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Regulation of epithelial Na(+) transport: implication of controlled ubiquitylation and ER-associated degradation

English title Regulation of epithelial Na(+) transport: implication of controlled ubiquitylation and ER-associated degradation
Applicant Staub Olivier
Number 125422
Funding scheme Project funding
Research institution Département de Pharmacologie & Toxicologie Faculté de Biologie et de Médecine Université de Lausanne
Institution of higher education University of Lausanne - LA
Main discipline Cardiovascular Research
Start/End 01.04.2009 - 31.03.2012
Approved amount 468'000.00
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Keywords (12)

ion channels; surface expression; ubiquitin; ERAD; sodium homeostasis; hypertension; deubiquitylation; multimeric membrane protein; sodium transport; kidney; ER-associated degradation; quality control

Lay Summary (English)

Lead
Lay summary
In most developed countries, excess salt intake is a major risk factor of hypertension, one of the most common diseases in the human population, affecting over 1 billion people worldwide.The kidney is able to precisely regulate the balance between uptake and excretion of sodium into and from the blood by regulating sodium transport in the kidney. This transport involves the regulated action of a sodium channel (called ENaC), which forms a pore across membranes and allows passage of sodium. Because sodium is the most important cation in the blood, changes in its concentration have direct consequences on blood volume and hence on the arterial blood pressure. The sodium channel is regulated by hormonal mechanisms, and can be strongly disturbed in diseases such as Liddle's syndrome, an inherited form of human hypertension We have largely contributed to the understanding of this regulation, by showing that the activity of this sodium channel is controlled by a mechanism called ubiquitylation, in which a small protein (named ubiquitin) is linked to the ENaC channel, promoting its internalization and degradation. Interestingly, ubiquitylation is reversible, and it is the balance between ubiquitylation and deubiquitylation that controls the channel's activity.Aim:In this project we want to study in detail the mechanisms of deubiquitylation both in vitro and in vivo, in epithelial cells derived from the kidney, and in mouse models, in which ENaC deubiquitylation has been inactivated. Moreover, we will investigate the role of ubiquitylation in the quality control of proper ENaC synthesis and surface expression.Significance:The process addresses fundamental issues with respect to the regulation of sodium balance (and its consequences for maintenance of blood pressure), and it treats novel biological issues with respect to the ubiquitylation/deubiquitylation and quality control of ion channels.
Direct link to Lay Summary Last update: 21.02.2013

