Oxidation and Inactivation of SERCA by Selective Reaction of

Elena S. Dremina,† Victor S. Sharov,† Michael J. Davies,‡ and Christian Schöneich*,†. Department of Pharmaceutical Chemistry, UniVersity of K...
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Chem. Res. Toxicol. 2007, 20, 1462–1469

Oxidation and Inactivation of SERCA by Selective Reaction of Cysteine Residues with Amino Acid Peroxides Elena S. Dremina,† Victor S. Sharov,† Michael J. Davies,‡ and Christian Schöneich*,† Department of Pharmaceutical Chemistry, UniVersity of Kansas, 2095 Constant AVenue, Lawrence, Kansas 66047, and Free Radical Group, The Heart Research Institute, 114 Pyrmont Bridge, Camperdown, Sydney, NSW, Australia ReceiVed April 6, 2007

The oxidative modification of proteins plays an important role in a wide range of pathological processes and aging. Proteins are modified by numerous biologic oxidants including hydrogen peroxide, peroxynitrite, singlet oxygen, and oxygen- and nitrogen-centered radicals. More recently, an additional class of physiologically important oxidants has been identified, peptide and protein peroxides. The latter react quite rapidly and selectively with protein cysteine residues. The sarco/endoplasmic reticulum Ca-ATPase (SERCA) is reversibly regulated through NO-dependent S-glutathiolation of specific cysteine residues. The irreversible oxidation of these cysteine residues could, therefore, impair NO-dependent muscle relaxation. Here, we show that specific protein-derived (amino acid) peroxides react selectively with a subset of the 22 reduced cysteine residues of SERCA1, including a peptide-containing Cys674 and Cys675, where Cys674 (in SERCA2) represents one of the targets for NO-dependent S-glutathiolation. Out of 11 tested amino acid, peptide, and protein peroxides, those derived from free tryptophan and free tyrosine showed the highest reactivity towards SERCA, while no oxidation under similar experimental conditions was detected through hydrogen peroxide. Among the peroxides from tryptophan, those of free tryptophan showed a significantly higher reactivity as compared to those from N- and C-terminally blocked tryptophan. Quantitative HPLC-MS/MS analysis demonstrated that the highest reactivity of the tryptophan-derived peroxides was observed for Cys774 and Cys938, cysteine residues, which are embedded within the transmembrane domains of SERCA1. This unusual reactivity of transmembrane domains cannot be solely rationalized by the hydrophobicity of the oxidant, as the peroxide from DL-tryptophan shows considerable higher reactivity as compared to the one derived from N-acetyl-tryptophan methyl ester. Our data demonstrate a potential role of peptide- and protein-derived peroxides as important mediators of oxidative stress in vivo, which may cause a selective oxidation of Cys residues leading to inactivation of membrane proteins. Introduction Oxidative modifications of proteins play an important role in a wide range of pathological processes associated with abnormal cell development, proliferation, and apoptosis (1). These post-translational modifications are carried out by various radical and nonradical oxidants, including peroxyl radicals, hydrogen peroxide, peroxynitrite, nitrogen dioxide, and singlet oxygen (1O2) (2). In general, oxidants that are less reactive but more selective towards specific amino acid residues have a greater potential for carrying out specific protein modifications, selectively affording protein activation, inactivation, and/or conformational changes. Protein and peptide peroxides belong to this class of oxidants, specifically targeting protein Cys residues (3). This feature motivated us to investigate their reaction(s) with the sarco/endoplasmic reticulum Ca-ATPase (SERCA),1 a membrane protein where the selective oxidation of specific Cys residues may lead to inactivation (4) or to the * To whom correspondence should be addressed. E-mail: schoneic@ ku.edu. † University of Kansas. ‡ The Heart Research Institute. 1 Abbreviations: BCA, bicinchoninic acid; BSA, bovine serum albumin; ESI, electrospray ionization; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GSH, glutathione; MS/MS, tandem MS; NO, nitric oxide; RPHPLC, reversed-phase HPLC; SERCA, sarcoplasmic/endoplasmic-reticulum Ca2+-ATPase; SR, sarcoplasmic reticulum; TG1, ThioGlo 1.

