Opposing effects of polysulfides and thioredoxin on apoptosis through caspase persulfidation

Hydrogen sulfide has been implicated in a large number of physiological processes including cell survival and death, encouraging research into its mechanisms of action and therapeutic potential. Results from recent studies suggest that the cellular effects of hydrogen sulfide are mediated in part by sulfane sulfur species, including persulfides and polysulfides. In the present study, we investigated the apoptosis-modulating effects of polysulfides, especially on the caspase cascade, which mediates the intrinsic apoptotic pathway. Biochemical analyses revealed that organic or synthetic polysulfides strongly and rapidly inhibit the enzymatic activity of caspase-3, a major effector protease in apoptosis. We attributed the caspase-3 inhibition to persulfidation of its catalytic cysteine. In apoptotically stimulated HeLa cells, short-term exposure to polysulfides triggered the persulfidation and deactivation of cleaved caspase-3. These effects were antagonized by the thioredoxin/thioredoxin reductase system (Trx/TrxR). Trx/TrxR restored the activity of polysulfide-inactivated caspase-3 in vitro, and TrxR inhibition potentiated polysulfide-mediated suppression of caspase-3 activity in situ. We further found that under conditions of low TrxR activity, early cell exposure to polysulfides leads to enhanced persulfidation of initiator caspase-9 and decreases apoptosis. Notably, we show that the proenzymes procaspase-3 and -9 are basally persulfidated in resting (unstimulated) cells and become depersulfidated during their processing and activation. Inhibition of TrxR attenuated the depersulfidation and activation of caspase-9. Taken together, our results reveal that polysulfides target the caspase-9/3 cascade and thereby suppress cancer cell apoptosis, and highlight the role of Trx/TrxR-mediated depersulfidation in enabling caspase activation.

Hydrogen sulfide has been implicated in a large number of physiological processes including cell survival and death, encouraging research into its mechanisms of action and therapeutic potential. Results from recent studies suggest that the cellular effects of hydrogen sulfide are mediated in part by sulfane sulfur species, including persulfides and polysulfides. In the present study, we investigated the apoptosis-modulating effects of polysulfides, especially on the caspase cascade, which mediates the intrinsic apoptotic pathway. Biochemical analyses revealed that organic or synthetic polysulfides strongly and rapidly inhibit the enzymatic activity of caspase-3, a major effector protease in apoptosis. We attributed the caspase-3 inhibition to persulfidation of its catalytic cysteine. In apoptotically stimulated HeLa cells, short-term exposure to polysulfides triggered the persulfidation and deactivation of cleaved caspase-3. These effects were antagonized by the thioredoxin/thioredoxin reductase system (Trx/TrxR). Trx/TrxR restored the activity of polysulfide-inactivated caspase-3 in vitro, and TrxR inhibition potentiated polysulfide-mediated suppression of caspase-3 activity in situ. We further found that under conditions of low TrxR activity, early cell exposure to polysulfides leads to enhancedpersulfidationofinitiatorcaspase-9anddecreasesapoptosis. Notably, we show that the proenzymes procaspase-3 and -9 are basally persulfidated in resting (unstimulated) cells and become depersulfidated during their processing and activation. Inhibition of TrxR attenuated the depersulfidation and activation of caspase-9. Taken together, our results reveal that polysulfides target the caspase-9/3 cascade and thereby suppress cancer cell apoptosis, and highlight the role of Trx/TrxR-mediated depersulfidation in enabling caspase activation.
Research conducted over the last two decades has established that hydrogen sulfide (H 2 S) acts as a signaling molecule that regulates various physiological and pathological responses (1)(2)(3)(4). Endogenous H 2 S is synthesized by cystathionine ␥-lyase and cystathionine ␤-synthase in the transsulfuration pathway and by 3-mercaptopyruvate sulfurtransferase involved in cysteine catabolism. H 2 S can be converted via enzymatic or nonenzymatic processes to different reactive sulfur species (RSS), 3 including sulfane sulfur species such as persulfides and polysulfides. These sulfur chemotypes exhibit rich chemistry and recent findings suggest their involvement in H 2 S-mediated responses (1)(2)(3)(4). In this context, H 2 S/RSS were found to either promote or inhibit cell death in a variety of cell types including cancer cells (reviewed in Ref. 5). The mechanisms responsible for these varied effects of H 2 S/RSS on cancer cell survival and death remain poorly characterized but likely depend on the chemical properties of the particular sulfur species generated in situ, their concentration, and the cellular context.
Apoptosis, the best-characterized form of regulated cell death, is a highly ordered process by which unwanted, defective, or damaged cells are swiftly and selectively eliminated from the body. Various stress stimuli activate the intrinsic apoptosis pathway leading to mitochondrial outer-membrane permeabilization, release of cytochrome c (Cyt c), and formation of the apoptosome. This then stimulates the oligomerization and autocatalytic activation of procaspase-9, which in turn cleaves and activates procaspase-3. Active caspase-3 then cleaves multiple substrates to execute apoptosis (6,7). There is considerable interest in understanding how H 2 S/RSS regulate or affect apoptosis of cancer cells, which appears to be complex and context-dependent. Indeed, multiple studies have shown that H 2 S/RSS can either promote or inhibit cancer cell apoptosis (5, 8 -13). These findings highlight the need for a better mechanistic understanding of the proapoptotic and antiapoptotic effects of H 2 S/RSS.
Emerging research indicates that cellular effects of H 2 S/RSS are mediated in part via protein persulfidation (Cys-SSH), a posttranslational modification of cysteine thiols (Cys-SH) (14,15). Protein persulfidation can lead to either gain or loss of function and modulates the activity of signaling proteins, transcription factors, and metabolic enzymes (14,15). RSS-mediated persulfidation of the transcription factor NF-B was shown to exert an antiapoptotic effect in nontumor cells (16).
However, how persulfidation may regulate the cellular apoptotic machinery remains poorly characterized. In addition, although recent studies have established that the thioredoxin/ thioredoxin reductase (Trx/TrxR) system catalyzes persulfide reduction in cells (17,18), whether and how RSS and the Trx system interact in the context of apoptosis regulation has not been investigated.
At present, it is not fully understood how H 2 S triggers Cys persulfidation in cells. H 2 S cannot directly react with Cys thiols because the sulfur atoms in both species have the same oxidation state (Ϫ2). Accordingly, de novo protein persulfidation is thought to occur via one of the following mechanisms: (i) the reaction of oxidized Cys derivatives with sulfide or (ii) sulfide oxidation products reacting with Cys thiols (19). Regarding the latter, polysulfides (R-S-S n -S-RЈ; nՆ1) are oxidation products of H 2 S and potential intermediates in persulfidation reactions. Polysulfides are abundant in some types of food such as garlic (20), can be detected in mammalian cells (21,22), and have recently been implicated in cellular regulation (23)(24)(25)(26).
In the present study we found that polysulfides inhibited apoptosis signaling in cancer cells through a mechanism that involves inhibitory persulfidation of apoptotic caspases, caspase-3 and -9. In addition, we provide evidence that procaspase-3 and -9 are constitutively persulfidated in resting cells and undergo depersulfidation during their activation. Caspase persulfidation is antagonized by the Trx system. Overall, our findings suggest that the balance between persulfidation and Trx-mediated depersulfidation can determine caspase activities and resultant apoptotic cell death.

