated phosphorylation of mTORC2 targets in the lung and liver tissues of the single Protor-2 -/- mice as well as the Protor-1 -/-Protor-2 -/-double knockout mice. This confirmed that there was no substantial phosphorylation of NDRG1 detected in these tissues and that phosphorylation of Akt and its substrates PRAS40 and FoxO1 was not reduced in the Protor-deficient animals (Supplementary Figure 2). IGF1-induced SGK1 phosphorylation is reduced in the kidney of Protor-1 -/- knockout mice. To investigate further the role that Protor-1 plays in regulating SGK1 activation in the kidney, single and double Protor-deficient mice were injected intravenously with 0.5 g/g IGF1 for 5min to activate the PI3K pathway in a wide variety of tissues (instead of limited insulin-sensitive tissues). Tissue extracts derived from these animals were probed using antibodies that recognise SGK1 phosphorylated at its hydrophobic motif (Ser422) as well as its T-loop residue (Thr256). While SGK1 hydrophobic motif phosphorylation was stimulated upon IGF1 treatment in the wild type control mice, phosphorylation of this site was barely detectable in the single Protor-1 -/- mice (Figure 4A) or Protor-1 -/-Protor-2 -/-double knockout mice (Figure 4C). Consistent with phosphorylation of Ser422 being required for the subsequent phosphorylation of Thr256 [START_REF] Biondi | The PIF-binding pocket in PDK1 is essential for activation of S6K and SGK, but not PKB[END_REF], there was also no phosphorylation of Thr256 in these animals. The loss of Ser422 and Thr256 phosphorylation would result in a lack of SGK1 activation, which accounts for the much-reduced phosphorylation of NDRG1. In contrast, SGK1 and NDRG1 were phosphorylated to the same extent in kidney extracts derived from wild type and single Protor-2 -/-knockout mice (Figure 4B). There was also no significant difference in the IGF1-induced phosphorylation of Akt at either Ser473 or Thr308 or that of PRAS40 in the kidney of Protor-1 and/or Protor-2 deficient mice (Fig 4).

NDRG1 phosphorylation was also assessed in response to IGF1 stimulation in the liver of the single and Protor-1 -/-Protor-2 -/-double knockout mice (Supplementary Fig 3). We observed that IGF1 did not lead to a marked increase in NDRG1 phosphorylation and consistent with this did not induce an electrophoretic band-shift of the total NDRG1 protein under conditions in which Akt and its substrate PRAS40 were phosphorylated (Supplementary Fig 3). We also generated mouse embryonic fibroblasts (MEFs) derived from wild type and Protor-1 -/-Protor-2 -/-double knockout mice and found that IGF1 induced a significant phosphorylation and an electrophoretic band-shift of NDRG1 protein in both wild type and Protor-1 -/-Protor-2 -/-double knockout MEFs (Fig 5). This observation is considered further in the Discussion. mTORC2 formation is unaffected by the loss of Protor-1 and Protor-2. Previously it was reported that loss of the mTORC2 component Sin1 affects the ability of mTOR and Rictor to interact [START_REF] Yang | Identification of Sin1 as an essential TORC2 component required for complex formation and kinase activity[END_REF][START_REF] Frias | mSin1 is necessary for Akt/PKB phosphorylation, and its isoforms define three distinct mTORC2s[END_REF][START_REF] Jacinto | SIN1/MIP1 maintains rictor-mTOR complex integrity and regulates Akt phosphorylation and substrate specificity[END_REF]. As reduced mTORC2 complex formation could potentially account for the decreased phosphorylation of SGK1, the ability of mTOR, Rictor and Sin1 to bind one another was investigated in the Protor-1 -/-and Protor-2 -/-single and double knockout animals. mTOR and Rictor still co-immunoprecipitated with Sin1, irrespective of the presence of either Protor-1 or Protor-2 in kidney (Figure 6A-C). This provides evidence that Protor-1 and Protor-2 are not essential for mTORC2 complex assembly.

