β-actin association with endothelial nitric-oxide synthase modulates nitric oxide and superoxide generation from the enzyme

Dmitry Kondrikov, Fabio V. Fonseca, Shawn Elms, David Fulton, Steven M. Black, Edward R. Block, Yunchao Su

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25 Scopus citations

Abstract

Protein-protein interactions represent an important post-translational mechanism for endothelial nitric-oxide synthase (eNOS) regulation. We have previously reported that β-actin is associated with eNOS oxygenase domain and that association of eNOS with β-actin increases eNOS activity and nitric oxide (NO) production. In the present study, we found that β-actin-induced increase in NO production was accompanied by decrease in superoxide formation. A synthetic actinbinding sequence (ABS) peptide 326 with amino acid sequence corresponding to residues 326-333 of human eNOS, one of the putative ABSs, specifically bound to β-actin and prevented eNOS association with β-actin in vitro. Peptide 326 also prevented β-actin-induced decrease in superoxide formation and increase in NO and L-citrulline production. A modified peptide 326 replacing hydrophobic amino acids leucine and tryptophan with neutral alanine was unable to interfere with eNOS-β-actin binding and to prevent β-actin-induced changes in NO and superoxide formation. Site-directed mutagenesis of the actin-binding domain of eNOS replacing leucine and tryptophan with alanine yielded an eNOS mutant that exhibited reduced eNOS-β-actin association, decreased NO production, and increased superoxide formation in COS-7 cells. Disruption of eNOS-β-actin interaction in endothelial cells using ABS peptide 326 resulted in decreased NO production, increased superoxide formation, and decreased endothelial monolayer wound repair, which was prevented by PEG-SOD and NO donor NOC-18. Taken together, this novel finding indicates that β-actin binding to eNOS through residues 326-333 in the eNOS protein results in shifting the enzymatic activity from superoxide formation toward NO production. Modulation of NO and superoxide formation from eNOS by β-actin plays an important role in endothelial function.

Original languageEnglish (US)
Pages (from-to)4319-4327
Number of pages9
JournalJournal of Biological Chemistry
Volume285
Issue number7
DOIs
Publication statusPublished - Feb 12 2010

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ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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