Effects of polysulfides on caspase-3 activity in vitro
We initiated this study by assessing the effects of several polysulfide compounds as well as a persulfide donor on the activity of purified caspase-3. For this, recombinant human caspase-3 was first subjected to DTT reduction and then the reduced protein (1 M) was incubated for 20 min at 37°C with SNAP-P* (S-methoxycarbonyl penicillamine disulfide, a persulfide donor) (27); the organic polysulfide diallyl trisulfide (DATS); or the synthetic polysulfides Na 2 S 2 , Na 2 S 3 , or Na 2 S 4 . Thereafter, caspase-3 activity was measured using a fluorogenic assay with the synthetic substrate Ac-DEVD-AMC. As shown (Fig. 1A), caspase-3 activity was effectively inhibited by either SNAP-P* or the polysulfide donors, most potently by Na 2 S 3 and Na 2 S 4 (Ն50% inhibition at 2 M of either compound). Time course experiments performed at room temperature using 0.1 M caspase-3 demonstrated rapid inactivation of the enzyme upon incubation with 0.5 or 1 M Na 2 S 4 (Fig. 1B). To ensure that residual polysulfide did not interfere with the caspase activity assay, we employed another assay in which caspase-3 was immobilized onto nickel-coated microplates, enabling the facile removal of excess reactants (see "Experimental procedures" for more details). This assay also showed that Na 2 S 4 potently and dose-dependently inhibited caspase-3 activity (Fig. 1C).

Mass spectrometric analysis of caspase-3 persulfidation
In view of these results, we next performed mass spectrometric (MS) analysis to identify cysteine residues in caspase-3 that could be persulfidated. For this purpose, untreated, Na 2 S 4treated, or SNAP-P*-treated caspase-3 were derivatized by iodoacetamide and subjected to tryptic digestion, and the Caspase activity was determined using fluorogenic substrate Ac-DEVD-AMC. B, caspase-3 (0.1 M) was incubated at room temperature with Na 2 S 4 for the indicated times. Activity was determined as in A. C, reduced caspase-3 was immobilized on nickel-coated plates and then incubated for 30 min at 37°C with the indicated concentration of Na 2 S 4 . After removal of excess sulfide, enzyme activity was determined as in A. Data in the graphs represent mean Ϯ S.D. (n Ն 3). *, p Ͻ 0.05; **, p Ͻ 0.01 versus vehicle control.

