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Cysteine/cystine couple is a newly recognized node in the circuitry for biologic redox signaling and control

DEAN P. JONES^*,1, YOUNG-MI GO^*, CORINNA L. ANDERSON^||, THOMAS R. ZIEGLER^*,, JOSEPH M. KINKADE, JR^, and WARD G. KIRLIN^¶

Departments of
^* Medicine,
Biochemistry and
Epidemiology,
Center for Clinical and Molecular Nutrition, and the Graduate Programs of
^|| Nutrition Health Sciences, Emory University; and
^¶ Department of Pharmacology and Toxicology, Morehouse School of Medicine, Atlanta, Georgia, USA

¹Correspondence: Department of Medicine, 4131 Rollins Research Center, Emory University, Atlanta, GA 30322, USA. Email: dpjones{at}emory.edu

SPECIFIC AIMS

Reversible oxidation of sulfur-containing side chains of cysteine and methionine in proteins functions in signaling and control of gene expression, cell proliferation, and apoptosis. Glutathione (GSH) and thioredoxin (Trx) systems maintain signaling components in a reduced state and are counterbalanced in signaling by oxidative mechanisms, typically thought to directly depend upon reactive oxygen species (ROS). However, in cisternae of the endoplasmic reticulum, an oxidase is used to generate an intermediate disulfide to oxidize target protein thiols by a thiol/disulfide exchange mechanism. In principle, similar systems could function elsewhere in redox signaling. Cystine is a ubiquitous disulfide generated from cysteine by an oxidase on extracellular surfaces, and cysteine/cystine redox state in human plasma is considerably oxidized relative to GSH/GSSG. If cysteine/cystine redox were similarly oxidized in cells, this system could function in redox signaling and control with cystine being an intermediate disulfide used to oxidize proteins by thiol/disulfide exchange. Such a mechanism would have an inherent advantage over ROS-dependent oxidation because of decreased risk of oxidizing proteins to non-reversible, higher oxidation states. The central position of cysteine as a precursor for protein and GSH synthesis suggests that cysteine/cystine could provide a suitable site for integration of redox control with other aspects of cellular homeostasis. Purposes of this study were to determine the redox state of cysteine/cystine in cells, to learn whether this thiol/disulfide couple is sufficiently oxidized to function as an oxidant in cell signaling, to explore whether cysteine/cystine redox is stable under differing physiologic conditions of cell growth and enzyme induction, and to determine whether the redox state of cysteine/cystine in cells is independent of the GSH/GSSG system.

PRINCIPAL FINDINGS

1. The redox state of Cys/CySS in proliferating HT29 cells was –145 ± 3 mV, a value considerably more oxidized than the value of –249 ± 2 mV for the GSH/GSSG couple
The redox state of Cys/CySS was calculated from cellular concentrations of Cys and CySS, which were determined to be 126 ± 13 µM and 31 ± 5 µM, respectively. Possible artifacts due to oxidation during sampling, carryover of cysteine or cystine from culture medium or sequestration of cysteine or cystine into subcellular compartments were ruled out by control experiments. Thus, although redox states of Cys/CySS and GSH/GSSG couples equilibrate in solution by thiol/disulfide exchange, this process is slow relative to metabolic flux and transport of these components in cells.

2. Differentiation of HT29 cells by treatment with sodium butyrate resulted in only 30 mV oxidation of the Cys/CySS couple while causing a 60 mV oxidation of the GSH/GSSG couple
Previous research shows that the GSH/GSSG redox state becomes oxidized in association with differentiation. At 48 h after addition of 5 mM butyrate, HT29 cells expressed differentiation markers alkaline phosphatase and -glutamyltranspeptidase and cell proliferation rate decreased substantially. Under these conditions, Cys and CySS concentrations were decreased compared with controls even though GSH concentration and protein synthesis were also decreased. The time course for oxidation of Cys/CySS was delayed relative to GSH/GSSG and magnitude of oxidation of Cys/CySS was considerably less, but general patterns of oxidation were similar. Data show that a substantial difference between redox states of Cys/CySS and GSH/GSSG is preserved during cell differentiation.

