Compartmentalization of Redox Regulation of Cell Responses
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《毒物学科学杂志》
In the November issue of Toxicological Sciences, Hansen et al. (2004) report results that they conclude show that a transcriptional regulator, NE-F2-related factor 2 (Nrf-2), exhibits compartmentally differential redox responses. The working model put forward by Hansen is conceptually straightforward. In the model, Nrf-2 is retained in the cytosol by Keap-1, but when critical thiols on Keap-1 are oxidized, Nrf-2 is released and translocated to the nucleus, where Nrf-2 participates in the transcriptional activation of numerous genes. For Nrf-2 to be fully functional in transcriptional activation, a key cysteine residue must be in the thiol (reduced) form. The model proposed by Hansen et al. overlooks the contributions of phosphorylation in activation of Nrf-2 (Huang et al., 2002; Nguyen et al., 2004) and other evidence that activation of Nrf-2 by metabolism of polyamines appears to be attributable to the actions of acrolein, which is a thiol alkylating agent, rather than through the production of H2O2 and presumed oxidation of cysteine residues (Kwak et al., 2003). Nevertheless, the model proposed by Hansen et al. is useful in expanding the discussion of oxidant stress responses and what advances will be needed to elucidate the critical elements of redox regulation of cell function and viability.
Separate from the ultimate requirement of oxidation to provide energy for cell functions, the basic idea that properties of proteins can be changed by oxidation is a valid working hypothesis. A similar redox mechanism was proposed for the transduction of NK-B, in that its translocation to the nucleus was suggested to be dependent on oxidation, whereas its binding to DNA depended on reduction (Droge et al., 1994). With regard to cytotoxicity, the concept that toxicants kill cells by substantial (>50%) depletion of cellular thiols (Di Monte et al., 1984) has not been substantiated in subsequent studies of oxidant cell killing, particularly in vivo (Smith et al., 1985), and contributions of thiol oxidation or alkylation to cell killing appear to be far more specific than some expected initially. More recently, the increased appreciation of specificity in modulation of cell function by modifications of protein thiols has been interpolated into redox regulation of thiol/disulfide status. In general, thiols are more readily oxidized than are other cellular components. The facile reversibility of S-thiolation reactions, whether formation of intra- or interchain disulfides (ProtS-SProt) or glutathione (GSH)-protein mixed disulfides (ProtS-SG) makes the participation of such modifications attractive in working hypotheses for mechanisms of regulation of a wide variety of cellular processes, from conception to death.
However, critical tests of hypotheses based upon thiol-disulfide-driven regulation of gene transcription, signal transduction, and related cell functions require that thiol redox status of specific thiols, meaning the specific modification of specific residues of specific proteins, be characterized and quantitated accurately, and with compartmental specificity. The challenges in meeting this goal are beyond present bioanalytical capabilities, but the necessity of developing and applying such methods and concepts should be recognized, so that the limitations of the implications of results not resolved at this level will not be so readily overlooked.
Many of the proteins that are most important in regulation of cell functions are present in relatively low abundance, and present bioanalytical methods are challenged, at best, to distinguish thiol from S-thiolated or S-alkylated forms of such proteins. Methods are being developed and reported, but at the present time, many of these reports are limited to detecting changes effected by large doses of diamide, H2O2, or other oxidants in vitro. However, the critical questions of changes occurring under physiological and even under relevant pathophysiological conditions in vivo are much more difficult to address and are limited by robust analytical methods required for such analyses.
In the absence of the ability to measure thiol status of specific residues in low abundance proteins, the more ready apparent availability of methods to measure GSH and GSSG contents are being used to link changes in cell functions with changes in GSH and GSSG contents (Kirlin et al., 1999; Schafer and Buettner, 2001). These studies are based principally on the assumption that the thiol/disulfide status of critical effector proteins are reflected by GSH and GSSG levels.
