Compartmentalization of Redox Regulation of Cell Responses

Abstract

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-kB, 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 (450%) 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 intraor 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 thioldisulfide-driven regulation of gene transcription, signal transduction, and related cell functions require that thiol redox status of specific thiols, meaning the specific modifications 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 1 For correspondence via fax: (614) 722-2774. E-mail: smithcv@chi. osu.edu