Affinage

NOS3

Nitric oxide synthase 3 · UniProt P29474

Round 2 corrected
Length
1203 aa
Mass
133.3 kDa
Annotated
2026-04-29
130 papers in source corpus 40 papers cited in narrative 39 extracted findings

Mechanistic narrative

Synthesis pass · prose summary of the discoveries below

NOS3 (eNOS) is a constitutive, Ca²⁺/calmodulin-dependent homodimeric enzyme that catalyzes the conversion of L-arginine to nitric oxide (NO) and citrulline in vascular endothelium and other cell types, serving as a central regulator of vascular tone, permeability, metabolism, and redox homeostasis (PMID:1378832, PMID:1379542). Its activity is governed by multisite phosphorylation—Akt/PKB-mediated Ser1177 phosphorylation activates the enzyme in a Ca²⁺-independent manner, while constitutive PKC/ROCK-mediated Thr495 phosphorylation inhibits calmodulin binding—and the balance of these marks, rather than either alone, determines NO output, as live-cell biosensor studies show that Ser1177 phosphorylation is insufficient to predict actual NO synthesis (PMID:10376603, PMID:11397791, PMID:31527268). Subcellular targeting to plasma membrane caveolae (via N-myristoylation, palmitoylation, and caveolin-1 interaction) and a less active trans-Golgi network pool creates spatially distinct NO signaling compartments with separable vascular functions (PMID:9188442, PMID:32152543, PMID:16679407). BH4 cofactor availability and redox post-translational modifications—S-glutathionylation of reductase-domain cysteines and S-nitrosylation at Cys94/Cys99—reversibly uncouple eNOS toward superoxide/peroxynitrite production, a pathological switch operative in hypertension, diabetes, and aging (PMID:21179168, PMID:16763164, PMID:29471036).

Mechanistic history

Synthesis pass · year-by-year structured walk · 14 steps
  1. 1992 High

    Molecular cloning of eNOS from human endothelium established it as a constitutive, Ca²⁺/calmodulin-dependent enzyme that converts L-arginine to NO and activates soluble guanylate cyclase, resolving the molecular identity of the endothelium-derived NO synthase.

    Evidence cDNA cloning, functional expression with L-arginine→citrulline conversion, co-culture guanylate cyclase reporter assay in two independent studies

    PMID:1378832 PMID:1379542

    Open questions at the time
    • Enzyme regulation beyond Ca²⁺/CaM not addressed
    • No information on post-translational modifications
    • No structural data
  2. 1993 High

    Characterization of the NOS3 gene structure and promoter revealed a TATA-less, Sp1/GATA-driven promoter with putative shear-stress and sterol-regulatory elements, providing the first framework for understanding how hemodynamic forces regulate eNOS transcription.

    Evidence Genomic cloning, FISH mapping to 7q35–36, promoter deletion/reporter assays

    PMID:7688726

    Open questions at the time
    • Functional significance of shear-stress elements not tested in vivo
    • No identification of trans-acting transcription factors beyond Sp1/GATA
  3. 1997 High

    Discovery that caveolin-1 tonically inhibits eNOS in a Ca²⁺/calmodulin-displaceable manner established the first protein–protein regulatory mechanism for eNOS and linked its activity to caveolar membrane microdomains.

    Evidence Co-immunoprecipitation, caveolin-1 overexpression, calmodulin displacement, NOS activity assays in endothelial cells

    PMID:8910295 PMID:9188442

    Open questions at the time
    • Structural basis of caveolin-1–eNOS interaction not resolved
    • Relative contribution of caveolar versus non-caveolar eNOS pools to NO output unclear
  4. 1998 High

    Crystal structures of NOS oxygenase domains revealed how BH4 and a zinc tetrathiolate stabilize the dimer interface and active-site channel, providing a structural foundation for understanding cofactor-dependent catalysis and the concept of eNOS uncoupling.

    Evidence X-ray crystallography at 2.4 Å (human eNOS) and iNOS oxygenase dimer with BH4/L-arginine co-crystals

    PMID:10074942 PMID:9516116

    Open questions at the time
    • Full-length eNOS structure including reductase domain not determined
    • Structural basis of electron transfer between reductase and oxygenase domains unresolved
  5. 1998 High

    Identification of Hsp90 as a dynamically recruited eNOS activator showed that chaperone association is required for agonist-stimulated NO production and vascular relaxation, adding a protein quality-control dimension to eNOS regulation.

