Design and Characterization of an Active Site Selective Caspase-3

Design and Characterization of an Active Site. Selective Caspase-3 Transnitrosating Agent. Douglas A. Mitchell†, Sarah U. Morton‡, and Michael A. ...
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Design and Characterization of an Active Site Selective Caspase-3 Transnitrosating Agent Douglas A. Mitchell†, Sarah U. Morton‡, and Michael A. Marletta†,§,¶,* †

Department of Chemistry, §Department of Molecular and Cell Biology, and ¶Division of Physical Biosciences, Lawrence Berkeley National Laboratory, University of California, Berkeley, California 94720-1460, and ‡Department of Biochemistry, University of California, San Francisco, California 94158

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n recent years, it has become clear that nitric oxide (NO) targets proteins other than soluble guanylate A B S T R A C T The oxidative addition of nitric oxide (NO) to a thiol, S-nitrosation, is cyclase (sGC) (1). S-Nitrosation, an example of sGC/ a focus of studies on cyclic guanosine monophosphate (cGMP)-independent NO cyclic guanosine monophosphate (cGMP)-independent signaling. S-Nitrosation of the catalytic cysteine of the caspase proteases has NO signaling, is a post-translational modification in important effects on apoptosis and consequently has received attention. Here we report on a small molecule that can directly probe the effects of S-nitrosation on which a cysteine thiol is converted to a nitrosothiol by the caspase cascade. This chemical tool is capable of permeating the mammalian the oxidative addition of NO. Modification of a cysteine cell membrane, selectively transnitrosating the caspase-3 active site cysteine, and residue that directly participates in an enzymatic reachalting apoptosis in cultured human T-cells. The efficacy of this reagent was comtion leads to inhibition of activity. The apoptotic propared with the commonly used reagent S-nitrosoglutathione and an esterified tease caspase-3 is one such protein, and a number of derivative. reports have recently begun to characterize the molecular mechanism of how NO inhibits the caspases and, thus, apoptosis (2–7). Procaspase-3 is S-nitrosated under basal conditions in human B- and T-cell lines on the catalytic cysteine, Cys163 (3). Similarly, the active site cysteine of procaspase-9 is S-nitrosated in human colon adenocarcinoma cells (8). The limited supply of NO (nanomolar under signaling concentrations), coupled with its promiscuous reactivity towards both thiol and non-thiol functionality, argues for the existence of molecular machinery that confers reaction specificity (5). For example, NO reacts indiscriminately and rapidly with heme proteins, reactive oxygen, and nitrogen species (9). Also, when a NO donor or transnitrosating agent is allowed to react with purified proteins in vitro, many cysteines are modified to nitrosothiols, even when a modest concentration of nitrosating *Corresponding author, reagent is used. In the case of caspase-3, multiple cys- [email protected] teines are modified to nitrosothiols (5, 7); however, it is known that only one cysteine is modified in cells (3). The same can be said for the ryanodine receptor (10, 11). To Received for review September 12, 2006 date, poly-S-nitrosation has only been described in cells and accepted October 11, 2006. Published online November 3, 2006 when an exogenous source of NO was applied. A rea10.1021/cb600393x CCC: $33.50 sonable hypothesis is that specificity is achieved by S-nitrosation being a protein-assisted process. Without © 2006 by American Chemical Society

