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

## **4.1. The nervous system**

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

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

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

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

the role of Grx2b and Grx2c in human cells still remain unsolved mysteries.

cluster and remains a monomer in non-stressed and stressed conditions [10, 21].

damage in these exposed tissues.

30 Glutathione in Health and Disease

**3.2. Grx2 structure**

**3.3. Grx2 mechanism**

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

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 disorders.

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 to mutant SOD1. However, Grx2 overexpression leads to the phosphorylation and activation of DRP1, restoring mitochondrial morphology and the mitochondrial GSH pool [27]. Overall, Grx2 was able to better reduce mutant SOD1 toxicity and increase neuronal cell survival, perhaps by working to correct oxidized disulfide bonds found in aggregated mutant SOD1.

Inhibition of Grx2 in zebrafish embryos' hearts prevent cardiac neural cells from entering the primary heart region [39]. This results in obstructed blood flow to the aorta and common cardinal vein. Proper heart looping and cardiac neural crest cell migration were also hindered by Grx2 knockdown. Because migration is highly dependent on actin polymerization, Grx2 knockdown would result in more globular actin (G-actin) production and prevent appropriate cell migration [37, 40]. Grx2 has also been attributed to vascular development in embryos. Sirtuin 1 (SIRT1) is prone to redox regulation with approximately 17 cysteines and 5 cysteines specifically known to be glutathionylated. Conserved Cys204 in the catalytic site is around the NAD+ binding site. NAD+ is a crucial cofactor that allows for SIRT1 functioning. Glutathionylation of the key Cys residue would block the cofactor binding site, leading to decreased SIRT1 activity [41]. Therefore, inhibition of Grx2 would promote glutathionylation

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

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33

*Grx2* gene Knockout (KO) mice have decreased aerobic respiration and ATP production due to complex I glutathionylation and inhibition [39]. As a side effect, hypertension ensued with increased cardiac glucose uptake. KO hearts also especially developed left ventricular hypertrophy and fibrosis, yet cardiac contraction and relaxation remained the same between

Usually, with aging, there is a decline in antioxidant expression and activity. Interestingly, Gao et al. first suggested that Grx2 is found to be increased nearly twofold in the mitochondrial matrix of cardiac muscle cells [43]. However, most of the Grx2 is prominently in the inactive dimer formation. Only by exogenous superoxide production by xanthine oxidase

When complex I inhibitor was used to induce endogenous ROS, there was not enough of an

Although there is no direct evidence hinting on the connection between Grx2 and immune cells, macrophages depend heavily on actin polymerization to migrate to areas of injury and inflammation. Actin has two prominent forms: filamentous F-actin and globular G-actin. G-actin is often glutathionylated, which prevents its conversion to F-actin [44]. Therefore, Grx2 activation can lead to increased F-actin production, which can cause increased cell

Research is still relatively scarce on how Grx2 may affect the cell bioenergetics, despite the consensus being that Grx2's regulation of complex I glutathionylation is a huge determinant

In skeletal muscle, the GSH/GSSG mitochondrial pool is relatively high to account for high respiratory rates, which may be a consequence of having somewhat high levels of Grx2 and Trx2 in the mitochondria. Uncoupling protein 3 (UCP3) reversible glutathionylation is carefully mediated by Grx2 and plays a crucial role in promoting glucose metabolism and controlling oxidative stress in skeletal muscle [45, 46]. On the other hand, brown adipose tissue (BAT) is specifically saturated with mitochondria rich in iron, giving BAT its unique color

O2

had no effect on Grx2 activation.

of the active site, causing SIRT1 inactivation and prevent angiogenesis [41].

was the dimer able to cleave to monomeric form [43]. H2

migration, differentiation, and proliferation [24, 37, 44].

wildtype and KO mice [39, 42].

ROS threshold to activate Grx2 [41, 43].

**4.3. Skeletal muscle and adipose tissue**

on total ATP production.

