Covalent modification of cytoskeletal proteins in neuronal cells by tryptamine-4,5-dione

Abstract

Serotonin, 5-hydroxytryptamine, is a systemic bioactive amine that acts in the gut and brain. As a substrate of myeloperoxidase in vitro, serotonin is oxidized to tryptamine-4,5-dione (TD), which is highly reactive with thiols. In this work, we successively prepared a monoclonal antibody to quinone-modified proteins and found that the antibody preferentially recognizes the TD–thiol adduct. Using the antibody, we observed that the chloride ion, the predominant physiological substrate for myeloperoxidase in vivo, is not competitive toward the enzyme catalyzed serotonin oxidation process, suggesting that serotonin is a plausible physiological substrate for the enzyme in vivo. Immunocytochemical analyses revealed that TD staining was observed in the cytosol of SH-SY5Y neuroblastoma cells while blot analyses showed that some cellular proteins were preferentially modified. Pull-down analyses confirmed that the cytoskeletal proteins tubulins, vimentin, and neurofilament-L were modified. When pure tubulins were exposed to micromolar levels of synthetic TD, self-polymerization was initially enhanced and then suppressed. These results suggest that serotonin oxidation by myeloperoxidase or the action of other oxidants could cause functional alteration of cellular proteins, which may be related to neurodegeneration processes or irritable bowel syndrome.

Neutrophil myeloperoxidase and its substrates: formation of specific markers and reactive compounds during inflammation

“…( 36 ) Chloride ions at physiological concentrations do not compete with TD-derived modifications, suggesting that serotonin is a promising physiological substrate of myeloperoxidase in vivo . ( 37 ) Cytoskeletal proteins, α- and β-tubulins, vimentin, and neurofilament-L, in SH-SY5Y neuroblastoma cells have been identified as target proteins. Self-polymerization of tubulins was modulated by the exposure of TD in vitro .…”

“…It has also been confirmed that the metabolite of serotonin, 5-hydroxyindoleacetic acid, is a favorable substrate and that the successive formation of quinone gives rise to the covalent conjugation of protein thiols. ( 37 ) It has been shown that urate is also a possible physiological substrate for myeloperoxidase. ( 38 ) These results suggest that myeloperoxidase may use many endogenous substrates in vivo .…”

A novel mechanism of vitamin D anti-inflammatory/antioxidative potential in type 2 diabetic patients on metformin therapy

“…Oxidative modification of intracellular and membrane components of vascular endothelial cells may lead to the development of endothelial dysfunction, vascular damage and atherosclerosis. A causal relationship was documented by the accumulation of MPO in atherosclerotic lesions [10][11][12][13][14][15].…”

Covalent adduction of endogenous and food-derived quinones to a protein: its biological significance

“…( 40 ) As shown below, the quinone-modified protein was immunochemically detected on a membrane or fixed tissue, indicating that adducted quinone on a protein is relatively stable. ( 41 , 42 )…”

“…( 9 , 19 , 40 ) Specific antibodies to quinone or quinone adduct have also been used for immunochemical detection. ( 42 , 49 , 50 )…”