Virginie Le Brun

Wolfgang Friess

Torsten Schultz-Fademrecht

Silke Muehlau

Patrick Garidel
email: patrick.garidel@boehringer-ingelheim.com

Lysozyme -Lysozyme self-interactions as assessed by the osmotic second virial coefficient: Impact for physical protein stabilization

Keywords: Boehringer Ingelheim, Process Science Protein-protein interaction, osmotic second virial coefficient, selfinteraction chromatography, B22, Lysozyme Protein-protein interaction, B 22 , self-interaction chromatography, osmotic second virial coefficient, Lysozyme, stabilisation osmotic second virial coefficient (B 22 ), selfinteraction chromatography (SIC)

Lysozyme -Lysozyme self-interactions as assessed by the

osmotic second virial coefficient: Impact for physical protein stabilization

Introduction

Currently most protein therapeutics are applied via the parenteral route [START_REF] Banga | Delivery of protein therapeutics[END_REF][START_REF] Daugherty | Formulation and delivery issues for monoclonal antibody therapeutics[END_REF]. Among the parenteral routes of administration, the development efforts for subcutaneous injection systems have increased in the last years. This is due to the fact that, especially for immunoglobulin therapeutics, relatively high doses are necessary, requiring high protein concentrations up to 100 mg/ml and higher. Furthermore, high protein concentrations are encountered in the downstream processing for protein purification. For such highly concentrated protein solutions, protein self-interactions and the formation of protein aggregates, reversible or irreversible, are critical issues [START_REF] Garidel | Impact of formulation design on stability and quality, chapter 7[END_REF]. The formed protein aggregates often lack bioactivity, may be immunogenic, can have altered half-lives and may serve as nucleation activators. Therefore, various excipients are added to the protein solution to mitigate or avoid protein aggregation [START_REF] Harn | Highly concentrated monoclonal antibody solutions: Direct analysis of physical structure and thermal stability[END_REF]. Up to now, it is often unclear how the different excipients and especially combination thereof affect physical protein stability and protein solubility [START_REF] Hall | Macromolecular crowding: qualitative and semiquantitative successes, quantitative challenges[END_REF][START_REF] Zimmerman | Macromolecular crowding: biochemical, biophysical, and physiological consequences[END_REF].

Protein-protein interactions are often more pronounced if irreversible alterations in protein secondary structure are observed [START_REF] Garidel | Fourier-transform midinfrared spectroscopy for analysis and screening of liquid protein formulations. Part 2: detailed analysis and applications[END_REF][START_REF] Garidel | Fourier-transform midinfrared spectroscopy for analysis and screening of liquid protein formulations. Part 1: understanding infrared spectroscopy of protein[END_REF], with the transition from alpha-helix to beta-sheet structure being the most prominent change. Such changes reduce protein activity and solubility [START_REF] Garidel | Impact of formulation design on stability and quality, chapter 7[END_REF].

Up to now it is still under debate whether further structural perturbations occur after protein self-association and aggregation [START_REF] Bajaj | Protein structural conformation and not second virial coefficient relates to long-term irreversible aggregation of a monoclonal antibody and ovalbumin in solution[END_REF], thus increasing protein aggregation kinetic. coefficient. Winzor et al. [START_REF] Winzor | A simpler analysis for the measurement of second virial coefficients by self-interaction chromatography[END_REF] have even proposed a new thermodynamic approach supporting a simplified procedure for determining the osmotic second virial coefficient by SIC using a reduced number of parameters. Thus, there are a lot of activities in order to introduce and improve self interaction chromatography for measuring the osmotic second virial coefficient.

The concept behind the above described approach is related to studies and observations made many years ago. In 1946, Bruno Zimm published a paper entitled "application of the methods of molecular distribution to solutions of large molecules" [START_REF] Zimm | Application of the methods of molecular distribution to solutions of large molecules[END_REF], where he stated that the objective of statistical mechanics is to link the knowledge of solution thermodynamics to the properties of the molecules that compose the solution. Zimm recognized that the large deviations from Raoult's law, which are especially observed for "large" solutes like polymers and proteins, are related to non-ideal properties of the solutes and this can be accounted for by means of methods using continuous molecular distributions functions.

The outcome of his investigation was the ability to interpret and better understand thermodynamic data derived from protein solutions. The thermodynamic property examined by Zimm was the osmotic pressure, and he considered the different molecular interactions between the solutes in solution. Zimm analyzed the dependency of the concentration with regard to the osmotic pressure. 1945 William McMillan and Joseph Mayer [START_REF] Mcmillan | The statistical thermodynamics of multicomponent systems[END_REF] derived a series of expansion for the osmotic pressure (Π) in terms of concentration. Under the consideration that the higher order terms can be neglected, the non-ideality of Π is given as: with R is the gas constant, T is the absolute temperature, c p is the protein concentration in mass units, M w is the molecular weight of the protein and B 22 the osmotic second virial coefficient. The parameter B 22 reflects the extent and direction of the non-ideal solution property, and thus proteinprotein interactions.

Π = R • T• c p (1/M w + B 22 • c p ) (1)
Various investigations have given evidence that the osmotic second virial coefficient is a valuable parameter for the optimization of crystallization solution conditions which are prone to the formation of crystals [START_REF] Bonnete | Second virial coefficient: variations with lysozyme crystallization conditions[END_REF][START_REF] Bonnete | Interest of the normalized second virial coefficient and interaction potentials for crystallizing large macromolecules[END_REF][START_REF] George | Second virial coefficient as predictor in protein crystal growth[END_REF][START_REF] Guo | Correlation of second virial coefficients and solubilities useful in protein crystal growth[END_REF][START_REF] Neal | Why is the osmotic second virial coefficient related to protein crystallization?[END_REF][START_REF] Tessier | Predictive crystallization of ribonuclease A via rapid screening of osmotic second virial coefficients[END_REF].

Such solution conditions bear a slightly negative B 22 value, i.e. conditions denoting weak attractive interactions [START_REF] Tessier | Self-interaction chromatography: A novel screening method for rational protein crystallization[END_REF][START_REF] Bhamidi | Protein crystal nucleation: Is the pair interaction potential the primary determinant of kinetics?[END_REF]. Various authors have shown that supersaturated protein solutions primary form crystals when the corresponding equilibrium solubility is such that the resulting data for the osmotic second virial coefficient is between -1⋅10 -4 and -8⋅10 -4 mol⋅ml⋅g -2 [START_REF] Bhamidi | Protein crystal nucleation: Is the pair interaction potential the primary determinant of kinetics?[END_REF][START_REF] Cheng | Salting-out of lysozyme and ovalbumin from mixtures: Predicting precipitation performance from proteinprotein interactions[END_REF][START_REF] Tessier | Measurements of protein self-association as a guide to crystallization[END_REF][START_REF] Ahamed | Design of self-interaction chromatography as an analytical tool for predicting protein phase behavior[END_REF][START_REF] Haas | Relation between the solubility of proteins in aqueous solutions and the second virial coefficient of the solution[END_REF].

When the negative B 22 value further decreases, solution conditions are obtained enhancing particle-particle attractions, and as a result in a number of cases amorphous protein precipitation is observed [START_REF] Ho | The likelihood of aggregation during protein renaturation can be assessed using the second virial coefficient[END_REF][START_REF] Neal | Molecular origins of osmotic second virial coefficients of proteins[END_REF][START_REF] Valente | Colloidal behavior of proteins: effects of the second virial coefficient on solubility, crystallization and aggregation of proteins in aqueous solution[END_REF].

This concept was applied for the prediction of solubility activity. Within the framework of the sticky hard sphere model [START_REF] Baxter | Percus-Yevick equation for hard spheres with surface adhesion[END_REF], Rosenbaum and colleagues [START_REF] Rosenbaum | Phase behavior of small attractive colloidal particles[END_REF][START_REF] Rosenbaum | Protein interactions and crystallization[END_REF] showed that the experimentally measured B 22 can be correlated with solubility data [START_REF] Haas | Relation between the solubility of proteins in aqueous solutions and the second virial coefficient of the solution[END_REF][START_REF] Lima | Osmotic second virial coefficients and phase diagrams for aqueous proteins from a much-improved Poisson-Boltzmann equation[END_REF][START_REF] Ruppert | Correlation between the osmotic second virial coefficient and the solubility of proteins[END_REF][START_REF] Stigter | Theory of the Donnan membrane equilibrium .2. calculation of the osmotic pressure and of the salt distribution in a Donnan system with highly charged colloid particles[END_REF][START_REF] Wanka | Die Bedeutung des zweiten osmotischen Virialkoeffizienten für die Proteinkristallization[END_REF]. This signifies that short range attractive interactions are the main interactions that describe the phase properties and behavior of a colloidal system [START_REF] Polyakov | An approach to determine the 2 nd virial-coefficient for calculation of phase-equilibria in water-protein-neutral polymer systems[END_REF]. Based on these considerations, the relation between B 22 and the solubility S is given by:

B 22 = (-∆µ 2 / R•T) • (1 / 2•M 2 •S) -(lnS / 2•M 2 •S) (2)
With ∆µ 2 = µ 2 0 (solution) -µ 2 0 (solid), µ 2 0 (solution) is the chemical potential of the protein in solution, µ 2 0 (solid) is the chemical potential of the protein in a solid form (e.g. crystal), R is the gas constant, T is the absolute temperature, M 2 is the molecular weight of the protein (denoted as 2) and S is the solubility expressed in g•ml -1 . This approach clearly shows that the second virial coefficient and the solubility are correlated [START_REF] Guo | Correlation of second virial coefficients and solubilities useful in protein crystal growth[END_REF][START_REF] Haas | Relation between the solubility of proteins in aqueous solutions and the second virial coefficient of the solution[END_REF]. Ruckenstein and Shulgin [START_REF] Ruckenstein | Effect of salts and organic additives on the solubility of proteins in aqueous solutions[END_REF] have recently reviewed the correlation between aqueous protein solubility and the osmotic second virial coefficient in the presence of salts, where good agreement were obtained between theoretical predictions and experimental results. When the cosolvent is an organic component, the situation is not so clear. Last year, Shulgin and Ruckenstein [START_REF] Shulgin | Various contributions to the osmotic second virial coefficient in protein-water-cosolvent solutions[END_REF] presented a study using the Kirkwood-Buff fluctuation theory [START_REF] Pierce | Recent applications of Kirkwood-Buff theory to biological systems[END_REF] to elucidate the effects of various contributions to the osmotic second virial coefficient in protein-water-cosolvent solutions and how to interpret this (for more details see reference).

