Oxidative Stress Studies in Yeast with a Frataxin Mutant: A Proteomics

Dec 3, 2009 - Duby et al., on the other hand, investigated the frataxin role in iron−sulfur .... (Rev A.03.02.060b) and peak lists were created with...
0 downloads 0 Views 3MB Size
Download PDF
Oxidative Stress Studies in Yeast with a Frataxin Mutant: A Proteomics Perspective Jin-Hee Kim,† Miroslav Sedlak,‡,§ Qiang Gao,† Catherine P. Riley,| Fred E. Regnier,†,| and Jiri Adamec*,| Department of Chemistry, Laboratory of Renewable Resources Engineering, Department of Agricultural and Biological Engineering, and Bindley Bioscience Center at Discovery Park, Purdue University, West Lafayette, Idiana 47907 Received June 19, 2009

Cellular response of wild-type Saccharomyces cerevisiae and the ∆yfh1 mutant to oxidative stress (OS) was examined by stressing cells through the addition of H2O2 to the growth medium. The ∆yfh1 mutant is unusual in that it accumulates iron in it is mitochondria. Wild-type growth was immediately arrested and recovered in 2 h following H2O2 treatment. No change in viability was observed. Growth of the mutant, on the other hand, was similar to wild-type yeast for 4 h but then rapidly declined, eventually reaching zero. Levels of carbonyl groups and reactive oxygen species (ROS) reached their maximum at 3 h following exposure. The impact of OS on protein function was also evaluated by proteomic techniques targeting protein carbonylation. Oxidized proteins were selected by affinity chromatography, and following trypsin digestion, peptide fragments were identified by RPLC-MS/MS. A total of 53 proteins were identified in both wild-type and mutant cells, respectively. Keywords: Oxidative stress • Friedreich ataxia • Frataxin • Protein damage • Cell viability

Introduction Although biological markers of oxidative stress have been described in disorders ranging from Alzheimer’s disease,1 Parkinson disease,2 and Friedreich ataxia3 to cancer,4 and diabetes mellitus,5 the molecular mechanism of stress propagation and stage(s) at which the phenomenon is most relevant remain unclear. Reactive Oxygen Species (ROS) including superoxide anions (O2•-) and hydroxyl radicals (OH · ) are very damaging to cells.6 Formation of superoxide anions in mitochondria of eukaryotes is primarily due to uncoupling of oxidative phosphorylation during oxidative breakdown of glucose and fatty acids to produce NADH and FADH2. Metabolic energy is derived from the reoxidation of NADH and FADH2 by O2 to produce ATP in the electron transport chain located in the inner mitochondrial membrane. ROS are generated as biproducts during this process through imperfections in electron transport that lead to “electron leakage” and yield the superoxide anion (O2•-). Superoxide anions are enzymatically detoxified by mitochondrial superoxide dismutase (MnSOD)7 and glutathione peroxidase (GPX)8 to give hydrogen peroxide (H2O2) and H2O. Hydroxyl radicals are generated from the decomposition of * To whom correspondence should be addressed: Jiri Adamec, Bindley Bioscience Center at Purdue University, 1203 W. State Street, West Lafayette, IN 47907, USA. Phone: 765-496-6148. Fax: 765-496-1518. E-mail: jadamec@ purdue.edu. † Department of Chemistry, Purdue University. ‡ Laboratory of Renewable Resources Engineering, Purdue University. § Department of Agricultural and Biological Engineering, Purdue University. | Bindley Bioscience Center at Discovery Park, Purdue University.

730 Journal of Proteome Research 2010, 9, 730–736 Published on Web 12/03/2009

H2O2 through transition metal catalysis as described by Fenton9 and later by Weiss.10 Friedreich ataxia (FA) is a rare but dramatic disease with an estimated prevalence of 1 per 50 000. FA is an autosomal recessive disease caused by a 6-30% reduction in the concentration of the mitochondrial protein frataxin (FXN). Early studies in Saccharomyces cerevisiae implicated the yeast homologue of frataxin (Yfh1p) in iron homeostasis and respiratory function.11 Moreover, Yfh1p deficiency results in alterations in protein function associated with oxidative damage. Both in vitro and in vivo studies demonstrated that frataxin is required ¨hlenhoff showed that Yfh1p depletion for heme synthesis.12 Mu caused a strong reduction in the assembly of mitochondrial iron-sulfur proteins (ISP),13 leading the authors to propose a specific role for frataxin in the biosynthesis of these proteins. This possibility is supported by the fact that frataxin was recently found to exist in complex with Isu1/Nfs1 proteins that are essential for iron sulfur cluster synthesis.14 An emerging picture is that frataxin functions as an iron maintenance protein by sequestering iron in ferrous form that can be utilized by ferrochelatase for heme synthesis, or by Isu1 for iron cluster synthesis. Duby et al., on the other hand, investigated the frataxin role in iron-sulfur assembly using ∆yfh1 strain with a stable mitochondrial genome.15 The authors compared the incorporation of Fe-S clusters into yeast mitochondrial ferredoxin under different physiological conditions and concluded that frataxin is not essential for ISP assembly but improves efficiency of the process. Although the precise function of frataxin is still unclear, cells with low levels of these proteins are well-known to accumulate iron in mitochondria,16 have 10.1021/pr900538e

