Modification of Tubulin Cysteines by Nitric Oxide and Nitroxyl Donors

modifying the cysteines of tubulin and at inducing the formation of tubulin interchain disulfide ... our work on brain microtubule proteins including ...
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Chem. Res. Toxicol. 2007, 20, 1693–1700

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Modification of Tubulin Cysteines by Nitric Oxide and Nitroxyl Donors Alters Tubulin Polymerization Activity† Lisa M. Landino,* Maria T. Koumas, Courtney E. Mason, and Jane A. Alston Department of Chemistry, The College of William and Mary, P.O. Box 8795, Williamsburg, Virginia 23187-8795 ReceiVed May 2, 2007

The modification of reduced cysteines of proteins by nitric oxide alters protein function, structure, and potentially, interactions with downstream signaling targets. We assessed the effect of the S-nitroso compounds S-nitrosoglutathione and S-nitroso-N-acetyl-penicillamine, the NO donor 2-(N,N-diethylamino)diazenolate 2-oxide, and the nitroxyl donor Angeli’s salt on the cysteines of the abundant cytoskeletal protein, tubulin. Total cysteine modification by each compound was quantitated and compared to peroxynitrite anion, an oxidant that we have studied previously. Angeli’s salt was most effective at modifying the cysteines of tubulin and at inducing the formation of tubulin interchain disulfide bonds followed by peroxynitrite anion, S-nitrosoglutathione, S-nitroso-N-acetyl-penicillamine, and 2-(N,Ndiethylamino)-diazenolate 2-oxide. S-nitrosation of tubulin by S-nitrosoglutathione and S-nitroso-N-acetylpenicillamine was detected by the Saville assay. Our data show that tubulin interchain disulfide bond formation by these molecules correlated with inhibition of tubulin polymerization. Closer examination of the reaction of tubulin with S-nitrosoglutathione showed a concentration-dependent shift in the type of cysteine modification detected. More tubulin disulfides were detected at lower concentrations of S-nitrosoglutathione than at higher concentrations, suggesting that reduced glutathione, generated by the reaction of S-nitrosoglutathione with tubulin cysteines, reduced disulfides initially formed by Snitrosoglutathione. Introduction In recent years, the potential role of nitric oxide (NO)2 as a reversible modulator of protein function has received considerable attention. Like phosphorylation, the chemical modification of reduced cysteines of proteins by NO, a modification called S-nitrosation or S-nitrosylation, alters protein function, structure, and consequently, interactions with downstream signaling targets (1–3). For example, one specific cysteine of the G-protein ras p21, cys118, is nitrosated and addition of NO to this residue serves as a “redox switch” to alter both the rate of guanine nucleotide exchange onto ras p21 and the interactions between ras p21 and MAP kinase, a protein activated by ras p21 (4). Among other well-studied protein targets for S-nitrosation are caspase-3 and the ryanodine receptor (2, 5–7). Our interest in protein thiol modification by NO stems from our work on brain microtubule proteins including tubulin and the microtubule-associated proteins (MAPs)1 tau and MAP2. We reported that mammalian brain microtubule protein is readily oxidized by peroxynitrite anion (ONOO-) in vitro. (8) We observed that the cysteines of microtubule proteins, rather than other amino acids, were most susceptible to oxidation by ONOO- and that increased thiol oxidation correlated with inhibition of microtubule polymerization. Interchain disulfides * To whom correspondence should be addressed. Telephone: (757) 2212554. Fax: (757) 221-2715. E-mail: [email protected]. † This manuscript is dedicated to Larry Marnett in honor of his 60th birthday. Thank you for your thoughtful mentorship, continued guidance, and enduring good humor. 1 The abbreviations used are as follows: GSNO, S-nitrosoglutathione; SNAP, S-nitroso-N-acetyl-penicillamine; AS, Angeli’s salt; DEA NONOate, 2-(N,N-diethylamino)-diazenolate 2-oxide; HNO, nitroxyl; TCEP, (tris(2carboxyethyl)phosphine hydrochloride); IAF, 5-iodoacetamido-fluorescein; MAPs, microtubule-associated proteins; ONOO-, peroxynitrite anion; PME buffer, 0.1 M PIPES, 1 mM MgSO4, 1 mM EGTA.