loss of nitric oxide (NO)-dependent activation, described in detail below. Protein peroxides have been detected in cells exposed to oxidative stress (5, 6). In addition, products (alcohols) consistent with the formation and subsequent decomposition of such peroxides have been detected in a wide range of diseased tissue samples at elevated levels over normal healthy tissues (7, 8). Thus, elevated levels of side-chain alcohols have been detected on protein-bound Val, Leu, and some other side chains from in vivo samples. Protein-derived peroxides generated in intact cells can be quite long-lived (half-lives of seveal hours) (5). The reasons for this are not entirely clear, but experiments show that most of the systems (both enzymatic and low molecular mass) that remove small molecular weight peroxides do not react rapidly with protein-derived species, possibly for steric or electronic reasons (9). Peroxides generated on bovine serum albumin (BSA) are poorly metabolized/detoxified, but when peroxidized BSA is exposed to proteases (e.g., pronase), there is a release of small peptides and amino acids, of which several retain the peroxide functions. These amino acid/peptide peroxides can be readily detected in test tube systems, and they are more reactive than those on the protein from which they were derived. The long lifetime of protein-derived peroxides suggest that cells have no efficient enzymatic defense against protein peroxides. The proteolysis of peroxidized proteins releases

10.1021/tx700108w CCC: $37.00  2007 American Chemical Society Published on Web 09/25/2007

Peptide Peroxides Oxidize Cysteine Residues of SERCA

peptide peroxides (9). The one-electron reduction of peroxides, for example, catalyzed by transition metals, can initiate chain oxidation reactions via the intermediary formation of alkoxyl radicals and can ultimately transfer the oxidative damage to other proteins and biomolecules (10, 11). In contrast, the two-electron reduction of peroxides will generate alcohols. Peptide and protein peroxides selectively oxidize cysteine residues and inactivate, for example, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (12), cellular caspases (13), and lysosomal thiol-dependent cathepsins (3) specifically through thiol oxidation. Therefore, the susceptibility of cells towards damage by peptide and protein peroxides is, in part, determined by the cellular content of reduced glutathione (GSH) (9). In general, peptide peroxides are rather long-lived and may confer a very selective damage to proteins. Such selective damage of cysteine residues of proteins by peptide and protein peroxides may lead to very specific biological responses. The SERCA plays a key role in calcium homeostasis. SERCA is responsible for the transport of Ca2+ into the endoplasmic or sarcoplasmic reticulum (ER/SR) and the maintenance of high luminal Ca2+ concentrations, necessary for triggering muscle contractions, cell signaling, and apoptosis (14, 15). Three genes for SERCA have been identified (SERCA1, SERCA2, and SERCA3), and different SERCA isoforms are expressed in tissue-specific and developmentally regulated patterns (16, 17). The fast-twitch muscle isoform SERCA1 is restricted to fasttwitch skeletal muscle, while SERCA2a is expressed in cardiac and slow-twitch skeletal muscle. The isoforms SERCA2b and SERCA3a-3f are present in a large variety of tissues, including nonmuscle tissues. SERCA2 activity is regulated, in part, through the NO-dependent reversible S-glutathiolation of cysteine residues at positions 669 and/or 674 (18). In SERCA1, a peptide containing Cys674 and Cys675, shows a significant agedependent irreversible oxidation in vivo (40%), suggesting that NO-dependent activation of SERCA may be impaired (19). The quantification of such individual Cys residues in SERCA1 was achieved with HPLC-electrospray-tandem mass spectrometry (MS/MS) of ThioGlo1 (TG1)-labeled Cys residues [ThioGlo1 ) methyl-10-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-9-methoxy-3-oxo-3H-benzo[f]chromene-2-carboxylate]. In addition, specifically, SERCA2 is sensitive to the nitration of Tyr residues in vivo (20). One potential mechanism of Tyr nitration involves the reaction of SERCA with peroxynitrite. However, peroxynitrite also effectively targets specific SERCA Cys residues such as Cys at positions 364, 417, 420, 498, 525, 674, 675, and 938 (in SERCA1). Considering the importance of selected Cys residues for NOdependent activation of SERCA, the present study was designed to test the selectivity of amino acid, peptide, and protein peroxides towards Cys residues in SERCA. Cys residues are the preferred targets of such peroxides in proteins, demonstrated for GAPDH (12), cellular caspases (13), and lysosomal thioldependent cathepsins (3). In contrast, Met residues are not a preferred site for oxidation by these peroxides (9), limiting the potential target to Cys-containing sequences. We will demonstrate that indeed Cys674 and Cys675 in SERCA1 are targets for such peroxides; however, surprisingly, a high reactivity towards these peroxides is also observed for Cys774 and Cys938 in the transmembrane domain, that is, a pattern different to that observed after peroxynitrite oxidation (19). Importantly, peptide and protein peroxides, especially from Trp and Tyr, are ca. 100fold more reactive towards SERCA as compared to hydrogen peroxide (H2O2).

