Are free radicals involved in thiol-based redox signaling?

Cells respond to many stimuli by transmitting signals through redox-regulated pathways. It is generally accepted that in many instances signal transduction is via reversible oxidation of thiol proteins, although there is uncertainty about the specific redox transformations involved. The prevailing view is that thiol oxidation occurs by a two electron mechanism, most commonly involving hydrogen peroxide. Free radicals, on the other hand, are considered as damaging species and not generally regarded as important in cell signaling. This paper examines whether it is justified to dismiss radicals or whether they could have a signaling role. Although there is no direct evidence that radicals are involved in transmitting thiol-based redox signals, evidence is presented that they are generated in cells when these signaling pathways are activated. Radicals produce the same thiol oxidation products as two electron oxidants, although by a different mechanism, and at this point radical-mediated pathways should not be dismissed. There are unresolved issues about how radical mechanisms could achieve sufficient selectivity, but this could be possible through colocalization of radical-generating and signal-transducing proteins. Colocalization is also likely to be important for nonradical signaling mechanisms and identification of such associations should be a priority for advancing the field.     

An expanding genetic code

More than 30 novel amino acids have been genetically encoded in response to unique triplet and quadruplet codons including fluorescent, photoreactive and redox active amino acids, glycosylated and heavy atom derived amino acids in addition to those with keto, azido and acetylenic chains. In this article, we describe recent advances that make it possible to add new building blocks systematically to the genetic codes of bacteria, yeast and mammalian cells. Taken together these tools will enable the detailed investigation of protein structure and function, which is not possible with conventional mutagenesis. Moreover, by lifting the constraints of the existing 20-amino-acid code, it should be possible to generate proteins and perhaps entire organisms with new or enhanced properties.     

Reversible disulfide bond formation of intracellular proteins probed by NMR spectroscopy

Reversible oxidation of amino acids within intracellular proteins leads to local and/or global conformational changes in protein structure. Thus, the enzymatic activity or binding properties of a protein might be regulated by local changes in a cell's redox potential, mediated by the availability of reducing/oxidizing equivalents. Whereas it is well established that intracellular pools of oxidizable groups compensate for oxidative stress, far less is known about the molecular mechanisms that accompany transient and reversible oxidation of cytoplasmic proteins. Therefore, the intrinsic redox properties of proteins amenable to reversible oxidation need to be determined. Here we describe the application of NMR spectroscopy to derive the redox properties of intracellular proteins. As exemplified for thioredoxin 1, the Tnk-1 kinase SH3 domain, and the hSH3(N) domain of the T cell protein ADAP, the conformational changes associated with disulfide bond formation can be followed directly upon titration with different ratios of reduced to oxidized glutathione. Redox potentials can be measured accurately in homogeneous solutions and define the conditions under which regulatory oxidation of the respective protein may occur in the living cell.     

Dynamic Redox Regulation of IL-4 Signaling

Quantifying the magnitude and dynamics of protein oxidation during cell signaling is technically challenging. Computational modeling provides tractable, quantitative methods to test hypotheses of redox mechanisms that may be simultaneously operative during signal transduction. The interleukin-4 (IL-4) pathway, which has previously been reported to induce reactive oxygen species and oxidation of PTP1B, may be controlled by several other putative mechanisms of redox regulation; widespread proteomic thiol oxidation observed via 2D redox differential gel electrophoresis upon IL-4 treatment suggests more than one redox-sensitive protein implicated in this pathway. Through computational modeling and a model selection strategy that relied on characteristic STAT6 phosphorylation dynamics of IL-4 signaling, we identified reversible protein tyrosine phosphatase (PTP) oxidation as the primary redox regulatory mechanism in the pathway. A systems-level model of IL-4 signaling was developed that integrates synchronous pan-PTP oxidation with ROS-independent mechanisms. The model quantitatively predicts the dynamics of IL-4 signaling over a broad range of new redox conditions, offers novel hypotheses about regulation of JAK/STAT signaling, and provides a framework for interrogating putative mechanisms involving receptor-initiated oxidation.     

