Beta-actin association with endothelial nitric-oxide synthase modulates nitric oxide and superoxide generation from the enzyme.
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.
Kondrikov
Dmitry
D
Department of Pharmacology and Toxicology, Medical College of Georgia, Augusta, Georgia 30912, USA.
Fonseca
Fabio V
FV
Elms
Shawn
S
Fulton
David
D
Black
Steven M
SM
Block
Edward R
ER
Su
Yunchao
Y
D000199:Actins / Q000378:metabolism*; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D002956:Citrulline / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D051379:Mice; D016297:Mutagenesis, Site-Directed; D009569:Nitric Oxide / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects / Q000502:physiology; D013481:Superoxides / Q000378:metabolism*
D000199:Actins; D010455:Peptides; D013481:Superoxides; D002956:Citrulline; D009569:Nitric Oxide; D052250:Nitric Oxide Synthase Type III