Researchers have identified an enzyme (NEDD4-2) that may help to curb chronic kidney disease

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University of South Australia researchers have identified an enzyme that may help to curb chronic kidney disease, which affects approximately 700 million people worldwide.

This enzyme, NEDD4-2, is critical for kidney health, says UniSA Centre for Cancer Biology scientist Dr. Jantina Manning in a new paper published this month in Cell Death & Disease.

The early career researcher and her colleagues, including 2020 SA Scientist of the Year Professor Sharad Kumar, have shown in an animal study the correlation between a high salt diet, low levels of NEDD4-2 and advanced kidney disease.

While a high salt diet can exacerbate some forms of kidney disease, until now, researchers did not realize that NEDD4-2 plays a role in promoting this salt-induced kidney damage.

“We now know that both a high sodium diet and low NEDD4-2 levels promote renal disease progression, even in the absence of high blood pressure, which normally goes hand in hand with increased sodium,” says Dr. Manning.

NEDD4-2 regulates the pathway required for sodium reabsorption in the kidneys to ensure correct levels of salt are maintained. If the NEDD4-2 protein is reduced or inhibited, increased salt absorption can result in kidney damage.

Even people on a low salt diet can get kidney damage if they have low levels of NEDD4-2 due to genetic variations or mutations in the gene.

Prof Kumar says the long-term goal is to develop a drug that can increase NEDD4-2 levels in people with chronic kidney disease (CKD).

“We are now testing different strategies to make sure this protein is maintained at a normal level all the time for overall kidney health,” Prof Kumar says.

“In diabetic nephropathy – a common cause of kidney disease – levels of NEDD4-2 are severely reduced. This is the case even when salt is not a factor.”

The study also revealed a surprising finding: that the high salt diet induced kidney disease is not always due to high blood pressure.

“In a lot of cases, kidney disease is exacerbated by hypertension, so we wanted to investigate that link in our study. In fact, we found the complete opposite—that a high salt diet caused excessive water loss and low blood pressure. This is significant because it means that kidney disease can also happen in people who don’t have high blood pressure,” Dr. Manning says.

A 2020 Lancet paper estimated that about 700 million people – or 10 percent of the world’s population – suffer from chronic kidney disease, which represents a 29 percent increase in the past 30 years.

The huge spike in CKD is mainly attributed to a global obesity epidemic in recent decades, leading to diabetes, one of the leading causes of chronic kidney disease along with high blood pressure.

World Health Organization statistics reveal a 300 percent increase in diabetes between 1980 and 2014, making it one of the top 10 causes of death worldwide and showing the gravity of the problem facing scientists trying to tackle kidney disease.

“Obesity and lifestyle are two main factors driving chronic kidney disease but there are other things at play as well,” says Dr. Manning. “Acute kidney injuries, drugs taken for other conditions, high blood pressure and a genetic predisposition can also cause it.”


Protein ubiquitination, a critical post translation modification, is necessary for ubiquitin-associated protein degradation by the ubiquitin proteasome system (UPS). UPS system comprise ubiquitin activating enzyme (E1), ubiquitin conjugating enzyme (E2), and ubiquitin ligase (E3). First, ubiquitin activation is performed by E1.

Then, E2 transfers ubiquitin to E3. Finally, E3 catalyses the covalent binding of ubiquitin to the target protein 1. E3 ligases are divided into three main families: zinc-binding RING finger adaptor (a recently discovered gene) 2, HECT (homologous with the carboxyl end of E6AP) catalytic 3 and U-box (a modified cyclic motif) families 4. E3 enzymes involve in substrate recognition and ubiquitin transfer to a single, multiple Lys (mono-/multi-ubiquitination) or as a poly-Ub chain (poly-ubiquitination) residues of the substrate (HECT family) 5 or easing ubiquitin transfer from E2 to the target protein (RING family).

All seven lysine (K6, K11, K27, K29, K33, K48 and K63) and the N-terminal M1 residues constitute linkage points during chain elongation 6, although lys48 and lys63 are mostly involved. K48-, K11-, K29 linked poly-Ub chains direct substrates to proteasomal degradation 7, 8, while mono-ubiquitination and K63-linked poly-Ub chains have non-proteolytic functions 9. In contrast, K63-linked chains control “proteasome-independent” events, including inflammation-related signaling pathways, DNA repair, endocytosis, and selective autophagy 10,11.

