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Published in final edited form as: Biochim Biophys Acta Gen Subj. 2023 Aug 29;1867(11):130452. doi: 10.1016/j.bbagen.2023.130452

Nitric Oxide and Thioredoxin modulate the activity of caspase 9 in HepG2 cells

Surupa Chakraborty a, Ankita Choudhuri a,≠, Akansha Mishra a,≠, Camelia Bhattacharyya a, Timothy R Billiar b, Detcho A Stoyanovsky †, Rajib Sengupta a,b,*
PMCID: PMC10592080  NIHMSID: NIHMS1928036  PMID: 37652366

Abstract

The interdependent and finely tuned balance between the well-established redox-based modification, S-nitrosylation, and its counteractive mechanism of S-nitrosothiol degradation, i.e., S-denitrosylation of biological protein or non-protein thiols defines the cellular fate in the context of redox homeostasis. S-nitrosylation of cysteine residues by S-nitrosoglutathione, S-nitroso-L-cysteine-like physiological and S-nitroso-L-cysteine ethyl ester-like synthetic NO donors inactivates Caspase-3, 8, and 9, thereby hindering their apoptotic activity. However, spontaneous restoration of their activity upon S-(de)nitrosylation of S-nitrosocaspases into their reduced, free thiol active states, aided by the members of the ubiquitous cellular redoxin (thioredoxin/ thioredoxin reductase/ NADPH) and low molecular weight dithiol (lipoic acid/ lipoamide dehydrogenase/ dihydrolipoic acid/ NADPH) systems imply a direct relevance to their proteolytic activities and further downstream signaling cascades. Additionally, our previous and current findings offer crucial insight into the concept of redundancy between thioredoxin and lipoic acid systems, and the redox-modulated control of the apoptotic and proteolytic activity of caspases, triggering their cyto- and neurotoxic effects in response to nitro-oxidative stress. Thus, this might lay the foundation for the exogenous introduction of precise and efficient NO. or related donor drug delivery system that can directly participate in catering to the S-(de)-nitrosylation-mediated functional outcomes of the cysteinyl proteases in pathophysiological settings.

1. Introduction

Apoptosis, alternatively known as ‘programmed cell death’ is an important cellular process that plays a pivotal role in a myriad of functions such as ensuring a normal cellular turnover, growth and development, proper functioning of the immune system, embryogenesis, cellular homeostasis, and age-, chemical-, disease-, or free radical accumulation and damage-induced cell arrest or atrophy, ideally characterized by distinctive morphological and biochemical features [1]. Given its widely attributed range of regulatory functions within a cell, apoptosis has been established as a highly coordinated cellular defense mechanism that involves caspases, a class of specific cysteine proteases, -mediated energy-dependent molecular cascade, triggered in response to a plethora of endogenous or exogenous (i.e., induced) stimuli, under both physiological and pathological conditions. Caspases are synthesized in vivo as relatively inactive zymogens or pro-caspases at the nanomolar (< 50 nM) range, and their protease-like enzymatic properties are necessarily governed by cysteine(yl) side chain residues for catalyzing peptide bond hydrolysis and an intrachain activation cleavage at aspartyl residues for most caspases, however with varying substrate specificities for each. Coupled with the activation of different initiator caspases (caspase 8, 9, 10) in the extrinsic or death receptor pathway and intrinsic or mitochondrial pathways of apoptosis, either by oligomerization or autocatalytic cleavage, caspases further activate downstream effector caspases (caspase 3 and 7) in a self-amplifying mechanism, ‘caspase cascade’ which is regarded as another cellular hallmark of apoptosis [2]. The activation of the intrinsic pathway leads to a loss of mitochondrial membrane potential that accelerates the sequestration of cyt c from the mitochondria into the cytosolic compartment, further promoting the oligomerization of a scaffolding protein, Apaf-1 (apoptotic protease-activating factor 1), and activating it in the presence of ATP or dATP; an activated Apaf-1 along with the procaspase-9 and cyt c concomitantly forms an active polymeric multiprotein apoptosome complex associated with an active caspase-9 [3,4]. Mature caspase 9, encoded by the CASP9 gene located on chromosome 1 is derived from its procaspase 9 monomeric precursor, harboring a characteristic N-terminal pro-domain which is recognized by the Apaf-1 caspase-recruitment domain (CARD) bearing a similar globular fold [5]. The in vitro assembly of cyt c, dATP, and a cytosolic extract (cell-free lysate) can form the active in vivo apoptosome complex [4–11].

