Proteins play essential roles in almost every aspect of a cell’s life, and also contribute to the structure and function of body tissues and organs. Cells and tissues adapt to their continuously changing environments by regulating the activity of their proteins. For example, proteins that are not fully active immediately after they are built instead require further ‘posttranslational’ modifications to become active.

Amino acids are the building blocks of proteins, and cysteine amino acids are frequent sites of posttranslational modifications because they are particularly chemically reactive. Under certain conditions inside the cell, the sulfur atom in a cysteine can bond with chemical group containing a second sulfur atom plus a hydrogen atom. This process, which is known as sulfhydration, can be triggered by the presence of the gas molecule, hydrogen sulfide (H₂S). The levels of hydrogen sulfide are highly regulated in the body, and it has been suggested to play a role in aging, environmental stress and many diseases. However, it is not clear whether sulfhydration plays a major role in disease conditions by modifying protein activity.

Efforts to address this question have been limited by a lack of methods that can measure the extent of sulfhydration of proteins. However, Gao et al. have now devised such a method. The approach takes steps to avoid false-positive and false-negative results, and can identify changes in the sulfhydration of cysteines across the entire complement of proteins produced by a cell, tissue or organ.

Gao et al. then used this new method to show that a master regulator of transcription (i.e. a protein that regulates the expression of many genes) causes large-scale changes in cysteine sulfhydration. These large-scale changes resulted in the reprogramming of the cell’s energy metabolism, and further experiments showed that hydrogen sulfide accumulation influences sulfhydration, protein activity and signaling pathways. The development of this new method may now lead to additional discoveries into the role of hydrogen sulfide as a signaling molecule.

Posttranslational modification is a fundamental mechanism in the regulation of structure and function of proteins. The covalent modification of specific amino acid residues influences diverse biological processes and cell physiology across species. Reactive cysteine residues in proteins have high nucleophilicity and low pKa values and serve as a major target for oxidative modifications, which can vary depending on the subcellular environment, including the type and intensity of intracellular or environmental cues. Oxidative environments cause different post-translational cysteine modifications, including disulfide bond formation (-S-S-), sulfenylation (-S-OH), nitrosylation (-S-NO), glutathionylation (-S-SG), and sulfhydration (-S-SH) (also called persulfidation) (Finkel, 2012; Mishanina et al., 2015). In the latter, an oxidized cysteine residue included glutathionylated, sulfenylated, and nitrosylated on a protein reacts with the sulfide anion to form a cysteine persulfide. The reversible nature of this modification provides a mechanism to fine tune biological processes in different cellular redox states.

Sulfhydration coordinates with other post-translational protein modifications such as phosphorylation and nitrosylation to regulate cellular functions (Altaany et al., 2014; Sen et al., 2012). Despite great progress in bioinformatics and advanced mass spectroscopic (MS) techniques, identification of different cysteine-based protein modifications has been slow compared to other post-translational modifications. In the case of sulfhydration, a small number of proteins have been identified, among them the glycolytic enzyme glyceraldehyde phosphate dehydrogenase, GAPDH (Mustafa et al., 2009). Sulfhydrated GAPDH at Cys¹⁵⁰ exhibits an increase in its catalytic activity, in contrast to the inhibitory effects of nitrosylation or glutathionylation of the same cysteine residue (Mustafa et al., 2009; Paul and Snyder, 2012). The biological significance of the Cys¹⁵⁰ modification by H₂S is not well-studied, but H₂S could serve as a biological switch for protein function acting via oxidative modification of specific cysteine residues in response to redox homeostasis (Paul and Snyder, 2012). Understanding the physiological significance of protein sulfhydration requires the development of genome-wide innovative experimental approaches. Current methodologies based on the modified biotin switch technique do not allow detection of a broad spectrum of sulfhydrated proteins (Finkel, 2012). Guided by a previously reported strategy (Sen et al., 2012), we developed an experimental approach that allowed us to quantitatively evaluate the sulfhydrated proteome and the physiological consequences of H₂S synthesis during chronic ER stress. The new methodology allows a quantitative, close-up view of the integrated cellular response to environmental and intracellular cues and is pertinent to our understanding of human disease development.

