Characterisation of the Cullin-3 mutation that causes a severe form of familial hypertension and hyperkalaemia

Deletion of exon 9 from Cullin‐3 (CUL3, residues 403–459: CUL3Δ403–459) causes pseudohypoaldosteronism type IIE (PHA2E), a severe form of familial hyperkalaemia and hypertension (FHHt). CUL3 binds the RING protein RBX1 and various substrate adaptors to form Cullin‐RING‐ubiquitin‐ligase complexes. Bound to KLHL3, CUL3‐RBX1 ubiquitylates WNK kinases, promoting their ubiquitin‐mediated proteasomal degradation. Since WNK kinases activate Na/Cl co‐transporters to promote salt retention, CUL3 regulates blood pressure. Mutations in both KLHL3 and WNK kinases cause PHA2 by disrupting Cullin‐RING‐ligase formation. We report here that the PHA2E mutant, CUL3Δ403–459, is severely compromised in its ability to ubiquitylate WNKs, possibly due to altered structural flexibility. Instead, CUL3Δ403–459 auto‐ubiquitylates and loses interaction with two important Cullin regulators: the COP9‐signalosome and CAND1. A novel knock‐in mouse model of CUL3WT/Δ403–459 closely recapitulates the human PHA2E phenotype. These mice also show changes in the arterial pulse waveform, suggesting a vascular contribution to their hypertension not reported in previous FHHt models. These findings may explain the severity of the FHHt phenotype caused by CUL3 mutations compared to those reported in KLHL3 or WNK kinases

Structural and biochemical characterization of the KLHL3-WNK kinase interaction important in blood pressure regulation

WNK1 [with no lysine (K)] and WNK4 regulate blood pressure by controlling the activity of ion co-transporters in the kidney. Groundbreaking work has revealed that the ubiquitylation and hence levels of WNK isoforms are controlled by a Cullin-RING E3 ubiquitin ligase complex (CRL3KLHL3) that utilizes CUL3 (Cullin3) and its substrate adaptor, KLHL3 (Kelch-like protein 3). Loss-of-function mutations in either CUL3 or KLHL3 cause the hereditary high blood pressure disease Gordon's syndrome by stabilizing WNK isoforms. KLHL3 binds to a highly conserved degron motif located within the C-terminal non-catalytic domain of WNK isoforms. This interaction is essential for ubiquitylation by CRL3KLHL3 and disease-causing mutations in WNK4 and KLHL3 exert their effects on blood pressure by disrupting this interaction. In the present study, we report on the crystal structure of the KLHL3 Kelch domain in complex with the WNK4 degron motif. This reveals an intricate web of interactions between conserved residues on the surface of the Kelch domain β-propeller and the WNK4 degron motif. Importantly, many of the disease-causing mutations inhibit binding by disrupting critical interface contacts. We also present the structure of the WNK4 degron motif in complex with KLHL2 that has also been reported to bind WNK4. This confirms that KLHL2 interacts with WNK kinases in a similar manner to KLHL3, but strikingly different to how another KLHL protein, KEAP1 (Kelch-like enoyl-CoA hydratase-associated protein 1), binds to its substrate NRF2 (nuclear factor-erythroid 2-related factor 2). The present study provides further insights into how Kelch-like adaptor proteins recognize their substrates and provides a structural basis for how mutations in WNK4 and KLHL3 lead to hypertension

RNAi screen for NRF2 inducers identifies targets that rescue primary lung epithelial cells from cigarette smoke induced radical stress

Chronic Obstructive Pulmonary Disease (COPD) is a highly prevalent condition characterized by inflammation and progressive obstruction of the airways. At present, there is no treatment that suppresses the chronic inflammation of the disease, and COPD patients often succumb to the condition. Excessive oxidative stress caused by smoke inhalation is a major driving force of the disease. The transcription factor NRF2 is a critical player in the battle against oxidative stress and its function is impaired in COPD. Increasing NRF2 activity may therefore be a viable therapeutic option for COPD treatment. We show that down regulation of KEAP1, a NRF2 inhibitor, protects primary human lung epithelial cells from cigarette-smoke-extract (CSE) induced cell death in an established in vitro model of radical stress. To identify new potential drug targets with a similar effect, we performed a siRNA screen of the 'druggable' genome using a NRF2 transcriptional reporter cell line. This screen identified multiple genes that when down regulated increased NRF2 transcriptional activity and provided a survival benefit in the in vitro model. Our results suggest that inhibiting components of the ubiquitin-proteasome system will have the strongest effects on NRF2 transcriptional activity by increasing NRF2 levels. We also find that down regulation of the small GTPase Rab28 or the Estrogen Receptor ESRRA provide a survival benefit. Rab28 knockdown increased NRF2 protein levels, indicating that Rab28 may regulate NRF2 proteolysis. Conversely ESRRA down regulation increased NRF2 transcriptional activity without affecting NRF2 levels, suggesting a proteasome-independent mechanism

New genetic loci link adipose and insulin biology to body fat distribution.

