Dissecting the molecular control of endothelial NO synthase by caveolin-1 using cell-permeable peptides

Bernatchez, Pascal; Bauer, Philip M.; Yu, Jun; Prendergast, Jay S.; He, Pingnian; Sessa, William C.

doi:10.1073/pnas.0407224102

Cited by 174 publications

(213 citation statements)

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“…Colocalization of caveolin-1 and eNOS was only partial in these cells, being mainly restricted to the PM and the perinuclear region ( Figure 6, A-D, colocalization pink), consistent with previous reports in different cell lines (Sowa et al, 2001;Bernatchez et al, 2005). On SFV-driven overexpression of NOSTRIN-GFP, membrane staining of both eNOS and caveolin was lost, and the triad of proteins colocalized almost exclusively at intracellular structures ( Figure 6, E-H, triple colocalization white), indicating that caveolin could be involved in the NOSTRIN-induced translocation of eNOS.…”

Section: Nostrin-induced Translocation Of Enos Involves Caveolin-1supporting

confidence: 80%

“…On in vivo delivery of the caveolin scaffolding domain, the peptide is taken up into endothelial cells where it attenuates NOmediated effects (Bucci et al, 2000). In spite of this strong effect of caveolin-1 on eNOS, association of the two proteins is only partial in microscopic as well as biochemical analyses (Sowa et al, 2001;Bernatchez et al, 2005) and varies between different types of blood vessels (Andries et al, 1998), suggesting that the eNOS-caveolin interaction might be subject to further regulation. In general, caveolae are viewed as rather rigid and immobile structures (Mundy et al, 2002;Thomsen et al, 2002).…”

Section: Introductionmentioning

confidence: 99%

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Translocation of Endothelial Nitric-Oxide Synthase Involves a Ternary Complex with Caveolin-1 and NOSTRIN

Schilling

Opitz

Wiesenthal

et al. 2006

MBoC

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Recently, we characterized a novel endothelial nitric-oxide synthase (eNOS)-interacting protein, NOSTRIN (for eNOStrafficking inducer), which decreases eNOS activity upon overexpression and induces translocation of eNOS away from the plasma membrane. Here, we show that NOSTRIN directly binds to caveolin-1, a well-established inhibitor of eNOS. Because this interaction occurs between the N terminus of caveolin (positions 1-61) and the central domain of NOSTRIN (positions 323-434), it allows for independent binding of each of the two proteins to eNOS. Consistently, we were able to demonstrate the existence of a ternary complex of NOSTRIN, eNOS, and caveolin-1 in Chinese hamster ovary (CHO)-eNOS cells. In human umbilical vein endothelial cells (HUVECs), the ternary complex assembles at the plasma membrane upon confluence or thrombin stimulation. In CHO-eNOS cells, NOSTRIN-mediated translocation of eNOS involves caveolin in a process most likely representing caveolar trafficking. Accordingly, trafficking of NOSTRIN/eNOS/ caveolin is affected by altering the state of actin filaments or cholesterol levels in the plasma membrane. During caveolar trafficking, NOSTRIN functions as an adaptor to recruit mediators such as dynamin-2 essential for membrane fission. We propose that a ternary complex between NOSTRIN, caveolin-1, and eNOS mediates translocation of eNOS, with important implications for the activity and availability of eNOS in the cell. INTRODUCTIONEndothelial nitric-oxide synthase (eNOS) is the major enzyme generating nitric oxide (NO) in endothelial and epithelial cells (Ortiz and Garvin, 2003;Sessa, 2004). Because NO is an extremely reactive signaling molecule its production needs to be tightly regulated. Regulation seems to take place at three levels: direct interaction of eNOS with accessory proteins such as caveolin and Ca 2ϩ /calmodulin, reversible phosphorylation, and differential localization of the enzyme within cells. Subcellular distribution of eNOS is in part governed by lipid modification, i.e., myristoylation and dual palmitoylation, which bring about the association of the enzyme with the Golgi and plasma membrane (PM), respectively (Govers and Rabelink, 2001). Importantly, eNOS seems to be regulated by different modes in different subcellular locations, e.g., Ca 2ϩ /calmodulin stimulation is mainly effective at the PM, whereas Akt-driven activation is most pronounced at the Golgi (Fulton et al., 2004). Differential subcellular localization of eNOS is subject to dynamic regulation, e.g., after certain stimuli, the enzyme also occurs at vesicular structures throughout the cytoplasm (Nuszkowski et al., 2001;Thuringer et al., 2002). At present, however, it remains largely unknown how the differential distribution of eNOS to various subcellular locales is achieved. Emerging determinants of eNOS trafficking are eNOS-interacting proteins, which may guide eNOS to a distinct destination within the cell. In support of this model, we described two novel eNOS-interacting proteins termed NOSIP (for eNOS-inter...

