title: 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.
authors.affiliation: School of Human Science and Environment, University of Hyogo, Hyogo 670-0092, Japan. Electronic address: yojikato@shse.u-hyogo.ac.jp.
authors.forename: Yoji
authors.initials: Y
authors.lastname: Kato
authors.affiliation: School of Human Science and Environment, University of Hyogo, Hyogo 670-0092, Japan. Electronic address: shigekix0505@gmail.com.
authors.forename: Shigeki
authors.initials: S
authors.lastname: Ono
authors.affiliation: School of Human Science and Environment, University of Hyogo, Hyogo 670-0092, Japan. Electronic address: kitamoto@shse.u-hyogo.ac.jp.
authors.forename: Noritoshi
authors.initials: N
authors.lastname: Kitamoto
authors.affiliation: Centre for Free Radical Research, Department of Pathology, University of Otago, Christchurch, New Zealand. Electronic address: tony.kettle@otago.ac.nz.
authors.forename: Anthony J
authors.initials: AJ
authors.lastname: Kettle
mesh_terms: D045744:Cell Line, Tumor; D003598:Cytoskeletal Proteins / Q000378:metabolism*; D006801:Humans; D045563:Indolequinones / Q000737:chemistry / Q000494:pharmacology*; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism*; D011499:Protein Processing, Post-Translational / Q000187:drug effects*; D014363:Tryptamines / Q000737:chemistry / Q000494:pharmacology*
chemical_list: D003598:Cytoskeletal Proteins; D045563:Indolequinones; D009419:Nerve Tissue Proteins; D014363:Tryptamines; C060213:tryptamine-4,5-dione
keywords: 5-Hydroxytryptamine; Adduct; Antibody; Neuronal cells; Quinone; Tryptamine 4,5-dione