Chapter Four - Measuring the Redox State of Cellular Peroxiredoxins by Immunoblotting
Introduction
The peroxiredoxins (Prxs) have received considerable attention in recent years as regulators of cellular H2O2 and modulators of H2O2-dependent signaling pathways (Hall et al., 2009, Rhee et al., 2005). Prxs are highly abundant proteins constituting up to 1% of the total soluble protein in most cells. Unlike other peroxidases that require a prosthetic heme group or selenocysteine moiety, the Prxs use a conserved cysteine residue present as a thiolate anion to decompose hydroperoxides. Recent kinetic studies have revealed that many Prxs react with H2O2 at rates comparable to catalase or glutathione peroxidase (k ~ 107–108 M− 1 s− 1) (Cox et al., 2009b, Ogusucu et al., 2007, Parsonage et al., 2008, Peskin et al., 2007). As such, the Prxs are increasingly being seen as primary targets of cellular H2O2 (Winterbourn and Hampton, 2008).
The initial reaction of the peroxidatic cysteine with H2O2 forms a sulfenic acid (CysP-SOH). The fate of this sulfenic acid differs depending on whether the Prx is a typical 2-Cys, atypical 2-Cys or 1-Cys enzyme. The most common class is the typical 2-Cys Prxs, which exist as obligate homodimers orientated in a head-to-tail manner. The sulfenic acid condenses with the resolving cysteine of the second subunit to form an intermolecular disulfide bond (Fig. 4.1A). The disulfide bonds in the oxidized dimer are reduced by thioredoxin (Trx) to complete the catalytic cycle. In contrast, atypical 2-Cys Prxs form an intramolecular disulfide bond upon oxidation (Seo et al., 2000), whereas the 1-Cys Prxs form a mixed disulfide with glutathione (Manevich et al., 2004).
One interesting aspect of these proteins is their sensitivity to hyperoxidation, with H2O2 reacting with the sulfenic acid intermediate to form a sulfinic acid (Fig. 4.1B). Intriguingly, eukaryotic typical 2-Cys Prxs have evolved structural features that dramatically enhance susceptibility to hyperoxidation compared to their prokaryotic counterparts (Wood et al., 2003). The hyperoxidized Prxs are enzymatically inactive, although they can be regenerated slowly by the sulfinyl reductase sulfiredoxin (Biteau et al., 2003, Jonsson & Lowther, 2007). It has been proposed that Prx hyperoxidation may enable H2O2 levels to accumulate and function as second messengers in cell signaling (Wood et al., 2003). Alternatively, studies suggest that the hyperoxidation of Prxs may represent a molecular switch that abolishes peroxidase activity, while enhancing chaperone activity (Jang et al., 2004).
Cellular Prxs are maintained in their reduced form by the Trx system. However, several studies have reported increases in the ratio of oxidized to reduced Prxs through increased exposure to H2O2 (Baty et al., 2005, Cox & Hampton, 2007, Cox et al., 2009a, Low et al., 2007), chloramines (Stacey et al., 2009), auranofin (Cox et al., 2008a), isothiocyanates (Brown et al., 2008), hexavalent chromium (Myers and Myers, 2009), and induction of receptor-mediated apoptosis (Cox et al., 2008b). Prx oxidation has also been observed in hearts during ischemia (Kumar et al., 2009) or upon perfusion with H2O2 (Schroder et al., 2008). Mammals have six Prxs that are widely distributed in different cellular compartments, including cytosol (Prx 1, 2, and 6), mitochondria (Prx 3 and 5), endoplasmic reticulum (Prx 4), and peroxisomes (Prx 5). In some studies where Prx oxidation was observed, it was restricted to a subset of Prxs, suggesting organelle specificity. Also, Prx oxidation occurred in the absence of widespread oxidative damage, for example, protein carbonylation or lipid peroxidation. Analysis of the redox transformations of endogenously expressed Prxs may provide valuable insight as a marker of disruptions in redox homeostasis. The methods described in this chapter provide a means to monitor the dimerization or hyperoxidation of individual typical 2-Cys Prxs in cells or tissue by immunoblotting under nonreducing conditions. These simple methods are well suited to the testing of multiple samples in parallel and overcome some of the technical challenges associated with previous methodologies.
Section snippets
Principle of the method
During their catalytic cycle, typical 2-Cys Prxs are oxidized to form an intermolecular disulfide bond, with the oxidized protein running as a dimer under nonreducing conditions (Fig. 4.2). The principle of the method is to trap the oxidized and reduced forms during cell or tissue extraction and monitor the proportions of monomer and dimer for each Prx with selective antibodies.
A critical step in the process is rapid alkylation of reduced cysteines to prevent artefactual Prx oxidation during
Principle of method
The hyperoxidation of typical 2-Cys Prxs can be monitored through an adaptation of the method described above. It takes advantage of the fact that in the absence of alkylation, reduced Prxs immediately dimerize following cell lysis. However, hyperoxidized Prxs are unable to dimerize, and will remain as monomers (Fig. 4.5).
We described above the difficulty in alkylating reduced Prxs before they are oxidized by adventitious peroxides. That complication is the basis of this method, and is what
Discussion
We have described a method for quantifying the interconversion of typical 2-Cys Prxs between their three main redox forms: reduced, oxidized, and hyperoxidized. The method was illustrated for cultured mammalian cells, but the principles have been adapted for use with purified protein (Cox et al., 2009a, Cox et al., 2009b, Peskin et al., 2007), isolated mitochondria (Requejo et al., 2010), and whole tissue (Kumar et al., 2009, Schroder et al., 2008). It should also be possible to explore this
Acknowledgments
The authors acknowledge the efforts and insight provided by past and present colleagues of the Free Radical Research Group, including Felicia Low, Alexander Peskin, James Baty, Andree Pearson, Kristin Brown, Sarah Cuddihy, Melissa Stacey, and Vikas Kumar. This project was supported by the Royal Society Marsden Fund, the Health Research Council of New Zealand, and the National Research Centre for Growth and Development. A. G. C. is a recipient of a Top Achiever Doctoral Scholarship from the
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2021, Free Radical Biology and MedicineCitation Excerpt :A significant increase in Prx2 dimer formation was detected within 5 min of treatment with D-ala, when compared to untreated conditions, and dimer formation was maintained over the time course (Fig. 9C and D). Under oxidative stress conditions and at later time points (30–60 min), we and others, have previously observed the disappearance of oxidized Prx dimers and the re-appearance of the Prx monomer, which indicates Prx hyperoxidation [31,32,45]. For both Prx1 and Prx2, hyperoxidized monomer formation was not observed, suggesting that 6 mM D-ala activation of NLS-HyPer-DAAO led to physiological levels of ROS that oxidize the Prxs but do not cause Prx hyperoxidation (Fig. 9C and D).