In earlier work, Jones reported that cysteine/cystine and thoredoxin [Trx(SH)2/Trx(SS)] redox pairs are not in thermodynamic equilibria with GSH/GSSG, or with each other (Jones et al., 2004). Why, then, would equilibration of one or more of these thiol/disulfide pairs with an effector ProtSH/ProtSSX be expected? The present report by Hansen et al. suggests one possibility, namely compartmentalization. One simplifying interpretation of the authors' report is that a shift in the redox status of the cytoplasm, as reflected in the GSH/GSSG couple, results in dissociation of Nrf-2 from Keap-1, whereas the ability of Nrf-2 to form transcriptional activation complexes in the nucleus is determined by Nrf-2 redox status in the nucleus. This latter activity is determined or reflected in part by [Trx(SH)2/Trx(SS)] redox status. This working hypothesis, which hopefully is not an egregious misinterpretation of the authors' intent, requires restriction of the exchange of mass, in this case more specifically GSH, between the cytosolic and nuclear compartments. This requirement is not consistent with evidence that the movement of GSH across the nuclear membrane is facile (Smith et al., 1996); however, it has been reported that GSH is not equally distributed throughout the cell, even within the cytosoplasm (Soderdalh et al., 2002). Even more problematic with the use of principles of chemical equilibration to explain mechanisms of regulation of dynamic processes in living systems, even GSH/GSSG and [Trx(SH)2/Trx(SS)] redox couples are not equilibrated with their respective NADPH/NADP+ ratios (Kirlin et al., 1999; Schafer and Buettner, 2001), which should ultimately drive the redox ratios of other couples, at the corresponding thermodynamic equilibrium states.
The application of the Nernst equation to assessments of thiol/disulfide status and defining a single redox poise, redox potential, or gas gauge level to a tissue, cell, or subcellular compartment is appealing, but the Nernst equation is derived from the Gibbs free energy change associated with a reaction. For the reaction
the Gibbs free energy change is
where the values represent the respective activities of the reactants and products. For reactants and products that do not dissociate, the activities are products of the respective concentrations and activity coefficients, and activity coefficients can approach unity at infinite dilution, meaning that concentrations can provide useful approximations of activities of non-dissociated species, for purposes of Nernst equation calculations. However, for species that dissociate in solution, the relationships between concentrations and activities are considerably more complicated (Daniels and Alberty, 1967). For example, the activity of something as simple as CaCl2 is proportional to the third power of the molal concentration ([Ca2+]3), and even that relationship assumes high dilution in homogeneous solution. The relationships between concentrations and activities of polyionic proteins or even simpler species like GSH and GSSG, would not be expected to be as simple as for CaCl2, especially in very heterogeneous mixtures that are characteristic of cells and tissues.
Faced with the enormous difficulty in determining the thiol/disulfide status of a specific thiol in a specific low abundance protein in a biologically relevant human sample or experimental animal model, Hansen et al. confront us with yet another challenge, that of distinguishing the subcellular compartment in which the protein is represented. Protein distributions can be studied by a variety of methods, such as confocal microscopy, but the ability to determine directly and definitively whether the redox status of a specific residue differs on a low abundance protein, such as Nrf-2, between two subcellular compartments will require ingenuity, methodological advancements and considerable work.
REFERENCES
Daniels, F., and Alberty, R. A. (1967). Physical Chemistry. John Wiley & Sons, New York.
Di Monte, D., Bellomo, G., Thor, H., Nicotera, P., and Orrenius, S. (1984). Menadione-induced cytotoxicity is associated with protein thiol oxidation and alteration in intracellular Ca2+ homostasis. Arch. Biochem. Biophys. 235, 343
Droge, W., Schulze-Osthoff, K., Mihm, S., Galter, D., Schenk, H., Ech, H. P., Roth, S., Gmunder, H. (1994). Functions of glutathione and glutathione disulfide in immunology and immunopathology. FASEB J. 8, 1131–1138.
Hansen, J. M., Watson, W. H., and Jones, D. P. (2004). Compartmentation of Nrf-2 redox control: Regulation of cytoplasmic activation by glutathione and DNA binding by thioredoxin-1. Toxicol. Sci. 82, 308–317.
Nguyen, T., Yang, C. S., and Pickett, C. B. (2004). The pathways and molecular mechanisms regulating Nrf2 activation in response to chemical stress. Free Radic. Biol. Med. 37, 433
Schafer, F. Q., and Buettner, G. R. (2001). Redox environment of the cell as viewed through the redox state of the glutathione disulfide/glutathione couple. Free Radic. Biol. Med. 30, 1191–1212.
Smith, C. V., Hughes, H., Lauterburg, B. H., and Mitchell, J. R. (1985). Oxidant stress and hepatic necrosis in rats treated with diquat. J. Pharmacol. Exp. Ther. 235, 172–177.
