Polynitrosylated proteins: characterization, bioactivity, and functional consequences

Area of Science:

• Biochemistry
• Molecular Biology
• Physiology

Background:

• Protein chemical modification is crucial for biological regulation.
• Nitrosylation of protein sulfhydryl groups confers nitric oxide (NO)-like activities and regulates protein function.
• Other nucleophilic side chains (hydroxyls, amines, aromatic carbons) are also susceptible to nitrosative attack.

Purpose of the Study:

• To investigate the reactivity and functional consequences of nitros(yl)ation at various nucleophilic centers in biological molecules.
• To determine the extent of nitrosation on different protein sites.
• To assess the biological activity of nitrosated proteins.

Main Methods:

• Chemical analysis and spectroscopic studies to identify nitrosation sites and quantify modification.
• Reactions with model proteins (BSA) and enzymes (tissue-type plasminogen activator) under varying nitrosating conditions.
• Assessment of antiplatelet, vasodilator, and enzymatic activities of modified proteins.

Main Results:

• Nitrosation occurs at sulfur, oxygen, nitrogen, and aromatic carbon centers, with thiols being most reactive.
• Polynitrosylated proteins were formed, with activity increasing with the degree of nitrosation.
• S-nitroso derivatives exhibited the greatest NO-related bioactivity, while polynitrosylation could attenuate enzymatic function.

Conclusions:

• Multiple protein sites are susceptible to nitrogen oxide attack, with thiols preferentially modified for functional regulation.
• Nitrosation at additional centers may have pathophysiological significance, facilitating NO bioactivity delivery.
• Polynitrosylated proteins found in stimulated macrophages and NO-treated subjects suggest physiological relevance.