Nedd4-family E3 members mostly synthesize K63-bound poly-Ub chains 12, 13, unlike other HECT ligases. This is followed by protein recognition and degradation by the 26S proteasome 5, 14. The neuronal precursor cell-expressed developmentally downregulated 4 (NEDD4) family constitutes an important group in the HECT group 15, 16.

NEDD4-1 and NEDD4-like (NEDD4L or NEDD4-2) enzymes represent E3 ubiquitin-protein ligases of the HECT family 17, 18. NEDD4 family enzymes are conserved from yeast to humans 19. As shown in Figure ​Figure11, the NEDD4 family contains nine members in humans: NEDD4-1 (RPF1), NEDD4L (NEDD4-2), ITCH/atropine-1 interaction protein 4 (AIP4), WW domain-containing E3 ubiquitin protein ligase 1 (WWP1), WWP2/Atropine-1-interacting protein 2 (AIP2), NEDL1 (HECW1), NEDL2 (HECW2), SMAD-specific E3 ubiquitin protein ligase 1 (SMURF1) and SMURF2 20. NEDD4 family enzymes structure consists of N-terminal C2 domain (Ca2+/phosphorlipase and membrane-binding; mediating Ca2+-associated targeting to the cell membrane substrate recognition 21), 2-4 WW domain and catalytic C-terminal domain (HECT; 350 residues controlling ubiquitin binding to e-NH2 groups of lysine residues on protein substrates 5, 18, 22). The WW domain catalyzes protein ubiquitination and catabolism (or endocytosis) 15.

The WW domain recognizes and binds to predominantly proline-rich sequences on substrate proteins 23, e.g., PPxY (where x represents any amino acid) 24, PPLP 25, PR 26 and phosphoserine/threonine (pS/pT) residues preceding proline 27. The catalytic HECT domain consists of an “n-leaf” and a catalytic cysteine residue in a “c-leaf” which confers the ligase its catalytic activity 28. NEDD4 E3 ligases differ in function because of distinct WW domains 29 and different substrates 16.

In the past few years, the ubiquitin proteasome system has been attributed key roles in regulating diverse cardiovascular disease 30, 31, including atherosclerosis, familial cardiopathy, idiopathic dilated cardiomyopathy and myocardial ischemia 32. Here we review the current evidence accumulated concerning the cardiovascular role of NEDD4 family members in cardiovascular disease.

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Figure 1
The ubiquitination-proteasome of NEDD4 E3 ligases pathway. Proteins targeted for UPS-induced degradation by NEDD4 E3 ligases comprise polyubiquitin chain by a process involving three proteins, including ubiquitin activating enzyme (E1), ubiquitin conjugating enzyme (E2), and ubiquitin ligase (E3). First, ubiquitin activation is performed by E1. Then, E2 transfers ubiquitin to E3. Finally, NEDD4 E3 ligases catalyses the covalent binding of ubiquitin to the target protein.

NEDD4-1
NEDD4-1 function

NEDD4-1 was first isolated from mouse neural progenitor cells in 1992, with reduced mRNA levels during mouse brain development. NEDD4-1 is widely expressed in the heart, lung, brain, somite, kidney, and other tissues. It may be involved in many human cell functions 33. NEDD4-1 catalyses the degradation of its substrate by polyubiquitination at K48 and K63 34-36 or by single ubiquitination of K6 or K27 37-38, indicating that NEDD4-1 plays a variety of regulatory roles through single/multiple ubiquitination.