 XXXX American Chemical Society

Oxidative Stress Studies in Yeast with a Frataxin Mutant decreased mitochondrial DNA levels and are sensitive to OS.11,17 This yeast model provides a unique opportunity to target OS in mitochondria and study the molecular mechanism(s) of this phenomenon in a biological system. In this study, we have evaluated differences in response of wild-type S. cerevisiae and the iron accumulating ∆yfh1 mutant strain to H2O2 induced OS. Beyond assessing traditional growth, viability and ROS concentration, recently developed proteomics techniques were used to identify oxidatively damaged proteins. Although carbonylated proteins related to cellular metabolism were found in both the wild-type and mutant strain, unique carbonylated proteins were identified in the mutant strain that are involved in protein synthesis, transcription, and frataxin function. This suggests a different mechanism of OS propagation in the mutant strain. The “discovery” approach using yeast as a model provides an important systematic first step to identifying potential protein candidates responsible for oxidation-related changes in cellular function and sets up the basis for validation of these changes in mammalian models and FA patients. The information is expected to establish a correlation between oxidative stress and changes in protein content. Ultimately, this information may lead to identify drug targets and to the development of therapeutic strategies aimed at preventing protein associated damage and the degenerative effects of oxidative stress.

Materials and Methods Strains and Growing Conditions. S. cerevisiae 10000 L (MATa ura3-52 leu2∆1 trp1∆63 HIS3 GAL2) and 10546A (∆yfh1 FY MATa ura3-52 HIS3 leu2∆1 LYS2 trp1∆63 YDL120w(4,531):: kanMX4) (EUROSCARF, Germany) were used in this study. An overnight yeast culture was inoculated into 100 mL of YEPD media (1% yeast extract, 2% peptone, 2% glucose) at OD ∼50 KU (Klett Unit) and cells were grown at 30 °C. To induce oxidative stress, cultures at OD ∼160 KU were treated with 20 or 100 mM H2O2 (final concentration). Viability Assay. Cell viability was determined using a red acid dye Phloxine B (Sigma-Aldrich).18 Aliquots of yeast culture were directly stained with Phloxine B solution (20 µg/mL)19 and analyzed in a hemacytometer (Hausser, PA) for the presence of red cells using light microscope (Bausch & Lomb, ×600 magnification). In each assay, 1000-1500 cells were inspected and viability was expressed as a percentage of viable, unstained cells. Detection of ROS Radicals. Yeast cells from a 10 mL of culture were harvested, washed with PBS buffer (pH 7.4) and resuspended in the same buffer containing 25 µM 2′7′-dichlorodihydroflurescein-diacetate (H2DCFDA, 10 mM stock solution in DMSO, Molecular Probes). Suspension was incubated at 30 °C for 60 min and directly observed using Eclipse TE 2000-U (Nikon, Japan) or Eclipse E600FN (Nikon, Japan) equipped with CoolSNAP-Pro cf: An Integrated Solution (Media Cybernetics, MD) under 485/22 nm (excitation wavelength/FWHM bandwidth). The number of fluorescent cells was counted in the field of view and ROS level was calculated as a percentage of fluorescent cells compared to the total number of cells counted under phase contrast. Determination of Carbonylated Protein Concentrations. Twenty milliliters aliquots of the culture were centrifuged at 3000g for 5 min at 4 °C, washed twice with cold water, and the pellet was resuspended in an equal volume to weight of lysis buffer (3.8 mM NaH2PO4 · H2O, 49.4 mM Na2HPO4 · H2O, 48.4 mM NaCl, 5 mM KCl, 20% glycerol, 1% 2-mercaptoethanol,

