Escobedo A, etal., Structure. 2014 Oct 7;22(10):1446-57. doi: 10.1016/j.str.2014.08.016.
We investigated the mechanisms of activation and degradation of the E3 ubiquitin ligase Nedd4L combining the available biochemical information with complementary biophysical techniques. Using nuclear magnetic resonance spectroscopy, we identified that the C2 domain binds Ca(2+) and inositol 1,4,5-
trisphosphate (IP3) using the same interface that is used to interact with the HECT domain. Thus, we propose that the transition from the closed to the active form is regulated by a competition of IP3 and Ca(2+) with the HECT domain for binding to the C2 domain. We performed relaxation experiments and molecular dynamic simulations to determine the flexibility of the HECT structure and observed that its conserved PY motif can become solvent-exposed when the unfolding process is initiated. The structure of the WW3 domain bound to the HECT-PY site reveals the details of this interaction, suggesting a possible auto-ubquitination mechanism using two molecules, a partially unfolded one and a fully functional Nedd4L counterpart.
OBJECTIVE: The capability of the protein NEDD4L to reduce renal tubular expression of epithelial Na+ channel (ENaC) is influenced by a functional rs4149601 G-->A NEDD4L polymorphism. As diuretics and beta-blockers inhibit re
nal sodium reabsorption and renin release, respectively, we hypothesized that the beta-blocker or diuretic-induced blood pressure reduction and prevention of cardiovascular disease would be greater in patients with the highest ENaC expression (rs4149601 G-allele), whereas there would be no such genetically mediated differences in treatment efficacy among patients treated with the vasodilator diltiazem. METHODS: We related rs4149601 status to 6-month blood pressure reduction and risk of cardiovascular events in 5152 hypertensive patients (DBP >/= 100 mmHg) from the Nordic Diltiazem Study (NORDIL) randomized to either beta-blocker and/or diuretic-based treatment or diltiazem-based treatment. RESULTS: In patients on beta-blocker or diuretic monotherapy, carriers of the G-allele had greater SBP reduction (19.5 +/- 16.8 vs. 15.0 +/- 19.3 mmHg, P < 0.001) and DBP reduction (15.4 +/- 8.3vs. 14.1 +/- 8.4 mmHg, P = 0.02) and during 4.5 years of follow-up among patients randomized to beta-blockers and/or diuretics, carriers of the G-allele had greater protection from cardiovascular events [relative risk (RR) = 0.52, 95% confidence interval (CI) = 0.36-0.74, P < 0.001] as compared to AA homozygotes. Within the diltiazem group, there was no difference in blood pressure reduction or risk of cardiovascular events according to genotype. CONCLUSION: The functional NEDD4L rs4149601 polymorphism influences the efficacy of beta-blocker and/or diuretic-based antihypertensive treatment both in terms of blood pressure reduction and cardiovascular disease protection, whereas diltiazem-based antihypertensive treatment efficacy is not influenced by this NEDD4L polymorphism.
OBJECTIVE: Single-nucleotide polymorphisms (SNPs) in NEDD4L may influence the ability of the NEDD4L protein to reduce epithelial sodium channel expression. A variant in NEDD4L, rs4149601
, was associated with antihypertensive response and cardiovascular outcomes during treatment with thiazide diuretics and beta-blockers in a Swedish population. We sought to further evaluate associations between NEDD4L polymorphisms, blood pressure response and cardiovascular outcomes with thiazide diuretics and beta-blockers. METHODS: Four SNPs, rs4149601, rs292449, rs1008899 and rs75982813, were genotyped in 767 patients from the Pharmacogenomic Evaluation of Antihypertensive Responses (PEAR) clinical trial and association was assessed with blood pressure response to hydrochlorothiazide and atenolol. One SNP, rs4149601, was also genotyped in 1345 patients from the International Verapmil SR Trandolapril Study (INVEST), and association was examined with adverse cardiovascular outcomes relative to hydrochlorothiazide treatment. RESULTS: Significant associations or trends were found between rs4149601, rs292449, rs75982813 and rs1008899 and decreases in blood pressure in whites on hydrochlorothiazide, and a significant association was observed with increasing copies of the GC rs4149601-rs292449 haplotype and greater blood pressure response to hydrochlorothiazide in whites (P = 0.0006 and 0.006, SBP and DBP, respectively). Significant associations were also seen with rs4149601 and an increased risk for adverse cardiovascular outcomes in whites not treated with hydrochlorothiazide [P = 0.022, odds ratio (95% confidence interval) = 10.65 (1.18-96.25)]. CONCLUSION: NEDD4L rs4149601, rs292449 and rs75982813 may be predictors for blood pressure response to hydrochlorothiazide in whites, and NEDD4L rs4149601 may be a predictor for adverse cardiovascular outcomes in whites not treated with hydrochlorothiazide.