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assistance and regulation, accounting for previous observations proves difficult. To further support a role for protein-mediated S-nitrosation and denitrosation reactions in vivo, the active site of caspase-3 is effective at excluding glutathione (GSH). Cellular concentrations of GSH (low millimolar) do not inhibit enzymatic activity and large excesses of GSH do not efficiently restore the activity of caspase-3-Cys163-SNO (5, 7, 12). Since the caspase active site nitrosothiol is not freely exchangeable with GSH, S-nitrosoglutathione (GSNO) reductase cannot be directly responsible for recovery of caspase-3 activity upon apoptotic stimulation (13–16). The mechanism of how cells that produce NO are capable of restoring caspase activity remains to be determined. To aid the study of how S-nitrosation regulates the caspase cascade and apoptosis, we have developed and characterized a peptide-based reagent (PepSNO) designed to carry out a specific transnitrosation reaction with the catalytic cysteine of caspase-3 in apoptotic human T-cells. Other small molecule reagents, for example, GSNO and S-nitroso-N-acetyl penicillamine (SNAP) , do not exhibit specificity with the multiple reactive cysteines within caspase-3 (5). Peptide nitrosothiol reagents designed from known protease specificities have been previously used in attempts to engineer faster-acting nitrosating agents for papain (17) and human rhinovirus 3C protease (HRV 3C) (18). The strategy worked in the case of HRV 3C where a nitrosating agent was developed that exhibited a second-order rate of deactivation 8-fold higher than GSNO. Specificity of the reagent for the catalytic cysteine over the other two cysteines was inferred but not explicitly demonstrated. The nitrosation rate enhancement in the case of papain was ⬍2-fold. In both studies, the reagents were tested only on purified enzyme. In this work, we implemented a strategy similar to that of Xian et al. (17, 18) and designed a peptide based on the optimized substrate recognition sequence of human caspase-3, DEVD (12). In the design employed here, the more tolerant P2 position was changed from valine to a thiol-containing amino acid (cysteine, C; homocysteine, hC; mercaptonorvaline, mnV; and penicillamine, penA) for subsequent conversion to the corresponding nitrosothiol. As anticipated, the distance from the electrophilic nitrogen of the nitrosothiol moiety to the nucleophilic sulfur of caspase-3 was a crucial design element. The crystal structure of caspase-3 bound to 660

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both a peptide aldehyde (19) and a chloromethyl ketone inhibitor (20) indicated that the distance from the C␤ of valine (P2 of the peptide) to the sulfur of caspase-3Cys163 is 4.3 Å. The side chain of the nitrosothiolcontaining amino acid must be capable of spanning this distance in order to react with the catalytic cysteine. The distances from C␤ to the electrophilic nitrogen of the nitrosothiol in the extended conformation of S-nitroso-C, S-nitroso-hC, and S-nitroso-mnV are ⬃2.7, 3.9, and 5.1 Å, respectively. The orthogonally protected L-amino acids C, hC, and penA were commercially available (Bachem, N-Fmoc, and S-Trityl) for use in solid-phase peptide synthesis, but the similarly protected mnV reagent was not. We therefore developed a synthetic route to L-5-[S-trityl]-[N9-fluorenylmethyloxycarbonyl]-mnV starting from L-glutamic acid (Supplementary Figure 1). Fmoc chemistry was then used to synthesize a panel of peptides that fit the caspase-3 recognition motif, AcDEXD, where X is the thiol-containing amino acid (Supplementary Methods). The thiol-containing peptides were converted to nitrosothiols by reaction with acidified nitrite. The purified peptides were studied for the ability to deactivate recombinant human caspase-3 using a fluorimetric activity assay (5). As illustrated (Figure 1, panel a), both the S-nitroso-C and S-nitroso-hC containing peptides were capable of deactivating caspase-3, while the corresponding thiol peptides were not. Both the S-nitroso-C and S-nitroso-hC peptides displayed a rapid rate of caspase-3 deactivation when compared with GSNO (6- and 18-fold, respectively) but were not as efficient as thioredoxin-Cys73-SNO or an irreversible inhibitor (AcDEVD-chloromethylketone, (1.4 ⫾ 0.1) ⫻ 105 M–1 s–1) under identical conditions. This suggested that the ratedetermining step was the transnitrosation reaction, not peptide binding. To further address the mechanism of deactivation, the biotin switch method was utilized to confirm S-nitrosation of caspase-3. When treated with the S-nitroso-C and S-nitroso-hC peptides, caspase-3 gave a strong signal on a horseradish peroxidase conjugated NeutrAvidin Western (NA-HRP), indicating S-nitrosation of the large subunit of caspase-3. Surprisingly, caspase-3 treated with the S-nitroso-mnV and S-nitroso-penA peptides did not yield a signal above controls (Figure 1, panel b). Additional evidence implicating transnitrosation as the mechanism for loss of activity, as opposed to active site occupation, was provided by a competitive inhibition www.acschemicalbiology.org