between the R- and β-tubulin subunits were detected by Western blot using antitubulin antibodies under nonreducing conditions. ONOO--induced disulfide bonds are at least partially responsible for the inhibition of microtubule polymerization that we observed, because the addition of reductase enzymes including thioredoxin reductase and the glutaredoxin reductase system restored a significant fraction of the polymerization activity that was lost following ONOO- addition (9, 10). Oxidative modification of tubulin and MAPs is expected to alter the dynamic assembly properties of microtubules and may affect the interactions of microtubules with auxiliary proteins. Tubulin, a heterodimer composed of similar 50 kDa R- and β-subunits, contains 20 reduced cysteines in its native state (11, 12). Our work and that of others shows that tubulin cysteine oxidation and/or modification inhibits microtubule polymerization (12–14). Given the knowledge that we have gained regarding the oxidation of tubulin and MAPs cysteines by ONOO-, we are interested in assessing the effects of NO on microtubule protein function. This is especially important because both oxidative modifications of proteins and cytoskeletal abnormalities have been detected in the brains of those afflicted with Alzheimer’s disease (15–17). Finally, cytoskeletal proteins such as tubulin and MAPs are among the most abundant proteins in a neuron; therefore, they are likely candidates for modification by oxidants (18, 19). In a study by Snyder and co-workers using mouse brain lysates and S-nitrosoglutathione (GSNO) as a NO donor, both R- and β-tubulin subunits were identified as sensitive targets for S-nitrosation using the biotin switch assay. (20) Furthermore, by comparing S-nitrosation in control mouse brain vs. mouse brain from neuronal nitric oxide synthase knockout mice, β-tubulin was identified as an endogenous target for S-

10.1021/tx7001492 CCC: $37.00  2007 American Chemical Society Published on Web 10/02/2007

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nitrosation in vivo. Thus, redox regulation of tubulin by NO is an area of research that deserves considerable attention. Herein, we investigate the effects of the S-nitroso compounds, GSNO, S-nitroso-N-acetyl-penicillamine (SNAP), the NO donor DEA NONOate, and the nitroxyl (HNO) donor Angeli’s salt on the cysteines of purified porcine tubulin. Total cysteine modification, the formation of tubulin disulfides, tubulin Snitrosation, and the effects on microtubule polymerization are presented and compared with those of ONOO-.

Experimental Procedures Materials. Porcine brains were obtained from Smithfield Packing Company in Smithfield, VA. Angeli’s salt was from Cayman Chemicals (Ann Arbor, MI). The NO donors, GSNO and SNAP, were either synthesized as described or purchased from Sigma (21). DEA NONOate was from Sigma-Aldrich. BCA protein assay reagent, West Pico chemiluminescence detection system, TCEP, and 5-iodoacetomido-fluorescein (IAF) were from Pierce. The mouse anti-β-tubulin antibody, clone 2.1, and the goat antimouse HRP conjugate were from Sigma. Synthesis of ONOO-. ONOO- was synthesized from acidified H2O2 and sodium nitrite as described in the literature (22).The concentration of ONOO- was determined by measuring the absorbance at 302 nm (ε302 ) 1670 M-1 cm-1) in 0.1 M NaOH. Purification of Porcine Brain Tubulin. Tubulin was purified from porcine brain by two cycles of temperature-dependent polymerization and depolymerization and subsequent phosphocellulose chromatography as described (23). Tubulin (typically 2.5–4.0 µg/µL) in PME buffer (0.1 M PIPES, pH 6.9, 1 mM MgSO4, 2 mM EGTA) containing 0.1 mM GDP was aliquoted and stored at -80 °C. Tubulin concentrations were determined by the bicinchoninic acid (BCA) protein assay (Pierce). Labeling of Tubulin Cysteines with IAF. Tubulin was diluted in either 0.1 M phosphate buffer pH 7.4 containing 1 mM DTPA or PME buffer pH 6.9 and then treated with each NO or HNO donor or ONOO- for 5–30 min at 37 °C in a total reaction volume of 30 µL (20 µg protein). ONOO- stock solutions were diluted with 0.1 M NaOH just prior to use and the volume of ONOOsolution added to achieve the indicated concentrations was normalized to avoid variations in pH. A solution of Angeli’s salt was prepared in 0.01 M NaOH. Solutions of SNAP, GSNO, or DEA NONOate were prepared in buffer. IAF in DMF was added to a final concentrations of 1.5 mM (2 µL) and samples were incubated at 37 °C for an additional 30 min. Proteins were resolved by SDSPAGE on a 7.5% gel under reducing conditions, and gel images were captured using a Kodak DC290 system and a UV transilluminator. The intensity of the fluorescein-labeled protein bands was measured using Kodak 1D Image Analysis software. Alternatively, fluorescein-labeled R- and β-tubulin bands were excised from the gel and placed in 200 µL 0.375 M Tris pH 8.8. After the protein had diffused from the gel, protein-bound fluorescein was quantitated at 494 nm. Detection of Interchain Disulfides by Western Blot. Following treatment with each NO or HNO donor or ONOO- as described above, tubulin species (10 µg total protein per lane) were separated by SDS-PAGE on 7.5% polyacrylamide gels under nonreducing conditions. Proteins were transferred to PVDF membranes, blocked with 3% milk for 30 min, and probed with Tub 2.1, a mouse monoclonal anti-β-tubulin antibody (1:2000) for two hours. The β-tubulin/antibody complex was visualized by chemiluminescence using the Pierce West Pico system. Microtubule Polymerization Assays. Purified tubulin (100 µL, 2.5 µg/µL) in PME was treated with each oxidant, NO, or HNO donor for 15 min at 25 °C. GTP (1 mM final) was added and the samples were incubated at 37 °C for another 20 min. Microtubule polymer was collected by centrifugation at 16000 g for 20 min. The supernatants were removed, and the protein concentration in each was determined by the BCA assay. Detection of Tubulin S-Nitrosation. Purified tubulin (60 µL, 0.75 µg/µL) was diluted with PB pH 7.4 and treated with GSNO