Various molecular theories of fluids can be used to develop a mean force expression for describing protein-protein interactions. The application of chromatography methods like self-interaction chromatography (SIC) [START_REF] Winzor | A simpler analysis for the measurement of second virial coefficients by self-interaction chromatography[END_REF][START_REF] Payne | Second virial coefficient determination of a therapeutic peptide by self-interaction chromatography[END_REF][START_REF] Tessier | Rapid measurement of protein osmotic second virial coefficients by self-interaction chromatography[END_REF] requires the correlation of the osmotic coefficient of diluted to concentrated aqueous solution conditions [START_REF] Haynes | Thermodynamic properties of aqueous alpha-chymotrypsin solutions from membrane osmometry measurements[END_REF]. solution conditions [START_REF] Rosenbaum | Phase behavior of small attractive colloidal particles[END_REF][START_REF] Rosenbaum | Protein interactions and crystallization[END_REF][START_REF] Rosenberger | Nucleation and crystallization of globular proteins -What we know and what is missing[END_REF][START_REF] Muschol | Lack of evidence for prenucleation aggregate formation in lysozyme crystal growth solutions[END_REF][START_REF] Muschol | Liquid-liquid phase separation in supersaturated lysozyme solutions and associated precipitate formation/crystallization[END_REF][START_REF] Muschol | Interactions in undersaturated and supersaturated lysozyme solutions: static and dynamic light scattering results[END_REF][START_REF] Rosenbaum | Protein interactions and phase behavior: Sensitivity to the form of the pair potential[END_REF].

In contrast to the typical B 22 target, i.e. the optimization of protein crystallization solution conditions, in the presented study, solution conditions that minimize attractive protein-protein interactions have to be identified, in order to avoid protein precipitation and aggregation. The procedure for the determination of the osmotic second virial coefficients as derived from SIC can be briefly summarized as follow. First, the protein of interest is covalently immobilized on chromatography particles. Then, a pulse of the protein of interest, free in solution, is injected and passed through the chromatography column filled with the chromatography beads carrying the protein on their surface. The obtained elution profile reflects the interaction of immobilized protein with protein that is free in solution, under the assumption that the immobilized protein retains its native threedimensional and secondary structure (this will be addressed in a subsequent paper, which is in preparation: comment of the authors).

Furthermore, it is assumed that the protein is immobilized to the chromatography particles in a broad range of orientations, avoiding a side specific interaction, which will not be representative for the interaction between two protein molecules, both free in solution. Under these conditions the measured protein-protein interaction reflects the ensemble The influence of various excipients with regard to protein-protein interactions can be screened using an automated chromatography system. Thus, the effectiveness of formulation solutions, containing e.g. different excipients at varying concentrations, can easily be assessed.

Solution conditions reducing attractive protein-protein interactions are characterized by a reduction of the retention volume, whereas the opposite is the case for solution conditions favoring attractive proteinprotein interactions.

The following solution conditions are investigated with regard to lysozymelysozyme interactions:

• protonation degree

• ionic strength • temperature
• salts of the Hofmeister series

• pharmaceutical excipients (sucrose, glycerol)

• PEGs (poly-ethylene glycol) of different molecular weights

In addition, the effect of chromatography bead surface coverage as well as storage stability of the functionalized chromatography particles with regard to the osmotic second virial coefficient was studied. B 22 is also correlated with protein solubility and protein physical stability. The pH of the solutions was adjusted using hydrochloric acid or sodium hydroxide and measured with a pH meter Inolab level 1 from WTW (Weilheim, Germany). The protein concentrations were evaluated with an UV-photometer UV1 from Thermo Spectronic (Dreieich, Germany).

F

Lysozyme immobilization

3 ml Toyopearl AF-Formyl 650M particles were washed on a glass frit with a 0.2 µm hydrophilic polyethersulfone membrane filter with first 250 ml deionized water and secondly with 50 ml of 0.1 M potassium phosphate with the same buffer 1 M NaCl, 5 mM acetic acid buffer pH 4.5 at a flow rate of 3 ml/min during 15 min using a FPLC system (Äkta Purifier, GE Healthcare, Uppsala, Sweden). At the end of the packing procedure the flow-rate was maintained at 0.75 ml/min during at least 30 min. The column integrity was tested by injecting 50 µl of 1 v% acetone solution.

Columns were stored at 4 °C in a 5 mM sodium phosphate buffer pH 7.0 containing 0.05 w% sodium azide. that measures the strength of interaction between the mobile phase protein and non-interacting species:

V r is the volume required to elute the protein in the mobile phase and V o the retention volume of non-interacting species (e.g. acetone). B 22 is related to the retention factor as follows [START_REF] Zimm | Application of the methods of molecular distribution to solutions of large molecules[END_REF][START_REF] Tessier | Rapid measurement of protein osmotic second virial coefficients by self-interaction chromatography[END_REF]: ρ s is the number of immobilized molecules per unit area, Ф the phase ratio, which is the total available surface available to the mobile phase protein, r the protein radius, N A Avogadro's number and M 2 the protein (index 2) molecular weight.

(3) V o -V r V o k' = B 22 = B HS - (4) k' ρ s . Ф B HS = π . r³.

Determination of lysozyme charges

Lysozyme molecule charges were calculated for the amino acid sequence of lysozyme from white chicken egg (EC 3.2.1.17) with the EMBOSSsoftware [START_REF] Rice | EMBOSS: the European Molecular Biology Open Software Suite[END_REF] based on single chain and 4 disulfide bonds.

Determination of protein solubility

350 µl of buffer solution were added to 100 mg lysozyme powder. The samples were first continually stirred during 24 h at 25°C to facilitate the powder dissolution and then centrifuged at 20,000 g during 1 h. After correction of the pH shift, the samples were stirred a second time 12 h at 25°C and finally centrifuged at 20,000 g during 1 h and pH controlled again. Lysozyme concentration was measured by UV spectroscopy at 280 nm with an extinction coefficient of 2.63 ml • mg -1 • cm -1 . Each determination was carried out in triplicate.

Determination of protein physical stability via stirring stress

Stirring stress was performed using 10 R glass vials (Schott, Mainz, Germany) filled with 5 ml of 10 mg/ml lysozyme solution at 25 °C. The

Results and Discussion

The surface charge of a protein, and related effects e.g. protein hydration, has a strong influence on protein-protein interactions. Therefore, the solubility of proteins is minimal at pH solution conditions close to their pI.

Lysozyme is a protein with an extremely basic pI (pI ≈11) [START_REF] Alderton | Isolation of lysozyme from egg white[END_REF][START_REF] Wetter | Immunological studies on egg white proteins .4. Immunochemical and physical studies of lysozyme[END_REF][START_REF] Zschornig | Modulation of lysozyme charge influences interaction with phospholipid vesicles[END_REF]. Its titration curve (in absence of any excipient, i.e. ion binding is not considered that may affect the surface charge in solution) was calculated using the EMBOSS-software [START_REF] Rice | EMBOSS: the European Molecular Biology Open Software Suite[END_REF]. At pH 4.5 lysozyme is positively charged, with 11 positive elementary charges. At pH 7.5 lysozyme is still positively charged, with 8 positive elementary charges. Charge neutralization is given for pH 11.

Correlation between protein surface coverage and B 22

As described above, the approach using SIC for the determination of B 22 based on the consideration of Zimm [START_REF] Zimm | Application of the methods of molecular distribution to solutions of large molecules[END_REF] takes account of an anisotropy interaction energy of a two-body interaction (i.e. interaction of two protein molecules). This means that the potential mean force for the description of the interaction depends on the separation of the two bodies and is a function of all orientations. Therefore, the concept just considers the interaction of a free protein molecule in solution with only one protein molecule immobilized on the chromatography particles. The interaction of e.g. a free protein molecule in solution with two immobilized protein molecules, i.e. a three-body interaction, is not considered (see equation 1). Therefore, the validity of the applied method depends on the chromatography particle surface coverage of the immobilized protein [START_REF] Tessier | Rapid measurement of protein osmotic second virial coefficients by self-interaction chromatography[END_REF].

This has also to be considered for the interaction in solution of the free protein molecules, namely interactions of free protein molecules with each other in solution should be strongly reduced. This can easily be realized using relatively diluted protein solutions. Thus, the free protein in solution interacts just with one immobilized protein molecule.

Three different degrees of protein surface coverage were tested: 18 mg/g, 21 mg/g and 56 mg/g (mg protein per g chromatography particles). The experiment with 21 mg/g was repeated with a similar surface coverage of 22 mg/g. The protein surface coverage was determined using UV difference measurement and via the BCA assay (see section Materials and Methods). The data reported in Figure 2 represent the average data of 6 independent experiments with their standard error of deviations.

As can be seen from Figure 2 M NaCl B 22 data are negative. Thus, at higher NaCl concentrations attractive protein-protein interactions are favored, because the protein charges become strongly screened with higher amounts of present NaCl [START_REF] Retailleau | No salting-in of lysozyme chloride observed at how ionic strength over a large range of pH[END_REF][START_REF] Retailleau | Importance of the nature of anions in lysozyme crystallisation correlated with protein net charge variation[END_REF].

With regard to the protein surface coverage (Figure 2) between 18 -56 mg/g the obtained B 22 data are quite similar with a slight trend of decreasing values for 56 mg/g. This is in accordance with literature [START_REF] Tessier | Rapid measurement of protein osmotic second virial coefficients by self-interaction chromatography[END_REF].

The comparison of two independent preparations of protein immobilization to the chromatography particles (21 and 22 mg lysozyme / g chromatography particles) shows a rather good reproducibility of B 22 values of ± 0.1 • 10 -4 mol • ml • g -2 at different NaCl concentrations.

Based on the derived theoretical approach, the osmotic second virial coefficient should be independent of protein concentration. This is the case when assuming no changes in protein conformation as well as neglecting higher-order processes [START_REF] Valente | Colloidal behavior of proteins: effects of the second virial coefficient on solubility, crystallization and aggregation of proteins in aqueous solution[END_REF]. Using relative low protein surface coverages, Teske et al. [START_REF] Teske | Measurement of lysozyme-lysozyme intrctions with quantitative affinity chromatography[END_REF] were able to show a good correlation between their results of osmotic second virial coefficient and results derived from static light scattering. Large deviations, compared to the results obtained by static light scattering, were observed for protein surface coverage up to 33 %. This implies multiple body interactions occur with increasing protein surface coverage. The presented result, is however not in accordance to the study presented by Tessier et al. [START_REF] Tessier | Self-interaction chromatography: A novel screening method for rational protein crystallization[END_REF] , who found that a coverage close to 33 % is required. The data presented in this study are in accordance to the SIC results presented by Tessier et al. [START_REF] Tessier | Rapid measurement of protein osmotic second virial coefficients by self-interaction chromatography[END_REF]. They have also shown a quantitative agreement between virial coefficients measured by SIC and by static light scattering. This is also evident from other studies [START_REF] Valente | Colloidal behavior of proteins: effects of the second virial coefficient on solubility, crystallization and aggregation of proteins in aqueous solution[END_REF][START_REF] Liu | Interactions of lysozyme in guanidinium chloride solutions from static and dynamic light-scattering measurements[END_REF][START_REF] Asthagiri | A consistent experimental and modeling approach to light-scattering studies of protein-protein interactions in solution[END_REF]. These examples show that the SIC method is a reliable technique for measuring virial coefficients.