    Evidence Co-immunoprecipitation after VEGF/histamine/shear stimulation, geldanamycin inhibition, isolated vessel relaxation

    PMID:9580552

    Open questions at the time
    • How Hsp90 promotes electron transfer or prevents uncoupling mechanistically undefined
    • Stoichiometry and binding interface with eNOS not resolved
  6. 1999 High

    The landmark discovery that Akt/PKB phosphorylates eNOS at Ser1177, rendering it active at sub-physiological Ca²⁺, established phosphorylation as a primary Ca²⁺-independent activation mechanism and connected eNOS to PI3K growth-factor signaling.

    Evidence In vitro kinase assay, S1177A mutagenesis, PI3K inhibitor, shear-stress stimulation in endothelial cells

    PMID:10376603

    Open questions at the time
    • Relative contribution of different Ser1177 kinases (Akt, AMPK, CaMKII, PKA) in specific physiological contexts not delineated
    • Phosphorylation-independent activation modes not addressed
  7. 2001 High

    Identification of Thr495 as a constitutively phosphorylated inhibitory site that blocks calmodulin binding established the dual-phosphorylation model: maximal eNOS activation requires coordinate Ser1177 phosphorylation and Thr495 dephosphorylation.

    Evidence Phosphospecific antibodies, PKC/PP1 inhibitors, T495A/T495D mutagenesis, calmodulin co-IP in endothelial cells

    PMID:11397791

    Open questions at the time
    • Identity of phosphatase(s) responsible for stimulus-specific Thr495 dephosphorylation incompletely characterized
    • Interplay between Thr495 and other inhibitory modifications (O-GlcNAc, S-nitrosylation) not integrated
  8. 2001 High

    Demonstration that hyperglycemia promotes O-GlcNAc modification at Ser1177, competing with activating Akt phosphorylation, provided the first mechanistic link between diabetic metabolic stress and eNOS inactivation.

    Evidence O-GlcNAc immunoblot, S1177 mutagenesis, GFAT antisense, MnSOD/UCP-1 overexpression in endothelial cells and diabetic mouse aorta

    PMID:11696579

    Open questions at the time
    • Quantitative stoichiometry of O-GlcNAc vs phosphorylation at Ser1177 in vivo not determined
    • Whether other eNOS residues undergo O-GlcNAcylation unknown
  9. 2006 High

    In vivo evidence that BH4 depletion causes eNOS to generate peroxynitrite instead of NO in diabetic mice established cofactor availability as the master switch of eNOS coupling state, with direct therapeutic implications.

    Evidence eNOS overexpression in db/db mice worsened hepatic I-R injury; BH4/sepiapterin supplementation recoupled eNOS and was protective

    PMID:16763164

    Open questions at the time
    • Relative contributions of BH4 oxidation vs de novo synthesis deficits not separated
    • Whether recoupling persists long-term in chronic disease not tested
  10. 2007 High

    In vitro kinase reconstitution showing ROCK directly phosphorylates eNOS at Thr495 identified a second kinase writer for this inhibitory site, connecting RhoA/ROCK vascular contractile signaling to eNOS inactivation.

    Evidence Constitutively active ROCK + recombinant eNOS in vitro kinase assay, Y27632 inhibitor in HUVECs, phosphospecific Western blot

    PMID:17651694

    Open questions at the time
    • Relative ROCK vs PKC contribution to Thr495 phosphorylation in specific vascular beds undefined
    • Whether ROCK phosphorylates other eNOS residues not tested
  11. 2010 High

    Discovery that S-glutathionylation of reductase-domain cysteines reversibly uncouples eNOS toward superoxide production in hypertension established a redox post-translational switch distinct from BH4 depletion-driven uncoupling.

    Evidence Site-directed Cys mutagenesis, EPR superoxide detection, vessel relaxation rescued by thiol-reducing agents, elevated S-glutathionylation in hypertensive vessels

    PMID:21179168

    Open questions at the time
    • Enzymes catalyzing eNOS S-glutathionylation/deglutathionylation not identified
    • Whether S-glutathionylation and BH4 loss cooperate or act independently unresolved
  12. 2019 High

    Live-cell multispectral biosensor imaging revealed that VEGF and insulin induce robust eNOS Ser1177 phosphorylation without detectable NO production, fundamentally demonstrating that phosphorylation status alone does not predict eNOS activation and that additional regulatory inputs are required.

