Balanced Regulation of Redox Status of Intracellular Thioredoxin

Mar 6, 2018 - In this study, we developed a method that can monitor the redox status of thioredoxin (Trx) and EGSH by direct NMR observation of Trx an...
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Balanced Regulation of Redox Status of Intracellular Thioredoxin Revealed by in-Cell NMR Ayano Mochizuki, Arata Saso, Qingci Zhao, Satoshi Kubo, Noritaka Nishida,* and Ichio Shimada* Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan S Supporting Information *

ABSTRACT: To understand how intracellular proteins respond to oxidative stresses, the redox status of the target protein, as well as the intracellular redox potential (EGSH), which is defined by the concentrations of reduced and oxidized glutathione, should be observed simultaneously within living cells. In this study, we developed a method that can monitor the redox status of thioredoxin (Trx) and EGSH by direct NMR observation of Trx and glutathione within living cells. Unlike the midpoint potential of Trx measured in vitro (∼ −300 mV), the intracellular Trx exhibited the redox transition at EGSH between −250 and −200 mV, the range known to trigger the oxidative stress-mediated signalings. Furthermore, we quantified the contribution of Trx reductase to the redox status of Trx, demonstrating that the redox profile of Trx is determined by the interplay between the elevation of EGSH and the reduction by Trx reductase and other endogenous molecules.

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apoptosis signaling.7,8 Therefore, it is crucial to elucidate the relationship between the redox status of Trx and the intracellular redox potential, in order to understand the redox-dependent signaling triggered by internal and external oxidative stresses. The in vitro redox profile of Trx has been characterized in previous studies. In particular, the quantitative NMR-based estimation of the reduced and oxidized states of Trx at various GSH/GSSG concentrations revealed that the midpoint potential (E0′) of Trx in vitro is about −270 mV at pH 7.5.9 However, this midpoint potential does not explain the role of Trx as a redox sensor, considering the intracellular redox potential that causes various cellular responses (EGSH > −250 mV). Generally, the oxidized/reduced ratios of intracellular redox proteins are determined not only by the difference (ΔE) between the intracellular redox potential (EGSH) and their own midpoint potential (E0′) but also by various endogenous intracellular molecules/proteins.10 Therefore, in order to determine how Trx responds to oxidative stress, the redox statuses of both Trx and glutathione should be measured under the intracellular conditions. In the conventional methods to evaluate the redox status, Trx and glutathione were extracted from the cell lysate and subjected to Western blot11 or HPLC12 analyses, respectively. However, these approaches were inherently inaccurate, due to the oxidation during cell lysis and the imperfect efficiency of chemical modifications to block the free thiol group. Several groups have developed redox-sensitive fluorescent proteins,

INTRODUCTION The intracellular redox environment is critically important for cellular homeostasis, and dysregulation of the redox environment by oxidative stress causes various diseases.1,2 The intracellular redox potential is a measure of the redox balance in the cell and is mainly defined by the ratio of glutathione couples (GSH/GSSG), the most abundant thiol compounds existing inside cells.3 Therefore, the glutathione redox potential (EGSH), calculated by the Nernst equation based on the concentrations of GSH and GSSG, is a good indicator of the intracellular redox environment. In a normally proliferating cell, the intracellular environment is maintained under reducing conditions with EGSH less than −300 mV.4 Internal or external oxidative stresses cause the oxidation of intracellular substances, triggering various intracellular redox signalings, including gene expression, cell differentiation, apoptosis, and necrosis.3 Thioredoxin (Trx) is a major redox-regulating protein, which is conserved from prokaryotes to vertebrae.5 Two conserved cysteine residues (C32, C35) in the active site of Trx mediate the reduction of the disulfide bond in the substrate proteins. The reduction of the oxidized Trx is mediated by Trx reductase (TrxR) and NADPH system. Besides its role as an antioxidant protein, Trx is involved in the redox-dependent signal transduction, through interactions with various proteins. For example, Trx activates several transcription factors, including NF-kB, Nrf2, and AP1, by reducing the disulfide bond that affects the DNA-binding affinity.6 ASK1 (apoptosis signalregulating kinase 1) is also reportedly inhibited in the reduced Trx-bound state, but becomes activated upon the release of Trx in the oxidized form, thereby allowing the recruitment of the downstream signaling molecules, TRAF-2/6, that activate the © XXXX American Chemical Society