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Materials and Methods Isolation of Sarcoplasmic Reticulum. Native SR vesicles (light fraction) were prepared from 6 month old Fisher 344 × Brown Norway F1 hybrid rat hindlimb skeletal muscle (fast-twitch fibers) as described earlier (20, 21). Briefly, muscles (usually 20–30 g) were homogenized in a Waring blender for 1 min at maximal speed at 4 °C in 3 volumes of buffer containing 0.1 M KCl, 0.1 mM EDTA, and 20 mM MOPS (pH 7.4). The homogenate was centrifuged at 5000g for 20 min to remove cell debris, the pellet was washed again under the same conditions, and the pooled supernatants were centrifuged at 11800g for 20 min to pellet the mitochondria. To dissolve myosin, the supernatant was filtered through six layers of cheesecloth and solid KCl was added to adjust a final concentration to 0.6 M. After 20 min of incubation, the SR was pelleted at 23500g for 1 h. The supernatant was decanted, and the pellets were resuspended in a medium containing 0.3 M sucrose and 20 mM MOPS (pH 7.0) and centrifuged for 30 min at 100000g. SR vesicles were resuspended in a small volume of medium consisting of 0.3 M sucrose and 20 mM MOPS (pH 7.0) using a dounce homogenizer, aliquoted, quickly frozen in liquid nitrogen, and stored at -70 °C. Protein concentrations were determined by the bicinchoninic acid (BCA) assay using BSA as a standard according to the manufacturer’s instructions (Pierce, Rockford, IL). Generally, SERCA represents ca. 40% of the protein in our SR preparation, as indicated by densitometric analysis of Coomassie Blue-stained gels. Generation of Amino Acid, Peptide, and Protein Peroxides. Amino acids and proteins were purchased from Sigma-Aldrich (Castle Hill, Australia), and peptides were supplied by Bachem (Switzerland). The procedures for peroxide preparation and characterization have been described in detail elsewhere (22–28). In brief, peroxides were generated from amino acids, peptides (all 2.5 mM), and proteins (50 mg/mL) by photoirradiation with visible light (from a Kodak S-AV 2050 slide projector) through a 345 nm cut-off filter in the presence of 10 µM rose bengal or by γ-radiation (12, 22). The amino acid and peptide substrates were chosen based on their documented yields of peroxides in vitro (28). Here, the side chains with large numbers of C–H bonds give the highest yields of hydroperoxides during γ-radiation, and for the photoxidation, only three amino acids give significant yields of peroxides, Trp, His, and Tyr. Samples were made up in water that had been passed through a four-stage Millipore Q system, equipped with a 0.2 µm pore size final filter. The pH of all solutions was checked prior to photolysis or radiation and adjusted, where necessary, to pH 7.0–7.5. Solutions were kept on ice during photoirradiation (60 min, except for Tyr, which was photoirradiated for 120 min) and were continually aerated. After cessation of photoirradiation, catalase (Sigma-Aldrich, bovine liver, 50 µg/mL ovalbumin, 250 µg/mL for amino acids and peptides) was added to remove H2O2 and the samples were incubated for 30 min at room temperature before freezing (-80 °C) in aliquots. For γ-irradiation, samples were subjected to a total radiation dose of 2000 Gray (determined by Fricke dosimetry (29)). Control solutions (nonphotoirradiated and non-γ-irradiated) were treated in the same manner, with the exception of the exposure to visible light or radiation. Peroxide concentrations were determined by a modified FOX (FeSO4/xylenol orange) assay, using H2O2 standards (23). This assay gives values comparable to iodometric analysis (22). The yields of peroxides formed on the various amino acids, peptides, and proteins examined are expressed as H2O2 equivalents and are given in Table 1. In general, between 7.6 and 62% of the original amino acids/peptides were converted to peroxides. Peroxide concentrations of all control (nonphotolysed) samples were also determined and found to be negligible (6 counts in a single scan were selected automatically for MS/MS analysis. Identified peptides were then quantified using the MassLinx 4.0 software (Micromass, Manchester, United Kingdom) by the integration of peaks in selected ion chromatograms obtained in additional HPLC-ESI-MS runs. Statistical Analysis. Quantitative results were obtained from the data for at least three independent experiments. Values are presented as means ( SD. Significance of a difference between two means was assessed by Student’s t test, calculated using a two-sample unequal variance and two-tailed distribution using Microsoft Excel XP Pro software.

