Sod1 integrates oxygen availability to redox regulate NADPH production and the thiol redoxome

Sod1 Regulates Glycolysis.

In many eukaryotes, including S. cerevisiae, glucose uptake negatively correlates with pO₂ (41⇓⇓–44). Indeed, we find that batch cultures of WT yeast cells grown anaerobically consume more glucose per cell than aerobically grown cultures (Fig. 1A). Media glucose concentration is plotted versus cell density, rather than time, to correct for differences in growth rate (SI Appendix, Fig. S1 A and B). Consistent with previous studies (20), aerobic cultures of sod1Δ strains consume more glucose than WT cells (Fig. 1A). However, in the absence of oxygen, both WT and sod1Δ cells consume similar amounts of glucose (Fig. 1A).

Fig. 1.

Cytosolic Sod1 interacts with and regulates glycolysis via the redox regulation of GAPDH. (A) Glucose consumption per cell for WT and sod1Δ cells grown aerobically (O₂) or anaerobically (N₂). The data are derived from the slope of the linear regions of plots measuring extracellular glucose as a function of culture density (SI Appendix, Fig. S1A). The data represent the average ± SD from two biological replicates. See also SI Appendix, Fig. S1B. (B and C) Time-resolved intracellular pH measurements of glucose starved cells upon pulsing WT, sod1Δ, or pma1-tap cells expressing a GFP-based pH sensor, pHluorin, with 2% or 0% glucose (GLU) (B). The cytosolic acidification rate, a proxy for glycolytic activity (phase 1), and the rate of realkalization of the cytosol, a proxy for Pma1 activity (phase 2), is shown for the indicated strains (C). The data represent the average ± SD from triplicate cultures. See also SI Appendix, Fig. S1C. (D and E) Analysis of Sod1-dependent GAPDH oxidation as assessed by labeling reduced GAPDH with thiol reactive mPEG-mal. (D) Representative immunoblot of GAPDH–mPEG-mal adducts in sod1Δ cells expressing yeast Sod1 (SOD1) or EV cultured in 2% GLU. (E) The difference in GAPDH oxidation in the indicated strains as assessed by quantifying the ratio of unlabeled GAPDH (oxidized GAPDH) to total GAPDH, both labeled (reduced GAPDH) and unlabeled GADPH. n = 17 from 6 independent experimental trials. (F) Measurements of GAPDH enzymatic activity in sod1Δ cells expressing SOD1 or EV. Data represent the average ± SD from quadruplicate cultures. See SI Appendix, Fig. S3 A and B for representative kinetics traces. (G) Lysates from tdh1Δ tdh2Δ tdh3Δ cells expressing HA-Tdh3 or Tdh3 were subjected to IP using α-HA antibody and the IP material was analyzed by immunoblot (IB) using α-HA or α-Sod1 antibody. The data are representative of three independent trials with another replicate shown in SI Appendix, Fig. S2I. Experimental details are presented in SI Appendix, SI Materials and Methods. (H) Assessment of GAPDH oxidation in sod1Δ cells expressing yeast Sod1 (SOD1), mitochondrial IMS targeted Sod1 (Sco2-SOD1), or EV. See SI Appendix, Fig. S3 F and G for representative mPEG-mal immunoblots and quantification of percent GAPDH oxidation. Data represent the average ± SD from three independent trials. The statistical significance is indicated by asterisks using two-tailed Student’s t tests for pairwise comparisons (E and F) or by one-way ANOVA for multiple comparisons with Dunett’s post hoc test (A, C and H): **P < 0.01, ***P < 0.001, ****P < 0.0001, n.s., not significant.

Since defects in glycolytic enzymes, including hexokinase 2 or GAPDH, decrease glucose uptake (27, 28), we sought to determine if the increased glucose consumption of sod1Δ cells was associated with altered glycolytic flux. Cytosolic pH is a reporter of glycolytic activity and can be monitored using the GFP-based ratiometric pH sensor, pHluorin. Supplementing glucose-starved WT yeast with glucose results in a rapid decrease in pH due to proton release from glycolytic phosphorylation reactions (Fig. 1 B and C, phase 1) (45), followed by a slower realkalization phase due to activation of Pma1, a cell surface H⁺-ATPase that pumps H⁺ into the extracellular space (Fig. 1 B and C, phase 2) (45). In response to glucose, sod1Δ cells exhibit more rapid rates of intracellular acidification, indicating that glycolysis is more active compared to WT cells. In contrast, sod1Δ cells exhibit a diminished rate of realkalization, indicating that Pma1 is less active in response to glucose, which is consistent with prior work that found that sod1Δ cells exhibit a defect in Pma1 activity (16, 46). To rule out that the intracellular acidification phase is affected by Pma1-dependent realkalization, glucose-dependent changes in intracellular pH were monitored in a strain expressing a hypomorphic allele of PMA1, pma1-tap, containing a C-terminal tandem affinity purification (TAP)-tag (SI Appendix, Fig. S1C). In pma1-tap cells, the rate of glucose-induced acidification is similar to WT cells (Fig. 1C, phase 1), but there is a significant decrease in the rate of realkalization (Fig. 1C, phase 2). Taken together, the data demonstrate that Sod1 negatively regulates glucose uptake and glycolytic activity.