research articles 0.3% IGEPAL CA-630, complete mini-protease inhibitor) and glass beads (425-600 µm, acid-washed), respectively. Cells were disintegrated by vortexing and the homogenate was centrifuged at 14 000g for 10 min. Protein concentration in the supernatant was determined by the Bradford protein assay (Bio-Rad, CA) and adjusted to 2 mg/mL with the lysis buffer. The sample (500 µL) was then mixed with 500 µL of 2,4-dinitrophenylhydrazine (2,4-DNPH; 10 mM in 2 M HCl) and concentrations of protein carbonyls were determined as described by Stadtman and colleagues.20 Isolation and Proteolysis of Carbonylated Proteins. Carbonylated proteins were isolated from 20 mL of culture by the method described above except EZ-Link biotinhydrazide (Thermo Fisher Scientific, Inc.) was added to the lysis buffer (5 mM final concentration). After cell debris removal by centrifugation, equal volumes of 30 mM sodium cyanoborohydride were mixed with supernatant and the mixture was incubated for 40 min on ice. The sample was then dialyzed against PBS buffer (pH 7.2) to remove excess biotin hydrazide and detergent and protein concentration was measured by Bio-Rad protein assay reagents. Dialyzed biotin hydrazide-labeled samples were loaded onto an avidin column packed with Ultralinked immobilized monomeric avidin (4.6 mm × 100 mm; stainless steel column, 100 psi packing pressure) and carbonylated proteins were collected according to the manufacturer’s guidelines. Isolated proteins were denaturated and reduced by adding urea (6 M final concentration) and TCEP (5 mM final concentration), respectively. The mixture was incubated for 1 h at 37 °C, diluted 6 times with ammonium bicarbonate buffer at pH 8.0 (ABC buffer) and digested with Glu-C (at 2:1 protein/Glu-C ratio) or trypsin (at 2:1 protein/trypsin ratio) overnight at 25 or 37 °C, respectively. The digested samples were acidified (pH less than 4) with 10% trifluoroacetic acid (TFA) and peptides were further purified using Reversed Phase Liquid Chromatography (RPLC) with a C-18 R1/10 column (4.6 × 100 mm; Applied Biosystems). Following injection, the column was washed with 12 mL of solvent A (3% acetonitrile; 0.1% TFA) and the peptides were eluted with 3 mL of solvent B (100% acetonitrile; 0.1% TFA). Collected fractions were dried under vacuum and the purified peptides were resuspended in 10-15 µL of 0.01% TFA. Identification of Carbonylated Proteins. Peptide separation and identification was carried out using an Agilent 1100 Series LC system equipped with an HPLC Chip interface (Agilent Technologies) and XCT Ultra ion trap mass spectrometer (Agilent Technologies). The system was controlled by ChemStation software (Agilent Technologies). Subsequent to injection, peptides were concentrated by on-chip 300SB-C18 enrichment and washed with solvent A (0.01% TFA in water) at a flow rate of 3 µL/min. After a 5 min wash segment, the enrichment column was switched into the nanoflow path of the on-chip C-18 reversed phase analytical column (150 mm × 75 µm; ZORBAX 300SB-C18; Agilent Technologies) coupled to the electrospray ion (ESI) source of the ion trap mass spectrometer. The column was eluted in a 55 min linear gradient from 5% to 35% solvent B (100% acetonitrile, 0.01% TFA) at a flow rate of 300 nL/min, followed by a 10 min gradient from 35% to 100% solvent B. The column was re-equilibrated with an isocratic flow (5% solvent B) at 300 nL/min. MS/MS data were acquired in the positive ion mode under the following conditions: a capillary voltage, 1850 V; end plate offset, 500 V; dry temperature, 300 °C; and dry gas flow, 6 L/min. Full scan MS data were collected in the range of Journal of Proteome Research • Vol. 9, No. 2, 2010 731

research articles

Kim et al.

Figure 1. Yeast cell growth curves after treatment of hydrogen peroxide. (A) Wild-type strain, (B) ∆yfh1 mutant. Black line graph shows the cell profile without induction. Red dotted line plot represent treatment of 20 mM of H2O2.

350-2000 m/z with 0.15 s maximum accumulation time and scan speed of 8100 m/z per second. Automated MS/MS spectra were acquired during the run in the data-dependent acquisition mode with the selection of the three most abundant precursor ions (0.5 min active exclusion; 2+ ions preferred). The MS/MS spectra were analyzed with the Spectrum Mill (Agilent Technologies) data analysis system (Rev A.03.02.060b) and peak lists were created with the Spectrum Mill Data Extractor program with the following attributes: scans with the same precursor (1.4 m/z were merged within a time frame of (15 s for ion trap data. Precursor ions needed to have a minimum signal-to-noise value of 25. Charges up to a maximum of +4 were assigned to the precursor ion, and the 12C peak was determined by Data Extractor. A search was performed against the NCBI database with a mass tolerance of (2.5 Da for the precursor ions, a tolerance of 0.7 Da for the fragment ions, and a minimum matched peak intensity of 50%. Up to two missed cleavages were allowed and only proteins with at least score of 8 and % score peak intensity (SPI)21,22 of 60 were considered. The identified proteins were further investigated according to their functions and localizations using the MIPS Saccharomyces cerevisiae genome database (http:// mips.gsf.de/genre/proj/yeast/).