The role of Grx2 in preventing Parkinson's pathology is also a recent research interest. The reduction of mitochondrial GSH pools may indirectly contribute to Grx2 impairment and the development of Parkinson's disease. Several studies have suggested that in dopaminergic neurons of the substantia nigra, the high GSSG/GSH ratio leads to Grx2 inhibition, causing a mitochondrial iron overload that deters the functions of complex I and aconitase, a tricarboxylic acid cycle (TCA) enzyme involved in converting citrate to isocitrate [29]. As a result, mitochondrial dysfunction due to reduced mitochondrial biogenesis and iron–sulfur cluster regeneration is rampant. When 1-methyl-4-phenyl-1,2,3,6,tetrahydropyridine (MPTP) is used to induce neurodegeneration in the extrapyramidal system, Grx2 responded with an increase in mRNA and protein levels after 4 h of treatment and helped alleviate MPTP toxicity and apoptosis [30]. Correspondingly, inhibition of Grx2 lead to compromised complex I activity, indicating Grx2's potential role in restoring complex I activity in times of oxidative stress [30, 31]. Another oxidative stress agent, 6-hydroxydopamine (6-OHDA), used to stimulate a Parkinson's disease model in neuroblastoma cell line, and *C. elegans* was found to increase the expression of Trx1, Trx2, Grx1, and Grx2. Grx2 was found to reduce 6-OHDA and thus prevent its cytotoxicity in dopaminergic neurons [29].

In an ischemia/reperfusion (I/R) model of the rat brain, Grx2 was the most affected, having rebounded from low levels during hypoxia to normal levels by reoxygenation [32]. When Grx2 was successively silenced, neuronal integrity was severely impacted, as there were more distinct areas of neuron damage and death. Furthermore, considering the use of perinatal rat brains, Grx2 was vital in the development of a normal neuronal phenotype [32].

Previous studies have also supported that Grx2 is fundamental for neuronal development. In a zebrafish model, Grx2c was found to be important for the development of the axonal scaffold, as silencing of Grx2c ultimately prevented the neuron differentiation and promoted neuron cell death in nearly 97% of embryos [33]. In particular, Grx2 mediated the redox regulation of collapsin response mediator protein 2 (CRMP2/*DPYSL2*), a protein that mediates axonal outgrowth, cytoskeleton remodeling, and neuronal cell migration [34]. However, their study also found that knockdown of Grx2 did not particularly induce carbonyl oxidative stress in the embryos. A follow-up study characterized the Cys-504-Cys-504 dithiol-disulfide switch as the regulatory mechanism for CRMP2 to control axonal development and neuronal differentiation [32]. Grx2 can reduce the two cysteines, causing CRMP2 conformational changes and activation for proper neuronal development [33, 34].

### **4.2. Cardiovascular and immune systems**

Cardiac cells depend on mitochondrial metabolism for the constant ATP supply necessary to pump blood throughout the body. Compared to the other body organs, the heart has the most number of mitochondria per cell with nearly 5000 mitochondria per cardiac muscle cell [38]. Considering the importance of mitochondria in cardiac function, it is hypothesized that Grx2 plays a major role in protecting the heart's mitochondria.

Inhibition of Grx2 in zebrafish embryos' hearts prevent cardiac neural cells from entering the primary heart region [39]. This results in obstructed blood flow to the aorta and common cardinal vein. Proper heart looping and cardiac neural crest cell migration were also hindered by Grx2 knockdown. Because migration is highly dependent on actin polymerization, Grx2 knockdown would result in more globular actin (G-actin) production and prevent appropriate cell migration [37, 40]. Grx2 has also been attributed to vascular development in embryos. Sirtuin 1 (SIRT1) is prone to redox regulation with approximately 17 cysteines and 5 cysteines specifically known to be glutathionylated. Conserved Cys204 in the catalytic site is around the NAD+ binding site. NAD+ is a crucial cofactor that allows for SIRT1 functioning. Glutathionylation of the key Cys residue would block the cofactor binding site, leading to decreased SIRT1 activity [41]. Therefore, inhibition of Grx2 would promote glutathionylation of the active site, causing SIRT1 inactivation and prevent angiogenesis [41].