Sod1 Interacts with and Regulates GAPDH Oxidation and Activity.

We hypothesized that Sod1-derived H₂O₂ may negatively regulate glucose uptake and glycolytic activity through the oxidative inactivation of GAPDH, which catalyzes a rate-determining step in glycolysis and contains a peroxide-sensitive active site Cys (23). S. cerevisiae encodes three GAPDH isoforms—TDH1, TDH2, and TDH3—with TDH3 being the most highly expressed in log-phase cultures, accounting for >50% of total cellular GAPDH (47). Yeast GAPDH has only two cysteines, catalytic C150 and C154, which sensitizes C150 to oxidation by H₂O₂ (SI Appendix, Fig. S1D). In order to probe the Sod1-dependence of GAPDH oxidation, we employed a thiol alkylation assay that exploits the reactivity of methoxypolyethylene glycol maleimide (mPEG-mal) with reduced but not oxidized thiols (48). The extent of GAPDH labeling with mPEG-mal, which is 5 kDa, is assessed by determining the changes in electrophoretic mobility of PEGylated GAPDH, corresponding to single- and double-labeled GAPDH at reduced C150 and/or C154 (Fig. 1D and SI Appendix, S1 E and F). Thus, the fraction of GAPDH oxidized in vivo can be determined by quantifying the ratio of the intensity of unlabeled GAPDH (oxidized GAPDH) to total GAPDH (SI Appendix, Fig. S1 F–O). The mPEG-mal approach was validated by treating cells with H₂O₂ and observing a twofold increase in GAPDH oxidation (SI Appendix, Fig. S1 F and G). Moreover, the identity of the specific sites of mPEG-mal labeling was confirmed by observing that a yeast strain expressing a single allele of Tdh3^C154S was found to have only two GAPDH proteoforms corresponding to single and unlabeled GAPDH (SI Appendix, Fig. S1P).

To determine if Sod1 oxidizes GAPDH in vivo, we analyzed GAPDH PEGylation in sod1Δ cells expressing empty vector (EV) (sod1Δ + EV) or WT SOD1 (sod1Δ + SOD1). sod1Δ + EV cells exhibit an increase in mPEG-mal labeling compared to sod1Δ + SOD1 cells (Fig. 1 D and E and SI Appendix, Fig. S2 A and B), indicating that GAPDH is more oxidized in cells expressing SOD1. Analysis of 17 replicates over 6 independent trials revealed that expression of SOD1 results in an approximately twofold increase in GAPDH oxidation, with the median and average GAPDH oxidation in cells expressing SOD1 being 26% and 35%, respectively, and 13% and 16%, respectively, in cells lacking SOD1 (Fig. 1E). The values of GAPDH oxidation in SOD1 expressing cells are on par with prior studies of glucose-grown WT yeast that found GAPDH is 26% oxidized (49). Due to variations in the absolute values of GAPDH oxidation across multiple trials (Fig. 1E), subsequent comparisons of GAPDH oxidation involved normalizing GAPDH oxidation to that of SOD1-expressing cells within each trial, though absolute values are indicated in SI Appendix.

In order to assess the extent to which Sod1 mediates the reversible oxidation of GAPDH, we employed an mPEG-mal labeling scheme that involved first capping free GAPDH thiols with N-ethylmaleimide, followed by DTT reduction of reversibly oxidized thiols and labeling them with mPEG-mal (SI Appendix, Fig. S2 C–G). Using this strategy, it was similarly found that Sod1 accounts for an approximately twofold increase in reversible GAPDH oxidation, with three independent trials giving an average GAPDH oxidation value of ∼50% in SOD1 expressing cells and 25% in cells lacking SOD1 (SI Appendix, Fig. S2 C–G). In addition, >95% of GAPDH is reducible and alkylatable in both WT and sod1Δ cells, indicating that differences in apparent GAPDH oxidation are not due to variations in the ability to reduce and alkylate GAPDH in the two strains (SI Appendix, Fig. S2H).

