Hereditary Form Of Hypertension Research Articles

Abstract LB031: WNK are drug targets for triple negative breast cancer

Abstract Triple negative breast cancer (TNBC) accounts for 10-20% of breast cancer cases, and is associated with poor diagnosis and prognostics. Due to the lack of therapeutics available, it is urgent to identify biomolecular targets to develop more efficient treatments [1]. Depletion and inhibition of the protein kinase With No Lysine (WNK) reduces migration of breast cancer cells, slowing down TNBC metastasis [2][3]. For this reason, WNKs are promising cancer drug targets. Other works suggest WNKs are also targets for the treatment of hypertension and stroke. The main focus of this work is to understand the molecular mechanisms of this family of proteins, which will help to develop more efficient drugs. WNKs are serine-threonine kinases important in ion transport regulation [4]. WNKs are critical intracellular osmosensors, with their function related to an equilibrium between an inactive dimer and an autophosphorylation-competent monomer. The dimer conformation covers phosphorylation sites [5], while simultaneously trapping water molecules [6]. The water trapping feature is hypothesized to be linked to ion pairs and clusters of charges between the Activation loop and the Catalytic loop. To access the role of these ion pairs, we expressed and purified seven mutants of key amino acids, analyzed their activity, phosphorylation state and sensitivity to ions and pressure. Some of these mutants are more monomeric, active and autophosphorylation competent than the wild-type, elucidating the role of these amino acids in the function of WNKs. Interestingly, some mutants are more sensitive to potassium, which confirms the WNK inhibition site for this ion [7]. [1]B. D. Lehmann et al., “Identification of human triple-negative breast cancer subtypes and preclinical models for selection of targeted therapies,” J. Clin. Invest., vol. 121, no. 7, pp. 2750–67, 2011.[2]S. Shyamasundar, et al., “miR-93 inhibits the invasive potential of triple-negative breast cancer cells in vitro via protein kinase WNK1,” Int. J. Oncol., vol. 49, no. 6, pp. 2629–36, 2016.[3]A. B. Jaykumas et al., “WNK1 Enhances Migration and Invasion in Breast Cancer Models,” Mol. Cancer Ther., vol. 20, no. 10, pp. 1800–08, 2021.[4]D. R. Alessi, et al., “The WNK-SPAK/OSR1 pathway: master regulator of cation-chloride cotransporters,” Sci. Signal., vol. 7, p. 334, 2014.[5]X. Min, et al., “Crystal structure of the kinase domain of WNK1, a kinase that causes a hereditary form of hypertension,” Structure, vol. 12, no. 7, pp. 1303-11, 2004.[6]R. Akella et al., “Osmosensing by WNK Kinases,” Mol. Biol. Cell, vol. 32, no. 18, pp. 1614–23, 2021.[7]J. M. Pleinis et al., “WNKs are potassium-sensitive kinases,” Am. J. Physiol Cell Physiol, vol. 320, no. 5, pp. C703-21, 2021. Citation Format: Liliana dos Reis Teixeira, John Humphreys, Haixia He, Radha Akella, Elizabeth Goldsmith. WNK are drug targets for triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB031.

Function and Regulation of the Epithelial Na+ Channel ENaC.