The ER is an organelle involved in the synthesis of proteins followed by various modifications. Disruption of this process results in the accumulation of misfolded proteins, causing ER stress (Tabas and Ron, 2011; Walter and Ron, 2011), which is associated with development of many diseases ranging from metabolic dysfunction to neurodegeneration (Hetz, 2012). ER stress induces transcriptional, translational, and metabolic reprogramming, all of which are interconnected through the transcription factor ATF4. ATF4 increases expression of genes promoting adaptation to stress via their protein products. One such gene is the H₂S-producing enzyme, cystathionine gamma-lyase (CTH) or γ-cystathionase , previously shown to be involved in the signaling pathway that negatively regulates the activity of the protein tyrosine phosphatase 1B (PTP1B) via sulfhydration (Krishnan et al., 2011). We therefore hypothesized that low or even modest levels of reactive oxygen species (ROS) during ER stress may reprogram cellular metabolism via H₂S-mediated protein sulfhydration (Figure 1A).

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We have previously shown that the insulin-producing mouse pancreatic β cells MIN6, known for their high metabolic activity, are very susceptible to ER stress (Guan et al., 2014; Krokowski et al., 2013). We tested whether or not MIN6 cells expressed the essential components of H₂S synthesis and protein sulfhydration in response to ER stress induced by thapsigargin (Tg, Figure 1). Upon Tg treatment, MIN6 cells exhibited higher levels of intracellular ROS, decreased GSH/GSSG (reduced/oxidized glutathione ratios), and increased CTH protein levels via transcriptional activation (Figure 1B–D, Figure 1—figure supplement 1 and 2A). This transcriptional reprogramming was also seen in mouse and human islets subjected to physiological or pharmacologically induced ER stress (Figure 1D, Figure 1—figure supplement 2B, C and 3). In agreement with increased CTH expression, H₂S levels increased during the chronic phase of the stress response (Figure 1E). Coordinated induction of expression of the gene encoding the glutamate/cystine exchanger, Slc7a11, also was increased (Figure 1D), and this induction was associated with increased glutamate/cystine flux (Figure 1—figure supplement 4). Slc7a11 mediates the exchange of oxidized extracellular cystine with intracellular glutamate. Once cystine is imported into the cells it gets reduced to cysteine, and then serves as a substrate for GSH and H₂S synthesis. These data support the idea that both increased uptake of the CTH substrate and increased levels of CTH contribute to increased H₂S levels in cells under ER stress.

The functional significance of increased H₂S synthesis was shown by measuring the catalytic activity of GAPDH (Mustafa et al., 2009), which gradually increased in response to elevated H₂S production in MIN6 cells during ER stress (Figure 1F). This increase in activity was independent of GAPDH protein levels (Figure 1F). As noted above, GAPDH has the unusual feature of being catalytically inactive when Cys¹⁵⁰ is oxidatively modified, except when it undergoes sulfhydration which restores/increases its activity (Mustafa et al., 2009). We therefore tested the protective effects of Cys¹⁵⁰ sulfhydration by H₂S on its catalytic activity in the presence of H₂O₂-induced oxidation. Recombinant GAPDH was incubated with H₂O₂ in the presence or absence of the H₂S donor, NaHS. The inhibition of GAPDH activity by H₂O₂ was significantly reversed by H₂S treatment (Figure 1G). Furthermore, incubation of purified GAPDH with oxidized glutathione (GSSG) resulted in formation of inactive glutathionylated GAPDH (Gao et al., 2010), which was significantly rescued by treatment with H₂S as well as DTT reduction (Figure 1H). HPLC-MS confirmed that recombinant GAPDH exposed to NaHS or GSSG was modified predominantly at Cys¹⁵⁰ (Figure 1—figure supplement 5, Figure 2—figure supplement 7B, C). These data confirm that H₂S is a positive regulator of GAPDH activity. Increased GAPDH activity is directly linked to H₂S production, as shown by the loss of induction (Figure 1I) upon treatment with the CTH inhibitor, propargylglycine (PAG). PAG inhibits H₂S synthesis and therefore is expected to decrease Cys¹⁵⁰ modification. Taken together, these results indicate that regulation of H₂S synthesis during ER stress might regulate the catalytic activity of other metabolic pathway proteins. The latter is raising the possibility that the ATF4-mediated sulfhydration of proteins is part of the integrated stress response (ISR), and has regulatory effects on cellular metabolism.

ATF4 increases gene expression of CTH (Dickhout et al., 2012), the cystine transporter Slc7a11, as well as the ROS-producing enzyme Ero1α (Han et al., 2013; Tabas and Ron, 2011). We hypothesized that during ER stress, this network of ATF4 target genes promotes protein sulfhydration (Figure 2A). Knockdown of ATF4 in MIN6 cells during Tg-induced ER stress caused inhibition of H₂S synthesis with a parallel loss of induction of CTH protein levels (Figure 2B). In contrast, in the absence of stress, ATF4 overexpression increased CTH and Ero1α levels and H₂S synthesis, in agreement with increased GAPDH activity (Figure 2C–E, Figure 2—figure supplement 1). The levels of GSH and the activity of the glutamate/cystine exchanger also increased with ATF4 overexpression (Figure 2F, G). These data support the hypothesis that ATF4 is a master regulator of protein sulfhydration in pancreatic β cells during ER stress.

