Blog pi

Overview

S-Nitrosylation is a post-translational modification in which a nitric oxide molecule is bound via a reactive thiol group of a cysteine residue. S-nitrosylation has various regulatory roles in bacteria, yeasts, plants and mammalian cells.

In-depth mechanism

S-Nitrosylation is a post-translational modification in which a nictric oxide (NO) group is coupled to a thiol group of a cysteine residue resulting in a S-nitrosothiol group^​1–5​. NO is an important short-lived physiological messenger that is highly reactive^​1,2​. It is generated through the conversion of L-arginine to L-citrulline and catalysed by the enzyme NO synthase^​2,3​. The signaling of NO can be categorised in classical and non-classical ways. In the classical way, NO binds to the heme group of guanylyl cyclase, which stimulates the transformation from GTP to cGMP. cGMP is a second messenger, which in turn activates cGMP-dependent protein kinase. cGMP-dependent protein kinase reduces the concentration of potassium and calcium ions in the cytosol. This causes a hyperpolarization of the membrane potential which triggers neurotransmission and vasodilation. The non-classical way describes the process of NO signaling via covalent post-translational modifications which includes among others S-nitrosylation^​3​. The frequency of S-nitrosylation depends on the local concentration of NO and affects intracellular traffic processes, protein phosphorylation and protein-protein interactions^​3–5​. Enzymes do not catalyse the reaction from the thiol to the S-nitrosothiol. However, the denitrosylation is regulated by two enzymes called S-nitrosoglutathione reductase and thioredoxin. If these two enzymes are lacking or inhibited, high concentrations of S-nitrosylated proteins occur^​2–4​. NO concentrations above 100 nM induce S-nitrosylation^​4​. The mechanism of S-nitrosylation is shown in figure 1.

Although S-nitrosylation is a non-enzymatic reaction (except prokaryotes), it is a highly selective modification and is dependent on different factors^​3,5​. Target cysteine residues must be in close proximity of the NO source, within a I/L-X-C-X2-D/E motif as well as within a highly hydrophobic region formed by tertiary protein structure or membranes. In addition, the environment is decisive whether a protein is S-nitrosylated or not. Since a thiolate is required for S-nitrosylation, the reaction is strongly dependent on the neighbouring amino acids, as these strongly influence the pKa of cysteine. Furthermore, S-nitrosylation is hindered by bulky amino acid residues such as phenylalanine, tyrosine, arginine and leucine. Over 3000 proteins with potential S-nitrosylation sites have been identified^​2,3​. S-Nitrosylation results in a molecular mass increase of 29 Da^​2​. Furthermore, the nitroso group causes an additional absorption in the range between 274 – 442 nm^​6​, the maximum being at 336 nm^​7​. The influence of the nitroso group on the absorbance was determined by the difference of the UV spectra of glutathione and S-nitrosoglutathione.