Redox Pioneer: Professor Sue Goo Rhee

Discovery of the peroxiredoxin family

While studying the regulation of glutamine synthetase in Saccharomyces cerevisiae in 1983, Kangwha Kim, a postdoctoral fellow in Rhee's laboratory, noticed that the yeast enzyme, which had been purified to apparent homogeneity, lost activity and underwent partial degradation when stored in a buffer containing dithiothreitol. Evidence suggested that the glutamine synthetase degradation was not due to protease contamination. Around this time, Earl Stadtman and Rodney Levine in the Laboratory of Biochemistry were investigating the effect of oxidative modification on protein degradation (26). Influenced by their studies, Rhee's group investigated whether reactive oxygen species were the culprit. Studies revealed that the damage to yeast glutamine synthetase was actually caused by hydroxyl radicals (HO^•). They were being generated from O₂ through the Fenton reaction, catalyzed by Fe²⁺ produced through a dithiothreitol-mediated reduction of trace amounts of Fe³⁺ in the buffer (22).

The fact that glutamine synthetase in crude extracts of yeast remained active in the presence of dithiothreitol and contaminating Fe³⁺, while the purified enzyme was rapidly inactivated, suggested the existence of a yeast component that protected glutamine synthetase from inactivation. This protective component was purified and found to be a 25-kDa protein (21). Rhee's group subsequently purified another protector protein from bovine brain on the basis of its ability to protect glutamine synthetase from the dithiothreitol–Fe³⁺ oxidation system. They cloned genes that encode the yeast and rat proteins and found similar amino acid sequences for the two protector proteins (6).

The 25-kDa protector proteins did not resemble any antioxidant known at the time: All known antioxidant enzymes were dependent on a cofactor such as a heme, flavin, manganese, or selenium, none of which was found in the 25-kDa yeast and rat proteins. Critical information needed for the characterization of the 25-kDa proteins came from studies of a seemingly unrelated enzyme, bacterial alkyl hydroperoxide reductase (AhpC), which had been shown in the laboratory of Bruce Ames to catalyze the conversion of alkyl hydroperoxides to their corresponding alcohols at the expense of nicotinamide adenine dinucleotide phosphate (NADPH) (13). Initial comparison of the amino acid sequences of 25-kDa proteins and AhpC exhibited similarity only within a small NH₂-terminal portion of the proteins. However, when Rhee's group resequenced the AhpC gene, similarity became apparent over their entire sequences, suggesting that the 25-kDa proteins also might be peroxidases (6).

Soon thereafter, Rhee's group found that the 25-kDa yeast protein reduces H₂O₂ and contains a cysteine residue at its active site. The electrons for the reduction of H₂O₂ were found to come from NADPH via thioredoxin reductase and thioredoxin. Serendipitously, dithiothreitol included in the homogenization buffer had provided the electrons in the initial studies. Rhee initially referred to this 25-kDa protein as thiol-specific antioxidant and subsequently as thioredoxin-dependent peroxidase, before settling on the name of peroxiredoxin, chosen because not all members of this family relied on thioredoxin as the electron donor (5).

A database search by Rhee's group subsequently found >25 protein sequences from a variety of organisms that showed similarity to the yeast/rat 25-kDa protein (6). None of these proteins had known antioxidant or other biochemical functions. Alignment of the sequences of the peroxiredoxin family members present in the database in 1994 revealed two highly conserved cysteine residues corresponding to Cys47 and Cys170 of yeast peroxiredoxin (6). The NH₂-terminal cysteine [later designated the peroxidatic cysteine (C_P)] was conserved in all family members and the COOH-terminal cysteine [designated the resolving Cys (C_R)] was present in most but not all members. On the basis of the location or absence of the C_R, peroxiredoxin enzymes were classified into 2-Cys, atypical 2-Cys, and 1-Cys peroxiredoxin subfamilies (34). Peroxiredoxin enzymes are present in multiple isoforms in most organisms (e.g., three in E. coli, five in S. cerevisiae, and nine in Arabidopsis thaliana).

In the early stage of peroxiredoxin studies, it was difficult to explain how the C_P–SH could be oxidized specifically by H₂O₂, given that many other proteins also contain cysteine residues. The unusual reactivity of peroxiredoxin toward H₂O₂ was later explained by the crystal structure of a peroxiredoxin, in which two basic amino acid residues interact with the sulfur atom of C_P–SH, thus promoting ionization of the thiol to the thiolate anion (8). It was known that only the thiolate anion was oxidized by peroxides. Structural studies carried out later in several laboratories revealed an extensive hydrogen bond network that lowers the activation energy for breaking the O–O bond of peroxides during the oxidation of C_P-S⁻ to sulfenic acid (C_P-SOH) (12).

