Regulated Protein Denitrosylation by Cytosolic and Mitochondrial Thioredoxins

Abstract

Nitric oxide acts substantially in cellular signal transduction through stimulus-coupled S-nitrosylation of cysteine residues. The mechanisms that might subserve protein denitrosylation in cellular signaling remain uncharacterized. Our search for denitrosylase activities focused on caspase-3, an exemplar of stimulus-dependent denitrosylation, and identified thioredoxin and thioredoxin reductase in a biochemical screen. In resting human lymphocytes, thioredoxin-1 actively denitrosylated cytosolic caspase-3 and thereby maintained a low steady-state amount of S-nitrosylation. Upon stimulation of Fas, thioredoxin-2 mediated denitrosylation of mitochondria-associated caspase-3, a process required for caspase-3 activation, and promoted apoptosis. Inhibition of thioredoxin-thioredoxin reductases enabled identification of additional substrates subject to endogenous S-nitrosylation. Thus, specific enzymatic mechanisms may regulate basal and stimulus-induced denitrosylation in mammalian cells.

Crystal structure of human thioredoxin revealing an unraveled helix and exposed S‐nitrosation site

“…Each of these has been implicated in S-nitrosation activities (also called S-nitrosylation), particularly with respect to regulation of apoptosis. [7][8][9][10][11][12][13][14] S-nitrosation/nitrosylation is the addition of NO to a cysteine residue, a redox reaction requiring one electron oxidation, and numerous proteins have been suggested to be regulated in this manner. 15 Removal of the nitroso group in target proteins, or transfer of the group to other proteins, is likely to involve hTrx1 in human cells.…”

How widespread is stable protein S-nitrosylation as an end-effector of protein regulation?

“…This includes the thioredoxin (Trx) system, comprising Trx and Trx reductase (TrxR) [22,42]. Denitrosation and subsequent activation of caspase-3 was shown by Benhar et al to be regulated by the Trx-TrxR system in vivo [43]. The ability of Trx to remove S-nitrosothiols is dependent on its active site Cys-X-X-Cys motif, which accepts the NO from the target by a trans-nitrosation reaction.…”

“…Is it possible that that these enzymes simply play their well-established roles as disulfide reductase enzymes after an Snitrosothiol transitions to the disulfide state [45]? Pharmacological inhibition of Trx with auranofin leads to the accumulation of S-nitrosated proteins, supposedly providing evidence of this enzyme as a denitrosylase [43]. However, blockade of this well-established, classical disulfide reductase activity is anticipated to generate the same result by limiting the resolution of S-nitrosothiols via disulfide formation.…”

“…In is quite typical for NO donors to be used at concentrations of 100-500 µM, including in studies published in prominent journals [43,129,130]. This adds significant complexity, especially in the context as to whether a protein is endogenously regulated by S-nitrosation.…”

Itinéraire d’un agent double

“…L'inhibition de l'activité enzymatique de la caspase-8 par S-nitrosylation protège les hépatocytes de rat de l'apoptose induite par le TNFα (tumor necrosis factor a) [14], tandis que dans des cellules de carcinome colique humain, la S-nitrosylation de la procaspase-9 prévient son clivage et son activation consécutive [15]. Cependant, la stimulation du récepteur apoptotique Fas permet la dénitrosylation de la caspase-3 par la thioredoxine réductase 2 (TrxR2) [16]. Dans ce contexte, il a été démontré plus récemment, que l'IL-24 jouait un rôle primordial, en amont [17].…”