We next found that Sod1 interacts with and regulates GAPDH activity. GAPDH catalyzes the oxidative phosphorylation of GAP to 1,3 BPG and requires Cys¹⁵⁰ for activity. Cells lacking SOD1 (sod1Δ + EV) exhibit threefold greater GAPDH activity (Fig. 1F and SI Appendix, Fig. S3 A and B), which correlates with the approximately twofold decrease in GAPDH thiol oxidation relative to SOD1-expressing cells (Fig. 1 D and E). Moreover, GAPDH and Sod1 interact in vivo as assessed by coimmunoprecipitation (co-IP) experiments (Fig. 1G and SI Appendix, Fig. S2I). IP of HA-Tdh3 using anti-HA antibody resulted in the detection of Sod1 by immunoblotting. In contrast, a control strain expressing untagged Tdh3 did not result in co-IP of Sod1, indicating that the apparent interaction between Sod1 and GAPDH is not due to Sod1 nonspecifically binding to the anti-HA resin.

Sod1-mediated GAPDH oxidation is not dependent on the carbon source. Galactose is a fermentable carbon source that alleviates glucose-mediated respiration repression, resulting in more mitochondrial respiratory activity (16). The absolute and relative amounts of GAPDH oxidation in sod1Δ + EV and sod1Δ + SOD1 cells cultured in 2% galactose is similar to cells cultured in 2% glucose (SI Appendix, Fig. S3 C–E).

The effect of Sod1 localization on GAPDH oxidation was also evaluated. Sod1 is primarily cytosolic but is also present in the mitochondrial intermembrane space (IMS). sod1Δ cells expressing an IMS-targeted allele of SOD1, Sco2-SOD1 (15, 16, 50), exhibit comparable GAPDH oxidation to sod1Δ + EV cells, both of which are significantly lower than cells expressing WT SOD1 (Fig. 1H and SI Appendix, Fig. S3 F and G). Altogether, these results indicate that extramitochondrial Sod1 interacts with and oxidatively inactivates GAPDH, thereby explaining the previous observations that sod1Δ cells exhibit increased glucose uptake (Fig. 1A) and glycolytic activity (Fig. 1 B and C).

Yno1 and Mitochondrial Respiration Are Sources of Superoxide for GAPDH Oxidation.

Sod1 requires a superoxide source to catalyze peroxide production for the control of GAPDH oxidation and activity. As with higher eukaryotes, two primary sources of superoxide in yeast include mitochondrial respiration and the yeast NADPH oxidase, Yno1 (51). Both sources contribute toward GAPDH oxidation as respiration-deficient rho⁰ and yno1Δ cells exhibit an approximately threefold lower degree of GAPDH oxidation relative to WT cells and phenocopy the sod1Δ mutant (Fig. 2A and SI Appendix, Fig. S4 A and B). Furthermore, overexpression of Yno1 on a galactose-inducible promoter (pYES-YNO1, 3%GAL), which resulted in a ∼30% increase in dihydroethidium (DHE)-detectable superoxide (SI Appendix, Fig. S4C) promoted GAPDH oxidation by twofold as compared to cells expressing EV (pYES2-EV, 0 and 3%GAL) or that were cultured in noninducing media (pYES2-YNO1, 0%GAL) (SI Appendix, Fig. S4 D–F). In total, these results indicate that both Yno1 and mitochondrial respiration are sources of the superoxide substrate required by Sod1 to drive the H₂O₂-dependent oxidation of GAPDH.

Fig. 2.

Aerobic GAPDH oxidation is dependent on and rate-limited by Sod1 and requires Yno1 and mitochondrial respiration as superoxide sources. (A) Analysis of GAPDH oxidation as assessed by mPEG-mal labeling of GAPDH in WT, rho⁰, yno1Δ, and sod1Δ cells cultured in 2% GLU. See SI Appendix, Fig. S4 A and B for representative mPEG-mal immunoblots and quantification of GAPDH oxidation. Data represent the average ± SD from three independent trials. (B and C) Assessment of the Sod1-dependence on the aerobic oxidation of GAPDH. (B) Relative Sod1 activity to assess aerobic and anaerobic Sod1 maturation in sod1Δ cells expressing EV, WT SOD1, or the P144S sod1 mutant. See SI Appendix, Fig. S4G for representative SOD activity gels and quantification of Sod1 activity. Data represent the average ± SD from three independent cultures. (C) Analysis of GAPDH oxidation as assessed by mPEG-mal labeling of GAPDH in aerobic or anaerobic sod1Δ cells expressing EV, WT SOD1, or the P144S sod1 mutant. See SI Appendix, Fig. S4 H and I for representative mPEG-mal immunoblots and quantification of GAPDH oxidation. Data represent the average ± SD from two or three independent trials. (D and E) Analysis of GAPDH oxidation in WT or sod1Δ cells expressing GAL-driven SOD1 cultured with increasing concentrations of galactose (GAL) (0%, 0.005%, 0.0075%, 0.01% and 0.1% GAL). (D) Titration of SOD1 reveals a positive correlation between Sod1 expression and GAPDH oxidation from two independent trials. (E) Representative mPEG-mal and Sod1 immunoblot from which the Sod1-dependence of GAPDH oxidation was assessed. In D, GAPDH oxidation was normalized to that of the WT cells and the linear regression analysis of the two trials gives coefficients of determination (r²) of 0.81 and 0.77, with P values of 0.01 and 0.02, respectively. See also SI Appendix, Fig. S4J. The statistical significance relative to WT (A) is indicated by asterisks using ordinary one-way ANOVA with Dunett’s post hoc test for the indicated pairwise comparison in A, **P < 0.01. The statistical significance relative to the aerobic SOD1 expressing cells is indicated by asterisks using two-way ANOVA for multiple comparisons with Tukey’s post hoc test for the indicated pairwise comparisons in B and C: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, n.s., not significant.