Rhee's group showed that most mammalian cells abundantly express six peroxiredoxin isoforms, four 2-Cys peroxiredoxin isoforms (peroxiredoxin I to peroxiredoxin IV), one atypical 2-Cys isoform (peroxiredoxin V), and one 1-Cys peroxiredoxin isoform (peroxiredoxin VI). Rhee's laboratory elucidated the catalytic mechanisms of peroxiredoxins I–V (34), and the reaction mechanism of peroxiredoxin VI was established by Fisher (10). All three types of peroxiredoxin enzymes share a common first step, in which the C_P-S⁻ is oxidized to C_P–SOH (Fig. 1). In peroxiredoxins I–IV, the C_P–SOH in the NH₂-terminal region of one subunit of the homodimer is attacked by C_R-SH located in the COOH-terminal region of the second subunit, resulting in the formation of an intersubunit disulfide bond that is subsequently reduced by thioredoxin (Trx) (Fig. 1a). In peroxiredoxin V, both C_P and C_R are present in the same polypeptide and, therefore, form an intrachain disulfide bond that is also reduced by thioredoxin (Fig. 1b). Peroxiredoxin VI lacks a C_R residue, and the C_P–SOH is resolved by a C_R–SH provided by a glutathione S-transferase (Fig. 1c).

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FIG. 1.

Reaction mechanisms of mammalian peroxiredoxin enzymes. All three types of peroxiredoxins share a common first step, in which the peroxidatic Cys (C_P) is oxidized to sulfenic acid (C_P–SOH). For 2-Cys peroxiredoxins (peroxiredoxins I–IV), the C_P–SOH in one subunit of the homodimer reacts with the resolving Cys (C_R) of the second subunit to form an intersubunit disulfide bond that is subsequently reduced by thioredoxin. The active sites of both subunits can form disulfide bonds, but only one is shown for clarity. C_P is Cys52 for Prx I, Cys51 for Prx II, Cys47 for Prx III, Cys87 for Prx IV, and Cys47 for both Prx V and Prx VI. C_R is Cys173 for Prx I, Cys172 for Prx II, Cys168 for Prx III, Cys 208 for Prx IV, and Cys151 for PrxV (a). In atypical 2-Cys peroxiredoxin (peroxiredoxin V), both C_P and C_R are present in the same polypeptide and, therefore, form an intrachain disulfide bond, which is also reduced by thioredoxin (b). The 1-Cys peroxiredoxin (peroxiredoxin VI) lacks a C_R residue, and its C_P–SOH is resolved by a cysteine in GST. The disulfide formed between the 1-Cys peroxiredoxin and GST is reduced by glutathione (c). GST, glutathione S-transferase.

Reversible hyperoxidation of 2-Cys peroxiredoxins and its significance

While studying the kinetics of a yeast peroxiredoxin and human peroxiredoxin I, Rhee and his colleagues observed that their peroxidase activity gradually decreased with time. They subsequently found that this inactivation was due to selective oxidation of C_P–SH to the sulfinic acid (C_P–SO₂H) (53). The Cys–SOH generated as an intermediate during catalysis thus occasionally underwent further oxidation to Cys–SO₂H, a reaction that cannot be reversed by thioredoxin and thus leads to peroxiredoxin inactivation. The presence of H₂O₂ alone was not sufficient to induce the oxidation of C_P–SH of peroxiredoxin I to C_P–SO₂H. All of the catalytic components (H₂O₂, thioredoxin, thioredoxin reductase, and NADPH) were required, indicating that hyperoxidation occurred only when peroxiredoxin I was engaged in the catalytic cycle. Kinetic analysis indicated that the enzyme was hyperoxidized at a rate of 0.07% per turnover, about 1 out of 1000.

In 2000, Hyun Ae Woo, a graduate student in Rhee's laboratory, showed that the sulfinic peroxiredoxin I produced during exposure of cells to H₂O₂ was gradually reduced back to the thiol form after removal of H₂O₂ (47). This was surprising because oxidation to the sulfinic state was thought to be an irreversible process in cells. Subsequently, Michel Toledano's laboratory identified sulfiredoxin as the enzyme responsible for the reduction of sulfinic peroxiredoxin in yeast and showed that this reaction utilized ATP to activate the reduction process (3) (Fig. 2).