    Evidence Genetically encoded NO and Ca²⁺ biosensors, chemogenetic eNOS activation, phosphospecific Western blot in endothelial cells

    PMID:31527268

    Open questions at the time
    • Identity of the additional regulatory input(s) gating NO production downstream of phosphorylation not determined
    • Whether this discordance operates in all endothelial beds unknown
  13. 2020 High

    Subcellular NO probes quantitatively demonstrated that Golgi-localized eNOS, though tenfold less active than plasma membrane eNOS, is required for Golgi structural integrity, establishing that spatially distinct eNOS pools serve non-overlapping cellular functions.

    Evidence DNA nanotechnology-based NO probes targeted to plasma membrane vs TGN, pharmacological eNOS inhibition, Golgi morphology assay in live cells

    PMID:32152543

    Open questions at the time
    • Molecular target(s) of Golgi-derived NO responsible for organelle maintenance unidentified
    • How eNOS trafficking between PM and Golgi is dynamically regulated remains unclear
  14. 2023 High

    Genetic dissection of the VEGFR2-Y1173/PLCγ/Ca²⁺/eNOS axis in knockin mice showed that eNOS-derived reactive nitrogen species nitrate and activate Src, which then phosphorylates VE-cadherin to drive junction disassembly, placing eNOS as a required intermediate in VEGF-induced vascular permeability.

    Evidence Vegfr2 Y1173F knockin mice, endothelial-specific Plcg1 KO, tyrosine nitration assay, VE-cadherin Y685 phosphorylation, vascular permeability assay

    PMID:37651195

    Open questions at the time
    • Whether NO or peroxynitrite mediates Src nitration not distinguished
    • Applicability beyond VEGF-driven permeability to other permeability-inducing stimuli not tested

Open questions

Synthesis pass · forward-looking unresolved questions
  • A full-length eNOS structure including the reductase domain, the structural basis for coupling/uncoupling transitions, and the identity of the regulatory gate that explains why phosphorylation at Ser1177 is necessary but insufficient for NO production remain unresolved.
  • No full-length eNOS structural model at atomic resolution
  • The additional regulatory input(s) beyond Ser1177 phosphorylation and Ca²⁺ that gate NO production are unknown
  • In vivo quantitative contributions of each uncoupling mechanism (BH4 loss, S-glutathionylation, S-nitrosylation) in specific disease states not determined

Mechanism profile

Synthesis pass · controlled-vocabulary classification · explore literature graph →
Molecular activity
GO:0016491 oxidoreductase activity 4 GO:0140096 catalytic activity, acting on a protein 2
Localization
GO:0005886 plasma membrane 4 GO:0005794 Golgi apparatus 2 GO:0005829 cytosol 1
Pathway
R-HSA-162582 Signal Transduction 4 R-HSA-109582 Hemostasis 3 R-HSA-1643685 Disease 3 R-HSA-382551 Transport of small molecules 1
Partners
AKT1ARRB2CAV1CTNNB1GIT1HSP90AA1KDRROCK1

