Intrinsic tryptophan fluorescence was used to estimate skBADH fluorescence quenching by enzyme substrates (BA and NAD+), products (GB and NADH) and inhibitors (H2O2 and copper) at the concentrations given in each figure legend. Another set of experiments was carried out in which the enzyme was incubated for 120 min in the presence of 100 µM H2O2 or 10 µM CuCl2. Fluorescence spectra were recorded at an excitation wavelength of 296 nm and emission wavelength of 300-400 nm (5 nm bandwidth) using a QM-2003 fluorometer (Photon Technology International) with a 75-W xenon lamp as the light source. The spectra were corrected by subtracting the spectrum obtained for the solvent under identical conditions. Three readings of emission data were accumulated. The assay was done using 2 µM of enzyme monomer in 100 mM Hepes-KOH buffer at pH 7.0 and then at pH 8.0. Fluorescence spectral centers of mass (intensity-weighted average emission wavelengths, λav) were calculated according to the following equation:

$$
\lambda\_{\rm av} = \Sigma \,\lambda I \, \langle \lambda \rangle / \,\Sigma I \,\langle \lambda \rangle \tag{3}
$$

where λ is the emission wavelength and *I*(λ) represents the fluorescence intensity at wavelength λ.

#### **2.7 Enzyme structural models**

The structural model of the skBADH tetramer was constructed using MOE v2009.10 software (Chemical Computing Group). The homology model was built using the cDNAdeduced swine kidney BADH amino acid sequence. The crystallographic structures from cod liver (1A4S, DOI:10.2210/pdb1a4s/pdb) and *Escherichia coli* BADH (1WNB, DOI:10.2210/pdb1wnb/pdb) were used as templates to generate the final homology model.

The hydrogen peroxide and copper interaction models with the skBADH active site were built using the PyMOL Molecular Graphics System, Version 1.3, Schrödinger, LLC. The model was designed based on previously described experimental evidence of the alteration of skBADH active site (Rosas-Rodríguez & Valenzuela-Soto, 2011).

#### **3. Results**

234 Oxidative Stress – Molecular Mechanisms and Biological Effects

contained 0.1 M Hepes-KOH, pH 8.0, 0.5 mM betaine aldehyde (BA) and 1.0 mM NAD+ in a final volume of 0.4 mL. SkBADH activity was assayed spectrophotometrically measuring NAD+ reduction by the increase in extinction at 340 nm using an Ultrospec 4000 (Pharmacia)

Prior to treatments skBADH (0.15 mg/mL) was dialyzed overnight at 4 °C against 10 mM Hepes-KOH buffer, pH 7.0, 1 mM EDTA, 10 mM 2-mercaptoethanol (buffer A, low ionic strength), and against buffer A plus 150 mM KCl (buffer B, physiological ionic strength).

SkBADH activity in the presence of hydrogen peroxide was evaluated at distinct pH values and ionic strengths. The enzyme was assayed in the presence of 0.1 mM of hydrogen peroxide in 10 mM Hepes buffer, pH 6.8, 7.0, 7.2, 7.4 and 8.0 and enzyme activity was measured. The effect of ionic strength was evaluated by incubating the enzyme at each pH

The enzyme was dialyzed with 10 mM Hepes buffer, pH 7 or pH 8.0, 10% glycerol (v/v), and 1 mM 2-mercaptoethanol. The enzyme was incubated for 120 min with 10 µM CuCl2, in the presence or absence of 0.15 M KCl, an aliquot was taken at different times and residual enzyme activity was measured by the standard assay. Rate constants for inactivation were calculated by fitting the data to a single exponential decay equation (Eq. 1) using OriginPro

 At = A∞ + A0e–kobs\*t (1) Where *At*, *A0* and *A∞* are enzyme activity at time t, zero, and infinite, respectively, expressed as a percentage of the initial activity; *k*obs is the observed pseudo-first order constant in

The reactivation of inactivated enzyme was carried out with 5 mM dithiothreitol (DTT), 10 mM glutathione (GSH) or 10 mM 2-mercaptoethanol. An aliquot was taken at different times and enzyme activity was measured using the standard assay. Activity data were fitted

Where *At*, *A0* and *A∞* are enzyme activity at time t, zero, and infinite, respectively, expressed as a percentage of the initial activity; *k*obs is the observed pseudo-first order constant in

Intrinsic tryptophan fluorescence was used to estimate skBADH fluorescence quenching by enzyme substrates (BA and NAD+), products (GB and NADH) and inhibitors (H2O2 and copper) at the concentrations given in each figure legend. Another set of experiments was

obs •t) + *A2* (1 *- e*–*k*

obs2 •t) (2)

to a double exponential growth equation (Eq. 2) using OriginPro 8.0.

tested with 0.15 M KCl for 60 minutes prior to the enzyme activity assay.

spectrophotometer.

**2.3 Effect of hydrogen peroxide in enzyme activity**

**2.4 Kinetics of inactivation by copper**

8.0 (OriginLab, Northampton, MA, USA).

monophasic inactivation.

monophasic inactivation.

**2.6 Enzyme fluorescence assay**

**2.5 Enzyme reactivation kinetics**

 *At = A1* (1 *- e*–*k*

#### **3.1 Effect of pH and hydrogen peroxide on enzyme activity**

To test the influence of pH on skBADH oxidation provoked by hydrogen peroxide, enzyme activity was assayed from pH 6.8 to 8.0. Enzyme activity was higher at pH 8.0 in the absence or presence of hydrogen peroxide (Fig. 1). SkBADH activity decreased 50% in the presence of 100 µM H2O2 at all pH values tested relative to the enzyme assays done with no hydrogen peroxide (Fig. 1A). Chemical modification studies carried out with BADH from *Pseudomonas aeruginosa* indicated that the catalytic cysteine residue exists as thiolate at pH values of 5.5 to 9.0 (González-Segura et al., 2002). Therefore, H2O2 is able to oxidize the catalytic cysteine in the skBADH at the pH values assayed in this work.

Physiological ionic strength obtained using 0.15 M KCl was tested to analyze its influence on skBADH oxidation by 100 µM H2O2. Enzyme activity decreased 50% when hydrogen peroxide was included in the activity assay medium (Fig. 1B). Physiologic ionic strength was not able to maintain enzyme activity in the presence of hydrogen peroxide at any of the pH values tested (Fig. 1B).

Previous studies indicated that enzyme exposure to Na+ or K+ ions maintains more than 50% of skBADH activity and that physiological ionic strength is necessary to maintain the tetrameric structure of skBADH (Guzman-Partida & Valenzuela-Soto, 1998; Valenzuela-

Structural and Activity Changes in Renal

Betaine Aldehyde Dehydrogenase Caused by Oxidants 237

Fig. 2. Fluorescence spectra for skBADH in the presence of hydrogen peroxide.

Fig. 3. Fluorescence spectra for skBADH incubated with 100 µM H2O2. *Inset:* SkBADH time

dependent quenching.

Soto et al., 2003). SkBADH is inhibited in the presence of 100 µM H2O2, a concentration that may be found in medulla cells during concentrated urine formation, and which requires high osmolarity and low oxygen tension (Burg & Ferraris, 2008; Neuhofer & Beck, 2006). In addition, these peroxide concentrations may be found in the medulla cells of hypertensive (Touyz & Briones, 2011; Thengchaisri & Kuo, 2003) and diabetic patients (Coughlan et al., 2009; Forbes et al., 2008), i.e. pathologies related to high ROS concentrations.

Fig. 1. Effect of pH and ionic strength on skBADH inactivation caused by hydrogen peroxide. Enzyme activity measured at: (A) low ionic strength; (B) physiological ionic strength (0.15 M KCl).

#### **3.2 The effect of hydrogen peroxide on enzyme fluorescence**

A methodical evaluation of the effect of hydrogen peroxide on enzyme tertiary structure was performed by incubating the enzyme in the presence of 0-0.5 mM H2O2. SkBADH maximum emission was detected at 333 nm (Fig. 2). Enzyme fluorescence was quenched by all hydrogen peroxide concentrations tested (Fig. 2), however fluorescence spectral centers of mass did not change and no blue or red shift was found.

A hydrogen peroxide concentration of 100 µM was chosen to analyze the effect of incubation time on enzyme quenching, because this concentration can be found in kidney. When the enzyme was incubated for 120 min in the presence of 100 µM H2O2, the fluorescence of skBADH was quenched in a time dependent manner (Fig. 3). The presence of hydrogen peroxide decreased the skBADH maximum emission by more than 50% at 120 min of incubation. Changes in enzyme emission fit a single decay equation with an R-square of 0.9893 (Fig. 3 Inset). It is noteworthy that skBADH fluorescence quenching was only detected in the presence of peroxide and the shape of the fluorescence spectra did not change. The fluorescence spectral centers of mass did not change either.

Soto et al., 2003). SkBADH is inhibited in the presence of 100 µM H2O2, a concentration that may be found in medulla cells during concentrated urine formation, and which requires high osmolarity and low oxygen tension (Burg & Ferraris, 2008; Neuhofer & Beck, 2006). In addition, these peroxide concentrations may be found in the medulla cells of hypertensive (Touyz & Briones, 2011; Thengchaisri & Kuo, 2003) and diabetic patients (Coughlan et al.,

2009; Forbes et al., 2008), i.e. pathologies related to high ROS concentrations.

Fig. 1. Effect of pH and ionic strength on skBADH inactivation caused by hydrogen peroxide. Enzyme activity measured at: (A) low ionic strength; (B) physiological ionic

A methodical evaluation of the effect of hydrogen peroxide on enzyme tertiary structure was performed by incubating the enzyme in the presence of 0-0.5 mM H2O2. SkBADH maximum emission was detected at 333 nm (Fig. 2). Enzyme fluorescence was quenched by all hydrogen peroxide concentrations tested (Fig. 2), however fluorescence spectral centers

A hydrogen peroxide concentration of 100 µM was chosen to analyze the effect of incubation time on enzyme quenching, because this concentration can be found in kidney. When the enzyme was incubated for 120 min in the presence of 100 µM H2O2, the fluorescence of skBADH was quenched in a time dependent manner (Fig. 3). The presence of hydrogen peroxide decreased the skBADH maximum emission by more than 50% at 120 min of incubation. Changes in enzyme emission fit a single decay equation with an R-square of 0.9893 (Fig. 3 Inset). It is noteworthy that skBADH fluorescence quenching was only detected in the presence of peroxide and the shape of the fluorescence spectra did not

**3.2 The effect of hydrogen peroxide on enzyme fluorescence**

of mass did not change and no blue or red shift was found.

change. The fluorescence spectral centers of mass did not change either.

strength (0.15 M KCl).

Fig. 2. Fluorescence spectra for skBADH in the presence of hydrogen peroxide.

Fig. 3. Fluorescence spectra for skBADH incubated with 100 µM H2O2. *Inset:* SkBADH time dependent quenching.

Structural and Activity Changes in Renal

**3.5 Enzyme reactivation kinetics**

obtained after long incubation periods.

kidney.

Betaine Aldehyde Dehydrogenase Caused by Oxidants 239

inactivation reached 59% (Fig. 5B). The inactivation process follows single exponential decay kinetics with *k*obs = 0.0412 ± 0.0057 min-1 for the enzyme incubated with KCl and *k*obs = 0.0147 ± 0.0048 min-1 for the enzyme incubated without KCl (Fig. 5B). Under renal pH and ionic strength conditions skBADH is inactivated by copper concentrations that can occur in

Fig. 5. Time courses of the inactivation of skBADH by copper. (A) Enzyme incubated in Hepes-KOH buffer at pH 8.0; (B) Enzyme incubated in Hepes-KOH buffer at pH 7.0. The lines are the best fit of the inactivation data to a single exponential decay equation.