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FIG. 2.

The catalytic cycle and reversible hyperoxidation of 2-Cys peroxiredoxin. The C_P thiolate of 2-Cys peroxiredoxin is oxidized by H₂O₂ to C_P–SOH, which, in turn, follows one of two pathways to either form a disulfide with C_R–SH or to undergo further oxidation to C_P–SO₂H. The inactivated hyperoxidized peroxiredoxin is reactivated through a reduction reaction catalyzed by the ATP-dependent activity of sulfiredoxin (Srx). The number of peroxiredoxin cysteine residues is for mammalian Prx I.

Critical cysteine residues of many other cysteine-containing proteins are also oxidized to sulfinic acid. Atypical 2-Cys peroxiredoxin (peroxiredoxin V) and 1-Cys peroxiredoxin (peroxiredoxin VI) also undergo hyperoxidation, although more slowly than 2-Cys peroxiredoxin. Rhee's group found that reduction of Cys–SO₂H catalyzed by sulfiredoxin is specific to 2-Cys peroxiredoxin isoforms (48). Furthermore, prokaryotic 2-Cys peroxiredoxin enzymes were found to be insensitive to hyperoxidation, and prokaryotes did not contain sulfiredoxin (51). The inactivation of 2-Cys peroxiredoxin via cysteine hyperoxidation was suggested to be a result of structural features acquired during evolution to support regulation of cellular function by H₂O₂ (51).

To this end, hyperoxidation of a yeast 2-Cys peroxiredoxin was shown to convert the dimeric protein to a decamer. The decamer undergoes further aggregation and gains a protein chaperone function (14). This gain of chaperone activity concomitant with the loss of peroxidase activity after 2-Cys peroxiredoxin hyperoxidation was also observed in other organisms, including mammalian cells (38).

The detection of hyperoxidized peroxiredoxin I had initially relied on complex proteomics analysis involving two-dimensional polyacrylamide gel electrophoresis followed by mass spectrometry. Rhee's group subsequently devised a simple immunoblot assay for the detection of hyperoxidized 2-Cys peroxiredoxins by employing antibodies that bind to the conserved sequence surrounding C_P–SO₂H of these enzymes (49). This simple assay made it possible to link the seemingly wasteful inactivation–reactivation cycle of 2-Cys peroxiredoxins to circadian rhythms. Daily timekeeping by living organisms is largely driven by transcriptional–translational feedback loops that give rise to rhythmic expression of clock genes. The remarkable story of the circadian oscillation of peroxiredoxin–SO₂H started with the discovery by O'Neill's laboratory of the hyperoxidation of peroxiredoxin as a transcription-independent circadian biomarker in a green algae and in red blood cells (28, 29). This was followed by the discovery in Rhee's group of the feedback control of steroidogenesis via peroxiredoxin III hyperoxidation in the mitochondria of adrenal gland (17).

In 2005, Rhee returned to Korea to lead a newly established research institute at Ewha Womans University and was named a National Honor Scientist of Korea. In Korea, Rhee's group showed that peroxiredoxin II undergoes an oscillatory hyperoxidation in red blood cells and that the rising phase of the oscillation is driven by H₂O₂ produced from the autoxidation of hemoglobin during O₂ transport. The decreasing phase is attributable to degradation of peroxiredoxin II–SO₂H by the 20S proteasome in the red blood cells (7) (Fig. 3a). Rhee's group also found that peroxiredoxin III, which is exclusively localized in the mitochondria, undergoes an oscillatory hyperoxidation in the adrenal gland, brown adipose tissue, and the heart (17, 18, 36) (Fig. 3b). The rising phase of the peroxiredoxin III–SO₂H oscillation is driven by H₂O₂ produced by cytochrome P450 during the conversion of cholesterol to corticosterone in mitochondria, whereas the decreasing phase is due to reduction of peroxiredoxin III–SO₂H catalyzed by sulfiredoxin. Rhee's group showed that the oscillation results in the release of mitochondrial H₂O₂ into the cytosol followed by an increase in the level of phosphorylated p38 mitogen-activated protein kinase (MAPK), both of which oscillate approximately in parallel with peroxiredoxin III–SO₂H (Fig. 3b). The level of sulfiredoxin in mitochondria also undergoes oscillation with a phase opposite to that of peroxiredoxin III–SO₂H (Fig. 3b). Rhee proposed that peroxiredoxin III–SO₂H formation and H₂O₂ release represent a circadian process that couples cholesterol metabolism (adrenal gland) or energy metabolism (brown adipose tissue and heart) to local clocks (35, 36).