ARTICLE Figure 1. S-Nitrosopeptides designed from the caspase-3 recognition sequence selectively S-nitrosate the caspase-3 catalytic cysteine in vitro. a) Recombinant caspase-3 was treated with the peptide nitrosating reagents, and the activity was measured. All peptides were based on the caspase-3 AcDEXD recognition motif. Assignments for the X axis are as follows: 1, S-nitrosocysteine; 2, cysteine; 3, S-nitrosohomocysteine; 4, homocysteine. Two transnitrosating agents not based on the AcDEXD motif were also studied under equivalent conditions: 9, S-nitrosoglutathione; 10, thioredoxin-C73-SNO. b) Caspase-3 was treated with the peptidic nitrosating agents and subjected to the biotin switch method. After SDS-PAGE and transfer to a nitrocellulose membrane, the blot was probed with NA-HRP. A loading control duplicate gel was stained with Coomassie. Assignments for AcDEXD are as follows: 1– 4, same as in panel a; 5, S-nitrosomercaptonorvaline; 6, mnV; 7, S-nitrosopenicillamine; 8, penA. c) Caspase-3 was treated with a mixture of the indicated nitrosating agent and the reversible and competitive peptide-aldehyde inhibitor AcDEVD-cho.

experiment, in which the Ki for the hC-containing peptide was determined to be 75 ␮M (Supplementary Figure 2). This inhibition constant is too weak to account for the observed loss of activity. Furthermore, competitive inhibition cannot explain the observed timedependent loss of activity. Finally, all of the AcDEXD reagents were tested for the ability to carry out an S-thiolation reaction (formation of a mixed disulfide with loss of HNO) with caspase-3-Cys163, but in no case was this product detected after trypsin digestion and matrixassisted laser desorption/ionization (MALDI) analysis (data not shown). Previous small molecule studies provide a possible explanation where transnitrosation reactions were shown to be ⬃100-fold faster than the related S-thiolation reaction (21, 22). Even so, our result was unexpected since similar conditions with GSNO led to detectable S-glutathiolation of caspase-3-Cys163 (5). Negative-mode MALDI and other sequencing-grade protease screens were also unsuccessful in detecting the AcDECD or AcDEhCD S-thiolation product. It is unlikely that the S-thiolation product was formed to a significant extent because MALDI readily detects the AcDECD and AcDEhCD peptides and the corresponding homodisulfides (data not shown). Two methods were employed to demonstrate that the transnitrosation reaction was specific for the caspase-3 active site cysteine, Cys163. First, a potent yet reversible caspase-3 peptide aldehyde inhibitor, AcDEVD-cho, was added with either the S-nitroso-C or S-nitroso-hC peptides to caspase-3, and the biotin switch method was carried out as usual. As shown (Figure 1, panel c), addiwww.acschemicalbiology.org

tion of AcDEVD-cho significantly reduced the Western signal, consistent with S-nitrosation at the catalytic cysteine. Additionally, MALDI mass spectrometry (MS) analysis on trypsin-digested caspase-3 that had undergone the biotin switch verified specificity for Cys163 (Supplementary Figure 3). It should be noted that selectivity for the active site cysteine is lost if caspase-3 is treated with ⬎3 equiv of the S-nitrosating agent. MALDI data confirmed that caspase-3 treated with 4 molar equiv of the S-nitroso-hC peptide formed a nitrosothiol on Cys264, a surface-accessible cysteine (data not shown) (20). The S-nitroso-hC peptide was additionally screened against papain, a much less specific cysteine protease, and caspase-7 to probe reagent specificity with other cysteine nucleophile enzymes. Reaction of papain with the S-nitroso-hC peptide (3 molar equiv) did not inhibit proteolysis of bovine serum albumin (data not shown). Because of the high sequence and structural homology of caspase-7 to caspase-3 (23) and the fact that both proteases optimally cleave after DEVD (12), it was anticipated that the S-nitroso-hC peptide would deactivate caspase-7. This peptide did react with caspase-7, albeit 30-fold slower (0.62 ⫾ 0.03 M–1 s–1) compared with caspase-3. The slower reaction of the S-nitroso-hC peptide with caspase-7 may stem from this enzyme being less efficient (kcat/KM (caspase-3) for AcDEVDpara-nitroanilide ⫽ 2.2 ⫻ 105 M–1 s–1; caspase-7 ⫽ 3.7 ⫻ 104 M–1 s–1) (24). Because of a faster transnitrosation reaction with caspase-3-Cys163, the S-nitroso-hC-containing peptide VOL.1 NO.10 • 659–665 • 2006