Received: January 16, 2018

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DOI: 10.1021/jacs.8b00426 J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

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Journal of the American Chemical Society such as Grx1-roGFP2,4 and organic compounds13 that can measure the intracellular EGSH within living cells. Recently, the fluorescent probe for measuring the redox status of Trx was also reported by fusion of Trx with the engineered redoxsensitive red fluorescent protein (TrxRFP1).14 However, these fluorescent-based methods require the introduction of artificial genes and organic compounds into the cells, which may cause unexpected cellular responses. Moreover, those probes detect the redox status of the Trx and glutathione indirectly based on the fluorescent intensity changes of the redox-sensitive reporter molecules, which could be affected by the interactions with other intracellular factors, and are susceptible for fluorescent quenching in the intracellular environments. Therefore, quantitative measurements of the redox status of the intracellular glutathione and Trx are difficult by conventional methods. In-cell NMR method is an emerging technique to monitor the protein structures and dynamics in the physiological intracellular environment by introducing stable isotope-labeled proteins into the cells using various methods.15,16 Our group also developed the bioreactor system, which perfuses the cells with fresh culture medium, thereby suppressing cell death during in-cell NMR measurements.25 Recent in-cell NMR studies demonstrated that the redox statuses of intracellular proteins can be measured in living cells, based on the signal intensities of the NMR signals derived from the oxidized and reduced states.17,18 In addition, in-cell NMR methods were reported that can measure the intracellular GSH/GSSG ratio by utilizing the in vivo isotope labeling of glutathione in the yeast and mammalian cells.19,20 In this study, we established an in-cell NMR method to directly observe the NMR signals of both Trx and glutathione within cells, by preparing cells in which both Trx and glutathione are isotopically labeled. In addition, we performed time-resolved NMR measurements in the presence of oxidative stress, by perfusing culture medium containing stress-imposing reagents using the bioreactor system. This method enabled the dual observations of the intracellular redox statuses of Trx and EGSH, allowing us to monitor how the redox status of intracellular Trx is affected by the elevation of EGSH induced by oxidative stresses.

Figure 1. NMR probes for detecting the redox statuses of glutathione and Trx and the redox profile of Trx in vitro. (a) The position of Ala29 in the Trx structure24 (PDB ID 3TRX) . The A29 signals of Trx in the HMQC spectrum. (b) The β methylene group of Cys in glutathione was used as the NMR probe for detecting the redox status of glutathione. The GSH and GSSG signals in the 1H−13C HSQC spectrum of glutathione in vitro. The cross sections of those signals are shown in insets. Note that the actual 13C chemical shift of the GSSG signals is 39.05 ppm (aliased on the 13C axis). (c) The in vitro redox profile and the midpoint redox potential (E0′) of Trx. The physiological EGSH ranges triggering various cell responses are indicated by arrows.

RESULTS AND DISCUSSION NMR Probes for Detecting the Redox Statuses of Trx and Glutathione. We searched for NMR probes that can detect the redox status of intracellular Trx with high sensitivity. Since the in-cell NMR observation of the 1H−15N heteronuclear single quantum coherence (HSQC) signals of Trx is reportedly difficult, due to severe line broadening in the intracellular environment,21 we utilized the methyl transverse relaxation optimized spectroscopy (TROSY) detection by observing the side-chain methyl signals.22 We found that the methyl group of Ala29, which is proximal to the cysteine residues, C32 and C35, at the active site exhibited two distinctive signals that correspond to the reduced and oxidized states (Figure 1a). Since the signals from A29 exhibited a marked upfield shift in the 1H chemical shift direction due to the ring current effect of W32, the A29 signal can be observed as a well-separated signal from the seven other Ala signals and the natural abundance signals derived from the medium and other cellular components in the in-cell NMR spectrum (Figure S1). Therefore, we utilized the A29 signals for detecting the redox status of Trx in the following in vitro and in-cell NMR experiments. In addition, we used a Trx mutant, in which the