O₂-Dependent GAPDH Oxidation Is Dependent on and Rate-Limited by Sod1.

All metabolic sources of superoxide and peroxide are ultimately derived from O₂. We therefore sought to determine if Sod1 is the sole enzymatic adapter that links oxygen availability to the control of GAPDH oxidation and if GAPDH oxidation is rate-limited by Sod1. Toward this end, we first asked if Sod1 mediates O₂-dependent GAPDH oxidation. Since WT Sod1 is transcriptionally and posttranslationally down-regulated in response to hypoxia and anoxia (52⇓⇓–55), we utilized sod1Δ cells expressing ADH1-driven Sod1^P144S, which is a mutant previously engineered to constitutively express mature enzymatically active Sod1 even in the absence of O₂ (16, 54). Indeed, the Sod1^P144S mutant is enzymatically active in lysates derived from both aerobic and anaerobic cultures, whereas WT Sod1 is only fully active in lysates derived from aerobically cultured cells (Fig. 2B and SI Appendix, Fig. S4G). sod1Δ cells expressing WT or Sod1^P144S exhibit a nearly twofold decrease in GAPDH oxidation when cultured anaerobically, consistent with the requirement for O₂ as the metabolic origin of superoxide and peroxide (Fig. 2C and SI Appendix, Fig. S4 H and I). However, remarkably, the O₂-dependence of GAPDH oxidation is completely lost in cells lacking SOD1. Furthermore, we determined that the oxidation of GAPDH is rate limited by Sod1, finding that GAL-regulated titration of Sod1 levels results in a dose-dependent increase in GAPDH oxidation (Fig. 2 D and E and SI Appendix, Fig. S4J). Altogether, these results indicate that the aerobic oxidation of GAPDH is dependent on and rate-limited by Sod1. It is also worth noting that there is a certain level of O₂-independent GAPDH oxidation that is not dependent on Sod1, albeit the nature of the oxidant in this case is not known.

Sod1-Mediated Oxidative Inactivation of GAPDH Results in Increased NADPH Production and Oxidative Stress Resistance.

We next determined if the Sod1-dependent oxidative inactivation of GAPDH results in rerouting of glycolytic metabolism toward oxPPP to produce NADPH and increase resistance to oxidative stress. Titration of Sod1 expression using a GAL-driven SOD1 allele results in both a dose-dependent increase in GAPDH oxidation as well as NADPH levels (Fig. 3 A and B and SI Appendix, Fig. S5 A–F). Control experiments in which GAL is titrated into cells expressing a non-GAL–driven SOD1 allele indicate that GAL alone does not alter NADPH levels (SI Appendix, Fig. S5G). Moreover, tdh3Δ cells, which express ∼60% less GAPDH than WT cells (Fig. 3C and SI Appendix, Fig. S5H) (47), exhibit increased NADPH levels (Fig. 3D), consistent with the finding that oxidative inactivation of GAPDH increases NADPH.

Fig. 3.

