Enzymatic mechanisms regulating protein S-nitrosylation: implications in health and disease

Abstract

Nitric oxide participates in cellular signal transduction largely through S-nitrosylation of allosteric and active-site cysteine thiols within proteins, forming S-nitroso-proteins (SNO-proteins). S-nitrosylation of proteins has been demonstrated to affect a broad range of functional parameters including enzymatic activity, subcellular localization, protein-protein interactions, and protein stability. Analogous to other ubiquitous posttranslational modifications that are regulated enzymatically, including phosphorylation and ubiquitinylation, accumulating evidence suggests the existence of enzymatic mechanisms for regulating protein S-nitrosylation. In particular, studies have led to the identification of multiple enzymes (nitrosylases and denitrosylases) that participate in targeted S-nitrosylation or denitrosylation of proteins in physiological settings. Nitrosylases are best characterized in the context of transnitrosylation in which a SNO-protein transfers an NO group to an acceptor protein (Cys-to-Cys transfer), but examples of transnitrosylation catalyzed by metalloproteins (Metal-to-Cys transfer) also exist. By contrast, denitrosylases remove the NO group from SNO-proteins, ultimately using reducing equivalents derived from NADH or NADPH. Here, we focus on the recent discoveries of nitrosylases and denitrosylases and the notion that their aberrant activities may play roles in health and disease.

Fig. 1

Metal-to-Cys and Cys-to-Cys nitrosylases. a The mechanism of action of Metal-to-Cys transnitrosylases may entail intramolecular NO group transfer (auto-S-nitrosylation) or intermolecular reactions with glutathione (not shown) to form SNO-proteins or GSNO, respectively. b The mechanism of action of Cys-to-Cys transnitrosylases exhibits similarities with enzymes involved in other ubiquitous posttranslational modifications. Transnitrosylases, palmitoyltransferases, and E2-conjugating enzymes participate in Cys-to-Cys transfer of NO, palmitate, or ubiquitin, respectively. E3 ubiquitin ligases transfer ubiquitin (ligated to cysteine) to target proteins

Fig. 2

Dysregulation of hemoglobin’s nitrosylase activity in sickle cell anemia. In RBCs from patients with sickle cell anemia, aberrant intramolecular transfer of NO from heme iron nitrosyl to Cys (impaired Metal-to-Cys nitrosylase activity) results in deficient formation of SNO-sickle hemoglobin (SNO-HbS). Additionally, aberrant docking of S-nitrosylated SNO-HbS to the membrane protein AE1 disrupts transnitrosylative transfer of the NO group to the membrane (impaired Cys-to-Cys nitrosylase activity). Decreased levels of membrane SNO are associated with decreased ability of RBCs to effect hypoxic vasodilation, which may contribute to the vaso-occlusive crisis of sickle cell anemia [27]

Fig. 3

Deficiency of cellular GSNOR activity in the etiology of hepatic carcinoma. The O⁶-alkylguanine-DNA alkyl transferase (AGT) protein transfers the alkyl group (shown as a methyl, CH3, group) from the O⁶ position of guanine to its active-site cysteine, thereby repairing DNA. However, AGT is S-nitrosylated in inflammatory conditions (in which iNOS is upregulated), and S-nitrosylation promotes ubiquitinylation and subsequently proteasomal degradation. GSNO is in equilibrium with SNO-AGT, and in wild-type mice, GSNOR regulates SNO-AGT denitrosylation by shifting the equilibrium towards GSNO, resulting in decreased SNO-AGT levels. However, in GSNOR^−/− mice, the absence of GSNOR activity results in increased S-nitrosylation and degradation of AGT. A chromosomal deletion, inclusive of the GSNOR gene, is found in 50% of patients with HCC. Thus, GSNOR denitrosylase activity may play a tumor suppressor function in the etiology of HCC