Introduction to the Thematic Minireview Series on Redox-active Protein Modifications and Signaling

The dynamics of redox metabolism necessitate cellular strategies for sensing redox changes and for responding to them. A common mechanism for receiving and transmitting redox changes is via reversible modifications of protein cysteine residues. A plethora of cysteine modifications have been described, including sulfenylation, glutathionylation, and disulfide formation. These post-translational modifications have the potential to alter protein structure and/or function and to modulate cellular processes ranging from division to death and from circadian rhythms to secretion. The focus of this thematic minireview series is cysteine modifications in response to reactive oxygen and nitrogen species.     

Mechanism-based Proteomic Screening Identifies Targets of Thioredoxin-like Proteins

Thioredoxin (Trx)-fold proteins are protagonists of numerous cellular pathways that are subject to thiol-based redox control. The best characterized regulator of thiols in proteins is Trx1 itself, which together with thioredoxin reductase 1 (TR1) and peroxiredoxins (Prxs) comprises a key redox regulatory system in mammalian cells. However, there are numerous other Trx-like proteins, whose functions and redox interactors are unknown. It is also unclear if the principles of Trx1-based redox control apply to these proteins. Here, we employed a proteomic strategy to four Trx-like proteins containing CXXC motifs, namely Trx1, Rdx12, Trx-like protein 1 (Txnl1) and nucleoredoxin 1 (Nrx1), whose cellular targets were trapped in vivo using mutant Trx-like proteins, under conditions of low endogenous expression of these proteins. Prxs were detected as key redox targets of Trx1, but this approach also supported the detection of TR1, which is the Trx1 reductant, as well as mitochondrial intermembrane proteins AIF and Mia40. In addition, glutathione peroxidase 4 was found to be a Rdx12 redox target. In contrast, no redox targets of Txnl1 and Nrx1 could be detected, suggesting that their CXXC motifs do not engage in mixed disulfides with cellular proteins. For some Trx-like proteins, the method allowed distinguishing redox and non-redox interactions. Parallel, comparative analyses of multiple thiol oxidoreductases revealed differences in the functions of their CXXC motifs, providing important insights into thiol-based redox control of cellular processes.     

谷氧还蛋白系统及其对细胞氧化还原态势的调控

细胞内氧化还原调控主要是由谷氧还蛋白系统和硫氧还蛋白系统完成。谷氧还蛋白属于硫氧还蛋白超家族,广泛分布在各种生物体内。作为一种巯基转移酶,它能够催化巯基．二硫键交换反应或者还原蛋白质谷胱甘肽二硫化物,以维持胞内的氧化还原态势。谷氧蛋白系统参与氧化胁迫、蛋白修饰、信号转导、细胞调亡和细胞分化等多种生物过程。对其体内作用靶蛋白的研究,有助于阐明谷氧还蛋白在整个细胞氧化还原网络的重要调控作用。     

Redox outside the box: linking extracellular redox remodeling with intracellular redox metabolism

Aerobic organisms generate reactive oxygen species as metabolic side products and must achieve a delicate balance between using them for signaling cellular functions and protecting against collateral damage. Small molecule (e.g. glutathione and cysteine)- and protein (e.g. thioredoxin)-based buffers regulate the ambient redox potentials in the various intracellular compartments, influence the status of redox-sensitive macromolecules, and protect against oxidative stress. Less well appreciated is the fact that the redox potential of the extracellular compartment is also carefully regulated and is dynamic. Changes in intracellular metabolism alter the redox poise in the extracellular compartment, and these are correlated with cellular processes such as proliferation, differentiation, and death. In this minireview, the mechanism of extracellular redox remodeling due to intracellular sulfur metabolism is discussed in the context of various cell-cell communication paradigms.     

Thioredoxin and thioredoxin reductase: Current research with special reference to human disease

Thioredoxin (Trx) and thioredoxin reductase (TrxR) plus NADPH, comprising the thioredoxin system, has a large number of functions in DNA synthesis, defense against oxidative stress and apoptosis or redox signaling with reference to many diseases. All three isoenzymes of mammalian TrxR contain an essential selenocysteine residue, which is the target of several drugs in cancer treatment or mercury intoxication. The cytosolic Trx1 acting as the cells’ protein disulfide reductase is itself reversibly redox regulated via three structural Cys residues. The evolution of mammalian Trx system compared to its prokaryotic counterparts may be an adaptation to the use of hydrogen peroxide and nitric oxide in redox regulation and signal transduction.     