Redox-induced transitions in bovine cytochrome bc1 complex studied by perfusion-induced ATR-FTIR spectroscopy

Redox transitions in a film of detergent-purified bovine cytochrome bc(1) complex were investigated by perfusion-induced attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy. The technique provides a flexible method for generating redox-induced IR changes of components of bovine cytochrome bc(1) complex at a high signal:noise ratio. These IR redox difference spectra arise from perturbations of prosthetic groups and surrounding protein. Visible difference spectra were recorded synchronously using a light beam reflected from the exposed prism surface and provided a quantitative means of determining the redox transitions that were occurring. IR and visible redox difference spectra of iron-sulfur protein/cytochrome c(1), heme b(H), and heme b(L) were separated by selective reduction and/or oxidation that extends published data on the homologous bacterial enzyme. Several bands could be tentatively assigned to redox-sensitive modes of hemes and ubiquinone and changes in the surrounding protein by comparison with available data for bacterial bc(1) complex, other related heme proteins, and model compounds. Some tentative assignments of further signals to specific amino acids are made on the basis of known crystal structures.     

Increased number of Arginine-based salt bridges contributes to the thermotolerance of thermotolerant acetic acid bacteria, Acetobacter tropicalis SKU1100

Constitution of oxidative defense systems and, correspondingly, oxidative stress prevention are highly dependent on amino acid supply. In vitro, experiments have demonstrated that amino acid availability participates to the homeostasis of reactive oxygen species. However the molecular mechanisms involved in the maintenance of redox homeostasis responsive to circulating amino acid levels remain unclear. As GCN2 is a protein kinase considered to be an important sensor for amino acids availability and a potential regulator of redox homeostasis, we hypothesized that this kinase can modulate redox homeostasis in vivo, in response to an amino acid-imbalanced diet.We investigated the response of GCN2+/+ and GCN2−/− mice to a long-term (24 weeks) leucine-imbalanced diet (EDΔLeu). In order to evaluate the oxidation level in each group of mice, we determined the degree of protein oxidation in the liver. Interestingly, GCN2−/− mice exhibited an increase in protein carbonylation, a marker of oxidative stress, in response to the EDΔLeu diet. These data correlate with a decrease in hepatic GPX1 expression, a major antioxidant enzyme, and a decrease in total GPX activity in the liver. Our results suggest that GCN2 and its downstream signaling pathway have an important role in the protection against oxidative injuries induced by an amino acid-imbalanced diet, and that it can play a critical role in the prevention of oxidative damage.     

Organic cofactors participated more frequently than transition metals in redox reactions of primitive proteins

Protein redox reactions are one of the most basic and important biochemical actions. As amino acids are weak redox mediators, most protein redox functions are undertaken by protein cofactors, which include organic ligands and transition metal ions. Since both kinds of redox cofactors were available in the pre‐protein RNA world, it is challenging to explore which one was more involved in redox processes of primitive proteins? In this paper, using an examination of the redox cofactor usage of putative ancient proteins, we infer that organic ligands participated more frequently than transition metals in redox reactions of primitive proteins, at least as protein cofactors. This is further supported by the relative abundance of amino acids in the primordial world. Supplementary material for this article can be found on the BioEssays website. BioEssays 30:766–771, 2008. © 2008 Wiley Periodicals, Inc.     

Influence of PAS Domain Flanking Regions on Oligomerisation and Redox Signalling By NifL

Per-ARNT-Sim (PAS) domains constitute a typically dimeric, conserved α/β tertiary fold of approximately 110 amino acids that perform signalling roles in diverse proteins from all kingdoms of life. The amino terminal PAS1 domain of NifL from Azotobacter vinelandii accommodates a redox-active FAD group; elevation of cytosolic oxygen concentrations result in FAD oxidation and a concomitant conformational re-arrangement that is relayed via a short downstream linker to a second PAS domain, PAS2. At PAS2, the signal is amplified and passed on to effector domains generating the ‘on’ (inhibitory) state of the protein. Although the crystal structure of oxidised PAS1 reveals regions that contribute to the dimerisation interface, 21 amino acids at the extreme N-terminus of NifL, are unresolved. Furthermore, the structure and function of the linker between the two PAS domains has not been determined. In this study we have investigated the importance to signalling of residues extending beyond the core PAS fold. Our results implicate the N-terminus of PAS1 and the helical linker connecting the two PAS domains in redox signal transduction and demonstrate a role for these flanking regions in controlling the oligomerisation state of PAS1 in solution.     