The S-nitrosylation, or the covalent attachment of an NO• moiety to specific cysteine thiol residues of both initiator and executioner cysteine caspases (CASP-SNO), upon subsequent exposure to NO, a simple, lipophilic, and highly diffusible molecule, regarded as a bioregulator of several cellular signal transduction processes, synthesized via NOS isoenzymes (nitric oxide synthase enzymatic pathway) or pH-dependent sGC-cGMP pathway (non-enzymatic pathway) can reversibly inhibit their activity and further apoptosis, as has been substantiated by experimental findings [12,13]. The cellular consequences of the reversible inhibition of caspase 9, 8, or 3 activities, are heightened by the possibilities of S-nitrosylation mediated hindrance of caspase cleavage and further reconstitution of their activity upon S-denitrosylation, which are markedly prominent to maintain their intracellular homeostasis in the face of changing nitrosative stress conditions. Endogenous NO production has antiapoptotic effects and NO-induced by S-nitrosothiol donors like S-nitrosoglutathione (GSNO) or S-Nitroso-N-acetyl-D,L-penicillamine (SNAP) directly inhibits caspase 9 activity, upstream of caspase 3, prevents the formation of caspase cleavage products and nuclear fragmentation, and eventually, abrogates the apoptosis-mediated cell death mechanisms via S-nitrosylation of caspases 9 and 3 that facilitates cell survival in human monocytes [14,15]. Although the implications and prospects of caspase 9 activity suppression via NO donor treatment are certainly conceivable, its catalytic activity response to NOS (specifically, iNOS) inactivation would pave the way for chemopreventive strategies to enhance certain oncogene-dependent apoptosis, mediated by an active caspase 9, thus attenuating carcinogenesis in the background of chronic inflammation [16,17]. S-nitrosylation of the active site cysteine (Cys 325) catalytic residue of an endogenous zymogen, or procaspase 9 indicated a constitutive level of S-nitrosoprocaspase 9 formation and its counteractive cellular activity rescue upon NOS inhibitor and TNF-α treatment-based depleting abundance of S-nitrosylated procaspase 9 have also been reported in scientific literature, with effects consistent with the previous findings [18,19].

Nonetheless, thioredoxin (both Trx1 and Trx2)-catalyzed S-denitrosylation prompts the conversion of inactive CASP-SNO (caspases 3 and 8) into their reduced, active counterparts (CASP-SH) in both recombinant purified forms and HepG2 cell lysates, thus lessening the burden of nitrosative stress and thereby adding zest to validate the findings with caspase 9 [20–23]. Additionally, significant emphasis was directed towards low molecular weight (LMW) dithiols, such as 6,8-dimercaptooctanoicacid (DHLA), the reduced form of lipoic acid (LA; 5-[1,2]dithiolan-3-yl-pentanoic acid) in the denitrosylation of caspase 9-SNO in thioredoxin reductase (TrxR) deficient HepG2 cells, consistent with our prior observations with other caspases [20–22]. LMW cellular dithiols (LA/DHLA system) can thus be regarded as feasible alternatives to the potential denitrosylases, such as the Trx system that results in a regain of caspase 9 activity [23,24].

In summary, the aim of our current work is to develop a rationale strong enough to contribute valuable insight into the S-(de)-nitrosylation mediated regulation of both extrinsic and intrinsic apoptotic pathways, that can pave the way for novel NO donors, drugs, LA supplements, and therapeutics with careful consideration of their possible impact on the activity and redox milieu of caspases (mainly caspase 3, 8, and 9). The focus on understanding the molecular mechanisms underpinning the anti-cancer effects of TrxR inhibition modulates redox signaling and oxidative defense in cancer cells, both in vitro and in patient samples. Understanding these complex interactions and their effects may lead to identifying new potential targets for drug therapy and beholds the potential to open new avenues in the field of medicine.