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Sulfhydrated proteins from MIN6 cells treated with Tg for 18 hr.

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To profile genome-wide changes in protein sulfhydration by ER stress and the integrated stress response, we exploited the different reactivity of cysteine persulfides (Cys-S-SH) and thiols (Cys-SH) to alkylating agents (Nishida et al., 2012; Pan and Carroll, 2013; Paul and Snyder, 2012), to develop a new thiol reactivity-based approach called BTA (Biotin Thiol Assay, (Figure 2H and Figure 2—figure supplement 2). We employed the following steps (Figure 2H): (1) labeling of reactive Cys-SH or Cys-S-SH groups by a biotin-conjugated maleimide (maleimide-PEG2-biotin, NM-biotin) (Weerapana et al., 2010), (2) binding of the biotin-labeled proteins on an avidin column, (3) elution of the retained proteins that contain a persulfide bridge using DTT, and (4) analysis of the eluted proteins.

The BTA approach was validated by testing the hypothesis that a lower concentration of the thiol-alkylating reagent (NM-biotin) will result in a selective labeling of highly reactive cysteine SH groups, and that this selectivity will be lost as the concentration of NM-biotin increases (Weerapana et al., 2010). We tested this hypothesis in extracts isolated from Tg-treated MIN6 cells in the presence of increasing concentrations of NM-biotin (0.05–1 mM). The eluate containing the sulfhydrated proteins was analyzed with SDS–PAGE and the proteins were visualized by Coomassie blue staining. Indeed, increasing the concentration of NM-biotin beyond 0.5 mM decreased the levels of eluted proteins (Figure 2—figure supplement 3), consistent with cysteine residues having free -SH groups becoming alkylated at high concentrations of NM-biotin. No protein was eluted from the column with the addition of DTT at high concentrations of NM-biotin (Figure 2—figure supplement 3), confirming that the biotin was attached to proteins via a disulfide bond (Figure 2—figure supplement 4A). This selectively labeling behavior not only relies on the probe concentration, but also is dependent on the protein structure conformations. We found that no protein was detected in the eluates if the BTA was performed on the denatured cell extracts (data not shown).

When Tg-treated MIN6 lysates were pretreated with DTT (to decrease the level of sulfhydrated proteins), the signal was significantly reduced compared to untreated lysates (Figure 2—figure supplement 4B), confirming that DTT reduces an intermolecular disulfide bond of cysteine persulfides labeled with biotin. These data show that sulfhydrated cysteine residues are the primary targets of NM-biotin at low concentrations, thus making the BTA a unique tool to identify the sulfhydrated proteome (Figure 2—figure supplement 2).

We next assessed the BTA assay using recombinant GAPDH, which contains six cysteine residues including the redox-regulated Cys¹⁵⁰. GAPDH was incubated with either H₂O₂ or NaHS. The presence of sulfhydrated GAPDH was evaluated by the BTA method following Western blot analysis. We found that only the NaHS-treated GAPDH was eluted as an H₂S-modified target, indicating that the assay distinguishes sulfhydration or free SH groups from other forms of cysteine modifications (Figure 2—figure supplement 5B). This was independently confirmed with the red malemide assay (Figure 2—figure supplement 6), which discriminates between free -SH groups and sulfhydrated-SH (Sen et al., 2012). We next tested if the BTA can identify modification of a specific cysteine on GAPDH by using recombinant wild-type and Cys¹⁵⁰Ser GAPDH mutant, which were incubated with H₂O₂ or NaHS. We found that NaHS treatment induced sulfhydration in the wild-type GAPDH (Figure 2—figure supplement 7A), a result that was also confirmed by high-resolution quadruple MS analysis (Figure 2—figure supplement 7B–C), and that this modification was absent in the Cys¹⁵⁰Ser mutant. Finally, a proteome-wide view of sulfhydration was obtained from Tg-treated MIN6 cell extracts subjected to BTA and further analyzed by LC-MS/MS (Figure 2—figure supplement 8). We identified 150 proteins, including several known targets for sulfhydration (Figure 2—figure supplement 8D) (Mustafa et al., 2009). Similar results were obtained from analysis of mouse liver (data not shown), a tissue known to exhibit high levels of H₂S synthesis (Kabil et al., 2011). Taken together, this shows that BTA discriminates between protein sulfhydration and other oxidative modifications.