Protein metabolism during endurance exercise

After reviewing all the available results from our laboratory and numerous reports in the literature concerning changes that have occurred in various aspects of protein metabolism during exercise, a number of conclusions can be drawn with some degree of confidence. During exercise, protein synthesis is depressed and this change leaves amino acids available for catabolic processes. The rate of leucine oxidation appears to be increased during exercise, and there is a movement of amino acids, mostly in the form of alanine, from muscle to liver where the rate of gluconeogenesis is increased as a result of exercise. These changes in protein metabolism are probably physiologically significant in at least three ways: amino acid conversion to citric acid cycle intermediates enhances the rate of oxidation of acetyl-CoA generated from glucose and fatty acid oxidation; increased conversion of amino acids to glucose helps to prevent hypoglycemia; oxidation of some amino acids may provide energy for muscular contraction.     

The physiological role of reversible methionine oxidation

Sulfur-containing amino acids such as cysteine and methionine are particularly vulnerable to oxidation. Oxidation of cysteine and methionine in their free amino acid form renders them unavailable for metabolic processes while their oxidation in the protein-bound state is a common post-translational modification in all organisms and usually alters the function of the protein. In the majority of cases, oxidation causes inactivation of proteins. Yet, an increasing number of examples have been described where reversible cysteine oxidation is part of a sophisticated mechanism to control protein function based on the redox state of the protein. While for methionine the dogma is still that its oxidation inhibits protein function, reversible methionine oxidation is now being recognized as a powerful means of triggering protein activity. This mode of regulation involves oxidation of methionine to methionine sulfoxide leading to activated protein function, and inactivation is accomplished by reduction of methionine sulfoxide back to methionine catalyzed by methionine sulfoxide reductases. Given the similarity to thiol-based redox-regulation of protein function, methionine oxidation is now established as a novel mode of redox-regulation of protein function. This article is part of a Special Issue entitled: Thiol-Based Redox Processes.     

The effect of acylcarnitines on the oxidation of branched chain alpha-keto acids in mitochondria

The oxidation of 14C-labelled branched-chain alpha-keto acids corresponding to the branched-chain amino acids valine, isoleucine and leucine has been studied in isolated mitochondria from heart, liver and skeletal muscle. 1. Heart and liver mitochondria have similar capacities to oxidize these alpha-keto acids based on protein content. Skeletal muscle mitochondria also show significant activity. 2. Half maximum rates are obtained with approximately 0.1 mM of the alpha-keto acids under optimal conditions. Added NAD and CoA had no effect on the oxidation rate, showing that endogenous mitochondrial NAD and CoA are required for the oxidation. 3. Addition of carnitine esters of fatty acids (C6--C16), succinate, pyruvate, or alpha-ketoglutarate inhibited the oxidation of the branched chain alpha-keto acids, especially in a high-energy state (no ADP added). In heart mitochondria the addition of AD (low-energy state) decreased the inhibitory effects of acylcarnitines of medium chain length or of pyruvate, and abolished the inhibitory effect of succinate. It is suggested that the oxidation rate is regulated mainly by the redox state of the mitochondria under the conditions used. 4. The results are discussed in relation to the regulation of branched-chain amino acid metabolism in the body.     