Sod1-mediated oxidative inactivation of GAPDH results in increased NADPH production and resistance to oxidative stress. (A and B) Sod1 expression positively correlates with GAPDH oxidation (A) and NADPH production (B). See SI Appendix, Fig. S5A for representative immunoblots assessing Sod1 expression and GAPDH oxidation by mPEG-mal labeling in sod1Δ cells expressing GAL-driven SOD1 cultured with increasing concentrations of galactose (GAL) (0.005%, 0.0075%, 0.01%, and 0.1% GAL). The NADP⁺/NADPH ratio inversely correlates with normalized Sod1 expression (A) and GAPDH oxidation (B), with linear regression analysis in A and B giving coefficient of determinations (r²) of 0.98 and 0.99 and P values of 0.01 and 0.007, respectively. See also SI Appendix, Fig. S5 B–G for representative correlation plots between Sod1 expression or GAPDH oxidation and NADP⁺/NADPH ratio and NADPH concentration. (C and D) Depletion of intracellular GAPDH decreases the NADP⁺/NADPH ratio. (C) GAPDH expression is reduced by ∼60% in tdh3Δ cells as assessed by immunoblot analysis (see SI Appendix, Fig. S5H for representative GPADH immunoblots). (D) Measurements of the NADP⁺/NADPH ratio in WT and tdh3Δ. Data represent the average ± SD from triplicate cultures. (E) Ablation of G6PD (Zwf1) increases the cellular NADP⁺/NADPH ratio. Data represent the average ± SD from triplicate cultures. (F and G) Ablation of Sod1 and Zwf1 increases DTT-reversible disulfide oxidation and dimerization of peroxiredoxin (HA-Tsa1x2). Representative immunoblot of HA-Tsa1 in WT, both treated and untreated with H₂O₂ or DTT, sod1Δ, and zwf1Δ cells. Tsa1 proteoforms identified include oxidized and reduced monomer and disulfide oxidized dimer (F). Quantification of the fraction of disulfide oxidized Tsa1 dimer over the total amount of Tsa1 from three independent trials (G). Data represent the average ± SD. Assignment of HA-Tsa1 proteoforms are derived from SI Appendix, Fig. S6. (H) Sod1 activity inversely correlates w+ith Tsa1 disulfide dimerization. Sod1 activity, as titrated using the copper chelator, BCS, negatively correlates with disulfide dimerized HA-Tsa1. Representative Sod1 activity gel and HA-Tsa1 immunoblot analysis are shown in SI Appendix, Fig. S7 F and G. (I–K) Aerobic and anerobic growth rates in 2% glucose media (I), aerobic growth rate in 2%GAL media (J), and diamide sensitivity (K) of WT and tdh3Δ cells. In K, growth after 9 h of exposure to diamide is reported. See SI Appendix, Fig. S7 H and I for complete growth curves. Data represent the average ± SD from triplicate cultures. The statistical significance relative to WT is indicated by asterisks using two-tailed Student’s t test for pairwise comparisons in C, D, E, and J: *P < 0.05, ***P < 0.001. The statistical significance relative to WT (I) is indicated by asterisks using ordinary one-way ANOVA with Dunett’s post hoc test for the indicated pairwise comparison in I, *P < 0.05. The statistical significance relative to the aerobic growth rate (I) or 0 µM diamide (K) is indicated by asterisks using two-way ANOVA for multiple comparisons with Dunett’s or Tukey’s post hoc test for the indicated pairwise comparisons in I and K, respectively; **P < 0.01, ***P < 0.001, ****P < 0.0001.

NADPH is required for the reduction and regeneration of numerous cellular antioxidants. Therefore, we determined if the alterations in Sod1-mediated NADPH production correlated with the oxidation state of Tsa1, a yeast peroxiredoxin that scavenges peroxide and that is maintained in a reduced state using NADPH. Peroxide oxidizes Cys⁴⁷ to a sulfenic acid, which in turn forms a disulfide oxidized dimer with Cys¹⁷⁰ on another Tsa1 monomer. At high peroxide concentrations, (>250 μM exogenous H₂O₂), Cys⁴⁷ is overoxidized to sulfinylated or sulfonylated states rather than becoming disulfide oxidized. To assess Tsa1 oxidation state, we expressed an N-terminal HA tagged allele of TSA1 in yeast and ascertained its oxidation state by immunoblotting for monomeric or disulfide oxidized dimeric states of Tsa1 using anti-HA antibodies or sulfonylated states using an anti–Prx-SO₃ antibody. HA-Tsa1 complements the H₂O₂ sensitivity of tsa1Δ cells (SI Appendix, Fig. S6A), and we can assign various proteoforms corresponding to reduced monomeric and reversibly oxidized monomeric and dimeric Tsa1 based on the electrophoretic mobility of HA-Tsa1 and HA-Tsa1^C47S expressed in WT and tsa1Δ cells on SDS/PAGE gels (SI Appendix, Fig. S6 B–D). zwf1Δ cells lacking G6PD, which catalyzes the first committed step of the oxPPP, exhibited decreased NADPH levels (Fig. 3E) and elevated disulfide oxidized Tsa1 dimers (Fig. 3 F and G), thereby confirming that NADPH limitation results in elevated Tsa1 oxidation. Likewise, sod1Δ cells also exhibited increased disulfide oxidized Tsa1 dimer formation (Fig. 3 F and G).

Since steady-state intracellular peroxide levels are similar between WT, sod1Δ, and zwf1Δ cells (SI Appendix, Fig. S6 E and F), we ascribe the increased Tsa1 disulfide oxidation as arising from diminished NADPH levels. Surprisingly, for reasons that are unclear at this time, tsa1Δ and sod1Δ cells also exhibited increased Tsa1 sulfonylation, despite these cells having similar steady-state H₂O₂ as WT cells (SI Appendix, Fig. S7 A–E). Titration of a copper chelator, bathocuproinedisulfonic acid (BCS), resulted in a dose-dependent decrease in Sod1 activity, without affecting expression, and consequent increase in the formation of disulfide-dimerized Tsa1 (Fig. 3H and SI Appendix, Fig. S7 F and G). Overall, these results are consistent with Sod1 mediated oxidative inactivation of GAPDH promoting NADPH production via the oxPPP, thereby allowing for the regeneration of peroxide scavengers like Tsa1.