The fully inactivated enzyme was incubated in the presence of 10 mM DTT, 10 mM GSH or 10 mM 2-mercaptoethanol. SkBADH incubated with DTT at pH 8.0 or pH 7.0 recovered 93% and 96% of its activity, respectively (Fig. 6). When the reducing agent used was 2 mercaptoethanol, under conditions of pH 8.0 or pH 7.0 the enzyme recovered 86% and 84% of its activity (Fig. 6). With GSH at pH 8.0, skBADH recovered 90% of its activity (Fig. 6B), while at pH 7.0 the enzyme recovered 30% of its activity (Fig. 6A). Enzyme recovery activity was faster at pH 8.0 than at pH 7.0, regardless of the reducing agent tested (Fig. 6). SkBADH incubated with reducing agents in the presence of 0.15 M KCl behaved very similarly to the curves in Figure 6 (data not shown). GSH was not able to recover enzyme activity when there was potassium in the assay medium. No significant skBADH activity changes were

The failure of GSH to reactivate skBADH and other BADHs inactivated by thiol-specific reagents had been reported (Rosas-Rodríguez & Valenzuela-Soto, 2011; Velasco-García et al., 2003; Vallari & Pietruszko, 1982); however, the reason for this failure is not yet clear and may be related to the larger size of GSH, which prevents it from accessing the active site.

In addition, with respect to the maximum emission wavelength, no blue/red shift could be detected which suggests the direct alteration of the enzyme site rather than a change in protein folding, and which also rules out any enzyme denaturation.

#### **3.3 The effect of ligands on enzyme quenching provoked by hydrogen peroxide**

The skBADH fluorescence spectra at both pH 7.0 and pH 8.0 showed that the maximum emission and the wavelength at which the maxima occurred were equal (Fig. 4). Fluorescence spectral centers of mass changed between 1 and 2 nm. At pH 7.0, hydrogen peroxide increased the enzyme's maximum emission but did not cause a blue or red shift (Fig. 4A). The presence of BA produced a red shift indicating a change in solvent exposition of tryptophans, caused by hydrogen peroxide and BA mix (Fig. 4A). However, the enzyme product GB had no effect on the quenching provoked by peroxide. At pH 8.0, as before, skBADH fluorescence was quenched by hydrogen peroxide without any blue or red shift (Fig. 4B). BA and GB did not play a role in the enzyme fluorescence quenching induced by hydrogen peroxide (Fig. 4B).

Fig. 4. Fluorescence spectra for skBADH incubated at pH 7.0 (A) and pH 8.0 (B) in the absence and presence of 100 µM H2O2, betaine aldehyde (BA) or glycine betaine (GB).

#### **3.4 The effect of copper on enzyme activity**

SkBADH incubated with 10 µM CuCl2 at pH 7.0 and 8.0 was inactivated in a time dependent manner (Fig. 5). In assays performed at pH 8.0, enzyme activity decreased 80%, however when KCl was included in the incubation assay, enzyme activity decreased 45% (Fig. 5A). Data analysis revealed single exponential decay kinetics with *k*obs = 0.0112 ± 0.005 min-1 for the enzyme incubated with KCl and *k*obs = 0.022 ± 0.002 min-1 for the enzyme incubated without KCl (Fig. 5A).

The enzyme incubated with 10 µM CuCl2 at pH 7.0 had a different inactivation pattern: skBADH lost 48% of its activity in absence of KCl, whereas in its presence enzyme

In addition, with respect to the maximum emission wavelength, no blue/red shift could be detected which suggests the direct alteration of the enzyme site rather than a change in

The skBADH fluorescence spectra at both pH 7.0 and pH 8.0 showed that the maximum emission and the wavelength at which the maxima occurred were equal (Fig. 4). Fluorescence spectral centers of mass changed between 1 and 2 nm. At pH 7.0, hydrogen peroxide increased the enzyme's maximum emission but did not cause a blue or red shift (Fig. 4A). The presence of BA produced a red shift indicating a change in solvent exposition of tryptophans, caused by hydrogen peroxide and BA mix (Fig. 4A). However, the enzyme product GB had no effect on the quenching provoked by peroxide. At pH 8.0, as before, skBADH fluorescence was quenched by hydrogen peroxide without any blue or red shift (Fig. 4B). BA and GB did not play a role in the enzyme fluorescence quenching induced by

**3.3 The effect of ligands on enzyme quenching provoked by hydrogen peroxide**

Fig. 4. Fluorescence spectra for skBADH incubated at pH 7.0 (A) and pH 8.0 (B) in the absence and presence of 100 µM H2O2, betaine aldehyde (BA) or glycine betaine (GB).

SkBADH incubated with 10 µM CuCl2 at pH 7.0 and 8.0 was inactivated in a time dependent manner (Fig. 5). In assays performed at pH 8.0, enzyme activity decreased 80%, however when KCl was included in the incubation assay, enzyme activity decreased 45% (Fig. 5A). Data analysis revealed single exponential decay kinetics with *k*obs = 0.0112 ± 0.005 min-1 for the enzyme incubated with KCl and *k*obs = 0.022 ± 0.002 min-1 for the enzyme incubated

The enzyme incubated with 10 µM CuCl2 at pH 7.0 had a different inactivation pattern: skBADH lost 48% of its activity in absence of KCl, whereas in its presence enzyme

protein folding, and which also rules out any enzyme denaturation.

hydrogen peroxide (Fig. 4B).

**3.4 The effect of copper on enzyme activity**

without KCl (Fig. 5A).

inactivation reached 59% (Fig. 5B). The inactivation process follows single exponential decay kinetics with *k*obs = 0.0412 ± 0.0057 min-1 for the enzyme incubated with KCl and *k*obs = 0.0147 ± 0.0048 min-1 for the enzyme incubated without KCl (Fig. 5B). Under renal pH and ionic strength conditions skBADH is inactivated by copper concentrations that can occur in kidney.

Fig. 5. Time courses of the inactivation of skBADH by copper. (A) Enzyme incubated in Hepes-KOH buffer at pH 8.0; (B) Enzyme incubated in Hepes-KOH buffer at pH 7.0. The lines are the best fit of the inactivation data to a single exponential decay equation.

#### **3.5 Enzyme reactivation kinetics**

The fully inactivated enzyme was incubated in the presence of 10 mM DTT, 10 mM GSH or 10 mM 2-mercaptoethanol. SkBADH incubated with DTT at pH 8.0 or pH 7.0 recovered 93% and 96% of its activity, respectively (Fig. 6). When the reducing agent used was 2 mercaptoethanol, under conditions of pH 8.0 or pH 7.0 the enzyme recovered 86% and 84% of its activity (Fig. 6). With GSH at pH 8.0, skBADH recovered 90% of its activity (Fig. 6B), while at pH 7.0 the enzyme recovered 30% of its activity (Fig. 6A). Enzyme recovery activity was faster at pH 8.0 than at pH 7.0, regardless of the reducing agent tested (Fig. 6). SkBADH incubated with reducing agents in the presence of 0.15 M KCl behaved very similarly to the curves in Figure 6 (data not shown). GSH was not able to recover enzyme activity when there was potassium in the assay medium. No significant skBADH activity changes were obtained after long incubation periods.

The failure of GSH to reactivate skBADH and other BADHs inactivated by thiol-specific reagents had been reported (Rosas-Rodríguez & Valenzuela-Soto, 2011; Velasco-García et al., 2003; Vallari & Pietruszko, 1982); however, the reason for this failure is not yet clear and may be related to the larger size of GSH, which prevents it from accessing the active site.

Structural and Activity Changes in Renal

Betaine Aldehyde Dehydrogenase Caused by Oxidants 241

The drop in the enzyme's maximum emission was similar at both pH values, though the wavelength at which that maximum occurred was different at pH 7.0 and pH 8.0. At pH 7.0 the maximum emission wavelength for the enzyme incubated only with copper was 327 nm (Fig. 7A). Copper caused a blue shift in the enzyme, implying that the tryptophans were less exposed to the solvent. The presence of 0.15 M KCl reversed the blue shift (Fig 7B). The skBADH fluorescence spectra at pH 8.0 with 10 µM CuCl2 did not exhibit a blue or red shift

Fig. 8. The effect of copper and incubation time on skBADH fluorescence spectra. Enzyme

SkBADH has four tryptophan residues per subunit, which means there are sixteen residues in the enzyme's active conformation (tetramer), making Trp fluorescence a useful technique for structure-function studies. Alvarez et al. (2010) report that the presence of reactive oxygen species could impact the fluorescent properties of a specific protein (Cyan Fluorescent Protein), due to chemical modifications that cause changes in the chromophore pocket (Alvarez et al., 2010). These authors suggest that the alterations could be related to

The quenching of skBADH fluorescence by hydrogen peroxide is consistent with data reported by Tsourkas et al. (2005) who detected a 50% decrease in fluorescent emission for Red Fluorescent Protein (DsRed) and 95% for Enhanced Yellow Fluorescent Protein (EYFP) in the presence of 50 µM hydrogen peroxide (Tsourkas et al., 2005). The authors attributed the quenching process to intramolecular cross-linking between two cysteines which puts

incubated at pH 8.0 with 10 µM CuCl2 (A), plus 0.15 M KCl (B).

the torsion of amino acid residues, which leads to a quenching process.

stress on the protein structure (Tsourkas et al., 2005).

**4. Discussion**

(Fig. 8A) and the presence of KCl did not cause any changes (Fig. 8B).

Fig. 6. Reactivation kinetics for skBADH inactivated by copper. Enzyme inactivated by 50 µM CuCl2 to a residual activity of 15% over a 150 min reaction. Enzyme incubated: at pH 7.0 (A) or pH 8.0 (B). The lines are the best fit of the inactivation data to a single exponential decay equation.

#### **3.6 The effect of copper on enzyme fluorescence**

The skBADH fluorescence spectra were obtained from enzyme incubation assays with 10 µM CuCl2 at pH 8.0 or pH 7.0. Enzyme fluorescence was quenched by copper at both pH values (Fig. 7 and 8). Enzyme maximum emission changed with respect to the time of incubation at both pH values. However, incubation time did not have any effect on the wavelength at which maximum emission occurred.

Fig. 7. The effect of copper and incubation time on skBADH fluorescence spectra. Enzyme incubated at pH 7.0 with 10 µM CuCl2 (A), plus 0.15 M KCl (B).

Fig. 6. Reactivation kinetics for skBADH inactivated by copper. Enzyme inactivated by 50 µM CuCl2 to a residual activity of 15% over a 150 min reaction. Enzyme incubated: at pH 7.0 (A) or pH 8.0 (B). The lines are the best fit of the inactivation data to a single exponential

The skBADH fluorescence spectra were obtained from enzyme incubation assays with 10 µM CuCl2 at pH 8.0 or pH 7.0. Enzyme fluorescence was quenched by copper at both pH values (Fig. 7 and 8). Enzyme maximum emission changed with respect to the time of incubation at both pH values. However, incubation time did not have any effect on the

Fig. 7. The effect of copper and incubation time on skBADH fluorescence spectra. Enzyme

incubated at pH 7.0 with 10 µM CuCl2 (A), plus 0.15 M KCl (B).

decay equation.

**3.6 The effect of copper on enzyme fluorescence**

wavelength at which maximum emission occurred.

The drop in the enzyme's maximum emission was similar at both pH values, though the wavelength at which that maximum occurred was different at pH 7.0 and pH 8.0. At pH 7.0 the maximum emission wavelength for the enzyme incubated only with copper was 327 nm (Fig. 7A). Copper caused a blue shift in the enzyme, implying that the tryptophans were less exposed to the solvent. The presence of 0.15 M KCl reversed the blue shift (Fig 7B). The skBADH fluorescence spectra at pH 8.0 with 10 µM CuCl2 did not exhibit a blue or red shift (Fig. 8A) and the presence of KCl did not cause any changes (Fig. 8B).

Fig. 8. The effect of copper and incubation time on skBADH fluorescence spectra. Enzyme incubated at pH 8.0 with 10 µM CuCl2 (A), plus 0.15 M KCl (B).

#### **4. Discussion**

SkBADH has four tryptophan residues per subunit, which means there are sixteen residues in the enzyme's active conformation (tetramer), making Trp fluorescence a useful technique for structure-function studies. Alvarez et al. (2010) report that the presence of reactive oxygen species could impact the fluorescent properties of a specific protein (Cyan Fluorescent Protein), due to chemical modifications that cause changes in the chromophore pocket (Alvarez et al., 2010). These authors suggest that the alterations could be related to the torsion of amino acid residues, which leads to a quenching process.