*Grx2* gene Knockout (KO) mice have decreased aerobic respiration and ATP production due to complex I glutathionylation and inhibition [39]. As a side effect, hypertension ensued with increased cardiac glucose uptake. KO hearts also especially developed left ventricular hypertrophy and fibrosis, yet cardiac contraction and relaxation remained the same between wildtype and KO mice [39, 42].

Usually, with aging, there is a decline in antioxidant expression and activity. Interestingly, Gao et al. first suggested that Grx2 is found to be increased nearly twofold in the mitochondrial matrix of cardiac muscle cells [43]. However, most of the Grx2 is prominently in the inactive dimer formation. Only by exogenous superoxide production by xanthine oxidase was the dimer able to cleave to monomeric form [43]. H2 O2 had no effect on Grx2 activation. When complex I inhibitor was used to induce endogenous ROS, there was not enough of an ROS threshold to activate Grx2 [41, 43].

Although there is no direct evidence hinting on the connection between Grx2 and immune cells, macrophages depend heavily on actin polymerization to migrate to areas of injury and inflammation. Actin has two prominent forms: filamentous F-actin and globular G-actin. G-actin is often glutathionylated, which prevents its conversion to F-actin [44]. Therefore, Grx2 activation can lead to increased F-actin production, which can cause increased cell migration, differentiation, and proliferation [24, 37, 44].

### **4.3. Skeletal muscle and adipose tissue**

to mutant SOD1. However, Grx2 overexpression leads to the phosphorylation and activation of DRP1, restoring mitochondrial morphology and the mitochondrial GSH pool [27]. Overall, Grx2 was able to better reduce mutant SOD1 toxicity and increase neuronal cell survival, perhaps by working to correct oxidized disulfide bonds found in aggregated mutant SOD1.

The role of Grx2 in preventing Parkinson's pathology is also a recent research interest. The reduction of mitochondrial GSH pools may indirectly contribute to Grx2 impairment and the development of Parkinson's disease. Several studies have suggested that in dopaminergic neurons of the substantia nigra, the high GSSG/GSH ratio leads to Grx2 inhibition, causing a mitochondrial iron overload that deters the functions of complex I and aconitase, a tricarboxylic acid cycle (TCA) enzyme involved in converting citrate to isocitrate [29]. As a result, mitochondrial dysfunction due to reduced mitochondrial biogenesis and iron–sulfur cluster regeneration is rampant. When 1-methyl-4-phenyl-1,2,3,6,tetrahydropyridine (MPTP) is used to induce neurodegeneration in the extrapyramidal system, Grx2 responded with an increase in mRNA and protein levels after 4 h of treatment and helped alleviate MPTP toxicity and apoptosis [30]. Correspondingly, inhibition of Grx2 lead to compromised complex I activity, indicating Grx2's potential role in restoring complex I activity in times of oxidative stress [30, 31]. Another oxidative stress agent, 6-hydroxydopamine (6-OHDA), used to stimulate a Parkinson's disease model in neuroblastoma cell line, and *C. elegans* was found to increase the expression of Trx1, Trx2, Grx1, and Grx2. Grx2 was found to reduce 6-OHDA and thus prevent its cytotoxicity in dopaminergic neurons [29].