three additional cysteine residues are mutated to Ser (C62S/ C69S/C73S), to observe the effects of oxidative stress at the active site. Redox Profiles of Trx and Glutathione in Vitro. The in vitro redox profile of Trx was examined by NMR. To observe the EGSH-dependent changes of the ratio of oxidized and reduced Trx, based on the A29 methyl group, band-selective optimized flip angle short transient (SOFAST)-HMQC spectra23 were measured for 5 min in the presence of the designated concentrations of GSH/GSSG, corresponding to EGSH values ranging from −330 mV to −255 mV (Figure S2). Since GSH gradually oxidizes to GSSG in the solution, the actual GSH/GSSG concentration was also quantified, based on the signal intensities of the cysteine β methylene group of glutathione in the 1H−13C constant time HSQC spectrum (Figure 1b), and EGSH was calculated, according to the Nernst equation (Figure S2). By curve-fitting the plot of the ratio of oxidized Trx at the corresponding EGSH using the Bolzmann function, the midpoint potential (E0′) of Trx in vitro was calculated to be −299 mV (Figure 1c). Therefore, in accordance with the previous report,9 we confirmed that the in vitro midpoint potential of Trx (−299 mV) is lower than the

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DOI: 10.1021/jacs.8b00426 J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

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signal-to-noise ratio of the GSH signal is ∼400, intracellular EGSH was estimated to be lower than −262 mV, assuming that the total concentration of intracellular glutathione is 4.6 mM.27 It should be noted that the glutathione signals were dramatically decreased if the introduction of Trx was performed after the in vivo labeling of glutathione, due to the leakage of glutathione during the SLO treatment (Figure S3). We also estimated that the intracellular Trx concentration to be ∼70 μM, based on the signal intensities of the cell lysate (Figure S4). During the 7 h of measurements, only the reduced signals were observed for both glutathione and Trx, indicating that the intracellular Trx and glutathione were maintained in the reduced state in the absence of oxidative stress (Figure 2b). More than 80% cells remained alive after the 7 h of measurements. In addition, the GSH signals showed a timedependent decrease, presumably due to metabolic turnover, indicating that cells were maintained in the physiologically active state. The Redox Status of Trx in the Presence of an Oxidant. To observe how the redox status of Trx is altered depending on the intracellular EGSH, we performed the in-cell NMR experiments in the presence of an oxidant, tert-butyl hydroperoxide (TBH). TBH is reportedly detoxicated by glutathione peroxidase (GPx), which produces GSSG as a side reaction, thereby causing the elevation of the intracellular redox potential28 (Figure 3a). We acquired in-cell NMR spectra of Trx and glutathione alternately, with the perfusion of medium with increasing concentrations of TBH from 1 mM to 3 mM (Figure 3b). In the presence of 1 mM TBH, NMR signals derived from the oxidized state were observed for both Trx and glutathione. From the Nernst equation, the intracellular EGSH was elevated to about −250 mV (Figure 3b, Figure S5), as the population of the oxidized Trx increased by about 20% (Figure 3c, Figure S5). In the presence of 3 mM TBH, the populations of the oxidized forms of glutathione and Trx gradually increased, and EGSH was elevated up to −210 mV (Figure 3b, Figure S5), as the oxidized Trx ratio reached 84% (Figure 3c, Figure S5). The plot of the redox status of Trx against EGSH indicated that the redox profile of intracellular Trx transitions from the reduced state to the oxidized state, at EGSH between −250 and −200 mV. Notably, the redox status of Trx in the cells is maintained in the reduced state, as compared to the in vitro status under the higher EGSH conditions (Figure 3d). In-Cell NMR Observation of Trx in the Presence of Trx Reductase Inhibitor. We investigated the underlying mechanism that generates the different redox profiles of Trx between in-cell and in vitro conditions. The activity of Trx is controlled by various endogenous molecules. In order to estimate the contributions of specific endogenous molecules to the Trx redox status, the in-cell NMR experiments can be performed using cells in which the activity of the target molecule is suppressed, by either the specific inhibitor or the knockout of the target gene. In the light of this, we examined the contribution of the Trx reductase (TrxR) by performing the in-cell NMR measurements in the presence of a TrxR inhibitor, aurothioglucose (ATG). ATG is a gold-containing compound that is used for the treatment of rheumatoid arthritis and is known to inhibit TrxR1 in the cytosol.29 The in-cell NMR experiments of Trx were performed in the presence of 1 mM ATG, in circulating medium without oxidative stress (Figure S6). During the 7 h measurements, a minimal GSSG signal was observed for glutathione, and the population of oxidized Trx

