Peroxide signaling through thiol switches: chemical and biological aspects.

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by Luis E. S. Netto

It is now well accepted that oxidants and other redox intermediates are not only damaging compounds, but also act as signaling molecules. This is especially evident for hydrogen peroxide, whose generation and degradation are finely regulated through multiple enzymatic systems. Proteins whose activities are based on Cysteine (Cys) residues are frequently reported to be oxidized in various biological systems in conditions where hydrogen peroxide is also generated. As the most parsimonious hypothesis, these proteins are frequently assumed to be directly oxidized by hydrogen peroxide, although this is not always supported by chemical data.

For instance, Protein Tyrosine Phosphatases (such as PTP1B and PTEN) are frequently proposed as the biological sensors of hydrogen peroxide, although these proteins react slowly with hydrogen peroxide (rate constant about 10 M-1 s-1) and are not abundant proteins. In contrast, peroxiredoxin enzymes are Cys-based peroxidases that react one million times faster with hydrogen peroxide and are very abundant. As an example, in red blood cells, Peroxiredoxin 2 (Prdx2) is the third most abundant protein.

Therefore, new proposals arose in an attempt to take into account both biological data (showing that Cys-based proteins are oxidized in cellular systems) and chemical information (low reactivity and low abundance of these Cys-based proteins). The so- called “floodgate hypothesis” is one of the most popular attempts. The basis of this proposal is that highly efficient peroxidases such as peroxiredoxins are temporally inactivated, allowing build-up of hydrogen peroxide levels within a given sub-cellular compartment, thus ensuing local redox-mediated signals to other messengers such as phosphatases. Indeed, peroxiredoxins can be reversibly inactivated by means of overoxidation (to sulfinic = Cys-SO2H or sulfonic = Cys-SO2H states) or phosphorylation [1]. A problem with this hypothesis is that even in the absence of highly reactive peroxidases, cells still have high amounts of GSH (mM levels) that probably outcompete protein tyrosine phosphatases for hydrogen peroxide.

Other models were raised taking into account that on chemical grounds, peroxiredoxins are better candidates as redox sensors or receptors for hydrogen peroxide. In this regard, the mechanisms by which oxidized peroxiredoxin transmits the signals coming from hydrogen peroxide to downstream steps are still being uncovered. Likely, hydrogen peroxide signaling is mediated through sequential transfers of oxidizing equivalents. Upon oxidation, all peroxiredoxin enzymes are oxidized to sulfenic acids (Cys-SOH) that in most cases can be converted to intra- or inter-molecular disulfides. Oxidized peroxiredoxins (Cys-SOH or Cys-SS-Cys) probably selectively transfers oxidizing equivalents to downstream regulatory proteins through selective protein-protein interactions and thiol-disulfide exchange reactions. The classical finding supporting this model came from yeast Saccharomyces cerevisiae in which the transcription factor Yap1 is activated by oxidation meditated by glutathione peroxidase 3 (Gpx3), also called “Oxidant Receptor Protein” (Orp1) [2]. In this signaling pathway, hydrogen peroxides oxidizes Gpx3/Orp1 into a sulfenic acid (Cys-SOH) that then condenses with a Cys residue of Yap1, giving rise to a transient, mixed disulfide between the two proteins. Through thiol – disulfide exchange reactions, an intra-molecular disulfide in formed in Yap1. This oxidized form of Yap1 has an altered structure that can not leave the nucleus. The nuclear accumulation of Yap1 facilitates its ability to induce the transcription of target genes, such as peroxiredoxins and catalase.

For some time, a challenge in the field was to identify similar pathways in mammalian systems. Recently, several targets for mammalian peroxiredoxin1 (Prdx1) were identified and this month the activation of a transcription factor (STAT3) by mammalian peroxiredoxin2 (Prdx2) was also reported [3]. In all cases, the transfer of oxidizing equivalents involves physical interaction between the two partners.

A third possible mechanism for hydrogen peroxide signaling would involve not only peroxiredoxin, but also an oxido-reductase, such as thioredoxin. Most peroxiredoxins are reduced by thioredoxin, which consequently becomes oxidized, in an intra-molecular disulfide state. Possibly, thioredoxin (or other oxido-reductases) could then transfer oxidizing equivalents to phosphatases or transcription factors. Indeed, it is known for a long time that several signal transduction pathways are activated by the oxidized, but not by the reduced form of thioredoxin. For instance, only reduced mammalian thioredoxin1 (Trx1) binds Apoptosis Signaling Kinase 1 (Ask-1), inhibiting its kinase activity. The oxidation of Trx1 leads to the physical dissociation of the complex and, consequently, to the activation of Ask-1, ensuing apoptosis. NF-κB is also redox-regulated in a similar way by Trx1. Our group has identified structural features in yeast peroxiredoxin (Tsa1) that are also conserved in mammalian peroxiredoxins and are responsible for its physical interaction with thioredoxin [4].

All the three models described here are not mutually exclusive and could potentially occur simultaneously in cells. Several new features are emerging and challenges in the field are the elaboration of models that can account for both chemical and biological aspects of redox signaling.

  1. H. A. Woo, S. H. Yim, D. H. Shin, D Kang, D. Y. Yu, S. G. Rhee.
    Inactivation of peroxiredoxin I by phosphorylation allows localized H₂O₂ accumulation for cell signaling.
    Cell, 140(4): 517-28, 2010. |
  2. A. Delaunay, D. Pflieger, M. B. Barrault, J. Vinh, M. B. Toledano.
    A thiol peroxidase is an H₂O₂ receptor and redox-transducer in gene activation.
    Cell, 111(4): 471-81, 2002. |
  3. M. C. Sobotta, W. Liou, S. Stöcker, D. Talwar, M. Oehler, T. Ruppert, A. N. Scharf, T. P. Dick.
    Peroxiredoxin-2 and STAT3 form a redox relay for H₂O₂ signaling.
    Nature Chemical Biology, 11: 64-70, 2015. |
  4. C. A. Tairum Jr, M. A. de Oliveira, B. B. Horta, F. J. Zara, L. E. S. Netto.
    Disulfide biochemistry in 2-cys peroxiredoxin: requirement of Glu50 and Arg146 for the reduction of yeast Tsa1 by thioredoxin.
    Journal of Molecular Biology, 424 (1-2): 28-41, 2012. |

Luis Eduardo Soares Netto, PhD.
Professor at Department of Genetics and Evolutionary Biology,
Institute of Biosciences, University of São Paulo, Brazil

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