2. Materials and methodology:

2.1. Reagents.

All reagents used and purified recombinant Caspase 9, Trx, and Trx reductase (TrxR) were purchased from Sigma Chemical Co. (St. Louis, MO). The solutions used in the experiments were prepared in deionized and Chelex-100-treated water or potassium phosphate buffer (pH = 7.4). S-nitroso-L-cysteine ethyl ester (SNCEE) preparation was carried out as outlined by Sengupta et al. (2009) [21].

2.2. Cell culture and treatments.

HepG2 cells were cultured in the Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum, 100 U/mL penicillin, 100 μg/mL streptomycin, and 2 mM L-glutamine in a humidified atmosphere in 5% CO2 at 37°C.

2.3. Activation of caspases in HepG2 cells.

Cells (2 × 106 cells/flask) were grown in T75 flasks for 24 h and then incubated for 6 h with medium (control) or with medium containing TNF-α (1–50 ng/mL; Fig. 3A, 3B)) and cycloheximide (40 μM) [22–24]. Whole-cell lysates for analysis of caspases 9 were harvested by repeated freeze and thaw cycles followed by centrifugation at 15,000g for 3 min at 4°C. Caspase 9 activity was measured fluorometrically on LS50B Perkin-Elmer spectrofluorimeter using 0.1 mM N-Ac-LEHD-AFC (N-Acetyl-Leu-Glu-His-Asp-7-amino-4-trifluoromethylcoumarin) (0.1 mM), as a substrate (λex = 380 nm; λem = 420 nm; excitation/emission slit, 5 nm). Activity calculations for caspases were corrected with the effect of specific inhibitors.

Figure 3:

Figure 3:

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Modulation of Caspase 9 activity upon incubation of (A) untreated and (B) TrxR-deficient HepG2 cells with RSNO and LA. Caspase 9 activity was stimulated with TNF-α (15 nM) and cycloheximide (40 μM) as described in Experimental procedures. The cells were pre-conditioned with SNCEE (50 μM) for 15 mins at 37°C, washed with PBS, and the Caspase 9 activity was henceforth recorded in SNCEE-free medium, either immediately or after an additional 60 mins incubation in the presence or absence of LA (50 μM). Untreated Caspase 9 in lysates of (TNF-α and cycloheximide treated)-HepG2 cells and lysates treated with Caspase-9 Inhibitor III (Sigma) (1 μM) were used as controls. Data represent the mean of three independent experiments ± S.E.

2.4. S-nitrosylation of caspase 9.

Recombinant purified caspase 9 was incubated with 1 mM DTT for 30 min at 25°C. DTT was removed via centrifugation using a Vivaspin 500 filter (3-kDa mol wt cutoff; Cole Parmer, Vernon Hills, IL); the first spin was followed by five wash cycles with chelex-purified phosphate buffer containing 1 mM EDTA. DTT-free Caspase 9 was incubated with 0.3 mM GSNO for 30 min at 25°C, and the excess GSNO was removed via ultrafiltration using a Vivaspin 500 filter with six wash cycles with phosphate buffer containing 1 mM EDTA. In the final reaction solution, the S-nitrosothiol content of Caspase 9-(SNO) was determined with a Sievers NO analyzer, with helium used as a gas carrier [20]. The reaction chamber of the analyzer contained 0.1 M phosphate buffer (pH 7.4), 0.2 mM CuCl2, and 50 mM ascorbic acid.

2.5. siRNA Transfection.

