Endothelium derived nitric oxide is a potent vasodilator and anti- inflammatory autocoid that has profound effects on basic cellular functions including gene expression, cell migration, proliferation and differentiation. Previous studies have demonstrated that compartmentalization of endothelial nitric oxide synthase (eNOS) is negatively regulated by a direct interaction with a coat protein of endothelial caveolae, caveolin- I (CAV- 1) and is activated by a direct interaction with a newly identified NOS regulatory protein, heat shock protein-90 (Hsp 90). With this background in mind, it is hypothesized that endothelium-derived NO production is regulated by dynamic protein- protein interactions between eNOS, CAV- 1 and Hsp9O and such interactions are responsible for the compartmentalization of eNOS activation. To examine the regulation of these important interactions the following specific aims are proposed.
Aim 1 will elucidate the importance of eNOS/CAV- 1 interactions in cellular trafficking and activation eNOS.
Aim 2 will characterize the molecular interactions of eNOS with Hsp 90 and elucidate the signaling pathways required for stimulus dependent association of the two proteins.
Aim 3 will examine the interrelationships between eNOS, CAV- 1 and Hsp9O. In all of the above AIMS, molecular and cell biological approaches to elucidate eNOS trafficking, fatty acylation and protein-protein interactions will be combined with functional analysis of eNOS activity and NO release. Collectively this work will facilitate our understanding of the molecular machinery required for eNOS activation in EC. The long-term goal is to utilize this information to gain a better understanding of the mechanisms of endothelial dysfunction, a common manifestation of a variety of cardiovascular disorders
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