BACKGROUND: Hypospadias, as a congenital disorder of the urethra, is the second most common birth abnormality of the male reproductive system. This study primarily investigates the effects of microRNA-494 (miR-494) on the transforming growth factor-β1 (TGF-β1)/Smads signaling pathway and
on the development of hypospadias by binding to neural precursor cell expressed developmentally downregulated gene 4-like (Nedd4L). METHODS: We induced a mouse model of hypospadias through di-(2-ethylhexyl) phthalate treatment. The underlying regulatory mechanisms of miR-494 in this model were analyzed upon treatment of miR-494 mimic, miR-494 inhibitor, or small interfering RNA against Nedd4L in urethral epithelial cells isolated from mice with hypospadias. We then verified the binding site between miR-494 and Nedd4L and applied a gain- and loss-of-function approach to determine the effects of miR-494 on cell proliferation, cycle distribution, and apoptosis. RESULTS: Male mice with hypospadias exhibited significantly higher miR-494 expression and lower Nedd4L expression in urethral tissues than normal male mice. Nedd4L was verified as a target gene of miR-494. Treatment with miR-494 inhibitor suppressed the activation of the TGF-β1/Smads signaling pathway, whereas down-regulation of miR-494 exerted protective effects on urethral epithelial cells by impeding cell proliferation and inducing cell apoptosis. CONCLUSIONS: The study indicates that downregulation of miR-494 inhibits the TGF-β1/Smads signaling pathway and prevents the development of hypospadias through upregulating Nedd4L.
NEDD4-2 alias NEDD4L (neural precursor cell expressed, developmentally downregulated) gene was reported as a candidate gene for epileptic photo-sensitivity. We aimed to investigate this possible association of NEDD4-2 variants with idiopathic photosensitive epil
epsy. Consecutive patients who had been followed up at our epilepsy center and diagnosed with idiopathic epilepsy according to ILAE criteria and clear-cut photoparoxysmal responses in their electroencephalograms and 100 ethnically matched healthy subjects were included in the study. The regions around previously reported three variants, namely, S233L, E271A and H515P were tracked with DHPLC and the samples showing variations were sequenced. 81 patients (63 females) aged between 12-63 years (45 had juvenile myoclonic epilepsy, 11 childhood absence epilepsy, 14 juvenile absence epilepsy, 7 late onset idiopathic generalized epilepsy, 1 unclassified idiopathic generalized epilepsy, and 3 patients with idiopathic photosensitive occipital lobe epilepsy) were included in this study. We found only one heterozygous S233L variant in a 23-year-old man who has photosensitive form of juvenile absence epilepsy and pattern sensitivity to striped carpets. Other two variants were not found in any of the other patients and controls. Our results suggest that three screened NEDD4-2 variants do not play a leading role in the pathogenesis of photosensitive epilepsy in the Turkish population.
Neurodevelopmental disorders with periventricular nodular heterotopia (PNH) are etiologically heterogeneous, and their genetic causes remain in many cases unknown. Here we show that missense mutations in NEDD4L mapping to the HECT domain of the encoded E3 ubiqui
tin ligase lead to PNH associated with toe syndactyly, cleft palate and neurodevelopmental delay. Cellular and expression data showed sensitivity of PNH-associated mutants to proteasome degradation. Moreover, an in utero electroporation approach showed that PNH-related mutants and excess wild-type NEDD4L affect neurogenesis, neuronal positioning and terminal translocation. Further investigations, including rapamycin-based experiments, found differential deregulation of pathways involved. Excess wild-type NEDD4L leads to disruption of Dab1 and mTORC1 pathways, while PNH-related mutations are associated with deregulation of mTORC1 and AKT activities. Altogether, these data provide insights into the critical role of NEDD4L in the regulation of mTOR pathways and their contributions in cortical development.
The kidney plays an important role in salt and blood pressure (BP) homeostasis. In previous studies, variants in the genes for alpha-adducin (ADD1), WNK1, and NEDD4L, which all regulate renal sodium absorption, have been associated with increased BP. However, fi
ndings have been inconsistent. We tested whether this is because of physiological interactions between the effects of variants in these genes. We assessed the single and combined effects of the ADD1 (Gly460Trp), WNK1 (rs880054 A/G), and NEDD4L (rs4149601 G/A) polymorphisms on renal and BP response to an acute Na load (n=344 subjects), BP decrease after 1 month of treatment with 12.5 mg of hydrochlorothiazide (n=193), and ambulatory 24-hour BP (n=690). Individually, the variants showed modest effects on some of the studied phenotypes. We found the ADD1 Trp allele to be permissive for the effects of variants of the other genes. In combination, the same variants (ADD1 Trp/WNK1 GG/Nedd4L GA+AA) showed a consistent effect on renal Na handling (P=0.009) and acute BP response to a saline infusion (P=0.021), BP lowering after thiazide treatment (P=0.008), and nocturnal systolic BP (P=0.044). Physiological interaction between the ADD1 and WNK1-NEDD4L pathways influences the effects of variants in these genes on sodium-related BP regulation. Relatively common alleles in the ADD1, WNK1, and NEDD4L genes when present in combination may have significant effects on renal sodium handling, BP, and antihypertensive response to thiazides.
