Nanoimages show disruption of tubulin polymerization by chlorpyrifos oxon

Implications for neurotoxicity

Hasmik Grigoryan, Oksana Lockridge

Research output: Contribution to journalArticle

38 Citations (Scopus)

Abstract

Organophosphorus agents cause cognitive deficits and depression in some people. We hypothesize that the mechanism by which organophosphorus agents cause these disorders is by modification of proteins in the brain. One such protein could be tubulin. Tubulin polymerizes to make the microtubules that transport cell components to nerve axons. The goal of the present work was to measure the effect of the organophosphorus agent chlorpyrifos oxon on tubulin polymerization. An additional goal was to identify the amino acids covalently modified by chlorpyrifos oxon in microtubule polymers and to compare them to the amino acids modified in unpolymerized tubulin dimers. Purified bovine tubulin (0.1 mM) was treated with 0.005-0.1 mM chlorpyrifos oxon for 30 min at room temperature and then polymerized by addition of 1 mM GTP to generate microtubules. Microtubules were visualized by atomic force microscopy. Chlorpyrifos oxon-modified residues were identified by tandem ion trap electrospray ionization and matrix-assisted laser desorption/ionization mass spectrometry of tryptic peptides. Nanoimaging showed that low concentrations (0.005 and 0.01 mM) of chlorpyrifos oxon yielded short, thin microtubules. A concentration of 0.025 mM stimulated polymerization, while high concentrations (0.05 and 0.1 mM) caused aggregation. Of the 17 tyrosines covalently modified by chlorpyrifos oxon in unpolymerized tubulin dimers, only 2 tyrosines were labeled in polymerized microtubules. The two labeled tyrosines in polymerized tubulin were Tyr 103 in EDAANNY*R of alpha tubulin, and Tyr 281 in GSQQY*R of beta tubulin. In conclusion, chlorpyrifos oxon binding to tubulin disrupts tubulin polymerization. These results may lead to an understanding of the neurotoxicity of organophosphorus agents.

Original languageEnglish (US)
Pages (from-to)143-148
Number of pages6
JournalToxicology and Applied Pharmacology
Volume240
Issue number2
DOIs
StatePublished - Oct 15 2009

Fingerprint

Tubulin
Polymerization
Microtubules
Dimers
Tyrosine
O,O-diethyl O-3,5,6-trichloro-2-pyridyl phosphate
Amino Acids
Electrospray ionization
Atomic Force Microscopy
Matrix-Assisted Laser Desorption-Ionization Mass Spectrometry
Cellular Structures
Guanosine Triphosphate
Ionization
Mass spectrometry
Axons
Atomic force microscopy
Desorption
Brain
Polymers
Proteins

Keywords

  • Chlorpyrifos oxon
  • Mass spectrometry
  • Nanoimages
  • Tubulin polymerization

ASJC Scopus subject areas

  • Toxicology
  • Pharmacology

Cite this

Nanoimages show disruption of tubulin polymerization by chlorpyrifos oxon : Implications for neurotoxicity. / Grigoryan, Hasmik; Lockridge, Oksana.

In: Toxicology and Applied Pharmacology, Vol. 240, No. 2, 15.10.2009, p. 143-148.

Research output: Contribution to journalArticle

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abstract = "Organophosphorus agents cause cognitive deficits and depression in some people. We hypothesize that the mechanism by which organophosphorus agents cause these disorders is by modification of proteins in the brain. One such protein could be tubulin. Tubulin polymerizes to make the microtubules that transport cell components to nerve axons. The goal of the present work was to measure the effect of the organophosphorus agent chlorpyrifos oxon on tubulin polymerization. An additional goal was to identify the amino acids covalently modified by chlorpyrifos oxon in microtubule polymers and to compare them to the amino acids modified in unpolymerized tubulin dimers. Purified bovine tubulin (0.1 mM) was treated with 0.005-0.1 mM chlorpyrifos oxon for 30 min at room temperature and then polymerized by addition of 1 mM GTP to generate microtubules. Microtubules were visualized by atomic force microscopy. Chlorpyrifos oxon-modified residues were identified by tandem ion trap electrospray ionization and matrix-assisted laser desorption/ionization mass spectrometry of tryptic peptides. Nanoimaging showed that low concentrations (0.005 and 0.01 mM) of chlorpyrifos oxon yielded short, thin microtubules. A concentration of 0.025 mM stimulated polymerization, while high concentrations (0.05 and 0.1 mM) caused aggregation. Of the 17 tyrosines covalently modified by chlorpyrifos oxon in unpolymerized tubulin dimers, only 2 tyrosines were labeled in polymerized microtubules. The two labeled tyrosines in polymerized tubulin were Tyr 103 in EDAANNY*R of alpha tubulin, and Tyr 281 in GSQQY*R of beta tubulin. In conclusion, chlorpyrifos oxon binding to tubulin disrupts tubulin polymerization. These results may lead to an understanding of the neurotoxicity of organophosphorus agents.",
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