**3. Glutaredoxin 2 (Grx2)**

ROS accumulation can be detrimental, as they can cause oxidative damage to proteins, lipids, DNA, and other crucial biological molecules [2]. Among these macromolecules, proteins are very sensitive to oxidative modification. Cysteine residues are particularly reactive with ROS due to the presence of thiol (-SH) group, which can be oxidized to sulfenic (SOH), sulfinic

defined as the reversible formation of a mixed disulfide (PSSG) between protein thiols (P-SH) and glutathione (GSH), appears to be the most important mode of thiol oxidation. GSH, normally an antioxidant molecule that scavenges free radicals, oxidizes to form glutathione mixed disulfide (GSSG). As the GSSG/GSH ratio increases, GSSG naturally adds to other proteins, causing protein glutathionylation. In most cases, the addition of GSSG to proteins renders them inactive, but for specific proteins such as heat shock proteins or peroxiredoxin 6, it may serve as an activation mechanism or a cell signaling event [3, 4]. In small amounts, PSSG may alert the cell that oxidative stress is present and may lead to certain signaling cascades to restore a healthy cellular redox state. Nonetheless, excessive PSSG can be lethal because of severe protein inactivation and damage [5]. Therefore, maintenance of a redox state can

To combat oxidative stress, the body is equipped with several antioxidant enzymes in order to restore the redox balance and protect the cell. Several antioxidant enzymes such as catalase, superoxide dismutase (SOD), and thioredoxin (Trx) have been thoroughly researched due to their effectiveness and ability to directly target and scavenge ROS [2, 6–8]. However, as it is becoming more clear that PSSG is an important post-translational modification linked to oxidative stress, recent research has highlighted the glutaredoxin (Grx) system, an antioxidant system capable of reversing PSSG formation. As shown in **Figure 1**, the Grx system

H), or formed disulfide bonds (S-S). Protein glutathionylation,

(SO2

H), sulfonic acids (SO3

28 Glutathione in Health and Disease

translate to increased cell survival and protection.

**Figure 1.** Glutaredoxin and thioredoxin system.

**2. Introduction to the glutaredoxin (Grx) system**

### **3.1. Grx2 localization and expression**

Grx2 was first discovered simultaneously by two research groups leading by Holmgren and Gladeshev in the same year 2001. Both teams characterized Grx2 as a 18 kDa oxidoreductase enzymes capable of reversing PSSG and working as electron donors for ribonucleotide reductase [9]. There are several alternative spliced forms in different types of organisms, but in humans specifically, there are two main subsets: the cytosolic Grx1 and the mitochondrial Grx2. Grx1 has also been implicated in the mitochondrial inner membrane space but in miniscule amounts. Grx2 is rather unique, in that depending on cell type and organism, may be present in different cellular components. Grx2 has three alternatively spliced forms: Grx2a, Grx2b, and Grx2c. Grx2a is the mitochondrial isoform, whereas Grx2b and Grx2c mainly reside in the cytoplasm and nucleus. Grx2a is ubiquitous in every cell, except in the testes [6, 10, 11]. In spermatids, Grx2c is prominently present in the cytoplasm, yet Grx2a is absent (found to be less than 1% in the mice testes) [12, 13]. In embryos, Grx2 expression tends to surpass other dominant antioxidant enzymes such as Trx1, Trx2, and Grx1. During the gestation of E11 embryos, more than 50% of Grx2 mRNA transcripts compose of Grx2a [13]. This is not remarkable when considering that aerobic respiration and mitochondria formation takes precedent in this stage of gestation. Surprisingly, Grx2b and Grx2c are rarely found in normal human cells, but its presence has been discovered in HeLa cells and certain cancer cell lines. In patients with hepatocellular carcinoma and underlying metabolic syndrome that increases a person's risk for diabetes, stroke, and heart disease, Grx2 expression was particularly high [14]. In non-small cell lung cancer and adenocarcinoma, proliferative effects are correlated with higher cytoplasmic and nuclear Grx2 levels [15]. Along with several studies supporting Grx2 is highly expressed in cancer cells, Grx2 may have a distinct and special function in cancer cells, possibly in enhancing cancer cell survival and proliferation [15–17].