Results and Discussion Effect of Oxidative Stress on Growth. To establish a growth pattern of both yeast strains under normal, not-stressed conditions YEPD media was individually inoculated with the wild-type and ∆yfh1 strains and cell density was measured every hour for 20 h. Both strains followed typical exponential growth curves and reached the maximum optical density of ∼500 and ∼350 KU, respectively (Figure 1). Addition of H2O2 to a final concentration of 20 mM using 30% solution (w/w) in H2O (Sigma-Aldrich) into cultures of the wild-type strain caused growth to cease. After a few hours, however, the cells resumed normal growth (Figure 1A). This recovery after a short period of arrested growth reflects relatively a low concentration of pro-oxidants and is in general agreement with previously reported studies.23-25 The exposure of the ∆yfh1 strain to 20 mM H2O2, on the other hand, caused unexpected changes in cell growth. Initial 732

Journal of Proteome Research • Vol. 9, No. 2, 2010

growth was not arrested as observed in the wild-type. Instead, growth of the ∆yfh1 strain was unchanged compared to unexposed cells for the first hour followed by short arrest for 1 h and accelerated growth similar to unexposed cells for an additional 2 h. Four hours after exposure, there was a rapid decline in cell density, most likely due to cell death and lysis (Figure 1B). One explanation for the unusual behavior of the ∆yfh1 strain at the early stages of H2O2 exposure is that these cells are already adapted to OS. Generally, adaptation to OS includes up-regulation of antioxidant defense enzymes and other stress response proteins in an effort to restore free radical/antioxidant balance. As the ∆yfh1 cells are experiencing higher levels of OS prior to H2O2 addition, the stimulation of stress response system is expected26,27 and the extra addition of pro-oxidant should not lead to the immediate growth arrest. At the 4 h following exposure, ∆yfh1 strain, however, reaches a “no return” point at which the cells cannot defend themselves and die. In wild-type, on the other hand, defense proteins are present in low levels before H2O2 addition and time is required for their synthesis after stress initiation. This would explain the temporary growth arrest after H2O2 addition and quick recovery at later stages of exposure. Yeast Viability and ROS Radical formation. Cell viability and ROS formation were measured in the ∆yfh1 strain after exposure to H2O2. In the control experiments (no added H2O2), both wild-type and the ∆yfh1 strain retained viability throughout the duration of the experiment (data not shown). Similar results were found for wild-type exposed to H2O2 (Figure 2B). Viability of the ∆yfh1 cells, on the other hand, showed a rapid decrease that corresponded to the decline in cell culture OD. (Figure 2C). In fact, the critical point was at 3 h; cell viability quickly decreased at 4 h. By the end of the experiment, the ∆yfh1 cultures consisted almost entirely of nonviable cells (Figure 2C). The formation ROS was determined before H2O2 addition and at 1 and 3 h after exposure (Figure 3A). The ROS level in wild-type cells (less than 1% cells) was insignificant, while the ∆yfh1 strain culture exhibited a significantly higher percentage of cells with ROS radicals. This increase in the cell population containing ROS is in agreement with other studies and is explained by iron mismanagement inside mitochondria due

Oxidative Stress Studies in Yeast with a Frataxin Mutant

research articles

Figure 2. Live and dead cell counting under oxidative stress. (A) A representative live and dead image at 3 h after treatment of H2O2 in ∆yfh1 mutant strains. Red arrow indicates nonviable cells which is shown as dark in this photograph (pinkish in visible light microscopy (×900 magnification, with oil immersion). The white bar represents 50 µm. (B and C) Yeast viability in wild-type and ∆yfh1 mutant cells, respectively. Line and dotted plots show yeast cell growth curve without (left) or with (right) an exposure to H2O2, respectively. Bar graph shows yeast live and dead counting.

to deletion of the YFH1 protein.26 The maximum concentration of ROS (18%) in wild-type was observed at 1 h following H2O2 addition. This time corresponds to the middle of growth arrest addition and suggests that cells are not fully adapted yet. Normal growth resumed in 3 h following exposure at which point ROS concentration returned close to the normal level (2.4%) due to ROS discharge and/or dilution. Unlike wild-type cells, the ∆yfh1 strain showed increasing levels in ROS concentration and reached 100% in 3 h following exposure (Figure 3A). This massive accumulation of ROS suggests the ∆yfh1 strain has difficulty removing/discharging ROS and the accumulation of ROS most likely contributes to subsequent cell death as seen in Figure 2C. Variation of Carbonyl Contents during Oxidative Stress. Protein carbonylation represents unique and highly specific indicators of OS induced protein modifications. Analysis of protein oxidation by carbonyl-derivatizing agents is widely used to assess oxidative damage at the molecular level. Levels of protein carbonylation were first measured spectrophotometrically using 2,4-DNPH at the time of H2O2 addition followed by progressive analysis for 15 h in ∆yfh1 strain cultures and 20 h in wild-type cultures (Figure 4A,B). Increased carbonylation was observed in wild-type cells during growth arrest, reaching a maximum at 1 h following exposure with average levels of carbonyl ∼1.7-fold higher compared to 0 h (Figure 4A). Immediately after growth recov-