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Figure 2. PepSNO inhibits apoptosis in Jurkat cells more effectively than GSNO. a) Structure of PepSNO. b) Etoposide (2.5 ␮g mL–1) was added to Jurkat cells under reduced light conditions for 5 h to induce apoptosis before the addition of peptide reagent (1 mM). The peptide reagents were allowed to react with the cells for an additional 5 h at 37 °C before harvesting, lysing, and measurement of the AcDEVD-ase activity by fluorimetry, reported as relative fluorescent units (RFUs): column 1, PepSH; column 2, PepSNO; column 3, PepSH and etoposide; column 4, PepSNO and etoposide. Error is standard deviation (SD) (n ⴝ 3). c) Sample preparation was the same as that for panel b. The activity of each sample was baseline subtracted from a sample that did not receive etoposide. The relative activities shown are normalized to the peptide thiol activity, which were equivalent within error (SD, n ⴝ 3): column 1, 2.5 mM GSH; column 2, 0.5 mM GSNO; column 3, 2.5 mM GSNO; column 4, 2.5 mM PepSH; column 5, 0.5 mM PepSNO; column 6, 2.5 mM PepSNO. d–f) Apoptosis was also quantified by the Vybrant flow cytometry assay. Apoptotic cells appear in the lower right quadrant: d) 0.5 mM PepSNO; e) 0.5 mM PepSH and etoposide; f) 0.5 mM PepSNO and etoposide.

was used in subsequent cellular experiments. Anticipating difficulties with transport into cells, we converted the four carboxylate moieties of the peptide to methyl esters (Figure 2, panel a). The efficacy of this esterified compound, hereafter “PepSNO”, was examined first by adding the reagent to intact Jurkat cells (human T-cell lymphoma) that were treated with either etoposide or a buffer control for 5 h before addition of PepSNO and the thiol control, PepSH. After an additional 5 h treatment, cells were lysed and tested for caspase-3 activity (Figure 2, panel b). The caspase-3/7 (AcDEVD-ase) activity remained virtually unchanged in the non-etoposidetreated cells, regardless of treatment with PepSH or PepSNO. In the cells that were treated with etoposide, however, treatment with PepSNO reduced the AcDEVDase activity to barely detectably levels, while the PepSHtreated cells exhibited a high level of activity. The effectiveness of PepSNO was then compared with GSNO at two different concentrations (Figure 2, panel c). PepSNO (0.5 mM) was capable of reducing AcDEVD-ase activity by almost 90%, while an equal concentration of GSNO reduced the activity by 35%. Each reagent at 2.5 mM completely inhibited AcDEVD-ase activity. The two most probable explanations for the increased potency of the PepSNO reagent are (i) a greater rate of association with caspase-3 and transnitrosation with Cys163 and (ii) increased cellular permeability. To address the contribution of permeability, the two carboxylates in GSH and 662