Protor isoforms are not required for mTORC2 kinase activity in vitro. We next investigated whether Protor isoforms are required for the kinase activity of mTORC2. mTORC2 was immunoprecipitated using the Sin1 antibody from either fed (Fig 7A &B) or IGF1-injected (Fig 7C &D) wild type or double Protor-1 -/-and Protor-2 -/-knockout mouse kidney or liver lysates and its ability to phosphorylate GST-Akt1 or GST-SGK1 assessed. These studies revealed that loss of Protor isoforms did not significantly impact on the ability of immunoprecipitated mTORC2 to phosphorylate GST-Akt1 or GST-SGK1 in cell free-assays. Similar results were obtained when mTORC2 activity was assayed in the Protor-1 -/-Protor-2 -/-double knockout MEFs (Fig 7E &F). These findings indicate that Protor isoforms are dispensable for regulating intrinsic mTORC2 kinase activity. The results presented in this study indicate that in contrast to the other mTORC2 specific components Sin1 and Rictor, Protor subunits are not essential for mouse viability, growth, mTORC2 complex assembly or phosphorylation of the hydrophobic and turn motifs of Akt. Studies using tissue-specific knockouts of Rictor in both muscle and adipose have demonstrated the key role mTORC2 plays in insulin signalling and glucose homeostasis. Muscle-specific Rictor -/-mice have defective insulin-stimulated glucose uptake in muscle and are glucose intolerant [START_REF] Kumar | Muscle-specific deletion of rictor impairs insulin-stimulated glucose transport and enhances Basal glycogen synthase activity[END_REF], while adipose-specific knockouts of Rictor display impaired glucose uptake and reduced whole body insulin sensitivity [START_REF] Kumar | Fat cell-specific ablation of rictor in mice impairs insulin-regulated fat cell and whole-body glucose and lipid metabolism[END_REF][START_REF] Cybulski | mTOR complex 2 in adipose tissue negatively controls whole-body growth[END_REF]. However we did not observe any significant defects in glucose clearance and insulin sensitivity, consistent with Akt Ser473 phosphorylation being unaffected in the single or Protor-1 -/-Protor-2 -/-double knockout mice. These observations indicate that the role of Protor-1 is distinct from that of Rictor and is not required for activation of Akt, at least under the conditions that we have studied.

Instead, our data suggest that Protor-1 is required for efficient activation of SGK1 in the kidney, the only tissue in which we were able to measure significant endogenous hydrophobic and T-loop motif phosphorylation of SGK1 and that of its substrate NDRG1. Interestingly, Protor-1 expression was previously shown to be high in the kidney, perhaps indicating the key role Protor-1 plays in this tissue [START_REF] Pearce | Identification of 13 Protor as a novel Rictor-binding component of mTOR complex-2[END_REF]. Our data also indicate that Protor-1 rather than Protor-2 is critical for efficient activation of SGK1 in the kidney, as defects in SGK1 and NDRG1 phosphorylation were not observed in the Protor-2 knockout animals. Moreover, the Protor-1 -/-Protor-2 -/-double knockout mice displayed identical reductions in SGK1 and NDRG1 phosphorylation to the single Protor-1 -/-deficient mice, emphasising that Protor-1 rather that Protor-2 is key for regulating SGK1 activation in the kidney.

In contrast to what was observed in kidney tissues, IGF1 induced a significant phosphorylation and an electrophoretic band-shift of NDRG1 protein in both wild type and Protor-1 -/-Protor-2 -/-double knockout MEFs, suggesting that the effects of Protor on SGK1 activation and NDRG1 phosphorylation may be specific to the kidney. In our hands, endogenous SGK1 protein in MEFs is undetectable using antibodies that readily detect SGK1 in the kidney. It is possible that when SGK1 levels are low, much higher levels of Akt or a distinct kinase contribute to observed NDGR1 phosphorylation in MEFs. Moreover, SGK2 or SGK3 which we did not assess, may not be regulated by Protor isoforms and if this was the case might account for observed NDGR1 phosphorylation in Protor deficient MEFs.