ery, the carbonyl level decreased linearly to prestress levels and remained constant. On the other hand, carbonyl content in ∆yfh1 strain progressively increased and reached a maximum of 5.2 nmol/mg of proteins at 3 h following H2O2 addition. The carbonyl level was 2.6-fold higher at this point compared to 0 h (Figure 4B). After an additional hour (maximum cell density during the growth), carbonylation rapidly decreased by 30% and remained almost unchanged until the end of the experiment. Carbonylation is an irreversible and irreparable modification, although it has been noted that carbonylated proteins can be more susceptible to proteolytic degradation than their nonoxidized counterparts.28 With more severe oxidation, proteins are more likely to form protease-resistant, high-molecular weight aggregates (also called “aggresomes”) that can inhibit proteasome activity.29,30 As a result, the damaged and potentially protease-susceptible substrates accumulated with time and caused long-term damage to cells. Because the DNPH method detects accessible carbonyls only (nondegraded and nonaggregated), the measured carbonyl content represents the result of competition between degradation and aggregation of carbonylated proteins under severe oxidative stress. This conclusion is also supported by the results from the cell viability assay of ∆yfh1 (Figure 2C), in which the number of viable cells Journal of Proteome Research • Vol. 9, No. 2, 2010 733

research articles

Kim et al.

Figure 3. ROS detection under oxidative stress. (A) Oxidative damage in yeast wild-type and ∆yfh1 mutant stressed by hydrogen peroxide. The formation of ROS in yeast was measured by the H2O2-dependent oxidation of H2DCFDA. (B) The representative capture of the fluorescence image of ∆yfh1 mutant cells (left) and phase contrast image (right) (×600 magnification; top, control; bottom, at 3 h).

rapidly decreased after 3 h in the case of 20 mM H2O2 induction, while the level of carbonyls remained constant (Figure 4B). Identification of Oxidized Proteins. Recent studies have indicated that, depending on the origin of the stress, oxidative damage may be accompanied by carbonylation of a specific set of proteins.27,31 This observation suggests that at early stages

Figure 4. Variation in carbonyl contents in yeast cells and identification of the carbonylated proteins. (A) Carbonyl changes in wild-type were measured at 0, 0.5, 1, 2, 3, 5, and 20 h, respectively. (B) Carbonyl contents in mutant ∆yfh1 were measured at 0, 0.5, 1, 2, 3, 4, 5, 7, and 15 h, respectively. (C) Identification of oxidized proteins using avidin affinity selection from wild-type (53 proteins) with treatment of 100 mM H2O2 for 2 h and ∆yfh1 mutant (53 proteins) with an exposure of 20 mM H2O2 for 3 h.

a small number of highly “stressor specific” molecules are altered resulting in activation of specific stress defense responses to prevent cellular malfunction. Once molecular damage reaches a “no return” point at which cells cannot defend themselves, damage is exponentially extended into a large number of biomolecules and triggers general apoptotic/ necrotic pathways leading to the cell death. Using ∆yfh1 strain and a combination of vitamin C and iron, Irazusta et al.27 showed that major targets of oxidative damage are magnesiumbinding proteins. In this case, oxidative damage at early stages will target the mitochondria primarily due to iron accumulation

Table 1. The Common 14 Carbonylated Proteins in Both Wild-Type and Mutant under Oxidative Stress protein name

scorea

CDC19, Pyruvate kinase ENO2, Enolase II, a phosphopyruvate hydratase TEF2, Translational elongation factor EF-1 alpha PDC1, Major of three pyruvate decarboxylase isozymes ADH1, Alcohol dehydrogenase TDH2, Glyceraldehyde-3-phosphate dehydrogenase PSA1, GDP-mannose pyrophosphorylase RPL14A, N-terminally acetylated protein component of the large (60S) ribosomal subunit TRX2, Cytoplasmic thioredoxin isoenzyme HSP26, Small heat shock protein (sHSP) FBA1, Fructose 1,6-bisphosphate aldolase CRN1, Coronin CCH1, Voltage-gated high-affinity calcium channel TOP2, DNA topoisomerase II

130.7 83.73 82.68 62.8 50.85 39.55 33.49 26.34

NCBI gi accession no.