The quenching of skBADH fluorescence by hydrogen peroxide is consistent with data reported by Tsourkas et al. (2005) who detected a 50% decrease in fluorescent emission for Red Fluorescent Protein (DsRed) and 95% for Enhanced Yellow Fluorescent Protein (EYFP) in the presence of 50 µM hydrogen peroxide (Tsourkas et al., 2005). The authors attributed the quenching process to intramolecular cross-linking between two cysteines which puts stress on the protein structure (Tsourkas et al., 2005).

Structural and Activity Changes in Renal

Betaine Aldehyde Dehydrogenase Caused by Oxidants 243

Fig. 9. Proposed mechanism for the reaction of the skBADH active site and H2O2. Hydrogen peroxide reacts with the thiol group (CSH) of the catalytic cysteine (C288) or the vicinal cysteine (C289) (1) leading to sulfenic acid formation (RSOH) (2), the proximity of a second thiolate group allows the intermediate sulfenic acid to combine with the thiolate group and

form a disulfide bond (RSSR) between cysteines (3).

A previous study reported that skBADH is inactivated by H2O2 in an oxygen independent modification process (Rosas-Rodríguez & Valenzuela-Soto, 2011). Our data for enzyme fluorescence quenching by peroxide indicates that the protein's tertiary structure was not changed (Fig. 2). Together, the inactivation and fluorescence data support the idea that enzyme inactivation is caused by a disulfide bond between vicinal cysteines (C288-C289) at the active site, as previously postulated (Rosas-Rodríguez & Valenzuela-Soto, 2011).

Based on the mechanism of the hydrogen peroxide and cysteine reaction (Luo & Anderson, 2008), we propose a structural model showing the interaction of peroxide with the skBADH active site that leads to enzyme inactivation (Fig. 9). The structural model of skBADH was compared with that of methanol dehydrogenase (pdb 1g72) to look for type VIII folding, because that kind of folding has been found in proteins forming disulfide bridges between vicinal cysteines (Carugo et al., 2003). Both models were very similar, thus we think that skBADH exhibits the type VIII β-turn folding that allows for disulfide bridge formation.

Copper had the strongest inactivation effect on skBADH under physiological pH and ionic strength conditions (Fig. 5). Because the potassium concentrations in kidney medulla cells are those tested in this study, skBADH is a target for copper oxidation which leads to a decrease in glycine betaine synthesis and accumulation. In addition to its function as an osmolyte, glycine betaine functions as an osmoprotector (Rosas-Rodríguez et al., 2010); the insufficient osmoprotection and osmoregulation of renal cells may be related to some kidney pathologies.

The highest levels of BADH activity and protein occur in human liver, the adrenal gland, and kidney (Izaguirre et al, 1991). Besides of the role played by BADH in kidney, mammalian BADH catalyzes the oxidation of different amino aldehydes in addition to betaine aldehyde, so this enzyme is thought to be involved in: (i) the biosynthesis of GB, which may function as a methyl donor for methionine synthesis in liver (duVigneaud, 1946, Muntz, 1950), (ii) polyamine catabolism (Ambroziak & Pietruszko, 1991), (iii) the synthesis of the inhibitory neurotransmitter γ-aminobutyrate (GABA) particularly in adrenal glands where putrescine is a source of γ-aminobutyric acid (Lin et al., 1996), and (iv) carnitine biosynthesis (Vaz et al., 2000). It is possible that all BADHs might be susceptible to copper inactivation, but it is important to conduct studies to test this idea.

SkBADH was inactivated by 10 µM CuCl2 as a consequence of its thiol groups being oxidized (Fig. 5). The oxidation process was reversed by thiol reducing agents, DTT and 2- ME and GSH (at pH 8.0) (Fig. 6B), whereas at pH 7.0 DTT and 2-ME were able to restore enzyme activity which is consistent with the generation of a disulfide bond in the enzyme (Fig. 6A). A disulfide bond could be formed between Cys 288 and Cys 289 at the active site (Fig. 10). GSH was not able to restore enzyme activity at pH 7.0, which suggests that GSH cannot prevent skBADH inactivation *in vivo*.

Inactivation by copper due to thiol oxidation has been observed for other enzymes such as rat and human aldose reductase, where inactivation results from disulfide bridge formation (Cecconi et al., 2002). Similarly, Hadizadeh et al. (2009) observed that the enzyme xanthine oxidase is inactivated by copper in a concentration and time dependent manner (Hadizadeh et al. 2009).

A previous study reported that skBADH is inactivated by H2O2 in an oxygen independent modification process (Rosas-Rodríguez & Valenzuela-Soto, 2011). Our data for enzyme fluorescence quenching by peroxide indicates that the protein's tertiary structure was not changed (Fig. 2). Together, the inactivation and fluorescence data support the idea that enzyme inactivation is caused by a disulfide bond between vicinal cysteines (C288-C289) at

Based on the mechanism of the hydrogen peroxide and cysteine reaction (Luo & Anderson, 2008), we propose a structural model showing the interaction of peroxide with the skBADH active site that leads to enzyme inactivation (Fig. 9). The structural model of skBADH was compared with that of methanol dehydrogenase (pdb 1g72) to look for type VIII folding, because that kind of folding has been found in proteins forming disulfide bridges between vicinal cysteines (Carugo et al., 2003). Both models were very similar, thus we think that skBADH exhibits the type VIII β-turn folding that allows for disulfide bridge formation.

Copper had the strongest inactivation effect on skBADH under physiological pH and ionic strength conditions (Fig. 5). Because the potassium concentrations in kidney medulla cells are those tested in this study, skBADH is a target for copper oxidation which leads to a decrease in glycine betaine synthesis and accumulation. In addition to its function as an osmolyte, glycine betaine functions as an osmoprotector (Rosas-Rodríguez et al., 2010); the insufficient osmoprotection and osmoregulation of renal cells may be related to some

The highest levels of BADH activity and protein occur in human liver, the adrenal gland, and kidney (Izaguirre et al, 1991). Besides of the role played by BADH in kidney, mammalian BADH catalyzes the oxidation of different amino aldehydes in addition to betaine aldehyde, so this enzyme is thought to be involved in: (i) the biosynthesis of GB, which may function as a methyl donor for methionine synthesis in liver (duVigneaud, 1946, Muntz, 1950), (ii) polyamine catabolism (Ambroziak & Pietruszko, 1991), (iii) the synthesis of the inhibitory neurotransmitter γ-aminobutyrate (GABA) particularly in adrenal glands where putrescine is a source of γ-aminobutyric acid (Lin et al., 1996), and (iv) carnitine biosynthesis (Vaz et al., 2000). It is possible that all BADHs might be susceptible to copper

SkBADH was inactivated by 10 µM CuCl2 as a consequence of its thiol groups being oxidized (Fig. 5). The oxidation process was reversed by thiol reducing agents, DTT and 2- ME and GSH (at pH 8.0) (Fig. 6B), whereas at pH 7.0 DTT and 2-ME were able to restore enzyme activity which is consistent with the generation of a disulfide bond in the enzyme (Fig. 6A). A disulfide bond could be formed between Cys 288 and Cys 289 at the active site (Fig. 10). GSH was not able to restore enzyme activity at pH 7.0, which suggests that GSH

Inactivation by copper due to thiol oxidation has been observed for other enzymes such as rat and human aldose reductase, where inactivation results from disulfide bridge formation (Cecconi et al., 2002). Similarly, Hadizadeh et al. (2009) observed that the enzyme xanthine oxidase is inactivated by copper in a concentration and time dependent manner (Hadizadeh

inactivation, but it is important to conduct studies to test this idea.

cannot prevent skBADH inactivation *in vivo*.

kidney pathologies.

et al. 2009).

the active site, as previously postulated (Rosas-Rodríguez & Valenzuela-Soto, 2011).

Fig. 9. Proposed mechanism for the reaction of the skBADH active site and H2O2. Hydrogen peroxide reacts with the thiol group (CSH) of the catalytic cysteine (C288) or the vicinal cysteine (C289) (1) leading to sulfenic acid formation (RSOH) (2), the proximity of a second thiolate group allows the intermediate sulfenic acid to combine with the thiolate group and form a disulfide bond (RSSR) between cysteines (3).

Structural and Activity Changes in Renal

**5. Conclusion**

**6. Perspectives**

**7. Acknowledgments**

improved the English.

**8. References**

Betaine Aldehyde Dehydrogenase Caused by Oxidants 245

Further studies are underway to determine if skBADH inactivation is dependent on copper concentration, to identify the time course of disulfide generation, and to detect whether

SkBADH was oxidized by hydrogen peroxide and by copper at concentrations that likely reflect physiologically relevant conditions (Atmane et al. 2003; Caselli et al. 1998; Kazi et al., 2008; Meng et al. 2002; Pravodh et al. 2011) in kidney, and the targets of oxidation were thiol groups, mainly those of the active site. Interestingly, the thiol groups were not oxidized to sulfenic or sulfinic acid, but rather a disulfide bond was formed between catalytic cysteine (Cys288) and a vicinal cysteine (C289). Potassium, under physiological conditions, increased the oxidation of the enzyme by copper, possibly because it maintains the enzyme active tetramer or enables the oxidation of the cysteine residues. Since GB synthesis is catalyzed by BADH and it is crucial for the kidney cells to be able to withstand osmotic stress conditions, this type of inhibition can have serious physiological implications. In addition, BADH is an enzyme that is also found in the liver and brain where its inactivation by oxidative stress is associated with other pathologies. In summary, identifying the specific modifications caused by ROS on key enzymes contributes to a greater understanding of the changes that occur during oxidative stress and provides insight into how oxidative stress can be prevented. Further work is underway to determine the mechanisms by which renal BADH can be

Hydrogen peroxide plays a role as an oxidant and as a secondary messenger, however the molecular basis of these roles and the concentration of H2O2 in cells has not been established. In addition, copper can interact with proteins, causing oxidation, or can generate oxidative stress. In light of this it is important to pursue this line of research, also because hydrogen peroxide and copper have been associated with some human pathologies that are characterized by some interaction between hydrogen peroxide and copper and enzymes and proteins. This opens up new areas for developing strategies to contend with

This study was supported by the Consejo Nacional de Ciencia y Tecnología (CONACYT). HFFM received a graduate scholarship from CONACYT. JARR is grateful for support from "Apoyos Complementarios para la Consolidación Institucional de Grupos de Investigación: Retención 149021" awarded by CONACYT. The authors are grateful to Bianca Delfosse, who

Ambroziak, W., & Pietruszko, R. (1991) Human aldehyde dehydrogenase. Activity with

*Biological Chemistry*, Vol.266, No.20, (July 1991), 13011-13018, ISSN 0021-924.

aldehyde metabolites of monoamines, diamines, and polyamines. *Journal of*

there are differences in inactivation when Cu(I) or Cu(II) is used in the treatments.

protected from the oxidative stress that occurs in renal medulla cells.

some of the diseases directly related to oxidative stress.

Fig. 10. Proposed mechanism for the reaction of the sKBADH active site and copper. Copper reacts with the thiol group (CSH) of the catalytic cysteine (C288) or the vicinal cysteine (C289) (A), leading to the formation of a disulfide bond (RSSR) between cysteines (B).

SkBADH fluorescence spectra with 10 µM CuCl2 at pH 7.0 and pH 8.0 exhibited differences in the wavelengths at which the maximum emission occurred (Fig. 7 and 8), demonstrating that copper is changing the tryptophans' access to the solvent. In addition, potassium seems to be playing an interesting role in exposing the tryptophans in the enzyme to the solvent. Previous studies have demonstrated that skBADH requires a monovalent cation to maintain its tetrameric conformation (Valenzuela-Soto et al., 2003). SkBADH crystallographic studies are needed to see whether there is a site on the enzyme for potassium or for a different monovalent cation, such as that found in *P. aeruginosa* BADH (González-Segura et al., 2009).

Further studies are underway to determine if skBADH inactivation is dependent on copper concentration, to identify the time course of disulfide generation, and to detect whether there are differences in inactivation when Cu(I) or Cu(II) is used in the treatments.