Figure 1. Modification of tubulin cysteines by NO and HNO donors. (A) Tubulin samples (8 µM protein; 160 µM cysteines) were treated with 100 or 500 µM donor for 30 min at 37 °C. IAF was then added to modify remaining thiols and samples were analyzed by SDS-PAGE under reducing conditions. The gel image was captured using the Kodak DC290 system and a UV transilluminator. (B) Quantitation of fluorescein labeling of tubulin cysteines. R- and β-tubulin bands were excised from the gel, and fluorescein was quantitated by measuring absorbance at 494 nm. The results represent the mean of at least three independent experiments.

or SNAP for 30 min. Ethanol was added to 80%, samples were left on ice for 15 min to precipitate protein, and the protein pellet was collected by centrifugation at 10000 g for 10 min. Pellets were washed with 80% ethanol and then resuspended in 60 µL of PB pH 7.4 containing 1% SDS. Samples were heated to 45–50 °C to aid in dissolving protein pellets. NO bound to protein was detected by the modified Saville assay described by Cook and co-workers (24). Neutral Greiss reagent and HgCl2 were added to the protein samples and the product derived from NOx was detected at 496 nm. Concentrations of SNO were calculated from a GSNO standard curve prepared in the presence of 1% SDS.

Results and Discussion Purified porcine tubulin was treated with GSNO, SNAP, and DEA NONOate, and their ability to modify tubulin cysteines was assessed. All reactions were performed in either phosphate buffer pH 7.4 or, in the case of polymerization assays, at pH 6.9 in PME buffer. All buffers contained 1 mM DTPA to prevent metal-catalyzed oxidation reactions. We routinely monitor cysteine oxidation and reduction by treating protein samples with a thiol-specific fluorescein labeling reagent, 5-iodoacetamido-fluorescein (IAF) (10, 25). Oxidized cysteines cannot be labeled; thus, as the concentration of NO donor or oxidant increases, the incorporation of fluorescein into tubulin decreases. Figure 1A shows a representative SDS-PAGE result using this methodology. Micromolar concentrations of tubulin were treated with either 100 or 500 µM concentrations of each compound or with ONOO-, an oxidant we have already studied in detail, for comparison. In Figure 1A, we compare the effect of GSNO and ONOO- on

Tubulin Cysteines and Nitric Oxide

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tubulin cysteine modification. The similar R- and β-tubulin subunits are resolved by SDS-PAGE. Both the R- and β-tubulin band intensities decreased at both concentrations of GSNO and ONOO- tested relative to the control, indicating that cysteines on both subunits are modified. ONOO- was more effective than GSNO at modifying the cysteines of tubulin as evidenced by the lighter bands in lanes 4 and 5 relative to those in lanes 2 and 3. Though the tubulin concentration was only 7 µM in these assays, the cysteine concentration is 140 µM because each tubulin dimer contains 20 reduced cysteines (12 in R-tubulin and 8 in β-tubulin) (11). The extent of modification of the individual tubulin subunits by GSNO, SNAP, DEA NONOate, ONOO- and the HNO donor, Angeli’s salt (labeled AS), was determined by measuring the amount of protein-bound fluorescein in each band. The results are summarized in Figure 1B. Angeli’s salt was by far the most effective at modifying the cysteines of tubulin with only 25% of cysteines remaining after treatment with 100 µM AS. This corresponds to the modification of 15 cysteines (9 in R-tubulin and 6 in β-tubulin), or 105 µM cysteine in tubulin, by 100 µM AS. Clearly HNO reacts in a manner different from NO and from the S-nitroso compounds. These data show that AS, a source of HNO, is one of the most thiophilic reagents known. After AS, ONOO- was the most effective followed by GSNO, SNAP, and lastly, DEA NONOate (labeled DN). It is notable that none of the compounds tested selectively modified R- or β-tubulin; in all cases, both subunits were modified to a comparable extent, suggesting similar cysteine reactivity. A direct comparison is challenging because of the differing chemical natures of the oxidants/NO donors used in Figure 1B. Both GSNO and SNAP are S-nitrosothiols that react with protein cysteines (R′SH) via transnitrosation as shown in eq 1 (26, 27).