Evidence

Reading pass · 39 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
1992 Human endothelial NOS (eNOS/NOS3) was cloned; it encodes a 1,203–1,294 amino acid calcium/calmodulin-dependent enzyme that converts L-arginine to NO and citrulline, is constitutively expressed in vascular endothelium, and produces NO that activates guanylate cyclase in co-culture assays. cDNA cloning, Northern blot, NADPH diaphorase histochemistry, L-arginine→citrulline conversion assay, co-culture guanylate cyclase reporter assay The Journal of biological chemistry / FEBS letters High 1378832 1379542
1993 The human NOS3 gene spans ~21 kb, contains 26 exons, maps to chromosome 7q35–7q36, has a TATA-less promoter with Sp1/GATA motifs, and contains putative shear-stress and sterol-regulatory cis-elements in its 5′-flanking region. Genomic cloning, Southern blot of somatic cell hybrids, fluorescence in situ hybridization, promoter deletion/reporter assays The Journal of biological chemistry High 7688726
1996 eNOS is tyrosine-phosphorylated in endothelial cells; increased tyrosine phosphorylation (induced by H₂O₂ or vanadate) decreases eNOS specific activity by ~50%. eNOS co-immunoprecipitates with caveolin-1, identifying caveolin-1 as the first eNOS-associated protein. ³²P metabolic labeling, phosphoamino acid analysis, phosphotyrosine Western blot, immunoNOS activity assay, co-immunoprecipitation The Journal of biological chemistry High 8910295
1997 Caveolin-1 tonically inhibits eNOS in a reciprocal, Ca²⁺-calmodulin-dependent manner: calmodulin addition disrupts the eNOS–caveolin complex in a Ca²⁺-dependent fashion; caveolin overexpression suppresses eNOS activity (not other NOS isoforms), and this inhibition is reversed by exogenous calmodulin. Co-immunoprecipitation, caveolin overexpression, in vitro calmodulin displacement, NOS activity assay The Journal of biological chemistry High 9188442
1998 Crystal structure of the NOS oxygenase dimer reveals how BH4 and L-arginine complete the catalytic center: BH4 binding refolds the dimer interface, creates a 30 Å active-center channel, causes a 35° helical tilt exposing heme edge, and heme propionate interactions with pterin and L-Arg suggest pterin exerts electronic influence on heme-bound oxygen activation. X-ray crystallography (iNOS oxygenase dimer structure used as the mechanistic template; eNOS structural comparison) Science High 9516116
1998 Hsp90 dynamically associates with eNOS and is rapidly recruited to the eNOS complex upon stimulation by VEGF, histamine, or shear stress; Hsp90 binding enhances eNOS activation, and pharmacological disruption of Hsp90 signaling attenuates agonist-stimulated NO production and endothelium-dependent vascular relaxation. Co-immunoprecipitation, Hsp90 inhibitor (geldanamycin) treatment, NOS activity assay, isolated vessel relaxation Nature High 9580552
1998 Crystal structure of human eNOS catalytic domain (2.4 Å) shows an unexpected structural zinc coordinated by four cysteines (two per monomer) at the dimer interface; active-site residues including a conserved glutamate (E361) bind L-arginine; active-site architecture is nearly identical between eNOS and iNOS, providing basis for selective inhibitor design. X-ray crystallography (2.4 Å resolution, co-crystal with L-arginine) Nature structural biology High 10074942
1998 VEGF upregulates eNOS mRNA and protein in human endothelial cells, producing a biphasic (acute 1 h and chronic >24 h) stimulation of NO production; increased eNOS enzyme content accounts for the chronic phase. Western blot, Northern blot, nitric oxide metabolite measurement, ionophore challenge The American journal of physiology Medium 9530221
1999 Akt/PKB directly phosphorylates eNOS at Ser1177; this phosphorylation activates eNOS in a Ca²⁺-independent manner and renders enzyme activity maximal at sub-physiological Ca²⁺ concentrations. PI3K/Akt pathway inhibition or Ser1177 mutation abrogates shear-stress-induced eNOS activation and NO production. In vitro kinase assay, site-directed mutagenesis (S1177A), PI3K inhibitor, eNOS phosphorylation Western blot, NO measurement Nature High 10376603
1999 The T-786C mutation in the eNOS promoter significantly reduces eNOS gene promoter activity as measured by luciferase reporter assays, and this variant is strongly associated with coronary spasm, linking reduced NO synthesis to vasospastic disease. Luciferase reporter gene assay, genetic association study Circulation Medium 10359729
2001 eNOS Thr495 in the calmodulin-binding domain is constitutively phosphorylated by PKC and is rapidly dephosphorylated by PP1 after bradykinin stimulation; dephosphorylation of Thr495 promotes calmodulin binding and enables subsequent CaMKII-mediated Ser1177 phosphorylation. The dual phosphorylation state (high Ser1177 + low Thr495) determines maximal agonist-induced NO output. T495A mutation increases CaM binding basally; T495D mutation nearly abolishes CaM binding. Phosphospecific Western blot, kinase/phosphatase inhibitors (Ro 31-8220, calyculin A, KN-93), site-directed mutagenesis, calmodulin co-immunoprecipitation Circulation research High 11397791
