Dissecting the molecular control of endothelial NO synthase by caveolin-1 using cell-permeable peptides.
In endothelia, NO is synthesized by endothelial NO synthase (eNOS), which is negatively regulated by caveolin-1 (Cav-1), the primary coat protein of caveolae. We show that delivery of Cav-1 amino acids 82-101 (Cav) fused to an internalization sequence from Antennapedia (AP) blocks NO release in vitro and inflammation and tumor angiogenesis in vivo. To characterize the molecular mechanism by which the AP-Cav peptide and Cav-1 mediate eNOS inhibition, we subdivided the Cav portion of AP-Cav into three domains (Cav-A, -B, and -C), synthesized five overlapping peptides (AP-Cav-A, -AB, -B, -BC, and -C), and tested their effects on eNOS-dependent activities. Peptides containing the Cav-B domain (amino acids 89-95) induced time- and dose-dependent inhibition of eNOS-dependent NO release in cultured endothelial cells, NO-dependent inflammation in the ear, and hydraulic conductivity in isolated venules. Alanine scanning of AP-Cav-B revealed that Thr-90 and -91 (T90,91) and Phe-92 (F92) are crucial for AP-Cav-B- and AP-Cav-mediated inhibition of eNOS. Mutation of F92 to A92 in the Cav-1 cDNA caused the loss of eNOS inhibitory activity compared with wild-type Cav-1. These data highlight the importance of amino acids 89-95 and particularly F92 in mediating eNOS inhibition by AP-Cav and Cav-1.
Bernatchez
Pascal N
PN
Department of Pharmacology and Program in Vascular Cell Signaling and Therapeutics, Boyer Center for Molecular Medicine, Yale University School of Medicine, New Haven, CT 06536, USA.
Bauer
Philip M
PM
Yu
Jun
J
Prendergast
Jay S
JS
He
Pingnian
P
Sessa
William C
WC
D000595:Amino Acid Sequence; D000818:Animals; D000893:Anti-Inflammatory Agents / Q000138:chemical synthesis; D000970:Antineoplastic Agents / Q000138:chemical synthesis; D002417:Cattle; D051242:Caveolin 1; D022461:Caveolins / Q000502:physiology*; D002463:Cell Membrane Permeability; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism; D004730:Endothelium, Vascular / Q000166:cytology; D007700:Kinetics; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors*; D052250:Nitric Oxide Synthase Type III; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000494:pharmacology*; D011993:Recombinant Fusion Proteins
D000893:Anti-Inflammatory Agents; D000970:Antineoplastic Agents; D051242:Caveolin 1; D022461:Caveolins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III