intracellular redox potential that triggers the cellular response (>−250 mV). Dual in-Cell NMR Measurements of Trx and Glutathione Signals. To measure the redox statuses of Trx and glutathione by the in-cell NMR method, we prepared cells, in which both Trx and glutathione are isotopically labeled (Figure 2a). The Ala methyl 13C-labeled Trx was introduced into HeLa

Figure 2. Preparation of the cells for dual in-cell NMR observations of Trx and glutathione. (a) Preparation of cells for in-cell NMR measurements. The isotope-labeled Trx and glutathione are colored red and cyan, respectively. (b) The time-resolved in-cell NMR measurements of glutathione and Trx. The cross sections of the GSH/ GSSG signals, and the A29 signal of Trx are shown on the left and right side of the experimental time axis, respectively.

S3 cells by reversible membrane permeabilization, using the pore forming toxin, streptolysin O (SLO).25 After resealing, the cells were cultured overnight in Dulbecco’s Modified Eagle’s medium (DMEM), containing 0.4 mM of 13C-labeled cysteine for overnight for in vivo labeling of glutathione, according to the method recently reported by Jin et al.19 The harvested cells were perfused with the fresh culture medium by the bioreactor system to maintain viability during the NMR measurements, as described previously.26 We acquired in-cell NMR spectra of Trx (for 30 min) and glutathione (for 20 min) alternately, in the absence of oxidative stress (Figure 2b). As a result, the Trx and glutathione signals were both observed only as the reduced state. Considering the fact that the signal of GSSG is below the noise level and the C

DOI: 10.1021/jacs.8b00426 J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

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Figure 3. In-cell NMR observations of redox statuses of Trx and glutathione under oxidative stresses. (a) The scheme of detoxification of TBH by glutathione peroxidase (GPx), which generates the GSSG. (b, c) The time-dependent changes of (b) EGSH and (c) the oxidized Trx ratio in the absence and presence of 1 mM and 3 mM TBH. One mM and 3 mM TBH were added to the medium at the time-points of 50 and 190 min, respectively, and perfused for 20 min before resuming the in-cell NMR measurements. (d) The plot of the redox profile of Trx in the intracellular environment. The redox profile of Trx in vitro is shown by the blue line.

was −250 mV), Trx is mostly in the reduced state (Figure 5c), owing to the TrxR activity, as indicated by the experiments using the TrxR inhibitor (Figure 5d). The addition of more extensive oxidative stress gradually increases the population of oxidized glutathione and Trx gradually (Figure 5e). The increase of the oxidized Trx seems to induce the activation of the endogenous enzymes that mediate the reduction of Trx (Figure 5f). These results indicate that the redox status of Trx is determined by the balance between intracellular redox potential and the endogenous molecules that maintain Trx in the reduced state. As a result, the redox profile of Trx exhibits the transition at EGSH between −250 mV and −200 mV, which is appropriate for responding to the intracellular oxidative stress that triggers various Trxmediated cell signaling events such as apoptosis.