A direct and continuous assay for the determination of thioredoxin reductase activity in cell lysates

Thioredoxin reductase (TR) is an oxidoreductase responsible for maintaining thioredoxin in the reduced state, thereby contributing to proper cellular redox homeostasis. The C-terminal active site of mammalian TR contains the rare amino acid selenocysteine, which is essential to its activity. Alterations in TR activity due to changes in cellular redox homeostasis are found in clinical conditions such as cancer, viral infection, and various inflammatory processes; therefore, quantification of thioredoxin activity can be a valuable indicator of clinical conditions. Here we describe a new direct assay, termed the SC–TR assay, to determine the activity of TR based on the reduction of selenocystine, a diselenide-bridged amino acid. Rather than being an end-point assay as in older methods, the SC–TR assay directly monitors the continuous consumption of NADPH at 340 nm by TR as it reduces selenocystine. The SC–TR assay can be used in a cuvette using traditional spectrophotometry or as a 96-well plate-based format using a plate reader. In addition, the SC–TR assay is compatible with the use of nonionic detergents, making it more versatile than other methods using cell lysates.     

Activity assays of mammalian thioredoxin and thioredoxin reductase: Fluorescent disulfide substrates,mechanisms, and use with tissue samples

Thioredoxin (Trx) is a protein disulfide reductase that, together with nicotinamide adenine dinucleotide phosphate (NADPH) and thioredoxin reductase (TrxR), controls oxidative stress or redox signaling via thiol redox control. Human cytosolic Trx1 has Cys32 and Cys35 as the active site and three additional cysteine residues (Cys62, Cys69, and Cys73), which by oxidation generates inactive Cys62 to Cys69 two-disulfide Trx. This, combined with TrxR with a broad substrate specificity, complicates assays of mammalian Trx and TrxR. We sought to understand the autoregulation of Trx and TrxR and to generate new methods for quantification of Trx and TrxR. We optimized the synthesis of two fluorescent substrates, di-eosin–glutathione disulfide (Di-E–GSSG) and fluorescein isothiocyanate-labeled insulin (FiTC–insulin), which displayed higher fluorescence on disulfide reduction. Di-E–GSSG showed a very large increase in fluorescence quantum yield but had a relatively low affinity for Trx and was also a weak direct substrate for TrxR, in contrast to GSSG. FiTC–insulin was used to develop highly sensitive assays for TrxR and Trx. Reproducible conditions were developed for reactivation of modified Trx, commonly present in frozen or oxidized samples. Trx in cell extracts and tissue samples, including plasma and serum, were subsequently analyzed, showing highly reproducible results and allowing measurement of trace amounts of Trx.     

Thioredoxin and Thioredoxin Reductase Control Tissue Factor Activity by Thiol Redox-dependent Mechanism

Abnormally enhanced tissue factor (TF) activity is related to increased thrombosis risk in which oxidative stress plays a critical role. Human cytosolic thioredoxin (hTrx1) and thioredoxin reductase (TrxR), also secreted into circulation, have the power to protect against oxidative stress. However, the relationship between hTrx1/TrxR and TF remains unknown. Here we show reversible association of hTrx1 with TF in human serum and plasma samples. The association is dependent on hTrx1-Cys-73 that bridges TF-Cys-209 via a disulfide bond. hTrx1-Cys-73 is absolutely required for hTrx1 to interfere with FVIIa binding to purified and cell-surface TF, consequently suppressing TF-dependent procoagulant activity and proteinase-activated receptor-2 activation. Moreover, hTrx1/TrxR plays an important role in sensing the alterations of NADPH/NADP⁺ states and transducing this redox-sensitive signal into changes in TF activity. With NADPH, hTrx1/TrxR readily facilitates the reduction of TF, causing a decrease in TF activity, whereas with NADP⁺, hTrx1/TrxR promotes the oxidation of TF, leading to an increase in TF activity. By comparison, TF is more likely to favor the reduction by hTrx1-TrxR-NADPH. This reversible reduction-oxidation reaction occurs in the TF extracellular domain that contains partially opened Cys-49/-57 and Cys-186/-209 disulfide bonds. The cell-surface TF procoagulant activity is significantly increased after hTrx1-knockdown. The response of cell-surface TF procoagulant activity to H₂O₂ is efficiently suppressed through elevating cellular TrxR activity via selenium supplementation. Our data provide a novel mechanism for redox regulation of TF activity. By modifying Cys residues or regulating Cys redox states in TF extracellular domain, hTrx1/TrxR function as a safeguard against inappropriate TF activity.     