Effect of temperature

The effect of temperature with regard to B 22 depends on the overall solution condition properties. Figure 3A shows the variation of B 22 as a function of temperature in the range between 5 and 35 °C at a NaCl concentration of 800 mM (pH = 4.5). The investigated temperature interval is well below the denaturation temperature of lysozyme [START_REF] Garidel | Fourier-transform midinfrared spectroscopy for analysis and screening of liquid protein formulations. Part 2: detailed analysis and applications[END_REF]. Thus the interactions between native structures are investigated. Under these solution conditions and in the investigated temperature interval all B 22 data are negative and lie between -7 and -1 • 10 -4 mol • ml • g -2 . These are solution conditions favoring crystallization [START_REF] Zhang | Precipitants and additives for membrane crystallization of lysozyme[END_REF][START_REF] Cheng | Salting-out of lysozyme and ovalbumin from mixtures: Predicting precipitation performance from proteinprotein interactions[END_REF][START_REF] Dumetz | Effects of pH on protein-protein interactions and implications for protein phase behavior[END_REF][START_REF] Dumetz | Protein phase behavior in aqueous solutions: crystallization, liquid-liquid phase separation, gels, and aggregates[END_REF][START_REF] Dumetz | Patterns of protein protein interactions in salt solutions and implications for protein crystallization[END_REF].

With increasing temperature repulsive interactions intensify. However, this trend depends on the overall solution conditions. Reducing the ionic strength from 0.8 M NaCl to 0.3 M NaCl, the temperature effect is rather small (Figure 3B) with a slight decrease in B 22 . Under these solution conditions B 22 is positive with values between 2.5 and 3.5

• 10 -4 mol • ml • g -2 .
F o r P e e r R e v i e w

Valente et al. [START_REF] Valente | Colloidal behavior of proteins: effects of the second virial coefficient on solubility, crystallization and aggregation of proteins in aqueous solution[END_REF] also observed the osmotic second virial coefficient becoming more positive as the temperature increases, which can be correlated with increasing protein solubility with increasing temperature.

Antipova et al. [START_REF] Antipova | Effect of sucrose on the thermodynamic properties of ovalbumin and sodium caseinate in bulk solution and at air-water interface[END_REF] reported an opposite effect, namely a decrease in the osmotic second virial coefficient as the temperature was increased up to 50 °C. Indeed, when the temperature increases, proteins begin to partially unfold, exposing hydrophobic regions that lead to more attractive interactions [START_REF] Valente | Colloidal behavior of proteins: effects of the second virial coefficient on solubility, crystallization and aggregation of proteins in aqueous solution[END_REF]. One important factor governing the trend of the osmotic second virial coefficient as a function of temperature increase is due to the fact whether the present excipients are able to solubilise or even stabilize the protein against thermal induced protein denaturation. This means, the comparison of such studies is only possible, when exactly the same formulations are used and tested. Tiny changes in the formulation composition seems to have a large effect on the osmotic second virial coefficient.

Effect of the protonation degree

Lysozyme has a strongly alkaline isoelectric point of ≈11 [START_REF] Alderton | Isolation of lysozyme from egg white[END_REF][START_REF] Wetter | Immunological studies on egg white proteins .4. Immunochemical and physical studies of lysozyme[END_REF][START_REF] Zschornig | Modulation of lysozyme charge influences interaction with phospholipid vesicles[END_REF].

Choosing pH conditions close to the pI would reduce protein solubility (see below) and thus favor protein precipitation. Increasing the pH substantially decreases B 22 (Figure 4). Under acidic pH conditions B 22 is positive, whereas at pH ≈7 B 22 becomes negative. Experiments for pH > 8 were not followed, because strongly alkaline solution conditions would damage the chromatography particles. In the presented case the dependency on pH In this context, one has to be aware that lysozyme has then tendency to self-associated depending on the pH of the solution [START_REF] Carlsson | Monte Carlo simulations of lysozyme selfassociation in aqueous solution[END_REF][START_REF] Hampe | A biophysical model of lysozyme self-association[END_REF][START_REF] Onuma | Lysozyme dimer association: similarities and differences compared with lysozyme monimer association[END_REF][93].

Correlation between B 22 and protein solubility

For protein formulation, solution conditions favoring protein solubility are of interest in order to stabilize the protein solution. Thus, the assay allows the identification of solution conditions leading to highest protein solubility.

Effect of ionic strength and various salts of the Hofmeister series

For more than 120 years (1888) it has been known from the pioneering work of Franz Hofmeister [94] that the presence of some salts is able to precipitate proteins, whereas other salts exert a salting in effect. The salt concentrations applied for e.g. protein precipitation by (NH 4 ) 2 SO 4 are often very high (up to 3 M). Especially at these high salt concentrations the effects and trends of the Hofmeister series become relevant [START_REF] Curtis | Protein-protein interactions in concentrated electrolyte solutions[END_REF]. As well as sulphate salts, the chloride salts of Na + , K + and Mg 2+ have a higher effect at large salt concentration, with MgCl 2 having the largest salting-in effect (Figure 6B1). At 0.8 M salt concentration one observed: (B 22 > 0) (see Figure 6B2), which is representative for repulsive interactions. This trend has also been described by Tessier et al. [START_REF] Tessier | Rapid measurement of protein osmotic second virial coefficients by self-interaction chromatography[END_REF]. The reason for this behavior is due to binding of the divalent magnesium cation to the acidic residues of lysozyme. This effect depends on the pH of the solution.

Effect of various pharmaceutical excipients (sucrose and glycerol)

In the presence of glycerol in the concentration range from 0 to 6 w% and in the presence of sucrose up to a concentration of 20 w%, the B 22 data are all strongly positive and nearly independent of excipient concentration (Figure 7). The reported data are between 8 and 10

• 10 -4 mol • ml • g -2 .
Thus, the presence of e.g. sucrose favors repulsive interactions between lysozyme molecules. The additional presence of high concentrations of NaCl (0.8 M) induces a strong decrease of B 22 . However, the presence of high sugar concentrations (up to 10 w% of sucrose) overcompensates the charge screening effect of the salt, which leads to a salting-out effect.

Thus the overall solution condition properties become positive in the sense of favoring protein solubility at sucrose concentration larger than 10 w%. The presence of glycerol also induces an increase of B 22 in presence of NaCl in protein solution (see Figure 7).

Valente et al. [START_REF] Valente | Second virial coefficient studies of cosolvent-induced protein self-interaction[END_REF] have reported that sugars (e.g. sucrose) influence the conformational dynamics of proteins, favoring the most compact conformation within the native-state ensembles. This reduction of protein volume is accompanied by a concomitant reduction in the hydrophobicity of the protein surface, thus reducing the tendency for protein-protein self association [START_REF] Valente | Second virial coefficient studies of cosolvent-induced protein self-interaction[END_REF]. Kaushik and Bhat [START_REF] Kaushik | Thermal stability of proteins in aqueous polyol solutions: Role of the surface tension of water in the stabilizing effect of polyols[END_REF] have pointed out to another possible stabilization mechanism that has to be considered. They explained the stabilization effect of certain polyols and sugars being related to the surface tension increase (for more details see [START_REF] Kaushik | Thermal stability of proteins in aqueous polyol solutions: Role of the surface tension of water in the stabilizing effect of polyols[END_REF]).

Effect of PEG molecular weight

Under the tested solution conditions, the B 22 values are all positive for the three tested PEG qualities with molecular weights of 6000, 4000 and 400 g/mol (Figure 8A). Under these conditions protein precipitation is unlikely.

The trend for the high molecular weight PEG is very similar. Increasing the

Storage stability of the functionalized chromatography column

Using the presented method as a screening assay, it is crucial that the column shows certain stability in order to be able to perform and evaluate various solution conditions with regard to B 22 . Therefore, the storage stability at 4 °C of the functionalized column was investigated (Figures 9).

The column was stored in a 5 mM sodium phosphate buffer at pH 7.0 containing 0.05% sodium azide at 4 °C up to 307 days. Figure 9A shows that the obtained B 22 data are very similar showing that the column still remains its functionality. However, analysis of the chromatograms (Figure 9B) shows the appearance of a shoulder at about 2.3 ml. This is especially observed after the column has been stored for 161 days. Based on the obtained data, stability of the lysozyme functionalized column, under the chosen storage conditions, is given for 3 months, which is in accordance to Tessier et al. [START_REF] Tessier | Rapid measurement of protein osmotic second virial coefficients by self-interaction chromatography[END_REF]. This allows SIC as being used as a high throughput screening method for the evaluation of a large number of solution conditions.

Physical protein stability via stirring stress

The physical protein stability was investigated under rapid stirring (1200 rpm) of 10 mg/ml lysozyme formulations in 300 mM NaCl at 25 °C as a function of pH. The stability of the different formulations was assessed by The overall protein solution conditions are dependent on a number of factors of the solution (e.g. pH, temperature, ionic strength, osmolarity)

and the presence of excipients and cosolutes. The presence of various ions/excipients may have a more or less pronounced impact on the protein solution and stability conditions, depending on whether unspecific or specific interactions are involved. The interactions of ions with proteins are governed by electrostatic interactions, but the influence of solvation (and solvation forces) also has to be considered [START_REF] Paliwal | Light-scattering studies of protein solutions: Role of hydration in weak protein-protein interactions[END_REF][START_REF] Valente | Second virial coefficient studies of cosolvent-induced protein self-interaction[END_REF]. The interactions of protein molecules in an aqueous environment are mediated by water molecules, and excipients and cosolutes can influence the protein surface characteristics, as well as the structure of the liquid water (kosmotropic and chaotropic agents). These effects on the water/protein interface influence directly the interfacial energies involved in the interaction and these interfacial energies can not be predicted so easily.

As described above, the effect of salts on the protein stability characteristics depends on the overall solution conditions. Yamasaki et al. [START_REF] Yamasaki | Differential scanning calorimetric studies on bovine serum albumin: II. Effects of neutral salts and urea[END_REF] have shown for BSA with regard to the thermally induced denaturation, that in the presence of kosmotrope salts, the protein structure is stabilized, whereas it becomes destabilized in the presence of chaotrope salts. This is especially the case at high ionic strengths, but a reversal of the stability effect is observed at low ionic strength (0.01-0.1 M). The reason for such a behavior lies in the screening of the electrostatic repulsions via the close interaction/binding of the anion to the In this study, it was shown that the effect of different salts/ions with respect to increasing protein solubility, reflected in an increase of B 22 , does not necessarily correlate with the Hofmeister series. In some cases a reverse effect is even observed. This was also described by Riès-Kautt & Ducruix [START_REF] Rieskautt | Relative effectiveness of various ions on the solubility and crystal-growth of lysozyme[END_REF]. The reason for this effect was attributed to the effectiveness of anions to promote protein crystallization, which is dependent on the net charge of the protein. Therefore, for acidic proteins the salting-in/saltingout behavior of ions follows the Hofmeister series, whereas the effect and order are reversed for basic proteins like lysozyme.

A strong impact on B 22 was observed as a function of the nature of the anion in solution.