The role of NEDD4-1 in myocardial reperfusion injury

Myocardial reperfusion injury involves tissue damage occurring with blood supply return to the cardiac tissue following ischemia or lack of oxygen, causing inflammation 39. NEDD4-1 expression is reduced in the late stage of ischemia/reperfusion (I/R), thereby attenuating its protective effects against cell death and cardiac I/R injury. In addition, activation of the AKT serine/threonine kinase (AKT) pathway protects the heart from I/R injury 40. NEDD4-1 promotes nuclear trafficking of active AKT. Phosphatase and Tensin Homolog (PTEN) represents a critical suppressor of Phosphatidylinositol-3-Kinase (PI3K) signaling and is controlled by NEDD4-1 via polyubiquitination 41-42. PTEN-associated AKT inhibition is suppressed by NEDD4-1, while AKT signaling is activated to protect against I/R-induced cell damage and apoptotic cell death, as demonstrated by decreased BAX and cleaved-caspase3/7 levels and elevated BCL2 amounts. Therefore, NEDD4-1 protects the myocardium from I/R induced apoptosis by activating PI3K/Akt pathway. This regulatory pathway provides a novel insight into reducing the intraoperative injury in patients with myocardial infarction.

The role of NEDD4-1 in heart development

In embryonic day 10.5 (E10.5) mice, NEDD4-1 is expressed in the pharyngeal and gill arches near the heart. Nedd4 knockout mice show severe heart and vascular defects in the second of pregnancy, leading to fetal death, accompanied by significant heart defects and vascular system abnormalities. In particular, outflow tract defects in knockout animals display a double outlet right ventricle 43. Nedd4 knockout mice also have endocardial cushion defects. Abnormalities are also present in the vascular system of Nedd4 knockout mice. In particular, the cephalic plexus vein in some knockout embryos is abnormal. In addition to these significant cardiovascular defects, Nedd4 knockout embryos exhibit delayed maturation of the lungs, where Nedd4 is highly expressed 44.

The role of NEDD4-1 in Vascular Calcification

Vascular calcification represents a major complication of atherosclerosis, chronic renal failure, diabetes, cardiovascular disease and other pathological conditions. More and more evidences show that vascular calcification likens osteogenesis 45-47. Among TGFβ superfamily proteins, BMP2 and TGFβ1 can be targeted for the treatment of multiple bone disorders. In previous study, NEDD4-1 suppression in SM22α+ mouse tissues resulted in deformed aortic structures. Meanwhile, Vitamin D-associated aorta vascular calcification was markedly elevated in Nedd4-null animals compared with wild type littermates. In addition, methylation of human NEDD4 gene promoter is remarkably enhanced in individuals with atherosclerosis. Further research indicated that NEDD4-1 E3 ligase is an important BMP/Smad signaling inhibitor, via polyubiquitination-associated degradation of C-terminal phosphorylated Smad1 (pSmad1) activated by TGF-β. Thus, dysregulated or dysfunctional NEDD4-1 E3 ligase could contribute to vascular calcification in VSMCs through induction of bone generating signals in the process of atherosclerosis progression 48.

NEDD4L
NEDD4L function

NEDD4L on human chromosome 18q21 contains the WW and HECT domains as similar to other NEDD4 genes. In mice, Nedd4L shows homology with Nedd4-1, but has no C2 domain in the N-terminus region. In addition, human NEDD4L and mouse nedd4-2 are homologous 49,50. Nedd4L is widely expressed during mouse development and in adult tissues, especially in the liver, kidney, heart, brain and lung 51,52. Furthermore, NEDD4L plays a vital role in hypertension and arrhythmia.

The role of NEDD4L in hypertension

Hypertension represents an important risk factor for cardiovascular ailments, including myocardial infarction, stroke, heart failure and kidney disease. It reflects salt-sensitivity or resistance when blood pressure response to high- or low-salt diets intake varies substantially 53-54. Epithelial sodium channel (ENaC) suppression in the kidney, lung via the NEDD4 family is important in fluid and electrolyte homeostasis. The ENaC significantly affects kidney Na+ reabsorption 55. It is a hetero-multimeric membrane protein consisting of the homologous subunits α, β, and γ, each comprising intracellular N and C terminal 55. NEDD4L was originally identified as a ligand of the ENaC; NEDD4L binds to β- and γ-subunits of the ENaC through the ENaC proline-tyrosine (PY) motif in the C-terminal region, interacting with the WW domain in NEDD4L, followed by ubiquitination and degradation 56-58. In Liddle’s syndrome, an ENaC incorporating variant β or γ subunits that lack the PY motif leads to deficient interaction with the WW domain of NEDD4L, resulting in excessive Na+ reabsorption and hypertension, with the characteristics of salt-sensitivity, hypokalaemia, metabolic alkalosis, and reduced renin activity and aldosterone amounts 22, 59-61.