Body fat distribution is a heritable trait and a well-established predictor of adverse metabolic outcomes, independent of overall adiposity. To increase our understanding of the genetic basis of body fat distribution and its molecular links to cardiometabolic traits, here we conduct genome-wide association meta-analyses of traits related to waist and hip circumferences in up to 224,459 individuals. We identify 49 loci (33 new) associated with waist-to-hip ratio adjusted for body mass index (BMI), and an additional 19 loci newly associated with related waist and hip circumference measures (P < 5 × 10(-8)). In total, 20 of the 49 waist-to-hip ratio adjusted for BMI loci show significant sexual dimorphism, 19 of which display a stronger effect in women. The identified loci were enriched for genes expressed in adipose tissue and for putative regulatory elements in adipocytes. Pathway analyses implicated adipogenesis, angiogenesis, transcriptional regulation and insulin resistance as processes affecting fat distribution, providing insight into potential pathophysiological mechanisms

Activation of the NRF2 transcription factor reporter system correlates with the induction of the NRF2 responsive gene NQO1.

<p>siRNAs of hit candidates of the primary RNAi screen were transfected into HEK-hNQ cells and luciferase activity or hNQO1 mRNA levels were determined 48 h post transfection. The log2 ratios of specific luciferase activity were plotted versus log2 ratios of normalized expression levels of human NQO1 of identical siRNA treated samples. Only results of significant differences (p≤0.05) in both readouts are shown. SiKEAP1 treated samples are highlighted in black.</p

Induction of CSE stress tolerance in HBEpC cell cultures by KEAP1 knock down.

<p>Cell cultures of primary human bronchial epithelial cells were transfected for 48h with positive and negative control siRNAs prior to stimulation with 3% CSE-conditioned medium. After stimulation for 24h cells were analyzed either by Zeiss Axioplan (20× magnification) microscopy (A) or for their vitality by the Cell Titer Glo assay (B). Downregulation of KEAP1 greatly improves the survival of cells after CSE exposure. An ATP standard was used for the calculation of ATP concentration per well. Data are represented as means of 3 independent biological replicates +SD. Significant differences of siKEAP1 compared to siControl treated samples are highlighted (* p≤0.05, ** p≤0.01, students T-test).</p

Activation of the NRF2 transcription factor reporter system by different agents of radical stress or KEAP1 modifier.

<p>(A) Cell cultures of HEK293-hNQ cells were treated for 24 h with tBHQ (3, 6, 12, 18, 25, 37, 50, 75, 100 [μM]), CSE (0.04, 0.08, 0.16, 0.32, 0.63, 1.25, 2.5, 5, 10 [% (v/v)]) or Paraquat (25, 50, 75, 100, 125, 150, 200, 250, 300 [μM]). Luciferase activity was expressed as % of untreated samples (Control). (B) Cultures of HEK293-hNQ and HEK293-hNQ-del cells were transfected with positive and negative control siRNAs and lysed 48 h later for the determination of luciferase activity. Treatment of these cells with the NRF2 activator tBHQ at 50μM was performed for 24h. All results were normalized to untreated HEK293-hNQ samples. Values are represented as means of independent biological experiments ± SD (n = 6).</p

RNAi screen to detect activators of NRF-2.

<p>(A) HEK-hNQ cells were transfected with siRNA pools (3 siRNAs per gene) in 384-well format. 48 hours post transfection, cells on three replicate plates were lysed and luminescence was measured. Cells on identical triplicate plates were stained with Hoechst dye and images were acquired using an automated microscope. (B) Performance of control siRNA pools. Average and standard deviations of normalized values for control siRNA pools on all plates. (C) Frequency distribution of coefficients of variance between three replicates indicates high reproducibility of screen results. Solid line, normalized luminescence measurements, dashed line, normalized number of nuclei. (D) Performance of control siRNA pools. Average and standard deviations of normalized values for control siRNA pools on all plates.</p

Western Blot analysis of NRF2 levels in selected screening hits.

<p>(A) Anti-NRF2 Western Blot of tissue cell lysates after siRNA to the indicated screening hits. Treatment of cells with MLN4924, a small molecule inhibitor of CUL3 and other Cullin-RING ligases, leads to a strong increase in NRF2 levels as compared to control siRNA cells (CTRL). siRNA downregulation of CUL3, KEAP1, PSMC2, PSMC4, PSMD6 and RAB28 also lead to increases in NRF2 levels. Poly-ubiquitylated species of NRF2 after downregulation of proteasomal subunits are also apparent (NRF2^ub). A Western Blot against Actin is included as loading control. All samples shown were run on the same gel. Dashed lines indicate where lanes have been removed for clarity. (B) Quantification of NRF2 Western Blots for the indicated siRNA samples relative to siRNA control samples. Asterisk indicates significant stabilization of NRF2 (p<0.05).</p

Confirmational screen with 219 genes using 3 individual siRNAs against each gene.

<p>Enrichment for luciferase-inducing genes. Frequency distribution of luciferase induction for all siRNA pools screened in pass 1 (dashed line) and the 657 individual siRNAs used in pass 2 (solid line).</p