In an ischemia/reperfusion (I/R) model of the rat brain, Grx2 was the most affected, having rebounded from low levels during hypoxia to normal levels by reoxygenation [32]. When Grx2 was successively silenced, neuronal integrity was severely impacted, as there were more distinct areas of neuron damage and death. Furthermore, considering the use of perinatal rat

Previous studies have also supported that Grx2 is fundamental for neuronal development. In a zebrafish model, Grx2c was found to be important for the development of the axonal scaffold, as silencing of Grx2c ultimately prevented the neuron differentiation and promoted neuron cell death in nearly 97% of embryos [33]. In particular, Grx2 mediated the redox regulation of collapsin response mediator protein 2 (CRMP2/*DPYSL2*), a protein that mediates axonal outgrowth, cytoskeleton remodeling, and neuronal cell migration [34]. However, their study also found that knockdown of Grx2 did not particularly induce carbonyl oxidative stress in the embryos. A follow-up study characterized the Cys-504-Cys-504 dithiol-disulfide switch as the regulatory mechanism for CRMP2 to control axonal development and neuronal differentiation [32]. Grx2 can reduce the two cysteines, causing CRMP2 conformational changes

Cardiac cells depend on mitochondrial metabolism for the constant ATP supply necessary to pump blood throughout the body. Compared to the other body organs, the heart has the most number of mitochondria per cell with nearly 5000 mitochondria per cardiac muscle cell [38]. Considering the importance of mitochondria in cardiac function, it is hypothesized that Grx2

brains, Grx2 was vital in the development of a normal neuronal phenotype [32].

and activation for proper neuronal development [33, 34].

plays a major role in protecting the heart's mitochondria.

**4.2. Cardiovascular and immune systems**

32 Glutathione in Health and Disease

Research is still relatively scarce on how Grx2 may affect the cell bioenergetics, despite the consensus being that Grx2's regulation of complex I glutathionylation is a huge determinant on total ATP production.

In skeletal muscle, the GSH/GSSG mitochondrial pool is relatively high to account for high respiratory rates, which may be a consequence of having somewhat high levels of Grx2 and Trx2 in the mitochondria. Uncoupling protein 3 (UCP3) reversible glutathionylation is carefully mediated by Grx2 and plays a crucial role in promoting glucose metabolism and controlling oxidative stress in skeletal muscle [45, 46]. On the other hand, brown adipose tissue (BAT) is specifically saturated with mitochondria rich in iron, giving BAT its unique color and is highly concentrated in GSSG rather than GSH. Grx2 is lacking in BAT mitochondria, whereas Trx2 has slightly greater expression to most likely compensate for the lack of Grx2 [45]. Uncoupling protein 1 (UCP1) is similarly regulated by glutathionylation for thermogenesis, but the smaller need for immediate regulation prompts Grx1 and Trx2 to perform UCP1 reduction, albeit at a much lower efficiency [45]. Differences in the choice of substrates, demand, and rate for metabolism in stages of fasting and exercise could explain the lesser role glutathionylation has on BAT mitochondria bioenergetics. Moreover, to maintain a steadfast high iron environment in mitochondria, Grx2 may have to be suppressed, considering Grx2's distinctive iron-sulfur core that differs from the rest of the thioredoxin superfamily. Also, an oxidizing GSSG pool is preferred in BAT mitochondria, and Grx2 expression could alter bioenergetics more to a reducing GSH pool and hinder BAT's ROS production to generate heat. These are all possibilities that could explain why Grx1 and Trx2 are favorably expressed in BAT mitochondria unlike Grx2's key localization in skeletal muscle mitochondria. The selectivity of cysteine glutathionylation in UCP1 and UCP3 provides a great insight into skeletal muscle and BAT functioning [45–47]. The mechanism or the relative importance of Grx2 in regulating these functions remains unseen but does highlight Grx2's potential role in curbing obesity and insulin resistance in diabetes.