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Section: Nostrin-induced Translocation Of Enos Involves Caveolin-1supporting

confidence: 80%

Section: Introductionmentioning

confidence: 99%

Translocation of Endothelial Nitric-Oxide Synthase Involves a Ternary Complex with Caveolin-1 and NOSTRIN

Schilling

Opitz

Wiesenthal

et al. 2006

MBoC

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“…Upon agonist (vascular endothelial growth factor acetylcholine)-induced increases in intracellular Ca 2+ , eNOS recruits calmodulin and HSP90, which facilitates the dissociation of Cav-1 from eNOS, resulting in the stimulus-response coupling of eNOS leading to the production of NO (20). We have shown that threonine 90,91 and especially phenylalanine 92 (T90,91,F92) in the Cav-1 scaffolding domain are responsible for inhibiting eNOS-derived NO release (21). Furthermore, this inhibitory domain is distinct from the binding domain because a mutated protein, F92A Cav-1, binds eNOS but fails to inhibit its activity (25,26).…”

mentioning

confidence: 99%

“…Furthermore, optimal NO release requires eNOS trafficking to caveolae, which are lipidenriched organelles at the plasma membrane known to maintain spatial organization of signaling complexes and participate in signal transduction events (20). Within caveolae, basal eNOS activity is maintained under tight inhibitory control by Caveolin-1 (Cav-1), the main coat protein of caveolae, through a direct protein-protein interaction between the scaffolding domain (amino acids 82-101) of Cav-1 and eNOS (21)(22)(23)(24). Upon agonist (vascular endothelial growth factor acetylcholine)-induced increases in intracellular Ca 2+ , eNOS recruits calmodulin and HSP90, which facilitates the dissociation of Cav-1 from eNOS, resulting in the stimulus-response coupling of eNOS leading to the production of NO (20).…”

mentioning

confidence: 99%

“…These observations revealed the strong inhibitory action of Cav-1 on eNOS and strengthened the concept of eNOS/Cav-1 antagonism: preventing eNOS binding to inhibitory Cav-1 results in increased basal NO release. Indeed, a cell-permeable, Cav-1-derived peptide with T90,91,F92 substituted to alanines, known as CavNOxin, can increase eNOS activity in reconstituted and endothelial cells (21,25) and reduce vascular tone ex vivo, an effect that was completely abolished in eNOS and Cav-1 knockout (KO) mice (25). These highly eNOS and Cav-1-specific biological functions of CavNOxin are the first to show increased NO release through regulated eNOS/Cav-1 interactions while preserving Cav-1-dependent biological activities such as caveolae formation (25).…”

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confidence: 99%

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Direct Endothelial Nitric Oxide Synthase Activation Provides Atheroprotection in Diabetes-Accelerated Atherosclerosis

et al. 2015

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Patients with diabetes have an increased risk of developing atherosclerosis. Endothelial dysfunction, characterized by the lowered bioavailability of endothelial NO synthase (eNOS)-derived NO, is a critical inducer of atherosclerosis. However, the protective aspect of eNOS in diabetes-associated atherosclerosis remains controversial, a likely consequence of its capacity to release both protective NO or deleterious oxygen radicals in normal and disease settings, respectively. Harnessing the atheroprotective activity of eNOS in diabetic settings remains elusive, in part due to the lack of endogenous eNOS-specific NO release activators. We have recently shown in vitro that eNOS-derived NO release can be increased by blocking its binding to Caveolin-1, the main coat protein of caveolae, using a highly specific peptide, CavNOxin. However, whether targeting eNOS using this peptide can attenuate diabetes-associated atherosclerosis is unknown. In this study, we show that CavNOxin can attenuate atherosclerotic burden by ∼84% in vivo. In contrast, mice lacking eNOS show resistance to CavNOxin treatment, indicating eNOS specificity. Mechanistically, CavNOxin lowered oxidative stress markers, inhibited the expression of proatherogenic mediators, and blocked leukocyte-endothelial interactions. These data are the first to show that endogenous eNOS activation can provide atheroprotection in diabetes and suggest that CavNOxin is a viable strategy for the development of antiatherosclerotic compounds.Diabetes is widely regarded as an independent risk factor for the development of cardiovascular diseases, with ;80% of cardiovascular mortality and morbidity being linked to macrovascular complications, such as atherosclerosis (1-3). The increased risk of development of vascular complications in individuals with type 1 (T1D) or type 2 diabetes (T2D) occurs despite intensive glycemic control, stressing the need for novel approaches to lessen the burden of diabetes-mediated macrovascular injury (4).The vascular endothelium plays a crucial role in diabetesassociated atherosclerosis through the regulation of vessel permeability, inflammation, coordination of leukocyte trafficking, and thrombosis (1,5). Indeed, the function of the vascular endothelium is significantly impaired during diabetes, a phenomena termed endothelial dysfunction and characterized by the reduced bioavailability of an important endothelial cell mediator, nitric oxide (NO). Such chronic attenuation of endothelial-derived NO release promotes platelet and leukocyte activation and adhesion, compromises endothelial cell barrier integrity, and causes the upregulation of proinflammatory genes (6-8). Moreover, reduced NO-dependent vasodilation and increased leukocyte adhesion to the endothelium, both of which are hallmarks of endothelial dysfunction, have been observed in patients with diabetes and diabetic animal models (9-12). Similarly, in cultured endothelial cells exposed to high glucose, lowered endothelial NO synthase (eNOS)-derived NO release was observed (13-15). In...

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Effects of Nitric Oxide on Atherosclerosis

Pong

Huang

2015

Atherosclerosis

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Dissecting the molecular control of endothelial NO synthase by caveolin-1 using cell-permeable peptides

Cited by 174 publications

References 21 publications

Translocation of Endothelial Nitric-Oxide Synthase Involves a Ternary Complex with Caveolin-1 and NOSTRIN

Translocation of Endothelial Nitric-Oxide Synthase Involves a Ternary Complex with Caveolin-1 and NOSTRIN

Direct Endothelial Nitric Oxide Synthase Activation Provides Atheroprotection in Diabetes-Accelerated Atherosclerosis

Effects of Nitric Oxide on Atherosclerosis

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