RNSO + R'SH f RSH + R'SNO

(1)

The NONOate class of NO donors are not S-nitroso compounds but rather release NO free radical as they decompose in solution. Samples containing tubulin and the oxidants/NO donors were incubated at 37 °C for 30 min, but ONOO(H) is very unstable at neutral pH and must be stored in dilute NaOH solution. Thus, a bolus addition of 100 or 500 µM ONOO- is misleading, because upon addition of a tiny volume of ONOO- stock solution to a buffered protein solution at physiological pH, ONOO- either reacts with protein target or isomerizes to form nitrate ion within seconds (28–30). Likewise, HNO cannot be stored and can only be studied using molecules such as Angeli’s salt, Na2N2O3, a commercially available HNO donor. The decomposition of Angeli’s salt to produce HNO proceeds as shown in equation 2 (31). The concentration of HNO produced in reactions with protein targets depends on both the pH and the presence of oxygen (32).

N2O32- + H+ f HNO + NO2-

(2)

A recent manuscript by Miranda and colleagues examined both the rate and mechanism of Angeli’s salt decomposition (32). They reported a rate constant of 2.7 × 10-3 s-1 at pH 7.4 under aerobic conditions (t1/2 ≈ 4 min). Their previous work had also compared the reactivity of Angeli’s salt with synthetic ONOO- and found them to be chemically different, though the reaction outcomes were often the same (33). IAF labeling as shown in Figure 1A tells us that cysteines of tubulin have been modified by the compounds tested, but it does not provide any information about the type of modification. In

Figure 2. Detection of NO and HNO-donor induced interchain tubulin disulfides. Tubulin samples (8 µM protein; 160 µM cysteines) were treated with 25 or 100 µM donor for 30 min at 37 °C. Protein species were separated by SDS-PAGE under nonreducing conditions on a 7.5% polyacrylamide gel and transferred to PVDF. Blocked membranes were incubated with anti-β-tubulin (1:2000) for 2 h. The tubulin–antibody complex was visualized using a chemiluminescence detection system. Monomeric β-tubulin (50 kDa) is labeled, as well as dimers and tetramers. (A) Tubulin samples were treated with 25, 50, and 100 µM GSNO (lanes 3–5) and SNAP (lanes 6–8). (B) Tubulin samples were treated with 25 or 100 µM GSNO (lanes 2 and 3), DEA NONOate (lanes 4 and 5), and Angeli’s salt (lanes 6 and 7). (C) Tubulin samples were treated with 25 and 100 µM GSNO (lanes 2 and 3), ONOO(lanes 4 and 5), and Angeli’s salt (lanes 6 and 7). Tubulin in lane 8 was treated with TCEP to reduce disulfides.

a protein like tubulin with 20 reduced cysteines, it is likely that disulfide bonds will form following S-nitrosation of a tubulin cysteine. For example, GSNO reacts with a protein thiol (PSH) to form a nitrosated protein intermediate according to eq 3. The intermediate can be attacked by an adjacent protein thiol(ate) to yield a disulfide bond (PSSP′) and HNO eq 4). Indeed, for proteins like tubulin with multiple cysteines, disulfides would be the expected product (1, 34).