The siRNA directed against TrxR was 5’-AGACCACGUUACUUGG GCAdTdT-3’ and the control was a scrambled sequence (5’-AGGCAAAUCACGGUGUCCUdTdT-3’) that does not match any sequence in the with the hypothesis that a time-dependent increase in caspase activity would reflect its endogenous S-denitrosylation. Treatment of control HepG2 cells with NO-donors led to 80% (5 min) and 5% (60 min in incubation medium without NO-donor) inhibition of caspase (Fig. 3A). GenBank human database for >16 nt (Dharmacon RNA Technologies; Chicago, IL). Approximately 2 × 105 HepG2 cells were plated per well in a six-well plate. The following day, cells were transfected with siRNA (30 pmol per well) in the presence of lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) by following the manufacturer’s recommendations [25]. Transfection with the same amount of nonspecific siRNA was performed as a control. The cells were harvested and analyzed at 48 h after transfection for cell viability and activity. TrxR assay. TrxR activity was determined in a coupled assay with Escherichia coli Trx (10 μM) and 5,5’-dithiobis(2-nitrobenzoic acid) (DTNB) as described in Ref. [20]. One unit of TrxR activity was defined as the formation of 74 μmol of 5-mercapto-2-nitrobenzoic acid (1 absorbance unit at 412 nm; ε412 = 13500 M−1 cm−1) per min per mL at pH 7.0 at 25 °C.

2.6. Caspase 9 Assay.

Subconfluent cultures of HepG2 cells were harvested by scraping on ice, washed in ice-cold phosphate-buffered saline (PBS), and resuspended in an equal volume of ice-cold isotonic lysis buffer (20 mM HEPES (pH 7.5), 10 mM KCl, 1 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 250 mM sucrose, 10 μg/ml of aprotinin, 1 μg/ml of leupeptin, 1 μg/ml of pepstatin A). After 30-min incubation on ice, the cells were lysed by freezing-thawing and centrifuged at 750 × g for 10 min. The supernatant obtained was further centrifuged at 10,000 × g for 10 min and at 20,000 × g for 30 min. The clarified supernatant was stored in aliquots at −80°C and used at protein concentrations ranging from 5 to 10 mg/ml. The apoptosome was activated by the addition of 1 mM dATP and 1 μM cytochrome c to the cytosolic cell extract (protein concentration, 5 to 10 mg/ml) containing 100 mM N-Ac-LEHD-AFC (Biomol). After 30-min incubation at 37°C, the liberation of AFC (excitation wavelength, 400 nm; emission wavelength, 489 nm) was measured for 30 to 45 min at 37°C with an LS50B Perkin-Elmer spectrofluorimeter.

3. Results and discussion:

3.1. S-denitrosylation of CASP-SNO by Trx system.

The impairment of Caspase 9 activity by physiological or synthetic NO donors upon S-nitrosylation can be reversed by Trx, the ubiquitous, dithiol redox protein, along with its other associated components, selenoenzyme TrxR, and NADPH. Follow-up attempts to unravel the role of the redox regulatory oxidoreductases in catalyzing the S-denitrosylation of CASP-SNOs (caspases 3, 8, and 9) yielded positive results, thereby extending their roles in harnessing the potential to trigger the expression of caspases in fundamental cellular processes like apoptosis. S-nitrosocaspase 9 did not readily decompose to any noteworthy extent in the absence of Trx [Figure 1A, control set].

Figure 1A:

Figure 1A:

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Trx-catalyzed S-denitrosylation of CASP9-SNO. The recombinant purified Caspase 9 (0.7 μM) was incubated with the complete Trx system (Δ) and the aliquots were charged into the NO analyzer. Untreated Caspase 9-SNO (▲) and Caspase 9-SNO treated with TrxR and NADPH (▯) were used as controls. The data are presented as a mean of three independent experiments ± S.E. Trx system contained 5 μM Trx, 0.08 μM (1 unit/ml) TrxR, and 0.4 mM NADPH.