In a renal I/R model, Grx2's increased expression in proximal tubular cells corresponded with less oxidative damage and injury after hypoxic conditions. Moreover, there was increased cellular survival and proliferation in HEK293 and HeLa cells after reoxygenation [16]. Considering Grx2's optimal localization in the kidney tubules, this may explain regeneration

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

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

35

Compared with other tissues, the ocular tissues are more susceptible to oxidative stress due to daily exposure of sunlight and relatively high oxygen consumption. Many major blindinducing diseases, like cataract, age-related macular degeneration (AMD), glaucoma, and diabetic retinopathy are all proved to be closely related with oxidative stress. Therefore, antioxidant enzymes play a fundamental role in regulating redox homeostasis and maintaining overall ocular health. Lou's group is the first research team to investigate Grx2 distribution and compare its enzyme activity in ocular tissues. Their data have shown that Grx2 is present in all ocular tissues except vitreous humor. Grx2 level was highest in the mitochondrial-rich and vascular-rich tissues such as the retina while lowest in the tissues that are non-vascular or low mitochondria presence, such as the lens [18]. Fernando et al. first explored the function of Grx2 in lens epithelial cells and determined that human Grx2 has direct peroxidase activity and is capable of using GSH and thioredoxin reductase (TR) as electron donors [35]. Mouse Grx2 did not have as much of a potent peroxidase activity. Moreover, Grx2 could directly

, tert-butyl hydroperoxide (tBHP), and cumene hydroperoxide with greater

affinity for tBHP and cumene hydroperoxide [23, 35]. *Grx2* KO in lens epithelial cells also has been shown to less cell viability, decreased ATP levels, and increased complex I inactivation

The main function of the lens is to maintain transparency so that the light can be transmitted and properly focused on the retina for vision. The eye lens contains high concentration GSH as the first line of defense against free radicals to protect critical enzymes and structural proteins from oxidation. However, with aging, the GSH pool decreases and oxidized GSH (GSSG) may attack protein thiols and formed PSSG. Many of lens structural proteins contain high level of thiol residues which are highly susceptible to oxidative damage. For example, alpha A-crystallin is one of the important and abundant structural proteins in the lens. It functions as a chaperone to facilitate protein folding and stabilize other lens proteins in a soluble form. However, under oxidative stress, many of its -SH groups could be glutathionylated, resulting in its chaperonelike function to be drastically lost. By using shotgun proteomic analysis, Giblin's group studied the glutathionylation sites of crystallins in the lens of the guinea pig under oxidative stress. Their data showed that almost all major crystallins, except αβ, were glutathionylated after the oxidative insult. More than 70% of the –SH groups were conjugated with GSH, cysteine, or both molecules. Interestingly, our previous data have shown that mice lacking Grx2 showed faster progression of cataract during aging. In the Grx2 null mice, the lens nuclear opacity began at 5 months, 3 months sooner than that of the control mice and the progression of cataract were also much faster than the agematched controls. Importantly, this early cataract formation is closely associated with alpha A-crystallin glutathionylation accumulation and compromised mitochondrial functions in

O2 [36].

and anti-apoptotic effects even after an ischemic attack.

**4.5. Cataract**

detoxify H2

O2

and glutathionylation when treated with H<sup>2</sup>

### **4.4. Cancer**

Cancer cells are in a constant oxidative stress environment. In order to combat excessive oxidative stress-induced cell death and increase survival, cancer cells often overexpress antioxidant enzymes [2, 14, 15]. With higher expression of antioxidant enzymes, there is increased drug resistance to anti-cancer drugs such as doxorubicin whose primary purpose is to increase oxidative stress and create DNA damage to cause cancer cell death. Several previous studies have highlighted that hepatocellular carcinoma and adenocarcinoma tend to have higher amounts of Grx2 compared to healthy tissue [14, 15]. The role of Grx2 in cytoprotection and promoting drug resistance is still uncertain, but some studies have attempted to decipher how the knockdown or inhibition of Grx2 would affect cancer cell survival.

Enoksson et al. overexpressed Grx2 in HeLa cells, which caused less sensitivity to doxorubicin and 2-deoxy-D-glucose (2-DG). Grx2 was found to inhibit cytochrome c release [48]. Cardiolipin binds to cytochrome c to the inner mitochondrial membrane as an anti-apoptotic measure. When cardiolipin is reduced or lost, cytochrome c is free to move to the cytoplasmic membrane and initiate apoptosis. Without cytochrome c release, caspase is not activated, and apoptosis is thwarted [49]. It is believed that Grx2 decreases cytochrome c release by enhancing cardiolipin expression through prevention of cardiolipin glutathionylation [48, 49].