The presented study shows that self-interaction chromatography can be used as a rapid development tool for biopharmaceuticals. It has to be emphasized that the used methods are based on the characterization of protein-protein interactions in their native state [START_REF] Bajaj | Protein structural conformation and not second virial coefficient relates to long-term irreversible aggregation of a monoclonal antibody and ovalbumin in solution[END_REF]. The second osmotic virial coefficient, which is derived from self-interaction chromatography, is a parameter of solution non-ideality that is useful for the prediction of solution conditions minimizing protein aggregation or solution conditions promoting protein solubility (see Figure 5). SIC allows rapid determination of B 22 under high throughput conditions using automated systems. A recent study [START_REF] Johnson | High-throughput selfinteraction chromatography: Applicationd in protein formulation prediction[END_REF] has presented a high-throughput self interaction chromatography application combined with chemometry (ANN: artifical Self interaction chromatography can be used for screening evaluations, with column stability given for at least 3 months stored at 4°C (for lysozyme). The analysis of B 22 determines protein-protein interactions and is an indicator for physical protein stability (see Figure 10) [START_REF] Bajaj | Protein structural conformation and not second virial coefficient relates to long-term irreversible aggregation of a monoclonal antibody and ovalbumin in solution[END_REF][START_REF] Hall | Macromolecular crowding: qualitative and semiquantitative successes, quantitative challenges[END_REF][START_REF] Chi | Roles of conformational stability and colloidal stability in the aggregation of recombinant human granulocyte colony-stimulating factor[END_REF].

Thus, for formulation and/or purification development, the determination of the second osmotic virial coefficient allows the identification of solution conditions promoting protein solubility and reducing the formation of aggregation due to protein-protein interactions. For the development of acceptable formulations one has additionally to consider solution conditions that are well tolerated for the intended medical application. For example, the pH of subcutaneous formulation should be close to the physiological pH, whereas the pH of infusion formulation can be much more acidic (pH of approximately 5). Thus, using additional excipients, for a given (acceptable) pH, one has to identify solution conditions leading to repulsive interactions between the protein molecules in order to avoid protein aggregation. It has to be mentioned excipients reducing the conformational stability of the protein in solution, should be avoided.

In future investigations, the presented method will be used for the analysis of protein-protein interactions of therapeutic proteins, e.g. antibodies, in (Injection of 20 µl lysozyme solution at 20 mg/ml, storage at 4°C in 5 mM sodium phosphate buffer pH 7.0 containing 0.05% sodium azide).

  Rosenberger and coworkers[START_REF] Rosenberger | Nucleation and crystallization of globular proteins -What we know and what is missing[END_REF] have shown by means of static and dynamic light scattering for undersaturated and supersaturated lysozyme solutions that B 22 values measured at low solution concentrations, which are prone for the formation of crystals, are indicative for supersaturated

  between two protein molecules under the investigated solution conditions.

5 . 1 M

 51 The washed particles were recovered and mixed to 10 ml lysozyme solution (6.5 mg/ml in 0.1 M potassium phosphate buffer pH 7.5) and 90 mg sodium cyanoborhydride used as activator of protein binding.The suspension was mixed over night (≈12 h) on a rotary mixer. At the end of the coupling reaction the particles were washed with 200 ml of 0.potassium phosphate buffer. After recovery they were added to 15 ml of 1 M ethanolamine pH 8.0 and 20 mg sodium cyanoborhydride to cap the remaining matrix reactive groups. The suspension was mixed on a rotary mixer during 4 h. At the end of the reaction the particles were washed with 200 ml of 1 M sodium chloride solution pH 7.0 to remove any unbound material. The amount of bound protein was determined by analyzing the absorbance of the initial protein solution and the wash solutions (A280), and by determining the protein quantity immobilized on the matrix by BCA (bicinchoninic acid) assay.The chromatography particles were prepared in the form of a 50%-slurry in 1 M NaCl, 5 mM acetic acid solution pH 4.5. Approximately 2.5 ml slurry was packed in a Tricorn ® 5/50 column (GE Healthcare, Uppsala, Sweden)

the osmotic second virial coefficient B 22

 22 All mobile phase solutions were buffered with 5 mM acetic acid at pH 3.0 and 4.5 or 5 mM sodium phosphate at pH 6 -8. Lysozyme was dissolved in the studied solutions at 20 mg/ml. B 22 measurements were realized with a FPLC Äkta Purifier system equipped with an UV detector (A280). Before each run the column was equilibrated with 10 ml of protein free mobile phase. The column dead volume was determined with the 1 v% acetone solution injection. All experiments were carried out at 25 °C and at a flow rate of 0.75 ml/min. 10 µl sample was injected; each sample was measured 6 times. Chromatogram peaks were analyzed with the UNICORN ® software (GE Healthcare, Uppsala, Sweden). The retention volume was determined at the peak maximum. The retention measurements were used to calculate the retention factor k' (equation 3)

B

  [START_REF] Winzor | Nonequivalence of second virial coefficients from sedimentation equilibrium and static light scattering studies of protein solutions[END_REF] was measured, for a constant protein surface coverage at constant temperature T = 25°C, 5 mM acetic acid pH = 4.5, as a function of ionic strength. The NaCl concentration was varied between 0 M to 0.8 M. Figure1shows exemplarily elution profiles of lysozyme at four different ionic strengths. The area under the curve (AUC) remains mainly unchanged.The retention volume increases with increasing ionic strength, reflecting changes in B 22 .

  an increase of the ionic strength induces a decrease of B 22 . At low ionic strength B 22 values are positive, and a change in sign is observed at a NaCl concentration of ≈0.6 M. Above 0.6

  B 22 is linear. At pH 11 one would obtain a B 22 of -3 • 10 -4 mol • ml • g -2 (data derived from extrapolation), reflecting conditions prone for precipitation. The explanation for the pH dependency with regard to B 22 is the change in protonation degree of lysozyme accompanied by changes of protein hydration. With decreasing pH the positive surface charge increases, i.e. the zetapotential and thus the solubility [58, 89]. These results are in accordance with Dumetz et al. [85]. Elcock and McCammon [14] have shown how to calculate and consider the pH dependency of the osmotic second virial coefficient by taking into account protein-protein interaction energies related to (a) electrostatic interactions based on the use of effective charges, (b) electrostatic desolvations occurring when charged groups are buried by an approaching protein partner and (c) van der Waals and hydrophobic interactions. The proposed calculations seems to be adequate for the description of weak protein-protein interactions.

  Figure 5A, represents the correlation between B 22 and protein solubility. The lowest protein solubility was set to 1, thus relative solubilities are reported in order to directly compare the impact on the formulation change. Protein solubility is the conditions because of the strong alkaline pI of lysozyme. Under these conditions negative B 22 are determined, which indicate a predominance of attractive protein-protein interactions and thus reduced solubility. Under acidic conditions, B 22 becomes positive and corresponds to an increase in solubility by a factor of 7. Increasing the ionic strength (Figure 5B) reduces strongly the protein solubility; B 22 is below -2 • 10 -4 mol • ml • g -2 . A removal of NaCl from the formulation induces a protein solubility increase of a factor of 20 (see Figure 5B). The correlation between B 22 and solubility as a function of pH was recently presented by Payne et al. [65] for a 36 amino acid therapeutic peptide. A good correlation between peptide solubility and B 22 was found in the pH range of 6 to 10, emphasizing the validity of the method (see equation 2, which relates solubility to B 22 ).

  However, for pharmaceutical applications such high salt concentrations are of minor importance. Therefore, the presented investigations are focused on the presence of lower salt concentrations. A parameter not further considered by Hofmeister was the pH of the solution, as the concept of pH was not known at that time. The concept of pH was introduced about 20 years later by Sørensen[START_REF] Sorensen | II: Mitteilung. Über die Messung und die Bedeutung der Wasserstoffionenkonzentration bei enzymatischen Prozessen[END_REF]. However, the solution pH and thus the overall surface charge of a particle have a strong impact on its interaction characteristics, and have to be considered. As ammonium sulphate has the property of being kosmotropic at high salt concentration (4 M), which favors protein precipitation[START_REF] Cacace | The Hofmeister series: salt and solvent effects on interfacial phenomena[END_REF], increasing the (NH 4 ) 2 SO 4 concentration should decrease B 22 . Figure6Ashows the variation of B 22 as a function of (NH 4 ) 2 SO 4 concentration between 0 and 0.8 M. Even if B 22 decreases by increasing salt concentration, B 22 data are still positive for the investigated (NH 4 ) 2 SO 4 salt concentrations. Negative B 22 could only be obtained when the (NH 4 ) 2 SO 4 concentration exceeds concentration of at least 3 M. Considering the B 22 data of two additional sulphate salts, namely K 2 SO 4 and Na 2 SO 4 , it can be seen that the B 22 data are very similar for salt concentrations between 0 and 0.3 M. Differences are observed for salt concentrations larger than 0.6 M, with the sodium salt resulting in more negative B 22 values.

  PEG concentration even leads to a salting-in effect, indicative of an increased and positive B 22 . The presence of PEG 400 shows basically no effect on the B 22 data, which lie between 7 and 10 • 10 -4 mol • ml • g -2 for the concentration range from 0 to 24 w% PEG 400. Depending on the overall solution conditions, PEG can also act as a precipitation reagent as shown by Tessier et al.[START_REF] Tessier | Predictive crystallization of ribonuclease A via rapid screening of osmotic second virial coefficients[END_REF]. For Ribonuclease A (pH 8 and 50 mM NaCl) with increasing the concentration of PEG 3350 g/mol B 22 decreases the presence of 15 w% PEG values of -18 • 10 - 4 mol • ml • g -2 are determined[START_REF] Tessier | Predictive crystallization of ribonuclease A via rapid screening of osmotic second virial coefficients[END_REF].

  measurement. The turbidity of the freshly prepared lysozyme formulations lies between 1.0-1.3 FNU, whereas the corresponding buffers show slightly reduced turbidities between 0.4-1.0 FNU. During the stress time of the experiment the turbidity of the buffer solutions is unchanged.For the stressed lysozyme formulations a strong increase of turbidity is observed that is intensified by increasing the solution pH. After 24 h stirring of the formulation at pH 8, the turbidity reached values of nearly 24 FNU. Furthermore visible, insoluble particles are observed. As further reference samples, lysozyme formulations at pH 3, 6, and 8 were stored at room temperature (no stirring) for 24 h. The turbidity was mainly unchanged with values between 1-2 FNU during the storage of 1 day at 25 °C. Thus, stirring induces the formation of protein aggregates, which is more pronounced with increasing pH. The figures 4 and 5A represent the B 22 data as a function of pH. Under acidic conditions positive B 22 data are measured, indicating solution conditions that favor protein-protein repulsion, whereas under alkaline pH conditions B 22 becomes negative. As stronger protein-protein repulsive interactions would be expected at high pH, the physical stability of the lysozyme correlates to B 22 data at high pH.This is in accordance with the study presented by Valente et al.[START_REF] Valente | Screening for physical stability of a Pseudomonas amylase using self-interaction chromatography[END_REF] for the physical stabilization of Pseudomonas amylase. Their conclusions was that self interaction chromatography measurements of total soluble amylase and enzymatic activity measurements correlated qualitatively with osmotic second virial coefficient except near the pI of the protein amlylase, where physical stability was minimal[START_REF] Valente | Screening for physical stability of a Pseudomonas amylase using self-interaction chromatography[END_REF].

  this effect is stronger with chaotrope than with kosmotrope ions.

  in order to predict protein-protein interactions based on the osmotic second virial coefficient. Additional efforts were undertaken to miniaturize the SIC technique. Deshpande et al.[START_REF] Deshpande | Protein self-interaction chromatography on a microchip[END_REF] showed a SIC microchip application, which allows a rapid screening of various solution conditions with a strongly reduced protein consumption.