Regulation of apoptotic caspases by thiol persulfidation
resulting peptides were analyzed by LC-MS/MS (see "Experimental procedures"). Iodoacetamide derivatization was performed to (i) quench thiol/persulfide reactions that may occur during sample processing and to (ii) generate carbamidomethylated persulfide derivatives (R-S-S-CH 2 -CO-NH 2 ) to help differentiate persulfides from sulfinic acid (both causing very similar mass changes). The MS analysis resulted in the identification of persulfidated cysteines in the polysulfide-and persulfide-treated, but not in the untreated, caspase-3 (Table S1, A and B). In both the Na 2 S 4 and SNAP-P* samples, the catalytic cysteine (Cys-163) was found to be the predominant site of persulfidation (Fig. 2, A and B; Table S1, A and B). Other, lowabundant modifications, were Cys-170 persulfide, Cys-264 persulfide, and Cys-163-Cys-170 disulfide (Tables S1, A and B).

Effects of polysulfides on cellular caspase-3 via persulfidation
We proceeded to examine possible effects of polysulfides on cellular caspase-3. For this purpose, we exposed HeLa cancer cells to the apoptosis inducer staurosporine (STS) in the presence or absence of Na 2 S 4 . We used an experimental design in which the cells were exposed to STS for 4 h with the polysulfide added for the final 1 h. Thereafter, the cells were lysed and caspase-3-like activity in cell lysates was measured. These experiments revealed that Na 2 S 4 dose-dependently inhibited STS-mediated induction of caspase-3 activity (Fig. 3A). Similar to Na 2 S 4 , DATS also effectively inhibited STS-dependent caspase activation (Fig. 3A). During its activation, caspase-3 undergoes maturation from its unprocessed, proform, to its processed, cleaved form. It was possible that the polysulfides inhibited the processing of procaspase-3 or disrupted another upstream event. However, immunoblot analysis showed that Na 2 S 4 or DATS did not block STS-induced cleavage of procaspase-3 ( Fig. 3B), suggesting that the polysulfides directly inhibited the activity of the processed enzyme. Considering these observations, we sought to examine if cellular caspase-3 was persulfidated under these experimental conditions. For this purpose, we employed the biotin thiol assay (BTA) for protein persulfidation (28). BTA results indicated that cellular caspase-3 was indeed persulfidated (Fig. 3B). Notably, procaspase-3 was found to be basally (i.e. constitutively) persulfidated in resting HeLa cells (Fig. 3B). Conversely, cleaved caspase-3 (after STS stimulation) was barely persulfidated; however, its persulfidation was markedly increased in cells exposed to Na 2 S 4 or DATS (Fig. 3B). Together, these cellular data suggest that polysulfides trigger the persulfidation of cleaved caspase-3, causing its deactivation. In addition, these results raise the intriguing possibility that endogenous persulfidation of procaspase-3 is involved in the regulation of this protease.
We then examined the effects of Na 2 S 4 on other cell types or apoptotic stimuli. Similar to our findings in HeLa cells, we observed that Na 2 S 4 inhibited caspase-3 activity in STS-treated THP-1 monocytic cells (Fig. 3C). Whereas STS activates the intrinsic apoptotic pathway, members of the TNF family of cytokines such as TNF and TRAIL trigger the extrinsic apoptotic pathway (7). We found that Na 2 S 4 effectively suppressed TRAIL-induced caspase-3 activity in HCT116 colon cancer cells (Fig. 3D). Thus, polysulfides can counteract caspase-3 acti-vation in different cell types, and in response to different apoptotic insults.

Co-regulation caspase-3 by persulfidation and thioredoxin
The extent of protein persulfidation reflects the balance between persulfidation and depersulfidation processes. It is known that the Trx/TrxR system mediates caspase-3 denitrosylation (29) and growing evidence suggests that Trx/TrxR acts as a persulfide reductase (17,18). Employing our in vitro assay with immobilized caspase-3 (as described above in Fig. 1C and depicted in Fig. 4A) we tested if Trx/TrxR could restore the activity of Na 2 S 4 -treated caspase-3. The results showed that a 30 min incubation with Trx/TrxR and NADPH resulted in near-complete recovery of caspase-3 activity, an effect almost equal to that achieved by the strong chemical reductant DTT and slightly better compared with the biologically relevant reductant GSH (Fig. 4B).
Next, to examine the role of the Trx system in regulating persulfidation in cells we employed auranofin (AUR), a highly potent and selective TrxR inhibitor (30). We found that AUR markedly potentiated the inhibitory effects elicited by Na 2 S 4 . Indeed, whereas treatment with 25 M Na 2 S 4 alone failed to inhibit caspase-3, in the presence of AUR ϳ75% inhibition was observed (Fig. 4C). Immunoblot analysis showed that cleavage of procaspase-3 was not inhibited by Na 2 S 4 in the presence of AUR (Fig. 4C), further arguing for a direct effect on the cleaved enzyme. We extended these findings by using Tri-1, a recently described potent and specific inhibitor of TrxR1 (31). We found that Tri-1, which has a different chemical structure and a different mechanism of action compared with AUR (31), also augmented Na 2 S 4 -mediated inhibition of caspase-3 ( Fig. S1), thus corroborating the role of TrxR1 in these effects. We then monitored the cleavage of a well-established caspase-3 substrate, inhibitor of caspase-activated DNase (ICAD). After its cleavage by caspase-3 the cleaved ICAD loses its affinity for CAD, and CAD is thereby released from ICAD and digests the chromosomal DNA (7). Despite the above-noted effects on caspase-3 activity, STS-dependent cleavage of ICAD was not altered in cells exposed to AUR and Na 2 S 4 . This lack of effect on ICAD processing is very likely because the polysulfide was added 3 h after STS stimulation, a time point where the key apoptotic processes (including ICAD cleavage) are well under way.