Similarly, Lillig et al. discovered that siRNA-mediated silencing of Grx2 sensitized HeLa cells to doxorubicin and phenylarsine oxide (PAO) severely. For doxorubicin, the ED50 dropped from 40 to 0.7 μM, whereas PAO's ED50 dropped from 200 to 5 nM [50]. When treated with a Grx1 inhibitor, HeLa sensitivity remained the same. Carbonyl stress was also slightly increased with Grx2 inhibition and doxorubicin treatment. This indicates that Grx2 potentially enhances drug resistance by altering the redox state of the HeLa cell. Conversely, it was also shown that Grx2 overexpression attenuated apoptosis by enhancing mitochondrial protection [48, 50].

In a renal I/R model, Grx2's increased expression in proximal tubular cells corresponded with less oxidative damage and injury after hypoxic conditions. Moreover, there was increased cellular survival and proliferation in HEK293 and HeLa cells after reoxygenation [16]. Considering Grx2's optimal localization in the kidney tubules, this may explain regeneration and anti-apoptotic effects even after an ischemic attack.

### **4.5. Cataract**

and is highly concentrated in GSSG rather than GSH. Grx2 is lacking in BAT mitochondria, whereas Trx2 has slightly greater expression to most likely compensate for the lack of Grx2 [45]. Uncoupling protein 1 (UCP1) is similarly regulated by glutathionylation for thermogenesis, but the smaller need for immediate regulation prompts Grx1 and Trx2 to perform UCP1 reduction, albeit at a much lower efficiency [45]. Differences in the choice of substrates, demand, and rate for metabolism in stages of fasting and exercise could explain the lesser role glutathionylation has on BAT mitochondria bioenergetics. Moreover, to maintain a steadfast high iron environment in mitochondria, Grx2 may have to be suppressed, considering Grx2's distinctive iron-sulfur core that differs from the rest of the thioredoxin superfamily. Also, an oxidizing GSSG pool is preferred in BAT mitochondria, and Grx2 expression could alter bioenergetics more to a reducing GSH pool and hinder BAT's ROS production to generate heat. These are all possibilities that could explain why Grx1 and Trx2 are favorably expressed in BAT mitochondria unlike Grx2's key localization in skeletal muscle mitochondria. The selectivity of cysteine glutathionylation in UCP1 and UCP3 provides a great insight into skeletal muscle and BAT functioning [45–47]. The mechanism or the relative importance of Grx2 in regulating these functions remains unseen but does highlight Grx2's potential role in curbing

Cancer cells are in a constant oxidative stress environment. In order to combat excessive oxidative stress-induced cell death and increase survival, cancer cells often overexpress antioxidant enzymes [2, 14, 15]. With higher expression of antioxidant enzymes, there is increased drug resistance to anti-cancer drugs such as doxorubicin whose primary purpose is to increase oxidative stress and create DNA damage to cause cancer cell death. Several previous studies have highlighted that hepatocellular carcinoma and adenocarcinoma tend to have higher amounts of Grx2 compared to healthy tissue [14, 15]. The role of Grx2 in cytoprotection and promoting drug resistance is still uncertain, but some studies have attempted to decipher how

Enoksson et al. overexpressed Grx2 in HeLa cells, which caused less sensitivity to doxorubicin and 2-deoxy-D-glucose (2-DG). Grx2 was found to inhibit cytochrome c release [48]. Cardiolipin binds to cytochrome c to the inner mitochondrial membrane as an anti-apoptotic measure. When cardiolipin is reduced or lost, cytochrome c is free to move to the cytoplasmic membrane and initiate apoptosis. Without cytochrome c release, caspase is not activated, and apoptosis is thwarted [49]. It is believed that Grx2 decreases cytochrome c release by enhancing cardiolipin expression through prevention of cardiolipin glutathionylation [48, 49].