Smith, C. V., Jones, D. P., Guenthner, T. M., Lash, L. H., and Lauterburg, B. H. (1996). Compartmentation of glutathione: Implications for the study of toxicity and disease. Toxicol. Appl. Pharmacol. 140, 1–12.
Soderdalh, T., Enokksson, M., Lundberg, M., Homlgren, A., Ottersen, O. P., Orrenius, S., Bolcsfoldi, G., and Cotgreave, I. A. (2003). Visualization of the compartmentalization of glutathione and protein-glutathione mixed disulfides in cultured cells. FASEB J. 17, 124–126.(Charles V. Smith1)
Separate from the ultimate requirement of oxidation to provide energy for cell functions, the basic idea that properties of proteins can be changed by oxidation is a valid working hypothesis. A similar redox mechanism was proposed for the transduction of NK-B, in that its translocation to the nucleus was suggested to be dependent on oxidation, whereas its binding to DNA depended on reduction (Droge et al., 1994). With regard to cytotoxicity, the concept that toxicants kill cells by substantial (>50%) depletion of cellular thiols (Di Monte et al., 1984) has not been substantiated in subsequent studies of oxidant cell killing, particularly in vivo (Smith et al., 1985), and contributions of thiol oxidation or alkylation to cell killing appear to be far more specific than some expected initially. More recently, the increased appreciation of specificity in modulation of cell function by modifications of protein thiols has been interpolated into redox regulation of thiol/disulfide status. In general, thiols are more readily oxidized than are other cellular components. The facile reversibility of S-thiolation reactions, whether formation of intra- or interchain disulfides (ProtS-SProt) or glutathione (GSH)-protein mixed disulfides (ProtS-SG) makes the participation of such modifications attractive in working hypotheses for mechanisms of regulation of a wide variety of cellular processes, from conception to death.
However, critical tests of hypotheses based upon thiol-disulfide-driven regulation of gene transcription, signal transduction, and related cell functions require that thiol redox status of specific thiols, meaning the specific modification of specific residues of specific proteins, be characterized and quantitated accurately, and with compartmental specificity. The challenges in meeting this goal are beyond present bioanalytical capabilities, but the necessity of developing and applying such methods and concepts should be recognized, so that the limitations of the implications of results not resolved at this level will not be so readily overlooked.
Many of the proteins that are most important in regulation of cell functions are present in relatively low abundance, and present bioanalytical methods are challenged, at best, to distinguish thiol from S-thiolated or S-alkylated forms of such proteins. Methods are being developed and reported, but at the present time, many of these reports are limited to detecting changes effected by large doses of diamide, H2O2, or other oxidants in vitro. However, the critical questions of changes occurring under physiological and even under relevant pathophysiological conditions in vivo are much more difficult to address and are limited by robust analytical methods required for such analyses.
In the absence of the ability to measure thiol status of specific residues in low abundance proteins, the more ready apparent availability of methods to measure GSH and GSSG contents are being used to link changes in cell functions with changes in GSH and GSSG contents (Kirlin et al., 1999; Schafer and Buettner, 2001). These studies are based principally on the assumption that the thiol/disulfide status of critical effector proteins are reflected by GSH and GSSG levels.
In earlier work, Jones reported that cysteine/cystine and thoredoxin [Trx(SH)2/Trx(SS)] redox pairs are not in thermodynamic equilibria with GSH/GSSG, or with each other (Jones et al., 2004). Why, then, would equilibration of one or more of these thiol/disulfide pairs with an effector ProtSH/ProtSSX be expected? The present report by Hansen et al. suggests one possibility, namely compartmentalization. One simplifying interpretation of the authors' report is that a shift in the redox status of the cytoplasm, as reflected in the GSH/GSSG couple, results in dissociation of Nrf-2 from Keap-1, whereas the ability of Nrf-2 to form transcriptional activation complexes in the nucleus is determined by Nrf-2 redox status in the nucleus. This latter activity is determined or reflected in part by [Trx(SH)2/Trx(SS)] redox status. This working hypothesis, which hopefully is not an egregious misinterpretation of the authors' intent, requires restriction of the exchange of mass, in this case more specifically GSH, between the cytosolic and nuclear compartments. This requirement is not consistent with evidence that the movement of GSH across the nuclear membrane is facile (Smith et al., 1996); however, it has been reported that GSH is not equally distributed throughout the cell, even within the cytosoplasm (Soderdalh et al., 2002). Even more problematic with the use of principles of chemical equilibration to explain mechanisms of regulation of dynamic processes in living systems, even GSH/GSSG and [Trx(SH)2/Trx(SS)] redox couples are not equilibrated with their respective NADPH/NADP+ ratios (Kirlin et al., 1999; Schafer and Buettner, 2001), which should ultimately drive the redox ratios of other couples, at the corresponding thermodynamic equilibrium states.