  Figures

  Elution profiles of lysozyme as a function of NaCl concentration in presence of 5 mM acetic acid at pH 4.5 at a loading of 18 mg/g: no salt (   ), 100 mM NaCl (---), 300 mM NaCl (---) and 800 mM NaCl (   )(Injection of 10 µl lysozyme solution at 20 mg/ml).

Figure 2 .

 2 Figure 2.

Figure 3 .

 3 Figure 3.

Figure 4 .

 4 Figure 4.

Figure 5 .

 5 Figure 5.

Figure 6 .

 6 Figure 6.

Figure 7 .

 7 Figure 7.

Figure 8 .

 8 Figure 8.

Figure 9 .

 9 Figure 9.

Figure 10 .

 10 Figure 10.

F

  B 22 (MgCl 2 ) > B 22 (KCl) > B 22(NaCl). At high ionic strength, the observed cation effect follows the trend of the Hofmeister series (see below).

	Biotechnology Journal	Page 22 of 49
	With increasing the MgCl 2 concentration, up to a concentration of
	approximately 0.3 M, first a decrease in B 22 is observed. However, further
	increasing the salt concentration above 0.3 M, induces an increase of B 22
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	Wiley-VCH

Wiley-VCH Biotechnology Journal

Acknowledgements

The authors would like to thank S. Bassarab for his interest, H. Schott, I.

Miller for technical aid and Boehringer Ingelheim Pharma GmbH & Co. KG for financial support.


I Coutinho

G Pereira

M F Simões

M Côrte-Real

J Gonçalves

L Saraiva

Selective activation of protein kinase C- and - by 6,11,12,14-tetrahydroxy-abieta-5,8,11,13-tetraene-7-one (coleon U)

Keywords: Coleon U, Selective PKC Activator, PKC-, PKC- Apoptosis, Yeast

-one (coleon U) is a diterpene compound isolated from Plectranthus grandidentatus with an antiproliferative effect on several human cancer cell lines. Herein, we studied the modulatory activity of coleon U on individual isoforms of the three protein kinase C (PKC) subfamilies, classical (cPKC- and -I), novel (nPKC- and -) and atypical (aPKC-), using a yeast PKC assay. The results showed that, whereas the PKC activator phorbol-12-myristate-13-acetate (PMA) activated every PKC tested except aPKC coleon U had no effect on aPKC and cPKCs. Besides, the effect of coleon U on nPKCs was higher than that of PMA. This revealed that coleon U was a potent and selective activator of nPKCs The isoform-selectivity of coleon U for nPKC- and - was confirmed using an in vitro PKC assay. Most importantly, while PMA activated nPKCs inducing an isoform translocation from the cytosol to the plasma membrane and a G2/M cell cycle arrest, coleon U induced nPKCs translocation to the nucleus and a metacaspase-and mitochondrial-dependent apoptosis. This work therefore reconstitutes in yeast distinct subcellular translocations of a PKC isoform and the subsequent distinct cellular responses reported for mammalian cells. Together, our study identifies a new isoform-selective PKC activator with promising pharmacological applications. Indeed, since coleon U has no effect on cPKCs and aPKC recognized as anti-apoptotic proteins, and selectively induces an apoptotic pathway dependent on nPKC- and - activation, it represents a promising compound for evaluation as an anti-cancer drug.

Introduction 6,11,12,14-tetrahydroxy-abieta-5,8,11,13-tetraene-7-one (coleon U) is an abietane diterpene compound isolated from Plectranthus grandidentatus, shown to inhibit the growth of several human cancer cell lines such as MCF-7 (breast), NCI-H460 (lung), SF-268 (CNS), TK-10 (renal) and UACC-62 (melanoma) in a dose-dependent manner [START_REF] Marques | Effect of abietane diterpenes from Plectranthus grandidentatus on the growth of human cancer cell lines[END_REF]. However, the mechanisms of action responsible for this coleon U-induced growth inhibition has not been elucidated. In a later study, a dose-dependent antiproliferative effect of coleon U on T-and Blymphocyte cells was also reported [START_REF] Cerqueira | Effect of abietane diterpenes from Plectranthus grandidentatus on Tand B-lymphocyte proliferation[END_REF]. Although this study established a relationship between the antiproliferative effect of coleon U and its capacity to induce apoptosis in lymphocyte cells, the molecular mechanisms associated with its antiproliferative effect remained unclear. However, the interference of this small-molecule with some cell signalling transduction kinases, such as protein kinase C (PKC) and/or protein tyrosine kinases, was hypothesised as a possible mechanism responsible for coleon U-induced growth inhibition [START_REF] Cerqueira | Effect of abietane diterpenes from Plectranthus grandidentatus on Tand B-lymphocyte proliferation[END_REF]. Since several diterpene compounds, such as phorbol esters, represent potent PKC activators [START_REF] Hofmann | Protein kinase C isozymes as potential targets for anti-cancer therapy[END_REF][START_REF] Battaini | Happy birthday protein kinase C: Past, present and future of asuperfamily[END_REF], we questioned whether coleon U could indeed modulate PKC activity.

PKC is considered an important family of signalling serine/threonine kinases with at least 10 isoforms grouped into three subfamilies based on their primary structure and cofactors required for activation: the classical PKCs (cPKCs: , I, II and ), activated by the second messengers Ca 2+ and diacylglycerol (DAG), the novel PKCs (nPKCs: , ,  and ) which respond only to DAG and the atypical PKCs (aPKCs:  and \) not responsive to either of the second messengers [START_REF] Battaini | Happy birthday protein kinase C: Past, present and future of asuperfamily[END_REF]. The PKC family is responsible for regulating a variety of physiological processes such as differentiation, proliferation, cell cycle and apoptosis, in an isoform specific manner. Hence, PKC isoforms represent key pharmacological targets for the treatment of numerous pathologies. For instance, some PKC isoforms have been recognised as important players in carcinogenesis, rendering them potentially suitable targets for anti-A c c e p t e d M a n u s c r i p t 4 cancer therapy [START_REF] Hofmann | Protein kinase C isozymes as potential targets for anti-cancer therapy[END_REF][START_REF] Gutcher | The isoform-specific regulation of apoptosis by protein kinase C[END_REF][START_REF] Koivunen | Protein kinase C (PKC) family in cancer progression[END_REF][START_REF] Irie | Toward the development of new medicinal leads with selectivity for protein kinase C isozymes[END_REF]. While PKC-- and - are frequently associated with proliferative effects being involved in the tumorigenesis of various cancers, PKC- is often linked to antiproliferative/pro-apoptotic effects in mammals [START_REF] Hofmann | Protein kinase C isozymes as potential targets for anti-cancer therapy[END_REF][START_REF] Gutcher | The isoform-specific regulation of apoptosis by protein kinase C[END_REF][START_REF] Koivunen | Protein kinase C (PKC) family in cancer progression[END_REF][START_REF] Irie | Toward the development of new medicinal leads with selectivity for protein kinase C isozymes[END_REF]. Consequently, selective activators of PKC-, - and - are considered tumour promoters, while selective activators of PKC- represent promising anti-cancer drugs [START_REF] Hofmann | Protein kinase C isozymes as potential targets for anti-cancer therapy[END_REF][START_REF] Gutcher | The isoform-specific regulation of apoptosis by protein kinase C[END_REF][START_REF] Koivunen | Protein kinase C (PKC) family in cancer progression[END_REF][START_REF] Irie | Toward the development of new medicinal leads with selectivity for protein kinase C isozymes[END_REF]. Although PKC- and - display a high degree of homology and similar substrate specificity, suggesting similar targets in signal transduction pathways for both nPKCs, they are frequently described as mediating quite contrasting physiological effects [START_REF] Hofmann | Protein kinase C isozymes as potential targets for anti-cancer therapy[END_REF][START_REF] Gutcher | The isoform-specific regulation of apoptosis by protein kinase C[END_REF][START_REF] Koivunen | Protein kinase C (PKC) family in cancer progression[END_REF]]. However, although several studies suggest that PKC- favours life over death [START_REF] Hofmann | Protein kinase C isozymes as potential targets for anti-cancer therapy[END_REF][START_REF] Gutcher | The isoform-specific regulation of apoptosis by protein kinase C[END_REF][START_REF] Koivunen | Protein kinase C (PKC) family in cancer progression[END_REF][START_REF] Irie | Toward the development of new medicinal leads with selectivity for protein kinase C isozymes[END_REF], recent research works also showed that PKC- activation can contribute to apoptosis. For example, it was demonstrated that ethanol induces apoptosis in hepatocytes via activation of the nPKC isoforms, PKC- and - [START_REF] Zhang | Ethanol induces apoptosis in hepatocytes by a pathway involving novel protein kinase C isoforms[END_REF].

In order to achieve the regulation of a particular PKC isoform, without affecting the activity of other isoforms also present in the cell, PKC isoform-selective activators are required. However, the complexity of the PKC family and the difficulty in carrying out independent analysis of an individual PKC isoform in mammalian cells may justify the low number of PKC isoform-selective modulators identified until now [START_REF] Hofmann | Protein kinase C isozymes as potential targets for anti-cancer therapy[END_REF][START_REF] Irie | Toward the development of new medicinal leads with selectivity for protein kinase C isozymes[END_REF][START_REF] Budas | Cardioprotective mechanisms of PKC isozyme-selective activators and inhibitors in the treatment of ischemia-reperfusion injury[END_REF].