With the aim of mimicking the in vivo induction of intrinsic pathway, an in vitro apoptosome-dependent caspase 9 activity assay was established to firmly ascertain the regulation of caspase activity post-incubation with S-nitrosothiol (RSNO) [Figure 1B and Figure 2]. The dose-dependent inhibition of its specific fluorogenic substrate catabolizing activity of caspase 9 can be attributed to its direct inhibition upon S-nitrosylation which could be reversed upon treatment with the complete Trx/TrxR/NADPH system, supporting the notion that the cellular redox milieu can be regarded as a crucial determinant for the activation of the cysteine protease under nitrosative stress conditions. The maximal rate (about 70%) of S-denitrosylation of S-nitrosocaspase 9 was achieved with 5 μM Trx, 0.08 μM TrxR, and 0.4 mM NADPH which are regarded well within the intracellular concentration range of Trx (5–50 μM). Thus, the capability of NO to reversibly inhibit these proteases (both upstream and downstream)and modulate the caspase 3/9,8,10 feedback amplification loop raises its likelihood of rescuing the cells from programmed cell death even after the induction of apoptotic stimuli and concomitant activation of upstream caspases.

Figure 1B:

Figure 1B:

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Effects of L-CysSNO (50 μM) and Trx system at the indicated concentrations, on the activity of recombinant purified Caspase 9 (Δ). The purified protein was incubated with its specific fluorogenic substrate, N-Ac-LEHD-AFC (0.1 mM), and its activity was assessed as relative fluorescence intensities (A.U.) upon the liberation of 7-AFC. A control experiment was carried out with Caspase 9 in the absence of its fluorogenic substrate (▯). Trx system contained 5 μM Trx, 0.08 μM (1 unit/ml) TrxR, and 0.4 mM NADPH.

Figure 2:

Figure 2:

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Modulation of Caspase 9 activity in NO donor-treated HepG2 cells. 0.03 mg protein/ml cell lysates were incubated with 50 uM (▯), 150 uM (o), and 500 uM (▲) RSNO over a 5 mins timeframe, and 0.1 mM Ac-LEHD-AFC substrate was added to initiate the reaction. Fluorescence spectra of untreated HepG2 cell lysate with the Caspase 9-specific substrate were considered as the control (Δ). The data are presented as a mean of three independent experiments ± S.E.

The caspase 9-attributed intrinsic pathway of apoptosis is also known as mitochondrial-dependent apoptosis owing to the central role of the organelle in mediating the intracellular interaction of pro-apoptotic proteins with its outer membrane, subsequently increasing its membrane permeability which further permeates the release of cyt c and pro-apoptotic factors into the cytoplasm. The effects of initiator protease (Caspase 9) inhibition subjected to NO• exposure can be countered by the Trx system upon reversible S-nitrosylation, also acknowledged as S-denitrosylation, in the mitochondria by human thioredoxin2 (hTrx2) and/or by human thioredoxin 1 (hTrx1) in the cytosolic compartment, coupled with their respective TrxRs [21–23, 26]. The Trx family of proteins (12kDa or larger) and its homologs (proteins with Trx fold, exhibiting high structural resemblances) harbor an evolutionarily conserved active site CXXC (Cys32-Gly-Pro-Cys35, in particular) sequence motif that necessarily exhibits its intracellular antioxidant functions as S-denitrosylases and thiol-disulfide oxidoreductases. A two-disulfide form of Trx1, possessing the oxidative modification between its catalytic and non-catalytic cysteine thiol residues, compromises its ability as a thiol oxidoreductase as well as a potential substrate for TrxR which ideally participates in reversibly recycling an oxidized Trx (Trx-S2) to its vicinal dithiol (Trx-(SH)2) form, utilizing NADPH as an electron donor [27]. Redox-active site cysteine thiols of Trx (Cys 32 and Cys 35), preferentially low pKa Cys 32 interact with S-nitrosylated protein, here, S-nitrosylated caspase 9, forming a transient HS-Trx-Cys(32)-SNO intermediate along with a reduced (active) caspase 9; HNO is concomitantly released leaving a deprotonated thiol (i.e., Cys 32 thiolate) which is a relatively weak base and induces an immediate nucleophilic attack on the neighboring vicinal Cys 35 thiol, thus forming another relatively stable disulfide Trx intermediate, only to be further reduced back to its active form utilizing TrxR and reducing equivalents of NADPH. It is thus comprehensible that Trx-catalyzed de novo S-denitrosylation of CASP 9-SNO augments the existing list of candidate PSNO (S-nitrosoprotein) substrates, theorizing Caspase 9-SNO as a plausible substrate for other cellular denitrosylases, besides the major Trx system [28–30].

