Fig. 1. Scheme for reversible redox modification of reactive cysteines. A reactive cysteine is a protein thiol (P-SH) that is ionized to the relatively nucleophilic thiolate anion ( P-S ) at physiological pH. The reactive cysteine can be readily oxidized to a thiyl radical (P-S' ) or sulfenic acid (P-SOH) under mild oxidative stress. These oxidized species are frequently unstable under physiological conditions and, if not enzymatically reduced, will react to form more stable intermediates. Further oxidation yields a sulfinic acid (P-SOOH), which is thought to be irreversible in biological systems. Alternatively, the thiyl radical or sulfenic acid can react with a low molecular weight thiol (RSH) to form a stable mixed disulfide (P-S-SR). The mixed disulfide can then be reduced by one of three known enzymes systems [thioredoxin (TRx), glutaredoxin (GRx), or protein disulfide-isomerase (PDI)] to restore the cysteine to its fully reduced state.

phosphatases8^'0 and small GTP-binding proteins.11 In most, if not all cases, thi-olates play an important role in the normal functioning of the protein. Therefore their modification would be expected to have functional consequences. Oxidation of a thiolate by superoxide or hydrogen peroxide produces a thiyl radical or sulfenic acid (Fig. 1). Under physiological conditions, these oxidation products are unstable and, unless enzymatically reduced back to the thiol, will rapidly react with other molecules to form more stable products. Further oxidation of a thiyl radical or sulfenic acid results in irreversible oxidation of the cysteine. Alternatively, these species can react with other thiols to form stable disulfides. In some instances the second thiol is contributed by another cysteine contained within the same protein or an associated protein, resulting in the formation of an intra- or intermolecular protein disulfide. If there are no protein cysteinyl thiols in the vicinity of the thiyl radical or sulfenic acid, they might react with one of several low molecular weight thiols that are present in the cell to form a mixed disulfide. Because of the relatively high concentration of glutathione (GSH) in the cell (1 to 10 m M),12 it is expected that the majority of protein mixed disulfides formed as a consequence of oxidative stress contain GSH. Indeed, the transient incorporation of glutathione into cellular protein is a well-established

8 G. Zhou, J. M. Denu, L. Wu, and J. E. Dixon, J. Biol. Chem. 269, 28084 (1994).

9 G. H. Peters, T. M. Frimurer, and O. H. Olsen, Biochemistry 37, 5383 (1998).

10 J. M. Denu and K. G. Tanner, Biochemistry 37, 5633 (1998).

11 H. M. Lander, D. P. Hajjar, B. L. Hempstead, U. A. Mirza, B. T. Chait, S. Campbell, and L. A. Quilliam, J. Biol. Chem. 272,4323 (1997).

response to oxidative challenge of intact tissues or cells in culture (reviewed in Refs. 13-15).

This process of protein thiolate oxidation and glutathiolation is intriguing in that it represents the reversible covalent modification of a protein attribute with functional importance. As such, the glutathiolation state of cellular proteins could serve to gauge the redox status of the intracellular environment, with the altered functional state of some modified proteins serving to transduce the oxidative stress into a biological response. Therefore a thorough accounting of proteins modified in this way and the functional consequences of modification could provide insight into the ways in which cells sense and respond to oxidative stress.

A number of techniques have been developed to study protein glutathiolation16-18 or redox-dependent modification of reactive cysteines.19'20 However, although these protocols can provide information regarding changes in the global glutathiolation or oxidation status of protein thiols, or can be used to study the glutathiolation state of individual proteins, it remains difficult to identify new glutathiolated proteins by these technologies. In addition, some of the available technologies are prone to artifacts arising from the need to inhibit protein synthesis while labeling the intracellular GSH pool, or the need to label proteins after cell lysis. We have described a novel reagent, biotinylated glutathione ethyl ester (BioGEE), and protocols that allows for the rapid purification of proteins that are oxidatively modified at reactive cysteine thiols in situ. This approach has a number of advantages over existing technologies. Because the tracer molecule can be incorporated into protein only as a consequence of cysteinyl thiol oxidation, the cell can be loaded without the need to inhibit protein synthesis. In addition, excess label is scavenged from the system before and during cell lysis so that incorporation of the label accurately reflects the glutathiolation state of the proteins before cell lysis. Finally, the use of biotin as a label allows sensitive nonradioactive detection and rapid affinity purification of labeled proteins with streptavidin conjugates.

By using BioGEE, we were able to demonstrate oxidative modification of several proteins in conjunction with tumor necrosis factor a (TNF-a)-stimulated apoptosis, and identified two proteins that had not previously been shown to be

13 P. Klatt and S. Lamas, Eur. J. Biochem. 267, 4928 (2000).

14 J. A. Thomas, B. Poland, and R. Honzatko, Arch. Biochem. Biophys. 319, 1 (1995).

15 I. A. Cotgreave and R. G. Gerdes, Biochem. Biophys. Res. Commun. 242, 1 (1998).

16 Y. C. Chai, S. Hendrich, and J. A. Thomas, Arch. Biochem. Biophys. 310, 264 (1994).

17 Y. C. Chai, S. S. Ashraf, K. Rokutan, R. B. Johnston, Jr., and J. A. Thomas, Arch. Biochem. Biophys. 310, 273 (1994).

18 J. A. Thomas, W. Zhao, S. Hendrich, and P. Haddock, Methods Enzymol. 251,423 (1995).

19 Y. Wu, K. S. Kwon, and S. G. Rhee, FEBS Lett. 440, 111 (1998).

20 J. R. Kim, H. W. Yoon, K. S. Kwon, S. R. Lee, and S. G. Rhee, Anal. Biochem. 283, 214 (2000).

0 0

Post a comment