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FIG. 3.

Scheme depicting the daily oscillation of peroxiredoxin II–SO₂H in red blood cells and of peroxiredoxin III–SO₂H in adrenal gland. (a) Peroxiredoxin II–SO₂H (PrxII–SO₂H) oscillation in mouse red blood cells. The rising phase of the oscillation is driven by H₂O₂ produced from the autoxidation of hemoglobin during O₂ transport, whereas the declining phase is attributable to degradation of peroxiredoxin II–SO₂H by the 20S proteasome and not to the reduction of peroxiredoxin II–SO₂H by sulfiredoxin. The amplitude of peroxiredoxin II–SO₂H oscillation corresponds to only ∼1% of total peroxiredoxin II. (b) Peroxiredoxin III–SO₂H oscillation in mouse adrenal gland. The rising phase of the oscillation is driven by H₂O₂ produced by cytochrome P450 during corticosterone production in mitochondria, whereas the declining phase is due to reduction of peroxiredoxin III–SO₂H (PrxIII–SO₂H) by sulfiredoxin. Because 70% of the total peroxiredoxin III is converted to the inactive SO₂H form, oscillation results in the release of mitochondrial H₂O₂ into the cytosol followed by an increase in the level of phosphorylated p38 MAPK (p-p38), both of which oscillate approximately in parallel with peroxiredoxin III–SO₂H. The level of sulfiredoxin (Srx) in mitochondria also undergoes oscillation with a phase opposite to that of peroxiredoxin III–SO₂H oscillation. The mechanism underlying daily oscillation of sulfiredoxin, abundance in mitochondria of the adrenal gland, has been elucidated (18). We modified this figure from Rhee and Kil (36), kindly provided by Dr. Rhee. MAPK, mitogen-activated protein kinase.

Intracellular messenger function of H₂O₂ and the localized regulation of peroxiredoxin

Through 1996, Rhee's research efforts were largely focused on the receptor-mediated activation of PLC, with only moderate attention being devoted to the identification and biochemical characterization of mammalian peroxiredoxin members. This pattern was slowly reversed as Rhee became interested in the potential signaling role of H₂O₂. Rhee reasoned that the role of multiple peroxiredoxin isoforms was not simply to scavenge peroxides, because cells are already equipped with efficient antioxidant enzymes such as catalase and glutathione peroxidase. He postulated that multiple peroxiredoxins would regulate the concentration of H₂O₂. This is analogous to the regulation of the local concentration of cyclic nucleotides via multiple isoforms of cyclic nucleotide phosphodiesterases. In support of this proposal, Yun Soo Bae, a postdoc in Rhee's laboratory, showed that epidermal growth factor (EGF) induced a transient accumulation of H₂O₂ in A431 cells. Blocking this accumulation resulted in the inhibition of EGF-dependent tyrosine phosphorylation of several proteins, including PLC-γ1 (2). Further, production of H₂O₂ had been detected in a variety of cell types stimulated with transforming growth factor-β1, interleukin-1, tumor necrosis factor-α, platelet derived growth factor (PDGF), or angiotensin II (33). Additionally, in 1999 David Lambeth's group identified a new type of NADPH oxidase isoform that could be coupled to the activation of growth factor receptors in nonphagocytic cells (42).

These findings led several groups including Rhee's to propose that H₂O₂ serves as an intracellular messenger (30, 31, 33). However, little was known about the molecules through which H₂O₂ acted to propagate the signal. Nor was it known how the H₂O₂ was removed in a timely manner to terminate the signal. Having learned through studies on peroxiredoxin enzymes that protein sulfhydryl groups with a low pK _a can be selectively oxidized by H₂O₂, Rhee postulated that proteins with low pK _a cysteines were the target of H₂O₂. He focused on protein tyrosine phosphatases, because the reversible inactivation of protein tyrosine phosphatase family members could explain why the production of H₂O₂ was required for growth factor-induced tyrosine phosphorylation, as shown by Toren Finkel's and Rhee's groups (2, 43). All protein tyrosine phosphatases share a Cys-X_5-Arg signature motif (where X is any amino acid), in which the invariant, low-pK _a cysteine residue functions as a nucleophile in catalysis.