The Epithelial Na+ Channel, ENaC, comprised of 3 subunits (αβγ, or sometimes δβγENaC), plays a critical role in regulating salt and fluid homeostasis in the body. It regulates fluid reabsorption into the blood stream from the kidney to control blood volume and pressure, fluid absorption in the lung to control alveolar fluid clearance at birth and maintenance of normal airway surface liquid throughout life, and fluid absorption in the distal colon and other epithelial tissues. Moreover, recent studies have also revealed a role for sodium movement via ENaC in nonepithelial cells/tissues, such as endothelial cells in blood vessels and neurons. Over the past 25 years, major advances have been made in our understanding of ENaC structure, function, regulation, and role in human disease. These include the recently solved three-dimensional structure of ENaC, ENaC function in various tissues, and mutations in ENaC that cause a hereditary form of hypertension (Liddle syndrome), salt-wasting hypotension (PHA1), or polymorphism in ENaC that contributes to other diseases (such as cystic fibrosis). Moreover, great strides have been made in deciphering the regulation of ENaC by hormones (e.g., the mineralocorticoid aldosterone, glucocorticoids, vasopressin), ions (e.g., Na+ ), proteins (e.g., the ubiquitin-protein ligase NEDD4-2, the kinases SGK1, AKT, AMPK, WNKs & mTORC2, and proteases), and posttranslational modifications [e.g., (de)ubiquitylation, glycosylation, phosphorylation, acetylation, palmitoylation]. Characterization of ENaC structure, function, regulation, and role in human disease, including using animal models, are described in this article, with a special emphasis on recent advances in the field. © 2021 American Physiological Society. Compr Physiol 11:1-29, 2021.

Unveiling the Distinct Mechanisms by which Disease-Causing Mutations in the Kelch Domain of KLHL3 Disrupt the Interaction with the Acidic Motif of WNK4 through Molecular Dynamics Simulation.

Kelch-like 3 (KLHL3) is a substrate adaptor of an E3 ubiquitin ligase complex that regulates the degradation of its substrates, including with-no-lysine [K] kinase 4 (WNK4). Mutations in KLHL3 are associated with pseudohypoaldosteronism type II (PHAII), a hereditary form of hypertension. Many PHAII-causing mutations are located in the Kelch domain of KLHL3 that binds with WNK4; however, detailed mechanisms by which these mutations disrupt the binding are not well-understood. In the present study we use molecular dynamics simulations and Western blot analyses to examine the effects of these mutations on the interaction between the Kelch domain of KLHL3 and the acidic motif (AM) of WNK4. The simulation results correlated well with those from Western blot analyses with the exception of the L387P mutation, which led to deregulation of AM degradation by KLHL3 but not recapitulated by simulations. On the basis of the simulation results, a mutation on the binding surface of the Kelch domain affected the Kelch-AM interaction through two major mechanisms: altering the electrostatic potential of the AM binding site and disrupting the Kelch-AM hydrogen bonds. The mutations buried inside the Kelch domain were predicted by our simulations to have no or modest effects on the Kelch-AM interaction. Buried mutations R384Q and S410L disrupted intramolecular hydrogen bonds within the Kelch domain and affected the Kelch-AM interaction indirectly. No significant effect of buried mutation A340V or A494T on the AM degradation or Kelch-AM interaction was observed, implying these mutations may disrupt mechanisms other than Kelch-AM interaction.

Phosphorylation of KLHL3 at serine 433 impairs its interaction with the acidic motif of WNK4: a molecular dynamics study.

Interaction between the acidic motif (AM) of protein kinase WNK4 and the Kelch domain of KLHL3 are involved in the pathogenesis of pseudohypoaldosteronism type II, a hereditary form of hypertension. This interaction is disrupted by some disease-causing mutations in either WNK4 or KLHL3, or by angiotensin II- and insulin-induced phosphorylation of KLHL3 at serine 433, which is also a site frequently mutated in patients. However, the mechanism by which this phosphorylation disrupts the interaction is unclear. In this study, we approached this problem using molecular dynamics simulation with structural, dynamical and energetic analyses. Results from independent simulations indicate that when S433 was phosphorylated, the electrostatic potential became more negative in the AM binding site of KLHL3 and therefore was unfavorable for binding with the negatively charged AM. In addition, the intermolecular hydrogen bond network that kept the AM stable in the binding site of KLHL3 was disrupted, and the forces for the hydrophobic interactions between the AM of WNK4 and KLHL3 were also reduced. As a result, the weakened interactions were no longer capable of holding the AM of WNK4 at its binding site in KLHL3. In conclusion, phosphorylation of KLHL3 at S433 disrupts the hydrogen bonds, hydrophobic and electrostatic interactions between the Kelch domain of KLHL3 and the AM of WNK4. This study provides a key molecular understanding of the KLHL3-mediated regulation of WNK4, which is an integrative regulator of electrolyte homeostasis and blood pressure regulation in the kidney. Significances Statement: WNK4 is an integrative regulator of electrolyte homeostasis, which is important in the blood pressure regulation by the kidney. Interaction between WNK4 and KLHL3 is a key physiological process that is impaired in a hereditary form of hypertension. This study provides substantial new insights into the role of phosphorylation of KLHL3 in regulating the interaction with WNK4, and therefore advances our understanding of molecular pathogenesis of hypertension and the mechanism of blood pressure regulation.