### **5. Conclusion**

244 Oxidative Stress – Molecular Mechanisms and Biological Effects

Fig. 10. Proposed mechanism for the reaction of the sKBADH active site and copper. Copper reacts with the thiol group (CSH) of the catalytic cysteine (C288) or the vicinal cysteine (C289) (A), leading to the formation of a disulfide bond (RSSR) between cysteines (B).

SkBADH fluorescence spectra with 10 µM CuCl2 at pH 7.0 and pH 8.0 exhibited differences in the wavelengths at which the maximum emission occurred (Fig. 7 and 8), demonstrating that copper is changing the tryptophans' access to the solvent. In addition, potassium seems to be playing an interesting role in exposing the tryptophans in the enzyme to the solvent. Previous studies have demonstrated that skBADH requires a monovalent cation to maintain its tetrameric conformation (Valenzuela-Soto et al., 2003). SkBADH crystallographic studies are needed to see whether there is a site on the enzyme for potassium or for a different monovalent cation, such as that found in *P. aeruginosa* BADH (González-Segura et al., 2009).

SkBADH was oxidized by hydrogen peroxide and by copper at concentrations that likely reflect physiologically relevant conditions (Atmane et al. 2003; Caselli et al. 1998; Kazi et al., 2008; Meng et al. 2002; Pravodh et al. 2011) in kidney, and the targets of oxidation were thiol groups, mainly those of the active site. Interestingly, the thiol groups were not oxidized to sulfenic or sulfinic acid, but rather a disulfide bond was formed between catalytic cysteine (Cys288) and a vicinal cysteine (C289). Potassium, under physiological conditions, increased the oxidation of the enzyme by copper, possibly because it maintains the enzyme active tetramer or enables the oxidation of the cysteine residues. Since GB synthesis is catalyzed by BADH and it is crucial for the kidney cells to be able to withstand osmotic stress conditions, this type of inhibition can have serious physiological implications. In addition, BADH is an enzyme that is also found in the liver and brain where its inactivation by oxidative stress is associated with other pathologies. In summary, identifying the specific modifications caused by ROS on key enzymes contributes to a greater understanding of the changes that occur during oxidative stress and provides insight into how oxidative stress can be prevented. Further work is underway to determine the mechanisms by which renal BADH can be protected from the oxidative stress that occurs in renal medulla cells.

#### **6. Perspectives**

Hydrogen peroxide plays a role as an oxidant and as a secondary messenger, however the molecular basis of these roles and the concentration of H2O2 in cells has not been established. In addition, copper can interact with proteins, causing oxidation, or can generate oxidative stress. In light of this it is important to pursue this line of research, also because hydrogen peroxide and copper have been associated with some human pathologies that are characterized by some interaction between hydrogen peroxide and copper and enzymes and proteins. This opens up new areas for developing strategies to contend with some of the diseases directly related to oxidative stress.

#### **7. Acknowledgments**

This study was supported by the Consejo Nacional de Ciencia y Tecnología (CONACYT). HFFM received a graduate scholarship from CONACYT. JARR is grateful for support from "Apoyos Complementarios para la Consolidación Institucional de Grupos de Investigación: Retención 149021" awarded by CONACYT. The authors are grateful to Bianca Delfosse, who improved the English.

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**Section 4**

**Reactive Species as Signaling Molecules**

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## **Section 4**

**Reactive Species as Signaling Molecules**

252 Oxidative Stress – Molecular Mechanisms and Biological Effects

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**14**

 *Spain*

**Signalling Oxidative Stress in**

Maria Angeles de la Torre-Ruiz1\*, Luis Serrano2, Mima I. Petkova1 and Nuria Pujol-Carrion1 *1Dept. Ciències Mèdiques Bàsiques-IRBLleida, Faculty of Medicine, University of Lleida 2Dept. Producció Vegetal & Ciència Forestal,*

Oxidative stress occurs in the natural environment with exposure to aerobic conditions and UV light. Reactive oxygen species (ROS) are also the consequence of normal cellular metabolism. Aerobic organisms sense redox perturbations and develop several different

The mitochondrial respiratory chain is the major ROS source of reactive oxygen species. ROS can damage a wide range of molecules, including nucleic acids, proteins and lipids. The accumulation of oxidised proteins, DNA damage and the increased production of ROS, concomitant with a depletion of antioxidant defences, seem to be key factors in aging and

Mitochondrial oxidation appears to be a major cause of signalling to different pathways; however it is still unclear which one of the inflammatory or the apoptotic signals plays a more relevant role in the mitochondrial generation of ROS. Starvation can increase ROS steady-state concentration and autophagy. Hydrogen peroxide appears to be the major oxidant in these conditions, and would oxidise specific cysteines in autophagyc genes leading to the increase in the autophagosome formation. However it is unknown how the

Mitogen activated protein kinases (MAPK) are required in all the eukaryotic cells to properly activate responses in order to allow cells to respond to the different external stresses. The finality of this is to assure cell survival (Wagner & Nebreda, 2009). Several stimuli, included oxidative stress are signalled to phosphorylate certain MAPK thus activating their kinase activity to phosporylate specific substrates (Shiozaki & Russell, 1995;

The eukaryotic microorganism Saccharomyces cerevisiae serves as a model system to study the signal transduction pathways involved in the response to oxidative stress. Thus, TOR,

adaptive mechanisms in order to acquire survival capacity (Zheng & Storz, 2000).

signal is transduced to specific targets (Reviewed in Finkel, 2011).

**1. Introduction**

cell death.

Nguyen et al., 2000).

\* Corresponding Author

*Saccharomyces cerevisiae*

 *ETSEA, University of Lleida,*

## **Signalling Oxidative Stress in**  *Saccharomyces cerevisiae*

Maria Angeles de la Torre-Ruiz1\*, Luis Serrano2, Mima I. Petkova1 and Nuria Pujol-Carrion1 *1Dept. Ciències Mèdiques Bàsiques-IRBLleida, Faculty of Medicine, University of Lleida 2Dept. Producció Vegetal & Ciència Forestal, ETSEA, University of Lleida, Spain*

#### **1. Introduction**

Oxidative stress occurs in the natural environment with exposure to aerobic conditions and UV light. Reactive oxygen species (ROS) are also the consequence of normal cellular metabolism. Aerobic organisms sense redox perturbations and develop several different adaptive mechanisms in order to acquire survival capacity (Zheng & Storz, 2000).

The mitochondrial respiratory chain is the major ROS source of reactive oxygen species. ROS can damage a wide range of molecules, including nucleic acids, proteins and lipids. The accumulation of oxidised proteins, DNA damage and the increased production of ROS, concomitant with a depletion of antioxidant defences, seem to be key factors in aging and cell death.

Mitochondrial oxidation appears to be a major cause of signalling to different pathways; however it is still unclear which one of the inflammatory or the apoptotic signals plays a more relevant role in the mitochondrial generation of ROS. Starvation can increase ROS steady-state concentration and autophagy. Hydrogen peroxide appears to be the major oxidant in these conditions, and would oxidise specific cysteines in autophagyc genes leading to the increase in the autophagosome formation. However it is unknown how the signal is transduced to specific targets (Reviewed in Finkel, 2011).

Mitogen activated protein kinases (MAPK) are required in all the eukaryotic cells to properly activate responses in order to allow cells to respond to the different external stresses. The finality of this is to assure cell survival (Wagner & Nebreda, 2009). Several stimuli, included oxidative stress are signalled to phosphorylate certain MAPK thus activating their kinase activity to phosporylate specific substrates (Shiozaki & Russell, 1995; Nguyen et al., 2000).

The eukaryotic microorganism Saccharomyces cerevisiae serves as a model system to study the signal transduction pathways involved in the response to oxidative stress. Thus, TOR,

\* Corresponding Author

Signalling Oxidative Stress in *Saccharomyces cerevisiae* 257

(2005). Importantly, Pascale et al. (2007) exposed in a review how alterations of the PKC cascade may have implications in physiological and pathological brain aging, such as

The cell-wall integrity pathway in budding yeast involves a protein kinase (MAPKs) cascade which participates in sensing and transmitting several extracellular signals and stresses, including: cell-wall, osmotic, mating and nutritional stress (for a review see Heinisch et al., 1999; Levin, 2005), oxidative (Vilella et al., 2005) and pH (Serrano et al., 2006) stresses. The PKC1-MAPK pathway is integrated by several cell-wall proteins that are putative cellmembrane receptors of different stimuli, they are: the Wsc1-Wsc4 family, Mid2 and Mtl1. They transmit signals to Rom2 which activates the G protein Rho1 which in its turn activates the kinase Pkc1 (this protein has high degree of homology with other isoforms of PKC in eukaryotic cells). Pkc1 activates a MAP kinase module: Bck1 (which is the MAPKKK) phosphorylates the redundant MAPKKs Mkk1 and Mkk2 and together they both activate Slt2, the last kinase member of the pathway. There are two downstream events which correlate to Slt2 activation: transcriptional activity driven by Rlm1 and Swi6 phosphorylation (Heinish et al., 1999; Levin, 2005). Swi6 is one output of Slt2 activity. Swi6 is phosphorylated by Slt2 via the CWI (Sidorova et al., 1995; Madden et al., 1997). However when the external imput is oxidative stress, *SWI6* acts as a sensor through the oxidation of its Cys-404 to a sulfenic residue affecting the cell capability to arrest the cell cycle in G1

The upper elements of the cell integrity pathway are involved in the organisation of the actin cytoskeleton under different conditions, including cell-wall and nutritional stresses (Helliwell et al., 1998; Delley & Hall, 1999; Torres et al., 2002), oxidative stress (Vilella et al.,

Exposure of *rom2* to oxidising agent results in diminished Slt2/Mpk1 phosphorylation (Vilella et al., 2005). Pkc1 is also required but the MAP kinase module, downstream of Pkc1, seems to be dispensable for this mechanism. Pkc1 overexpression confers cells with more resistance to oxidising agents. It has been demonstrated that upon oxidative stress Pkc1 translocates to the cell periphery. However, Pkc1 transmits the signal to Slt2/Mpk1 if cells

The Pkc1 pathway is also related to the TOR pathway. Budding yeast have two different TOR genes: *TOR1* and *TOR2*, which share 67% sequence identity and are partly redundant in function (Helliwell et al., 1994). Loewith et al. (2002) purified and identified the components of two distinct TOR complexes, TORC1 and TORC2. TORC1 modulates translation initiation, inhibits protein turnover and represses the transcription of genes related to nutrient starvation. Early studies in *Saccharomyces cerevisiae* indicated that TOR has at least two functions: one regulated by the TORC1 complex which is sensitive to rapamycin and the other which is driven by the TORC2 complex and closely related to the organisation of the actin cytoskeleton and independent of rapamycin inhibition (Loewith et al., 2002; and for reviews Wullschleger et al., 2006; Inoki et al., 2005; Inoki & Guan, 2006). Tor2 functions in both complexes while Tor1 only participates in the TORC1 complex. The unique Tor2 function is related to Pkc1 and the organisation of the actin cytoskeleton (Helliwell et al., 1998). However, the rapamycininsensitive Tor2-unique function has not been described in other eukaryotic model systems (Crespo and Hall, 2002). Rapamycin also induces depolarisation of the actin cytoskeleton

Alzheimer's disease.

(Chiu et al., 2011).

2005) and pH (Motizuki et al., 2008) among others.

have intact secretory machinery (Vilella et al., 2005)

RAS and CWI pathways are the best characterised routes known to play a relevant role in transducing the oxidative signal in budding yeast.

#### **2. Sensing oxidative stress**

Mitochondria are believed to be the major factories of ROS as a byproduct of the respiratory metabolism. However recent studies indicate that ROS produced in mitochondria are signalling molecules capable to activate proteins such as the stress c-Jun N terminal kinase (Finkel, 2011). The activation of the kinase occurs because ROS in fact, inactivate a cysteine dependent phosphatase that regulates c-Jun (Kamata, 2005). The major source of ROS in mitochondrial respiratory chain are complexes I and III. Therefore, mitochondria are ROS generators and consequently act as sensors/transmissors of oxidative stress in eukaryotic cells.