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GSNO were converted to their methyl esters (GSHe and GSNOe, respectively, Supplementary Methods) and examined under identical conditions. Treatment of intact Jurkat cells with GSNOe (0.5 mM) inhibited AcDEVD-ase activity (65%) to a greater extent than treatment with GSNO but still not as efficiently as treatment with PepSNO, P ⬍ 0.005 (Student’s t-test, Supplementary Figure 4, panel a). AcDEVD-ase activity is a relatively early stage marker for apoptosis. A later marker in Jurkat cells is the translocation of phosphatidylserine (PS) to the cell surface (25). Using the Vybrant apoptosis assay kit (Invitrogen), we quantified the extent of apoptosis (annexin V, binds specifically to PS) and necrosis (propidium iodide) in cells treated with PepSH and PepSNO (Figure 2, panels d–f). Without etoposide treatment, the percentage of apoptotic Jurkat cells was 1% (Figure 2, panel d). Cells that were stimulated to undergo apoptosis with etoposide and were treated with PepSH (0.5 mM) contained a high percentage of apoptotic cells (Figure 2, panel e), but the same treatment with PepSNO reduced the extent of apoptosis by almost two-thirds (Figure 2, panel f). The same analysis was carried out for GSH, GSNO, GSHe, and GSNOe (Supplementary Table 1). Treatment of cells with GSNO and GSNOe led to an increase in the percentage of apoptotic cells in the absence of etoposide, while treatment of cells with GSH and GSHe inhibited etoposide-induced apoptosis, which was in accord with earlier observations (26). www.acschemicalbiology.org

ARTICLE Figure 3. PepSNO transnitrosates the caspase-3 active site cysteine in apoptotic Jurkat cells. a) Relative S-nitrosation of caspase-3 in etoposide treated Jurkat cells. After a 5 h treatment with etoposide, the indicated peptide reagent (0.5 mM) was added for an additional 5 h. Anti-caspase-3 was used to purify caspase-3 by immunoprecipitation, which was then subjected to the biotin switch. Blots were probed with NA-HRP and anticaspase-3 to ascertain the extent of caspase-3 S-nitrosation and to normalize for total caspase-3: column 1, GSH; column 2, GSNO; column 3, GSHe; column 4, GSNOe; column 5, PepSH; column 6, PepSNO. P-values were calculated by the Student’s t-test: (gray) P ⴝ 0.02; (white) P ⴝ 0.10; (black) P < 0.001; (#) P ⴝ 0.006. b) A cell-permeable, caspase-3 reversible inhibitor (CPCI) competes with PepSNO for the active site of caspase-3. Cells were treated with etoposide and PepSH or PepSNO as usual, but 30 min prior to addition of PepSH or PepSNO, CPCI or DMSO was added to the cells: lane 1, PepSNO without CPCI; lane 2, PepSNO with CPCI; lane 3, PepSH without CPCI; lane 4, PepSH with CPCI.

These results underscore the necessity for more selective reagents to study the effects of S-nitrosation in apoptosis and other apoptotic processes (27). While we cannot rule out the possibility that PepSNO is participating in side reactions at this concentration and time course, the data confirm that this reagent is superior to GSNO and GSNOe in the inhibition of caspase-3dependent apoptosis via S-nitrosation of the catalytic cysteine. A further demonstration that PepSNO is a more selective reagent than GSNO and GSNOe is observed when the biotin switch method is performed on the entire cellular lysate using 0.5 mM of each peptide reagent. This method detects only abundant S-nitrosated proteins; therefore, caspase-3 is not detected (28). After treatment with GSNOe, the lysate contained numerous bands reactive toward NA-HRP, while the bands from GSH-, GSNO-, and GSHe-treated lysates were significantly more faint (Supplementary Figure 4, panel b). An identical assay using PepSH and PepSNO did not have any visible bands (data not shown), indicating that even though PepSNO was used at a higher concentration in the cellular experiments than in the in vitro experiments (0.5 mM vs 20 ␮M), specificity for the caspase active site is retained within our detection. When one considers that PepSNO must penetrate the cell membrane and be processed by intracellular esterases before binding to the caspase-3 active site, it is understandable that a higher concentration is required in cells to attain inhibition of enzyme activity. Despite the nonselective nature of GSNO and GSNOe, we were able to detect caspase-3-SNO in apoptotic Jurkat cells treated with these reagents using antiwww.acschemicalbiology.org

