Protein Nitrosylation

Protein Nitrosylation

There are a number of mechanisms through which the effects of NO are mediated, but the reaction of NO with cysteine residues in proteins, a process known as nitrosylation, is emerging as one of the most important mechanisms. Nitrosylation is a physiologically important post-translational modification that affects a wide variety of proteins involved in a number of cellular processes. The role of nitrosylation in regulating signal transduction has been largely overlooked until relatively recently. This is because the production of the small, highly reactive NO molecule had been thought to lack the specificity and control observed in other post-translational modifications such as phosphorylation. However, evidence has now emerged that suggests that in fact nitrosylation shares many properties with phosphorylation. Both modifications exhibit substrate specificity, strict spatial and temporal regulation and are reversible.

The functional consequences of protein nitrosylation depends on the protein that is affected. In many cases, such as the caspases, nitrosylation has been shown to inhibit enzyme activity. In other proteins, such as matrix metalloproteinases, it is thought that nitrosylation increases the activity of the enzymes. Despite its importance in many aspects of cell biology our understanding of protein nitrosylation and its regulation remains poorly understood.

Recently a technique was developed that has greatly increased our ability to study nitrosylation. This technique is known as the biotin-switch technique, and exploits differences in chemistry between NO-reacted cysteines (nitrosocysteines) and normal, unmodified cysteines to specifically label the nitrosocysteines with biotin. Following biotinylation the proteins can be easily isolated from the non-nitrosylated proteins using streptavidin beads. The biotin-switch technique is outlined below.


Isolation of proteins using this method allows us to determine which proteins are nitrosylated in cells either by using specific antibodies in Western blots or by using a proteomic approach to identify them. If nitrosylated proteins are isolated from trophoblast cells using this approach and then run on a 2D gel, the results can be seen in the figure below.


Approximately 60 spots, corresponding to around 60 different nitrosylated proteins can be seen in this gel. These proteins were isolated from untreated cells, but if NO activity is increased several hundred spots can be seen, suggesting that many more proteins are capable of being nitrosylated.

It has recently been demonstrated that there is specificity of nitrosylation within a protein so that although a target protein may possess many available cysteines only one of them, and always the same one, is nitrosylated. It is not entirely clear how this specificity is achieved, but it has been suggested that there may be a consensus motif around the cysteine to be nitrosylated that facilitates the reaction. Recently it has been shown that such consensus motifs may not always be found in the primary amino sequence, but in many cases may be a product of the tertiary structure of the protein.

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