In budding yeast, actin appears to be another possible candidate for sensing oxidative stress (discussed in part 6.2 below)

Transmembrane proteins act also as sensors of a number of stimuli. These proteins can function as detectors of environmental changes and also as a potential transmission molecules, to perhaps downstream signal transduction pathways. Rajavel et al. (1999) identified Mtl1 protein, a Mid2 homologue with a role in cell integrity signalling in vegetative growth. Mtl1 is an element of the cell integrity pathway, overexpression of *MTL1* supress defects in Rho1 function (Sekiya-Kawasaki et al., 2002). In the context of oxidative stress, the most likely candidate for being a transmembrane protein sensor is Mtl1 protein. Vilella et al. (2005) showed that Mtl1 is a cell surface sensor for oxidative stress. Mtl1 functions as oxidatives stress sensor since its function is necessary to survive in response to oxidants and to transmit the oxidative signal to the downstream elements of the cell integrity pathway (Vilella et al., 2005; Petkova et al., 2010a). Mtl1 is a transmembrane protein and localises to the cell periphery in all the stress conditions tested (submitted), supporting the hypothesis that Mtl1 acts as a cell-wall sensor, specifically as an oxidative stress sensor, given that mtl1 mutant cells are only sensitive to oxidative conditions and nutritional starvation (both conditions generate ROS production in the cells). Whether Mtl1 detects extracellular or/and intracellular oxidative stress is still unknown.

#### **3. Transducing oxidative stress**

#### **3.1 CWI, TOR and RAS pathways**

Signal transduction pathways function as transmissors of environmental estimuli to specific substrates. In budding yeast characteristic routes involved in this processes are CWI, TOR and RAS.

Protein kinase C (PKC) is a protein with a role in oxidative stress response (for a review, Nitti et al., 2008).

In human cells, PKC is involved in the protection against oxidative stress in the heart. The knowledge of this signalling is essential to the development of drugs to treat stroke and cardiac arrithmias (Barnett et al., 2007). Another important feature of the activation of the PKC signal transduction pathway is its role in aging, as reported by Battaini & Pascale

RAS and CWI pathways are the best characterised routes known to play a relevant role in

Mitochondria are believed to be the major factories of ROS as a byproduct of the respiratory metabolism. However recent studies indicate that ROS produced in mitochondria are signalling molecules capable to activate proteins such as the stress c-Jun N terminal kinase (Finkel, 2011). The activation of the kinase occurs because ROS in fact, inactivate a cysteine dependent phosphatase that regulates c-Jun (Kamata, 2005). The major source of ROS in mitochondrial respiratory chain are complexes I and III. Therefore, mitochondria are ROS generators and consequently act as sensors/transmissors of oxidative stress in eukaryotic

In budding yeast, actin appears to be another possible candidate for sensing oxidative stress

Transmembrane proteins act also as sensors of a number of stimuli. These proteins can function as detectors of environmental changes and also as a potential transmission molecules, to perhaps downstream signal transduction pathways. Rajavel et al. (1999) identified Mtl1 protein, a Mid2 homologue with a role in cell integrity signalling in vegetative growth. Mtl1 is an element of the cell integrity pathway, overexpression of *MTL1* supress defects in Rho1 function (Sekiya-Kawasaki et al., 2002). In the context of oxidative stress, the most likely candidate for being a transmembrane protein sensor is Mtl1 protein. Vilella et al. (2005) showed that Mtl1 is a cell surface sensor for oxidative stress. Mtl1 functions as oxidatives stress sensor since its function is necessary to survive in response to oxidants and to transmit the oxidative signal to the downstream elements of the cell integrity pathway (Vilella et al., 2005; Petkova et al., 2010a). Mtl1 is a transmembrane protein and localises to the cell periphery in all the stress conditions tested (submitted), supporting the hypothesis that Mtl1 acts as a cell-wall sensor, specifically as an oxidative stress sensor, given that mtl1 mutant cells are only sensitive to oxidative conditions and nutritional starvation (both conditions generate ROS production in the cells). Whether Mtl1

Signal transduction pathways function as transmissors of environmental estimuli to specific substrates. In budding yeast characteristic routes involved in this processes are CWI, TOR

Protein kinase C (PKC) is a protein with a role in oxidative stress response (for a review,

In human cells, PKC is involved in the protection against oxidative stress in the heart. The knowledge of this signalling is essential to the development of drugs to treat stroke and cardiac arrithmias (Barnett et al., 2007). Another important feature of the activation of the PKC signal transduction pathway is its role in aging, as reported by Battaini & Pascale

detects extracellular or/and intracellular oxidative stress is still unknown.

transducing the oxidative signal in budding yeast.

**2. Sensing oxidative stress**

(discussed in part 6.2 below)

**3. Transducing oxidative stress 3.1 CWI, TOR and RAS pathways**

and RAS.

Nitti et al., 2008).

cells.

(2005). Importantly, Pascale et al. (2007) exposed in a review how alterations of the PKC cascade may have implications in physiological and pathological brain aging, such as Alzheimer's disease.

The cell-wall integrity pathway in budding yeast involves a protein kinase (MAPKs) cascade which participates in sensing and transmitting several extracellular signals and stresses, including: cell-wall, osmotic, mating and nutritional stress (for a review see Heinisch et al., 1999; Levin, 2005), oxidative (Vilella et al., 2005) and pH (Serrano et al., 2006) stresses. The PKC1-MAPK pathway is integrated by several cell-wall proteins that are putative cellmembrane receptors of different stimuli, they are: the Wsc1-Wsc4 family, Mid2 and Mtl1. They transmit signals to Rom2 which activates the G protein Rho1 which in its turn activates the kinase Pkc1 (this protein has high degree of homology with other isoforms of PKC in eukaryotic cells). Pkc1 activates a MAP kinase module: Bck1 (which is the MAPKKK) phosphorylates the redundant MAPKKs Mkk1 and Mkk2 and together they both activate Slt2, the last kinase member of the pathway. There are two downstream events which correlate to Slt2 activation: transcriptional activity driven by Rlm1 and Swi6 phosphorylation (Heinish et al., 1999; Levin, 2005). Swi6 is one output of Slt2 activity. Swi6 is phosphorylated by Slt2 via the CWI (Sidorova et al., 1995; Madden et al., 1997). However when the external imput is oxidative stress, *SWI6* acts as a sensor through the oxidation of its Cys-404 to a sulfenic residue affecting the cell capability to arrest the cell cycle in G1 (Chiu et al., 2011).

The upper elements of the cell integrity pathway are involved in the organisation of the actin cytoskeleton under different conditions, including cell-wall and nutritional stresses (Helliwell et al., 1998; Delley & Hall, 1999; Torres et al., 2002), oxidative stress (Vilella et al., 2005) and pH (Motizuki et al., 2008) among others.

Exposure of *rom2* to oxidising agent results in diminished Slt2/Mpk1 phosphorylation (Vilella et al., 2005). Pkc1 is also required but the MAP kinase module, downstream of Pkc1, seems to be dispensable for this mechanism. Pkc1 overexpression confers cells with more resistance to oxidising agents. It has been demonstrated that upon oxidative stress Pkc1 translocates to the cell periphery. However, Pkc1 transmits the signal to Slt2/Mpk1 if cells have intact secretory machinery (Vilella et al., 2005)

The Pkc1 pathway is also related to the TOR pathway. Budding yeast have two different TOR genes: *TOR1* and *TOR2*, which share 67% sequence identity and are partly redundant in function (Helliwell et al., 1994). Loewith et al. (2002) purified and identified the components of two distinct TOR complexes, TORC1 and TORC2. TORC1 modulates translation initiation, inhibits protein turnover and represses the transcription of genes related to nutrient starvation. Early studies in *Saccharomyces cerevisiae* indicated that TOR has at least two functions: one regulated by the TORC1 complex which is sensitive to rapamycin and the other which is driven by the TORC2 complex and closely related to the organisation of the actin cytoskeleton and independent of rapamycin inhibition (Loewith et al., 2002; and for reviews Wullschleger et al., 2006; Inoki et al., 2005; Inoki & Guan, 2006). Tor2 functions in both complexes while Tor1 only participates in the TORC1 complex. The unique Tor2 function is related to Pkc1 and the organisation of the actin cytoskeleton (Helliwell et al., 1998). However, the rapamycininsensitive Tor2-unique function has not been described in other eukaryotic model systems (Crespo and Hall, 2002). Rapamycin also induces depolarisation of the actin cytoskeleton

Signalling Oxidative Stress in *Saccharomyces cerevisiae* 259

In recent years several studies there have been published that demonstrate the relationship that exists between the TOR and cAMP-PKA pathways. Schmelzle et al. (2004) suggested that the RAS/cAMP pathway could be a novel TOR effector branch. More recently, Chen & Powers (2006) have demonstrated that the TOR and PKA-cAMP pathways coregulate different biosynthetic pathways which control the expression of genes involved in fermentation and aerobic respiration. Both the TOR and cAMP-PKA pathways regulate the expression of genes needed to overcome the diauxic and stationary phases (Cardenas et al., 1999; Garreau et al., 2000), and in whose regulation Msn2/Msn4 transcriptional activity has been reported to be essential (Powers et al., 2006) (Figure 1). Stationary is a phase prone to

Fig. 1. Oxidative response signalling network in *Saccharomyces cerevisiae*. Oxidative stress negatively regulates TORC1 and RAS activities. TORC1 responds to nutrient availability and is inhibited by rapamycin. This complex when activated promotes the cytoplasm sequestration of specific transcription factors depicted in the figure. TORC1 and RAS/cAMP pathways activate Sfp1 transcription factor inducing ribosomal gene expression. Both TORC1 and RAS converge in *SCH9.* RAS signals to PKA kinase that inhibits both *YAK1* kinase and *MSN2/MSN4* by promoting *MSN2/MSN4* cytoplasm sequestration. *YAK1* in its turn activates *SKN7/HSF1* transcription factor that is required for the oxidative response. For this response TORC1 signals to RAS activation. TORC1 inhibits the CWI activity. However, TORC2 complex signals to cytoplasm elements of the CWI pathway to organise actin

cytoskeleton. Red circles containing letters depict phosphorylated amino acid residues. CWI, cell wall integrity; STRE, stress-responsive element; DAL, degradation of urea and allatoin;

NDP, nitrogen discrimination pathway; RTG, retrograde pathway.

through Sit4 and Tap42, two downstream elements of the TORC1 complex (Torres et al., 2002). TOR function controls a variety of cellular activities. In a global sense TOR inhibits transcription of stress-responsive elements, the nitrogen pathway, starvation-genes, and genes involved in the retrograde response. In this regulation there is a general mechanism: the sequestration of the transcription factors: Msn2/Msn2, Gln3 (Beck & Hall, 1999) and Rtg1/Rtg3 (Crespo et al., 2002; Dilova et al., 2004) in the cytoplasm.

Mitochondrial retrograde signalling (RTG) is a pathway of communication from mitochondria to the nucleus under normal and pathophysiological conditions. The best understood of this pathway is in the budding yeast *Saccharomyces cerevisiae*. It involves multiple factors that sense and transmit mitochondrial signals to induce changes in nuclear gene expression. These changes lead to a reconfiguration of metabolism to accommodate cells to defects in mitochondria that provoke abnormal ROS production. RTG is linked to aging, chronological life span, mitochondrial DNA maintenance, TOR signalling, and nutrient sensing pathways, and is conserved in other fungal species (Liu & Butow, 2006). Lst8 is an integral component of TOR kinase complex. It negatively regulates the RTG pathway at the level of Rtg2. The critical regulatory step of the RTG pathway is the dynamic interaction between Rtg2 and Mks1. The prototypical target of the RTG pathway is *CIT2* (encoding a peroxisomal isoform of citrate synthase, which enables cells to utilize two carbon compounds, such as acetate and ethanol, as sole carbon sources) under the control of Rtg3/Rtg1 heterodimer (Liao et al., 1991).