In order to determine if the oxidative inactivation of GAPDH by Sod1 provides a physiological benefit for cells, we measured the aerobic growth and oxidative stress resistance of cells expressing low and high levels of GAPDH. Although WT and tdh3Δ cells have similar aerobic and anaerobic growth rates in 2% GLU (Fig. 3I), when cultured in 2% GAL, a fermentable carbon source that promotes respiration in yeast, tdh3Δ cells have a marked enhancement in growth rate compared to WT cells (Fig. 3J). Moreover, tdh3Δ cells exhibit greater resistance to peroxide (SI Appendix, Fig. S7H) and diamide induced oxidative stress (Fig. 3K and SI Appendix, Fig. S7I). Altogether, the enhanced aerobic fitness and oxidative stress resistance of cells depleted of GAPDH is consistent with a beneficial role for the Sod1-mediated inactivation of GAPDH.

Sod1 Regulates GAPDH Oxidation in Human Cells.

Since both Sod1 and GAPDH are highly conserved, from yeast to humans, we sought to establish if Sod1 regulates GAPDH oxidation in human embryonic kidney HEK293 cells. Human GAPDH has an additional peroxide reactive Cys, which would result in three PEGylated proteoforms, corresponding to triple-, double-, and single-labeled GAPDH in immunoblots (SI Appendix, Fig. S8A). As expected, cells treated with H₂O₂ exhibited less mPEG-mal labeling, indicating a larger fraction of oxidized GAPDH compared to nontreated cells (SI Appendix, Fig. S8 B and C). To determine if Sod1 promoted GAPDH oxidation, we depleted Sod1 in HEK293 cells using small-interfering RNA against Sod1 (siSOD1) or scrambled control RNAi (siCTRL). Across various trials, we consistently observed a depletion of ∼60 to 80% of Sod1 (SI Appendix, Fig. S8 D and E) and a corresponding decrease in GAPDH oxidation (SI Appendix, Fig. S8 F–H). Sod1 increases GAPDH by ∼7 to 16%, which is comparable to the contribution observed from exogenous peroxide treatment (SI Appendix, Fig. S8B). Moreover, as with yeast, we find that Sod1 expression levels across multiple trials positively correlate with GAPDH oxidation (SI Appendix, Fig. S8I). Notably, the y-intercept of the linear regression (r² = 0.76; P = 0.002) is close to 0, indicating that in the complete absence of Sod1, GAPDH oxidation is expected to be ∼0% (SI Appendix, Fig. S8I). Furthermore, cell lines that overexpress Sod1, such as the breast cancer cell line MCF7 (56), exhibit a nearly threefold increase in GAPDH oxidation compared to HEK293 cells (SI Appendix, Fig. S8 J and K). Altogether, these results indicate that Sod1-mediated oxidation of GAPDH is conserved in humans.

Redox Proteomics Identifies Additional Putative Targets of Sod1 Redox Regulation.

The high abundance and broad cellular distribution of Sod1 suggests that it may function as a redox regulator of a broad variety of substrates in addition to GAPDH. To test this, we conducted a high-powered quantitative redox proteomics screen of WT and sod1Δ yeast to identify Sod1-dependent redox substrates. We used a combined stable isotope labeling with amino acids in cell culture (SILAC)-tandem mass tags (TMT) approach, whereby sod1Δ-dependent changes in protein abundance are quantified through SILAC and changes in reversible cysteine oxidation are quantified through cysteine-reactive iodoacetyl TMT (iodo-TMT) before and after reduction with DTT (Fig. 4A) (57). In this case, the same peptide from WT and sod1Δ cells is distinguished by a mass shift, allowing quantitative comparison of the peptide’s abundance in each strain [protein expression difference = log₂(sod1Δ_light/WT_heavy)]. Subsequent fragmentation/sequencing of each peptide reveals the peptide/protein identity and, if cysteine is present, releases TMT reporter ions enabling quantification of the cysteine oxidation percentage in each strain (oxidation difference = sod1Δ%Ox − WT%Ox) (Materials and Methods). Consequently, the study reveals a broad range of proteins that undergo significant changes in abundance, cysteine oxidation, or both (Fig. 4B and SI Appendix, Fig. S9A).

Fig. 4.