2001 Hyperglycemia inhibits eNOS activity by promoting O-linked N-acetylglucosamine (O-GlcNAc) modification at Ser1177, competing with activating phosphorylation at the same Akt site; antisense inhibition of GFAT or blockade of mitochondrial superoxide reversed both O-GlcNAc modification and eNOS inhibition. O-GlcNAc immunoblot, phosphospecific Western blot, antisense GFAT, UCP-1/MnSOD overexpression, S1177 mutant eNOS, citrulline activity assay The Journal of clinical investigation High 11696579
2002 Rho GTPase/ROCK pathway negatively regulates eNOS phosphorylation at Ser1177 by suppressing PKB/Akt activation; constitutively active PKB rescues eNOS phosphorylation but not eNOS gene expression in RhoA/ROCK-expressing cells, revealing two separable regulatory arms. Adenovirus-mediated gene transfer, phosphospecific Western blot, NO measurement, dominant-negative/constitutively active constructs Molecular and cellular biology High 12446767
2002 eNOS is targeted to caveolae by co-translational N-myristoylation and post-translational palmitoylation; caveolin-1 interacts with eNOS via its scaffolding domain and inhibits NOS activity; this inhibition is reversed by excess Ca²⁺/calmodulin and by Akt-induced Ser1177 phosphorylation. Triton-insoluble fraction isolation, co-immunoprecipitation, lipid modification mutants, NOS activity assay American journal of physiology. Renal physiology High 12060581
2003 Gene transfer of eNOS selectively to the thick ascending limb (THAL) of eNOS-knockout mice (using an NKCC2 promoter-driven adenovirus) restores L-arginine-induced NO production and inhibition of NaCl absorption, demonstrating that eNOS is the essential isoform mediating NO-dependent regulation of THAL NaCl transport. Adenoviral gene transfer in vivo, DAF-2DA fluorescence (NO indicator), isolated THAL chloride flux measurement Hypertension High 12913056
2004 eNOS subcellular localization to the trans-Golgi network (TGN) versus cytosol determines the specificity of vascular responses: ACh-induced eNOS translocation preferentially to the TGN correlates with vasodilation, whereas PAF-induced translocation to the cytosol correlates with hyperpermeability, despite both agonists causing eNOS phosphorylation and NO release. eNOS-GFP stable transfection, lipid raft fractionation, immunofluorescence microscopy, endothelial permeability assay American journal of physiology. Heart and circulatory physiology Medium 16679407
2005 Laminar shear stress increases eNOS mRNA stability and translational efficiency by lengthening the 3′ poly(A) tail through increased nuclear polyadenylation; shear-induced long-poly(A) eNOS transcripts shift into more translationally active polysome fractions (t½ increased from 6 h to 18 h). H₂O₂ and statins also induce this 3′ polyadenylation. Poly(A) tail length assay, polysome fractionation, mRNA half-life measurement, nuclear run-on Circulation research Medium 15905462
2005 Red blood cells express a functional eNOS localized to the plasma membrane and cytoplasm; RBC-NOS is regulated by L-arginine, calcium, and PI3-kinase-dependent phosphorylation. RBC eNOS activity regulates RBC membrane deformability and inhibits platelet activation. eNOS-/- mice RBCs lack NOS protein and activity. Western blot, immunofluorescence, L-arginine→citrulline assay, PI3K inhibitor, eNOS-/- mouse RBCs, platelet aggregation assay, RBC deformability Blood High 16368881
2005 Hyperhomocysteinemia impairs eNOS activity primarily through PKC activation, which increases Thr495 phosphorylation and decreases eNOS protein expression; a PKC inhibitor (GFX) reversed Hcy-mediated eNOS inactivation and Thr495 phosphorylation. Antioxidant and BH4 supplementation failed to rescue the defect. Aortic ring relaxation, intravital microscopy, eNOS activity assay, phosphospecific Western blot, PKC inhibitor (GFX), CBS-/- mice Arteriosclerosis, thrombosis, and vascular biology Medium 16210565
2005 PKCα isoform specifically activates eNOS: PKCα overexpression increases eNOS Ser1179 phosphorylation and NO production; PKCα siRNA or dominant-negative mutant decreases FGF2-induced eNOS activation. In vivo PKCα transduction of rat femoral arteries increases resting blood flow in an L-NAME-sensitive manner. PKCα overexpression/siRNA/dominant-negative in primary endothelial cells, phosphospecific Western blot, NO measurement, in vivo blood flow (Doppler), L-NAME inhibition Circulation research Medium 16081872
2006 Hydroxyurea increases eNOS-derived NO in endothelial cells via PKA-dependent rapid and transient phosphorylation of eNOS at Ser1177; PKB/Akt contributes partially. Hydroxyurea also elevates intracellular cAMP, cGMP, and Ca²⁺ upstream of eNOS activation. NOS inhibitor competition, PKA/PKB inhibitors, phosphospecific Western blot, DAF-2DA NO fluorescence, cAMP/cGMP measurement Blood Medium 16527893
