Metabolism of nitric oxide: NOS3 activation and regulation

Nitric oxide (NO), a multifunctional second messenger, is implicated in physiological processes in mammals that range from immune response and potentiation of synaptic transmission to dilation of blood vessels and muscle relaxation. NO is a highly active molecule that diffuses across cell membranes and cannot be stored inside the producing cell. Its signaling capacity is controlled at the levels of biosynthesis and local availability. Its production by NO synthases is under complex and tight control, being regulated at transcriptional and translational levels, through co- and posttranslational modifications, and by subcellular localization. NO is synthesized from L-arginine by a family of nitric oxide synthases (NOS). Three NOS isoforms have been characterized: neuronal NOS (nNOS, NOS1) primarily found in neuronal tissue and skeletal muscle; inducible NOS (iNOS, NOS2) originally isolated from macrophages and later discovered in many other cell types; and endothelial NOS (eNOS, NOS3) present in vascular endothelial cells, cardiac myocytes, and in blood platelets. The enzymatic activity of all three isoforms is dependent on calmodulin, which binds to nNOS and eNOS at elevated intracellular calcium levels, while it is tightly associated with iNOS even at basal calcium levels. As a result, the enzymatic activity of nNOS and eNOS is modulated by changes in intracellular calcium levels, leading to transient NO production, while iNOS continuously releases NO independent of fluctuations in intracellular calcium levels and is mainly regulated at the gene expression level (Pacher et al. 2007).<p>The NOS enzymes share a common basic structural organization and requirement for substrate cofactors for enzymatic activity. A central calmodulin-binding motif separates an NH2-terminal oxygenase domain from a COOH-terminal reductase domain. Binding sites for cofactors NADPH, FAD, and FMN are located within the reductase domain, while binding sites for tetrahydrobiopterin (BH4) and heme are located within the oxygenase domain. Once calmodulin binds, it facilitates electron transfer from the cofactors in the reductase domain to heme enabling nitric oxide production. Both nNOS and eNOS contain an additional insert (40-50 amino acids) in the middle of the FMN-binding subdomain that serves as autoinhibitory loop, destabilizing calmodulin binding at low calcium levels and inhibiting electron transfer from FMN to the heme in the absence of calmodulin. iNOS does not contain this insert. <p>In this Reactome pathway module, details of eNOS activation and regulation are annotated. Originally identified as endothelium-derived relaxing factor, eNOS derived NO is a critical signaling molecule in vascular homeostasis. It regulates blood pressure and vascular tone, and is involved in vascular smooth muscle cell proliferation, platelet aggregation, and leukocyte adhesion. Loss of endothelium derived NO is a key feature of endothelial dysfunction, implicated in the pathogenesis of hypertension and atherosclerosis. The endothelial isoform eNOS is unique among the nitric oxide synthase (NOS) family in that it is co-translationally modified at its amino terminus by myristoylation and is further acylated by palmitoylation (two residues next to the myristoylation site). These modifications target eNOS to the plasma membrane caveolae and lipid rafts. <p>Factors that stimulate eNOS activation and nitric oxide (NO) production include fluid shear stress generated by blood flow, vascular endothelial growth factor (VEGF), bradykinin, estrogen, insulin, and angiopoietin. The activity of eNOS is further regulated by numerous post-translational modifications, including protein-protein interactions, phosphorylation, and subcellular localization. <p>Following activation, eNOS shuttles between caveolae and other subcellular compartments such as the noncaveolar plasma membrane portions, Golgi apparatus, and perinuclear structures. This subcellular distribution is variable depending upon cell type and mode of activation. <p>Subcellular localization of eNOS has a profound effect on its ability to produce NO as the availability of its substrates and cofactors will vary with location. eNOS is primarily particulate, and depending on the cell type, eNOS can be found in several membrane compartments: plasma membrane caveolae, lipid rafts, and intracellular membranes such as the Golgi complex.

一氧化氮（NO），作为一种多功能性第二信使，在哺乳动物体内参与多种生理过程，其范围涵盖免疫反应、突触传递的增强、血管舒张以及肌肉松弛。NO是一种高度活跃的分子，能够穿过细胞膜，且不能在其产生细胞内储存。其信号传导能力在生物合成和局部可用性层面受到调控。NO合酶的生产过程受到复杂而严格的控制，通过转录和翻译水平的调控、共翻译和后翻译修饰以及亚细胞定位进行调节。NO由一氧化氮合酶（NOS）家族通过L-精氨酸合成。已鉴定出三种NOS异构体：神经元型NOS（nNOS，NOS1），主要存在于神经元组织和骨骼肌中；诱导型NOS（iNOS，NOS2），最初从巨噬细胞中分离出来，后来在许多其他细胞类型中也被发现；以及内皮型NOS（eNOS，NOS3），存在于血管内皮细胞、心肌细胞和血小板中。三种异构体的酶活性均依赖于钙调蛋白，钙调蛋白在细胞内钙离子浓度升高时与nNOS和eNOS结合，而在基础钙离子浓度时则与iNOS紧密关联。因此，nNOS和eNOS的酶活性受细胞内钙离子水平变化的影响，导致NO的短暂产生，而iNOS则独立于细胞内钙离子水平的波动持续释放NO，其主要调节机制在基因表达水平（Pacher et al. 2007）。

NOS酶类共享一种基本的结构组织和底物辅助因子对酶活性的需求。一个中央的钙调蛋白结合基序将氨基末端氧合酶结构域与羧基末端还原酶结构域分开。辅因子NADPH、FAD和FMN的结合位点位于还原酶结构域内，而四氢生物蝶呤（BH4）和血红素的结合位点位于氧合酶结构域内。一旦钙调蛋白结合，它将促进还原酶结构域中的辅因子向血红素转移电子，从而实现一氧化氮的产生。nNOS和eNOS在FMN结合亚结构域的中间部分包含一个额外的插入序列（40-50个氨基酸），该序列作为自抑制环，在低钙离子水平时破坏钙调蛋白的结合，并抑制在无钙调蛋白的情况下FMN向血红素的电子转移。iNOS不含有此插入序列。

在本Reactome通路模块中，详细描述了eNOS的激活和调控。最初被识别为内皮源性舒张因子，eNOS产生的NO是血管稳态中的关键信号分子。它调节血压和血管张力，并参与血管平滑肌细胞增殖、血小板聚集和白细胞粘附。内皮源性NO的丢失是内皮功能障碍的关键特征，与高血压和动脉粥样硬化的发病机制有关。在内皮型NOS（eNOS）是NOS家族中独特的，它在氨基末端通过肉豆蔻酰化进行共翻译修饰，并且进一步通过棕榈酰化（两个位于肉豆蔻酰化位点旁边的残基）进行酰化。这些修饰将eNOS靶向到质膜微囊泡和脂筏。

刺激eNOS激活和一氧化氮（NO）产生的因素包括血流产生的流体切应力、血管内皮生长因子（VEGF）、缓激肽、雌激素、胰岛素和血管生成素。eNOS的活性还受到多种翻译后修饰的调节，包括蛋白质-蛋白质相互作用、磷酸化和亚细胞定位。

激活后，eNOS在微囊泡和其他亚细胞区室之间穿梭，如非微囊泡的质膜部分、高尔基体和核周结构。这种亚细胞分布取决于细胞类型和激活方式。

eNOS的亚细胞定位对其产生NO的能力有深远影响，因为其底物和辅因子的可用性将随位置而变化。eNOS主要存在于颗粒中，并且根据细胞类型，eNOS可以存在于几个膜区室中：质膜微囊泡、脂筏和细胞内膜，如高尔基体复合物。