The S-nitrosylation of non-catalytic structural thiols (either Cys69 or Cys73) upon pre-incubation with excess GSNO leads to the formation of S-nitrosothioredoxin (Trx-SNO), which has been suggested to trans-S-nitrosylate procaspase 3 and caspase 3 at a rate of 196 M−1s−1 [31–33]. Wu et al. demonstrated the GSNO-mediated S-nitrosylation at Cys73 of Trx after the formation of a Cys32/Cys35 disulfide bond, upon which Trx1 disulfide reductase and denitrosylase activities were attenuated [32]. This would naturally question the theory of S-denitrosylation mediated safeguarding and protective mechanism of Trx in the face of nitrosative stress, as envisioned and experimentally evidenced in a vast repertoire of S-nitrosylated PSNO substrates. It is, however, noteworthy to mention that specific PSNO substrates, such as S-nitrosylated caspase 3 are relatively stable in the presence of 5–10 mM GSH and can be exclusively S-denitrosylated by the complete Trx system, which gives an additional impetus to the endogenous S-nitroso modification of Trx as well as the specificity of Trx system as a robust and major S-denitrosylase [34–35]. Interestingly, it has been later reported that Trx-SNO is readily S-denitrosylated in the presence of either reduced thioredoxin (active site thiols are in reduced form) or 5–10 mM GSH [20]; reduced Trx can readily S-denitrosylate Trx-SNO at a rate of 309 M−1s−1. Thus, S-denitrosylation of Trx-SNO by GSH and reduced Trx will be the dominant processes as the cytosolic concentrations of Trx and GSH are higher than that of caspase 3 [20]. The insights of experimental evidence provided, clarify the situation considerably by suggesting that both Trx-SNO and CASP-SNO, if formed in cells, will undergo GSH and Trx/TrxR system-dependent S-denitrosylation and thus, the homeostatic balance will shift in favor of an apoptotic profile.

3.2. S-denitrosylation of CASP-SNO by LMW dithiols.

LMW dithiols such as DHLA share the same goal of lessening the nitro-oxidative stress burden on cells, thus allowing us to leverage their redox properties as a potent, ubiquitous, lipophilic LMW Trx mimetic with comparable kinetic profiles and substrate specificities [36]. While the susceptibility of LA-driven metal chelation, broad-spectrum antioxidant activity, and thiol/disulfide exchange reactions have been regarded as critical for transcription factors and proteins involved in signaling processes, the theory of LA-catalyzed S-denitrosylation was purely speculative until Stoyanovsky et al. (2005) presented the experimental evidence for the catabolism of S-nitrosothiols as well as S-nitrosoproteins by Trx and LA/DHLA system [26, 37–39]. In addition to the ubiquitous oxidoreductases like the Trx system, LA and/or its reduced counterpart DHLA exhibit protective antioxidant potency comparable to that of other antioxidants such as Vitamin E and glutathione (GSH), demonstrated by their ability as a sulfhydryl reductant to reduce oxidized glutathione (GSSG) to GSH and as candidate denitrosylase by exhibiting hydrophobic binding to RSNOs. Under the aforementioned experimental conditions, DTT (5 mM) and LA (50 μM) triggered the S-denitrosylation of S-nitrosylated caspase 9 (CASP9-SNO) in untreated HepG2 cells (control) and TrxR-deficient HepG2 cells respectively, post-treatment with 50 μM SNCEE, a synthetic, highly lipophilic and freely permeable NO donor for 15 mins; concentration of SNCEE was standardized so as to be sufficient for S-nitrosylating caspases, here, caspase 9 which are present in the nanomolar range of concentration in in vivo systems without causing any significant cytotoxicity [20, 40, Figure 3A, 3B].