Next, the BTA methodology identified the sulfhydrated proteome downstream of the transcription factor ATF4 during ER stress (Figure 2A). MIN6 cells treated with Tg increased GAPDH levels in the DTT eluate, that was abolished by knocking down ATF4 (Figure 2I). In the absence of stress, ATF4 knockdown resulted in an increase in sulfhydrated GAPDH in MIN6 cells (Figure 2I). Because ATF4-deficient cells have decreased levels of sulfur-containing amino acids (Harding et al., 2003), and sulfur amino acid restriction is linked to an increase in the transulfuration pathway (Hine et al., 2015), it is possible that the increase in GAPDH sulfhydration in ATF4-depleted cells is due to activation of CBS (cystathionine-β-synthase) (Niu et al., 2015), the second cytosolic H₂S-producing enzyme. Moreover, we found that in the absence of stress ATF4 overexpression induced GAPDH sulfhydration that is dependent on the CTH activity (Figure 2J–K). The use of PAG decreased GAPDH sulfhydration (Figure 2K). We conclude that sulfhydration of proteins during ER stress is part of the ISR and is controlled by the transcription factor, ATF4.

The BTA requires determining the concentration of NM-Biotin for selectively labeling proteins with reactive, sulfhydrated cysteines rather than the relatively high abundant and less reactive, unmodified (with free SH groups) cysteine residues. However, this labeling step has some limitations under certain circumstances. For example, if free Cys-SH groups are biotinylated on the same protein containing one or more Cys-S-SH groups (Figure 2H and Figure 2—figure supplement 2), then the protein will not be eluted with DTT and will not be identified as a target for sulfhydration. In addition, if a protein contains sulfhydrated cysteines with low reactivity for the probe, this protein will not be captured and identified as an H₂S-modified target. Due to those limitations and in order to extend the capability of the BTA approach, we introduced a proteolytic digestion step before applying the avidin column step. This added step provided not only the isolation of cysteine-containing peptides with persulfide bonds, but also increased the identification of sulfhydrated proteins. The eluted peptides were then sequenced and identified by LC-MS/MS analysis, thereby identifying the modified cysteines on proteins. By using the modified BTA technique, we have identified over ~ 1000 novel sulfhydrated cysteines in MIN6 cells treated with Tg, corresponding to about 820 proteins (Figure 2—figure supplement 9 and Figure 2—source data 1), including GAPDH, wherein two cysteine-containing peptides were captured: Cys¹⁵⁰ and Cys²⁴⁵. Remarkably, the Cys¹⁵⁰ active-site peptide was highly enriched as compared to the C-terminal of Cys²⁴⁵, supporting prior mutagenesis studies that have shown Cys¹⁵⁰ as the primary H₂S-modified site on GAPDH, both in vitro and in vivo. One of novel targets including phosphoglycerate dehydrogenase (PHGDH) was also confirmed by Western blot analysis (Figure 2—figure supplement 10). CTH Knockdown mediated by shRNA decreased GAPDH and PHGDH sulfhydration levels, confirming that those proteins identified by the BTA method are bona-fide targets for sulfhydration in vivo (Figure 2—figure supplement 11). Sulfhydrated peptides do not reveal a consensus protein abundance and sulfhydrome (Figure 3—figure supplement 6,7and Figure 3—source data 2), supporting that lower concentrations of the NM-biotin labeling reveal reactivity of cysteine residues rather than protein abundance.

In order to obtain genome-wide stress-induced changes in the sulfhydrome and individual cysteine residues within these proteins, we devised a modified BTA protocol (Figure 3A) by introducing a stable isotope-labeling step after the DTT elution step. This protocol uses (1) digestion of biotinylated cell extracts with trypsin before avidin capture, (2) labeling of free Cys-SH groups on peptides in the column eluent by mass-difference cysteine-alkylating reagents with either NEM-H₅ (Light) or NEM-D₅ (Heavy), and (3) quantification by LC-MS/MS analysis of H/L ratios of the individual pair-labeled cysteines in the identified peptides based on a mass-difference labeling. In addition to quantifying changes in protein sulfhydration, this modified BTA approach allows the detection of additional proteins (Figure 2—figure supplement 11 and Figure 2—source data 1). We used as an experimental system the ATF4-expressing MIN6 cells to profile quantitatively the sulfhydrated proteome. As shown in Figure 2A, ATF4-mediated signaling triggers the cellular response, which leads to increased protein sulfhydration. Using the experimental plan in Figure 3A, we identified over 834 cysteine-containing peptides (Figure 3B and Figure 3—source data 1). Of these peptides, 771 exhibited pair-labeling with an overall average H/L ratio 2.6, and 348 peptides (45%) displayed high ratios (H/L>2). These findings confirmed that ATF4 drives global changes in protein sulfhydration in MIN6 cells.