GSNO + PSH f GSH + PSNO PSNO + P'SH f PSSP' + HNO

(3) (4)

Our work with ONOO- showed that interchain disulfides between tubulin subunits form readily and these can be detected by Western blot analysis of samples run on SDS-PAGE under nonreducing conditions (8). Both dimers and tetramers of approximately 100 and 200 kDa, respectively, are observed. Figure 2A shows a Western blot of 7 µM tubulin (140 µM cysteine) treated with 25, 50, or 100 µM GSNO or SNAP for

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30 min. The control in lane 1 shows a major band at 50 kDa corresponding to β-tubulin and a small amount of tubulin dimer. Tubulin used in these experiments is purified in the absence of a reducing agent; thus, some air oxidation does occur. When TCEP, a phosphine-based disulfide reducing agent, is added as shown in lane 2, the higher-molecular-weight dimer disappears. GSNO induced a dose-dependent increase in the formation of both tubulin dimers and tetramers. Tubulin disulfides were detected when tubulin was treated with as little as 10 µM GSNO (data not shown). Of note, as tubulin becomes oxidized, the main β-tubulin band at 50 kDa broadens because of an increase in intrachain disulfides. Likewise, as tubulin is reduced, the band tightens. This is attributed to changes in SDS binding as the protein is oxidized or reduced. Following separation under nonreducing conditions, the R- and β-tubulin bands are more diffuse and several dimer and tetramer bands are observed. The protein tubulin, as purified from brain tissue, is composed of multiple individual R- and β-tubulin gene products that vary slightly in molecular weight (35). Thus, different Rβ dimer combinations yield variations in the mass of interchain disulfide-linked dimers and tetramers. Also of note, not all tubulin disulfides are interchain; intrachain disulfides would not yield higher-molecular weight species on a Western blot. However, in our experience, the relative amount of higher-molecular-weight species observed on a Western blot is proportional to total disulfides. Unlike GSNO, SNAP did not induce the formation of interchain tubulin disulfides significantly (Figure 2A). Though the amount of dimers and tetramers formed by all concentrations of SNAP tested is greater than that of the control, the increase in disulfides does not appear to be dose-dependent. In fact, SNAP was tested at concentrations up to 600 µM and little to no additional interchain disulfides were observed (data not shown). One suggestion is that SNAP does not react to the same extent as GSNO because it is more hindered (see Scheme 1). The SNO group of SNAP is bound to a tertiary carbon whereas the SNO in GSNO is bound to a primary carbon. This difference would be expected to affect the reactivity of a protein thiol with the sulfur of the SNO group. Total cysteine modification by SNAP is certainly less than that induced by GSNO (Figure 1B). When creatine kinase (CK) was treated with GSNO or SNAP, Konorev and colleagues noted a difference in the type of cysteine modification induced by each. GSNO induced Sglutathionylation of CK, whereas the predominant modification by SNAP was S-nitrosation (36). Because SNAP is more hindered, a protein thiol(ate) would react with the nitrogen of the NO donor to yield S-nitrosated protein. Conversely, a protein

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thiol(ate) would react with the sulfur of GSNO to yield glutathionylated protein, PSSG. A second protein thiol could then react with PSSG to yield a protein disulfide and GSH. The rates of reaction of a protein with GSNO vs. SNAP would be expected to vary as well. Also of note, GSNO has multiple charges at physiological pH, which may enhance its interaction with tubulin. In Figure 2B, the extent of interchain disulfides induced by 25 and 100 µM GSNO, DEA NONOate, and AS are compared. Tubulin disulfide bond formation was greatest in those samples treated with AS. In fact, with the higher dose of 100 µM AS tested, the tubulin is oxidized to such an extent that the majority of it fails to enter the separating gel and only a small band of monomeric β-tubulin remains (Figure 2B, lane 7). DEA NONOate induces some interchain disulfide bond formation but it was barely greater than control levels. Even a dose as high as 600 µM DEA NONOate failed to induce significant tubulin oxidation (data not shown). Thus, NO free radical, even in the presence of oxygen, does not oxidize tubulin cysteines or regulate tubulin function. Lastly, tubulin interchain disulfide formation induced by ONOO- was compared to GSNO and AS in Figure 2C. Again, interchain disulfides were greatest in those samples treated with AS, and ONOO- was more effective than GSNO. The most effective compound tested was clearly AS, followed by ONOO-, GSNO, SNAP, and DEA NONOate. This ranking is identical to that seen for total cysteine modification as shown in Figure 1B. This work shows that not all S-nitroso compounds and NO donors will have equivalent effects on protein thiols. GSNO reactivity with tubulin is different from SNAP, even though they are both S-nitroso compounds. Likewise, treatment with a true NO donor, DEA NONOate, has a different effect on tubulin thiols. The effect of these compounds on microtubule polymerization was also studied. ONOO- induced disulfides inhibit polymerization and the extent of cysteine oxidation correlates with inhibition (8). In this assay, we measured the supernatant tubulin concentration after treatment with each compound, polymerization with GTP at 37 °C, and centrifugation to collect the microtubule pellet. We chose this assay because both GSNO and SNAP absorb at wavelengths that interfered with typical light scattering assays. Figure 3 plots the percent activity remaining as a function of the concentration of each compound. Concentrations up to 1 mM were tested in some cases because the concentration of tubulin was higher in this assay than in the IAF labeling or Western blot assays shown in Figures 1 and 2. Thus, we wanted to maintain the relative ratio of tubulin to NO donor/oxidant. The higher concentration of tubulin was required to ensure high polymer yield. Given that AS and ONOO- induced interchain disulfide bond formation to the greatest extent, it is not surprising that they were most effective as inhibitors of microtubule polymerization. When tubulin was treated with 1 mM ONOO-, polymerization activity was reduced to only 27% of control, whereas addition of 1 mM AS yielded no microtubule polymer. Polymerization activity was reduced to 80% of control by only 100 µM AS. The polymerization results obtained with SNAP and DEA NONOate were not unexpected given the modest amount of tubulin disulfides detected in Figures 2A and 2B. GSNO inhibited microtubule polymerization to a greater extent than SNAP or DN with a 30% reduction in activity when tubulin