Results Inactivation of Ca2+-ATPase Activity of SERCA by Peroxides. The incubation of rat skeletal muscle SR with 100 µM various peroxides (obtained by appropriate dilution of the representative peroxide sample listed in Table 1) at 37 °C for 1 h resulted in an inhibition of SERCA activity only for Trpand Tyr-derived peroxides (Figure 1). In contrast, under the same conditions, H2O2 did not cause significant SERCA inactivation. Blocking of the N- and C-termini of Trp and Tyr resulted in a decrease of SERCA inhibition, evident from a comparison of the peroxides from N-Ac-Trp-OMe and DL-Trp or the peroxides from Gly-Tyr-Gly and L-Tyr. Peroxides from proteins (BSA and ovalbumin) did not inactivate SERCA under the selected reaction conditions. Quantification of SR Thiols and Enzyme Activity. The quantification of modified SR thiols was selectively carried out for the most reactive peroxide, derived from DL-Trp. The titration of native SR vesicles with TG1 yielded 20.6 ( 1.2 mol Cys/

Peptide Peroxides Oxidize Cysteine Residues of SERCA

Figure 1. Inhibition of Ca-ATPase activity of SERCA by amino acids, peptides, and peroxides. A 100 µM concentration of peroxides (a dagger indicates that peroxides were generated by γ-radiation) or H2O2 was added to skeletal muscle SR vesicles obtained from 6 month old rats (1.5 mg/mL protein in 50 mM MOPS buffer, pH 7.0, containing 120 mM KCl and 10 mM MgCl2) and incubated in the darkness at 37 °C for 1 h. A 100 µM concentration of control samples (nonphotolysed and nonradiated) was incubated with SR, and the measured values of activity were used as controls for each individual peroxide. For H2O2, a sample of SR without any further addition was used as a control. Statistical significance (*, P > 0.9; **, P > 0.95) was calculated using values measured for SR sample from 6 month old animals as a control (1 ( 0.1).

Figure 2. Loss of Ca-ATPase activity and reactive Cys residues in rat skeletal muscle SR after exposure to DL-Trp peroxides. Ca-ATPase activity (open circles) and loss of reactive Cys content (closed circles) in skeletal muscle SR vesicles (1.5 mg/mL protein in 50 mM MOPS buffer, pH 7.0, containing 120 mM KCl and 10 mM MgCl2) after incubation in the darkness at 37 °C for 1 h with different concentrations of DL-Trp peroxides. DL-Trp peroxides were synthesized by the photooxidation of DL-Trp (see Materials and Methods and Table 1) with a conversion of ca. 60%. Hence, the used stock solutions of DLTrp contained unreacted DL-Trp (at a level of ca. 0.67-fold the amount of DL-Trp peroxide).

mol SERCA in good agreement with our earlier measurements (19). Figure 2 shows that the exposure of SR to the peroxide from DL-Trp caused a concentration-dependent decrease of CaATPase activity in parallel with a decrease in the total number of TG1-reactive Cys residues. In fact, the incubation of SR for 1 h at 37 °C with 300 µM DL-Trp-derived peroxides resulted in a ca. 80% inhibition of SERCA activity, paralleled by the loss of 6 mol of TG1-reactive Cys residues per mol of SERCA. Importantly, treatment with peroxides and/or labeling with TG1 did not affect the electrophoretic mobility of SERCA (data not shown) necessary for mapping of Cys residues after SDS-PAGE

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Figure 3. Effect of GSH on the inactivation of SERCA by 100 µM DL-Trp peroxide. Skeletal muscle SR vesicles from 6 month old rats (1.5 mg/mL protein in 50 mM MOPS buffer, pH 7.0, containing 120 mM KCl and 10 mM MgCl2) were incubated in the darkness at 37 °C for 1 h alone or with 100 µM of DL-Trp peroxides in the presence of different concentrations of GSH. DL-Trp peroxides were synthesized by the photooxidation of DL-Trp (see Materials and Methods and Table 1) with a conversion of ca. 60%. Hence, the used stock solutions of DL-Trp contained unreacted DL-Trp (at a level of ca. 0.67-fold the amount of DL-Trp peroxide).