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Sulfhydrated proteins from ATF4 overexpressed MIN6 cells.

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Relative abundance of proteins in MIN6 cells treated with Tg for 18 hr.

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Sulfhydrated cysteine residues in proteins may contribute to their biological activities, especially when these modified cysteine residues reside within functional domains. We thus queried the Uniprot database to retrieve functional annotations for the aforementioned 827 peptides. The analysis revealed that 28% of the peptides were localized to protein regions whose structural and functional properties are known (Figure 3C, Figure 3—figure supplement 1). An additional 18% were found within functional regions of proteins with cysteine residues of unknown significance. Our finding of sulfhydration of specific cysteine residues within functional domains of proteins suggests that the cysteine modification influences the activity of proteins. In contrast, 4.2% of the peptides in question contained cysteine residues in experimentally proven active sites of enzymes or cysteine residues involved in disulfide bond formation (Figure 3C). This percentage of active cysteine-containing peptides from the BTA assay is significantly larger than the 0.2% of all cysteines in the Uniprot database that have been assigned to experimentally characterized active cysteines (Weerapana et al., 2010). Finally, 2.4% of the annotated peptides were found in known nucleotide binding domains of proteins, suggesting the potential for regulation of gene expression by H₂S cysteine-modified proteins. None of these proteins have been reported as targets of sulfhydration. Although not found among the 827 peptides in this study, the NF-κB RelA transcription factor has been shown to be sulfhydrated in the DNA-binding domain, resulting in increased DNA-binding activity (Sen et al., 2012). We also found by querying the redox modification databases (RedoxDB and GPS-SNO) that 11% of the 834 peptides from the RedoxDB and 36% from the GPS-SNO corresponded to cysteine residues that are known to be modified by nitrosylation or glutathionylation (Figure 3B, Figure 3—figure supplement 2). Taken together, these data indicate that protein sulfhydration not only influences the catalytic activity of enzymes like GAPDH, but also can potentially regulate a broad range of biological processes.

We noted a wide range of H/L ratios in the identified peptides, reflecting a large difference in the reactivity of individual cysteines to sulfhydration in MIN6 cells that overexpress ATF4 (Figure 3B–D). In addition, labeled cysteines on the same protein exhibited remarkably different ratios (Figure 3—figure supplement 3). For example, the protein, PHGDH, was labeled on two cysteines, Cys²⁸¹ had an H/L ratio of 5.8, whereas Cys²⁵⁴ displayed a ratio of 17. To identify a sulfhydration motif in proteins, all cysteine-containing peptides (Figure 3B) were analysed by the pLogo motif analyzer program. This analysis did not reveal conserved residues surrounding the modified cysteine site (Figure 3—figure supplement 4A), a result consistent with other enzyme-independent oxidative modifications of cysteines (Weerapana et al., 2010) (Marino and Gladyshev, 2011). However, when we selected only the peptides with high H/L ratios (>2), and searched for a sequence motif at the cysteine modification sites (Figure 3—figure supplement 4B), an Arg residue was significantly enriched at the +1 position of the modified cysteine (Figure 3—figure supplement 4B). Additionally, structure motif and surface accessibility analysis revealed that the modified cysteine is highly accessible and positioned at the N-terminal alpha-helices (Figure 3—figure supplement 4C–D). This is consistent with previous reports suggesting that a reactive cysteine thiolate anion is stabilized by interaction with alpha-helix dipoles (Kortemme and Creighton, 1995; Weerapana et al., 2010).

Bioinformatics clustering with two pathway annotation programs DAVID and Ingenuity Pathway Analysis (IPA) revealed an enrichment of sulfhydrated proteins in glycolysis and mitochondrial oxidative metabolism (Figure 3E, Figure 3—figure supplement 5). Data from this analysis are in agreement with increased activity of the glycolytic enzyme, GAPDH, by sulfhydration, and prompted the question as to whether or not glycolytic flux is regulated by the H₂S-dependent modification of enzymes involved in intermediary metabolism. Also in agreement with this hypothesis, we found that ATF4-overexpressing MIN6 cells had higher glycolytic rates as evaluated with a Seahorse analyzer (Figure 4—figure supplement 1). Furthermore, the activity of the rate-limiting glycolytic enzyme, pyruvate kinase 2 (PKM2), also was increased in ATF4 overexpressing MIN6 cells, in a manner dependent on CTH activity (Figure 4—figure supplement 2). We therefore returned to the induction of ER stress by Tg-treatment of MIN6 cells and evaluated glycolytic flux rates in the absence or presence of PAG. The advantage of using PAG instead of genetic manipulation such as gene knockdown is that the inhibitor could be added at the same time as labeled glucose, thus allowing assessment of the inhibitor’s effect on glycolytic flux rates.