Tor function also regulates ribosomal protein expression in response to environmental conditions via PKA. This regulation involves the Forkhead factor, *FHL1*, and two cofactors: *IFH1* and *CRF1* (Martin et al., 2004). Tor controls ribosomal gene transcription by maintaining *CRF1* in the cytoplasm, then upon Tor inhibition, *CRF1* translocates to the nucleus and inhibits ribosomal expression, though it is probable that other target transcription factors and different regulatory mechanisms are also involved in this signalling.

The Msn2/Msn4 transcription factor binds and activates genes containing the stress response element (STRE: CCCCT) in response to a wide variety of stresses, including nutritional, osmotic, acidic and oxidative stress (Martínez-Pastor et al., 1996; Schmitt & McEntee, 1996; Beck & Hall, 1999; Hasan et al., 2002). The Ras-cAMP-PKA pathway also negatively regulates Msn2/Msn4 nuclear localisation (Martínez-Pastor et al., 1996; Boy-Marcotte et al., 1998; Görner et al., 1998).

RAS/cAMP pathway is activated when exposed of an optimal carbon source (Broach et al., 1990; Thevelein, 1994). In budding yeast there are two RAS proteins Ras1 and Ras2, both of them are GTPases that signal to the protein kinase PKA and cAMP production (Broach 1991). In optimal growth conditions RAS/cAMP pathway is activated and repress the function of the general stress transcription factor Msn2/Msn4 (Martínez-Pastor et al., 1996; Boy-Marcotte et al., 1998; Görner et al., 2002). On the contrary, nutrient starvation and oxidative stress conditions (Petkova et al., 2010a) are concomitant with RAS/cAMP repression.

*SCH9* encodes for a protein kinase involved in life span regulation. Sch9 activates respiratiory metabolism in quiescent phase thus provoking increase in ROS concentration, this effect induces a decrease in life span and increases DNA damage (Madia et al., 2009). Sch9 negatively regulates PKA activity (Zhang et al., 2011). To extend life span it is necessary to reduce TOR activity leading to a decrease of mitochondrial activity, but it is necessary to signal to Sch9 downregulation (Pan & Shadel, 2009).

through Sit4 and Tap42, two downstream elements of the TORC1 complex (Torres et al., 2002). TOR function controls a variety of cellular activities. In a global sense TOR inhibits transcription of stress-responsive elements, the nitrogen pathway, starvation-genes, and genes involved in the retrograde response. In this regulation there is a general mechanism: the sequestration of the transcription factors: Msn2/Msn2, Gln3 (Beck & Hall, 1999) and

Mitochondrial retrograde signalling (RTG) is a pathway of communication from mitochondria to the nucleus under normal and pathophysiological conditions. The best understood of this pathway is in the budding yeast *Saccharomyces cerevisiae*. It involves multiple factors that sense and transmit mitochondrial signals to induce changes in nuclear gene expression. These changes lead to a reconfiguration of metabolism to accommodate cells to defects in mitochondria that provoke abnormal ROS production. RTG is linked to aging, chronological life span, mitochondrial DNA maintenance, TOR signalling, and nutrient sensing pathways, and is conserved in other fungal species (Liu & Butow, 2006). Lst8 is an integral component of TOR kinase complex. It negatively regulates the RTG pathway at the level of Rtg2. The critical regulatory step of the RTG pathway is the dynamic interaction between Rtg2 and Mks1. The prototypical target of the RTG pathway is *CIT2* (encoding a peroxisomal isoform of citrate synthase, which enables cells to utilize two carbon compounds, such as acetate and ethanol, as sole carbon sources) under the control of

Tor function also regulates ribosomal protein expression in response to environmental conditions via PKA. This regulation involves the Forkhead factor, *FHL1*, and two cofactors: *IFH1* and *CRF1* (Martin et al., 2004). Tor controls ribosomal gene transcription by maintaining *CRF1* in the cytoplasm, then upon Tor inhibition, *CRF1* translocates to the nucleus and inhibits ribosomal expression, though it is probable that other target transcription factors and different

The Msn2/Msn4 transcription factor binds and activates genes containing the stress response element (STRE: CCCCT) in response to a wide variety of stresses, including nutritional, osmotic, acidic and oxidative stress (Martínez-Pastor et al., 1996; Schmitt & McEntee, 1996; Beck & Hall, 1999; Hasan et al., 2002). The Ras-cAMP-PKA pathway also negatively regulates Msn2/Msn4 nuclear localisation (Martínez-Pastor et al., 1996; Boy-

RAS/cAMP pathway is activated when exposed of an optimal carbon source (Broach et al., 1990; Thevelein, 1994). In budding yeast there are two RAS proteins Ras1 and Ras2, both of them are GTPases that signal to the protein kinase PKA and cAMP production (Broach 1991). In optimal growth conditions RAS/cAMP pathway is activated and repress the function of the general stress transcription factor Msn2/Msn4 (Martínez-Pastor et al., 1996; Boy-Marcotte et al., 1998; Görner et al., 2002). On the contrary, nutrient starvation and oxidative stress

*SCH9* encodes for a protein kinase involved in life span regulation. Sch9 activates respiratiory metabolism in quiescent phase thus provoking increase in ROS concentration, this effect induces a decrease in life span and increases DNA damage (Madia et al., 2009). Sch9 negatively regulates PKA activity (Zhang et al., 2011). To extend life span it is necessary to reduce TOR activity leading to a decrease of mitochondrial activity, but it is

conditions (Petkova et al., 2010a) are concomitant with RAS/cAMP repression.

necessary to signal to Sch9 downregulation (Pan & Shadel, 2009).

Rtg1/Rtg3 (Crespo et al., 2002; Dilova et al., 2004) in the cytoplasm.

Rtg3/Rtg1 heterodimer (Liao et al., 1991).

Marcotte et al., 1998; Görner et al., 1998).

regulatory mechanisms are also involved in this signalling.

In recent years several studies there have been published that demonstrate the relationship that exists between the TOR and cAMP-PKA pathways. Schmelzle et al. (2004) suggested that the RAS/cAMP pathway could be a novel TOR effector branch. More recently, Chen & Powers (2006) have demonstrated that the TOR and PKA-cAMP pathways coregulate different biosynthetic pathways which control the expression of genes involved in fermentation and aerobic respiration. Both the TOR and cAMP-PKA pathways regulate the expression of genes needed to overcome the diauxic and stationary phases (Cardenas et al., 1999; Garreau et al., 2000), and in whose regulation Msn2/Msn4 transcriptional activity has been reported to be essential (Powers et al., 2006) (Figure 1). Stationary is a phase prone to

Fig. 1. Oxidative response signalling network in *Saccharomyces cerevisiae*. Oxidative stress negatively regulates TORC1 and RAS activities. TORC1 responds to nutrient availability and is inhibited by rapamycin. This complex when activated promotes the cytoplasm sequestration of specific transcription factors depicted in the figure. TORC1 and RAS/cAMP pathways activate Sfp1 transcription factor inducing ribosomal gene expression. Both TORC1 and RAS converge in *SCH9.* RAS signals to PKA kinase that inhibits both *YAK1* kinase and *MSN2/MSN4* by promoting *MSN2/MSN4* cytoplasm sequestration. *YAK1* in its turn activates *SKN7/HSF1* transcription factor that is required for the oxidative response. For this response TORC1 signals to RAS activation. TORC1 inhibits the CWI activity. However, TORC2 complex signals to cytoplasm elements of the CWI pathway to organise actin cytoskeleton. Red circles containing letters depict phosphorylated amino acid residues. CWI, cell wall integrity; STRE, stress-responsive element; DAL, degradation of urea and allatoin; NDP, nitrogen discrimination pathway; RTG, retrograde pathway.

Signalling Oxidative Stress in *Saccharomyces cerevisiae* 261

redox state regulation of cysteines in Sty1 is needed for the hydrogen-peroxide induced increment of Aft1 mRNA levels. This leads to the transcriptional activation of specific genes and the subsequent augment in the oxidative stress survival (Degols & Russell, 1997).

Also in *S. pombe* Veal et al. (2004) showed the existence of an interesting mechanism by which a peroxiredoxin acts a redox sensor specifically required to activate Sty1. Sty1 is a MAPK responsive to all the stresses in *S. pombe*. High concentrations of hydrogen peroxide promote Sty1 activation by the peroxiredoxine, whereas at low concentrations of the oxidising agent this peroxiredoxin also regulates Pap1 nuclear accumulation (Veal et al., 2007). All these studies show that hydrogen peroxide is capable of oxidising specific kinases, and that certain molecules involved in oxidative repair, such as peroxiredoxins, are required

Grx3 and Grx4, two monothiol glutaredoxins of *S. cerevisiae*, regulate Aft1 nuclear localisation and negatively regulate its function. PICOT thioredoxin has a high degree of sequence homology with both Grx3 and Grx4 proteins of *S. cerevisiae*. The absence of both proteins makes the cells sensitive to hydrogen peroxide. In the absence of both Grx3 and Grx4 there is a constitutive oxidative stress induced, in part, by the deregulation of iron homeostasis (Pujol-Carrion et al., 2006). There are some other reports demonstrating similar regulations of other protein kinases. Therefore, there is important to isolate and characterise proteins involved in regulating the redox state of protein kinases in order to maintain an oxidatory equilibrium which allows correct cellular function. It will also be interesting to elucidate the degree of conservation of these proteins and their regulatory functions in

Actin is a target for oxidative stress. Actin cytoskeleton is of enormous relevance in *S cerevisiae*. It is responsible for all the morphogenetic processes, stress responses, organelles delivery etc. There are two potential cysteines more susceptible for being oxidised. Moreover, other regulatory molecules such as Grx3 and Grx4 regulate actin function in oxidative conditions (Pujol-Carrion et al., 2010). Grx3 and Grx4 are two putative glutaredoxins but there have not been described any enzymatic property related to that putative function. They are involved in the regulation of Aft1 a transcription factor required for the correct iron homeostasis (Pujol-Carrion et al., 2006; Ojeda et al., 2006). Grx4 is required for the maintenance of cable structure. Grx3 plays a redundant role in conjunction with Grx4. Both Grx3 and Grx4 have two theorethical domains, one Trx domain close to the C terminus and a Glutaredoxin domain. Both glutaredoxins through their Trx domains are required to repolarise the actin cytoskeleton in oxidative conditions and also for survival in oxidative stress conditions. Interestingly, Grx4 plays a more direct role in the defence against oxidative stress since Grx4 overproduction increases cell survival when cells are

Different transcription factors regulate the adaptive response to oxidative stress conditions: the general stress response is mediated by the Msn2/Msn4 transcription factor, whereas specific responses are mediated by Yap1, Skn7 and Hsf1. Msn2/Msn4 nuclear localisation and activity are regulated by both TORC1 and PKA (detailed formerly). For the induction of many antioxidant genes, Skn7 and Yap1 act cooperatively upon oxidative stress (Lee et al.,

in order to achieve a correct signalling response.

exposed to oxidants (Pujol-Carrion et al., 2010).

**4. Transcriptional regulation**

evolution.

generate ROS species given that yeast are committed to a respiratory metabolism.The cell integrity pathway is also required for viability in quiescence since Slt2 phosphorylation is necessary for cells to survive in stationary phase and upon rapamycine treatment (Krause & Gray, 2002, Torres et al., 2002). Moreover, rapamycine treatment induces depolarisation of the actin cytoskeleton in a cell-integrity pathway-dependent way (Torres et al., 2002) involving the participation of Wsc1 transmembrane proteins.

#### **3.2 Phospatases as regulatory elements**

Although oxidative stress is able to induce MAP kinase cascades, reversal of MAPK activation requires the transcriptional induction of specialised cysteine-based phosphatases that mediate MAPK dephosphorylation. In some occasions, oxidative stress inactivates phosphatases by thiol modification leading to abnormal MAPK upregulation. Recently, Fox et al. (2007) described a mechanism by which the stress inducible MAPK phosphatase Sdp1 acquired enhanced catalytic activity under oxidative conditions. Sdp1 uses an intramolecular disulphide bridge and an invariant histidine side chain to recognise a tyrosine-phosphorylated MAPK substrate in oxidant conditions. The disulphide bridge seems to be essential in order to reach a maximum activity. It is well known that yeast develop several strategies to recognize and adapt to oxidative stress. Reversible formation of disulphide bonds is a major way to regulate oxidative stress response in prokaryotic and eukaryotic microorganisms as well as higher eukaryotes (Lushchak, 2011). This must be one of the most efficient mechanisms in the oxidative context given that it is conserved in regulatory proteins, such as the phosphatase Sdp1. The reverse oxidation seems to be a rapid and effective activating mechanism to regulate stress responsive MAPK proteins.