Thioredoxin-dependent Redox Regulation of Chloroplastic Phosphoglycerate Kinase from Chlamydomonas reinhardtii

In photosynthetic organisms, thioredoxin-dependent redox regulation is a well established mechanism involved in the control of a large number of cellular processes, including the Calvin-Benson cycle. Indeed, 4 of 11 enzymes of this cycle are activated in the light through dithiol/disulfide interchanges controlled by chloroplastic thioredoxin. Recently, several proteomics-based approaches suggested that not only four but all enzymes of the Calvin-Benson cycle may withstand redox regulation. Here, we characterized the redox features of the Calvin-Benson enzyme phosphoglycerate kinase (PGK1) from the eukaryotic green alga Chlamydomonas reinhardtii, and we show that C. reinhardtii PGK1 (CrPGK1) activity is inhibited by the formation of a single regulatory disulfide bond with a low midpoint redox potential (−335 mV at pH 7.9). CrPGK1 oxidation was found to affect the turnover number without altering the affinity for substrates, whereas the enzyme activation appeared to be specifically controlled by f-type thioredoxin. Using a combination of site-directed mutagenesis, thiol titration, mass spectrometry analyses, and three-dimensional modeling, the regulatory disulfide bond was shown to involve the not strictly conserved Cys²²⁷ and Cys³⁶¹. Based on molecular mechanics calculation, the formation of the disulfide is proposed to impose structural constraints in the C-terminal domain of the enzyme that may lower its catalytic efficiency. It is therefore concluded that CrPGK1 might constitute an additional light-modulated Calvin-Benson cycle enzyme with a low activity in the dark and a TRX-dependent activation in the light. These results are also discussed from an evolutionary point of view.     

The Redox Proteome

The redox proteome consists of reversible and irreversible covalent modifications that link redox metabolism to biologic structure and function. These modifications, especially of Cys, function at the molecular level in protein folding and maturation, catalytic activity, signaling, and macromolecular interactions and at the macroscopic level in control of secretion and cell shape. Interaction of the redox proteome with redox-active chemicals is central to macromolecular structure, regulation, and signaling during the life cycle and has a central role in the tolerance and adaptability to diet and environmental challenges.     

Mutation in the cysteine bridge domain of the γ-subunit affects light regulation of the ATP synthase but not photosynthesis or growth in Arabidopsis

The chloroplast ATP synthase synthesizes ATP from ADP and free phosphate coupled by the electrochemical potential across the thylakoid membrane in the light. The light-dependent regulation of ATP synthase activity is carried out in part through redox modulation of a cysteine disulfide bridge in CF1 gamma-subunit. In order to investigate the function of the redox regulatory domain and the physiological significance of redox modulation for higher plants, we designed four mutations in the redox regulatory domain of the gamma-subunit to create functional mimics of the permanently reduced form of the gamma-subunit. While the inability to reduce the regulatory disulfide results in lower photosynthesis and growth, unexpectedly, the results reported here show that inability to reoxidize the dithiol may not be of any direct detriment to plant photosynthetic performance or growth.     

Colour As a Signal for Entraining the Mammalian Circadian Clock

Twilight is characterised by changes in both quantity (“irradiance”) and quality (“colour”) of light. Animals use the variation in irradiance to adjust their internal circadian clocks, aligning their behaviour and physiology with the solar cycle. However, it is currently unknown whether changes in colour also contribute to this entrainment process. Using environmental measurements, we show here that mammalian blue–yellow colour discrimination provides a more reliable method of tracking twilight progression than simply measuring irradiance. We next use electrophysiological recordings to demonstrate that neurons in the mouse suprachiasmatic circadian clock display the cone-dependent spectral opponency required to make use of this information. Thus, our data show that some clock neurons are highly sensitive to changes in spectral composition occurring over twilight and that this input dictates their response to changes in irradiance. Finally, using mice housed under photoperiods with simulated dawn/dusk transitions, we confirm that spectral changes occurring during twilight are required for appropriate circadian alignment under natural conditions. Together, these data reveal a new sensory mechanism for telling time of day that would be available to any mammalian species capable of chromatic vision.     