We directly measured changes in metabolic flux by utilizing stable isotope label incorporation and mass isotopomer analyses. MIN6 cells were treated with Tg for 18 hr; the growth media was changed to (D-glucose-¹³C₆) media in the presence or absence of PAG during the last 3.5 hr of treatment. Tg-treatment significantly augmented glycolytic flux as determined by the increase in the glycolytic intermediate, 3 phosphoglyceric acid (3PG), as well as lactate and alanine (Figure 4A–C, Supplementary file 1). The flux was consistent with an increase in the relative concentrations of 3PG and alanine (Figure 4B, Supplementary file 1). However, lactate levels were decreased significantly despite the increased flux. A decrease in cellular lactate levels supports the idea that there is high consumption of pyruvate by the mitochondria to generate oxaloacetate (OAA). In contrast to the increased glycolytic rates, flux to tricarboxylic acid cycle (TCA) intermediates was significantly reduced by Tg-treatment as evidenced by the low ¹³C label incorporation of both acetyl-CoA and OAA moieties of citrate, fumarate, and malate (Figure 4A–B, Supplementary file 1). When the cells were exposed to PAG along with Tg-treatment, the increase in glycolytic flux was prevented, as shown by the decrease in ¹³C-labeling of 3PG, lactate, and alanine. In contrast, TCA cycle flux was restored, as exemplified by the increase in ¹³C-labeling of both OAA and acetyl-CoA moieties of citrate, succinate, fumarate, and malate, and a decrease in their concentrations, suggesting utilization. ¹³C-labeling of aspartate and glutamine also increased significantly, indicating increased cataplerosis of TCA cycle intermediates. Moreover, we determined the activity of PKM2 in MIN6 cells treated with Tg in the presence or absence of PAG. Tg-treatment increased PKM2 activity, but PAG addition inhibited the increase, without affecting PKM2 protein levels (Figure 4—figure supplement 3). These data suggest that ER stress, via H₂S-mediated signaling, promotes glycolysis and decreases mitochondrial oxidative metabolism.

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In summary, sulfhydration of specific cysteines in proteins is a key function of H₂S (Kabil and Banerjee, 2010; Paul and Snyder, 2012; Szabo et al., 2013). Thus, the development of tools that can quantitatively measure genome-wide protein sulfhydration in physiological or pathological conditions is of central importance. However, a significant challenge in studies of the biological significance of protein sulfhydration is the lack of an approach to selectively detect sulfhydrated cysteines from other modifications (disulfide bonds, glutathionylated thiols and sulfienic acids) in complex biological samples. In this study, we introduced the BTA approach that allowed the quantitative assessment of changes in the sulfhydration of specific cysteines in the proteome and in individual proteins. BTA is superior to other reported methodologies that aimed to profile cysteine modifications, such as the most commonly used, a modified biotin switch technique (BST). BST was originally designed to study protein nitrosylation and postulated to differentiate free thiols and persulfides (Mustafa et al., 2009). A key advantage of BTA over the existing methodologies is that the experimental approach has steps to avoid false-positive and negative results, as target proteins for sulfhydration. BST is commonly generating such false targets for cysteine modifications (Forrester et al., 2009; Sen et al., 2012).

Using mutiple validations, our data support the specificity and reliability of the BTA assay for analysis of protein sulfhydration both in vitro and in vivo. With this approach, we found that ATF4 is the master regulator of protein sulfhydration in pancreatic β cells during ER stress, by means of its function as a transcription factor. A large number of protein targets have been discovered to undergo sulfhydration in β cells by the BTA approach. Almost 1000 sulfhydrated cysteine-containing peptides were present in the cells under the chronic ER stress condition of treatment with Tg for 18 hr. Combined with the isotopic-labeling strategy, almost 820 peptides on more than 500 proteins were quantified in the cells overexpressing ATF4. These data show the potential of the BTA method for further systematic studies of biological events. To our knowledge, the current dataset encompasses most known sulfhydrated cysteine residues in proteins in any organism. Our bioinformatics analyses revealed sulfhydrated cysteine residues located on a variety of structure–function domains, suggesting the possibility of regulatory mechanism(s) mediated by protein sulfhydration. Structure and sequence analysis revealed consensus motifs that favor sulfhydration; an arginine residue and alpha-helix dipoles are both contributing to stabilize sulfhydrated cysteine thiolates in the local environment.