Regulation of apoptosis signaling via reversible persulfidation of caspase-9
Based on the above observations, we next sought to investigate cellular effects induced by polysulfide exposure that precedes or coincides with the initiation of apoptosis. Accordingly, cells were exposed simultaneously to STS and different combinations of Na 2 S 4 and AUR. As shown in Fig. 4D, in the absence of AUR, an early exposure to Na 2 S 4 did not affect STS-induced caspase-3 activation. In contrast, in the presence of AUR, Na 2 S 4 treatment effectively and dose-dependently inhibited caspase activation. Importantly, in this setting, the cleavage of procaspase-3 was also attenuated (Fig. 4D). These observations suggest that under conditions of suppressed Trx/TrxR activity, early administration of Na 2 S 4 disrupts the apoptotic signaling upstream of caspase-3. Of note, analysis of ICAD processing

Regulation of apoptotic caspases by thiol persulfidation
showed that AUR/Na 2 S 4 co-treatment led to decreased ICAD cleavage, indicating a weaker apoptotic response (Fig. 4D).
In light of these observations, we postulated that AUR/Na 2 S 4 treatment may disrupt the activity of caspase-9, the initiator caspase that cleaves and activates procaspase-3 during intrinsic apoptosis (7). Supporting this notion, we observed that Na 2 S 4 dose-dependently inhibited caspase-9 activity in apoptotic cell lysates, which was quantitatively similar to the effect toward caspase-3 (Fig. 5A). Next, we found that although cell treatment with Na 2 S 4 alone had very little effect on STS-induced caspase-9 activity, in the presence of AUR, Na 2 S 4 effectively inhibited the enzyme (Fig. 5B). Immunoblot analysis showed that treatment with AUR/Na 2 S 4 (but not Na 2 S 4 alone) partially inhibited the cleavage of procaspase-9 (Fig. 5C); however, the inhibitory effect on caspase-9 activity was far greater than the effect on proenzyme cleavage (Fig. 5, B and C). For example, treatment with 250 M Na 2 S 4 (in the presence of AUR) led to Ͼ90% inhibition of caspase-9 activity as compared with ϳ50% decrease in procaspase processing. These data suggested the possibility that caspase-9 persulfidation could be involved in these effects. We therefore performed BTA analysis, the results of which provided support and further insight about this hypothesis. Specifically, the data showed that procaspase-9 was constitutively persulfidated in resting cells whereas after STS stimulation the cleaved caspase-9 was mostly nonpersulfidated (Fig. 5C, bottom blot, compare lanes 1 and 2). Notably, treatment with AUR alone attenuated STS-induced depersulfidation of cleaved caspase-9 (Fig. 5C, compare lanes 2 and 5). This result combined with the activity data ( Fig. 5B) suggests that Trx/TrxR mediated stimulus-coupled depersulfidation of caspase-9, promoting its activation. In addition, AUR enhanced Na 2 S 4 -dependent persulfidation of cleaved caspase-9 (Fig. 5C,  compare lanes 3 and 6).
The partial inhibition of procaspase-9 processing noted above (Fig. 5C) prompted us to assess the possible effect of Na 2 S 4 /AUR on release of Cyt c from mitochondria, a process required for apoptosome formation and activation of procaspase-9 (7). The results showed that STS-induced release of Cyt c to the cytosol was largely intact (albeit slightly reduced) when comparing AUR-or AUR/Na 2 S 4 -treated cells to their