Similarly, Lillig et al. discovered that siRNA-mediated silencing of Grx2 sensitized HeLa cells to doxorubicin and phenylarsine oxide (PAO) severely. For doxorubicin, the ED50 dropped from 40 to 0.7 μM, whereas PAO's ED50 dropped from 200 to 5 nM [50]. When treated with a Grx1 inhibitor, HeLa sensitivity remained the same. Carbonyl stress was also slightly increased with Grx2 inhibition and doxorubicin treatment. This indicates that Grx2 potentially enhances drug resistance by altering the redox state of the HeLa cell. Conversely, it was also shown that Grx2 overexpression attenuated apoptosis by enhancing mitochondrial

the knockdown or inhibition of Grx2 would affect cancer cell survival.

obesity and insulin resistance in diabetes.

**4.4. Cancer**

34 Glutathione in Health and Disease

protection [48, 50].

Compared with other tissues, the ocular tissues are more susceptible to oxidative stress due to daily exposure of sunlight and relatively high oxygen consumption. Many major blindinducing diseases, like cataract, age-related macular degeneration (AMD), glaucoma, and diabetic retinopathy are all proved to be closely related with oxidative stress. Therefore, antioxidant enzymes play a fundamental role in regulating redox homeostasis and maintaining overall ocular health. Lou's group is the first research team to investigate Grx2 distribution and compare its enzyme activity in ocular tissues. Their data have shown that Grx2 is present in all ocular tissues except vitreous humor. Grx2 level was highest in the mitochondrial-rich and vascular-rich tissues such as the retina while lowest in the tissues that are non-vascular or low mitochondria presence, such as the lens [18]. Fernando et al. first explored the function of Grx2 in lens epithelial cells and determined that human Grx2 has direct peroxidase activity and is capable of using GSH and thioredoxin reductase (TR) as electron donors [35]. Mouse Grx2 did not have as much of a potent peroxidase activity. Moreover, Grx2 could directly detoxify H2 O2 , tert-butyl hydroperoxide (tBHP), and cumene hydroperoxide with greater affinity for tBHP and cumene hydroperoxide [23, 35]. *Grx2* KO in lens epithelial cells also has been shown to less cell viability, decreased ATP levels, and increased complex I inactivation and glutathionylation when treated with H<sup>2</sup> O2 [36].

The main function of the lens is to maintain transparency so that the light can be transmitted and properly focused on the retina for vision. The eye lens contains high concentration GSH as the first line of defense against free radicals to protect critical enzymes and structural proteins from oxidation. However, with aging, the GSH pool decreases and oxidized GSH (GSSG) may attack protein thiols and formed PSSG. Many of lens structural proteins contain high level of thiol residues which are highly susceptible to oxidative damage. For example, alpha A-crystallin is one of the important and abundant structural proteins in the lens. It functions as a chaperone to facilitate protein folding and stabilize other lens proteins in a soluble form. However, under oxidative stress, many of its -SH groups could be glutathionylated, resulting in its chaperonelike function to be drastically lost. By using shotgun proteomic analysis, Giblin's group studied the glutathionylation sites of crystallins in the lens of the guinea pig under oxidative stress. Their data showed that almost all major crystallins, except αβ, were glutathionylated after the oxidative insult. More than 70% of the –SH groups were conjugated with GSH, cysteine, or both molecules. Interestingly, our previous data have shown that mice lacking Grx2 showed faster progression of cataract during aging. In the Grx2 null mice, the lens nuclear opacity began at 5 months, 3 months sooner than that of the control mice and the progression of cataract were also much faster than the agematched controls. Importantly, this early cataract formation is closely associated with alpha A-crystallin glutathionylation accumulation and compromised mitochondrial functions in *Grx2* gene KO mice [37]. These data suggested that oxidative stress-induced thiol/disulfide imbalance at the stage of PSSG accumulation is the first critical checkpoint to prevent the cascading events leading to cataract formation. The findings also suggest that Grx2 protein may be a potential anticataract agent for delaying or slowing down the age-related cataracts in humans.