The best-characterised role of SGK1 in vivo is in the kidney where it stimulates sodium transport into epithelial cells by enhancing the stability and expression of the epithelial sodium channel (ENaC) (reviewed in [START_REF] Loffing | Sgk kinases and their role in epithelial transport[END_REF]). The expression of ENaC is typically maintained at a low level by ubiquitination and degradation. SGK1 phosphorylates the HECT E3 ubiquitin ligase, Nedd4.2, preventing its interaction with ENaC and hence preventing ENaC degradation [START_REF] Debonneville | Phosphorylation of Nedd4-2 by Sgk1 regulates epithelial Na(+) channel cell surface expression[END_REF][START_REF] Snyder | Serum and glucocorticoidregulated kinase modulates Nedd4-2-mediated inhibition of the epithelial Na+ channel[END_REF][START_REF] Ichimura | 14-3-3 proteins modulate the expression of epithelial Na+ channels by phosphorylation-dependent interaction with Nedd4-2 ubiquitin ligase[END_REF]. Consistent with this, SGK1 -/-mice have a salt wasting phenotype, excreting more Na + than wild-type mice when on a low salt diet, leading to reduced blood pressure [START_REF] Wulff | Impaired renal Na(+) retention in the sgk1-knockout mouse[END_REF][START_REF] Fejes-Toth | Epithelial Na+ channel activation and processing in mice lacking SGK1[END_REF]. SGK1 -/-mice are also resistant to dexamethasone-induced increases in blood pressure [START_REF] Boini | Role of serum-and glucocorticoid-inducible kinase SGK1 in glucocorticoid regulation of renal electrolyte excretion and blood pressure[END_REF] and display reduced glucocorticoid-induced intestinal glucose transport [START_REF] Grahammer | Intestinal function of gene-targeted mice lacking serum-and glucocorticoid-inducible kinase 1[END_REF]. As SGK1 activity is reduced in the Protor-1 -/-Protor-2 -/-double knockout mice, ENaC expression may also be lowered. However, we observed similar levels of ENaC and ENaC subunits in membrane fractions derived from the kidney tissue of wild type and Protor-1 -/-Protor-2 -/-double knockout mice (Supplementary Fig 4), suggesting that Protor isoforms are not essential for normal regulation of basal ENaC expression in otherwise healthy unstressed animals. It would be interesting to test whether under conditions of salt challenge or in It will also be necessary to address the mechanism by which Protor regulates efficient activation of SGK1 in the kidney. Our studies suggest that loss of Protor isoforms does not impact on the ability of immunoprecipitated mTORC2 to phosphorylate SGK1 or Akt. This raises the possibility that Protor-1 may instead act as a scaffolding component to localise SGK1 to a particular location within the cell and/or to present SGK1 to mTORC2. In S. cerevisiae TORC2 there are two non-essential components termed Bit61 and AVO2 [START_REF] Loewith | Two TOR complexes, only one of which is rapamycin sensitive, have distinct roles in cell growth control[END_REF]43], that have been implicated in binding two substrates of TORC2 (Slm1 and Slm2) [START_REF] Audhya | Genome-wide lethality screen identifies new PI4,5P2 effectors that regulate the actin cytoskeleton[END_REF][START_REF] Fadri | The pleckstrin homology domain proteins Slm1 and Slm2 are required for actin cytoskeleton organization in yeast and bind phosphatidylinositol-4,5-bisphosphate and TORC2[END_REF]. Although there is no obvious sequence similarity between Protor isoforms and Bit61 and AVO2, it would be interesting to investigate whether Protor isoforms performed a similar function. Thus far we have been unable to demonstrate a convincing co-immunoprecipitation of endogenous Protor-1 and SGK1 derived from kidney extracts. However, we cannot rule out the possibility that SGK1 and Protor-1 may interact with one another with low micromolar affinity. This would not be observed in co-immunoprecipitation studies, as the subunits would dissociate under the conditions and timeframe of such experiments.

