**5. Grx2 interactions and protein targets**

Ribonucleotide reductase has been classified as a primary target for electron donation from several members of the thioredoxin superfamily including Trx1, Trx2, Grx1, and Grx2. In DNA nucleotide synthesis, ribonucleotide reductase performs the rate-limiting step by converting ribonucleotides to deoxyribonucleotides. dNTPs are crucial for DNA reproduction and repair. Therefore, by mediating ribonucleotide reductase, Grx2 essentially allows for sustained DNA production even in times of oxidative damage. Evidence shows that Grx2 uses a monothiol mechanism to donate electrons with activity highly dependent on its cofactor, GSH, concentration.

Several studies have demonstrated Grx2's ability to protect classical mitochondrial targets, such as complex I and IV. Particularly, Wu et al. have shown that complex I and IV glutathionylation is reversed by Grx2 in retinal pigment epithelial (RPE) cells and furthermore, prevents hydrogen peroxide (H2 O2 )-induced damage [31]. UCP1 in BAT and UCP3 in skeletal muscle are involved in thermogenesis and ROS production and signaling [45, 46]. Allosteric control of these proteins relies on Grx1 and Grx2's reversible glutathionylation. *Grx2* KO mice displayed slightly significant lower body mass, accompanied by denser gonadal white adipose tissue and less reactivity to H2 O2 -induced activation of UCP3 [46]. Without Grx2, UCP3 activation ceased. TCA cycle metabolites such as succinate and pyruvate were decreased in the liver, compared to a decrease in 2-oxoglutarate in skeletal muscle [51]. 2-Oxoglutarate dehydrogenase (Ogdh) is another enzyme that has been shown to be directly regulated by ROS-induced glutathionylation. Grx2 works to deglutathionylate Ogdh, and Ogdh in return also controls mitochondrial redox balance by preserving normal ROS levels. With H2 O2 treatment, GSH initially feeds the amplifying cascade of ROS production by Ogdh, but ROS levels are strictly controlled by Grx2's activation and direct peroxidase activities [51]. The inhibition of Grx2 leads to the limitation of Ogdh activity and could explain lower amounts of 2-oxoglutarate in *Grx2* KO mice.

Grx2 has also been implicated to interact with TR to reduce certain substrates. With the help of TR, Grx2's primary protein targets are coenzyme A (CoA) and other small molecule disulfides [7, 22, 23]. Because of Grx2's high affinity to oxidized targets, CoA mixed disulfide and glutathione disulfide were easily reduced due to TR's ability to reduce both disulfide bridges and mixed glutathione disulfide at Grx2's catalytic Cys sites [22, 52, 53]. Interestingly, there is also evidence that supports that Grx2 may also use TR as an electron donor to reduce the GSSG mitochondrial pool especially in times of GSH deficiency or mitochondrial dysfunction [26, 35].

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

*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

Ribonucleotide reductase has been classified as a primary target for electron donation from several members of the thioredoxin superfamily including Trx1, Trx2, Grx1, and Grx2. In DNA nucleotide synthesis, ribonucleotide reductase performs the rate-limiting step by converting ribonucleotides to deoxyribonucleotides. dNTPs are crucial for DNA reproduction and repair. Therefore, by mediating ribonucleotide reductase, Grx2 essentially allows for sustained DNA production even in times of oxidative damage. Evidence shows that Grx2 uses a monothiol mechanism to donate electrons with activity highly dependent on its cofactor,

Several studies have demonstrated Grx2's ability to protect classical mitochondrial targets, such as complex I and IV. Particularly, Wu et al. have shown that complex I and IV glutathionylation is reversed by Grx2 in retinal pigment epithelial (RPE) cells and furthermore,

muscle are involved in thermogenesis and ROS production and signaling [45, 46]. Allosteric control of these proteins relies on Grx1 and Grx2's reversible glutathionylation. *Grx2* KO mice displayed slightly significant lower body mass, accompanied by denser gonadal white adi-

activation ceased. TCA cycle metabolites such as succinate and pyruvate were decreased in the liver, compared to a decrease in 2-oxoglutarate in skeletal muscle [51]. 2-Oxoglutarate dehydrogenase (Ogdh) is another enzyme that has been shown to be directly regulated by ROS-induced glutathionylation. Grx2 works to deglutathionylate Ogdh, and Ogdh in return

ment, GSH initially feeds the amplifying cascade of ROS production by Ogdh, but ROS levels are strictly controlled by Grx2's activation and direct peroxidase activities [51]. The inhibition of Grx2 leads to the limitation of Ogdh activity and could explain lower amounts of 2-oxoglu-

Grx2 has also been implicated to interact with TR to reduce certain substrates. With the help of TR, Grx2's primary protein targets are coenzyme A (CoA) and other small molecule disulfides [7, 22, 23]. Because of Grx2's high affinity to oxidized targets, CoA mixed disulfide and glutathione disulfide were easily reduced due to TR's ability to reduce both disulfide bridges and mixed glutathione disulfide at Grx2's catalytic Cys sites [22, 52, 53]. Interestingly, there is also evidence that supports that Grx2 may also use TR as an electron donor to reduce the GSSG mitochondrial pool especially in times of GSH deficiency or mito-

also controls mitochondrial redox balance by preserving normal ROS levels. With H2

)-induced damage [31]. UCP1 in BAT and UCP3 in skeletal


O2 treat-

O2

O2

in humans.

36 Glutathione in Health and Disease

GSH, concentration.

prevents hydrogen peroxide (H2

pose tissue and less reactivity to H2

tarate in *Grx2* KO mice.

chondrial dysfunction [26, 35].

**5. Grx2 interactions and protein targets**

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 vitally important in activating crucial transcription factors or signaling pathways [2, 11, 50]. The screening-based assay in Schutte's study highlighted multiple key transcription factors including ruvB-like 1 (RUVB1), elongation factor tu (EFTU), and heterogeneous nuclear ribonucleoprotein F (HNRPF) [24]. Although some of these targets have not been confirmed, it still provides insightful information about how Grx2 may facilitate cellular survival and protection.

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