Tubulin Cysteines and Nitric Oxide

Figure 3. Effect of NO and HNO donors on microtubule polymerization. Purified tubulin (100 µL, 2.5 µg/µL) was treated with each oxidant, NO, or HNO donor for 15 min at 25 °C. GTP (1 mM final) was added and the samples were incubated at 37 °C for 20 min. Microtubule polymer was collected by centrifugation at 16000 g for 20 min. The protein concentration in each supernatant was determined by the BCA method. The results represent the mean of at least three independent experiments performed in duplicate.

was treated with 1 mM GSNO. The inhibition results for GSNO are consistent with tubulin disulfides, not other modifications, as the primary mechanism of inhibition of polymerization. On the basis of Figure 2B, 100 µM GSNO induced tubulin disulfide formation to a similar extent as 25 µM AS. Several observations led us to explore the reaction of GSNO with tubulin more closely. When tubulin was treated with a broader range of GSNO concentrations (greater than those used in Figures 2A–C), we observed that tubulin interchain disulfides increased relative to control at 50 and 100 µM but decreased at 250 and 500 µM GSNO (Figure 4A). This experiment was repeated several times with the same outcome. Of note, samples treated with 250 and 500 µM GSNO showed less tubulin oxidation than the control. Careful examination of Figure 4A shows traces of tetramer in lane 4 (250 µM sample) that are not present in the control. This suggests an initial oxidation event to form the tetramers followed by a repair step. The samples in Figure 4A were incubated at 37 °C for 30 min. When tubulin was treated with 250 µM GSNO for varying lengths of time ranging from 5 to 30 min, we observed maximal oxidation at 15 min with less damage observed at 30 min (data not shown). This result also supports the hypothesis that tubulin was initially oxidized by GSNO and then some component of the reaction mixture repaired the interchain tubulin disulfides. However, IAF labeling data in Figure 1B show that 500 µM GSNO modified tubulin cysteines to a greater extent than 100 µM GSNO. We repeated the IAF labeling assay using the same tubulin preparation and the same GSNO stock solution as used in Figure 4A. Figure 4B shows that when tubulin was treated with 50 – 500 µM GSNO for 30 min, there was a dosedependent decrease in fluorescein labeling, indicative of an increase in total cysteine modification. At this point, we have two questions to address: (1) what component of the reaction mixture is repairing tubulin disulfides in those samples treated with 250 or 500 µM GSNO (Figure 4A); and (2) how is it possible that total cysteine modification

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Figure 4. Tubulin cysteine modification by GSNO. Tubulin samples (7 µM protein, 140 µM cysteines) were treated with 50, 100, 250, and 500 µM GSNO for 30 min at 37 °C. (A) Tubulin samples were separated by SDS-PAGE under nonreducing conditions, and tubulin interchain disulfides were detected by Western blot. (B) Samples were treated with IAF and separated by SDS-PAGE under reducing conditions. The fluorescein-labeled protein bands were visualized using the Kodak DC290 system and a UV transilluminator.

increases at high GSNO (Figures 1B and 4B), even though disulfides are being repaired? To address the first question, we examined the products generated from the reaction of protein cysteines with GSNO. As shown in eqs 3 and 4, GSH is a product of the NO transfer from GSNO to a protein cysteine PSH. A second product generated during disulfide bond formation is HNO. Doyle and co-workers first reported the reaction of thiophenol with HNO to yield disulfide and hydroxylamine NH2OH (37). Given that Angeli’s salt is an HNO donor and Angeli’s salt induces the formation of tubulin disulfides, the following outcome is expected:

HNO + 2PSH f PSSP (disulfide) + NH2OH

(5)

We attempted to measure NH2OH following reactions of tubulin with GSNO and AS. However, the colorimetric assay described to quantitate NH2OH is affected by the presence of thiols (38). Given that the thiol concentrations (both protein and small molecule) change as our reactions proceed, we could not obtain consistent data at this time. We treated control tubulin and ONOO- damaged tubulin with 100 or 250 µM NH2OH and analyzed the tubulin by Western blot. As ONOO- induces tubulin disulfides and reacts rapidly and completely with either protein target or via isomerization to nitrate ion, we did not have to consider any subsequent ONOO- reaction. No reduction of tubulin disulfides by NH2OH was detected. In a similar experiment, we treated control tubulin, containing a detectable amount of tubulin dimers, with 50 and 100 µM GSH. The results of this experiment are shown in Figure 5. Tubulin dimers are detected in the control sample and those dimers decreased as the concentration of GSH increased. Likewise, when 100-500 µM GSH was added to ONOOdamaged tubulin, there was a substantial decrease in interchain tubulin disulfides relative to the sample treated with ONOOalone (data not shown). These results lend support to the hypothesis that GSH, formed from the initial reaction of GSNO with tubulin cysteines (eq 3), repairs tubulin interchain disulfides induced by GSNO via thiol/disulfide exchange.

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Figure 5. Reduction of tubulin interchain disulfides by GSH and DN. Tubulin samples (7 µM protein, 140 µM cysteines) was treated with 50 or 100 µM GSH (lanes 2 and 3) or with 100 or 500 µM DEA NONOate (lanes 4 and 5) for 30 min at 37 °C. Samples were separated by SDS-PAGE under nonreducing conditions and tubulin interchain disulfides were detected by Western blot.

Figure 6. S-Nitrosation of tubulin. Purified tubulin (60 µL, 0.75 µg/ µL) was diluted with PB pH 7.4 and treated with GSNO or SNAP for 30 min. Ethanol was added to 80%, samples were left on ice for 15 min to precipitate protein and the protein pellet was collected by centrifugation at 10000 g for 10 min. Pellets were washed with 80% ethanol and then resuspended in 60 µL of PB at pH 7.4 containing 1% SDS. NO bound to protein was detected by a modified Saville assay (24). Concentrations of SNO were calculated from a GSNO standard curve. These data represent the results of three independent trials.

In Figure 5, we also revisited the reaction of control tubulin with DEA NONOate and confirm that this compound did not induce the formation of tubulin interchain disulfides. It appears that the disulfides in control tubulin decreased slightly in the presence of NO gas from DEA NONOate, suggesting that NO has a slight reductive ability and may generate NO+.

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To quantitate S-nitrosation of tubulin, we performed a modified version of the Saville assay as described by Cook and co-workers (24). GSNO, SNAP, DN, and AS were tested for their ability to S-nitrosate tubulin using the same protein preparation as used for the IAF labeling and Western blots. Figure 6 shows the results of this assay for GSNO and SNAP. As the concentration of GSNO increased from 50 to 500 µM, the yield of tubulin S-nitrosation increased from 0.5 to 2.3 mol SNO/mol tubulin. At all concentrations of SNAP tested, only very low yields of protein SNO ranging from 0.45 to 0.98 mol/ mol protein were detected. Data for DN and AS are not shown because only trace amounts of S-nitrosation were detected with these compounds. To reconcile the data in Figures 6, 1B and 4B (IAF labeling) with that in Figure 4A showing a decrease in tubulin disulfides at high GSNO concentrations, we developed the model shown in Scheme 2. The Rβ tubulin dimer contains multiple cysteines that can be oxidized to disulfides or S-nitrosated by GSNO. Transnitrosation of a tubulin cysteine by GSNO yields GSH in the local environment that may reduce a tubulin disulfide. Reduction of a tubulin disulfide by GSH yields additional thiols that may be S-nitrosated by excess GSNO still present in the reaction mixture. If two adjacent cysteines are both S-nitrosated, then disulfide bond formation is not possible. The tubulin S-nitrosation data in Figure 6 clearly shows increased SNO modification as the concentration of GSNO increases. Our observations and this model suggest an interesting potential mode of regulation for microtubule polymerization. GSNO, serving as the physiological NO carrier, may react with tubulin to yield oxidized protein with reduced ability to polymerize. GSH generated in the local environment from GSNO could repair tubulin disulfides via thiol/disulfide exchange. GSH concentrations in most eukaryotic cells are in the 1–10 mM with a typical GSH:GSSG ratio of 100 to 400 (39). Under normal conditions, GSH would be present in sufficient quantities to protect proteins from oxidation by reactive oxygen species. For example, an oxidant such as H2O2 would likely react with GSH rather than with a protein cysteine. However, GSNO can persist even in a sea of GSH (40). Once GSNO encounters a protein thiol, it would react according to eq 3 and, in the case of tubulin, generate a protein disulfide. Rapid reduction of the protein disulfide by GSH, either derived from GSNO or present in solution would yield GSSG via thiol/ disulfide exchange (39). The addition of a reductant such as TCEP or DTT to GSNOtreated tubulin restores all cysteines to control levels. Thus, no higher oxidation states of sulfur are generated. Of note, GSNO was treated with up to 10 equiv of TCEP and the reaction was