4180 6321968 6319594 6323073 6324486 3724 6320148 6322847 6321648 6319546 6322790 6323460 6321656 19880950

23.35 22.74 21.24 16.61 16.56 15.72

functionb

Metabolism Metabolism Protein synthesis Metabolism Metabolism Metabolism Metabolism Protein synthesis Cell defense Protein folding Metabolism Cell growth Cellular transport Cell cycle and DNA processing

a Identification score of the overall protein, calculated by adding scores of all the peptides detected for the protein. identified proteins using the MIPS Functional Catalogue (http://mips.helmholtz-muenchen.de/projects/funcat).

734

Journal of Proteome Research • Vol. 9, No. 2, 2010

b

Functional classification of

research articles

Oxidative Stress Studies in Yeast with a Frataxin Mutant Table 2. The Carbonylated Proteins Related to Mitochondrial Function or Localization NCBI gi accession no.

6324343 6325326 6322988 19880950 6319558 6319837 6323880 6324591 6320695 6319986 6322338 6322352 6322188 6322142 6319646 6322637 6324045 6324344

protein name

scorea

wild-type

ACC1, Acetyl-CoA carboxylase SPE3, Spermidine synthase VPS13,YLL040c TOP2, Essential type II topoisomerase MIS1, Mitochondrial C1-tetrahydrofolate synthase ILV6, Regulatory subunit of acetolactate synthase MRE11, Subunit of a complex with Rad50p and Xrs2p PET127, Protein with a role in mitochondrial RNA stability and/or processing RIB3, DHBP synthase GDH2, NAD(+)-dependent glutamate dehydrogenase MTC1, YJL123C UTP10, Nucleolar protein CFD1, Iron-sulfur cluster binding protein SYG1, Plasma membrane protein SSE2, HSP70 family SAC1, Phosphatidylinositol (PI) phosphatase MRPL10, Mitochondrial ribosomal protein of the large subunit TIM23, Essential protein of the mitochondrial inner membrane

27.86 19.95 16.5 15.72 15.38 12.87 12.67 12.17

• •

10.17 9.96 8.88 8.75 8.7 8.7 8.43 8.42 8.19 8.05

• •

•

∆yfh1

• • •

• • •

• • • • • • • •

functionb

localizationc

1 4 6 4 1 1 3 2

C, ER, N, M C, N, M C, M, E N, M M M C, N, M M

1 1 7 2 1 4 3 6 5 6

C, N, M C, M C, G, ER, M N, M C, M MEM, C, M, V C, M CP, C, ER, G, M, V M M

a Identification score of the overall protein, calculated by adding scores of all the peptides detected for the protein. b Functional classification of identified proteins using the MIPS Functional Catalogue (http://mips.helmholtz-muenchen.de/projects/funcat); 1, metabolism; 2, transcription; 3, cell defense and protein folding; 4, cell cycle and growth/budding; 5, protein synthesis; 6, cell transport; 7, others and unknown. c C, Cytoplasm; E, Endosome; ER, Endoplasmic Reciculum; G, Golgi; N, Nucleus; M, Mitochondria; P, Plasmic membrane; V, Vacuolar.

within the mitochondria. Hydrogen peroxide molecules, on the other hand, diffused throughout the entire cell and can generate a different pattern of damaged proteins. Carbonylated protein identification was achieved by growing wild-type yeast or the ∆yfh1 strain to midlogarithmic phase and exposing them to 100 mM H2O2 for 2 h or 20 mM H2O2 for 3 h, respectively. In this case, both wild-type and the ∆yfh1 strain exhibited similar levels of carbonylation, ROS, and viability. Overall, we found the same number (53) of carbonylated proteins in wild-type and the ∆yfh1 strain. The identified proteins were categorized by their functions and localization (Figure 3C). A complete list of proteins is given in Supplementary Table S1. Of these, 14 proteins were found in both strains suggesting common pathways of oxidative damage (Table 1). Half of them were carbonylated glycolytic enzymes, suggesting a possible slowdown of glycolysis and the TCA cycle. Other common proteins found were involved in protein synthesis and folding (3), cellular defense, growth, transport, and DNA processing. Interestingly, the differentiators between wild-type and ∆yfh1 strain were functionality and localization of damaged proteins. While a vast majority of proteins correspond to transcription in the nucleus in wild-type, damage in the ∆yfh1 strain was primarily targeted to protein synthesis within the cytosol. Only 18 proteins (9 unique to the wild-type, 8 to the ∆yfh1 strain, 1 found in both strains) were associated with mitochondrial function (Table 2). Comparing our data to the proteins damaged by iron-induced stress in the ∆yfh1 strain,27 we found only 2 common proteins including pyruvate kinase and translational elongation factor 1-alpha. On the other hand, we identified additional magnesium-/zinc-binding proteins such as pyruvate decarboxylase and alcohol dehydrogenase, fructose 1, 6-bisphosphate aldolase, pyruvate carboxylase. The finding of these proteins suggests that oxidative damage is mediated by replacement of magnesium or zinc ions by iron in their binding sites27 or by direct interaction of transition metals with H2O2.

