Polynitrosylated proteins: characterization, bioactivity, and functional consequences.
Chemical modification of proteins is a common theme in their regulation. Nitrosylation of protein sulfhydryl groups has been shown to confer nitric oxide (NO)-like biological activities and to regulate protein functions. Several other nucleophilic side chains -- including those with hydroxyls, amines, and aromatic carbons -- are also potentially susceptible to nitrosative attack. Therefore, we examined the reactivity and functional consequences of nitros(yl)ation at a variety of nucleophilic centers in biological molecules. Chemical analysis and spectroscopic studies show that nitrosation reactions are sustained at sulfur, oxygen, nitrogen, and aromatic carbon centers, with thiols being the most reactive functionality. The exemplary protein, BSA, in the presence of a 1-, 20-, 100-, or 200-fold excess of nitrosating equivalents removes 0.6 +/- 0.2, 3.2 +/- 0.4, 18 +/- 4, and 38 +/- 10, respectively, moles of NO equivalents per mole of BSA from the reaction medium; spectroscopic evidence shows the proportionate formation of a polynitrosylated protein. Analogous reaction of tissue-type plasminogen activator yields comparable NO protein stoichiometries. Disruption of protein tertiary structure by reduction results in the preferential nitrosylation of up to 20 thus-exposed thiol groups. The polynitrosylated proteins exhibit antiplatelet and vasodilator activity that increases with the degree of nitrosation, but S-nitroso derivatives show the greatest NO-related bioactivity. Studies on enzymatic activity of tissue-type plasminogen activator show that polynitrosylation may lead to attenuated function. Moreover, the reactivity of tyrosine residues in proteins raises the possibility that NO could disrupt processes regulated by phosphorylation. Polynitrosylated proteins were found in reaction mixtures containing interferon-gamma/lipopolysaccharide-stimulated macrophages and in tracheal secretions of subjects treated with NO gas, thus suggesting their physiological relevance. In conclusion, multiple sites on proteins are susceptible to attack by nitrogen oxides. Thiol groups are preferentially modified, supporting the notion that S-nitrosylation can serve to regulate protein function. Nitrosation reactions sustained at additional nucleophilic centers may have (patho)physiological significance and suggest a facile route by which abundant NO bioactivity can be delivered to a biological system, with specificity dictated by protein substrate.
Simon
D I
DI
Department of Medicine, Cardiovascular Division, Brigham and Women's Hospital, Boston, MA 02115, USA.
Mullins
M E
ME
Jia
L
L
Gaston
B
B
Singel
D J
DJ
Stamler
J S
JS
D000818:Animals; D001665:Binding Sites; D002021:Buffers; D002417:Cattle; D005342:Fibrinolysis / Q000187:drug effects; D006801:Humans; D066298:In Vitro Techniques; D009682:Magnetic Resonance Spectroscopy; D015394:Molecular Structure; D009569:Nitric Oxide / Q000737:chemistry / Q000378:metabolism; D009603:Nitroso Compounds / Q000737:chemistry* / Q000378:metabolism* / Q000494:pharmacology; D010974:Platelet Aggregation / Q000187:drug effects; D010975:Platelet Aggregation Inhibitors / Q000737:chemistry / Q000494:pharmacology; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism* / Q000494:pharmacology; D011817:Rabbits; D012710:Serum Albumin, Bovine / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D013053:Spectrophotometry; D013438:Sulfhydryl Compounds / Q000737:chemistry; D010959:Tissue Plasminogen Activator / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D014664:Vasodilation / Q000187:drug effects
D002021:Buffers; D009603:Nitroso Compounds; D010975:Platelet Aggregation Inhibitors; D011506:Proteins; D013438:Sulfhydryl Compounds; D012710:Serum Albumin, Bovine; D009569:Nitric Oxide; D010959:Tissue Plasminogen Activator