Scheme 2. Proposed Mechanism of GSNO-Mediated Tubulin Cysteine Modification

Tubulin Cysteines and Nitric Oxide

monitored by UV/vis. The characteristic 335 nm absorbance of GSNO decreased to zero within 5 min. Therefore, the only possible cysteine modifications by GSNO are oxidation to disulfides, S-nitrosation and S-glutathionylation. The biotin switch assay, developed in the Snyder laboratory, originally caught our attention because their work suggested that R- and β-tubulin were S-nitrosated by GSNO (20). The biotin switch assay depends on ascorbic acid as a selective reductant of protein S-nitrosothiols. Our research took an unexpected turn when we observed that ascorbic acid can reduce tubulin and MAPs disulfides that were formed by ONOO- (25). This result was unexpected because numerous researchers claimed that ascorbic acid reduces only S-nitroso compounds and not disulfides (20, 41). As a result, we cannot perform the biotin switch assay to determine the extent of tubulin Snitrosation because both disulfides and S-nitrosated cysteines would be reduced by ascorbic acid. In past work, we detected S-glutathionylation of tubulin when ONOO- damaged tubulin, containing disulfides, was treated with GSH (via thio/disulfide exchange) (9). In this work, we used an antiglutathione antibody to assay for S-glutathionylation. We treated tubulin samples with GSNO over a range of concentrations and times but failed to detect any glutathionylation by dot or Western blot. We have assessed the effect of several different classes of compounds including an NO donor, an HNO donor and S-nitroso compounds on tubulin cysteines and on their ability to inhibit tubulin polymerization. Previously, we determined that ONOO- oxidizes tubulin cysteines to disulfides and cysteine oxidation correlated with inhibition of polymerization (8). Tyrosine nitration was also detected and partially responsible for the inhibition we observed. Angeli’s salt, a HNO donor, oxidizes tubulin cysteines to disulfides exclusively, all reducible by TCEP or DTT, and is a more effective inhibitor of tubulin polymerization than ONOO-. It is especially noteworthy that HNO, released from AS, gave nearly stoichiometric oxidation of tubulin cysteines. Competition studies in which tubulin and GSH were combined and then treated with AS were attempted by Western blot. However, the results were inconclusive. For samples containing both tubulin and GSH, we did not observe oxidation of tubulin to disulfides but rather reduction of tubulin disulfides presumably by GSH (as shown in Figure 5) (data not shown). The fact that GSH was able to reduce tubulin disulfides implies that AS oxidized tubulin in the presence of GSH and then GSH served as the reducing agent. Competition experiments need to be explored in greater detail. In the case of GSNO, multiple cysteines are modified and we detected moderate levels of tubulin disulfides and Snitrosation. Though total cysteine modification by GSNO is only slightly less than by ONOO- (Figure 1B), GSNO does not inhibit tubulin polymerization to the same extent. This suggests that S-nitrosation of tubulin does not inhibit polymerization, at least not as effectively as tubulin disulfides. Likewise, total cysteine modification by SNAP is less than GSNO and ONOO- (Figure 1B) and SNAP does not induce tubulin disulfide formation to the same extent as GSNO. SNAP is a poor inhibitor of tubulin polymerization, which supports our conclusion that disulfides, not S-nitrosation of tubulin cysteines, inhibit polymerization.The NONOate class of NO donors are not S-nitroso compounds but rather they release NO free radical in solution. The modest levels of tubulin cysteine modification and inhibition of polymerization were not unex-

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pected given that the reaction between free NO and a protein thiol proceeds by a different mechanism (42). Acknowledgment. The authors acknowledge support from the National Institute of Neurological Disorders and Stroke (R15-NS38885 to L.M.L.), the Petroleum Research Fund (Grant 44091 B4 to L.M.L.), and the Jeffress Memorial Trust (Grant J-670 to LML).

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