Nitric oxide and thioredoxin modulate the activity of caspase 9 in HepG2 cells

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 inactivate Caspase-3, 8, and 9, thereby hindering their apoptotic activity. However, spontaneous restoration of their activity upon S-denitrosylation 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 systems that can directly participate in catering to the S-(de)-nitrosylation-mediated functional outcomes of the cysteinyl proteases in pathophysiological settings.

Keywords: Caspase 9; Lipoic acid; Nitric oxide; S-nitrosylation; Thioredoxin.

Figure 1A:

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.

Figure 1B:

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:

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.

Figure 3:

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.

Figure 4:

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.