#### **3.3 ROS detoxifier proteins acting as signalling regulatory molecules**

The elements that form part in signalling pathways that are involved in the response to oxidative stress are susceptible to be oxidised and consequently to be impaired in their functions. Therefore, molecules that repair oxidised proteins are likely to be associated to signalling proteins.

In mammals, there are repairing molecules that interplay in the oxidative stress mechanism. These are: thioredoxins, glutaredoxins, peroxiredoxins and other enzymes with an important role on tumourgenesis and oxidative damage resistance. In yeast some peroxiredoxins have been described to play a role in the regulation of the expression of specific stress genes (Ross et al., 2000).

Watson et al. (1999) have demonstrated the interaction between thioredoxins (proteins that reduce oxidised proteins) and PKC. Kahlos et al. (2003) also demonstrated the functional interaction between oxidoreductases and PKC (from endothelial pulmonary cells). In particular, these authors determined that these oxydoreductases were able to reduce disulphide bonds formed in the PKC protein as a consequence of nitric oxide treatment. PICOT protein, is a PKC interacting protein that negatively regulates its function (Witte et al., 2000).

Another form to activate and therefore also regulate MAPK upon oxidative stress has been described in *Schizosaccharomyces pombe* (Day & Veal 2010). These authors demonstrated that

generate ROS species given that yeast are committed to a respiratory metabolism.The cell integrity pathway is also required for viability in quiescence since Slt2 phosphorylation is necessary for cells to survive in stationary phase and upon rapamycine treatment (Krause & Gray, 2002, Torres et al., 2002). Moreover, rapamycine treatment induces depolarisation of the actin cytoskeleton in a cell-integrity pathway-dependent way (Torres et al., 2002)

Although oxidative stress is able to induce MAP kinase cascades, reversal of MAPK activation requires the transcriptional induction of specialised cysteine-based phosphatases that mediate MAPK dephosphorylation. In some occasions, oxidative stress inactivates phosphatases by thiol modification leading to abnormal MAPK upregulation. Recently, Fox et al. (2007) described a mechanism by which the stress inducible MAPK phosphatase Sdp1 acquired enhanced catalytic activity under oxidative conditions. Sdp1 uses an intramolecular disulphide bridge and an invariant histidine side chain to recognise a tyrosine-phosphorylated MAPK substrate in oxidant conditions. The disulphide bridge seems to be essential in order to reach a maximum activity. It is well known that yeast develop several strategies to recognize and adapt to oxidative stress. Reversible formation of disulphide bonds is a major way to regulate oxidative stress response in prokaryotic and eukaryotic microorganisms as well as higher eukaryotes (Lushchak, 2011). This must be one of the most efficient mechanisms in the oxidative context given that it is conserved in regulatory proteins, such as the phosphatase Sdp1. The reverse oxidation seems to be a rapid and effective activating mechanism to regulate stress responsive MAPK proteins.

involving the participation of Wsc1 transmembrane proteins.

**3.3 ROS detoxifier proteins acting as signalling regulatory molecules**

The elements that form part in signalling pathways that are involved in the response to oxidative stress are susceptible to be oxidised and consequently to be impaired in their functions. Therefore, molecules that repair oxidised proteins are likely to be associated to

In mammals, there are repairing molecules that interplay in the oxidative stress mechanism. These are: thioredoxins, glutaredoxins, peroxiredoxins and other enzymes with an important role on tumourgenesis and oxidative damage resistance. In yeast some peroxiredoxins have been described to play a role in the regulation of the expression of

Watson et al. (1999) have demonstrated the interaction between thioredoxins (proteins that reduce oxidised proteins) and PKC. Kahlos et al. (2003) also demonstrated the functional interaction between oxidoreductases and PKC (from endothelial pulmonary cells). In particular, these authors determined that these oxydoreductases were able to reduce disulphide bonds formed in the PKC protein as a consequence of nitric oxide treatment. PICOT protein, is a PKC interacting protein that negatively regulates its function (Witte et

Another form to activate and therefore also regulate MAPK upon oxidative stress has been described in *Schizosaccharomyces pombe* (Day & Veal 2010). These authors demonstrated that

**3.2 Phospatases as regulatory elements**

signalling proteins.

al., 2000).

specific stress genes (Ross et al., 2000).

redox state regulation of cysteines in Sty1 is needed for the hydrogen-peroxide induced increment of Aft1 mRNA levels. This leads to the transcriptional activation of specific genes and the subsequent augment in the oxidative stress survival (Degols & Russell, 1997).

Also in *S. pombe* Veal et al. (2004) showed the existence of an interesting mechanism by which a peroxiredoxin acts a redox sensor specifically required to activate Sty1. Sty1 is a MAPK responsive to all the stresses in *S. pombe*. High concentrations of hydrogen peroxide promote Sty1 activation by the peroxiredoxine, whereas at low concentrations of the oxidising agent this peroxiredoxin also regulates Pap1 nuclear accumulation (Veal et al., 2007). All these studies show that hydrogen peroxide is capable of oxidising specific kinases, and that certain molecules involved in oxidative repair, such as peroxiredoxins, are required in order to achieve a correct signalling response.

Grx3 and Grx4, two monothiol glutaredoxins of *S. cerevisiae*, regulate Aft1 nuclear localisation and negatively regulate its function. PICOT thioredoxin has a high degree of sequence homology with both Grx3 and Grx4 proteins of *S. cerevisiae*. The absence of both proteins makes the cells sensitive to hydrogen peroxide. In the absence of both Grx3 and Grx4 there is a constitutive oxidative stress induced, in part, by the deregulation of iron homeostasis (Pujol-Carrion et al., 2006). There are some other reports demonstrating similar regulations of other protein kinases. Therefore, there is important to isolate and characterise proteins involved in regulating the redox state of protein kinases in order to maintain an oxidatory equilibrium which allows correct cellular function. It will also be interesting to elucidate the degree of conservation of these proteins and their regulatory functions in evolution.

Actin is a target for oxidative stress. Actin cytoskeleton is of enormous relevance in *S cerevisiae*. It is responsible for all the morphogenetic processes, stress responses, organelles delivery etc. There are two potential cysteines more susceptible for being oxidised. Moreover, other regulatory molecules such as Grx3 and Grx4 regulate actin function in oxidative conditions (Pujol-Carrion et al., 2010). Grx3 and Grx4 are two putative glutaredoxins but there have not been described any enzymatic property related to that putative function. They are involved in the regulation of Aft1 a transcription factor required for the correct iron homeostasis (Pujol-Carrion et al., 2006; Ojeda et al., 2006). Grx4 is required for the maintenance of cable structure. Grx3 plays a redundant role in conjunction with Grx4. Both Grx3 and Grx4 have two theorethical domains, one Trx domain close to the C terminus and a Glutaredoxin domain. Both glutaredoxins through their Trx domains are required to repolarise the actin cytoskeleton in oxidative conditions and also for survival in oxidative stress conditions. Interestingly, Grx4 plays a more direct role in the defence against oxidative stress since Grx4 overproduction increases cell survival when cells are exposed to oxidants (Pujol-Carrion et al., 2010).

#### **4. Transcriptional regulation**

Different transcription factors regulate the adaptive response to oxidative stress conditions: the general stress response is mediated by the Msn2/Msn4 transcription factor, whereas specific responses are mediated by Yap1, Skn7 and Hsf1. Msn2/Msn4 nuclear localisation and activity are regulated by both TORC1 and PKA (detailed formerly). For the induction of many antioxidant genes, Skn7 and Yap1 act cooperatively upon oxidative stress (Lee et al.,

Signalling Oxidative Stress in *Saccharomyces cerevisiae* 263

that repair possible DNA lesions. UV response is characterised in yeast (Engelberg 1994). Oxidative stress is also known to provoke a wide number of genetic anomalies leading to genome instability cancer and inflammatory diseases. DNA damage checkpoints protect genome integrity (Latif et al., 2001). The DNA damage checkpoint is activated in response to

Since MAPKs are activated in response to several stresses, they are also involved in the UV and oxidative response. In particular JNKs and p38 (Engelberg, 1994; Rouse et al., 1994) are involved in these responses. Both kinases are conserved in yeast. Sty1 in *S pombe* and Hog1 in *S cerevisiae* have a high degree of similarity with JNK and p38 mammalian kinases and are known to be involved in the UV and oxidative stress responses (Haghnazari & Heyer,

Among the possible MAP kinases involved in the UV response, Slt2 and Hog 1 are required for survival whereas Mlp1 and Fus3 are dispensable, (Brian et al, 2004). These authors propose that Slt2 is specifically required for survival in front of UV, based on the observation that *slt2* mutant is not significantly sensitive to MMS (Methyl methanesulfonate) treatment. They demonstrate that addition of sorbitol suppressed the requirement for Slt2, what suggests that probably UV is provoking damage at the level of the cell surface. 8-MOP 8-furocoumarin 8-methoxypsolaren is a chemical agent used in the treatment of psoriasis and other skin diseases. The combination of 8-MOP plus UVA, causes DNA double strand breaks, being Slt2 function required for survival (Dardalhon et al., 2009). At present it is unknown which is the precise role of Slt2 and the other members of the CWI pathway in these responses (Bryan et al., 2004). One possible explanation would be that 8-MOP+UV induces increase in ROS steady-state levels. Another interpretation is that oxygen reacts with 8-MOP and components of the cell membrane leading to lipid peroxidation. This would be the starting point to signal to the CWI (Dall'Acqua & Martelli,

In a recent report, Bandyopadhyay et al. (2010) have used a new methodological approach called differential epistasis mapping. By doing so they have also found a genetic interaction between Slt2 and DNA repair genes. Moreover, MMS treatment induced Slt2 translocation

There exist cross-talk between DNA damage and oxidative stress since the UV response in mammals is believed to be induced at the cell membrane through the peroxidation of lipids. On the contrary, alkylating agents might induce the UV response through the oxidation of SH free groups and subsequent glutathione pool depletion (Devary et al., 1993). In addition, the UV transcription response mediated by AP-1 in mammals induces the expression of genes required for the response to oxidative damage (van Dam et al., 1995). In conclusion,

In mammals oxidative stress activates all the known MAPK. ERK activation promotes cell survival whereas JNK and p38 suppress apoptosis and induce cellular responses to stress

In mammals a crosstalk between DNA damage checkpoint genes and certain MAPK has been well described (Shafman et al., 1995; Bulavin et al., 2001). In an attempt to describe an equivalent crosstalk in yeast Haghnazari & Heyer (2004) analysed the possible relationship

to the nucleus and the transcriptional activation of ribonucleotide reductase genes.

there exists some evidence that UV response helps to combat oxidative stress.

oxidative stress (Leroy et al., 2001).

2004; Alao & Sunnerhagen, 2008).

1991; Zarebska, 2000; Dardalhon et al., 2009)

(Runchel et al., 2011).

1999; Brombacher et al., 2006; He et al., 2005). The contribution of Skn7 to the oxidative stress response does not occur through any of the cysteines of the protein. In addition, SKN7 phosphorylation does not seem to be required in the oxidative stress response (Morgan et al., 1997). It has been proposed a model in which Skn7, when located in the nucleus, cooperates with oxisided Yap1 . The association of Yap1 with Skn7 is a prerequisite for Skn7 phosphorylation and the activation of oxidative stress response genes (He et al., 2009). Skn7 interacts also with Hsf1 and both cooperate to induce heat shock genes specifically in response to oxidative stress (Raitt et al., 2000). Hsf1, like Msn2/Msn4, is negatively regulated by PKA via Yak1 kinase. Sfp1 is a transcriptional factor that induces ribosomal gene expression when located in the nucleus. It is positively regulated by either TOR or RAS activities. In response to oxidative stress and DNA damage Sfp1 translocates to the citoplasm with the consequent adaptive downregulation of ribosomal gene expression (Marion et al., 2004) (Figure 1).