Disease-causing mutations in KLHL3 impair its effect on WNK4 degradation

Mutations in with-no-lysine (K) kinase 4 (WNK4) and a ubiquitin E3 ligase complex component kelch-like 3 (KLHL3) both cause pseudohypoaldosteronism II (PHAII), a hereditary form of hypertension. We determined whether WNK4 or its effector is regulated by KLHL3 in Xenopus oocytes. KLHL3 inhibited the positive effect of WNK4 on Na+–Cl− cotransporter (NCC) by decreasing WNK4 protein abundance without decreasing that of NCC and the downstream kinase OSR1 directly. Ubiquitination and degradation of WNK4 were induced by KLHL3. The effect of KLHL3 on WNK4 degradation was blocked by a dominant negative form of cullin 3. All five PHAII mutations of KLHL3 tested disrupted the regulation on WNK4. We conclude that KLHL3 is a substrate adaptor for WNK4 in a ubiquitin E3 ligase complex.

Kelch‐like 3 (KLHL3) controls WNK4 ubiquitination and degradation

Mutations in with‐no‐lysine (K) kinase 4 (WNK4) and ubiquitin E3 ligase components KLHL3 and Cullin 3 (CUL3) cause pseudohypoaldosteronism II (PHAII), a hereditary form of hypertension. WNK4 regulates Na‐Cl cotransporter (NCC) and so does KLHL3. However, it is unclear whether NCC or a regulator of NCC is a substrate of the KLHL3‐CUL3 E3 ligase complex. The objective of this study is to identify which protein is the true substrate of KLHL3‐CUL3 E3 ligase. We expressed KLHL3 and/or CUL3 together with Na‐K‐2Cl cotransporter 2 (NKCC2) and WNK4 in Xenopus oocytes. KLHL3 significantly decreased NKCC2‐mediated Na uptake and the positive effect of WNK4 on NKCC2. However, the protein abundance of NKCC2 was not decreased, but the level of WNK4 was robustly decreased by KLHL3. KLHL3 also did not substantially reduce the protein abundance of NCC or oxidative stress‐responsive 1 (OSR1) in a similar setting. KLHL3 significantly increased the ubiquitination and degradation of WNK4 protein. Although CUL3 alone didn't have significant effect on WNK4, a dominant negative CUL3 construct alleviated the effect of KLHL3 on WNK4, suggesting that CUL3 works together with KLHL3 to regulate WNK4 stability. All five PHAII mutations in KLHL3 tested decreased the ability of KLHL3 to reduce WNK4 protein abundance. We conclude that WNK4 is a substrate of KLHL3‐CUL3 ubiquitin E3 ligase complex and PHAII mutations in KLHL3 affect electrolyte transporters indirectly via WNK4.

A Case of Liddle Syndrome

Pediatric hypertension is usually secondary to an underlying identifiable cause, most often renal. Hypertension with low Plasma Rennin Activity (PRA), although rare, is important as it is often familial and is associated with single gene disorders (monogenic). It hence carries greater genetic implications for the family. The authors' hereby report a case of low PRA hypertension which was diagnosed as Liddle Syndrome, an autosomal dominant form of hereditary hypertension. Early detection and appropriate treatment may help to improve the long term morbidity and mortality in children with this condition.