2006 eNOS exists in an 'uncoupled' state in diabetic db/db mice, generating peroxynitrite rather than NO; eNOS gene therapy exacerbates hepatic ischemia-reperfusion injury in diabetes. BH4 or sepiapterin supplementation recouples eNOS and is protective, establishing BH4 availability as the key determinant of eNOS coupling status. eNOS transgenic/adenovirus overexpression in db/db mice, hepatic I-R model, serum ALT, peroxynitrite measurement, BH4/sepiapterin treatment Circulation research High 16763164
2007 Rho-kinase (ROCK) directly phosphorylates eNOS at Thr495 in vitro and in cells; constitutively active RhoA or ROCK increases Thr495 phosphorylation; the ROCK inhibitor Y27632 suppresses thrombin-induced Thr495 phosphorylation, identifying ROCK as a direct kinase writer for this inhibitory eNOS site. In vitro kinase assay (constitutively active ROCK + recombinant eNOS), COS-7 cell overexpression, Y27632 inhibitor in HUVECs, phosphospecific Western blot Biochemical and biophysical research communications High 17651694
2008 eNOS is required for tumor maintenance downstream of the oncogenic Ras–PI3K–AKT pathway; AKT-mediated eNOS phosphorylation promotes S-nitrosylation and activation of endogenous wild-type Ras, creating a feed-forward loop required throughout tumorigenesis. Blocking eNOS phosphorylation inhibits tumor initiation and maintenance. Oncogenic Ras/PI3K/AKT pathway manipulation, eNOS phosphorylation block, S-nitrosylation assay, tumor xenograft models, genetic epistasis Nature High 18344980
2010 S-glutathionylation of eNOS at two conserved cysteine residues in the reductase domain reversibly uncouples the enzyme, switching from NO production to superoxide generation primarily from the reductase domain. This modification is increased in hypertensive vessels and impairs endothelium-dependent vasodilation, which is restored by thiol-reducing agents that reverse S-glutathionylation. S-glutathionylation assay, site-directed mutagenesis (Cys residues), EPR-based superoxide detection, NO measurement, isolated vessel relaxation, thiol-reducing agent treatment Nature High 21179168
2010 PKCζ binds to ERK5 under basal conditions, phosphorylates ERK5 at S486 upon TNFα stimulation, thereby inhibiting the ERK5/KLF2 pathway and decreasing eNOS protein stability. DN-PKCζ reverses TNFα-mediated suppression of eNOS, placing PKCζ upstream of ERK5 as a regulator of eNOS protein turnover. Co-immunoprecipitation, mammalian 2-hybrid assay, in vitro phosphorylation, ERK5 S486A mutant, DN-PKCζ adenovirus, siRNA, eNOS protein stability assay Blood Medium 20538799
2010 Endocardial Tbx5 deletion causes atrial septal defects through increased Nos3-mediated apoptosis of endocardial cells; compound Tbx5/Nos3 haploinsufficiency worsens cardiac phenotype, establishing a genetic epistatic interaction where endocardial Nos3 activation (downstream of Tbx5 loss) mediates cell death in atrial septum formation. Endocardial-specific Cre-mediated deletion, compound haploinsufficiency mouse genetics, TUNEL apoptosis assay Proceedings of the National Academy of Sciences of the United States of America Medium 20974940
2013 eNOS Ser1176 phosphorylation (mouse equivalent of human Ser1177) regulates insulin sensitivity and energy metabolism in vivo: knockin mice with unphosphorylatable SA mutation develop hypertension, insulin resistance, hyperinsulinemia, and adiposity, while phosphomimetic SD mice are protected from high-fat-diet-induced weight gain, revealing eNOS phosphorylation as a metabolic regulatory node. Knockin mouse generation (S1176A and S1176D), metabolic phenotyping, blood pressure measurement, vascular reactivity assay Biochemical and biophysical research communications High 23291238
2014 PKC-dependent phosphorylation of eNOS at T495 drives eNOS uncoupling in lung microvascular endothelial cells exposed to Gram-positive pore-forming toxins (LLO, PLY): PKCα activation promotes T495 phosphorylation, dissociation of Hsp90 and caveolin-1 from eNOS, and superoxide/peroxynitrite production instead of NO, increasing endothelial permeability. A caveolin-1 scaffolding domain peptide blocks PKCα-driven superoxide generation. Phosphomimetic eNOS T495D mutant, PKCα overexpression, NOS inhibitor, superoxide/peroxynitrite assays, co-immunoprecipitation (Hsp90, caveolin-1), transendothelial permeability PloS one Medium 25020117
2017 Nox4-driven oxidative stress in aging endothelial cells activates PDI and induces ER stress, causing dissociation of Hsp90 from eNOS and thereby promoting eNOS uncoupling (shift from NO to superoxide) during endothelial senescence; Nox4 siRNA or chemical Nox4 inhibition restores eNOS coupling. Serial passage aging model, Nox4 siRNA, Nox4/PDI/Hsp90/eNOS Western blot and co-IP, EPR superoxide detection, NO assay Free radical biology & medicine Medium 28916474
2018 Caveolin-1 stabilizes eNOS expression and regulates its activity: Cav-1 siRNA reduces eNOS protein and gene expression but increases per-molecule eNOS phosphorylation and NO production. Conversely, eNOS-derived NO promotes Cav-1 S-nitrosylation and destabilizes Cav-1 oligomers, and eNOS activity is required for caveola-mediated endocytosis of albumin and insulin. siRNA knockdown, Adv-Cav-1-GFP overexpression, S-nitrosylation assay, eNOS Ser1177 phosphorylation, live-cell immunofluorescence, endocytosis assay Molecular biology of the cell Medium 29563255