Supporting Information Available: Lists of total carbonylated proteins identified in both wild-type and ∆yfh1

strains. This material is available free of charge via the Internet at http://pubs.acs.org.

References (1) Aliev, G.; Smith, M. A.; Seyidova, D.; Neal, M. L.; Lam, B. T.; Nunomura, A.; Gasimov, E. K.; Vinters, H. V.; Perry, G.; LaManna, J. C.; Friedland, R. P. The role of oxidative stress in the pathophysiology of cerebrovascular lesions in Alzheimer’s disease. Brain Pathol. 2002, 12 (1), 21–35. (2) Berg, D.; Gerlach, M.; Youdim, M. B.; Double, K. L.; Zecca, L.; Riederer, P.; Becker, G. Brain iron pathways and their relevance to Parkinson’s disease. J. Neurochem. 2001, 79 (2), 225–236. (3) Schulz, J. B.; Dehmer, T.; Scho¨ls, L.; Mende, H.; Hardt, C.; Vorgerd, M.; Bu ¨rk, K.; Matson, W.; Dichgans, J.; Beal, M. F.; Bogdanov, M. B. Oxidative stress in patients with Friedreich ataxia. Neurology 2000, 55 (11), 1719–1721. (4) Akman, S. A. Overview of oxidative stress and cancer. In Critical Reviews of Oxidative Stress and Aging: Advances in Basic Science, Diagnostics, and Intervention; World Scientific Publishing: New Jersey, 2003; pp 925-954. (5) Maritim, A. C.; Sanders, R. A.; Watkins, J. B., III. Diabetes, oxidative stress, and antioxidants: A review. J. Biochem. Mol. Toxicol. 2003, 17 (1), 24–38. (6) Stadtman, E. R.; Berlett, B. S. Reactive oxygen-mediated protein oxidation in aging and disease. Chem. Res. Toxicol. 1997, 10 (5), 485–494. (7) Fridovich, I. Superoxide radical and superoxide dismutases. Annu. Rev. Biochem. 1995, 64, 97–112. (8) Ursini, F.; Maiorino, M.; Brigelius-Flohe´, R.; Aumann, K. D.; Roveri, A.; Schomburg, D.; Flohe´, L. Diversity of glutathione peroxidases. Methods Enzymol. 1995, 252, 38–53. (9) Fenton, H. J. H. Oxidation of tartaric acid in presence of iron. J. Chem. Soc., Trans. 1894, 65, 899–910. (10) Haber, F.; Weiss, J. The catalytic decomposition of hydrogen peroxide by iron salts. Proc. R. Soc. London, Ser. A 1934, 147, 332– 351. (11) Babcock, M.; de Silva, D.; Oaks, R.; Davis-Kaplan, S.; Jiralerspong, S.; Montermini, L.; Pandolfo, M.; Kaplan, J. Regulation of mitochondrial iron accumulation by Yfh1, a putative homolog of frataxin. Science 1997, 276 (5319), 1709–1712. (12) Lesuisse, E.; Santos, R.; Matzanke, B. F.; Knight, S. A.; Camadro, J. M.; Dancis, A. Iron use for haeme synthesis is under control of the yeast frataxin homologue (Yfh1). Hum. Mol. Genet. 2003, 12 (8), 879–889. (13) Mu ¨ hlenhoff, U.; Richhardt, N.; Ristow, M.; Kispal, G.; Lill, R. The yeast frataxin homolog Yfh1p plays a specific role in the maturation of cellular Fe/S proteins. Hum. Mol. Genet. 2002, 11 (17), 2025– 2036.