Regulation of apoptotic caspases by thiol persulfidation
respective (minus AUR) controls (Fig. 5D). Taken together, the data thus suggest that AUR/Na 2 S 4 treatment targets the apoptotic cascade mainly at the level of caspase-9, which in turn results in diminished activation of caspase-3 and cleavage of ICAD. To further assess the functional relevance of these findings, we measured DNA fragmentation, a hallmark of apoptotic cell death. Consistent with our findings regarding the activities of caspase-9 and -3 and ICAD cleavage, we found that, AUR/ Na 2 S 4 co-treatment resulted in decreased DNA fragmentation, indicative of attenuated apoptosis (Fig. 5E).
Mammalian TrxR enzymes are susceptible to inactivation by various oxidants (32), yet it is unknown if their activity is modulated by polysulfides. To address this issue, we measured the enzymatic activity of cellular TrxR under different treatment combinations. As expected, we found that AUR treatment sub-stantially inhibited TrxR activity. Of note, cell treatment with Na 2 S 4 did not affect TrxR1 activity; however, the polysulfide markedly potentiated the inhibitory effect of AUR (Fig. 5F). Specifically, whereas AUR treatment resulted in ϳ50% inhibitory effect on TrxR activity, ϳ90% inhibition was seen in cells exposed to AUR plus Na 2 S 4 (Fig. 5F). In conjunction with the previous results, these new data highlight a correlation between the extent of apoptosis and the level of TrxR activity, supporting the notion that a functional Trx/TrxR system is required for efficient transmission of the apoptotic signal.
The last set of experiments pointed to caspase-9 as an important target of polysulfide action. To further examine how polysulfides affect caspase-9 we employed a well-established cellfree assay for Cyt c-dependent activation of the caspase-9 and -3 cascade (33). The results showed that Na 2 S 4 effectively Figure 4. Regulation of caspase-3 persulfidation by the thioredoxin system. A, schematic illustration of the assay used to assess the effects of polysulfides and reductants on caspase-3 activity. DTT-reduced caspase-3 (harboring a hexahistidine-tag) is immobilized on nickel-coated Microwell plates followed by treatment with polysulfides (step 1) and reductants (step 2). Thereafter, caspase activity is determined by measuring cleavage of the fluorogenic substrate, Ac-DEVD-AMC (step 3). B, recombinant caspase-3 was immobilized on nickel-coated plates and then treated with 1 M Na 2 S 4 for 30 min at 37°C. After removal of excess sulfide, the enzyme was incubated for 30 min at room temperature with 5 mM glutathione (GSH), complete Trx system (5 M Trx, 0.1 M TrxR, 200 M NADPH) or 5 mM DTT. After removal of excess reductants, enzymatic activity was determined using a fluorescent assay. **, p Ͻ 0.01 versus Na 2 S 4 alone. C, HeLa cells were treated Ϯ 2 M AUR and 1 M STS (4 h) with Na 2 S 4 added for the final hour. Thereafter, caspase-3-like activity in cell lysates was determined. Cleavage of procaspase-3 or ICAD was determined by immunoblotting. D, HeLa cells were simultaneously treated for 4 h with AUR, STS, and Na 2 S 4 as indicated. Caspase-3 activation and cleavage of ICAD were determined as in C. Caspase activities reported in C and D are expressed as percent of STS samples. **, p Ͻ 0.01 versus vehicle control unless noted. Data in the graphs represent mean Ϯ S.D. (n Ն 3).

Regulation of apoptotic caspases by thiol persulfidation
suppressed Cyt c-induced increase in caspase-9 activity without interfering with the cleavage of procaspase-9 (Fig. 5G). These data reinforce the idea that the primary target of exogenously applied polysulfides is the cleaved enzyme, rather than its precursor. Notably, an additional proteolytic product (ϳ13 kDa) was readily detected upon Cyt c activation of caspase-9, the level of which decreased as a function of Na 2 S 4 exposure (Fig. 5G, lower band marked by an asterisk). This small fragment most likely represents the cleavage of caspase-9 by caspase-3, which removes the prodomain, as part of a positive feedback loop (34,35). Accordingly, disruption of this amplification loop may contribute to the antiapoptotic action of polysulfides.

Discussion
A large body of evidence has shown that H 2 S serves as signaling molecule in various cellular processes and cell types (1-4).
Recently, there has been growing interest in sulfane sulfur species, including polysulfides, as possible mediators of H 2 S signaling (3,4,36). There is accumulating evidence for the presence and effects of polysulfides in various cell types, including cancer cells, provoking a range of mechanistic and analytical investigations (21,(23)(24)(25)(26). Nonetheless, the role of endogenous polysulfides in cellular signaling remains a subject of considerable debate and ongoing investigation (15). Alongside this, there is also increased interest in the application of exogenous polysulfides for modulating cellular processes and pathophysiological responses. For example, a recent study demonstrated that administration of exogenous polysulfides inhibits Toll-like receptor signaling in macrophages and reduces endotoxic shock in mice (37). These advances highlight the need to further characterize the effects of polysulfides on cellular physiology and elucidate the underlying mechanisms. In the present study, we revealed that polysulfides suppress apoptosis signaling in cancer cells via inhibitory persulfidation of apoptotic caspases.
Redox regulation of mammalian caspases has been the subject of previous investigations. Among caspases, the redox control of caspase-3, an enzyme that bears a highly nucleophilic thiol at its active site, has been mostly studied. It was discovered that either S-nitrosylation or S-glutathionylation negatively regulates caspase-3 activation (38, 39). Studies in lymphocytes have shown that cytosolic procaspase-3 is non-nitrosylated under basal steady-state conditions, whereas mitochondria-associated procaspase-3 is constitutively nitrosylated (40). It was further established that denitrosylation and activation of mitochondrial procaspase-3 is triggered by certain apoptotic stimuli such as Fas, and that Trx proteins mediate both basal and stimulus-induced denitrosylation (29,40). Notably, in the present study, we found that procaspases-3 and -9 (which are mostly localized in the cytoplasm) are basally persulfidated in unstimulated HeLa cells, and that upon apoptotic stimulation they undergo depersulfidation. Furthermore, during