Redox proteomics identifies additional putative targets of Sod1 redox regulation. (A) SILAC-TMT redox proteomics workflow. SILAC labels distinguish the cellular origin of each peptide. Cysteine-reactive and isobaric iodo-TMT reporters, undetectable during the first MS stage (MS1) and released during peptide fragmentation in MS2, enable unique cysteine %oxidation calculation in WT and sod1Δ cells. (B) Plot of aggregated average cysteine oxidation difference relative to protein expression difference in sod1Δ vs. WT cells. Points near the origin (0,0) undergo less change in relative abundance and cysteine oxidation compared to points further from the origin. Cysteine coverage indicates the fraction of cysteine residues detected for each protein. (C) Volcano plot of −log₁₀(P value) relative to protein expression difference [log₂(sod1Δ/WT)] calculated from aggregate SILAC data. Proteins above −log₁₀(P value) 1.3 are significantly different in abundance between the two strains (P < 0.05). Positive and negative values of log₂(sod1Δ/WT) indicate proteins that are more expressed in sod1Δ and WT, respectively. Proteins with a single detected label are indicated as ± infinity. (D) Subset of data from C, showing expression change for proteins associated with bioprocesses functionally associated with loss of SOD1. See also SI Appendix, Fig. S9B. (E) Median FE values for GO bins observed for proteins undergoing greater than twofold change in abundance between sod1Δ and WT cells. (F) Volcano plot of −log₁₀(P value) relative to protein expression difference [%Ox_sod1Δ − %Ox_WT] calculated from iodo-TMT data aggregated at the cysteine level. −Log₁₀(P value) of 1.3 or above indicate significantly different cysteine oxidation between the two strains (P < 0.05). See also SI Appendix, Fig. S10A. (G) Kyoto Encyclopedia of Genes and Genomes (KEGG) Brite and Pathway enrichment analysis for the 96 proteins harboring oxidation sites that change significantly in the absence of SOD1. Number of associated proteins shown in parentheses. See also SI Appendix, Fig. S9D. (H) Median FE values for GO bins observed for proteins with at least one cysteine undergoing >5% difference in oxidation between WT and sod1Δ. (I) Rank-ordered plot of cysteine residues from proteins that exhibit statistically significant differences in oxidation between sod1Δ and WT. Statistical outliers in oxidation change between strains (circle size) and with respect to other cysteine residues contained within share bioprocess (blue halo, blue text) are indicated. (J) Deconvoluted oxidation differences for proteins in which a single cysteine was found to be a statistical outlier from other observed cysteine oxidation sites in the same protein. See also SI Appendix, Fig. S10B.

Focusing first on the effects of SOD1 deletion on proteome-wide protein abundance, we independently analyzed the SILAC mass spectrometry (MS) data. A total of 4,409 proteins were confidently detected and quantified, and ∼9% of these (373) exhibited significant differences in abundance between the two strains (Fig. 4C and Dataset S1). Of these, 114 (30.6%) exhibit a significant decrease in protein abundance in sod1Δ cells compared to 259 (69.4%) proteins that exhibit a significant increase in abundance. The changes in protein expression reflect known metabolic defects associated with loss of SOD1, including alleviation of glucose repression, increased mitochondrial mass, induction of the iron starvation and antioxidant responses, and diminished plasma membrane casein kinase expression (Fig. 4D and SI Appendix, Fig. S9B). Gene ontology (GO) enrichment analysis of proteins undergoing a statistically significant change in abundance revealed a set of outlier ontologies (i.e., GO terms falling outside the 90th percentile in the distribution of fold-enrichment) that could be clustered into discrete bins. Notably, we observed the greatest enrichment for ontologies linked to glycolysis and pentose phosphate pathways. This included chitin synthesis (median fold-enrichment [FE]: 29.6), which feeds directly from D-fructose-6-phosphate, a precursor to the GAPDH substrate D-glyceraldehyde-3-phosphate and a key metabolite in the PPP; cellular polysaccharide catabolism (starch and sucrose catabolism) (FE: 21.1); amino acid biosynthesis (FE: 20.6), which relies on several glycolytic intermediates; as well as purine biosynthesis/metabolism that feeds on D-ribose-5-phosphate from the PPP (FE: 14.1) (Fig. 4E and Dataset S1). We observed lower but still significant GO enrichment of proteins involved in redox processes and functions (FE: 14.1), including ontologies encompassing oxidoreductase activity, NADH oxidation, and ROS metabolic processes; and enrichment of proteins associated with ATP synthase/proton pumping (FE: 17.6).

Next, we focused on the effects of SOD1 deletion on proteome-wide cysteine oxidation as revealed by iodo-TMT MS data. A total of 2,077 cysteine residues were confidently detected ranging in oxidation level from 1 to 100% and a median level of 28.9% across all sites measured. Cross-referencing all sites against protein functional residues revealed 54 of these sites associated with metal binding (38 sites, 74%), active sites (11 sites, 20%), and protein or DNA binding (5 sites, 9%) and cysteine residues involved in these functions also exhibited statistically higher overall oxidation levels in both WT and sod1Δ (44.6%, P < 0.0001) (SI Appendix, Fig. S9C and Dataset S1). Mean global oxidation levels were nearly identical between both strains (32.8% vs. 32.9%) (SI Appendix, Fig. S9A).