Crystal structure of the kinase domain of WNK1, a kinase that causes a hereditary form of hypertension.

WNK kinases comprise a small group of unique serine/threonine protein kinases that have been genetically linked to pseudohypoaldosteronism type II, an autosomal dominant form of hypertension. Here we present the structure of the kinase domain of WNK1 at 1.8 A resolution, solved in a low activity conformation. A lysine residue (Lys-233) is found in the active site emanating from strand beta2 rather than strand beta3 as in other protein kinases. The activation loop adopts a unique well-folded inactive conformation. The conformations of the P+1 specificity pocket, the placement of the conserved active site threonine (Thr-386), and the exterior placement of helix C, contribute to the low activity state. By homology modeling, we identified two hydrophobic residues in the substrate-binding groove that contribute to substrate specificity. The structure of the WNK1 catalytic domain, with its unique active site, may help in the design of therapeutic reagents for the treatment of hypertension.

Epithelial sodium channel, salt intake, and hypertension.

The epithelial sodium channel (ENaC) is a membrane protein made of three different but homologous subunits (a, b, and g) present in the apical membrane of epithelial cells of, for example, the distal nephron. This channel is responsible for salt reabsorption in the kidney and can cause human diseases by increasing channel function in Liddle's syndrome, a form of hereditary hypertension, or by decreasing channel function in pseudohypoaldosteronism type I, a salt-wasting disease in infancy. This review briefly discusses recent advances in understanding the implication of ENaC in Liddle's syndrome and in pseudohypoaldosteronism type I, both caused by mutations in the SCNN1 (ENaC) genes. Furthermore, it is still an open question to which extent SCNN1 genes coding for ENaC might be implicated in essential hypertension. The development of Scnn1 genetically engineered mouse models will provide the opportunity to test the effect of environmental factors, like salt intake, on the development of this kind of salt- sensitive hypertension.

Solution structure of a Nedd4 WW domain-ENaC peptide complex.

Nedd4 is a ubiquitin protein ligase composed of a C2 domain, three (or four) WW domains and a ubiquitin ligase Hect domain. Nedd4 was demonstrated to bind the epithelial sodium channel (alphabetagammaENaC), by association of its WW domains with PY motifs (XPPXY) present in each ENaC subunit, and to regulate the cell surface stability of the channel. The PY motif of betaENaC is deleted or mutated in Liddle syndrome, a hereditary form of hypertension caused by elevated ENaC activity. Here we report the solution structure of the third WW domain of Nedd4 complexed to the PY motif-containing region of betaENaC (TLPIPGTPPPNYDSL, referred to as betaP2). A polyproline type II helical conformation is adopted by the PPPN sequence. Unexpectedly, the C-terminal sequence YDSL forms a helical turn and both the tyrosine and the C-terminal leucine contact the WW domain. This is unlike other proline-rich peptides complexed to WW domains, which bind in an extended conformation and lack molecular interactions with residues C-terminal to the tyrosine or the structurally equivalent residue in non-PY motif WW domain targets. The Nedd4 WW domain-ENaC betaP2 peptide structure expands our understanding of the mechanisms involved in WW domain-ligand recognition and the molecular basis of Liddle syndrome.

Effect of subunit composition and Liddle's syndrome mutations on biosynthesis of ENaC.