separation and in-gel digestion coupled to HPLC-ESI-MS/MS analysis (see below). Effect of GSH on the Inhibition of Ca-ATPase Activity of SERCA by DL-Trp Peroxide. GSH protected SERCA from oxidative damage induced by DL-Trp-derived peroxides, as displayed in Figure 3. For example, the addition of 100 µM GSH prior to the incubation of SERCA with 100 µM DL-Trpderived peroxide resulted in a ca. 36% reduced SERCA inactivation. This can be quantitatively rationalized by the efficient competition of SERCA thiols and GSH for peroxide (with a similar rate constant for both of sets of thiols): On the basis of 22 reduced Cys residues in SERCA and a content of SERCA of 40% relative to total protein in our SR preparation, 1.5 mg/mL SR protein corresponds to 180 µM SERCA thiols. Hence, the addition of 100 µM GSH to 1.5 mg/mL SR protein predicts a 36% reduction of the addition of SERCA thiols assuming that the DL-Trp-derived peroxide reacts equally fast with GSH and specific SERCA thiols critical for activity. To determine whether the protection of SERCA by GSH was through direct reaction of peroxides with GSH or through S-glutathiolation of SERCA, we analyzed for bound GSH according to our published procedure (18). We incubated 1 mg/ mL SR protein with 200 µM DL-Trp-derived peroxide in the absence and presence of 0.5 mM GSH and subsequently spun the protein down by centrifugation at 14000g for 15 min to remove all small molecular weight components including free GSH. The centrifugation was repeated three times, after repeated resolubilization of the pellet in 50 mM MOPS buffer, pH 7.0 (containing 120 mM KCl and 10 mM MgCl2). The protein was ultimately resolubilized in a buffer containing 4% (w/v) SDS, 120 mM Tris, pH 8.8, 7 M urea, and 2 M thiourea, and disulfidebound GSH was released through a 10 min reaction with 1 mM dithiothreitol, prior to derivatization with TG1 and HPLC analysis with fluorescence detection. GSH concentrations were determined by comparison with authentic standards in the same buffer. The incubation of SERCA with peroxide in the presence of GSH did not lead to any increase in SERCA-bound GSH as compared to a control experiment in the absence of GSH, indicating that S-glutathiolation is of minor importance under the selected reaction conditions.

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Dremina et al.

Figure 4. Effect of DL-Trp peroxide on the content of reduced Cys12 SERCA residue. This figure illustrates principles of quantitative mapping of oxidation-sensitive cysteine residues in SERCA (16). Following incubation with peroxide, SR is labeled with TG1 and SERCA in-gel digests were analyzed by HPLC-electrospray-MS. Panels a, b, and c show selected mass (m/z 958.8) chromatograms for a TG1-Cys adduct of a representative SERCA peptide, STEEC12LSYFGVSETTGLTPDQVK, from samples incubated with 200, 100, or 0 µM DL-Trp peroxide, respectively. Integration of chromatographic peaks at 37 min for the peptide ion MH33+ 958.8 (panels d–f) demonstrates DL-Trp peroxide concentration-dependent loss of TG1-reactive Cys12 (see respective intensity labels below mass labels). To normalize yields of Cys-containing peptides in individual HPLC-MS analyses, integration of internal standard peptide ions from SERCA sequence-containing nonoxidizable residues such as DIVPGDIVEVAVGDK (MH22+ 763.4, shown in panels g–i) was used for relative quantitation of reactive Cys residues. Sequences of all peptides are confirmed by HPLCMS/MS analysis in separate experiments.