Regulation of apoptotic caspases by thiol persulfidation
apoptosis, TrxR inhibition attenuated caspase depersulfidation and activation. These results support the notion that reversible persulfidation regulates the activation of cytosolic caspase-9 and -3. Moreover, to our knowledge, these findings provide the first demonstration of stimulus-coupled protein depersulfidation. In addition, we showed that cell exposure to exogenous polysulfide promotes the persulfidation of cleaved caspase-3 and -9, blocking protease activities.
We determined that polysulfides very potently inhibited caspases under cell-free conditions, with effects observed in the low micromolar range. Higher concentrations of polysulfides were needed to effectively inhibit caspases in intact cells, likely reflecting both the presence of competing targets and the robust activities of thiol/persulfide reducing systems. Indeed, we demonstrated that inhibition of the cellular Trx system greatly sensitized caspases to inactivation by polysulfides. Collectively, our results indicate that the effects of polysulfides on caspases depend upon both the timing of cell exposure to the polysulfide and the activity of the Trx system. Effective blockade of the apoptotic process occurred upon early administration of polysulfides (i.e. concurrently with application of the apoptotic stimulus) and under conditions of low TrxR activity. In this setting, caspase-9 was markedly persulfidated and repressed, resulting in diminished activation of caspase-3 and downstream molecules. It is noteworthy that, even without addition of exogenous polysulfide, TrxR inhibition had functional consequences, evident by the attenuation of caspase-9 depersulfidation and activation after apoptotic stimulation (Fig. 5, B and C). These observations lend support to the idea that Trx/TrxR-mediated depersulfidation plays a regulatory role in caspase-9 activation. More generally, the current results expand the emerging concept that the Trx system plays an important role in sulfide homeostasis and signaling (15,17,18).
The cellular mechanism that mediates endogenous persulfidation of procaspases is currently unknown. More generally, fundamental mechanistic questions remain unresolved regarding the formation and targeting of persulfides in cells (15,23,24). In this regard, a recent study suggested a model whereby H 2 S oxidation by Cyt c may facilitate protein persulfidation, with procaspase-9 being studied as a potential target protein (41). It was reported that cell treatment with the H 2 S donor GYY4137 caused elevated persulfidation and decreased activation of procaspase-9 (41). However, the functional downstream consequences of procaspase-9 persulfidation were not analyzed, nor was the mechanism of depersulfidation addressed. The present study, by revealing dynamic caspase persulfidation and its regulation by Trx, significantly extends upon previous observations. Of note, our cell-based and cell-free assays indicate that endogenous and exogenous RSS have distinct effects, targeting the inactive procaspase precursors and the active (processed) enzymes, respectively. Additional research is necessary to elucidate the cellular mechanism mediating persulfidation of procaspases-9 and -3 and address the question of persulfide targeting and turnover. Our findings nonetheless reveal rapid turnover of persulfides which allows dynamic regulation of caspase signaling. They further indicate decreased stability and faster turnover of persulfide on cleaved caspases as compared with the proenzymes. On the basis of the present data, we can speculate that structural changes that occur upon the proteolytic maturation of caspase-3 and -9 render the active site thiol more accessible to react with polysulfides on the one hand and Trx on the other hand. This notion is consistent with structural analyses, which have shown that caspase processing involves significant rearrangements in the active site and its surroundings, rendering the catalytic cysteine more exposed to the solvent (42). Altogether, our observations may help guide future experimental approaches to manipulate caspases using polysulfides or related compounds.
In conclusion, our findings show that polysulfides exert an antiapoptotic effect through persulfidation of components of the core apoptotic machinery, namely, caspase-3 and -9. The results further reveal that persulfidation and Trx antagonistically regulate apoptotic signaling in cancer cells. Altogether, the new findings advance a mechanistic understanding of sulfidebased regulation of cellular apoptosis. We should emphasize that the present studies do not exclude the possibility that the effects of H 2 S/RSS on apoptosis involve other targets and mechanisms. Indeed, recent proteomic studies indicate that a large number of cellular proteins are susceptible to persulfidation (28,43,44). Hence, additional roles of persulfidation in apoptotic signaling are likely to be discovered in the future. Further elucidation of the mechanisms by which H 2 S/RSS regulate apoptosis and other cell death pathways may ultimately enable the development of new therapeutic approaches for cancer treatment.