In conclusion, our results establish that Protor-1 is required for SGK1 phosphorylation and signalling in the kidney. Further work is required to delineate the mechanism by which Protor-1 regulates efficient activation of SGK1 in the kidney and whether Protor isoforms are required for the activation of SGK1 and other SGK isoforms in other tissues and cell types. In addition, our data indicate that Protor-1 and Protor-2 play distinct roles and further work will be required to uncover the function of Protor-2. It is also possible that Protor isoforms play a key role in regulating mTORC2 function under conditions that we have not investigated. It is also possible that mTORC2 phosphorylates substrates other than Akt, SGK, and perhaps PKC isoforms, so in the future it will be important to investigate the role of Protor isoforms in controlling these, as yet uncharacterised, functions of mTORC2. The availability of Protor knockout mouse models and MEFs should prove useful for further studies to define the roles of Protor subunits in regulating mTORC2.

Table 1 Breeding strategy used to generate Protor-1 -/-, Protor-2 -/-and Protor-1 -/-Protor-2 -/-knockout mice. Single homozygous Protor-1 -/-, Protor-2 -/-as well as Protor-1 -/-Protor-2 -/-double knockout mice were bred using depicted strategy. The number and the percentage of each genotype are indicated followed by its expected Mendelian frequency. In the case of the Protor-1 -/- Protor-2 -/-double knockout mice, as wild-type littermate and double knock-in mice can only be obtained at a ratio of 1 in 16 in the same cross (Table 1), we interbred Protor-1 -/- Protor-2 -/-mice as well as Protor-1 +/+ Protor-2 +/+ animals. In the case of the 5' probe, the wild type allele produces a 4.9kb fragment while the conditional knockout allele generates a 7kb fragment. The 3' probe detects a fragment of 25.5kb from the wild type allele and a 15.1kb fragment from the conditional knockout allele. (C) Genomic DNA was PCR amplified with Protor-1 primers P1, P2 and P6. The wild-type allele (detected using P1 and P2) generates a 290bp product while the knockout allele (detected using P1 and P6) generates a 559bp product. (D) Protor-1 was immunoprecipitated from each of the indicated mouse tissue lysates and HEK293 cell lysate and immunoblotting carried out using the same antibody. (E) Diagram depicting the Protor-2 targeting construct, the endogenous Protor-2 allele, the targeted allele obtained following homologous recombination, the Protor-2 conditional knockout allele and the constitutive knockout allele. The positions of the Protor-2 PCR primers (P1, P2 & P4) used for genotyping are indicated with arrows. The translation initiation site for Protor-2 is within exons 2. (F) Genomic DNA purified from the targeted ES cells of the indicated genotypes was digested with either EcoRI or HindIII and subjected to Southern analysis with the corresponding DNA probes. In the case of the 5' probe, the wild type allele produces a 6.8kb fragment while the conditional knockout allele generates a 9.8kb fragment. The 3' probe detects a fragment of 12kb from the wild type allele and a 10.6kb fragment from the conditional knockout allele. (G) Genomic DNA was PCR amplified with Protor-2 primers P1, P2 and P4. The wild-type allele (detected using P1 and P2) generates a 221bp product while the knockout allele (detected using P1 and P4) generates a 527bp product. (H) Protor-2 was immunoprecipitated from each of the indicated mouse tissue lysates and immunoblotting carried out using the same antibody. (A) Kidney lysates were generated from fed Protor-1 +/+ , Protor-1 +/-and Protor-1 -/-mice and subjected to immunoblotting with the indicated antibodies. (B) As in (A) except lysates were from Protor-2 +/+ , Protor-2 +/-and Protor-2 -/-mice. (C) As in (A) except lysates were from Protor-1 +/+ Protor-2 +/+ and Protor-1 -/-Protor-2 -/-mice. (A) Protor-1 +/+ Protor-2 +/+ and Protor-1 -/-Protor-2 +/+ mice were deprived of food for 3hrs and intravenously injected with 0.5 g/g body weight IGF1 or the equivalent amount of PBS as a negative control. After 5min, tissues were rapidly removed and frozen in liquid nitrogen. Kidney lysates were subjected to immunoblotting with the indicated antibodies and each lane represents a sample derived from a different mouse. (B) As in (A) except Protor-1 +/+ Protor-2 +/+ and Protor-1 +/+ Protor-2 -/-were injected with IGF1. (C) As in (A) except Protor-1 +/+ Protor-2 +/+ and Protor-1 -/-Protor-2 -/-were injected with IGF1. The wild type and Protor-1 -/-Protor-2 -/-deficient MEF cells were serum-starved overnight and stimulated with 50 ng/ml IGF1 for 30 min prior to lysis and immunoblot analysis with the indicated antibodies. Similar results were obtained in two separate experiments.

