Nitric oxide synthase generates nitric oxide locally to regulate compartmentalized protein S-nitrosylation and protein trafficking

Abstract

Nitric oxide (NO) is a highly diffusible and short-lived physiological messenger. Despite its diffusible nature, NO modifies thiol groups of specific cysteine residues in target proteins and alters protein function via S-nitrosylation. Although intracellular S-nitrosylation is a specific posttranslational modification, the defined localization of an NO source (nitric oxide synthase, NOS) with protein Snitrosylation has never been directly demonstrated. Endothelial NOS (eNOS) is localized mainly on the Golgi apparatus and in plasma membrane caveolae. Here, we show by using eNOS targeted to either the Golgi or the nucleus that S-nitrosylation is concentrated at the primary site of eNOS localization. Furthermore, localization of eNOS on the Golgi enhances overall Golgi protein S-nitrosylation, the specific S-nitrosylation of N-ethylmaleimidesensitive factor and reduces the speed of protein transport from the endoplasmic reticulum to the plasma membrane in a reversible manner. These data indicate that local NOS action generates organelle-specific protein S-nitrosylation reactions that can regulate intracellular transport processes.endothelial nitric oxide synthase ͉ Golgi ͉ targeting N itric oxide (NO), produced by the nitric oxide synthase (NOS) family of proteins, regulates a variety of important physiological responses, including vasodilation, respiration, cell migration, and apoptosis (1-5). NO has been considered to mediate these responses by activating the primary NO effector soluble guanylyl cyclase to produce cGMP (6) or by NO-based chemical modifications of proteins or perhaps lipids. One clear example of cGMPindependent actions of NO is protein thiol group modification by NO known as S-nitrosylation (7). This posttranslational control mechanism regulates important physiological activities of proteins in response to endogenously or exogenously generated NO (8). Thus, S-nitrosylation of proteins is an emerging area of investigation for NO-mediated physiological responses (8).NO is a lipophillic, highly diffusible, and short-lived physiological messenger (9). On the one hand, NO is thought to diffuse over a wide area (100 m), moving freely through membranes of neighboring cells (9). On the other hand, given the apparent promiscuity of NO, the question arises as to how S-nitrosylation might occur in a precisely regulated manner, i.e., protein S-nitrosylation occurs on specific thiol residues in proteins that are targeted to specific organelles in cells (10), and low concentrations of NO activate the ryanodine receptor via thiol modification, whereas higher concentrations inhibit the receptor (11). There are several compelling arguments in favor of the concept that all sources of NO are not bioequivalent. (i) In cardiac myocytes, which express two forms of NOS, gene deletion of either neuronal NOS or endothelial NOS (eNOS) exerts isoform-specific phenotypes, arguing that the source of NO favors local control of different cellular functions (12). Moreover, in many circumstances, the actions of endogenously gener...

Redox-based regulation of signal transduction: Principles, pitfalls, and promises

“…NOS enzyme) [18], IV) reversibility; and V) enzymatic control (e.g. S-nitrosoglutathione; SNO) reductase ( [1,19]) controls S-nitrosothiol tone [9,20]).…”

“…Stimulation of growth factor receptors, toll like receptors (TLR), and cytokine receptors, also leads to the activation of signaling cascades that are regulated by hydrogen peroxide (H 2 O 2 ) as a result of activation of non-phagocytic oxidases (NOX), or dual oxidase (DUOX) (See Table 1) [18,19,[21][22][23][24]. The expression of multiple isoforms of these enzymes in a variety of tissues provides evidence that the deliberate production of low levels of oxidants is a feature of many cells [25].…”

Vascular biology and pathobiology of the liver: Report of a single-topic symposium

“…eNOS-based nitrosylation at sites of eNOS localization is important for EC biology because nitrosylation of specific targets has recently been demonstrated to regulate exocytosis in ECs. 1,10 Collectively, these observations suggest that the location of NOS determines S-nitrosylation of specific proteins by creating a high NO concentration near the target proteins, thereby favoring the S-nitrosylation reaction. 10 Model Organisms for the Study of Vascular Biology.…”

“…1,10 Collectively, these observations suggest that the location of NOS determines S-nitrosylation of specific proteins by creating a high NO concentration near the target proteins, thereby favoring the S-nitrosylation reaction. 10 Model Organisms for the Study of Vascular Biology. The zebrafish (Danio rerio) is a powerful model organism for the study of vascular biology due to a rapid development cycle that is easily visualized and readily amenable to morpholino-based gene knockdown technology.…”

Dynamic regulation of endothelial NOS mediated by competitive interaction with α‐actinin‐4 and calmodulin

“…As a highly lipophilic and diffusible molecule, the subcellular localization of eNOS is critical for its function, both intercellularly and intracellularly (26). Indeed, eNOS that is restricted to the plasma membrane appears to be more sensitive to calcium-and Akt-dependent agonists than is Golgi-localized eNOS (27).…”