S-Denitrosylation: A Crosstalk between Glutathione and Redoxin Systems

Abstract

S-nitrosylation of proteins occurs as a consequence of the derivatization of cysteine thiols with nitric oxide (NO) and is often associated with diseases and protein malfunction. Aberrant S-nitrosylation, in addition to other genetic and epigenetic factors, has gained rapid importance as a prime cause of various metabolic, respiratory, and cardiac disorders, with a major emphasis on cancer and neurodegeneration. The S-nitrosoproteome, a term used to collectively refer to the diverse and dynamic repertoire of S-nitrosylated proteins, is relatively less explored in the field of redox biochemistry, in contrast to other covalently modified versions of the same set of proteins. Advancing research is gradually unveiling the enormous clinical importance of S-nitrosylation in the etiology of diseases and is opening up new avenues of prompt diagnosis that harness this phenomenon. Ever since the discovery of the two robust and highly conserved S-nitrosoglutathione reductase and thioredoxin systems as candidate denitrosylases, years of rampant speculation centered around the identification of specific substrates and other candidate denitrosylases, subcellular localization of both substrates and denitrosylases, the position of susceptible thiols, mechanisms of S-denitrosylation under basal and stimulus-dependent conditions, impact on protein conformation and function, and extrapolating these findings towards the understanding of diseases, aging and the development of novel therapeutic strategies. However, newer insights in the ever-expanding field of redox biology reveal distinct gaps in exploring the crucial crosstalk between the redoxins/major denitrosylase systems. Clarifying the importance of the functional overlap of the glutaredoxin, glutathione, and thioredoxin systems and examining their complementary functions as denitrosylases and antioxidant enzymatic defense systems are essential prerequisites for devising a rationale that could aid in predicting the extent of cell survival under high oxidative/nitrosative stress while taking into account the existence of the alternative and compensatory regulatory mechanisms. This review thus attempts to highlight major gaps in our understanding of the robust cellular redox regulation system, which is upheld by the concerted efforts of various denitrosylases and antioxidants.

Keywords: S-(de)nitrosylation; S-nitrosoglutathione; S-nitrosoproteins; antioxidant systems; glutaredoxin; glutathione; glutathione peroxidase; glutathione reductase; glutathione transferase; glutathionylation; nitric oxide; nitric oxide synthases; nitro-oxidative stress; oxidoreductases; peroxiredoxin; reactive nitrogen species; reactive oxygen species; redox homeostasis; redundancy; thiol disulfides; thioredoxin; thioredoxin interacting protein; thioredoxin reductase; thioredoxin-related protein 14.

Figure 1

The figure shows the correlation between glutathione and its derivatives. 1.GSH can be synthesized from its precursor, γ-glutamylcysteine, derived from glutamate and cysteine.2. Most known proteins can react with NO-donors or NO_x species, forming labile intermediates, such as S-nitrosylated proteins (PSNOs). 3. PSNOs, except a few such as Caspase 3, can be readily denitrosylated in the presence of an abundant physiological concentration (5–10 mM) of GSH. 4. S-nitrosoglutathione reductase (GSNOR) can convert GSNO, produced by the addition of an NO donor to GSH, into oxidized glutathione (GSSG), which is recycled back to GSH by GR, utilizing NADPH as an electron source; 5. or even take part in catalyzing the trans-nitrosylation of proteins by GSNO, thus playing contrasting yet significant roles.

Figure 2

Mechanism of GSH-mediated denitrosylation of PSNOs. S-nitrosylated proteins can be denitrosylated by reduced glutathione (GSH), forming protein with reduced thiol(s) and GSNO, which is metabolized by ubiquitously expressed GSH into a stable disulfide form (GSSG) at the expense of HNO. GSSG (oxidized GSH) can be further reduced back to GSH by NADPH-dependent GR. Alternatively, GSNO can also be irreversibly metabolized by GSNOR to a complex N-hydroxysulphenamide (GSNHOH) form, utilizing reducing equivalents from NADH [3,42].

Figure 3

Grx-catalyzed denitrosylation of PSNOs via dithiol and monothiol mechanisms. Two distinct yet functionally similar Grx-dependent mechanisms have been proposed that can suffice for protein denitrosylation [13]; (a) the dithiol mechanism wherein Grx shuttles between three competent oxidant states, namely the reduced dithiol Grx-(SH)₂, an intermediate Grx-(SNO)(SH), and the oxidized disulfide Grx-S₂ form, (b) the monothiol mechanism involving Grx with only an active N-terminal cysteine residue Grx-(SH) and the Grx-SNO form. While the figure shows GSH dependency in both systems for continuous enzymatic turnover, it also highlights additional disparities between the mechanisms, such as the coupling of GSH with monothiol Grx for exhibiting denitrosylase activity, unlike its dithiol form.

Figure 4

Proposed schematic representation predicting parallels between GSH and redoxin systems. Oxidized Trx (Trx-S₂) is reduced back to its vicinal dithiol (Trx-(SH)₂) form by TrxR, utilizing NADPH as an electron donor. TRP14, with a redox potential similar to that of Trx1, proceeds in a similar way that uses both its active site thiols in a TrxR1-dependent NADPH oxidation. GSH (5–10 mM), stimulated by the Grx/GR system, can directly reduce Trx in the absence of TrxR, thus acting as a cellular backup, whereas Txnip inhibits the reducing activity of Trx via a disulfide exchange reaction [77,78]. This figure also hypothesizes GSH-catalyzed reduction and Txnip-mediated inhibition of TRP14 activity in a manner analogous to that of Trx.