Detection of Reactive Oxygen Species-sensitive Thiol Proteins by Redox Difference Gel Electrophoresis

Abstract

Reactive oxygen species (ROS) produced by the mitochondrial respiratory chain can be a redox signal, but whether they affect mitochondrial function is unclear. Here we show that low levels of ROS from the respiratory chain under physiological conditions reversibly modify the thiol redox state of mitochondrial proteins involved in fatty acid and carbohydrate metabolism. As these thiol modifications were specific and occurred without bulk thiol changes, we first had to develop a sensitive technique to identify the small number of proteins modified by endogenous ROS. In this technique, redox difference gel electrophoresis, control, and redox-challenged samples are labeled with different thiol-reactive fluorescent tags and then separated on the same two-dimensional gel, enabling the sensitive detection of thiol redox modifications by changes in the relative fluorescence of the two tags within a single protein spot, followed by protein identification by mass spectrometry. Thiol redox modification affected enzyme activity, suggesting that the reversible modification of enzyme activity by ROS from the respiratory chain may be an important and unexplored mode of mitochondrial redox signaling.Reactive oxygen and nitrogen species (ROS 2 and RNS) are used by mammalian cells as signaling molecules for redox regulation (1, 2). Examples are hydrogen peroxide (H 2 O 2 ) and nitric oxide (NO), which are weak oxidants that can cross membranes and react with defined targets (2, 3). Modifications to protein thiols by ROS/RNS are an important aspect of redox signal transduction (2, 4, 5). These thiol modifications include oxidation to sulfenic acids, intra-and intermolecular disulfides, glutathionylation, sulfenyl-amide linkages, and S-nitrosation (6 -9). As many of these modifications can be reversed through interaction with glutathione or thioredoxin, protein thiol redox state can respond to the redox environment (10, 11). The mitochondrial respiratory chain is the major source of ROS in most cells (12,13) and is involved in redox signaling (14). The proximity of mitochondrial protein thiols to the major ROS source and the high matrix pH (ϳ8.0) make modification of mitochondrial protein thiols by ROS more likely than for cytosolic thiols (15). Some mitochondrial proteins have shown ROS-dependent thiol oxidation:peroxiredoxin III (PrxIII) (16), NADP ϩ -dependent isocitrate dehydrogenase (17), and complex I (18, 19). The role of protein thiol modification in mitochondrial redox regulation, however, remains unclear, in large part because of the difficulty of identifying mitochondrial thiol proteins responsive to the low ROS fluxes found in vivo.To explore the role of mitochondrial protein thiols in redox signaling, we developed a sensitive proteomic approach to identify thiol proteins that are modified by low levels of endogenous ROS. We adapted the difference gel electrophoresis (DIGE) technique (20, 21) labeling protein thiols in control and redox-challenged samples with two different fluorescent dyes containing thiol-reactive mal...

Mitochondrial redox signalling at a glance

“…However, assessing changes in ROS and protein redox modifications in biological systems is technically demanding and requires an understanding of the underlying chemistry . Despite this, considerable evidence demonstrates the presence of protein thiols within mitochondria that can be modified by H 2 O 2 and S-nitrosating agents (Chouchani et al, 2010;Hurd et al, 2005a;Hurd et al, 2005b;Hurd et al, 2007;Prime et al, 2009;Sun et al, 2007). There are now a variety of methods that can be used to assess the levels of particular ROS within mitochondria, and these include mitochondria-targeted small-molecule fluorescence probes (Dickinson et al, 2010a;Dickinson et al, 2010b;Robinson et al, 2006), the use of mitochondria-targeted proteins derived from green fluorescent protein -whose fluorescence is redox sensitive (Meyer and Dick, 2010), and mitochondria-targeted mass spectrometry probes that enable mitochondrial ROS levels to be estimated in vivo (Cochemé et al, 2011).…”

“…There are now a variety of methods that can be used to assess the levels of particular ROS within mitochondria, and these include mitochondria-targeted small-molecule fluorescence probes (Dickinson et al, 2010a;Dickinson et al, 2010b;Robinson et al, 2006), the use of mitochondria-targeted proteins derived from green fluorescent protein -whose fluorescence is redox sensitive (Meyer and Dick, 2010), and mitochondria-targeted mass spectrometry probes that enable mitochondrial ROS levels to be estimated in vivo (Cochemé et al, 2011). The proteins modified and the nature of the thiol modification can also be determined by using a number of redox proteomic techniques (Chouchani et al, 2010;Dahm et al, 2006;Danielson et al, 2011;Taylor et al, 2003;Held et al, 2010;Hurd et al, 2007).…”

Nrf2 activation in the treatment of neurodegenerative diseases: a focus on its role in mitochondrial bioenergetics and function

“…It has been described that mitochondrial ROS can reversibly modify thiol groups present in several enzymes implicated in carbohydrate and lipid metabolism, affecting their activity (Hurd et al, 2007). Most of the enzymes found in the cited study are involved in fatty acid oxidation (FAO) (carnitine acetyltransferase, very long chain acylCoA dehydrogenase, propionyl-CoA carboxylase); and also in the regulation of pyruvate dehydrogenase (PDH) by pyruvate dehydrogenase kinase 2, which inhibits PDH and prevents the entry of pyruvate from glycolysis into the Krebs cycle (Hurd et al, 2007). It has also been well documented that glyceraldehyde-3-phosphate dehydrogenase (GAPDH), an enzyme that catalyzes the sixth step of glycolysis, possesses a cysteine in its active site, which can be oxidized by reactive oxygen and nitrogen species .…”

A FRET-based method to study protein thiol oxidation in histological preparations

“…In particular, several spectrophotometry-based protocols were developed to measure thiol levels. More recently, proteomic methods to identify disulfides in proteins have been established [6][7][8]. However, means to image exact loci of thiol oxidation in cells and tissues are quite limited [9].…”