Biochemical

  Journal Immediate Publication. Published on 18 Mar 2011 as manuscript BJ20102103 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20102103

Biochemical

  Journal Immediate Publication. Published on 18 Mar 2011 as manuscript BJ20102103 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20102103 A c c e p t e d M a n u s c r i p t

Biochemical

  Journal Immediate Publication. Published on 18 Mar 2011 as manuscript BJ20102103 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20102103

  Biochemical Journal Immediate Publication. Published on 18 Mar 2011 as manuscript BJ20102103 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20102103 A c c e p t e d M a n u s c r i p t

Biochemical

  Journal Immediate Publication. Published on 18 Mar 2011 as manuscript BJ20102103 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20102103 A c c e p t e d M a n u s c r i p t

Biochemical

  Journal Immediate Publication. Published on 18 Mar 2011 as manuscript BJ20102103 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20102103 A c c e p t e d M a n u s c r i p t 10 Discussion.

Biochemical

  Journal Immediate Publication. Published on 18 Mar 2011 as manuscript BJ20102103 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20102103 A c c e p t e d M a n u s c r i p t hypertensive models, Protor isoforms play a more dominant role in the control of ENaC expression.

Figure 1

 1 Figure 1 Generation of Protor-1 -/-and Protor-2 -/-knockout mice.(A) Diagram depicting the Protor-1 targeting construct, the endogenous Protor-1 allele, the targeted allele obtained following homologous recombination, the Protor-1 conditional knockout allele with the neomycin cassette removed by Flp recombinase and the constitutive knockout allele obtained following Cre-mediated recombination. The light grey rectangles represent exons, black rectangles indicate the targeted exons and the dark grey and light grey triangles represent loxP and FRT sites, respectively. The positions of the 5' and 3' probes used for Southern blot analysis and the Protor-1 PCR primers (P1, P2 & P6) used for genotyping are indicated. The translation initiation sites for Protor-1 / and Protor-1 are within exons 2 and 4 respectively. (B) Genomic DNA purified from the targeted ES cells of the indicated genotypes was digested with either BamHI or AseI and subjected to Southern analysis with the corresponding DNA probes. In the case of the 5' probe, the wild type allele produces a 4.9kb fragment while the conditional knockout allele generates a 7kb fragment. The 3' probe detects a fragment of 25.5kb from the wild type allele and a 15.1kb fragment from the conditional knockout allele. (C) Genomic DNA was PCR amplified with Protor-1 primers P1, P2 and P6. The wild-type allele (detected using P1 and P2) generates a 290bp product while the knockout allele (detected using P1 and P6) generates a 559bp product. (D) Protor-1 was immunoprecipitated from each of the indicated mouse tissue lysates and HEK293 cell lysate and immunoblotting carried out using the same antibody. (E) Diagram depicting the Protor-2 targeting construct, the endogenous Protor-2 allele, the targeted allele obtained following homologous recombination, the Protor-2 conditional knockout allele and the constitutive knockout allele. The positions of the Protor-2 PCR primers (P1, P2 & P4) used for genotyping are indicated with arrows. The translation initiation site for Protor-2 is within exons 2. (F) Genomic DNA purified from the targeted ES cells of the indicated genotypes was digested with either EcoRI or HindIII and subjected to Southern analysis with the corresponding DNA probes. In the case of the 5' probe, the wild type allele produces a 6.8kb fragment while the conditional knockout allele generates a 9.8kb fragment. The 3' probe detects a fragment of 12kb from the wild type allele and a 10.6kb fragment from the conditional knockout allele. (G) Genomic DNA was PCR amplified with Protor-2 primers P1, P2 and P4. The wild-type allele (detected using P1 and P2) generates a 221bp product while the knockout allele (detected using P1 and P4) generates a 527bp product. (H) Protor-2 was immunoprecipitated from each of the indicated mouse tissue lysates and immunoblotting carried out using the same antibody.