PERIOD–TIMELESS Interval Timer May Require an Additional Feedback Loop

In this study we present a detailed, mechanism-based mathematical framework of Drosophila circadian rhythms. This framework facilitates a more systematic approach to understanding circadian rhythms using a comprehensive representation of the network underlying this phenomenon. The possible mechanisms underlying the cytoplasmic “interval timer” created by PERIOD–TIMELESS association are investigated, suggesting a novel positive feedback regulatory structure. Incorporation of this additional feedback into a full circadian model produced results that are consistent with previous experimental observations of wild-type protein profiles and numerous mutant phenotypes.     

Thioredoxin targets in Arabidopsis roots

The importance of redox‐regulation in Arabidopsis thaliana roots has been investigated through the identification of the proteins interacting with thioredoxin (TRX), an ubiquitous thiol‐disulfide reductase. We have applied a proteomic approach based on affinity chromatography on a monocysteinic mutant of plastidial y‐type TRX used as a bait to trap putative partners in a crude extract of root proteins. Seventy‐two proteins have been identified, functioning mainly in metabolism, detoxification and response to stress, protein processing and signal transduction. This study allowed us to isolate 24 putative new targets and to propose the mevalonic acid‐dependent biosynthesis of isoprenoids as a new redox‐mediated process. The redox‐regulation of phenylpropanoid biosynthesis is also suggested, three enzymes of this pathway being retained on the column. We also provided experimental evidence that phenylammonia‐lyase was enzymatically more active when reduced by TRXy in root crude extract. Among the high number of partners involved in defense against stress we isolated from the column, we focused on plastidial monodehydroascorbate reductase and showed that its activity was dramatically increased in vitro in the presence of DTT‐reduced TRXy1 in root crude extracts. Our data strongly suggest that TRXy1 could be the physiological regulator of monodehydroascorbate reductase in root plastids.     

Modulating effects of hesperidin on circadian pattern indices of rotenone induced redox homeostasis in clock mutant (cryb) of Drosophila melanogaster

The circadian timing system controls drug metabolism and cellular processes over the 24 h period in every cell. Impaired redox homeostasis is a casual factor for a number of diseases and it is desirable to understand the orchestration of circadian clock under oxidative stress in the model organism, Drosophila melanogaster. This study evaluates the effect of hesperidin on the circadian rhythms of lipid peroxidation products and antioxidants during rotenone-induced oxidative stress in fruit fly. The characteristics of temporal rhythms (acrophase, amplitude, and mesor) of glutathione peroxides (GPx), reduced glutathione (GSH)), were markedly declined in rotenone-treated flies when compared to other groups. Treatment of hesperidin to rotenone-treated flies significantly increased the mesor and modified the amplitudes of antioxidants. Further, delays in acrophase in rotenone-induced flies were reversed by hesperidin treatment. Thus, treatment of hesperidin results in normalization of the altered rhythms of these indices plausibly by its cytoprotective and antioxidant effects. Impairment of 24 h rhythms in oxidative stress markers and antioxidants were observed during rotenone treatment and the impairment is severe in circadian clock mutant cry^b flies. A reversibility of rhythms was prominent consequent to hesperidin treatment in wild-type flies than cry^b flies. These findings revealed a role of circadian clock in redox homeostasis and the use of Drosophila model in screening putative antioxidative phytomedicines earlier to their use in mammalian systems.     

Effect of hesperidin on the temporal regulation of redox homeostasis in clock mutant (Cryb) of Drosophila melanogaster

Disorganized redox homeostasis is a main factor causing a number of diseases and it is imperative to comprehend the orchestration of circadian clock under oxidative stress in the organism, Drosophila melanogaster. This investigation analyses the influence of hesperidin on the circadian rhythms of lipid peroxidation products and antioxidants during rotenone-stimulated oxidative stress in fruit fly. The characteristics of rhythms of thiobarbituric acid reactive substances (TBARS), antioxidants (superoxide dismutase (SOD) and catalase (CAT)) were noticeably decreased in rotenone administered flies. Supplementation of hesperidin to rotenone-treated flies increased the mesor and modulated the amplitudes of antioxidants and conspicuously decreased the mesor values of TBARS. In addition, delays in acrophase in rotenone-induced flies were reversed by hesperidin treatment. Thus, treatment of hesperidin caused normalization of the altered rhythms. Disorganization of 24 h rhythms in markers of redox homeostasis was observed during rotenone treatment and the impairment is severe in circadian clock mutant (Cryb) flies. Reversibility of rhythms was prominent subsequent to hesperidin treatment in wild-type flies than (Cryb) flies. These observations denote a role of circadian clock in redox homeostasis and the use of Drosophila model in screening putative antioxidative phytomedicines prior to their usage in mammalian systems.