Patterns of Grx2 expression vary from species to species. Unsurprisingly, Grx2's localization in mice showed that Grx2a was present in all tissue types. However, it was also identified that Grx2b and Grx2c mRNA transcripts were found at various expression levels in several tissues with higher concentrations in the heart, liver, kidney, and eyes [12, 13]. Another study looking at porcine ocular tissues showed that Grx2 is abundantly present in the ciliary body and retina but is lacking in the vitreous humor [18]. Both the ciliary body and retina have direct access to blood vessels and are also known to be more sensitive to oxidative stress, increasing the risk in these areas for a variety of oxidative stress-induced eye diseases. Therefore, Grx2's concentration in these areas indicate Grx2's potential role in preventing oxidative-induced damage in these exposed tissues.

Grx2 employ the same mechanism to remove GSH from its protein targets: it accepts the GSH from the protein adduct and becomes Grx-SSG only to be returned back to its reduced form (Grx-SH) by glutaredoxin reductase (GR) and the reducing power of NADPH. GSSG is

The Important Functions of GSH-Dependent Enzyme Glutaredoxin 2 (Grx2)

http://dx.doi.org/10.5772/intechopen.78653

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The cysteines in the active site of Grx2 play a major role for Grx2 to function. Along with active site Cys residues, there are several disulfide bridges between non-active site Cys residues that promote further stability in Grx2 structure [25]. This attribute has been remarkably the only conserved feature found in all vertebrate-specific Grx2 forms, indicating the necessity of this disulfide bridge in promoting Grx2's potency. However, other modifications of Cys residues may inhibit Grx activity. A study performed by Hashemy et al. indicated that compared to its counterpart Grx1, Grx2 enzyme activity was relatively initiated by S-nitrosylation, leading to the conversion of Grx2 into its active monomeric form [17]. Grx1's cysteine oxidation either by S-nitrosylation or intradisulfide bonds only lead to significant structural changes and inactivation. Therefore, compared to Grx1, Grx2 is more robust in highly oxidative stressed environments and is still able to function despite Cys modifications [17]. Moreover, this evidence highlights the ability of Grx2 to be exclusively activated under

further processed and recycled to GSH by the enzyme glutathione reductase [23, 24].

**4. Grx2 is a potent antioxidant and anti-apoptotic enzyme in** 

Numerous research studies have defined Grx2 as a potent antioxidant and anti-apoptotic enzyme in the brain. Compared to other tissues, the brain is more vulnerable and sensitive to oxidative stress. Moreover, mitochondrial dysfunction is a similar theme found in several neurodegenerative diseases, including Parkinson's, amyotrophic lateral sclerosis (ALS), and Alzheimer's disease [26]. Because mitochondria are heavily involved in the apoptotic pathway, the ability to prevent mitochondrial induction of cell death and repair oxidatively damaged neurons are methods believed to be the future treatments for neurodegenerative

In motor neurons, aberrant SOD1, misfolded protein aggregation, oxidative stress, and mitochondrial dysfunction are all key players in ALS pathogenesis [27]. Ferri et al. was able to determine that Grx1 overexpression may increase mutant SOD1 solubility in the cytosol but has no effect in its solubility in the mitochondria or motor neuron apoptosis [27]. The accumulation of mutant SOD1 is attributed to higher rates of apoptosis due to ATP and GSH depletion and unusual mitophagy. A previous study in yeast has shown that GSH is a main contributor for SOD1 activation and glutathionylation especially when using oxidative stress agents like menadione are used and is crucial for lifespan development [28]. Rather, Grx2 overexpression cleared mutant SOD1 in the mitochondria, resulting in decreased mitochondrial fragmentation and abolishing neuron apoptosis [27]. Optic atrophy 1 (OPA1), a pro-fusion protein, and dynamin-related protein 1 (DRP1), a mitochondrial fragmentation protein, are often imbalanced due

oxidative stress conditions.