Figure 5. Regulation of the redox status of intracellular Trx. (a) In the absence of oxidative stress, the redox status of Trx is maintained in the reduced state even in the presence of the TrxR inhibitor (b), indicating that the intracellular EGSH is lower than −320 mV. (c) In the presence of weak oxidative stress, EGSH is elevated to ∼ −250 mV, which is higher than the in vitro midpoint potential of Trx. However, the redox status of Trx is mostly maintained in the reduced state. (d) In contrast, when TrxR is inhibited, the population of oxidized Trx increased significantly. (e) In the presence of strong oxidative stress (EGSH ∼ − 200 mV), the redox status of Trx is shifted to the oxidized state, because the oxidation of Trx surpasses the activity of TrxR. (f) The increase of oxidized Trx may induce the up-regulation of TrxR and other endogenous enzymes that mediate the reduction of oxidized Trx.

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CONCLUSION The redox statuses of intracellular proteins cannot be predicted from the midpoint potential (E0′) measured in vitro, since various intracellular redox-regulating molecules exist within cells. Therefore, in order to reveal how intracellular proteins respond to oxidative stresses, the redox statuses of proteins, as well as the redox potential (i.e., GSH/GSSG balance), should be observed within living cells. In this study, we utilized the inE

DOI: 10.1021/jacs.8b00426 J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

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of glutathione. The harvested cells were subjected to density gradient centrifugation using 20% Percoll, for the removal of dead cells. The populations of the Trx-introduced cells and the dead cells were analyzed by a CytoFLEX flow cytometer (Beckman Coulter), as described previously.26 NMR Measurement Using the Bioreactor System. All in-cell NMR experiments were performed at 37 °C using an Avance III 800 spectrometer equipped with a cryogenic probe (Bruker Biospin). The Trx introduced cells (3 × 107) were encapsulated within the 8% Mebiol gel and perfused with DMEM at a flow rate of 2.75 mL/h in the 5 mm Shigemi tube, as described previously.26 To add the oxidative stress, the bioreactor system was detached from the NMR tube and refilled with the DMEM containing 1 or 3 mM TBH, and the NMR measurements resumed after perfusion at least for 10 min (500 μL). After the in-cell NMR measurement, the cells encapsulated in the Mebiol gel were released by lowering the temperature, and the supernatant was collected as described above. The intracellular GSH and GSSG concentration was determined based on the signal intensities of β-methylene resonance of Cys moiety of glutathione as described above, assuming the total concentration of intracellular glutathione with 4.6 mM.27

cell NMR method to directly observe the redox statuses of Trx and glutathione within living cells, by introducing isotopelabeled Trx and glutathione. In addition, the in-cell NMR measurements in the presence of oxidative stress-inducing reagents, using the bioreactor system, enabled the observation of the redox statuses of glutathione and Trx in a real-time manner, upon the addition of oxidative stresses and an inhibitor of endogenous regulatory proteins. The result revealed how the redox status of Trx changes in response to the EGSH elevation induced by oxidative stress. The development of redox-sensitive fluorescent probes needs laborious optimization of the probe design, which is not always successful. In contrast, the method developed in this study is generally applicable for many other intracellular redox proteins and can be used to elucidate how those proteins respond to alteration of the intracellular redox potential, induced by external or internal oxidative stresses. TrxR is considered as a prominent target for cancer and rheumatoid arthritis therapeutics. We expect that the in-cell NMR method, established in this study, will become a valuable tool for evaluating the efficacies of inhibitor of TrxR in physiological and pathological contexts.