Loss of Specific Cys Residues upon Exposure to Peroxide from Trp. TG1 labeling of SERCA Cys residues allows for the mapping of their differential reactivity towards peroxides by HPLC-ESI-MS/MS analysis (19). The method of quantification of individual Cys-containing peptides has been described in detail (19) and will be only briefly summarized in the following, referring to Figure 4. All quantitation is based on the loss of TG1 labeling of specific peptides as a result of oxidation, as compared to the TG1 labeling of the same peptides in nonoxidized SERCA1. After TG1 labeling of SERCA1, the protein is digested and the individual peptides are analyzed by HPLC-MS. Selected mass chromatograms (Figure 4, panels a–c) for specific TG1 adducts are integrated (Figure 4, panels d–f), where the reaction with increasing concentrations of peroxide is indicated by a decrease in the respective peak areas. These values are normalized on the peak areas of a group of SERCA1 peptides, which contain neither oxidizable nor hydrolyzable (Asn, Gln) amino acids and, therefore, serve as internal standards (to correct for possible variations of protein amounts due to sample preparation). Figure 4, panels g–i shows representative data for the SERCA peptide DIVPGDIVEVAVGDK. In agreement with our earlier measurements, a total of 16 different Cys residues can be labeled by TG1 and the respective peptides detected by ESI-MS/MS in native SR samples. Only the peptides containing Cys318, Cys70, Cys910, Cys670, Cys876, Cys888, Cys344, and Cys349 are not detected by TG1 labeling (19, 32). Cys70, Cys318, and Cys910 are located in large, hydrophobic transmembrane sequences, which are difficult to recover in sufficient quantities. The residues Cys876 and Cys888 are involved in a disulfide bond. Surprisingly, a TG1-labeled peptide containing both Cys344 and Cys349 could not be recovered after in-gel

digestion of SERCA, consistent with our previous data (19). However, the total labeling of ca. 21 Cys residues, indicated by fluorescence quantification of the entire nondigested protein (see above), suggests that both Cys344 and Cys349 exist predominantly in their reduced form and support our previous conclusion (19) that the TG1-labeled peptide containing Cys344 and Cys349 cannot be recovered in sufficient quantities from the gel. Therefore, the peroxide-dependent oxidation of Cys344 and Cys349 was quantified via an alternative method (see below). Figure 4 illustrates the HPLC-MS approach to assess sequencespecific loss of TG1-reactive Cys residues due to reaction with 12 DL-Trp for a representative residue, Cys . Table 2 summarizes the data on the molar content of specific Cys residues in SERCA1 after incubation of SR with 100 µM and 200 µM Trpderived peroxides for 1 h at 37 °C. We observe a partial loss of nine Cys residues (positions 674, 675, 12, 377, 268, 417, 420, 938, and 774). Integration over all Cys residues quantifiable by mass spectrometry yields a total loss of 1.73 mol Cys/mol SERCA for 100 µM and 3.12 mol Cys/mol SERCA for 200 µM DL-Trp-derived peroxide. These yields correspond to ca. 60% of those quantified by fluorescence analysis of the entire protein (Figure 2). The discrepancy of ca. 2 mol Cys/mol SERCA, quantified by TG1 labeling, is likely due to oxidation of Cys344 and Cys349 (see below) (32). Oxidation Products. Cys sulfenic acid (CysSOH) represents a prominent product of the reaction of protein Cys residues with H2O2 and organic peroxides (33). A representative example relevant to the present work is the oxidation of Enterococcus faecalis NADH oxidase Cys42 to CysSOH by C(4a)-peroxyflavin (34). Unless located in inaccessible domains within proteins, sulfenic acids are rather unstable towards reaction with ad-

Peptide Peroxides Oxidize Cysteine Residues of SERCA Table 2. Content of specific Cys Residues in Rat Skeletal Muscle SERCA1 (mol/mol Protein) after Exposure to DL-Trp Peroxidea DL-Trp

Cys residue 525 561 636 471 674, 675 498 614 364 12 377 268 417, 420 938 774 total lossb

peroxide

100 µM

200 µM

0.93 ( 0.15 1.07 ( 0.10 1.21 ( 0.22 1.20 ( 0.21 0.92 ( 0.05* 1.23 ( 0.25 1.15 ( 0.21 1.04 ( 0.06 0.81 ( 0.15* 0.90 ( 0.26 0.89 ( 0.10* 0.83 ( 0.10* 0.66 ( 0.29* 0.33 ( 0.14** 1.73

0.9 ( 0.12 1.20 ( 0.23 1.31 ( 0.32 1.02 ( 0.21 0.73 ( 0.11** 1.16 ( 0.20 1.20 ( 0.25 1.22 ( 0.25 0.66 ( 0.12** 0.64 ( 0.14** 0.60 ( 0.21** 0.67 ( 0.14** 0.39 ( 0.15** 0.29 ( 0.15** 3.12