Based on these data, we exploited a yeast PKC expression system to study the modulatory activity of coleon U on individual PKC isoforms of the three PKC subfamilies, cPKCs ( and I), nPKCs ( and ) and aPKCs (), considered as major isoforms in carcinogenesis. The conservation of many pathways and cellular processes in yeast, such as apoptosis [START_REF] Madeo | Apoptosis in yeast[END_REF], has allowed transposing the knowledge obtained in yeast to mammalian cells and vice-versa. Additionally, yeast expressing human proteins has been used as a valuable tool to elucidate the role of these proteins in complex cellular processes, and to screen for their pharmacological modulators [START_REF] Barberis | Yeast as a screening tool[END_REF][START_REF] Petranovic | Can yeast systems biology contribute to the understanding of human disease?[END_REF]. Furthermore, since the yeast PKC (Pkc1p in Saccharomyces cerevisiae) is a structural but not a functional homologue of mammalian PKC A c c e p t e d M a n u s c r i p t 5 isoforms [START_REF] Perez | Yeast protein kinase C[END_REF], yeast has been considered a well-suited organism to study individual mammalian PKC isoforms. In fact, it was demonstrated that mammalian PKCs expressed in yeast have functional characteristics similar to those found in mammalian cells [START_REF] Parissenti | Yeast as a Host to Screen for Modulators and Regulatory Regions of Mammalian Protein Kinase C Isoforms[END_REF][START_REF] Sprowl | Changes in expression of cell wall turnover genes accompany inhibition of chromosome segregation by bovine protein kinase C alpha expression in Saccharomyces cerevisiae[END_REF]. It was also showed that the yeast PKC expression system was a potential in vivo assay for the screening of PKC modulators [START_REF] Parissenti | Yeast as a Host to Screen for Modulators and Regulatory Regions of Mammalian Protein Kinase C Isoforms[END_REF][START_REF] Keenan | Isoform specificity of activators and inhibitors of protein kinase C gamma and delta[END_REF]. Based on these data, this yeast PKC assay has been used by our group not only to study the role of PKC isoforms on apoptosis regulation [START_REF] Saraiva | Specific modulation of apoptosis and Bcl-xL phosphorylation in yeast by distinct mammalian protein kinase C isoforms[END_REF], but also to search for isoform-selective PKC activators [START_REF] Saraiva | Synthesis and in vivo modulatory activity of protein kinase C of xanthone derivatives[END_REF][START_REF] Saraiva | Characterization of phorbol esters activity on individual mammalian protein kinase C isoforms, using the yeast phenotypic assay[END_REF] and inhibitors [START_REF] Saraiva | Inhibition of protein kinase C by synthetic xanthone derivatives[END_REF][START_REF] Saraiva | Inhibition of alpha, betaI, delta, eta and zeta protein kinase C isoforms by xanthonolignoids[END_REF]. This simpler eukaryotic cell model, established and validated in such studies, allows the effect of small-molecules on each PKC isoform to be analysed without the genetic complexity of the PKC family, specifically the coexistence of multiple PKC isoforms in the same mammalian cells and the extensive cross-talk amongst numerous mammalian signalling pathways.

In the present study, we found that coleon U is a potent and selective activator of nPKC- and -. Besides, several experiments were carried out in order to elucidate the molecular mechanisms of action behind the observed coleon U-induced growth inhibition in yeast expressing the nPKC- or -. Another relevant point arising from this work was the validation of the yeast assay to reconstitute the distinct subcellular translocations of a specific mammalian PKC isoform and the subsequent different cellular responses described in higher eukaryotes.

A c c e p t e d M a n u s c r i p t 6

Materials and Methods

Plant material, isolation and identification of coleon U

Details concerning the extraction, isolation and structure elucidation of the abietane diterpene coleon U from Plectranthus grandidentatus Gürke (Fig. 1) have previously been described [START_REF] Marques | Effect of abietane diterpenes from Plectranthus grandidentatus on the growth of human cancer cell lines[END_REF][START_REF] Teixeira | Abietane diterpenoids from Plectranthus grandidentatus[END_REF].

Plasmids

Constructed yeast expression plasmids YEplac181-LEU2 with the cDNA encoding for bovine PKC-, rat PKC-I or PKC-mouse PKC- or PKC-, under control of a galactoseinducible GAL1 promote, were kindly provided by Dr. Nigel Goode (The Royal Veterinary College, Hawkshead Lane, Hertfordshire, UK). Constructed yeast expression plasmid pOW4-URA3 with the cDNA encoding for human Bcl-xL, under control of an ADH1 promoter, was kindly provided by Dr. Charles Rudin (Cancer Research Building, Baltimore, USA).

Constructed yeast expression plasmid pCLbGFP-TRP3 with the cDNA encoding for mitochondria-localised green fluorescent protein (mt-GFP), under control of a GAL1-10

promoter, was kindly provided by Dr. Stéphen Manon (Université de Bordeaux, Bordeaux, France). All the plasmids used were amplified in Escherichia coli DH5 and confirmed by restriction analysis.

Yeast strain, growth conditions and yeast expression of a mammalian protein

For yeast expression studies Saccharomyces cerevisiae strain CG379 ( ade5 his7- raffinose (Sigma-Aldrich, Sintra, Portugal) selective medium and grown, at 30 ºC under continuous shaking, to 0.5 OD 600 (mid-log phase; about 42 h incubation), as described [START_REF] Saraiva | Specific modulation of apoptosis and Bcl-xL phosphorylation in yeast by distinct mammalian protein kinase C isoforms[END_REF].

Yeast expression of a mammalian PKC isoform and/or human Bcl-xL was previously confirmed by Western blot analysis [START_REF] Saraiva | Specific modulation of apoptosis and Bcl-xL phosphorylation in yeast by distinct mammalian protein kinase C isoforms[END_REF][START_REF] Saraiva | Characterization of phorbol esters activity on individual mammalian protein kinase C isoforms, using the yeast phenotypic assay[END_REF].

Effect of compounds on yeast cell growth

All compounds tested were prepared in dimethyl sulfoxide (DMSO; Sigma-Aldrich, Sintra, Portugal). To analyse the effect of compounds on yeast growth, transformed cells were incubated in galactose selective medium with 0.1 -10 M coleon U, 1 M phorbol 12myristate 13-acetate (PMA; Sigma-Aldrich, Sintra, Portugal or 0.1% DMSO only to 0.5 OD 600 . Cell growth was determined by colony forming unit (c.f.u.) counts, as described [START_REF] Saraiva | Specific modulation of apoptosis and Bcl-xL phosphorylation in yeast by distinct mammalian protein kinase C isoforms[END_REF].

The percentage of growth inhibition was estimated considering 100% growth as the number of c.f.u. obtained with yeasts incubated with DMSO only.

In vitro PKC assay

The in vitro PKC assay was performed using the non-radioactive kit MESACUP Protein Kinase Assay System and purified PKC enzymes from Upstate (Grupotaper, Sintra, Portugal): cPKCs (mixture of classical PKC isoforms, ,  and , obtained from rat brain; 5 ng per assay); nPKC- (50 ng per assay); nPKC- (12.5 ng per assay); aPKC- (12.5 ng per assay), basically according to the kit procedure. Briefly, this ELISA-based detection method uses a peptide pseudosubstrate, pre-coated on a 96-well plate that can be phosphorylated by PKC.

Samples containing the active PKC enzyme were transferred to the pseudosubstrate-coated wells. A biotinylated monoclonal antibody that recognises the phosphorylated form of the pseudosubstrate was added to the wells and detected using HRP-conjugated streptavidin.

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

Cell cycle analysis

Flow cytometric analysis of DNA content was obtained using Sytox Green Nucleic Acid from Molecular Probes (Alfagene, Carcavelos, Portugal), basically as reported [START_REF] Fortuna | Cell cycle analysis of yeasts using Syber Green I[END_REF]. Briefly, about 10 7 cells incubated in galactose selective medium with 1 M PMA, 1 M coleon U or DMSO only were fixed in 70% (v/v) ethanol overnight at 4 ºC, treated with 250 g/ml RNase A (DNase-free; Sigma-Aldrich, Sintra, Portugal) for 3 h at 50 ºC and thereafter with 1 mg/ml Proteinase K (Sigma-Aldrich, Sintra, Portugal) for 3 h at 37 ºC. Subsequently, cells were incubated with 10 M Sytox Green overnight at 4 ºC. Fluorescence from at least 30 000 cells was analysed using the FL1 detector in linear amplification from a flow cytometer. Yeast cell cycle phases were quantified using ModFit LT TM software (Verity Software House, Inc., Topsham, USA).

Analysis of plasma membrane integrity, DNA fragmentation and chromatin condensation

Propidium iodide (PI) and TUNEL staining to monitor plasma membrane integrity and DNA fragmentation respectively were carried out as described [START_REF] Saraiva | Specific modulation of apoptosis and Bcl-xL phosphorylation in yeast by distinct mammalian protein kinase C isoforms[END_REF]. Briefly, about 10 7 cells incubated in galactose selective medium with 1 M coleon U or DMSO only were collected and incubated with 5 g/ml PI (Sigma-Aldrich, Sintra, Portugal) for 10 min at room for 15 min at room temperature, as described [START_REF] Silva | Hyperosmotic stress induces metacaspase-and mitochondria-dependent apoptosis in Saccharomyces cerevisiae[END_REF]. At least 600 cells per sample were analysed under a fluorescence microscope.

Assessment of yeast metacaspase activity

Yeast metacaspase (Yca1p) activation was analysed basically as described [START_REF] Saraiva | Specific modulation of apoptosis and Bcl-xL phosphorylation in yeast by distinct mammalian protein kinase C isoforms[END_REF]. Flow cytometric analysis: about 10 6 cells incubated in galactose selective medium with 1 M coleon U or DMSO only were collected and incubated with 12.5 M of FITC-VAD-fmk from Promega (VWR International Material de Laboratório, Lda., Lisboa, Portugal) for 1 h at 30 ºC; fluorescence from at least 10 000 cells was analysed using the FL1-H detector from a flow cytometer. Cell death assay: cells were incubated in galactose selective medium with 1 M coleon U and 20 M of the caspase inhibitor z-VAD-fmk from Promega (VWR International Material de Laboratório, Lda., Lisboa, Portugal). Cell death was assessed by c.f.u. counts, considering 100% survival as the number of c.f.u. obtained with cells incubated with DMSO only.

Assessment of reactive oxygen species (ROS) production

ROS production was monitored by flow cytometry using dihydroethidium (DHE) from

Molecular Probes (Alfagene, Carcavelos, Portugal) as described [START_REF] Madeo | Oxygen stress: a regulator of apoptosis in yeast[END_REF]. About 10 6 cells, incubated in galactose selective medium with 1 M coleon U or DMSO only, were collected and incubated with 5 g/ml DHE for 30 min at 30 ºC. Fluorescence from at least 10 000 cells was analysed using the FL2-H detector from a flow cytometer. Fluorescence from at least 10 000 cells was analysed using the FL1-H and FL2-H detectors from a flow cytometer.

Assessment of mitochondrial fragmentation

Analysis of mitochondrial fragmentation of transformed yeast was carried out using the plasmid pCLbGFP encoding for mt-GFP, as described [START_REF] Okamoto | The sorting of mitochondrial DNA and mitochondrial proteins in zygotes: preferential transmission of mitochondrial DNA to the medial bud[END_REF]. Cells incubated in galactose selective medium with 1 M coleon U or DMSO only were observed under a fluorescence microscope.

Immunofluorescence assay

Flow cytometric data acquisition and analysis

Flow cytometric analysis was performed using a FACSCalibur TM flow cytometer and the CellQuest software (BD Biosciences, San José, CA, USA).

Fluorescence microscopy

For fluorescent microscopic examination, samples were observed under an Eclipse E400 fluorescence microscope (Nikon, Japan) equipped with a 100 W mercury lamp and appropriate filter setting. Yeast cells were observed with an oil immersion lens (Plan Fluor 100/1.30) and images were captured by a Digital Sight camera system (Nikon DS-5Mc, Japan) carrying built-in software for image acquisition (Nikon ACT-2U, Japan).