3. Culture of HT29 cells in Cys-free medium resulted in little change in Cys/CySS redox
A marked decrease in cellular GSH concentration and an associated oxidation of GSH/GSSG (90 mV) were observed when cells were cultured in a cysteine-deficient medium. Addition of either 100 µM Cys or 50 µM CySS resulted in rapid recovery of redox of the GSH/GSSG couple. Under these conditions, there was little oxidation of Cys/CySS redox, showing that Cys/CySS redox state was considerably more stable than GSH/GSSG in cells under these conditions. Because redox states of Cys/CySS did not change in parallel with GSH/GSSG redox, these results show that Cys/CySS redox is controlled independently of GSH/GSSG redox under conditions of Cys deficiency.

4. Inhibition of GSH synthesis with buthionine sulfoximine (BSO) caused a marked decline in GSH concentration and an associated 40 mV oxidation of GSH/GSSG redox but had no effect on the Cys/CySS redox state
Cysteine deficiency not only alters GSH homeostasis but also results in inhibition of protein synthesis. An alternate way to change cellular GSH concentration is to use BSO, an inhibitor of the first step in GSH synthesis. Results with BSO showed that GSH concentration is substantially decreased with little effect on concentration of Cys. The redox state of the GSH/GSSG couple was considerably oxidized while the Cys/CySS couple was not. These results confirm the finding that with Cys deficiency the redox state of Cys/CySS in cells is regulated independently of the redox state of the GSH/GSSG couple.

5. Benzyl isothiocyanate (BIT), an inducer of GSH synthesis, caused a decreased Cys concentration and a 25 to 30 mV oxidation of Cys/CySS that was sustained for 24 h even though GSH concentration was increased with little change in the GSH/GSSG redox state
BIT is a Michael acceptor that increases expression of detoxification enzymes by transcriptional activation mediated though the antioxidant response element (ARE) in control regions of target genes. Glutamate:cysteine ligase catalytic and regulatory subunit genes, which determine rates of GSH synthesis, have AREs in their respective control regions and are responsive to BIT. Cells treated with BIT had increased GSH concentration but GSH/GSSG redox exhibited a sustained, modest 8–10 mV oxidation. In contrast, Cys concentration was decreased and the Cys/CySS couple was substantially oxidized. Under the condition of enzyme induction by BIT, the Cys/CySS system is threefold more responsive than the GSH/GSSG system.

CONCLUSIONS AND SIGNIFICANCE

The present study shows that steady-state redox potential of the cysteine/cystine couple (E_h = –160 to –125 mV) in cells is independent of the redox state of GSH/GSSG and is sufficiently oxidized relative to the GSH/GSSG redox couple for the cysteine/cystine couple to function as an oxidant in redox switching. The redox state of Trx1 is maintained at –270 to –280 mV in proliferating cells and is oxidized little in response to differentiation, apoptosis, or sulfur amino acid deficiency. In contrast, the redox state of the GSH/GSSG couple is variable in cells, with approximate values of –260 mV in rapidly proliferating cells, –200 in differentiated cells, –160 in apoptotic cells and –135 mV in cysteine-starved cells. Presence of Cys/CySS as a third independent thiol/disulfide couple, indicates that multiple discrete redox circuits can occur simultaneously and independently within cells. A model incorporating Cys/CySS as a distinct oxidizing node for redox signaling, along with Trx/TrxSS as a reducing node, and GSH/GSSG as an intermediate, switchable node, is illustrated in Fig. 1 . The character of the thiol/disulfide nodes proposed in this model to allow distinct redox signaling and control functions, can be viewed in analogy to electron carriers NADH/NAD⁺ and NADPH/NADP⁺. NADH/NAD⁺ and NADPH/NADP⁺ couples are maintained at different steady-state redox potentials in cells to provide different pathways of electron flow to support catabolism and anabolism simultaneously within the same aqueous compartment.