2018 OxLDL induces eNOS S-nitrosylation at Cys94 and Cys99 (but not Cys441) via an iNOS-dependent mechanism; S-nitrosylated eNOS shows enhanced interaction with β-catenin, increased β-catenin transcriptional activity, and endothelial dysfunction (increased migration and adhesion molecule expression). Specific iNOS inhibitor 1400W reduces eNOS S-nitrosylation and blocks β-catenin activation. Biotin-switch S-nitrosylation assay, eNOS Cys→Ala site-directed mutagenesis, co-immunoprecipitation, iNOS inhibitor 1400W, β-catenin reporter Biochimica et biophysica acta. Molecular basis of disease Medium 29471036
2019 Multispectral biosensor imaging reveals a striking discordance between eNOS phosphorylation and actual NO synthesis: ATP and histamine promote both eNOS phosphorylation and robust NO/Ca²⁺ increases; insulin and VEGF elicit strong eNOS Ser1177 phosphorylation but cause no detectable increase in intracellular NO or Ca²⁺, demonstrating that phosphorylation alone is insufficient to predict eNOS activation. Genetically encoded NO biosensor (multispectral imaging), Ca²⁺ biosensor, chemogenetic eNOS activation, phosphospecific Western blot Proceedings of the National Academy of Sciences of the United States of America High 31527268
2020 A DNA-based fluorescent probe (NOckout) targeted to either the plasma membrane or the TGN quantitatively maps endogenous NOS3 activity at these two distinct subcellular locations in live cells. Although Golgi-localized NOS3 is tenfold less active than plasma-membrane NOS3, Golgi NOS3 activity is essential for structural integrity of the Golgi apparatus. DNA nanotechnology-based subcellular NO probe, live-cell fluorescence imaging, pharmacological NOS3 inhibition, Golgi morphology assay Nature chemical biology High 32152543
2020 β-Arrestin2 (β-Arr2) is an integral component of the GPCR–eNOS signalosome in liver sinusoidal endothelial cells: β-Arr2 localizes with eNOS and GIT1, directly stimulates eNOS activity, and its expression is reduced during liver injury. β-Arr2-mediated GIT1/eNOS complex formation is dependent on Erk1/2 and Src. β-Arr2 knockout mice show dramatically increased portal hypertension after bile-duct ligation due to reduced eNOS activity. Co-immunoprecipitation, β-Arr2 KO mice, adenoviral β-Arr2 overexpression, NOS activity assay, portal pressure measurement, Erk1/2 and Src inhibitors Proceedings of the National Academy of Sciences of the United States of America Medium 32404425
2021 Hepatocyte-specific eNOS is a key regulator of NAFLD/NASH susceptibility: genetic deletion or viral knockdown of hepatocyte eNOS exacerbates steatosis, inflammation, and mitochondrial dysfunction (decreased fatty acid oxidation, increased H₂O₂ emission, impaired mitophagy via BNIP3/LC3II); eNOS overexpression attenuates Western diet-induced NASH. Human NASH liver shows reduced eNOS correlating with reduced mitochondrial oxidation and BNIP3. Hepatocyte-specific KO (Cre-lox), adenoviral knockdown/overexpression, mitochondrial respiration (Seahorse), H₂O₂ emission, mitophagy markers, human tissue correlation Diabetes Medium 34380696
2022 Endothelial BACE1 cleaves tight junction protein occludin; elevated BACE1 causes membranal accumulation of caveolin-1, which increases caveolin-1 binding to eNOS and attenuates eNOS activity, resulting in endothelial dysfunction. Endothelial-specific BACE1 transgenic mice display reduced eNOS activity, cerebral blood flow reduction, BBB leakage, and cognitive impairment; BACE1 inhibition rescues eNOS activity. Mass spectrometry substrate identification, co-immunoprecipitation (Cav-1/eNOS), BACE1 transgenic mice, eNOS activity assay, cerebral blood flow measurement, cognitive testing Circulation research Medium 35382554
2022 DF-induced endothelial TXNDC5 promotes proteasome-mediated degradation of HSF1, reducing Hsp90 expression and accelerating eNOS protein degradation. Endothelium-specific Txndc5 deletion markedly reduces atherosclerosis in ApoE⁻/⁻ mice and increases eNOS protein, while Txndc5-targeting CRISPR nanoparticles replicate this protection, placing TXNDC5 upstream of Hsp90–eNOS stability. Endothelium-specific KO (ApoE⁻/⁻ background), CRISPR nanoparticle delivery, eNOS protein quantification, HSF1/Hsp90 Western blot, atherosclerotic lesion quantification Science advances Medium 35061532
2023 VEGFR2 pY1173/PLCγ signaling activates Ca²⁺/PKC-dependent eNOS, which is required for tyrosine nitration and activation of Src; Src-mediated VE-cadherin Y685 phosphorylation then drives endothelial junction disintegration and vascular leakage. Vegfr2Y1173F/+ and Plcg1iECKO mice show decreased PLCγ/eNOS activation and stabilized endothelial barrier. VEGFR2 Y1173F knockin mice, endothelial-specific Plcg1 KO, eNOS activity assay, tyrosine nitration assay, Src phosphorylation, VE-cadherin phosphorylation, vascular permeability assay The Journal of clinical investigation High 37651195