caspase-3 immunoprecipitation and the biotin switch method. The relative amount of caspase-3-SNO, which was normalized to total caspase-3, was determined for cells treated with 0.5 mM of the following: GSH, GSNO, GSHe, GSNOe, PepSH, and PepSNO (Figure 3, panel a). Cells treated with the thiol version of each reagent contained a basal amount of caspase-3-SNO, while the nitrosothiol derivative increased the percentage of caspase-3-SNO. The trend observed, PepSNO ⬎ GSNOe ⬎ GSNO, complied with the prediction based on the AcDEVD-ase activity of similarly treated cells (Figure 2, panel c and Supplementary Figure 4, panel a). Selectivity for the catalytic cysteine of caspase-3 in a cellular context was demonstrated for PepSNO by pretreating Jurkat cells with a cell-permeable, C-terminal aldehyde (reversible), caspase-3-selective active site inhibitor (Figure 3, panel b). The level of caspase-3 S-nitrosation was highest in cells treated with PepSNO lacking the inhibitor, while cells treated with PepSNO and the inhibitor had levels of caspase-3-SNO comparable to controls (PepSH with or without inhibitor). A low but detectable amount of procaspase-3 was found to be S-nitrosated. However, S-nitrosation on the processed caspase was much greater. In summary, we have developed a cell-permeable peptide nitrosothiol (PepSNO) that inhibits etoposidestimulated apoptosis in Jurkat cells via S-nitrosation of the catalytic cysteine of caspase-3. We characterized this process using a fluorimetric caspase-3/7 activity assay, flow cytometry, and the biotin switch method on purified enzymes, Jurkat cell lysate, and immunoprecipitated caspase-3. Specificity for the active site cysteine of caspase-3 was shown by active site competitive inhiVOL.1 NO.10 • 659–665 • 2006

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bition experiments and MS. In each of these studies, the efficacy of PepSNO was superior to the commonly used biological transnitrosating agent GSNO and a more cellpermeable version, GSNOe. Derivatives of PepSNO may be tailored in future studies to be selective for individual caspase isoforms. If the optimal recognition sequences

are used as a blueprint (12), reagents could be designed with the expectation to be as caspase-selective as the related proteolytic substrate or aldehyde inhibitor (24). Reagents such as these will help elucidate the mechanism of NO- and nitrosothiol-dependent regulation of the caspase cascade.

METHODS

AcDEVD-ase Activity of T-Cells. Jurkat cells were lysed under reduced light in RIPA/HEN buffer (100 mM 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid, 150 mM NaCl, 1 mM EDTA, 1 mM NaF, 100 ␮M neocuproine, 5% glycerol, 1% Triton X-100, 1% sodium deoxycholate, 0.05% SDS, pH 7.4), containing Complete (Roche) protease inhibitor cocktail, by mild vortexing at 4 °C. The insoluble fraction was removed by centrifugation (14,000 rpm, 15 min, 4 °C). Total soluble protein was quantified by the Bradford assay and normalized to 1.0 –2.0 mg mL–1 by dilution into RIPA/HEN, and the solution was supplemented with AcDEVD-amc (75 ␮M). AcDEVD-ase (caspase-3/7) activity was measured on a Molecular Devices Spectra Max Gemini XS (␭ex ⫽ 380 nm, ␭em ⫽ 460 nm) using 96-well black fluorimetry plates (Corning) in triplicate. Immunoprecipitation. Under reduced light, Jurkat cells were lysed, and the insoluble fraction was removed as above. The supernatants were then precleared by addition protein G–Sepharose 4B (Invitrogen, 30 ␮L, equilibrated in RIPA/HEN) for 30 min at 4 °C with rocking. The beads were pelleted by centrifugation, and the supernatant was transferred to a clean tube containing the anti-caspase-3 polyclonal antibody (Cayman Chemical, 30 ␮L). The antibody was allowed to bind for 90 min at 4 °C with rocking, after which the protein G beads were again added (40 ␮L, equilibrated in RIPA/HEN) for an additional 90 min binding at the same temperature (reduced light). The immunoprecipitate was pelleted by centrifugation (2000 rpm, 2 min), and the supernatant was decanted. The beads were washed three times with RIPA/HEN (1 mL) before elution of the immunocomplex from the beads using the biotin switch blocking buffer (contains 2.5% w/w SDS and 30 mM N-ethylmaleimide). The elution was carried out under reduced light at 55 °C for 25 min with frequent vortexing. After removal of the beads by centrifugation, the supernatant was subjected to the biotin switch method as described above.