In the pathogen yeast *Candida albicans* there has been characterised a gene *CAP1,* which is homologue to the transcription factor Yap1 and has a role in oxidative stress resistance (Alarco et al., 1999). In *C. albicans* Hog1 pathway is also required for a correct oxidative stress response though through a different pathway than that used by *CAP1* gene (Alonso-Monge et al., 2003).

#### **5. Signal crosstalk events in the oxidative transduction**

Mtl1 is a transmembrane cell-wall protein required for cell survival upon oxidative conditions (Vilella et al., 2005). Mtl1 belongs to the CWI pathway and its essential function is to inhibit Tor1 and Ras2 function in conditions of oxidative stress and nutrient depletion. This signal is transduced through Rom2 and Rho1 (both elements of the CWI pathway), however, the rest of the downstream components of the mentioned pathway are dispensable for this essential function (Petkova et al., 2010a) (Figure 2). Consequently, upon oxidative stress both Tor1 and Ras2 functions must be transiently repressed. Downstream outputs of this signal are transcriptional induction mediated by Msn2/Msn4 and ribosomal gene repression, probably due in part to the regulation of Sfp1. In this study the authors propose two possible models: a) the oxidative signal from Mtl1 flows to Tor1 and Ras2 independently and converge in a common pathway; b) this signal flows to Tor1 and then to inhibit Ras2 for the regulation of Msn2/Msn4 activity and the downregulation of ribosomal gene expression. There is another crosstalk involving CWI elements, TOR and RAS, In this case the signal flows from RAS2 and TOR1 inactivation to induce the phosphorylation of Slt2 after treatment with hydrogen hydroxide and upon glucose starvation. Interestingly this backwards signal occurs in the absence of Mtl1 protein (Petkova et al., 2010b).

#### **6. Cellular functions affected by oxidative stress**

#### **6.1 Cell cycle oxidation and DNA damage**

There exist a wide variety of DNA damaging agents. They provoke different DNA lesions (reviewed in Sage & Harrison, 2011). UV light is a well known DNA damaging agent inducing the formation of thymidin dimmers. DNA damaging agents activate a number of checkpoint genes (Lowndes & Murguia, 2000) in order to transiently block cell cycle progression and simultaneously to activate (transcriptional and/or postraductional) genes

1999; Brombacher et al., 2006; He et al., 2005). The contribution of Skn7 to the oxidative stress response does not occur through any of the cysteines of the protein. In addition, SKN7 phosphorylation does not seem to be required in the oxidative stress response (Morgan et al., 1997). It has been proposed a model in which Skn7, when located in the nucleus, cooperates with oxisided Yap1 . The association of Yap1 with Skn7 is a prerequisite for Skn7 phosphorylation and the activation of oxidative stress response genes (He et al., 2009). Skn7 interacts also with Hsf1 and both cooperate to induce heat shock genes specifically in response to oxidative stress (Raitt et al., 2000). Hsf1, like Msn2/Msn4, is negatively regulated by PKA via Yak1 kinase. Sfp1 is a transcriptional factor that induces ribosomal gene expression when located in the nucleus. It is positively regulated by either TOR or RAS activities. In response to oxidative stress and DNA damage Sfp1 translocates to the citoplasm with the consequent adaptive downregulation of ribosomal gene expression

In the pathogen yeast *Candida albicans* there has been characterised a gene *CAP1,* which is homologue to the transcription factor Yap1 and has a role in oxidative stress resistance (Alarco et al., 1999). In *C. albicans* Hog1 pathway is also required for a correct oxidative stress response though through a different pathway than that used by *CAP1* gene (Alonso-

Mtl1 is a transmembrane cell-wall protein required for cell survival upon oxidative conditions (Vilella et al., 2005). Mtl1 belongs to the CWI pathway and its essential function is to inhibit Tor1 and Ras2 function in conditions of oxidative stress and nutrient depletion. This signal is transduced through Rom2 and Rho1 (both elements of the CWI pathway), however, the rest of the downstream components of the mentioned pathway are dispensable for this essential function (Petkova et al., 2010a) (Figure 2). Consequently, upon oxidative stress both Tor1 and Ras2 functions must be transiently repressed. Downstream outputs of this signal are transcriptional induction mediated by Msn2/Msn4 and ribosomal gene repression, probably due in part to the regulation of Sfp1. In this study the authors propose two possible models: a) the oxidative signal from Mtl1 flows to Tor1 and Ras2 independently and converge in a common pathway; b) this signal flows to Tor1 and then to inhibit Ras2 for the regulation of Msn2/Msn4 activity and the downregulation of ribosomal gene expression. There is another crosstalk involving CWI elements, TOR and RAS, In this case the signal flows from RAS2 and TOR1 inactivation to induce the phosphorylation of Slt2 after treatment with hydrogen hydroxide and upon glucose starvation. Interestingly

this backwards signal occurs in the absence of Mtl1 protein (Petkova et al., 2010b).

There exist a wide variety of DNA damaging agents. They provoke different DNA lesions (reviewed in Sage & Harrison, 2011). UV light is a well known DNA damaging agent inducing the formation of thymidin dimmers. DNA damaging agents activate a number of checkpoint genes (Lowndes & Murguia, 2000) in order to transiently block cell cycle progression and simultaneously to activate (transcriptional and/or postraductional) genes

**6. Cellular functions affected by oxidative stress**

**6.1 Cell cycle oxidation and DNA damage**

**5. Signal crosstalk events in the oxidative transduction**

(Marion et al., 2004) (Figure 1).

Monge et al., 2003).

that repair possible DNA lesions. UV response is characterised in yeast (Engelberg 1994). Oxidative stress is also known to provoke a wide number of genetic anomalies leading to genome instability cancer and inflammatory diseases. DNA damage checkpoints protect genome integrity (Latif et al., 2001). The DNA damage checkpoint is activated in response to oxidative stress (Leroy et al., 2001).

Since MAPKs are activated in response to several stresses, they are also involved in the UV and oxidative response. In particular JNKs and p38 (Engelberg, 1994; Rouse et al., 1994) are involved in these responses. Both kinases are conserved in yeast. Sty1 in *S pombe* and Hog1 in *S cerevisiae* have a high degree of similarity with JNK and p38 mammalian kinases and are known to be involved in the UV and oxidative stress responses (Haghnazari & Heyer, 2004; Alao & Sunnerhagen, 2008).

Among the possible MAP kinases involved in the UV response, Slt2 and Hog 1 are required for survival whereas Mlp1 and Fus3 are dispensable, (Brian et al, 2004). These authors propose that Slt2 is specifically required for survival in front of UV, based on the observation that *slt2* mutant is not significantly sensitive to MMS (Methyl methanesulfonate) treatment. They demonstrate that addition of sorbitol suppressed the requirement for Slt2, what suggests that probably UV is provoking damage at the level of the cell surface. 8-MOP 8-furocoumarin 8-methoxypsolaren is a chemical agent used in the treatment of psoriasis and other skin diseases. The combination of 8-MOP plus UVA, causes DNA double strand breaks, being Slt2 function required for survival (Dardalhon et al., 2009). At present it is unknown which is the precise role of Slt2 and the other members of the CWI pathway in these responses (Bryan et al., 2004). One possible explanation would be that 8-MOP+UV induces increase in ROS steady-state levels. Another interpretation is that oxygen reacts with 8-MOP and components of the cell membrane leading to lipid peroxidation. This would be the starting point to signal to the CWI (Dall'Acqua & Martelli, 1991; Zarebska, 2000; Dardalhon et al., 2009)

In a recent report, Bandyopadhyay et al. (2010) have used a new methodological approach called differential epistasis mapping. By doing so they have also found a genetic interaction between Slt2 and DNA repair genes. Moreover, MMS treatment induced Slt2 translocation to the nucleus and the transcriptional activation of ribonucleotide reductase genes.

There exist cross-talk between DNA damage and oxidative stress since the UV response in mammals is believed to be induced at the cell membrane through the peroxidation of lipids. On the contrary, alkylating agents might induce the UV response through the oxidation of SH free groups and subsequent glutathione pool depletion (Devary et al., 1993). In addition, the UV transcription response mediated by AP-1 in mammals induces the expression of genes required for the response to oxidative damage (van Dam et al., 1995). In conclusion, there exists some evidence that UV response helps to combat oxidative stress.

In mammals oxidative stress activates all the known MAPK. ERK activation promotes cell survival whereas JNK and p38 suppress apoptosis and induce cellular responses to stress (Runchel et al., 2011).

In mammals a crosstalk between DNA damage checkpoint genes and certain MAPK has been well described (Shafman et al., 1995; Bulavin et al., 2001). In an attempt to describe an equivalent crosstalk in yeast Haghnazari & Heyer (2004) analysed the possible relationship

Signalling Oxidative Stress in *Saccharomyces cerevisiae* 265

complex mediates tolerance to DNA damage and the absence of *tor1* confers cells with more sensitivity to hydroxiurea and MMS, both of them DNA damaging agents. Upon the cell cycle arrest characteristic of the DNA damage checkpoint, *tor1* is required to dephosphorylate and

The actin molecule is sensitive to oxidative stress (Dalle-Donne et al., 2001). Upon oxidative conditions the actin molecule can be oxidised and a disulphide bond can be formed between

Cysteine is one amino acid prone to be oxidised when ROS levels increase. In actin Cysteine 374 is the most susceptible to oxidation, according to Takashi (1979). Investigations with erythrocytes from patients suffering sickle cell anemina, demonstrated that these cells posses actin molecules oxidised and forming intramolecular disulphide bonds between C284-C373 (Shartava et al., 1995, 1997; Bencsath et al., 1996). This modification correlates with a decrease in actin polymerisation rates. In yeast these cysteines are homologous to C285 and C374. Recently, Farah et al. (2011) in an elegant study, described the formation of oxidation induced actin bodies (OABs) upon oxidative stress by using budding yeast as a cellular model. These bodies resemble big patches and contain proteins and oxidised actin with intramolecular disulphide bonds between C285 and C374. These authors demonstrated that the formation of C285-374 responds to a protective mechanism against actin oxidation. OABs come from cortical patches. C285-374 are required for the adaptive response and recovery in front to oxidative damage. If actin again is a sensor for oxidative stress, it remains to be elucidated which are all the signalling outputs that govern the cellular responses to oxidative stress once actin oxidation starts the signalling process in the cells. Actin oxidation accelerates cell death in yeast (Dalle-Donne et al., 2001). Studies in eukaryotic model *S. cerevisiae* have allowed the identification of the oxidoreductase *OYE2*  (Old Yellow Enzyme 2) that is important to protect actin molecules from being oxidised in Cys285 and Cys374 (Haarer et al., 2004). A deletion in the *OYE2* gene induces an increase in ROS steady-state levels and makes cells more sensitive to oxidation (Farah et al., 2007; Odat et al., 2007). Although Oye enzymes are placed in the signalling network that governs ROS, actin cytoskeleton and survival, it remains unknown at the molecular level which is the connection between any specific signal transduction pathway and Oye2 in response to the

Vilella et al. (2005) describe a role for CWI pathway in connecting oxidative stress stimulus with the actin cytoskeleton. This study reveals that oxidative stress depolarises the actin cytoskeleton. None of the CWI elements is required to mediate this depolarisation, however Pkc1 is essential in order to restore the organisation of the actin cytoskeleton in oxidative

In a recent work (Pujol-Carrion et al., 2010) it has been demonstrated that actin polymerisation is a target of hydrogen peroxide. The authors develop an assay based on total protein extracts obtained from different strains of *S. cerevisiae*. These protein extracts are used as polymerisation seeds to study actin assembly. Actin filaments are detected by means of the technique of fluorescence recovery after photobleaching (FRAP). The rationale of this assay is that the association of small amounts of protein extracts with actin monomers

conditions, concomitantly with an increase in cell viability (Vilella et al., 2005).

reactivate Cdc2, what elicits the resumption of mitosis (Schonbrun et al., 2009).

**6.2 Actin cytoskeleton organisation**

redox signal.

cysteins 284 and 373 (Dalle-Donne et al., 2001, 2003).