Preparation of recombinant proteins
Human caspase-3 was expressed in Escherichia coli strain BL21(DE3). The His 6 -tagged protein was purified by affinity chromatography on nickel-nitrilotriacetic acid-agarose as described elsewhere (29). Briefly, bacterial lysates were prepared in buffer (50 mM Tris, 100 mM NaCl, pH 8.0) and incubated with nickel-nitrilotriacetic acid-agarose for 1 h. Caspase-3 was eluted with an imidazole linear gradient from 0 -200 mM. Fractions containing caspase-3 were pooled and concentrated by ultrafiltration. For the experiments, the protein was reduced by incubation with 50 mM DTT for 30 min at room temperature followed by removal of excess reductant by gel filtration using Sephadex G-25 columns (GE Healthcare).
Human Trx was prepared as described previously (45). Briefly, E. coli cells expressing His-tagged Trx were cultured in 500 ml LB and induced by the addition of 0.5 mM isopropyl-␤d-1-thiogalactopyranoside for 6 h. Cells were harvested by centrifugation at 3000 rpm for 10 min at 4°C, resuspended in 10 ml of extraction buffer (300 mM KCl, 50 mM KH 2 PO 4 , 5 mM imidazole, with protease inhibitors, pH 7.4) and disrupted by ultrasonication. After centrifugation at 12,000 ϫ g for 30 min at 4°C, the supernatant was applied to nickel-nitrilotriacetic acid beads overnight at 4°C. The beads were washed with extraction buffer containing imidazole (10 mM) and finally the protein was eluted with 250 mM imidazole. Fractions were analyzed by SDS-PAGE, pooled and stored at Ϫ80°C. Protein purity was at least 95% as judged by SDS-PAGE.

Assessment of the effects of polysulfides on caspase activities
The effects of polysulfides on recombinant caspase-3 were analyzed as follows. DTT-reduced caspase-3 (1 M) was incubated at 37°C for 20 min with persulfide/polysulfide donors as indicated in the legend to Fig. 1. The activity of caspase-3 (diluted to 5 nM) was determined using fluorogenic substrate Ac-DEVD-AMC (100 M) and quantified by fluorescent detection of free AMC with excitation at 360 nm and emission at 465 nm.
Assays using immobilized caspase-3 were performed as follows. Reduced caspase-3 (0.5 M) was incubated in 96-well His-Grab plates (Pierce) in 50 l buffer H (50 mM Hepes, 100 mM NaCl, and 0.1% CHAPS, pH 7.5) for 1 h at room temperature. Unbound material was removed by three washes with buffer H and the immobilized enzyme was incubated with Na 2 S 4 (concentrations and durations as indicated in the legends). In some experiments, after sulfide treatment, samples were subjected to additional incubation with reductants. Each incubation step was followed by three washes with buffer H. Finally, caspase-3 activity was determined using fluorogenic substrate Ac-DEVD-AMC (100 M).

Cell-free assay of Cyt c-induced caspase activation
The assay was performed using THP-1 cells according to Ref 46. Briefly, cells were harvested in PBS by centrifugation at 1500 rpm for 5 min at 4°C. Cells were then incubated for 15 min in three volumes of ice-cold cell extract buffer (20 mM Hepes, pH 7.5, 10 mM KCl, 1.5 mM MgCl 2 , 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 100 M PMSF, 10 g/ml leupeptin, and 2 g/ml aprotinin), followed by homogenization using a B-pestle. Lysates were then centrifuged at 15,000 ϫ g for 20 min to remove nuclei, mitochondria, and cellular debris. To analyze the effects of polysulfides on caspase activation, DTT was first removed by ultrafiltration using Amicon Ultra Filters (10 kDa, Millipore). Then, extracts (50 g, 2 mg/ml) were incubated with bovine heart Cyt c (50 g/ml) and dATP (1 mM) and with different concentration of Na 2 S 4 for 60 min at 37°C. Caspase activation was analyzed by immunoblotting or enzyme activity assay.