Statistical analysis

Data were analysed statistically using the SigmaStat 3.

Coleon U is a potent and selective activator of nPKC- and -

In order to analyse a possible modulation of PKC isoforms by coleon U (Fig. 1), we used a yeast PKC expression system that consists of the use of yeast cells expressing an individual mammalian PKC-, -I, -, - or -. In this assay, it was demonstrated that PKC activators inhibit the growth of yeast expressing a PKC isoform without interfering with the growth of yeast transformed with the empty vector (control yeast) [START_REF] Parissenti | Yeast as a Host to Screen for Modulators and Regulatory Regions of Mammalian Protein Kinase C Isoforms[END_REF][START_REF] Saraiva | Synthesis and in vivo modulatory activity of protein kinase C of xanthone derivatives[END_REF][START_REF] Saraiva | Characterization of phorbol esters activity on individual mammalian protein kinase C isoforms, using the yeast phenotypic assay[END_REF].

Hence, we began by analysing the effect of several concentrations of coleon U, 0.1 -10 M, on the growth of yeast expressing PKC-, -I, -, - or - and control yeast (Fig. 2A).

Yeast cell growth was analysed by c.f.u. counts and the effects obtained with coleon U were compared to those obtained with the standard PKC activator, PMA (Fig. 1; an activator of cPKCs and nPKCs). We detected that 0.1 and 1 M coleon U significantly inhibited the growth of yeast expressing PKC- or - without affecting the growth of control yeast and yeast expressing PKC-, -I or - (Fig. 2A) This selectivity of coleon U to inhibit the growth of yeast expressing PKC- or - was particularly evident for 1 M (Fig. 2B). In fact, for this concentration, and in opposition to PMA that activated every PKC tested except aPKC-coleon U had no effect on cPKCs as well as on aPKC- (Fig. 2B andC). In addition, the effect of coleon U on yeast expressing the nPKCs, PKC- or - was shown to be higher than that obtained with PMA (Fig. 2B andC). These results therefore indicated that coleon U was a potent and selective activator of the two nPKCs, PKC- and - Direct activation of PKC- and - by coleon U and the selectivity of this smallmolecule for these nPKCs was further confirmed using an in vitro protein kinase assay system and purified PKC enzymes: cPKCs (mixture of classical PKC isoforms, ,  and ), nPKC-, nPKC- and aPKC-. In the in vitro PKC assay, PKC activators increase the phosphatidylserine (PS; an endogenous PKC activator) effect. In fact, in this assay, it was 3).

Coleon U-induced growth inhibition in yeast expressing nPKC- or - is associated with the occurrence of a metacaspase-and mitochondrial-dependent apoptotic cell death

It was showed that the expression of bovine PKC- in S. cerevisiae causes growth inhibition associated with the occurrence of a G2/M arrest, which is markedly increased by treatment of these cells with PMA [START_REF] Sprowl | Changes in expression of cell wall turnover genes accompany inhibition of chromosome segregation by bovine protein kinase C alpha expression in Saccharomyces cerevisiae[END_REF]. However, as previously reported by us [START_REF] Saraiva | Specific modulation of apoptosis and Bcl-xL phosphorylation in yeast by distinct mammalian protein kinase C isoforms[END_REF], under our experimental conditions and with the yeast strain used, single expression of a mammalian PKC-, -, - or - in yeast did not significantly interfere with cell growth and survival. Even so, in agreement with that reported for PKC- [START_REF] Sprowl | Changes in expression of cell wall turnover genes accompany inhibition of chromosome segregation by bovine protein kinase C alpha expression in Saccharomyces cerevisiae[END_REF], treatment of yeast expressing nPKC- or - with PMA caused significant growth inhibition related to a G2/M cell cycle arrest (Fig. 4A,B). This blockage in cell cycle progression by PMA was not accompanied by the occurrence of cell death, as revealed by the preservation of plasma membrane integrity, the absence of DNA fragmentation and chromatin condensation in PMA-treated yeast cells (data not shown).

With a view to elucidating the molecular mechanisms behind coleon U-induced growth inhibition in yeast cells expressing nPKC- or - we began by analysing the effect of this small-molecule on the cell cycle progression of these transformed yeast. In contrast to PMA, coleon U only slightly interfered with the cell cycle progression of yeast expressing nPKC- or - (Fig. 4A andB). This small effect of coleon U on cell cycle could not explain per se the marked growth inhibition induced by this small-molecule on yeast expressing nPKC- or -.

Hence, we next addressed whether coleon U-induced growth inhibition could reflect cell death. With this goal, several apoptotic markers were investigated. We verified that 1 M coleon U caused in yeast expressing nPKC- or - but not in control yeast and yeast (Fig. 5A-C). These results indicated that coleon U-induced growth inhibition in yeast expressing PKC- or - was linked to the activation of an apoptotic cell death.

Additionally, flow cytometric analysis of FITC-VAD-fmk stained cells revealed the occurrence of yeast metacaspase (Yca1p) activation in coleon U-treated yeast cells expressing PKC- or -, but not in control yeast and yeast expressing PKC-, -I or - (Fig. 6A andB).

This was further confirmed by the marked decrease in the percentage of dead cells expressing nPKC- or - obtained when coleon U treatment was carried out in the presence of the caspase inhibitor z-VAD-fmk (Fig. 6C).

We further observed that 1 M coleon U markedly increased mitochondrial ROS production (Fig. 7A andB), decreased  m (Fig. 8A andB) and increased mitochondrial fragmentation (Fig. 8C) in yeast expressing nPKC- or -, but not in control yeast and yeast expressing PKC-, -I or -.

It was showed that human Bcl-xL is also a mitochondrial anti-apoptotic protein in yeast, inhibiting yeast apoptosis induced by stress stimuli such as hydrogen peroxide, menadione [START_REF] Chen | Bcl-2 family members inhibit oxidative stressinduced programmed cell death in Saccharomyces cerevisiae[END_REF] and acetic acid [START_REF] Saraiva | Specific modulation of apoptosis and Bcl-xL phosphorylation in yeast by distinct mammalian protein kinase C isoforms[END_REF]. Moreover, a previous work performed by our group showed that co-expression of human Bcl-xL with a mammalian PKC isoform did not significantly interfere with yeast cell growth and survival [START_REF] Saraiva | Specific modulation of apoptosis and Bcl-xL phosphorylation in yeast by distinct mammalian protein kinase C isoforms[END_REF]. Based on these data, the -, - or - (Fig. 8D).

Together, the results obtained corroborate the activation by coleon U of a metacaspase-and mitochondrial-dependent apoptotic pathway dependent on nPKC- or - expression in yeast

Coleon U-induced apoptosis is associated with the translocation of nPKC- and - from the cytosol to the nucleus of yeast cells

In mammalian cells, it was reported that distinct subcellular translocations of a specific PKC isoform lead to different cellular responses [START_REF] Shirai | Activation mechanisms of protein kinase C: maturation, catalytic activation, and targeting[END_REF]. Since PMA and coleon U induced in yeast expressing nPKC- or - different cellular responses, we questioned whether these two PKC activators could also induce distinct subcellular translocations of these nPKCs in yeast cells.

With this goal, immunofluorescence studies were carried out with yeast cells expressing nPKC- or - treated with 1 M PMA, 1 M coleon U or DMSO only. Since the results obtained with PKC- were not significantly different from those obtained with PKC-, the PKC- results were used as representative of the subcellular distribution obtained with the two nPKCs (Fig. 9A andB). The results showed that in the absence of activators, nPKCs were predominantly located at the cytosol of yeast cells (Fig. 9A, B; DMSO). In agreement with that reported for mammalian cells [START_REF] Shirai | Activation mechanisms of protein kinase C: maturation, catalytic activation, and targeting[END_REF], PMA caused a marked translocation of nPKCs to the yeast plasma membrane (Fig. 9A andB; PMA). On the other hand, coleon U caused a pronounced translocation of nPKCs to the nucleus of yeast cells (Fig. 9A andB; coleon U).

Together, the results obtained showed that PMA and coleon U induced translocation of nPKCs to distinct yeast subcellular compartments.

A c c e p t e d M a n u s c r i p t 16

Discussion

Previous studies from our group exploited the yeast PKC expression system not only in searching for isoform-selective PKC modulators [START_REF] Saraiva | Synthesis and in vivo modulatory activity of protein kinase C of xanthone derivatives[END_REF][START_REF] Saraiva | Characterization of phorbol esters activity on individual mammalian protein kinase C isoforms, using the yeast phenotypic assay[END_REF][START_REF] Saraiva | Inhibition of protein kinase C by synthetic xanthone derivatives[END_REF][START_REF] Saraiva | Inhibition of alpha, betaI, delta, eta and zeta protein kinase C isoforms by xanthonolignoids[END_REF], but also in studying the role of several PKC isoforms in apoptosis regulation [START_REF] Saraiva | Specific modulation of apoptosis and Bcl-xL phosphorylation in yeast by distinct mammalian protein kinase C isoforms[END_REF]. In the present work, this yeast PKC assay was used to study the modulatory activity of coleon U on several PKC isoforms of the three PKC subfamilies, cPKCs ( and I), nPKCs ( and ) and aPKC (), considered as the most relevant isoforms in carcinogenesis. With this cell system, it was possible to analyse the effect of coleon U on each PKC isoform expressed in the same cellular background and without the genetic complexity of mammalian pathways and interference from other PKC family isoforms.

In agreement with that reported for human cells [START_REF] Marques | Effect of abietane diterpenes from Plectranthus grandidentatus on the growth of human cancer cell lines[END_REF][START_REF] Cerqueira | Effect of abietane diterpenes from Plectranthus grandidentatus on Tand B-lymphocyte proliferation[END_REF], we observed that 0.1 -1 M coleon U also induced yeast growth inhibition but only in cells expressing the nPKC- or -.

