A noninhibitory mutant of the caveolin-1 scaffolding domain enhances eNOS-derived NO synthesis and vasodilation in mice
Pascal Bernatchez, Arpeeta Sharma, Philip M. Bauer, Ethan Marin, William C. Sessa
Pascal Bernatchez, Arpeeta Sharma, Philip M. Bauer, Ethan Marin, William C. Sessa
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Research Article Cell biology

A noninhibitory mutant of the caveolin-1 scaffolding domain enhances eNOS-derived NO synthesis and vasodilation in mice

Abstract

Aberrant regulation of eNOS and associated NO release are directly linked with various vascular diseases. Caveolin-1 (Cav-1), the main coat protein of caveolae, is highly expressed in endothelial cells. Its scaffolding domain serves as an endogenous negative regulator of eNOS function. Structure-function analysis of Cav-1 has shown that phenylalanine 92 (F92) is critical for the inhibitory actions of Cav-1 toward eNOS. Herein, we show that F92A–Cav-1 and a mutant cell–permeable scaffolding domain peptide called Cavnoxin can increase basal NO release in eNOS-expressing cells. Cavnoxin reduced vascular tone ex vivo and lowered blood pressure in normal mice. In contrast, similar experiments performed with eNOS- or Cav-1–deficient mice showed that the vasodilatory effect of Cavnoxin is abolished in the absence of these gene products, which indicates a high level of eNOS/Cav-1 specificity. Mechanistically, biochemical assays indicated that noninhibitory F92A–Cav-1 and Cavnoxin specifically disrupted the inhibitory actions of endogenous Cav-1 toward eNOS and thereby enhanced basal NO release. Collectively, these data raise the possibility of studying the inhibitory influence of Cav-1 on eNOS without interfering with the other actions of endogenous Cav-1. They also suggest a therapeutic application for regulating the eNOS/Cav-1 interaction in diseases characterized by decreased NO release.

Authors

Pascal Bernatchez, Arpeeta Sharma, Philip M. Bauer, Ethan Marin, William C. Sessa

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Figure 2

F92A and WT Cav-1 show similar biochemical properties.

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F92A and WT Cav-1 show similar biochemical properties. (A) BAECs were in...
(A) BAECs were infected with adenoviruses (25 MOI) encoding for WT (myc tagged) and F92A–Cav-1 (HA tagged) for 48 hours and lysates subjected to sucrose gradient fractionation. Top panel depicts relative distribution of proteins across the gradient (quantified by densitometry), and bottom panels show levels of endogenous Cav-1 (top), WT Cav-1 (myc), F92A–Cav-1 (HA), and HSP90 as a control Immunoblot analysis from fractions collected. Fractions 2 to 5 represent buoyant, CEM fractions, and fractions 8–12 bulk proteins with HSP90 as a marker. (B) HEK293 cells were transfected with WT and F92A–Cav-1 cDNAs and lysates subjected to sucrose fractionation and Western blotting as in A. (C) BAECs were infected as described in A, lysed, and subjected to velocity gradient centrifugation to compare the relative MW of Cav-1 container oligomers. (quantified by densitometry above). Gradients were calibrated with known MW standards. Fractions were blotted for endogenous Cav-1 (top), WT Cav-1 (middle), and F92A–Cav-1 (bottom). The peak level of Cav-1 was in fraction 6 consistent with greater than 10 monomers of Cav-1 (each monomers in 22 kDa). (D) Expression of WT and F92A–Cav-1 cDNAs in HEK293 cells that express low levels of endogenous Cav-1. Samples were processed as described in C. Similar results were obtained in 2 additional experiments.