Source papers

Stage 0 corpus · 130 papers · ranked by NIH iCite citations
Year Title Journal Citations PMID
1993 The L-arginine-nitric oxide pathway. The New England journal of medicine 5250 7504210
1999 Activation of nitric oxide synthase in endothelial cells by Akt-dependent phosphorylation. Nature 2980 10376603
2002 Generation and initial analysis of more than 15,000 full-length human and mouse cDNA sequences. Proceedings of the National Academy of Sciences of the United States of America 1479 12477932
2014 A proteome-scale map of the human interactome network. Cell 977 25416956
2020 A reference map of the human binary protein interactome. Nature 849 32296183
1998 Dynamic activation of endothelial nitric oxide synthase by Hsp90. Nature 837 9580552
2001 Hyperglycemia inhibits endothelial nitric oxide synthase activity by posttranslational modification at the Akt site. The Journal of clinical investigation 700 11696579
1993 Structure and chromosomal localization of the human constitutive endothelial nitric oxide synthase gene. The Journal of biological chemistry 692 7688726
2002 Demonstration that C-reactive protein decreases eNOS expression and bioactivity in human aortic endothelial cells. Circulation 658 12234944
2011 Phylogenetic-based propagation of functional annotations within the Gene Ontology consortium. Briefings in bioinformatics 656 21873635
1999 T-786-->C mutation in the 5'-flanking region of the endothelial nitric oxide synthase gene is associated with coronary spasm. Circulation 656 10359729
2001 Phosphorylation of Thr(495) regulates Ca(2+)/calmodulin-dependent endothelial nitric oxide synthase activity. Circulation research 618 11397791
2015 Vascular nitric oxide: Beyond eNOS. Journal of pharmacological sciences 568 26499181
1998 Structure of nitric oxide synthase oxygenase dimer with pterin and substrate. Science (New York, N.Y.) 564 9516116
1992 Cloning and expression of a cDNA encoding human endothelium-derived relaxing factor/nitric oxide synthase. The Journal of biological chemistry 531 1378832
1998 VEGF upregulates ecNOS message, protein, and NO production in human endothelial cells. The American journal of physiology 512 9530221
1994 Oligo-capping: a simple method to replace the cap structure of eukaryotic mRNAs with oligoribonucleotides. Gene 492 8125298
2011 Mechanisms underlying adverse effects of HDL on eNOS-activating pathways in patients with coronary artery disease. The Journal of clinical investigation 476 21701070
1997 Reciprocal regulation of endothelial nitric-oxide synthase by Ca2+-calmodulin and caveolin. The Journal of biological chemistry 468 9188442
2010 S-glutathionylation uncouples eNOS and regulates its cellular and vascular function. Nature 465 21179168
1996 Endothelial nitric oxide synthase is regulated by tyrosine phosphorylation and interacts with caveolin-1. The Journal of biological chemistry 445 8910295
2005 Red blood cells express a functional endothelial nitric oxide synthase. Blood 442 16368881
2004 The status, quality, and expansion of the NIH full-length cDNA project: the Mammalian Gene Collection (MGC). Genome research 438 15489334
1992 Molecular cloning and characterization of human endothelial nitric oxide synthase. FEBS letters 431 1379542
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