Pathway analyses showed that H₂S-mediated sulfhydration of cysteine residues is the part of the ISR with the highest enrichment in proteins involved in energy metabolism. The metabolic flux revealed that H₂S promotes aerobic glycolysis associated with decreased oxidative phosphorylation in the mitochondria during ER stress in β cells. The TCA cycle revolves by the action of the respiratory chain that requires oxygen to operate. In response to ER stress, mitochondrial function and cellular respiration are down-regulated to limit oxygen demand and to sustain mitochondria. When ATP production from the TCA cycle becomes limited and glycolytic flux increases, there is a risk of accumulation of lactate from pyruvate. One way to escape accumulation of lactate is the mitochondrial conversion of pyruvate to oxalacetic acid (OAA) by pyruvate carboxylase. This latter enzyme was found to be sulfhydrated, consistent with the notion that sulfhydration is linked to metabolic reprogramming toward glycolysis.

The switch of energy production from mitochondria to glycolysis is known as a signature of hypoxic conditions. This metabolic switch has also been observed in many cancer cells characterized as the Warburg effect, which contributes to tumor growth. The Warburg effect provides advantages to cancer cell survival via the rapid ATP production through glycolysis, as well as the increasd conversion of glucose into anabolic biomolecules (amino acid, nucleic acid, and lipid biosynthesis) and reducing power (NADPH) for regeneration of antioxidants. This metabolic response of tumor cells contributes to tumor growth and metastasis (Vander Heiden et al., 2009). By analogy, the aerobic glycolysis trigged by increased H₂S production could give β cells the capability to acquire ATP and nutrients to adapt their cellular metabolism toward maintaining ATP levels in the ER (Vishnu et al., 2014), increasing synthesis of glycerolphospholipids, glycoproteins and protein (Krokowski et al., 2013), all important components of the ISR. Similar to hypoxic conditions, a phenotype associated with most tumors, the decreased mitochondria function in β cells during ER stress, can also be viewed as an adaptive response by limiting mitochondria ROS and mitochondria-mediated apoptosis. We therefore view that the H₂S-mediated increase in glycolysis is an adaptive mechanism for survival of β cells to chronic ER stress, along with the improved ER function and insulin production and folding, both critical factors controlling hyperglycemia in diabetes. Future work should determine which are the key proteins targeted by H₂S and thus contributing to metabolic reprogramming of β cells, and if and how insulin synthesis and secretion is affected by sulfhydration of these proteins during ER stress.

Abnormal H₂S metabolism has been reported to occur in various diseases, mostly through the deregulation of gene expression encoding for H₂S-generating enzymes (Wallace and Wang, 2015). An increase of their levels by stimulants is expected to have similar effects on sulfhydration of proteins like the ATF4-induced CTH under conditions of ER stress. It is the levels of H₂S under oxidative conditions that influence cellular functions. In the present study, ER stress in β cells induced elevated CTH levels, whereas CBS was unaffected. The deregulated oxidative modification at cysteine residues by H₂S may be a major contributing factor to disease development. In this case, it would provide a rationale for the design of therapeutic agents that would modulate the activity of the involved enzymes.

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Author details

1. Xing-Huang Gao

   Department of Genetics and Genome Sciences, Case Western Reserve University, Cleveland, United States

   Contribution

   X-HG, Conceived the study; Designed and performed the experiments; Prepared the manuscript and analysed and interpreted the data

   Competing interests

   The authors declare that no competing interests exist.

2. Dawid Krokowski

   Department of Genetics and Genome Sciences, Case Western Reserve University, Cleveland, United States

   Contribution

   DK, Contibuted expertise on the stress response and amino acid uptake studies; Additionally, he has actively participated in the experimental design and data interpretation during the entire study

   Competing interests

   The authors declare that no competing interests exist.

3. Bo-Jhih Guan

   Department of Genetics and Genome Sciences, Case Western Reserve University, Cleveland, United States

   Contribution

   B-JG, Contributed RNA analysis and manuscript preparation

   Contributed equally with

   Ilya Bederman

   Competing interests

   The authors declare that no competing interests exist.