Biochemical

  Journal Immediate Publication. Published on 18 Mar 2011 as manuscript BJ20102103 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20102103 A c c e p t e d M a n u s c r i p t

Figure 2 NDRG1

 2 Figure 2 NDRG1 phosphorylation is reduced in the kidney of Protor-1 -/-knockout mice. Tissue lysates from (A) Kidney, (B) Adipose, (C) Brain (D) Heart, (E) Liver (F) Lung (G) Muscle, (H) Spleen, or (I) Testis were generated from fed mice of the indicated genotype and subjected to immunoblotting with the indicated antibodies.

Figure 3 NDRG1

 3 Figure 3 NDRG1 phosphorylation is reduced in the kidney of Protor-1 -/-and Protor-1 -/-Protor-2 -/-knockout mice.(A) Kidney lysates were generated from fed Protor-1 +/+ , Protor-1 +/-and Protor-1 -/-mice and subjected to immunoblotting with the indicated antibodies. (B) As in (A) except lysates were from Protor-2 +/+ , Protor-2 +/-and Protor-2 -/-mice. (C) As in (A) except lysates were from Protor-1 +/+ Protor-2 +/+ and Protor-1 -/-Protor-2 -/-mice.

Figure 4

 4 Figure 4 Phosphorylation of SGK1 is reduced in the kidney of Protor-1 -/-knockout mice.(A) Protor-1 +/+ Protor-2 +/+ and Protor-1 -/-Protor-2 +/+ mice were deprived of food for 3hrs and intravenously injected with 0.5 g/g body weight IGF1 or the equivalent amount of PBS as a negative control. After 5min, tissues were rapidly removed and frozen in liquid nitrogen. Kidney lysates were subjected to immunoblotting with the indicated antibodies and each lane represents a sample derived from a different mouse. (B) As in (A) except Protor-1 +/+ Protor-2 +/+ and Protor-1 +/+ Protor-2 -/-were injected with IGF1. (C) As in (A) except Protor-1 +/+ Protor-2 +/+ and Protor-1 -/-Protor-2 -/-were injected with IGF1.

Figure 5

 5 Figure 5 NDRG1 phosphorylation is normal in Protor deficient mouse embryonic fibroblasts (MEFs) .The wild type and Protor-1 -/-Protor-2 -/-deficient MEF cells were serum-starved overnight and stimulated with 50 ng/ml IGF1 for 30 min prior to lysis and immunoblot analysis with the indicated antibodies. Similar results were obtained in two separate experiments.

Figure 6

 6 Figure 6Protor-1 and Protor-2 are not required for mTORC2 complex formation in vivo.(A to C) Sin1 was immunoprecipitated from kidney lysates obtained from mice of the indicated genotype and subjected to immunoblotting with the indicated antibodies. Similar results were obtained in two separate experiments.