By taking advantage of the fact that reduced but not oxidized thiol can be alkylated with ¹⁴C-labeled iodoacetic acid, Seung Rock Lee in Rhee's laboratory showed that protein tyrosine phosphatase1B is reversibly oxidized by H₂O₂ produced in response to EGF stimulation in A431 cells (25). Rhee's group also showed that the tumor suppressor protein phosphatase and tensin homolog (PTEN), another member of the protein tyrosine phosphatase family, is an effector of H₂O₂ in PDGF-stimulated cells (23). On the basis of these findings, Rhee proposed that the activation of protein tyrosine kinases and phosphoinositide 3-kinase in growth factor-stimulated cells was not sufficient to increase the steady state level of protein tyrosine phosphorylation and phosphatidylinositol 3, 4, 5-trisphosphate (PIP₃). Rather, concurrent inhibition of protein tyrosine phosphatases and PTEN by H₂O₂ was also required for these effects (30) (Fig. 4). Subsequently, not only protein tyrosine phosphatases but also many other signaling proteins including protein kinases, ion channels, and transcription factors have been shown to be regulated through the reversible oxidation of their cysteine residues by H₂O₂ (39).

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FIG. 4.

The role of tyrosine phosphorylation of peroxiredoxin I in H₂O₂ accumulation around lipid rafts and in intracellular signaling. Activated RPTK induce the production of H₂O₂ through activation of NADPH oxidase (Nox) located near the receptor. The activated receptor also induces the tyrosine phosphorylation of lipid raft-associated peroxiredoxin I (PrxI) through activation of an Src family protein tyrosine kinase. The phosphorylation causes inactivation of peroxiredoxin I and accumulation of Nox-generated H₂O₂ in the submembrane compartment. The accumulated H₂O₂ inactivates protein tyrosine phosphatases in the vicinity of the activated receptor by oxidizing their active-site cysteine thiols (-SH) to various oxidation states (-S_OX). This inhibition of protein tyrosine phosphatases by H₂O₂ is critical for growth factor signaling because activation of RPTK alone is not sufficient to achieve the levels of protein tyrosine phosphorylation necessary for signaling. The concurrent inhibition of protein tyrosine phosphatases by H₂O₂ also prevents a futile cycle of phosphorylation and dephosphorylation. For a similar reason, the receptor-induced activation of phosphoinositide 3-kinase is not sufficient for the accumulation of PI(3,4,5)P₃ (PIP₃). H₂O₂-dependent inactivation of PTEN through oxidation of its active-site cysteine thiol to disulfide is also necessary. PTEN, phosphatase and tensin homolog; RPTK, receptor protein tyrosine kinases.

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Professor Rhee and colleagues in 2019. From left to right: Rodney L. Levine, Sue Goo Rhee, and P. Boon Chock.

Even though the target thiols of H₂O₂ effector proteins often have a low pK _a, they react with H₂O₂ several orders of magnitude slower than C_P–SH of peroxiredoxin (45). Furthermore, peroxiredoxins are abundant proteins with a high-affinity binding site for H₂O₂. Rhee thus felt that it was necessary to explain how the effector proteins can overcome such a competitive disadvantage and be oxidized by H₂O₂ that is produced transiently. Rhee launched an investigation into how the kinetic disadvantage of H₂O₂ effector proteins relative to peroxiredoxin can be overcome. He hypothesized that peroxiredoxin molecules near these effector proteins must be transiently inactivated to allow the effectors to react with H₂O₂. His studies uncovered two examples of such a scenario.

One is the reversible inactivation of mammalian peroxiredoxin I through phosphorylation on tyrosine-194. This is mediated by Src-family protein tyrosine kinases in cells stimulated by growth factors or by activation of T cell and B cell receptors (50). Only ∼0.3% of total peroxiredoxin I was found to be phosphorylated in PDGF-stimulated NIH 3T3 cells, because phosphorylation is confined to peroxiredoxin I molecules associated with lipid rafts in the plasma membrane. The spatially confined inactivation of peroxiredoxin I thus provides a means for generating a favorable H₂O₂ gradient around lipid rafts, where signaling proteins are concentrated, while preventing the global accumulation of H₂O₂ (Fig. 4).