Analysis of modifications of caspase-3 by MS
Reduced caspase-3 (1 M) was incubated with Na 2 S 4 (1 or 2 M) or with SNAP-P* (50 or 100 M) for 15 min at 37°C. Thereafter, samples were subjected to alkylation with 10 mM iodoacetamide for 2 h at room temperature. The proteins in the samples were separated on a nonreducing SDS-PAGE gel. A gel slice corresponding to molecular weights from 10 to 50 kDa was excised and the proteins in the gel were digested in 10% acetonitrile and 10 mM ammonium bicarbonate with modified trypsin (Promega) at a 1:10 enzyme-to-substrate ratio, overnight at 37°C. The resulting peptides were desalted using C18 tips (homemade stage tips) and then subjected to LC coupled with tandem MS (LC-MS/MS). The peptides were resolved by reverse-phase chromatography on 0.075 ϫ 180-mm fused silica capillaries (J&W) packed with Reprosil reversed phase material (Dr. Maisch GmbH, Germany). The peptides were then eluted over 30 min with a linear gradient of 5 to 28% acetonitrile with 0.1% formic acid in water ,15 min gradient of 28 to 95% acetonitrile with 0.1% formic acid in water, and 15 min at 95% acetonitrile with 0.1% formic acid in water, at a flow rate of 0.15 l/min. The MS/MS analyses were performed with a Q Exactive Plus mass spectrometer (Thermo Fisher Scientific) in a positive mode using repetitively full MS scan followed by collision inducing dissociation of the 10 most dominant ions selected from the first MS scan. Raw data were subjected to the SEQUEST search algorithm using Proteome Discoverer Software version 1.4 (Thermo Fisher Scientific). Detected peptides were searched against human and E. coli UniProt databases as fully tryptic peptides and in addition against the human caspase-3 sequence as semi-tryptic peptides. All the analysis was performed with mass tolerance of 15 ppm for the precursor masses and 0.05 atomic mass units for the fragment ions. Oxidation of Met and several modifications of Cys (oxidation, dehydro, carbamidomethyl, carbamidomethylated persulfide, and propionamidation) were accepted as variable modifica-

Regulation of apoptotic caspases by thiol persulfidation
tions. Minimal peptide length was set to six amino acids and a maximum of two miscleavages were allowed. Peptide-and protein-level false discovery rates were filtered to 1% using the target-decoy strategy. Semi-quantitation was done by calculating the peak area of each peptide based on its extracted ion currents.

Assessment of caspase persulfidation using BTA
The BTA was performed according to Ref. 28 with minor modifications. In brief, whole cell lysates were prepared using RIPA buffer (100 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 0.5% Triton X-100, 0.5% deoxycholic acid, cOmplete EDTAfree protease inhibitors, pH 7.5) and protein concentrations were adjusted to 0.8 mg/ml. A total of 1 mg protein was used for each reaction. Thiol and persulfide groups were alkylated with biotin-maleimide for 30 min at room temperature. After acetone precipitation, biotinylated proteins were captured by streptavidin-agarose resin at 4°C overnight. After extensive washes of the beads, disulfide-linked proteins (formerly persulfidated) were eluted with 20 mM DTT for 30 min at room temperature. Eluates were subjected to immunoblot analysis using anti-caspase-3 or anti-caspase-9 antibodies.

Analysis of cytochrome c release from mitochondria
After treatments, cells were harvested, washed in PBS, resuspended in hypotonic lysis buffer (10 mM Hepes, 3 mM MgCl 2 ,10 mM KCl, 0.1 mM EDTA, pH 7.5), and disrupted by passage (five times) through a 25-gauge needle. Lysates were centrifuged at 600 ϫ g for 10 min at 4°C to remove nuclei and unbroken cells. Supernatants were then centrifuged at 15,000 ϫ g for 30 min at 4°C. Supernatants from this step were designated as cytosolic fractions and subjected to immunoblot analysis using anti-Cyt c antibody.

Analysis of DNA fragmentation
After treatments, cells were collected by centrifugation and washed with PBS and then lysed with lysis buffer (10 mM Tris-HCl, 10 mM EDTA, 0.5% Triton X-100, pH 8.0). The lysates were incubated with proteinase K (0.1 g/l) for 2 h at 50°C. Thereafter, the lysates were incubated with RNase (0.1 g/l) for an additional 2 h at 37°C. After isopropanol precipitation, DNA was extracted and subjected to electrophoresis in a 1% (w/v) agarose gel in 40 mM Tris acetate buffer (pH 7.4) at 100 V. After electrophoresis, DNA was visualized by staining with ethidium bromide.

Determination of activity of cellular TrxR
TrxR activity in cell lysates was measured using the insulin reduction end point assay as detailed before (47). In brief, 25 g of protein cell lysate was incubated in a final volume of 50 l containing 0.3 mM insulin, 660 M NADPH, 2.5 mM EDTA, and 5 M human Trx1 in 85 mM Hepes (pH 7.5) for 20 min at room temperature. Control reactions excluding Trx1 were used for background subtraction. Then, 250 l of 1 mM DTNB, 240 M NADPH, and 200 mM Tris-HCl, pH 8, in 6 M guanidine hydrochloride was added and the absorbance was determined at 412 nm.

Statistical analysis
All data are presented as the mean Ϯ S.D. Statistical differences were analyzed by one-way analysis of variance followed by Tukey post-test using Prism software (GraphPad). A p value Ͻ 0.05 was considered statistically significant.

Data Availability
The mass spectrometry data have been deposited to the Pro-teomeXchange Consortium via the PRIDE (48) partner repository with the dataset identifier PXD017144.