Oxidative stress may lead to inactivation of other fundamental antioxidant enzymes. Compared to other antioxidant enzymes, Grx2 is less prone to inactivation and has higher affinity to glutathionylated substrates, making it ideal antioxidant enzyme in terms of scavenging free radicals in highly oxidizing environments [22]. Other antioxidant enzymes like TR has a sensitive selenocysteine at the active site that makes it susceptible to electrophilic oxidation and inactivation with abundant oxidative stress [54]. A study by Zhang et al. used TR inhibitors such as auranofin and 4-hydroxynonenal (HNE) to determine the effect on Grx2 activity. Seemingly, Grx2 and cytosolic Trx1 and mitochondrial Trx2 act as back-up systems to each other [23]. When Trx1 and Trx2 are compromised, Grx2 expression is increased, and vice-versa. GSH was also shown to directly reduce Trx1 and Trx2, and the addition of Grx2 increased Trx's repair and enzyme activity [7, 55]. When Grx2 overexpression was induced in HeLa cells, HeLa cells were resistant to auranofin and HNE's cytotoxic effects by promoting anti-apoptotic effects and dually reducing and activating oxidized Trx [55]. Interestingly, considering Trx2 and Grx2's principal mitochondrial localization, the two enzymes may also share common targets. Peroxiredoxin 3 (Prx3) is especially located in the mitochondria and has direct peroxidase and signaling activities. Prx3 has two catalytic cysteines, as do most of the typical members of the peroxiredoxin family, but can also be regulated by oxidation of certain structural Cys residues [56]. Hanschmann et al. were able to determine that both Grx2 and Trx2 work as electron donors for Prx3 [57]. Inhibition of either Grx2 or Trx2 does not affect the expression or activation of Prx3, reinforcing the idea that Grx2 and Trx2 may work as back-up systems for each other [7, 23, 56]. However, among dual inhibition, oxidized Prx3 accumulates in the mitochondria. Grx2 also may have functions in preventing the oxidation of ascorbate or vitamin C. Grx2 acts as an electron donor to dehydroascorbate (DHA) reductase, an enzyme responsible for the conversion of DHA into ascorbate [11]. Vitamin C can also then potentially

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

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With Grx2's mitochondrial and nuclear localization, Grx2 has the potential to regulate the activity and expression of several different proteins. Unfortunately, there is limited research on Grx2 protein targets, yet screening-based assays help identify certain possibilities that may explain why Grx2 is anti-apoptotic or capable of enhancing cytoprotection in oxidative stress environments. Schutte et al. identified that cytoskeleton, chaperone, proteolysis, metabolic, and transcription factor proteins were the most likely candidates to be regulated by Grx2 [24]. Considering Grx2's effects on proliferation and migration, proteins like actin, tubulin, and dihydropyrimidinase-related protein 2 (DPYL2) with key cysteine residues are regulated by Grx2c. Chaperone proteins have multiple functions including proper folding of proteins and response to ER and oxidative stress, so heat shock proteins may be critically controlled by Grx2 enzymatic activity. Grx2's mitochondrial localization makes it a prime candidate for ensuring metabolic activity. Previous studies have highlighted Grx2's management of TCA cycle metabolites and ATP production in the cell [51, 58]. This may explain why such as glyceraldehyde-3-phosphate (G3PD), enoyl CoA hydratase (ECHM), and glycine N-methyltransferase (GNMT) are specific Grx2 targets [24]. Moreover, ubiquitination is closely linked with oxidative stress and autophagy, which may explain why 26S proteasome subunits and certain E3 ubiquitin ligases may be possible Grx2 targets [24]. Regarding Grx2's nuclear localization especially in HeLa cells, this definitely suggests that Grx2 may be

work as an antioxidant and scavenge free radicals in the cell.
