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UBC Theses and Dissertations

Identifying and characterizing caveolin-1 derived peptides : a novel approach to promoting nitric oxide release from the endothelium Trane, Andy E.


Cardiovascular diseases are one of the largest causes of mortality globally. One of the hallmarks of cardiovascular diseases is a reduction in systemic endothelial nitric oxide synthase (eNOS)-derived Nitric Oxide (NO), a critical regulator of vascular homeostasis. eNOS regulation is complex, involving phosphorylation and direct protein interactions. The main negative regulator is caveolin-1 (Cav-1), the homo-oligomeric coat protein of caveolae, which interacts with eNOS via its scaffolding domain (CAV). Studies have shown that alanine substitution of F92 in CAV can lead to abolishment of the inhibitory effect on eNOS; furthermore, CAV peptides with the F92A substitution can be used as an antagonist to promote basal eNOS-derived NO to reduce blood pressure and reduce cardiovascular disease progression. We hypothesized that identification of the eNOS binding motif in CAV could be used as the basis for a pharmacophore to develop antagonists aimed at increasing vascular NO. We performed a protein interaction study to identify a 10 residue ‘binding site peptide’ (BSP) in CAV that could account for the majority of eNOS binding. Both BSP and its F92A counterpart (BSPF92A) bound eNOS with similar affinity as the full CAV sequence as confirmed by polarization assay, while computational modeling suggested that the peptides inserted themselves in to a hydrophobic pocket in eNOS. While substitution of F92 prevents inhibition of activated eNOS, we found that both BSP and BSPF92A could promote basal NO release from resting endothelial cells (ECs), independent of cell permeabilization sequence used. Furthermore, BSP and BSPF92A generated NO in an eNOS and lipid raft dependent manner. Subsequently, we found that neither BSP nor BSPF92A affected basic biochemical properties of eNOS and Cav-1, such as oligomerization, subcellular targeting and co-localization. Instead, the presence of F92 was found to promote phosphorylation of eNOS, an important step in its activation. As a result of this finding, we have identified the basis for two different pharmacophores that increase NO in different manners. One that promotes activity indirectly (BSP) while the other one acts as an antagonist (BSPF92A). We hope to use this as the beginnings for a therapeutics development platform to promote cardioprotective NO.

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