The rationale for this experimental design was based on previously established kinetics of S-denitrosylation of caspase-SNO by the Trx/TrxR/NADPH system with the hypothesis that a time-dependent increase in caspase activity would reflect its endogenous S-denitrosylation [20,21]. Treatment of control HepG2 cells with NO-donors led to 80% (5 min) and 5% (60 min in incubation medium without NO-donor) inhibition of caspase (Fig. 3A). This would reflect the endogenous S-denitrosylation (without the addition of exogenous NADPH). The caspase 9 assay was performed with its specific fluorogenic substrate, N-Ac-LEHD-AFC (0.1 mM), and its activity was assessed as relative fluorescence intensities (A.U.) upon the liberation of 7-AFC. The regeneration of caspase activity (after the additional 60 mins incubation in a medium without NO-donor) was insignificant in TrxR-deficient cells (Fig. 3B), which suggests the requirement for Trx catalysis in this process. However, substitution with LA followed by incubation for 60 min led to a marked reactivation of Caspase 9 activity in TrxR-deficient HepG2 cells (Fig. 3B), presumably via intracellular reduction of LA to DHLA and the reaction of the latter with CASP-SNO. The data presented herein provide proof of concept that LMW dithiols can mimic the activity of Trx in reactions of protein S-denitrosylation.

An initial inhibition of caspase 9 specific Ac-LEHD-AFC substrate was followed by restoration of its catalytic activity either immediately (after 5 mins of cell lysate preparation) or after an additional incubation time for 60 mins at 37°C. The findings observed are consistent with our hypothesis postulating that LA exposure into the culture medium containing HepG2 cells with impaired TrxR, aids in about 70–75% activity restoration of caspase 9, closely mimicking the in vivo intracellular milieu. The simultaneous administration of a non-physiological or synthetic reductant, DTT, in the wild-type HepG2 cell lysate, expectedly reflects a better restoration of (active) caspase 9 activity (about 90–95%) due to the more effective decay of PSNOs (CASP9-SNO) in presence of TrxR. It could thus be speculated that a dysfunctional Trx system or the deficiency of TrxR or any of the Trx system components renders the caspases (8, 9, 3) inactive in intracellular nitrosative stress conditions which could be a deciding factor for the cell(s) to put forth a candidate denitrosylase such as LA/DHLA to readily restore the homeostatic balance in favor of apoptosis and downstream signaling cascades.

The outlines of the process of S-denitrosylation of CASP9-SNO mediated by the Trx system and LA system are very similar, however with comparable efficiencies, as experimentally evidenced by utilizing 10 equivalents of LA relative to Trx to catalyze the reaction mechanism. DHLA, the reduced form of LA potentially aids in the S-denitrosylation of caspase(s) alongside or in the absence of the pronounced denitrosylases, i.e., Trx/TrxR, with the formation of an intramolecular disulfide ring closure via a transient intermediate, concomitantly releasing HNO in the process. Lipoamide dehydrogenase (LD) utilizes the NADH-dependent electron transfer to the catalytic site of oxidized DHLA, allowing its conversion into dithiol form. Redox-active vicinal dithiols of LA, an eight-carbon fatty acid molecule, can reversibly undergo oxidation forming a disulfide-containing dithiolane form which explains its ability as a redox cofactor that covalently binds to cognate (multi)enzyme complexes, allowing the interconversion between free thiol, disulfide ring form, and other reaction intermediates [41]. The physiological concentrations of LA within a cell lie in the range of 5–30 nmol/g and its reduction to DHLA is accomplished by NADH/NADPH-driven enzymes or oxidoreductases including LD, TrxR, GR, etc.; LA/DHLA thus acts as an antioxidant redox couple that shuttles the reduced form (DHLA) into the extracellular milieu, further reflecting their activity as S-denitrosylases and disulfide reductants [36, 39, 42].

