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

Kato Y, Ono S, Kitamoto N, Kettle AJ - Redox Biol (2014)

Bottom Line: In this work, we successively prepared a monoclonal antibody to quinone-modified proteins and found that the antibody preferentially recognizes the TD-thiol adduct.Pull-down analyses confirmed that the cytoskeletal proteins tubulins, vimentin, and neurofilament-L were modified.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.

View Article: PubMed Central - PubMed

Affiliation: School of Human Science and Environment, University of Hyogo, Hyogo 670-0092, Japan. Electronic address: yojikato@shse.u-hyogo.ac.jp.

No MeSH data available.


Related in: MedlinePlus

Immunocytostaining of neuroblastoma SH-SY5Y cells incubated with myeloperoxidase/acetaldehyde/xanthine oxidase/serotonin or synthetic TD. The cells were treated in serum-free medium for 30 min with the enzyme system (complete, A), the omission of the component (C–F), or 50 µM synthetic TD (G). The untreated cells are also shown in B. After the treatment, cells were fixed with methanol and permeabilized with 0.5% Triton X-100. The cells were then incubated with the antibody (1B7), followed by secondary antibody (FITC labeled, green). The nuclei were stained with peritoneum iodide (red). A, complete; B, control (untreated cells); C, without serotonin; D, without acetaldehyde; E, without myeloperoxidase; F, without xanthine oxidase; and G, TD (50 µM). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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f0020: Immunocytostaining of neuroblastoma SH-SY5Y cells incubated with myeloperoxidase/acetaldehyde/xanthine oxidase/serotonin or synthetic TD. The cells were treated in serum-free medium for 30 min with the enzyme system (complete, A), the omission of the component (C–F), or 50 µM synthetic TD (G). The untreated cells are also shown in B. After the treatment, cells were fixed with methanol and permeabilized with 0.5% Triton X-100. The cells were then incubated with the antibody (1B7), followed by secondary antibody (FITC labeled, green). The nuclei were stained with peritoneum iodide (red). A, complete; B, control (untreated cells); C, without serotonin; D, without acetaldehyde; E, without myeloperoxidase; F, without xanthine oxidase; and G, TD (50 µM). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Mentions: TD may be a neurotoxin because it has a highly reactive quinone moiety. Proteins in SH-SY5Y neuroblastoma cells were modified by the serotonin oxidation system, which included the complete enzyme system and substrates (Fig. 4). This suggested that the serotonin oxidation product, TD, modified cellular proteins. Positive immunostains were also observed in cell lysates and some bands seemed to be selectively modified by TD in a dose-dependent manner (Fig. 5). The intensities of positive bands by immunostaining were not matched with those by protein staining, indicating that the adduction onto proteins was not random.


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

Kato Y, Ono S, Kitamoto N, Kettle AJ - Redox Biol (2014)

Immunocytostaining of neuroblastoma SH-SY5Y cells incubated with myeloperoxidase/acetaldehyde/xanthine oxidase/serotonin or synthetic TD. The cells were treated in serum-free medium for 30 min with the enzyme system (complete, A), the omission of the component (C–F), or 50 µM synthetic TD (G). The untreated cells are also shown in B. After the treatment, cells were fixed with methanol and permeabilized with 0.5% Triton X-100. The cells were then incubated with the antibody (1B7), followed by secondary antibody (FITC labeled, green). The nuclei were stained with peritoneum iodide (red). A, complete; B, control (untreated cells); C, without serotonin; D, without acetaldehyde; E, without myeloperoxidase; F, without xanthine oxidase; and G, TD (50 µM). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4215393&req=5

f0020: Immunocytostaining of neuroblastoma SH-SY5Y cells incubated with myeloperoxidase/acetaldehyde/xanthine oxidase/serotonin or synthetic TD. The cells were treated in serum-free medium for 30 min with the enzyme system (complete, A), the omission of the component (C–F), or 50 µM synthetic TD (G). The untreated cells are also shown in B. After the treatment, cells were fixed with methanol and permeabilized with 0.5% Triton X-100. The cells were then incubated with the antibody (1B7), followed by secondary antibody (FITC labeled, green). The nuclei were stained with peritoneum iodide (red). A, complete; B, control (untreated cells); C, without serotonin; D, without acetaldehyde; E, without myeloperoxidase; F, without xanthine oxidase; and G, TD (50 µM). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Mentions: TD may be a neurotoxin because it has a highly reactive quinone moiety. Proteins in SH-SY5Y neuroblastoma cells were modified by the serotonin oxidation system, which included the complete enzyme system and substrates (Fig. 4). This suggested that the serotonin oxidation product, TD, modified cellular proteins. Positive immunostains were also observed in cell lysates and some bands seemed to be selectively modified by TD in a dose-dependent manner (Fig. 5). The intensities of positive bands by immunostaining were not matched with those by protein staining, indicating that the adduction onto proteins was not random.

Bottom Line: In this work, we successively prepared a monoclonal antibody to quinone-modified proteins and found that the antibody preferentially recognizes the TD-thiol adduct.Pull-down analyses confirmed that the cytoskeletal proteins tubulins, vimentin, and neurofilament-L were modified.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.

View Article: PubMed Central - PubMed

Affiliation: School of Human Science and Environment, University of Hyogo, Hyogo 670-0092, Japan. Electronic address: yojikato@shse.u-hyogo.ac.jp.

No MeSH data available.


Related in: MedlinePlus