Oxygen metabolism and the toxicity of activated oxygen species, the hub of action for many aggressors

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The second lecture (March 6, 2014) focused on redox signaling. After highlighting the role of ROS in hearing loss associated with aminoglycoside (and cisplatin) administration, this lecture focused on a novel aspect of the effects of oxygen species, redox signaling.

While increased _O2 triggers an adaptive response involving redox signaling mediated mainly by hydrogen peroxide (_H2O2), its failure results in the production of ROS, including free radicals (such as OH, _O2-^,_RO2), and non-radical products (such as _H2O2, _RO2H) (Gardes-Albert et al., 2003), leading to oxidative stress, characterized by irreversible cellular damage. For a long time, redox signalling was masked by the effect of ROS on all biological macromolecules: DNA, proteins and lipids. Discovered some fifteen years ago in the field of immunity, its ubiquitous nature has since been recognized. Strictly speaking, _H2O2 is not a reactive species, as it has no unpaired electrons. Its ability to signal takes advantage of this absence of reactivity, which enables it to diffuse and therefore act at a distance. Superoxide anion (_O2-⁾ is considered a potential messenger of this signaling. Recent data suggest that, by inactivating a set of proteins, it could lead to the activation of signalling pathways that either promote adaptation to _H2O2, or trigger a first step towards cell death. Redox signaling, set up during the transition to aerobic life, plays an essential role, as demonstrated by the fact that the absence of glutathione, a major reducing agent in this signaling, is incompatible with life.

_H2O2_comeseither_fromthe dismutation of ^O2- by superoxide dismutases (SOD), or from the activity of various oxidases. In humans, there are three SODs: two of the CuZn-SOD type, SOD1 (cytoplasmic and mitochondrial) and SOD3 (extracellular), and one of the Mn-SOD type, SOD2 (mitochondrial). A variety of oxidases produce _H2O2: long-chain and branched-chain fatty acid oxidases, located in the peroxisome, xanthine and hypoxanthine oxidases, in the cytoplasm and peroxisome, monoamine oxidases, in the mitochondria.. _O2-^, for its part, is formed either in the mitochondria by the capture by O2_ofan electron from the activity of mitochondrial complexes I and III, or in the plasma membrane and endoplasmic reticulum by the oxidation of NADPH by NADPH oxidases (NOX and DUOX). Little is known about the regulation of NADPH production in the mitochondria. The same applies to the role of_availableO2, since both hypoxia and hyperoxia lead to excessive ROS production.

_H2O2_isreduced to_H2Oby three pathways involving three different peroxidases (Bindoli et al., 2008): thioredoxin (Trx)-dependent peroxyredoxins (Prx), glutathione cycle (GSH)-dependent glutathione peroxidases (GPx), and peroxisome catalase. There is competition between _H2O2 degradation and its role in redox signalling.

Redox signaling consists in the reversible conversion of cysteine residues of proteins (several hundred) or peptides, from a reduced to an oxidized form. The cellular concentration of thiol groups in proteins that may be involved in this signaling is estimated at 5 mM. Induced conformational changes result in modifications of activity (e.g., increased or decreased enzymatic activity), biophysical properties (e.g., protein stability), subcellular localization and molecular interaction. In the presence of _H2O2, the thiol group of cysteines in the form of thiol anion (Cys-S-⁾ or protonated thiol (Cys-SH) can take on a sulfenic form (Cys-SOH) (Finkel, 2011). This conversion depends on the state of the thiols (thiol anions are more reactive than protonated thiols), and on the nature of the protein or peptide carrying them (the rate of conversion is very different from one protein or peptide to another). The sulfenic form of cysteine thiols can lead to a change in the catalytic activity of certain enzymes or in the activity of transcription factors involved in the response to oxidative stress. Cysteines in sulfenic form are also intermediates in the formation of disulfide bridges (intra- or intermolecular), which are much more stable and play an essential role in protein folding. Methionine, another sulfur-containing amino acid, could also be the origin of a redox system. However, its oxidation rate is lower than that of cysteine. Recent data suggest that actin polymerization and CAMKII(Ca2+/Calmodulin-dependent kinase) activity are regulated by the oxidation of some of their methionines. The return to the reduced state (thiolate anion) of cysteine thiols in the form of sulfenes or disulfide bridges requires a reducing agent, glutathione, or an enzymatic activity that exchanges oxidized and reduced thiols, whether disulfide reductases, thioredoxins (Trx) or glutaredoxins (Grx). Grx and Trx have comparable three-dimensional structures. These small molecules are powerful antioxidants, possessing several CXXC-type motifs in their active site (C = cysteine and X = any amino acid), which act as oxidation state detectors. Finally, further oxidation of the sulfenic forms of cysteines leads to sulfinic forms (_SO2H), whose reversion involves sulfiedoxins, and then sulfonic forms (_SO3H), an irreversible oxidation state that alters proteins.