Reciprocal Control of the Circadian Clock and Cellular Redox State - a Critical Appraisal

Redox signalling comprises the biology of molecular signal transduction mediated by reactive oxygen (or nitrogen) species. By specific and reversible oxidation of redox-sensitive cysteines, many biological processes sense and respond to signals from the intracellular redox environment. Redox signals are therefore important regulators of cellular homeostasis. Recently, it has become apparent that the cellular redox state oscillates in vivo and in vitro, with a period of about one day (circadian). Circadian time-keeping allows cells and organisms to adapt their biology to resonate with the 24-hour cycle of day/night. The importance of this innate biological time-keeping is illustrated by the association of clock disruption with the early onset of several diseases (e.g. type II diabetes, stroke and several forms of cancer). Circadian regulation of cellular redox balance suggests potentially two distinct roles for redox signalling in relation to the cellular clock: one where it is regulated by the clock, and one where it regulates the clock. Here, we introduce the concepts of redox signalling and cellular timekeeping, and then critically appraise the evidence for the reciprocal regulation between cellular redox state and the circadian clock. We conclude there is a substantial body of evidence supporting circadian regulation of cellular redox state, but that it would be premature to conclude that the converse is also true. We therefore propose some approaches that might yield more insight into redox control of cellular timekeeping.     

Proton release from water oxidation by photosystem II: similar stoichiometries are stabilized in thylakoids and PSII core particles by glycerol

During the four-stepped catalytic cycle of water oxidation by photosystem II (PSII) molecular oxygen is released in only one of the four reaction steps whereas the release of four protons is distributed over all steps. In principle, the pattern of proton production could be taken as indicative of the partial reactions with bound water. In thylakoids the extent and rate of proton release varies as function of the redox transition and of the pH without concomitant variations of the redox pattern. The variation has allowed to discriminate between deprotonation events of peripheral amino acids (Bohr effects) as opposed to the chemical deprotonation of a particular redox cofactor, and of water. In contrast, in thylakoids grown under intermittent light, as well as in PSII core particles the pattern of proton release is flat and independent of the pH. This has been attributed to the lack in these materials of the chlorophyll a,b-binding (CAB) proteins. We now found that a thylakoid-like, oscillatory pattern of proton release was restored simply by the addition of glycerol which modifies the protein–protein interaction. Being a further proof for the electrostatic origin of the greater portion of proton release, this effect will serve as an important tool in further studies of water oxidation.     

Efficient Intracellular Delivery of GFP by Homeodomains of Drosophila Fushi-tarazu and Engrailed Proteins

The 60 amino acid long homeodomain of Antennapedia (Antp), either alone or as a fusion protein with 30-40 amino acid long foreign polypeptides, has been reported to cross biological membranes by an energy- and receptor-protein-independent mechanism. Moreover, the 16 amino acid long third helix of the Antp homeodomain, so-called penetratin, possesses translocation properties when fused to fewer than 100 amino acids as well. These findings led us to study whether such a protein tansduction property is shared by other homeodomains. We report here that homeodomains of two homeoproteins, Fushi-tarazu and Engrailed, are able to transduce a 238 amino acid long green fluorescent protein into cultured cells as efficiently as other well-known protein transduction domains, such as an internal oligopeptide of Tat and penetratin. These findings suggest that such transduction activity of homeodomains might have some physiological roles and that it can be exploited for development of efficient transduction vectors for research use and protein therapy.     

Efficient Intracellular Delivery of GFP by Homeodomains of Drosophila Fushi-tarazu and Engrailed Proteins

The 60 amino acid long homeodomain of Antennapedia (Antp), either alone or as a fusion protein with 30–40 amino acid long foreign polypeptides, has been reported to cross biological membranes by an energy- and receptor-protein-independent mechanism. Moreover, the 16 amino acid long third helix of the Antp homeodomain, so-called penetratin, possesses translocation properties when fused to fewer than 100 amino acids as well. These findings led us to study whether such a protein tansduction property is shared by other homeodomains. We report here that homeodomains of two homeoproteins, Fushi-tarazu and Engrailed, are able to transduce a 238 amino acid long green fluorescent protein into cultured cells as efficiently as other well-known protein transduction domains, such as an internal oligopeptide of Tat and penetratin. These findings suggest that such transduction activity of homeodomains might have some physiological roles and that it can be exploited for development of efficient transduction vectors for research use and protein therapy.