Statistical significance (*, P > 0.9; **, P > 0.95) was calculated using values measured for the SR sample from 6 month old animals as a control (1 ( 0.15). b Total loss is calculated as a sum of individual losses for all displayed Cys residues. a

ditional thiols (to yield disulfides) or further oxidation to sulfinic and sulfonic acids. After the exposure of SERCA1 to Trpderived peroxides, we detected a disulfide bridge between Cys344 and Cys349 on the same tryptic peptide, for which quantification is described below. We have not attempted to locate all possible disulfide bridges between different tryptic peptides containing one of the 24 possible Cys residues in SERCA1 as the theoretical number of possibilities is unreasonably high. However, we have located Cys sulfonic acids (CysSO3H) on Cys377, Cys674, and Cys675. For all three Cys residues, exposure to increasing levels of Trp-derived peroxides resulted in an increasing yield of CysSO3H, quantified through integration of the peak area of the respective mass spectrometric peaks displayed in selected ion chromatograms. Quantification of Oxidation of Cys344 and Cys349. The extent of Cys344 and Cys349 oxidation was quantified by mass spectrometric analysis of their oxidation product. Cys344 and Cys349 are located within a tryptic octadecapeptide of the sequence SLPSVETLGCTSVICSDK. A single oxidation product of this peptide, containing the intramolecular disulfide, SLPSVETLGCTSVICSDK, was detected by MS/MS analysis after oxidation of SERCA1 with DL-Trp-derived peroxide and

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tryptic digestion in the gel (we specifically searched for other oxidation products such as sulfenic, sulfinic, and sulfonic acid on either of the two Cys residues, or both, but detected none). Baseline levels of the disulfide-containing peptide were detected for native SERCA1, which was not exposed to peroxides, consistent with our earlier results (19). However, when native SERCA1 was exposed to increasing concentrations of DL-Trpderived peroxide, between 0 and 300 µM, we quantified an increasing yield of the disulfide-containing peptide with the maximal yields leveling for 200 and 300 µM peroxide. These maximal yields represent a stoichiometric conversion of the reduced peptide to the disulfide-containing peptide and can be used as an authentic standard for quantitation. In a representative experiment, the maximal yield of the disulfide, obtained through oxidation with 200 µM peroxide, was 3.6fold higher than the baseline level of the disulfide in native SERCA1. Maximal yields of the disulfide correspond to the loss of 2 mol of Cys, generating 1 mol of disulfide. Therefore, on the basis of the observed 3.6-fold difference, the baseline level of disulfide in native SERCA1 corresponds to 1/3.6 ) 0.28 mol of disulfide. With this internal calibration, we calculate that the oxidation of SERCA1 with 100 and 200 µM DL-Trp-derived peroxide leads to the formation of 0.16 and 0.72 mol disulfide/ mol SERCA, respectively, accounting for the oxidation of 0.32 and 1.44 mol Cys residues per peptide (Cys344 and Cys349). The total yield of Cys oxidation is then represented by the sum of (i) loss of TG1 labeling and (ii) formation of disulfide between Cys344 and Cys349. SERCA oxidation by 100 µM DLTrp-derived peroxide leads to the oxidation of 2.05 mol Cys/ mol SERCA1, where 1.73 mol of Cys is quantified through the analysis of individual TG1-labeled peptides and 0.32 mol of Cys is quantified through the formation of disulfide between Cys344 and Cys349. Hence, we account for a total of 68% of the lost Cys residues, quantified by fluorescence analysis of the entire protein (see Figure 2). The analysis for 200 µM DL-Trpderived peroxides yields a loss of 3.12 mol of TG1-labeled peptides and a loss of 1.44 mol of Cys344/Cys349, that is, a total loss of 4.56 mol Cys/mol SERCA1. This accounts for 89% of the lost Cys residues quantified by fluorescence analysis of the entire protein. We conclude that our quantitative MS analysis of individual Cys covers nearly 90% of the Cys residues targeted specifically by a high level of peroxides.

Figure 5. Cartoon of SERCA1 indicating the location of Cys residues through numbers. Cys residues found to be modified by age-dependent oxidation in vivo (19) are shown in bold, those modified by peroxinitrite oxidation in vitro (19) are shown underlined, and those modified by 351 DL-Trp peroxides in vitro (current data) are shown in boxes. The phosphorylation site at Asp is also shown.