4. Ilya Bederman

   Department of Pediatrics, Case Western Reserve University, Cleveland, United States

   Contribution

   IB, Performed and analysed the metabolic flux studies

   Contributed equally with

   Bo-Jhih Guan

   Competing interests

   The authors declare that no competing interests exist.

5. Mithu Majumder

   Department of Pharmacology, Case Western Reserve University, Cleveland, United States

   Contribution

   MM, Contributed RNA analysis

   Competing interests

   The authors declare that no competing interests exist.

6. Marc Parisien

   Alan Edwards Centre for Research on Pain, McGill University, Montreal, Canada

   Contribution

   MP, Performed bioinformatics tasks and assisted in data interpretation

   Competing interests

   The authors declare that no competing interests exist.

7. Luda Diatchenko

   Alan Edwards Centre for Research on Pain, McGill University, Montreal, Canada

   Contribution

   LD, Performed bioinformatics tasks and assisted in data interpretation

   Competing interests

   The authors declare that no competing interests exist.

8. Omer Kabil

   Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, United States

   Contribution

   OK, Performed the H2S measurements

   Competing interests

   The authors declare that no competing interests exist.

9. Belinda Willard

   Mass Spectrometry Laboratory for Protein Sequencing, Lerner Research Institute, Cleveland Clinic, Cleveland, United States

   Contribution

   BWi, Performed the proteomics analysis with LC-MS/MS

   Competing interests

   The authors declare that no competing interests exist.

10. Ruma Banerjee

    Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, United States

    Contribution

    RB, Advised on H2S signaling and the chemistry of modifications of cysteine residues; Manuscript editing

    Competing interests

    The authors declare that no competing interests exist.

11. Benlian Wang

    1. Center for Proteomics and Bioinformatics, School of Medicine, Case Western Reserve University, Cleveland, United States
    2. Center for Synchotron Biosciences, School of Medicine, Case Western Reserve University, Cleveland, United States

    Contribution

    BWa, Participated in the proteomics analysis of GAPDH and initial studies of the identification of the sulfhydrated proteome

    Competing interests

    The authors declare that no competing interests exist.

12. Gurkan Bebek

    1. Center for Proteomics and Bioinformatics, School of Medicine, Case Western Reserve University, Cleveland, United States
    2. Center for Synchotron Biosciences, School of Medicine, Case Western Reserve University, Cleveland, United States

    Contribution

    GB, Performed the IPA analysis

    Competing interests

    The authors declare that no competing interests exist.

13. Charles R. Evans

    Department of Internal Medicine, Division of Metabolism, Endocrinology & Diabetes, University of Michigan Medical School, Ann Arbor, United States

    Contribution

    CRE, Analyzed the Metabolomics data

    Competing interests

    The authors declare that no competing interests exist.

14. Paul L. Fox

    Department of Cellular and Molecular Medicine, Lerner Research Institute, Cleveland Clinic, Cleveland, United States

    Contribution

    PLF, Advised on cysteine modifications by nitrosylation

    Competing interests

    The authors declare that no competing interests exist.

15. Stanton L. Gerson

    Department of Medicine, Division of Hematology/Oncology, School of Medicine, Case Western Reserve University, Cleveland, United States

    Contribution

    SLG, Assisted with the Seahorse analyzer

    Competing interests

    The authors declare that no competing interests exist.

16. Charles L. Hoppel

    Department of Pharmacology, Case Western Reserve University, Cleveland, United States

    Contribution

    CLH, Provided expertise on cellular metabolism; Mitochondrial function and metabolic data analysis

    Competing interests

    The authors declare that no competing interests exist.

17. Ming Liu

    Department of Internal Medicine, Division of Metabolism, Endocrinology & Diabetes, University of Michigan Medical School, Ann Arbor, United States

    Contribution

    ML, Provided expertise on β cell biology and data analysis concerning β cell function.

    Competing interests

    The authors declare that no competing interests exist.

18. Peter Arvan

    Department of Internal Medicine, Division of Metabolism, Endocrinology & Diabetes, University of Michigan Medical School, Ann Arbor, United States

    Contribution

    PA, Provided expertise on β cell biology and data analysis concerning β cell function

    Competing interests

    The authors declare that no competing interests exist.

19. Maria Hatzoglou

    Department of Genetics and Genome Sciences, Case Western Reserve University, Cleveland, United States

    Contribution

    MH, Conceived the study; Designed the experiments; Prepared the manuscript and analysed and interpreted the data

    For correspondence

    mxh8@case.edu

    Competing interests

    The authors declare that no competing interests exist.

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