The second example is the reversible inactivation of peroxiredoxin I through phosphorylation on threonine-90 by cyclin-dependent kinase1 (Cdk1) during cell cycle progression (Fig. 5). The threonine-phosphorylated peroxiredoxin I was found at the centrosome during early stages of mitosis but not during interphase or late mitotic stages (27). The accumulation of H₂O₂ around the centrosome that results from the inactivation of centrosome-associated peroxiredoxin I facilitates oxidative inactivation of centrosome-bound dual specificity phosphatase cell division cycle 14B (Cdc14B), a protein tyrosine phosphatase family member. Inactivation prevents premature degradation of mitotic activators (27).

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FIG. 5.

Transient exposure of the centrosome to H₂O₂ through phosphorylation of centrosome-associated peroxiredoxin I (Prx I) and the oxidative inactivation of centrosome-bound Cdc14B. The centrosome (circle) is shielded from high levels of H₂O₂ (gray shading) by centrosome-associated peroxiredoxin I in the G₂ phase of the cell cycle. During early mitosis, pericentrosomal peroxiredoxin I is inactivated through threonine phosphorylation by cyclin-dependent kinase I (to Prx I-T_pi), allowing exposure of the centrosome to H₂O₂. The centrosome is then shielded from H₂O₂ again when phosphorylated Prx I is dephosphorylated during late mitosis. Increased H₂O₂ at the centrosome during early mitosis causes oxidative inactivation of centrosome-bound Cdc14B (dual specificity phosphatase cell division cycle 14B), thus preventing the premature degradation of several mitotic activators including cyclin B (27).

Development of blot procedures to detect H₂O₂-sensitive proteins

After learning that the low pK _a cysteines of protein tyrosine phosphatase1B and PTEN are targets of H₂O₂ generated in response to stimulation with PDGF or EGF, Rhee's group developed a procedure to detect proteins with reactive cysteine residues susceptible to oxidation by intracellularly generated H₂O₂. This approach was based on the labeling of cysteine residues with fluorescein-conjugated iodoacetamide (52) or biotin-conjugated iodoacetamide (20) followed by blotting of the labeled proteins with antifluorescein antibodies or with streptavidin.

Identification and characterization of thioredoxin-related protein 14 proteins

Using the blot analysis method, Rhee's group identified a 14-kDa rat protein as a target of H₂O₂ and named it thioredoxin-related protein 14 (TRP14) (16). TRP14 constitutes a family of proteins present from bacteria to mammals. All members possess a WCPDC motif, whereas all thioredoxin subfamily proteins possess a WCGPC motif. Rhee's group demonstrated that analogous to thioredoxin proteins, TRP14 functions as a disulfide reductase, although it acts on different substrates (15).

Identification and characterization of mitochondria-specific thioredoxin reductase

Peroxiredoxin III and thioredoxin 2 were known to be mitochondria-specific proteins. However, only one isoform of thioredoxin reductase had been identified in mammals. Rhee's group purified and cloned from rat liver a second type of thioredoxin reductase, named thioredoxin reductase 2 (24). They demonstrated that thioredoxin reductase 2 is specifically expressed in mitochondria and that together, peroxiredoxin III, thioredoxin 2, and thioredoxin reductase 2 provide a primary line of defense against H₂O₂ produced by the mitochondrial respiratory chain.

Identification of sestrin as a positive regulator of the Nrf2 pathway

A year after the discovery of the reversible hyperoxidation of 2-Cys peroxiredoxin and its catalysis by sulfiredoxin, it was reported that sestrin also catalyzed the reduction of 2-Cys peroxiredoxin–SO₂H (4). Sestrin and sulfiredoxin show no sequence similarity, although sestrin expression is induced in response to oxidative stress. Rhee's group presented several lines of evidence that challenged the putative reductase function of sestrin (46). Subsequently, his group found that the sulfiredoxin gene is one of the targets of the major antioxidant transcription factor Nrf2 (nuclear factor erythroid 2-related factor 2) (1). Rhee's group also found that sestrin promotes the autophagic degradation of Keap1 (Kelch-like ECH-associated protein 1), a repressor of Nrf2 activity, and thereby upregulates Nrf2 signaling. This causes the induction of genes for antioxidant enzymes, including sulfiredoxin. Thus, Rhee established that the antioxidant function of sestrin is not due to sulfinic acid reductase activity, but rather to its role as a positive regulator of the Nrf2 pathway.