Understanding this redox signaling is still in its infancy. Many questions remain to be answered. In particular, how can this signaling be specific when the signaling agent,_H2O2_, diffuses, including across membranes, and can interact with multiple targets?

To answer these questions, redox "biodetector" probes have been developed for use in in vivo imaging. They enable real-time monitoring of the redox status of various cellular compartments: mitochondria, nucleus and cytoplasm, under experimental conditions of interest. We have seen how these tools have revealed the existence of a dialogue between mitochondria and the nucleus, involving redox signalling. In fine, it results in transcriptional modifications (Al-Mehdi et al., 2012).

_H2O2signalling_operatesdirectly on a variety of targets: (i) enzymes, Prx and GPx (which reduce_H2O2_to_H2O) and tyrosine phosphatase 1B (PTP1B); following modification of the thiols on their cysteines to a sulfenic form, these enzymes become inactive. The oxidation target cysteine is located in the catalytic site of PTP1B; the receptor on which it acts then remains phosphorylated and active; this cysteine can be reactivated by Trx. _H2O2, also acting directly on Trx, has an effect on numerous Trx targets, including p53, NFkB.. (ii) proteins involved in the control of transcription, such as the oxidation of the cysteines of KEAP1, the protein which retains the transcription factor NRF2 in the cytoplasm; under the effect of the oxidation of its cysteines sensitive to redox status, KEAP1 releases NRF2, which then enters the nucleus; after dimerization with the small MAF protein, it binds to the promoter sequences, known as antioxidant response elements (AREs : antioxidant response elements), of genes coding for antioxidants; (iii) cysteine proteases (ATG-4, involved in autophagy, for example); (iv) finally, _H2O2 oxidizes glutathione, which has effects on Grx (which have numerous targets), glutathione S-transferases (which catalyze the conjugation of glutathione to endogenous or exogenous substrates) and GPx. Cellular compartmentalization plays an essential role in the implementation and regulation of these various signalling pathways. Finally, the Prx reductases and Trx reductases required to maintain these two peroxidases in reduced form have NADPH as their cofactor, supplied by the pentose phosphate pathway and mitochondrial respiration. Several of these enzymes carry a selenocysteine, a selenated analogue of cysteine in which the thiol group is replaced by a selenol group. Selenocysteine is the 21st amino^acid. Rarely used, it is encoded by the UGA stop codon. It is also oxidized by _H2O2.

The above elements can be compared with two forms of deafness. One is DFNB25, an isolated autosomal recessive deafness whose gene codes for GRXCR1, which includes a Grx-like domain. The other is Wolfram syndrome, with deafness associated with diabetes; this gene is also responsible for DFNA6/14/38 dominant isolated deafness. The gene responsible codes for a mitochondrial protein, Miner1, involved in the redox status of thiol groups, the cellular response tounfolded proteins(unfolded protein response), and calcium homeostasis.