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enzymatic reduction reactions

Source: From various sources.

Source: From various sources.

antioxidant systems. Scavenging and enzymatic systems to protect cells from oxidative stress are found in almost all organisms; however, anaerobic and microaerophilic bacteria and Archaea have an oxidoreductase not found anywhere else (Lumppio et al. 2001). Several antioxidant enzymes are localized in specific organelles of the cell (Table 6.3).

Among the different protective systems listed in Table 6.3 we briefly highlight the role of glutathione, which acts both as a scavenger on its own and as a substrate in enzymatic reactions. Glutathione is a tripeptide, g-glutamyl-cysteinyl-glycine, which in reduced form (written GSH) has a free thiol group on the cysteine residue. In the transition to the oxidized form (GSSG) two thiol groups react with each other, losing two electrons, as in the reaction catalysed by glutathione peroxidase:

Oxidized glutathione is then again reduced at the expense of reduction equivalents from NADPH by means of glutathione reductase:

Since glutathione is responsible for the majority of ROS-protection reactions and it is also involved in conjugating lipophilic substances in xenobiotic biotransformation reactions (see below), the maintenance of a large pool of reduced glutathione is very important for the vitality of the cell. Cellular stress due to ROS, altered redox state, and xeno-biotic metabolism may cause glutathione depletion.

To be effective as protective mechanisms, the antioxidant systems listed in Table 6.3 must be upregulated when the cell perceives oxidative stress. This is indeed the case. Recent research has shown that genes encoding antioxidant protective enzymes all have a characteristic sequence in their promoter, designated the antioxidant-respons-ive element (ARE). This element is also called the electrophile-responsive element (EpRE), after researchers discovered that not only oxidative stress but also electrophile chemicals could activate the element. As in the case of heat-shock proteins, the sequence serves to bind a transcription factor which is activated by stress. The factor binding to AREs has been identified as Nrf2, also called NF-E2 (an abbreviation of nuclear factor erythroid 2-related factor 2; Nguyen et al. 2003, 2004; Jaiswal 2004; Kobayashi and Yamamoto 2005). Under normal physiological conditions, Nrf2 is continuously degraded under the influence of a protein known as Keap1 (Kelch-like ECH-associating protein 1). This protein supports the tagging of Nrf2 with ubiquitin, after which it is destined for cytoplasmic proteolysis. Degradation of Nrf2 is compensated by continuous synthesis, but under normal conditions the pool of Nrf2 in the cytoplasm is very small. Under conditions of oxidative stress Keap1 is inactivated and Nrf2 is stabilized (Fig. 6.5). Presumably, the oxidative-stress signal is transduced through the SAPK pathway leading to activation of protein kinase C (PKC) and other cytosolic factors. Phosphorylation of Nrf2 by PKC results in the release of Nrf2 from its repressor. Nrf2 may then translocate to the nucleus, and will bind to an ARE. However, before becoming fully active it needs to undergo heterodimerization. This is comparable to the formation of activator protein AP-1 by heterodimerization of c-Jun and c-Fos (see Section 6.2.1 on SAPK signalling). In the case of Nrf2, the partner protein is suggested to be a member of the family of Maf proteins. Maf proteins are nuclear factors that like Nrf2 may bind to ARE. In the absence of Nrf2, they exert negative control over ARE-mediated gene expression. How this negative control is lifted by Nrf2 is not known

Antioxidant and xenobiotic

Degraded A

Antioxidant and xenobiotic

Thiol Nrf2 Cfos

Figure 6.5 Scheme of Nrf2 activation and induction of antioxidant genes by ARE binding. Nrf2 in the cytoplasm is bound to a protein, Keap1, which promotes its degradation by ubiquitination. An oxidative-stress signal, transduced via SAPK, activates protein kinases that may phosphorylate Nrf2 and liberate it from inactivation of Keap1. In addition, Keap1 may be destabilized by reactive chemicals, for example through alkylation of cysteine residues critical for its activity. Either mechanism allows Nrf2 to translocate to the nucleus. There it undergoes dimerization with an as-yet unknown partner (X) and triggers expression of genes with AREs in their promoter. A variety of nuclear factors (Maf G/K and others) normally inhibit ARE-mediated gene expression, but are removed by the Nrf2/X heterodimer. From Jaiswal (2004), with permission from Elsevier.

Figure 6.5 Scheme of Nrf2 activation and induction of antioxidant genes by ARE binding. Nrf2 in the cytoplasm is bound to a protein, Keap1, which promotes its degradation by ubiquitination. An oxidative-stress signal, transduced via SAPK, activates protein kinases that may phosphorylate Nrf2 and liberate it from inactivation of Keap1. In addition, Keap1 may be destabilized by reactive chemicals, for example through alkylation of cysteine residues critical for its activity. Either mechanism allows Nrf2 to translocate to the nucleus. There it undergoes dimerization with an as-yet unknown partner (X) and triggers expression of genes with AREs in their promoter. A variety of nuclear factors (Maf G/K and others) normally inhibit ARE-mediated gene expression, but are removed by the Nrf2/X heterodimer. From Jaiswal (2004), with permission from Elsevier.

precisely. Anyway, expression of the antioxidant system seems to be regulated by both negative and positive agents and this could allow fine-tuning to the physiological needs of the cell.

Our present knowledge of the regulation of antioxidant systems is almost completely limited to human cells and Drosophila. One of the reasons for this is that the issue raises a good deal of interest in medical research: tumor cells are characterized by an upregulated oxidative-stress response. How the knowledge generated in the medical sector translates to an ecological context of animals and plants in the wild is difficult to evaluate at the moment; however, it may be expected that, like the stress-activated signalling pathways and the stress proteins, most of components of the oxidative-stress response are evolu-tionarily conserved. In the genome-wide studies discussed later in this chapter we will discuss some evidence supporting this statement.

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