Regulated protein denitrosylation by cytosolic and mitochondrial thioredoxins

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.