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

Abstract

Protein-protein interactions represent an important post-translational mechanism for endothelial nitric-oxide synthase (eNOS) regulation. We have previously reported that beta-actin is associated with eNOS oxygenase domain and that association of eNOS with beta-actin increases eNOS activity and nitric oxide (NO) production. In the present study, we found that beta-actin-induced increase in NO production was accompanied by decrease in superoxide formation. A synthetic actin-binding sequence (ABS) peptide 326 with amino acid sequence corresponding to residues 326-333 of human eNOS, one of the putative ABSs, specifically bound to beta-actin and prevented eNOS association with beta-actin in vitro. Peptide 326 also prevented beta-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-beta-actin binding and to prevent beta-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-beta-actin association, decreased NO production, and increased superoxide formation in COS-7 cells. Disruption of eNOS-beta-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 beta-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 beta-actin plays an important role in endothelial function.

• Authors: Dmitry Kondrikov, Fabio V Fonseca, Shawn Elms, David Fulton, Steven M Black, Edward R Block, Yunchao Su
• Journal: The Journal of biological chemistry (J Biol Chem) Vol. 285 Issue 7 Pg. 4319-27 (Feb 12 2010) ISSN: 1083-351X [Electronic] United States
• PMID: 19946124 (Publication Type: Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't)

Chemical References

• Actins
• Peptides
• Superoxides
• Citrulline
• Nitric Oxide
• Nitric Oxide Synthase Type III

Topics

• Actins (metabolism)
• Animals
• COS Cells
• Chlorocebus aethiops
• Citrulline (metabolism)
• Humans
• Immunoprecipitation
• Mice
• Mutagenesis, Site-Directed
• Nitric Oxide (metabolism)
• Nitric Oxide Synthase Type III (chemistry, genetics, metabolism)
• Peptides (chemical synthesis, chemistry, pharmacology)
• Protein Binding (drug effects, physiology)
• Superoxides (metabolism)