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METHODS

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/jacs.8b00426. Further information on the NMR probe to detect the redox status of Trx (S1), measurement of the redox profiles of Trx in vitro (S2), in vivo labeling of glutathione (S3), measurements of intracellular Trx concentration (S4), in-cell NMR spectra of Trx and glutathione under oxidative stresses (S5), in-cell NMR spectra of Trx in the presence of the TrxR1 inhibitor (S6), concentration dependency of the redox profile of intracellular Trx (S7), measurements of TrxR activity in cell lysate (S8) (PDF)

Protein Expression and Preparation. The cDNA encoding human full length thioredoxin 1 (residues 1−104) was cloned into the pET-15b vector. The plasmid was transformed into E. coli strain BL21(DE3), and protein expression was induced by 1 mM IPTG for 4 h at 37 °C. The harvested cells were lysed by sonication, and the supernatant was purified by nickel affinity chromatography. After the removal of the histidine tag by thrombin (GE Healthcare), Trx was further purified by Hi-TrapQ anion exchange chromatography. Selective 1H−13C labeling of the Ala methyl groups of Trx in a deuterated background ([u-2H, Ala-[13CH3]]Trx) was performed, as described.30 The Trx mutant was generated according to the QuikChange (Stratagene) protocol. In Vitro NMR Experiments. The resonance assignments of Trx were achieved using the following triple resonance experiments, recorded with uniformly 13C, 15N-labeled Trx: HNCACB, CBCA(CO)NH, and HBHA(CO)NH. The in vitro redox profiles of Trx and glutathione were examined by recording the SOFAST HMQC and constant time (ct) HSQC spectra, respectively. The actual concentrations of GSH and GSSG were estimated based on the signal intensities, and the EGSH was calculated according to the Nernst eq 1 based on the signal intensities of the β-methylene resonances of GSH and GSSG in the constant time HSQC spectrum.

EGSH

ASSOCIATED CONTENT

S Supporting Information *

RT [GSH]2 = E0 − ln zF [GSSG]

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AUTHOR INFORMATION

Corresponding Authors

*[email protected] *[email protected] ORCID

Ichio Shimada: 0000-0001-9864-3407 Notes

The authors declare no competing financial interest.

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(2)

ACKNOWLEDGMENTS We thank Prof. Y Urano for helpful discussion. This work was supported by grants for the Development of core technologies for innovative drug development based upon IT from the Japan Agency for Medical Research and Development, AMED (to I.S.), and the Ministry of Education, Culture, Sports, Science and Technology (MEXT)/Japan Society for the Promotion of Science KAKENHI grant no. 26119005 (to N.N.), and grant nos. 21121002 and 17H06097 (to I.S.).

where IRox is the intensity ratio of Trx in the oxidized form and d is the steepness of the curve. Preparation of Cells for in-Cell NMR Measurements. HeLa S3 cells were cultured in suspension in DMEM containing 10% FCS, under a 5% CO2 atmosphere. [u-2H, Ala-[13CH3]] Trx was introduced into HeLaS3 cells using the SLO protocol as described previously. The Trx-introduced cells were cultured in DMEM (cysteine free Gibco #21013) supplemented with 0.4 mM [u-13C] Cys, for in vivo labeling

(1) Townsend, D. M.; Tew, K. D.; Tapiero, H. Biomed. Pharmacother. 2003, 57 (3−4), 145−155. (2) Balendiran, G. K.; Dabur, R.; Fraser, D. Cell Biochem. Funct. 2004, 22 (6), 343−352. (3) Schafer, F. Q.; Buettner, G. R. Free Radical Biol. Med. 2001, 30 (11), 1191−1212.

where R is the gas constant (8.3145 J K−1 mol−1), T is the temperature in Kelvin, z = 2 for the two-electron reduction, and F is the Faraday constant (9.6485 × 104 C mol−1). The standard midpoint potential of glutathione is −262 mV at pH 7.5, 25 °C.3 All spectra were processed by Topspin (Bruker Biospin) and analyzed by UCSF Sparky.31 The midpoint potential (E0′) of Trx was determined by curve fitting of eq 2, using the least-squares method.

IR ox =

1 (EGSH − E 0′)/ d

1+e

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DOI: 10.1021/jacs.8b00426 J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

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DOI: 10.1021/jacs.8b00426 J. Am. Chem. Soc. XXXX, XXX, XXX−XXX