**different systems**

disorders.

**4.1. The nervous system**

By using artificial cloning of recombinant human Grx2 (hGrx2) in *E. coli* and HeLa cells, Grx2 was able to be fully expressed in the mitochondria and nucleus. Closer analysis suggests that Grx2a has a distinctive mitochondrial N-terminal signal sequence, yet Grx2a and Grx2b both have an arginine and lysine rich C-terminal sequence that highly resembles a nuclear translocation sequence [11]. Staining also suggested that Grx2a was found exclusively in the mitochondria, whereas Grx2b had a strong presence in the cytoplasm and a weaker presence in the nucleus. This contrasted to data previously shown that in Jurkat cells, Grx2 preferentially is found in the nucleus compared to the mitochondria [11]. Nonetheless, the reasons for these differences are unclear, other than the obvious alternative splicing of Grx2 transcripts and the absence of a translocation signal in Grx1 transcripts. Moreover, what prompts the predilection of Grx2a in the mitochondria despite having a nuclear translocation signal and the role of Grx2b and Grx2c in human cells still remain unsolved mysteries.

### **3.2. Grx2 structure**

Compared to the exclusively monomeric and constitutively expressed Grx1, Grx2 is normally 18 kDa and found in an inactive dimeric form with an iron-sulfur cluster [19, 20]. As shown in **Figure 2**, the iron-sulfur cluster is crucial for Grx2's typical functioning, as it is able to detect oxidative stress in the environment. In terms of voltage differences, one study claimed that Grx2's dimeric form only disassociated with pulses greater than 0.5 V [21]. When oxidative stress is present, Grx2 cleaves to its active 16 kDa monomeric form and directly scavenges free radicals and reverses PSSG. Of all the isoforms, Grx2b does not have the specific iron–sulfur cluster and remains a monomer in non-stressed and stressed conditions [10, 21].

### **3.3. Grx2 mechanism**

Grx1 and Grx2 have a particular active site template: Cys-X-Tyr-Cys. Grx1 has a proline residue after the first cysteine residue, whereas Grx2 has a serine instead. Although this gives Grx2 a slightly slower reaction rate, the serine essentially gives Grx2 a higher affinity for glutathionylated substrates [22]. Both monothiol and dithiol mechanisms can be performed, yet the monothiol mechanism using the N-terminal Cys is more accepted. Both Grx1 and

**Figure 2.** Glutaredoxin 2 as an iron-sulfur (Fe-S) protein.

Grx2 employ the same mechanism to remove GSH from its protein targets: it accepts the GSH from the protein adduct and becomes Grx-SSG only to be returned back to its reduced form (Grx-SH) by glutaredoxin reductase (GR) and the reducing power of NADPH. GSSG is further processed and recycled to GSH by the enzyme glutathione reductase [23, 24].

The cysteines in the active site of Grx2 play a major role for Grx2 to function. Along with active site Cys residues, there are several disulfide bridges between non-active site Cys residues that promote further stability in Grx2 structure [25]. This attribute has been remarkably the only conserved feature found in all vertebrate-specific Grx2 forms, indicating the necessity of this disulfide bridge in promoting Grx2's potency. However, other modifications of Cys residues may inhibit Grx activity. A study performed by Hashemy et al. indicated that compared to its counterpart Grx1, Grx2 enzyme activity was relatively initiated by S-nitrosylation, leading to the conversion of Grx2 into its active monomeric form [17]. Grx1's cysteine oxidation either by S-nitrosylation or intradisulfide bonds only lead to significant structural changes and inactivation. Therefore, compared to Grx1, Grx2 is more robust in highly oxidative stressed environments and is still able to function despite Cys modifications [17]. Moreover, this evidence highlights the ability of Grx2 to be exclusively activated under oxidative stress conditions.