The application of the Nernst equation to assessments of thiol/disulfide status and defining a single redox poise, redox potential, or gas gauge level to a tissue, cell, or subcellular compartment is appealing, but the Nernst equation is derived from the Gibbs free energy change associated with a reaction. For the reaction
the Gibbs free energy change is
where the values represent the respective activities of the reactants and products. For reactants and products that do not dissociate, the activities are products of the respective concentrations and activity coefficients, and activity coefficients can approach unity at infinite dilution, meaning that concentrations can provide useful approximations of activities of non-dissociated species, for purposes of Nernst equation calculations. However, for species that dissociate in solution, the relationships between concentrations and activities are considerably more complicated (Daniels and Alberty, 1967). For example, the activity of something as simple as CaCl2 is proportional to the third power of the molal concentration ([Ca2+]3), and even that relationship assumes high dilution in homogeneous solution. The relationships between concentrations and activities of polyionic proteins or even simpler species like GSH and GSSG, would not be expected to be as simple as for CaCl2, especially in very heterogeneous mixtures that are characteristic of cells and tissues.
Faced with the enormous difficulty in determining the thiol/disulfide status of a specific thiol in a specific low abundance protein in a biologically relevant human sample or experimental animal model, Hansen et al. confront us with yet another challenge, that of distinguishing the subcellular compartment in which the protein is represented. Protein distributions can be studied by a variety of methods, such as confocal microscopy, but the ability to determine directly and definitively whether the redox status of a specific residue differs on a low abundance protein, such as Nrf-2, between two subcellular compartments will require ingenuity, methodological advancements and considerable work.
REFERENCES
Daniels, F., and Alberty, R. A. (1967). Physical Chemistry. John Wiley & Sons, New York.
Di Monte, D., Bellomo, G., Thor, H., Nicotera, P., and Orrenius, S. (1984). Menadione-induced cytotoxicity is associated with protein thiol oxidation and alteration in intracellular Ca2+ homostasis. Arch. Biochem. Biophys. 235, 343
Droge, W., Schulze-Osthoff, K., Mihm, S., Galter, D., Schenk, H., Ech, H. P., Roth, S., Gmunder, H. (1994). Functions of glutathione and glutathione disulfide in immunology and immunopathology. FASEB J. 8, 1131–1138.
Hansen, J. M., Watson, W. H., and Jones, D. P. (2004). Compartmentation of Nrf-2 redox control: Regulation of cytoplasmic activation by glutathione and DNA binding by thioredoxin-1. Toxicol. Sci. 82, 308–317.
Nguyen, T., Yang, C. S., and Pickett, C. B. (2004). The pathways and molecular mechanisms regulating Nrf2 activation in response to chemical stress. Free Radic. Biol. Med. 37, 433
Schafer, F. Q., and Buettner, G. R. (2001). Redox environment of the cell as viewed through the redox state of the glutathione disulfide/glutathione couple. Free Radic. Biol. Med. 30, 1191–1212.
Smith, C. V., Hughes, H., Lauterburg, B. H., and Mitchell, J. R. (1985). Oxidant stress and hepatic necrosis in rats treated with diquat. J. Pharmacol. Exp. Ther. 235, 172–177.
Smith, C. V., Jones, D. P., Guenthner, T. M., Lash, L. H., and Lauterburg, B. H. (1996). Compartmentation of glutathione: Implications for the study of toxicity and disease. Toxicol. Appl. Pharmacol. 140, 1–12.
Soderdalh, T., Enokksson, M., Lundberg, M., Homlgren, A., Ottersen, O. P., Orrenius, S., Bolcsfoldi, G., and Cotgreave, I. A. (2003). Visualization of the compartmentalization of glutathione and protein-glutathione mixed disulfides in cultured cells. FASEB J. 17, 124–126.(Charles V. Smith1)