Figure 7

 7 Figure 7 Protor isoforms are not required for mTORC2 kinase activity in vitro.(A) Immunoprecipitates were prepared using either pre-immune IgG (negative control) or Sin1 antibodies from fed wild type or Protor-1 -/-Protor-2 -/-double knockout mouse kidney or liver lysates. Immunoprecipitates were incubated with GST-Akt1 in the presence of 0.1mM ATP and 10mM MgCl 2 for 30min at 30°C. Kinase reactions were analysed by immunoblotting with the indicated antibodies. (B) As in (A) except GST-N-SGK1 [61-431] was used as the substrate in the mTORC2 kinase assay. (C) As in (A) except immunoprecipitates were generated from kidney or liver lysates from mice injected with IGF1 for 5min. (D) As in (C) except GST-N-SGK1 [61-431] was used as the substrate in the mTORC2 kinase assay. (E) as in (A) except immunoprecipitates were prepared from wild type or Protor-1 -/-Protor-2 -/-double knockout MEF cells. (F) as in (E) except GST-N-SGK1 [61-431] was used as the substrate in the mTORC2 kinase assay. Similar results were obtained in two separate experiments.

C

  FIGURE 1

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© 2011 The Authors Journal compilation © 2011 Portland Press Limited

Acknowledgements.

We thank Gail Fraser for genotyping of the mice, the Sequencing Service (College of Life Sciences, University of Dundee, Scotland) for DNA sequencing and the protein production and antibody purification teams [Division of Signal Transduction Therapy (DSTT), University of Dundee] co-ordinated by Hilary McLauchlan and James Hastie. LRP is funded by an MRC UK Studentship and EMS is supported by an AstraZeneca supported BBSRC-CASE PhD Studentship. We thank the Medical Research Council, and the pharmaceutical companies supporting the Division of Signal Transduction Therapy Unit (AstraZeneca, Boehringer-Ingelheim, GlaxoSmithKline, Merck KgaA and Pfizer) for financial support.