Nedd4 knockout mice display high blood pressure even with normal diets, an aliment exacerbated with high-salt diets 62. The IL17A-SGK1/NEDD4L-dependent pathway modulates renal sodium transport by improving renal function in hypertension and other autoimmune disorders. Glucocorticoid Regulated Kinase 1 (SGK1) induced by serum and glucocorticoids triggers a cascade that leads to hypertension by ENaC activation 61-62. SGK1 phosphorylation of NEDD4L promotes its interaction with the chaperone 14-3-3, thus eliminates the ubiquitination of its target substrates 63-64.

SGK1 phosphorylates NEDD4L on serine 444 65, which is necessary for the binding of NEDD4L to 14-3-3, and results in a reduction of ENaC ubiquitination and an enhancement of ENaC activity mediated by NEDD4L. SGK1 is activated by WNK1 (WNK Lysine Deficient Protein Kinase 1), which is involved in pseudo-hypoaldosteronism type II hypertension. WNK1 activates SGK1, inhibits NEDD4L, enhances ENaC activity and leads to hypertension 66. ENaC activity was also observed during the inhibition of NEDD4L by AMP-activated Protein Kinase (AMPK) 65.

These results indicate that IL17A induces an increase in NCC activity through SGK1 phosphorylation and the inhibition of NEDD4L-mediated ubiquitination and NCC degradation. These studies provide a mechanistic link by which the IL17A-SGK1/NEDD4L-dependent pathway modulates renal sodium transport, which may improve renal function in hypertension and other autoimmune disorders 67.

The role of NEDD4L in arrhythmia

Reduced voltage-gated sodium channel (Nav1.5) function and expression supply a slowed conduction substrate for heart arrhythmias. Calcium-associated increases in NEDD4L reduces Nav1.5 levels by ubiquitination. Nav1.5 co-localizes with NEDD4L, and ubiquitin is downregulated in the failing heart in rats. The above findings indicate an important role for NEDD4L in Nav1.5 suppression in heart failure (HF) 68. In addition, defection of the NEDD4L C2 isoform changes cardiac conduction in the resting state as well as pro-arrhythmic alterations upon acute myocardial infarction (MI).

Studies indicated that reduced NEDD4L function results in serious heart arrhythmia via modifications of cardiac ion-channels after transcription. Patients with salt sensitivity and hypertension because of NEDD4L anomalies may be more prone to severe cardiovascular ailments compared with individuals without NEDD4L anomalies. MicroRNA-1(miR-1) regulates genes in the heart and skeletal muscle. Analysis of potential miR-1 target genes identified miR-1 as a direct Nedd4 regulator; in addition, NEDD4L modulates cardiac development in Drosophila 69. Furthermore, miR-1-associated Nedd4 regulation might help control the trafficking and catabolism of cardiac NEDD4L substrates in the cardiac tissue. However, further study is needed to clarify which substrate ubiquitinated by NEDD4L is involved in arrhythmia.

The role of NEDD4L in cardiac regenerative repair

CircRNAs (circular RNAs) represent potent modulators of cardiac development and disease. Studies have found a new circRNA termed Nfix circRNA (circNfix). Down-regulation of circNfix can promote proliferation and angiogenesis in cardiomyocytes, inhibit the apoptosis of cardiomyocytes after myocardial infarction, alleviate cardiac insufficiency and improve patient prognosis. It was found that circNfix enhances Ybx1 (Y-box binding protein 1) interaction with NEDD4L, and induces Ybx1 degradation by ubiquitination via NEDD4L, which inhibits the expression of cyclin A2 and cyclin B1. In addition, suppression of superenhancer modulated circNfix promotes cardiac regeneration and enhances heart function following myocardial infarction via degradation of Ybx1, which may provide a promising strategy to improve prognosis after MI 70.

reference link : https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7586430/


More information: Jantina A. Manning et al. The ubiquitin ligase NEDD4-2/NEDD4L regulates both sodium homeostasis and fibrotic signaling to prevent end-stage renal disease, Cell Death & Disease (2021). DOI: 10.1038/s41419-021-03688-7

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