Journal of Proteome Research • Vol. 9, No. 2, 2010 735

research articles (14) Gerber, J.; Mu ¨ hlenhoff, U.; Lill, R. An interaction between frataxin and Isu1/Nfs1 that is crucial for Fe/S cluster synthesis on Isu1. EMBO Rep. 2003, 4 (9), 906–911. (15) Duby, G.; Foury, F.; Ramazzotti, A.; Herrmann, J.; Lutz, T. A nonessential function for yeast frataxin in iron-sulfur cluster assembly. Hum. Mol. Genet. 2002, 11 (21), 2635–2643. (16) Adamec, J.; Rusnak, F.; Owen, W. G.; Naylor, S.; Benson, L. M.; Gacy, A. M.; Isaya, G. Iron-Dependent self-assembly of recombinant yeast frataxin: Implications for Friedreich Ataxia. Am. J. Hum. Genet. 2000, 67 (3), 549–562. (17) Foury, F.; Cazzalini, O. Deletion of the yeast homologue of the human gene associated with Friedreich’s ataxia elicits iron accumulation in mitochondria. FEBS Lett. 1997, 411 (2-3), 373–377. (18) Lee, S. S.; Robinson, F. M.; Wang, H. Y. Rapid determination of yeast viability. Biotechnol. Bioeng. Symp. 1981, 11, 641–649. (19) IJpma, A. S.; Greider, C. W. Short telomeres induce a DNA damage response in Saccharomyces cerevisiae. Mol. Biol. Cell 2003, 14 (3), 987–1001. (20) Levine, R. L.; Garland, D.; Oliver, C. N.; Amici, A.; Climent, I.; Lenz, A. G.; Ahn, B. W.; Shaltiel, S.; Stadtman, E. R. Determination of carbonyl content in oxidatively modified proteins. Method. Enzymol. 1990, 186, 464–478. (21) Moulder, R.; File´n, J. J.; Salmi, J.; Katajamaa, M.; Nevalainen, O. S.; Oresic, M.; Aittokallio, T.; Lahesmaa, R.; Nyman, T. A. A comparative evaluation of software for the analysis of liquid chromatography-tandem mass spectrometry data from isotope coded affinity tag experiments. Proteomics 2005, 5 (11), 2748–2760. (22) Kapp, E. A.; Schutz, F.; Connolly, L. M.; Chakel, J. A.; Meza, J. E.; Miller, C. A.; Fenyo, D.; Eng, J. K.; Adkins, J. N.; Omenn, G. S. An evaluation, comparison, and accurate benchmarking of several publicly available MS/MS search algorithms: sensitivity and specificity analysis. Proteomics 2005, 5 (13), 3475–3490.

736

Journal of Proteome Research • Vol. 9, No. 2, 2010

Kim et al. (23) Yoo, B.-S.; Regnier, F. E. Proteomic analysis of carbonylated proteins in two-dimensional gel electrophoresis using avidinfluorescein affinity staining. Electrophoresis 2004, 25 (9), 1334– 1341. (24) Demasi, A. P.; Pereira, G. A.; Netto, L. E. Yeast oxidative stress response: influences of cytosolic thioredoxin peroxidase I and of the mitochondrial functional state. FEBS J. 2006, 273 (4), 805–816. (25) Flattery-O’Brien, J. A.; Dawes, I. W. Hydrogen peroxide causes RAD9-dependent cell cycle arrest in G2 in Saccharomyces cerevisiae whereas menadione causes G1 arrest independent of RAD9 function. J. Biol. Chem. 1998, 273 (15), 8564–8571. (26) Bulteau, A. L.; Dancis, A.; Gareil, M.; Montagne, J. J.; Camadro, J. M.; Lesuisse, E. Oxidative stress and protease dysfunction in the yeast model of Friedreich ataxia. Free Radical Biol. Med. 2007, 42 (10), 1561–1570. (27) Irazusta, V.; Moreno-Cermen ˜ o, A.; Cabiscol, E.; Ros, J.; Tamarit, J. Major targets of iron-induced protein oxidative damage in frataxindeficient yeasts are magnesium-binding proteins. Free Radical Biol. Med. 2008, 44 (9), 1712–1723. (28) Bota, D. A.; Davies, K. J. Lon protease preferentially degrades oxidized mitochondrial aconitase by an ATP-stimulated mechanism. Nat. Cell Biol. 2002, 4 (9), 674–680. (29) Grune, T.; Jung, T.; Merker, K.; Davies, K. J. Decreased proteolysis caused by protein aggregates, inclusion bodies, plaques, lipofuscin, ceroid, and ‘aggresomes’ during oxidative stress, aging, and disease. Int. J. Biochem. Cell Biol. 2004, 36 (12), 2519–2530. (30) Bota, D. A.; Van Remmen, H.; Davies, K. J. Modulation of Lon protease activity and aconitase turnover during aging and oxidative stress. FEBS Lett. 2002, 532 (1-2), 103–106. (31) Mirzaei, H.; Regnier, F. E. Identification of yeast oxidized proteins Chromatographic top-down approach for identification of carbonylated, fragmented and cross-linked proteins in yeast. J. Chromatogr., A 2007, 1141 (1), 22–31.

PR900538E