This isoform-selectivity was further observed for 0.3 M coleon U using an in vitro PKC assay. Together, these results led us to discover that coleon U is a selective activator of nPKC- and -. This work also studied the molecular mechanisms of action underlying coleon U-induced growth inhibition in yeast expressing nPKC- or -. We demonstrated that this coleon U antiproliferative effect was not linked to the induction of cell cycle arrest, but instead to the activation of an apoptotic cell death. This is in agreement with a previous study performed in lymphocyte cells, where detection of PS externalisation by annexin V allowed the antiproliferative effect of coleon U to be associated with apoptosis induction [START_REF] Cerqueira | Effect of abietane diterpenes from Plectranthus grandidentatus on Tand B-lymphocyte proliferation[END_REF]. Herein, it is believed that translocation of a specific PKC isoform to a cellular compartment is the major determinant of its specificity and function [START_REF] Shirai | Activation mechanisms of protein kinase C: maturation, catalytic activation, and targeting[END_REF]. Indeed, similarly to results from mammalian cells [START_REF] Shirai | Activation mechanisms of protein kinase C: maturation, catalytic activation, and targeting[END_REF][START_REF] Wang | The V5 domain of protein kinase C plays a critical role in determining the isoform-specific localization, translocation, and biological function of protein kinase C-delta and -epsilon[END_REF], we found that in the absence of activators both nPKC- and - were localised in the cytosol of yeast cells. Additionally, as reported for PKC- [START_REF] Shirai | Activation mechanisms of protein kinase C: maturation, catalytic activation, and targeting[END_REF][START_REF] Wang | The V5 domain of protein kinase C plays a critical role in determining the isoform-specific localization, translocation, and biological function of protein kinase C-delta and -epsilon[END_REF] and PKC- [START_REF] Wang | The V5 domain of protein kinase C plays a critical role in determining the isoform-specific localization, translocation, and biological function of protein kinase C-delta and -epsilon[END_REF] in mammalian cells, treatment of yeast cells with PMA induced translocation of these isoforms to the plasma membrane. On the other hand, coleon U treatment stimulated translocation of nPKC- and - to the nucleus of yeast cells. In accordance with the results obtained in yeast, recent studies performed with mammalian cells revealed that several proapoptotic kinases, specifically PKC-, undergo cytoplasmic-nuclear shuttling in response to DNA damage [30][START_REF] Reyland | Protein kinase Cdelta and apoptosis[END_REF][START_REF] Yoshida | Nuclear trafficking of pro-apoptotic kinases in response to DNA damage[END_REF]. These studies demonstrated that upon exposure to a genotoxic stress, such as etoposide, PKC- accumulates in the nucleus. Whereas retention of PKC- in the cytoplasm is compatible with cell survival, its nuclear retention is required for commitment to apoptosis, showing that cellular localisation of PKC- regulates the survival/death pathway [30][START_REF] Reyland | Protein kinase Cdelta and apoptosis[END_REF][START_REF] Yoshida | Nuclear trafficking of pro-apoptotic kinases in response to DNA damage[END_REF]. Indeed, nuclear targeting of kinases such as PKC- is considered a new and essential regulatory mechanism that directly influences the induction of apoptosis [START_REF] Yoshida | Nuclear trafficking of pro-apoptotic kinases in response to DNA damage[END_REF]. Thus, while modulation of nPKC- and - nuclear translocation by coleon U remains an open issue that deserves further studies, we corroborated previous data from mammalian cells showing that the correct cellular localisation is critical to the function of these two kinases. detection of PKC- mitochondrial localisation [START_REF] Goerke | Induction of apoptosis by protein kinase C delta is independent of its kinase activity[END_REF]. Another work also showed that although induction of apoptosis via activation of PKC- and - increased cytochrome c release and accumulation of ROS, no PKC- and - were detected in mitochondria [START_REF] Zhang | Ethanol induces apoptosis in hepatocytes by a pathway involving novel protein kinase C isoforms[END_REF]. Supporting these data, we showed that also in yeast, under apoptotic conditions, PKC- and - seem to activate a mitochondrial pathway without directly interfering with this organelle. As stressed by Yoshida [START_REF] Yoshida | Nuclear trafficking of pro-apoptotic kinases in response to DNA damage[END_REF], the molecular devices that allow various stimuli, such as coleon U, to be transmitted from the nucleus into the mitochondrion and which represents a point of integration for the different apoptotic signalling cascades are still unclear. Further studies are underway in yeast to clarify this issue.

In conclusion, this work identifies a new isoform-selective small-molecule with potential pharmacological applications. Indeed, as a potent and selective activator of the nPKC- and -, coleon U represents a promising tool to further understand the nPKC- and - cellular signalling pathways in mammalian cells. Furthermore, the absence of an effect on cPKC- and - and aPKC- reported as anti-apoptotic proteins, and its selectivity to induce an apoptotic pathway dependent on nPKC- and - activation, indicate that coleon U is a promising compound for evaluation as an anti-cancer drug. This work also shows, for the first time, that the yeast PKC expression system allows reconstituting distinct subcellular translocations of a specific PKC isoform and the subsequent different cellular responses previously reported for mammalian systems. Considering these similarities and the advantages provided by the genetic tractability of yeast, a broader outcome of this study is the

A c c e p t e d M a n u s c r i p t 9 temperature.

 9 TUNEL was performed using the In Situ Cell Death Detection Kit, Fluorescein (Roche Diagnostics, Amadora, Portugal). For chromatin condensation analysis, cells were incubated with 4 g/ml DAPI (4,6-diamido-2-phenyl-indole; Sigma-Aldrich, Sintra, Portugal)

A c c e p t e d M a n u s c r i p t 10 2. 10 .

 1010 Assessment of mitochondrial membrane potential ( m )Modification of  m in transformed yeast was monitored by flow cytometry using 3,3'dihexyloxacarbocyanine iodide (DiOC 6 (3)) from Molecular Probes (Alfagene, Carcavelos, Portugal). About 10 6 cells, incubated in galactose selective medium with 1 M coleon U or DMSO only were incubated with 1 nM DiOC 6[START_REF] Hofmann | Protein kinase C isozymes as potential targets for anti-cancer therapy[END_REF] for 30 min at 30 ºC. Thereafter, 2 g/ml PI was added to exclude necrotic cells. Carbonyl cyanide 3-chlorophenylhydrazone (CCCP; Sigma-Aldrich, Sintra, Portugal) was used as positive control; after treatment with DiOC 6 (3), cells incubated with DMSO only were treated with 0.4 nM CCCP for 15 min at 30 ºC.

About 10 7

 7 cells grown in galactose selective medium with 1 M PMA, 1 M coleon U or DMSO only were fixed with 3.7% formaldehyde (Sigma-Aldrich, Sintra, Portugal) for 30 min at 30 ºC. For spheroplasts formation, cells were treated with Zymolyase Solution, containing 0.1 M Potassium Phosphate pH 7.5, 2 l/ml 2-Mercaptoethanol (Sigma-Aldrich, Sintra, Portugal), 1 mg/ml Zymolyase 20 T (MP Biomedicals Solon, Fountain Parkway Solon, USA), at 37 ºC for 25 min. Spheroplasts were transferred to polylysine-coated glass slides (Sigma-Aldrich, Sintra, Portugal) and permeabilised with 1% Triton X-100 (Sigma-Aldrich, Sintra, A c c e p t e d M a n u s c r i p t 11 Portugal) for 2 min at 4 ºC before incubation with the mouse monoclonal antibody anti-PKC- or anti-PKC- (1:50) from Santa Cruz Biotechnology (Frilabo, Porto, Portugal) for 2 h at room temperature. This was then followed by incubation with the anti-mouse Alexa Fluor 488 secondary antibody (1:200) from Molecular Probes (Alfagene, Carcavelos, Portugal) for 2 h at room temperature. Mounting medium containing 1.5 g/ml DAPI was used to visualise nuclear DNA. Samples were observed under a fluorescence microscope.

A c c e p t e d M a n u s c r i p t 13 observed that 0. 3

 133 M coleon U significantly increased the PS effect on nPKC- and - In opposition, 0.3 M coleon U did not interfere with the PS effect on cPKCs and aPKC-Fig.

A c c e p t e d M a n u s c r i p t 14 expressing

 14 PKC-, -I or - a marked increase in the percentage of cells with chromatin condensation and DNA fragmentation (TUNEL-positive cells), without loss of plasma membrane integrity as revealed by the low percentage of PI-positive cells (absence of necrosis)

  interference of human Bcl-xL with coleon U-induced cell death in yeast expressing PKC-, -I, -, - or - and control yeast was analysed. The results obtained showed that Bcl-xL completely abolished the coleon U-induced apoptosis in yeast expressing nPKC- or - A c c e p t e d M a n u s c r i p t 15 without interfering with the coleon U effect in control yeast and yeast expressing PKC-, -I,

  we have further shown that this coleon U-induced apoptosis involved the activation of a metacaspase-and mitochondrial-dependent pathway in yeast expressing nPKC- or -, as revealed by the increase in mitochondrial dysfunctions such as fragmentation, depolarization and ROS accumulation and by the complete abolishment of coleon U cytotoxic effect by the mitochondrial anti-apoptotic protein Bcl-xL.

A c c e p t e d M a n u s c r i p t 17 Another

 17 relevant point from this work is the corroboration that a specific PKC isoform can induce, in the same cellular background, different cellular responses depending on the stimulus applied. In fact, comparing the effects of PMA and coleon U, we detected that these two PKC activators induced different effects in yeast due to the activation of the same PKC isoform. While PMA caused a G2/M cell cycle arrest, coleon U induced an apoptotic cell death in yeast expressing nPKC- or -. This can be explained by the ability of different stimuli to selectively translocate a PKC isoform to distinct subcellular compartments. In fact,

A c c e p t e d M a n u s c r i p t 18 Though

 18 several evidences supporting the involvement of mitochondria in coleon Uinduced apoptosis are presented herein, we could not demonstrate mitochondrial accumulation of nPKC- or - after coleon U treatment. Interestingly, other authors demonstrated the involvement of mitochondria in PKC--induced apoptosis in a rat vascular smooth muscle cell line based on the decrease of apoptosis by Bcl-2 and loss of  m without

A c c e p t e d M a n u s c r i p t 19 validation

 19 of this cell model to unravel the intra-organelle communication systems and their roles in the PKC isoform apoptotic signalling network, as well as to study the molecular mechanisms of action of compounds with potential therapeutic applications. Acknowledgments We are grateful to Dr. Nigel Goode for providing YEplac181-PKC- PKC-, PKC-, -PKC- and -PKC-; to Dr. Heimo Riedel for providing YEp52-PKC-and Yep51-PKC-; to Dr. Charles Rudin for providing pOW4-Bcl-xL; to Dr. Stéphen Manon for providing pCLbGFP-mt-GFP; to Joana Tavares for her help and technical advice in some experiments; to Cristina G-Marques for the previous isolation of coleon U; to Helena Vasconcelos for critical reading of the manuscript. We thank REQUIMTE/CEQUP and FCT (I&D No 8/94), POCTI (QCA III) and FEDER for financial support. I. Coutinho is recipient of a PhD fellowship from FCT (SFRH/BD/36066/2007).

Fig. 1 -

 1 Fig. 1 -Chemical structures of coleon U, an abietane diterpene isolated from P.

Fig. 2 -Fig. 3 - 2 A c c e p t e d M a n u s c r i p tFig. 4 -Fig. 5 -

 23245 Fig. 2 -Effect of PMA and coleon U on the growth of control yeast and yeast expressing

Fig. 6 -Fig. 7 -A c c e p t e d M a n u s c r i p t 28 Fig. 8 -

 67288 Fig. 6 -Effect of coleon U on metacaspase activation of control yeast and yeast expressing

Fig. 9 - 1 A c c e p t e d M a n u s c r i p t 1 Figure 1

 9111 Fig. 9 -Effect of PMA and coleon U on yeast subcellular localisation of PKC-. Analysis of

  Figure 1

  Figure 5