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Discussion The exposure of proteins to endogenous or exogenous radical sources generates protein peroxides (10, 28). Proteolysis of peroxidized proteins results in the formation of relatively longlived peptide peroxides (9). Oxidative modifications of biomolecules by such peroxides represent an important pathway towards potential pathological dysfunctions (2, 3, 5, 6, 9–13). For example, Trp- and Tyr-derived peroxides inhibit GAPDH, cellular caspases, and lysosomal cathepsins (3), suggesting that they target critically important cysteine residues of the enzymes (3, 12, 13). In the present study, we investigated the differential sensitivity of SERCA cysteine residues towards amino acid, peptide, and protein peroxides, motivated by our earlier studies on the functional importance of specific cysteine residues for SERCA activity. Our data suggest that the structures of the peptide-derived peroxides control some of the reactivity towards the protein: For example, the inhibition of the Ca-ATPase activity of SERCA by Trp- and Tyr-derived peroxides was considerably more effective as compared to that by H2O2 under the same conditions (Figure 1). The Trp-derived peroxides were more effective than Tyr-derived peroxides (Figure 1), as expected based on previous results on the inactivation of cellular caspases (13). Blocking the N and C termini of the Trp and Tyr residues reduces the inhibitory effect of their peroxides on SERCA, in contrast to data with caspases. Peroxidation of Tyr yields predominantly structure 2, while peroxidation of Trp yields structure 4 (Chart 1). The higher reactivity of Trp-derived peroxides as compared to Tyr-derived peroxides must, therefore, originate from the structural differences of 2 and 4. Blocking of the C and N termini of Tyr and Trp prevents cyclization of 1 to 2 and of 3 to 4, suggesting that the lower reactivity of peroxides derived from N- and C-blocked Tyr and Trp as compared to those from free Tyr and Trp originates from the absence of cyclization. Out of 16 SERCA1 Cys residues quantifiable through TG1 labeling, nine are reactive towards DL-Trp-derived peroxides (Table 2). The peroxide displays a strong reactivity towards Cys774 and Cys938, both located in the transmembrane domain of SERCA, whereas a lower reactivity is observed towards the Cys residues present at positions 12, 268, 377, 417, 420, 674, and 675 (Figure 5). The rather high reactivity of the Trp-derived peroxides towards Cys residues located in the transmembrane domain (Cys268, Cys774, and Cys938) is interesting. This may, to some extent, be due to the hydrophobicity of peroxide 4. The crystal structures of SERCA1 in the absence and presence of Ca2+ (35, 36) show that Cys938 is more exposed on the surface of the protein as compared to Cys774 and Cys268, which rather point towards the interior of the transmembrane domain. Hence, the observed reactivity of Trp-derived peroxides with Cys residues of the transmembrane domain is not consistent with their surface exposure. On the other hand, specifically, the more buried Cys residues may participate in hydrogen bonding, resulting in a partial negatively charged sulfur, which should be easier to oxidize. Interestingly, Cys938, but not Cys774, represents also a target for the water-soluble anion peroxynitrite (19). In addition, the peroxide shows a high reactivity towards Cys344 and Cys349, quantifiable through analysis of an intrachain disulfide as the single oxidation product. Mechanistically, the formation of the intrachain disulfide can be rationalized through an intermediate sulfenic acid, which cleaves off water through reaction with the second thiol. Together, all Cys residues quantified through MS analysis of either TG1-labeled peptides or the disulfide between Cys344 and Cys349 account for up to 90% of the lost thiols quantified by TG1 fluorescence of the

Dremina et al.

entire protein. With such a manifold of targets, it is difficult to correlate the oxidation of one specific Cys residue by DL-Trpperoxides with SERCA inactivation: It is likely that oxidation of each of the individual Cys residues is associated with a degree of inactivation and that the measured SERCA inactivation represents the sum of all of the partial inactivations caused by the oxidation of individual Cys residues. In addition, at least four Cys residues could not be quantified by TG1 labeling and oxidation of the latter may contribute to the overall SERCA inactivation. An important detail is the oxidative modification of Cys674,675 by DL-Trp peroxides. In smooth muscle SERCA2, Cys674 is reversibly modified through S-glutathiolation induced by NO, resulting in SERCA activation (18). Any irreversible oxidation of this Cys residue would lead to a loss of NOdependent activation. Our present data on SERCA1 suggest that amino acid-derived peroxides could target Cys674 of the highly homologous isoform SERCA2, potentially affecting NO-derived smooth muscle relaxation. Acknowledgment. We thank Dr. Philip Morgan for the synthesis and assay of the peroxide samples. This research was supported by a grant from the NIH (PO1AG12993) and the Australian Research Council through the ARC Centres of Excellence program.

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