Adipose IB: pNDRG1 IB: NDRG1 IB: Akt pS473 IB: Akt1 IB: pPRAS40 IB: PRAS40 IB: pNDRG1 IB: NDRG1 IB: Akt pS473 IB: Akt1 IB: pPRAS40 IB: PRAS40 IB: pNDRG1 IB: NDRG1 IB: Akt pS473 IB: Akt1 IB: pPRAS40 IB: PRAS40 Protor-1 +/+ +/--/-Brain Protor-1 Heart Protor-1 A B C IB: pNDRG1 IB: NDRG1 IB: Akt pS473 IB: Akt1 IB: pPRAS40 IB: PRAS40 IB: pNDRG1 IB: NDRG1 IB: Akt pS473 IB: Akt1 IB: pPRAS40 IB: PRAS40 +/+ +/--/-Muscle IB: pNDRG1 IB: NDRG1 IB: Akt pS473 IB: Akt1 IB: pPRAS40 IB: PRAS40 IB: pNDRG1 IB: NDRG1 IB: Akt pS473 IB: Akt1 IB: pPRAS40 IB: PRAS40 IB: pNDRG1 IB: NDRG1 IB: Akt pS473 IB: Akt1 IB: pPRAS40 IB: PRAS40 Protor-1 +/+ +/--/-+/+ +/--/-+/+ +/--/-+/+ +/--/-Kidney IB: pNDRG1 IB: NDRG1 IB: Akt pS473 IB: Akt1 IB: pPRAS40 IB: PRAS40 Protor-1 IB: pFoxO1 IB: FoxO1 IB: pFoxO1 IB: FoxO1 IB: pFoxO1 IB: FoxO1 IB: pFoxO1 IB: FoxO1 IB: pFoxO1 IB: FoxO1 IB: pFoxO1 IB: FoxO1 IB: pFoxO1 IB: FoxO1 IB: pFoxO1 IB: FoxO1 IB: pFoxO1 IB: FoxO1 Biochemical Journal Immediate Publication. Published on 18 Mar 2011 as manuscript BJ20102103 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20102103 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. © 2011 The Authors Journal compilation © 2011 Portland Press Limited IB: pNDRG1 IB: NDRG1 IB: Akt pT308 IB: Akt pS473 IB: Akt1 IB: pPRAS40 IB: PRAS40 IB: pNDRG1 IB: NDRG1 IB: Akt pS473 IB: Akt1 IB: pPRAS40 IB: PRAS40 IB: pNDRG1 IB: NDRG1 IB: Akt pS473 IB: Akt1 IB: pPRAS40 IB: PRAS40 IB: Akt pT308 IB: Akt pT308 A B IB: Akt pT450 IB: Akt pT450 IB: Akt pT450 IB: pNDRG1 IB: NDRG1 IB: pIGFR IB: IGFR IB: Akt pT308 IB: Akt pS473 IB: Akt1 IB: pPRAS40 IB: PRAS40 IB: SGK1 pS422 IB: SGK1 pT256 -IGF1: -+ + + --+ + + IB: SGK1 pS422 IB: SGK1 pT256 IB: pNDRG1 IB: NDRG1 IB: Akt pT308 IB: Akt pS473 IB: Akt1 IB: pPRAS40 IB: PRAS40 IB: pIGFR IB: IGFR -IGF1: -+ + + --+ + + IB: mTOR IB: Rictor IB: Sin1 IB: Protor-1 IB: Protor-2 Kidney IP: Sin1 IB: mTOR IB: Rictor IB: Sin1 IB: Protor-1 IB: Protor-2 Kidney IP: Sin1 IB: mTOR IB: Rictor IB: Sin1 IB: Protor-1 IB: Protor-2 Kidney IP: Sin1 IB: Akt pS473 IB: Akt1 IB: mTOR IB: SGK1 pS422 IB: SGK1 IB: mTOR IB: mTOR IB: Akt pS473 IB: Akt1 IB: mTOR IB: SGK1 pS422 IB: SGK1 IB: Akt pS473 IB: Akt1 IB: mTOR IB: SGK1 pS422 IB: SGK1 IB: mTOR Akt-mTORC2 activity assay SGK1-mTORC2 activity assay IB: mTOR IB: Akt pS473 IB: Akt1 IB: mTOR IB: SGK1 pS422 IB: SGK1

from Cell Signaling Technology (#9379).

Author Contributions.

LRP with the help of EMS performed most experiments, KS performed IGF1 injections and provided critical advice. LRP & SW performed the experiments shown in Supplementary Figure 1. LRP, EMS and DRA planned experiments, analysed the data and wrote the manuscript.

Genotype

Number Expected Frequency

Protor-1 +/+ 163 (31.6%) 25% Protor-1 +/- 249 (48.3%) 50% Protor-1 -/- 104 (20.1%) 25%

Protor-1 +/+ 8 (14.5%) 6.25% Protor-1 -/ - 4 (7.3%) 6.25% Protor-1 +/- 7 (12.7%) 25%

Cross

Protor-1 +/-X Protor- Protor-2 +/-X Protor-2 +/- X Protor-1 +/-Protor-2 +/- Protor-1 +/-Protor-2 +/- X Protor-1 +/+ Protor-2 +/+ Protor-1 +/+ Protor-2 +/+ X Protor-1 -/-Protor-2 -/- Protor-1 -/-Protor-2 -/-

TABLE 1

Protor-1 +/- 3 (5.5%) 12.5% Protor-1 +/+ 14 (25.3%) 12.5% Protor-1 -/- 3 (5.5%) 6.25% Protor-1 +/+ 3 (5.5%) 6.25% Protor-1 -/- 9 (16.4%) 12.5% Protor-1 +/- Protor-2 +/+ Protor-2 -/ - Protor-2 +/- Protor-2 +/+ Protor-2 +/- Protor-2 +/+ Protor-2 -/- Protor-2 +/- Protor-2 -/- 4 (7.3%) 12.5% Protor-1 +/+ Protor-2 +/+ 100%

Protor-1 -/-Protor-2