Approximately 5% of the quantified cysteine sites (99 sites, 96 proteins) undergo significant changes in oxidation between WT and sod1Δ cells, and of these, we found that 65% exhibit reduced oxidation levels in the absence of SOD1 (Fig. 4F and Dataset S1). GAPDH peptides, and C150/C154 specifically, were readily detected but failed to undergo iodo-TMT reporter release under any of the three collision energies used for peptide fragmentation, preventing quantitation of SOD1-dependent oxidation in these experiments. However, we were able to detect DTT-irreversible GAPDH thiol oxidation products, including sulfinic acid (SO₂) and dehydroalanine, and found they were elevated up to 1.6-fold in WT cells relative to sod1Δ cells (Dataset S1). These results support the conclusion that Sod1 contributes to both reversible and irreversible GAPDH oxidation as detected by targeted mPEG-mal/GAPDH immunoblotting experiments and MS, respectively.

On average, cysteine residues in sod1Δ cells appear to be less oxidized compared to WT cells, consistent with a role for Sod1 in providing a source of peroxides for thiol oxidation. On the other hand, cysteine residues that are more oxidized in sod1Δ cells compared to WT may be due to oxidative stress, in part mediated by the decrease in NADPH production due to diminished Sod1-mediated GAPDH oxidation and the subsequent reduction in flux through the oxPPP.

Over half of the proteins harboring the 99 significantly changing oxidation sites are classified as enzymes, with the next most populous class represented at less than 10% in comparison (Fig. 4G and Dataset S1). Most of these proteins are also associated with metabolism, synthesis of secondary metabolites, and biosynthesis of amino acids. Clustering of ontologies that were statistical outliers for FE supported these findings and produced three distinct ontology bins. Ontologies associated with amino acid biosynthesis were predominantly enriched (FE: 133.9) and to a far greater extent than what we observed for Sod1-dependent protein abundance changes (Fig. 4H and Dataset S1). Moreover, many of the proteins within this bin exhibit reduced oxidation in the absence of SOD1, and map to multiple different amino acid biosynthesis/metabolism pathways including those for lysine (Lys20/21), arginine (Car1), threonine (Hom2), and s-adenosyl methionine (Sam1/2), to name a few (SI Appendix, Fig. S9 D and E). Ontologies encompassing redox processes and functions were also highly enriched (FE: 90.4), and include proteins involved in catalysis of redox reactions in which a CH-NH2 group acts as a hydrogen or electron donor and reduces NAD⁺ or NADP⁺. Finally, we also found significant enrichment of proteins involved in Ras and Rho signal transduction (median FE: 36.5), which hint at a unique and unrealized connection between SOD1, redox homeostasis, actin mobilization, and cell motility.

Considering the role of Sod1 as a direct regulator of GAPDH through C150 oxidation, we asked whether our proteomic data might contain evidence of a similar relationship between Sod1 and other proteins, wherein: 1) the protein is specifically affected at one out of multiple possible cysteine residues; and 2) oxidation of this cysteine is significantly reduced in the absence of SOD1. To create this filter, we binned each of the 99 sites based on whether the change in oxidation was a statistical outlier (>90th percentile) from other cysteine residues in the protein, revealing 18 proteins (Fig. 4 I and J). In seven of these cases, the change in oxidation was also found to be an outlier with respect to other oxidized cysteine residues found in other proteins within the associated bioprocess for the given protein (Fig. 4I, blue halos, SI Appendix, Fig. S10A, and Dataset S1). Deconvolution of their site-specific changes in oxidation revealed a range of responses reflecting the sensitivity of specific cysteine residues to the presence or absence of SOD1 (Fig. 4J). Some of these coincided with proteins enriched previously through ontology, most notably Sfa1 and Gfa1 involved in pyruvate metabolism and conversion of D-fructose-6-phosphate, respectively. Sfa1-C12 and Gfa1-C562, but not other detected cysteine residues in either protein, undergo a significant decrease in oxidation in the absence of SOD1 (Dataset S1). We also observed that in one case, Nbp35 (required for maturation of extramitochondrial Fe-S proteins), the site of dynamic oxidation is a functional metal binding site in the protein (Dataset S1). Finally, other proteins not revealed by previous analyses were also evident, most notably Uba1, which is the sole enzyme necessary for activating ubiquitin in yeast. Uba1-C447, which is immediately adjacent to the ATP binding site for the protein, undergoes a large drop in oxidation in the absence of SOD1, going from a maximum ∼89% in WT cells down to ∼28% in sod1Δ cells (SI Appendix, Fig. S10B and Dataset S1). In comparison, C600, which is the site of ubiquitin attachment, undergoes very little change in oxidation upon deletion of SOD1. Collectively, these data provide an unparalleled look into the dynamic oxidation of specific cysteine thiols as well as protein abundance changes in response to the loss of SOD1.