The epithelial Na+ channel (ENaC) is comprised of three homologous subunits: alpha, beta, and gamma, all of which are required for formation of the fully functional channel. This channel is responsible for salt reabsorption in the kidney, the airway, and the large bowel. Mutations in ENaC can cause human disease by increasing channel function in Liddle's syndrome, a form of hereditary hypertension, or by decreasing channel function in pseudohypoaldosteronism type I, a salt-wasting disease of infancy. We previously showed that ENaC is expressed on the cell surface as a minimally glycosylated, Triton-insoluble protein. In the present study we found that ENaC existed initially as a Triton-soluble protein that contained high-mannose glycosylation, presumably in the endoplasmic reticulum. This form of the protein disappeared as the Triton-insoluble, minimally glycosylated form became the more prevalent species. In pulse-chase studies of individually expressed subunits, we found that the Triton-soluble form of beta-ENaC accumulated initially, whereas the Triton-soluble form of alpha-ENaC decreased throughout the time course. However, when all three subunits were coexpressed, the alpha- and beta-subunits showed a similar pattern. The complex became Triton insoluble at some point after the endoplasmic reticulum, as incubation at 15 degrees C blocked the conversion to the insoluble form. Deletion of the carboxy-terminal tail of beta-ENaC causes Liddle's syndrome. This mutation increased the amount of newly synthesized Triton-insoluble ENaC heteromultimers but did not affect the half-life of insoluble protein. Therefore, subunit composition and mutations in individual subunits can influence biosynthesis of the ENaC complex.

WW domains of Nedd4 bind to the proline-rich PY motifs in the epithelial Na+ channel deleted in Liddle's syndrome.

The amiloride-sensitive epithelial sodium channel (ENaC) plays a major role in sodium transport in kidney and other epithelia, and in regulating blood pressure. The channel is composed of three subunits (alphabetagamma) each containing two proline-rich sequences (P1 and P2) at its C-terminus. The P2 regions in human beta and gammaENaC, identical to the rat betagammarENaC, were recently shown to be deleted in patients with Liddle's syndrome (a hereditary form of hypertension), leading to hyperactivation of the channel. Using a yeast two-hybrid screen, we have now identified the rat homologue of Nedd4 (rNedd4) as the binding partner for the P2 regions of beta and gammarENaC. rNedd4 contains a Ca2+ lipid binding (CaLB or C2) domain, three WW domains and a ubiquitin ligase (Hect) domain. Our yeast two-hybrid and in vitro binding studies revealed that the rNedd4-WW domains mediate this association by binding to the P2 regions, which include the PY motifs (XPPXY) of either betarENaC (PPPNY) or gammarENaC (PPPRY). SH3 domains were unable to bind these sequences. Moreover, mutations to Ala of Pro616 or Tyr618 within the betarENaC P2 sequence (to PPANY or PPPNA, respectively), recently described in Liddle's patients, led to abrogation of rNedd4-WW binding. Nedd4-WW domains also bound to the proline-rich C-terminus (containing the sequence PPPAY) of alpharENaC, and endogenous Nedd4 co-immunoprecipitated with alpharENaC expressed in MDCK cells. These results demonstrate that the WW domains of rNedd4 bind to the PY motifs deleted from beta or gammaENaC in Liddle's syndrome patients, and suggest that Nedd4 may be a regulator (suppressor) of the epithelial Na+ channel.

Age-related hair cell loss in spontaneously hypertensive and normotensive rats

In the spontaneously hypertensive (SH) rat, a hereditary form of hypertension and widespread vascular abnormalities develop. In the present study the inner ear degeneration pattern in SH and normotensive (N) rats not exposed to noise was investigated and correlated with frequency-specific auditory brainstem response (ABR) thresholds. Rats of both strains were found to have a spontaneous loss of apical hair cells, but this was slightly more pronounced in SH. In SH rats, but not in those of the N strain, there was an age-related progressive loss of outer hair cells in the region 3–5 mm from the stapes, and a slight additional loss at the most basal end. According to available frequency maps, the hair cell loss should correspond to hearing loss between 6 and 24 kHz. In the SH rats hearing loss was not observed in the whole of this range, only at 16 and 20 kHz. It cannot be determined to what extent the localized spontaneous loss of hair cells is caused by vascular factors and what other mechanisms are involved, but vascular factors probably only play a minor role.