General. Purification of recombinant human caspase-3 was carried out as previously described (5). Jurkat cells were maintained in a 5% CO2, water-saturated atmosphere at 37 °C in RPMI-1640 with glutamine (Gibco) supplemented with fetal bovine serum (FBS, 10%), penicillin (100 U mL–1), and streptomycin (100 ␮g mL–1). Rate of in Vitro Caspase Deactivation. Recombinant caspase-3 and caspase-7 (0.1–5 ␮M) were reacted with varying concentrations of peptide nitrosating reagents (1–300 ␮M, dark, 25 °C, pH 7.4) under nonreducing conditions. At timed intervals, caspase activity was measured by diluting the above solution to a final caspase concentration (20 –30 nM) in assay buffer supplemented with the profluorescent aminomethylcoumarin substrate AcDEVD-amc (75 ␮M, Calbiochem). Rate of deactivation was quantified by plotting the remaining AcDEVD-ase activity versus time of reaction. Rate constants were calculated using the bimolecular rate equation, V ⫽ k[caspase][inhibitor]. Due to the rapid deactivation kinetics exhibited by the irreversible chloromethylketone inhibitor, AcDEVD-cmk (Calbiochem), a lower concentration of enzyme (10 –50 nM) and inhibitor (5–50 nM) were used. Biotin Switch Method. Recombinant caspase-3 (8 ␮M) was allowed to react under reduced light with various peptide nitrosating agents (20 ␮M) for 20 min at RT and then subjected to the biotin switch method (5, 29). To demonstrate specificity for the active site cysteine of caspase-3 (Cys163), a separate experiment included a reversible peptide-aldehyde inhibitor, AcDEVDcho (100 ␮M, Calbiochem). The biotin switch method was also used to show S-nitrosation of caspase-3 in human T-cells. Whole-cell lysates and caspase-3 immunoprecipitates (see below), using GSNO, GSNOe, and PepSNO as transnitrosating agents, were analyzed as previously described (30 –32). The selectivity of PepSNO for the active site cysteine of caspase-3 was demonstrated in Jurkat cells using cells pretreated with the cell-permeable, reversible caspase-3 inhibitor CPCI, AcAAVALLPAVLLALLAP-DEVD-cho (50 ␮M, Alexis Biochemicals), 30 min prior to the addition of PepSH/SNO. After the NA-HRP Westerns were imaged, blots were stripped (Restore, Pierce), blocked, and probed with anti-caspase-3 (Cayman Chemical) to ensure equal loading. Induction of Apoptosis and Peptide Treatment. Jurkat cells were harvested and resuspended in media containing reduced serum (1% FBS) and etoposide (2.5 ␮g mL–1, Sigma). After 5 h at 37 °C, PepSNO, PepSH, GSNO, GSH, GSNOe, and GSHe (0.5 and 2.5 mM) were added to the cultures (10 mL) under reduced light conditions. Cells were harvested by centrifugation after a further 5 h incubation and washed twice (PBS) prior to lysis. Flow Cytometry. Flow cytometry was performed with a FACSCalibur flow cytometer (BD Biosciences) using CellQuest software. Apoptosis and necrosis were quantified using 50,000 intact cells per sample that were labeled with propidium iodide and Alexa Fluor 488 conjugated annexin V as suggested by the manufacturer’s instructions (Vybrant Kit no, 2, Invitrogen).

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Acknowledgments: We thank Phung Gip and Carolyn Bertozzi (UC–Berkeley) for the use of equipment, Debajyoti Datta and James Wells (UC–San Francisco) for caspase-7, Nathaniel Martin, Joshua Woodward, and Jacquin Niles (UC–Berkeley) for helpful discussions, and finally, members of the Marletta laboratory for critical review of the manuscript. This work was supported in part by grants from the National Institutes of Health and DeBenedictis Fund of UC–Berkeley to M.A.M. D.A.M. is supported by an American Heart Association predoctoral fellowship. Supporting Information Available: This material is free of charge via the Internet. Competing interests statement: The authors declare that they have no competing financial interests.

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