4. Conclusion:

After years of diligently experimenting with thiol-dependent caspases, the scientific riddle entails their catalytic deactivation in the presence of physiological or synthetic NO donors, or at the onset of nitrosative stress and further S-denitrosylation-mediated activity rescue. It still remained unresolved to firmly elucidate the orderly execution of the evolutionarily conserved apoptotic signaling cascade. However, in the context of validating the previous findings of caspases 3 and 8, in caspase 9, the results reflect an analogy between the extrinsic and intrinsic apoptotic pathways based on their redox-mediated regulation, thereby establishing a complete picture to comprehend this process in a dynamic cellular milieu [Figure 4]. The scope of our work concerns the identification of initiator (Caspase 8 and 9) and executioner caspases (Caspase 3), which are the pivotal players driving the apoptotic pathways, as a candidate or substrate PSNOs for Trx-mediated S-denitrosylation. The present study, being a prolongation or continuation of our previous works, rationally uncovers the reversible S-nitrosylation of caspase 9 in conjunction with solidifying the role of the major S-denitrosylases, as predominant regulators of redox signal transduction via the mitochondrial-triggered intrinsic apoptotic pathway. Secondly, there has been a copious number (over 10,000) of S-nitrosylated proteins reported in the scientific literature as an essential influence in defining or structuring an S-nitrosoproteome and deciphering an NO-mediated apoptotic inhibitory mechanism in all reported caspases would not necessarily determine their substrate specificity for Trx-mediated S-denitrosylation. This allowed us to explore the stringencies of redox regulation in the intrinsic pathway separately and provided us the ground for developing our hypothesis that tinkering with the redox switch of S-nitrosylation and S-denitrosylation of caspase 9, upon exposure to physiological levels of the Trx system could elucidate its protective cellular rescue functions in basal and nitrosative stress conditions. Thirdly, a major part of our study was also aimed at contrasting the accepted dogma or notion that exclusively implicates Trx-catalyzed S-denitrosylation. Besides, characterizing yet another PSNO, here caspase 9-SNO, to enrich the expanding S-nitrosoproteome records, we have presented experimental evidence with both recombinant purified protein and TrxR-deficient HepG2 (human hepatocarcinoma) cells for a clear synergism between the two ubiquitous cellular dithiols, Trx and DHLA/LA in mediating S-denitrosylation of S-nitrosocaspase 9. This would undoubtedly reinforce the idea of exploring favoring roles for additional enzymes/proteins as cellular S-denitrosylases or backup system(s) in imparting PSNO specificity which may lead to further studies with LMW drug designing.

Figure 4:

Figure 4:

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Trx/TrxR/NADPH and LA/DHLA-catalyzed S-denitrosylation of S-nitrosylated caspases in response to nitrosative stress. The activity of caspases 8, 9, and 3 gets inhibited under nitrosative stress conditions upon S-nitrosylation by NO or NO• moiety, further compromising their potential to participate in apoptosis. This condition can be remarkably reversed by the ubiquitous Trx system (Trx/TrxR/NADPH) and both the LA system (LA/LD/NADH) and DHLA with comparable efficiencies [21], thereby establishing a novel feedback control mechanism to rescue the cellular proteases and restoring their catalytic activity in executing the extrinsic and/or intrinsic pathways of apoptosis.

The S-(de) nitrosylation-based inactivation or reconstitution of the enzyme (caspase 9, here) has been correlated with its protease-like activity utilizing their highly specific activity-based assays, and this unprecedented versatility of the S-nitrosylation and S-denitrosylation-based posttranslational modifications of caspases (caspase 9, here), alternatively reflects a distinct ‘redox switch’ mechanism that can potentially be entrenched as a piece of stronger evidence for the regulation of cellular S-nitrosoproteases in the apoptotic cascade of signal transduction and would further help us with the global mapping of PSNO substrates for the Trx and LMW dithiol-catalyzed denitrosylation. Thus, the biochemical interplay between S-(de)-nitrosylation and modulation of caspase activity seemingly suggest parallelism with an increased need for fine-tuning the redox control of the respective caspase(s), when pushed to the limits of nitrosative stress with moderate to high levels of pathophysiological significance.

Acknowledgments:

This work is dedicated to the memory of Prof. Detcho A. Stoyanovsky (deceased November 2019), a passionate biochemist who build the foundation of this project. We are grateful to Prof. Stoyanovsky for his enormous help, support, and guidance in providing resources, funding acquisition, and oversight of the experiments. This work was funded by U.S. Public Health Service Grants ES09648.

Footnotes

Conflict of Interest Disclosure:

The authors declare no conflict of interest.

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