As the literature swelled with examples of intracellular signaling triggered or affected by ROI and RNI, it became increasingly difficult to know how to relate these findings to the classical model of second messengers or to its successor, the contemporary model of molecular handshakes. Several recent studies ratcheted the problem to a new level.
First, Stamler, Lamas, and Fang collected nearly 100 examples of proteins that are regulated posttranslationally by nitrosylation (13
). Most were intracellular, and there were examples from almost every functional class. Second, Jaffrey et al. confirmed that at least some of these proteins are nitrosylated in healthy animals (52
). Third, using macrophages from mice deficient in iNOS, phox, or both, Ehrt et al. demonstrated that iNOS and phox exert widespread effects on gene expression in activated macrophages (25
). Macrophage activation by a cytokine (IFN-γ) and a microbe (the tubercle bacillus) led to altered expression of about 25% of the monitored macrophage genome and about 40% of the monitored transcriptome. Of the genes affected by macrophage activation, 58% were significantly impacted in their expression by iNOS and/or phox. Remarkably, there was functional selectivity among the genes affected by iNOS and phox. Genes involved in immunity and inflammation were almost completely spared (that is, regulated by macrophage activation independently of iNOS and phox), while many others were suppressed in an iNOS- and/or phox-dependent manner. The authors hypothesized that “a self-induced state of metabolic stringency within the activated, infected macrophage may necessitate suppression of the expression of those genes not essential to the emergency at hand” (25
). Taking the three reports together, and considering that production of ROI and RNI is nearly ubiquitous in the aerobic biome, it is difficult to escape the conclusion that the impact of ROI and RNI on intracellular signal transduction is physiologic and widespread.
Investigators have offered several different characterizations of intracellular signaling by ROI/RNI. First, ROI and RNI have been described as first messengers triggering private pathways. For example, Zheng et al. described OxyR as a bacterial transcription factor acting as an H2
receptor by undergoing oxidation and allosteric activation, leading to expression of antioxidant defenses and other adaptations to oxidative stress (53
). Helmann and colleagues extended the concept to include not just cysteine-dependent proteins but also metalloproteins as bacterial peroxide sensors (54
). Delaunay et al. presented a more complex version of the same concept, in which a specific enzyme serves as an H2
receptor in yeast. The enzyme undergoes allosteric changes when oxidized and then specifically transfers the oxidized status and a corresponding allosteric change to a transcription factor (56
) in a manner analogous to a kinase cascade causing successive phosphorylations. For RNI, the classic example has already been mentioned: extracellular NO acts as a first signal to activate intracellular soluble guanylate cyclase, triggering a phosphorylation cascade (2
). In other examples, Kim et al. (57
) argued that OxyR serves not only as an H2
receptor but also as an NO receptor, undergoing either nitrosylation or oxidation. Demple and Demple showed that another bacterial transcription factor, SoxR, can act as an NO receptor. SoxR’s iron-sulfur clusters are nitrosylated by NO, leading to a conformational change in the protein that allows it to transactivate the gene encoding another transcription factor, SoxS. SoxS in turn controls induction of antioxidant enzymes (58
). Thus, ROI and RNI can signal by initiating pathways that operate with type I specificity. However, in these situations, ROI and RNI themselves are the agonists, not the downstream mediators.
In another characterization of signaling by RNI, nitrosylation has been likened to phosphorylation (13
). However, phosphorylation and dephosphorylation convey specificity because cells express an array of kinases and phosphatases, each of which binds transiently to a restricted set of client proteins. In contrast, there does not appear to be a corresponding array of nitrosylases or denitrosylases individually dedicated to modifying specific client proteins. Thus, reversible nitrosylation of signaling intermediates that have oligomolecular specificity is not by itself an indication that RNI act with type I specificity.
Describing a very different set of circumstances, Reth wrote that H2
acts as a second messenger (16
). He described “a cloud of H2
” emanating from receptors whose activation by other agonists is coupled both to phosphorylation and to ROI production (16
). The best-described impact of H2
on signaling initiated by other agonists is that H2
transiently inactivates tyrosine phosphatases via reversible oxidation of their active-site cysteinyl residues (59
) and thereby augments phosphokinase-mediated signal transmission. Because the specificity is imparted by the receptor and is not preserved by the H2
in this situation is not a second messenger in Sutherland’s sense. Rather, H2
is acting in this case as a secondary messenger, modifying the extent or duration of a reaction initiated by another signal. This regulatory role, executed through a reversible covalent interaction, typifies type III specificity.
Carbon monoxide (CO) is another example of a mediator that can signal with type III specificity. Heme synthesis is under circadian control (62
). Heme oxygenase, an enzyme with constitutive as well as ROI- and RNI-induced isoforms, breaks down heme to give rise to CO. CO activates a mammalian transcription factor, NPAS2, that regulates circadian rhythm by binding covalently to the heme in its PAS domain (63
In a special case analogous to ATP acting as a signal to mTOR, O2
acts a covalent mediator by serving as a substrate for two regulatory amino acid hydroxylases that are sensitive to physiologic variations in O2
concentration. Iron-dependent prolyl hydroxylase uses O2
to hydroxylate certain prolyl residues in hypoxia-inducible transcription factor-1α (HIF-1α). This leads to the recognition of HIF-1α by the von Hippel–Lindau tumor suppressor gene product. The result is ubiquitin ligation of HIF-1α and its proteasomal degradation. Another O2
-using enzyme hydroxylates an asparagine residue in HIF-1α, preventing p300/CBP from binding there to support HIF-1α’s transactivating activity. Under hypoxic conditions, both hydroxylations are diminished. Activated HIF-1α accumulates, binds HIF-1β, and drives gene transcription (64
). The process may be regulated or mimicked by ROI and RNI (69