View larger version (24K): [in this window] [in a new window]   Figure 1. Circuitry model for cellular redox signaling and control by reversible oxidation of thiols. Reversible oxidation-reduction of possible classes of signaling proteins (l–6) can occur via combinations of opposing reduction and oxidation reactions. In this scheme, electron flow is from the most reduced component (NADPH) on the top to electron acceptors on the bottom. Reduction is driven by NADPH-dependent thioredoxin reductase 1 (TR1) and glutathione disulfide reductase (GR) through redox control nodes represented by Trx1 and GSH. Trx1 and GSH are located schematically according to respective measured steady-state redox values, given in the scale on the left. Oxidation of signaling proteins can occur by reactive oxygen species (ROS) or by O2 catalyzed by sulfhydryl oxidases (SO). The present study shows that the Cys/CySS couple can provide a third thiol/disulfide node for cellular redox signaling in which oxidation can occur without direct coupling to ROS or O2. Oxidation of Cys/CySS occurs extracellularly catalyzed by a membranal thiol oxidase (TO). Redox circuits for proteins of classes 1 and 2 include Trx1 as reducing node and ROS or O2 as oxidant, respectively. Redox circuits for proteins of classes 4 and 5 depend upon GSH as reductant and O2 or ROS as oxidant. Circuits for proteins of classes 3 and 6 depend upon Trx1 and GSH, respectively, for reduction and are coupled to Cys redox state for oxidation. Because GSH redox state changes as a function of cell growth state, proteins with intermediate Eo values (e.g., 6a and 6b) could be differentially regulated by change in GSH/GSSG redox state during cell growth and growth arrest.

In the model in Fig. 1 , activity of a protein can be switched by either 1) a dithiol/disulfide switch, or 2) a thiol/S-thiyl switch, with directionality controlled by coupling to a reduced (Trx or GSH) or oxidized (ROS, O₂ or CySS) redox node:

1) Pr-SS + reductant— Pr-(SH)₂ ("on" or "off", depending upon protein)

Pr-(SH)₂ + oxidant— Pr-SS (activity state reversed from above)

2) Pr-SS-R + R’SH— Pr-SH + RSSR’ ("on" or "off", depending upon protein)

Pr-SH + RSSR— Pr-SS-R (activity state reversed from above)

In this model, six different classes of signaling/control proteins can exist in terms of the reductant, oxidant combination: 1) Trx, ROS; 2) Trx, O₂; 3) Trx, CySS; 4) GSH, ROS; 5) GSH, O₂; and 6) GSH, CySS (Fig. 1) . Peroxiredoxins are reduced by Trx and oxidized by H₂O₂, and have the character of a Class 1 redox signaling protein. Class 4 proteins include some glutathione S-transferases, which have a peroxidase activity that allows for sulfhydryl switching which could control processes such as binding to JunK. Sulfhydryl oxidases could function in signaling through sulfhydryl switching proteins coupled with either Trx (Class 2) or GSH (Class 5).

Present results show that S-cysteinylation of proteins is likely to provide new classes of signaling proteins with Trx (Class 3) or GSH (Class 6) as a reductant:

PrSH + CySS— PrSS-Cys + Cys (activity "on" or "off")

Pr-SS-Cys + GSH— PrSH + CySSG (activity opposite of above).

For such a scheme, CySSG, which is maintained at a very low concentration in cells, would be rapidly removed by reaction with GSH, Trx, or GSSG reductase. Equivalent reaction schemes in which CySS is used to oxidize dithiols to disulfides could also occur, countered by Trx or GSH.

Present results show that the cellular Cys/CySS couple is considerably more oxidized than the GSH/GSSG couple and varies independently of the GSH/GSSG couple. Together with recent findings that the redox state of Trx1 varies independently of that for the GSH/GSSG couple, results show that Cys/CySS provides a distinct node in the circuitry for thiol-disulfide control. This third node expands the dynamic network for signaling by providing additional connections for controlling antioxidant defenses and specific growth and apoptosis pathways. Qualitative implications of this research are that S-cysteinylation of protein, often ignored because it can so readily occur as an artifact of protein extraction, may represent a common covalent modification that has a central role in regulating physiological processes.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0971fje; doi: 10.1096/fj.03-0971fje

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