Oxidative Thiol Modifications in Pro- And Eukaryotic Organisms

For many years, oxidative thiol modifications in cytosolic proteins were largely disregarded as in vitro artifacts, and considered unlikely to play significant roles within the reducing environment of the cell. Recent developments in in vivo thiol trapping technology combined with in vivo mass spectrometric analysis have now provided convincing evidence that thiol-based redox switches are used as molecular tools in many proteins to regulate their activity in response to reactive oxygen and nitrogen species. Reversible oxidative thiol modifications have been found to modulate the function of proteins involved in many different pathways, starting from gene transcription, translation and protein folding, to metabolism, signal transduction, and ultimately apoptosis. This introduction will focus on three well-characterized eukaryotic proteins that use thiol-based redox switches to influence gene transcription, metabolism and signal transduction. The transcription factor Yap1 is a good illustration of how oxidative modifications affect the function of a protein without changing its activity. We use glyeraldehyde-3-phosphate dehydrogenase to demonstrate how thiol modification of an active site cysteine re-routes metabolic pathways and converts a metabolic enzyme into a pro-apoptotic factor. Finally, we introduce the redox-sensitive protein tyrosine phosphatase PTP1B to illustrate that reversibility is one of the fundamental aspects of redox-regulation. Chapter 1: Introduction 8 1.1 Reactive oxygen species and oxidative stress Reactive oxygen species (ROS) include a broad variety of different chemical oxidants including hydrogen peroxide (H2O2), superoxide anion (•O2-), singlet oxygen ( O2), and hydroxyl radicals (•OH). Inside the cells, reactive oxygen species are constantly generated as side products of neutrophil-mediated phagocytosis [2], cellular respiration [3], and various NADPH oxidases [4]. This ROS production appears not to be just a harmful byproduct of aerobic metabolism, but seems to involve a tightly regulated process [5]. It allows low levels of ROS to serve as important signaling molecules in processes such as gene transcription, protein tyrosine deand phosphorylation, stress protection, apoptosis, and metabolism [5-8]. Similarly, reactive nitrogen species (RNS), which include the free radical nitric oxide (•NO), peroxynitrite (ONOO) and nitrogen dioxide (NO2), also play significant roles as redox-active molecules [9]. In the presence of oxygen, endogenously produced •NO can rapidly lead to S-nitrosation of protein thiols (SNO) and glutathione (GSNO), presumably via the formation of other reactive nitrogen species, such as N2O3, NO2 and thiyl radicals [10]. Over 200 different proteins have been found to be targeted by S-nitrosation, including metabolic enzymes, phosphatases, transcription factors, and others [11]. To maintain redox homeostasis under conditions where ROS and RNS concentrations begin to increase (see below), cells can draw on a wide arsenal of enzymatic and non-enzymatic defense and repair strategies. For rapid ROS detoxification and scavenging, most cells use a combination of antioxidant enzymes with very high catalytic activity, such as superoxide dismutase (Sod), catalase (Cat), and glutathione peroxidase (Gpx), or of high cellular abundance, such as peroxiredoxins (>0.5% of total cell protein) [12]. The original oxidation status of cytosolic protein thiols is maintained and rapidly restored by the action of two redoxbalancing systems, thioredoxin (Trx) and glutaredoxin (Grx), which draw their reducing power ultimately from NADPH [13]. The overall reducing environment of the cytosol of −220 to −260 mV, which represents the cellular standard redox potential, is preserved by the equilibrium between the small tripeptide glutathione (GSH) and its oxidized form (GSSG) [14,15]. The GSH/GSSG couple constitutes the redox buffer of most eukaryotic and prokaryotic cells. The low reactivity of the GSH thiol at Chapter 1: Introduction 9 physiological conditions (pKa of 9.4) [16] is counterbalanced by its high intracellular concentration (~1-10 mM) and rapid enzymatic regeneration by glutathione reductase