Responsible applicant and co-applicants

Employees

Publications

Publication
Aldosterone paradox: differential regulation of ion transport in distal nephron.
Arroyo Juan Pablo, Ronzaud Caroline, Lagnaz Dagmara, Staub Olivier, Gamba Gerardo (2011), Aldosterone paradox: differential regulation of ion transport in distal nephron., in Physiology (Bethesda, Md.), 26(2), 115-23.
Alveolar epithelial CNGA1 channels mediate cGMP-stimulated, amiloride-insensitive, lung liquid absorption.
Wilkinson William J, Benjamin Audra R, De Proost Ian, Orogo-Wenn Maria C, Yamazaki Yasuo, Staub Olivier, Morita Takashi, Adriaensen Dirk, Riccardi Daniela, Walters Dafydd V, Kemp Paul J (2011), Alveolar epithelial CNGA1 channels mediate cGMP-stimulated, amiloride-insensitive, lung liquid absorption., in Pflugers Archiv : European journal of physiology, 462(2), 267-79.
Deubiquitylating enzyme USP2 counteracts Nedd4-2-mediated downregulation of KCNQ1 potassium channels.
Krzystanek Katarzyna, Rasmussen Hanne Borger, Grunnet Morten, Staub Olivier, Olesen Søren-Peter, Abriel Hugues, Jespersen Thomas (2011), Deubiquitylating enzyme USP2 counteracts Nedd4-2-mediated downregulation of KCNQ1 potassium channels., in Heart rhythm : the official journal of the Heart Rhythm Society, 9(3), 440-8.
Intracellular ubiquitylation of the epithelial Na+ channel controls extracellular proteolytic channel activation via conformational change.
Ruffieux-Daidié Dorothée, Staub Olivier (2011), Intracellular ubiquitylation of the epithelial Na+ channel controls extracellular proteolytic channel activation via conformational change., in The Journal of biological chemistry, 286(4), 2416-24.
Nedd4-2 modulates renal Na+-Cl- cotransporter via the aldosterone-SGK1-Nedd4-2 pathway.
Arroyo Juan Pablo, Lagnaz Dagmara, Ronzaud Caroline, Vázquez Norma, Ko Benjamin S, Moddes Lauren, Ruffieux-Daidié Dorothée, Hausel Pierrette, Koesters Robert, Yang Baoli, Stokes John B, Hoover Robert S, Gamba Gerardo, Staub Olivier (2011), Nedd4-2 modulates renal Na+-Cl- cotransporter via the aldosterone-SGK1-Nedd4-2 pathway., in Journal of the American Society of Nephrology : JASN, 22(9), 1707-19.
Role of the ubiquitin system in regulating ion transport.
Rotin Daniela, Staub Olivier (2011), Role of the ubiquitin system in regulating ion transport., in Pflugers Archiv : European journal of physiology, 461(1), 1-21.
Ubiquitin-specific protease 2-45 (Usp2-45) binds to epithelial Na+ channel (ENaC)-ubiquitylating enzyme Nedd4-2.
Oberfeld Benjamin, Ruffieux-Daidié Dorothée, Vitagliano Jean-Jacques, Pos Klaas Martinus, Verrey François, Staub Olivier (2011), Ubiquitin-specific protease 2-45 (Usp2-45) binds to epithelial Na+ channel (ENaC)-ubiquitylating enzyme Nedd4-2., in American journal of physiology. Renal physiology, 301(1), 189-96.
Intracellular ubiquitylation of the epithelial Na+ channel controls extracellular proteolytic channel activation via conformational change.
Ruffieux-Daidié Dorothée, Staub Olivier (2010), Intracellular ubiquitylation of the epithelial Na+ channel controls extracellular proteolytic channel activation via conformational change., in The Journal of biological chemistry, 286(4), 2416-24.
Mineralocorticoid receptor degradation is promoted by Hsp90 inhibition and the ubiquitin-protein ligase CHIP.
Faresse Nourdine, Ruffieux-Daidie Dorothée, Salamin Mélanie, Gomez-Sanchez Celso E, Staub Olivier (2010), Mineralocorticoid receptor degradation is promoted by Hsp90 inhibition and the ubiquitin-protein ligase CHIP., in American journal of physiology. Renal physiology, 299(6), 1462-72.
Role of the ubiquitin system in regulating ion transport.
Rotin Daniela, Staub Olivier (2010), Role of the ubiquitin system in regulating ion transport., in Pflugers Archiv : European journal of physiology, 461(1), 1-21.
Stimulation of ENaC activity by rosiglitazone is PPARγ-dependent and correlates with SGK1 expression increase.
Renauld Stephane, Tremblay Karine, Ait-Benichou Siham, Simoneau-Roy Maxime, Garneau Hugo, Staub Olivier, Chraïbi Ahmed (2010), Stimulation of ENaC activity by rosiglitazone is PPARγ-dependent and correlates with SGK1 expression increase., in The Journal of membrane biology, 236(3), 259-70.
Characterization and binding affinities of SmLANP: a new Schistosoma mansoni member of the ANP32 family of regulatory proteins.
Pinheiro C S, Staub O, Mulvenna J, Loukas A, Jones M K, Rabelo E M (2009), Characterization and binding affinities of SmLANP: a new Schistosoma mansoni member of the ANP32 family of regulatory proteins., in Molecular and biochemical parasitology, 165(2), 95-102.
hShroom1 links a membrane bound protein to the actin cytoskeleton.
Dye D E, Karlen S, Rohrbach B, Staub O, Braathen L R, Eidne K A, Coombe D R (2009), hShroom1 links a membrane bound protein to the actin cytoskeleton., in Cellular and molecular life sciences : CMLS, 66(4), 681-96.

Collaboration

Group / person Country
Types of collaboration
University of Mexico Mexico (North America)
- in-depth/constructive exchanges on approaches, methods or results
- Publication

Associated projects

Number Title Start Funding scheme
128693 Etudes de génomique, de protéomique et études fonctionnelles sur des segments de néphrons isolés 01.12.2009 R'EQUIP
128784 Protein Analysis Facility of the University of Lausanne : renewal of a Quadrupole-Time-Of-Flight (Q-TOF) mass spectrometer for proteomics 01.01.2010 R'EQUIP
103779 Regulation of transepithelial NA+ transport in the distal bephron by ubiquitination and de-ubiquitination 01.04.2004 Project funding
141013 Integrated kinase and ubiquitylation networks coordinating balanced Na+ homeostasis and blood pressure regulation by the kidney 01.04.2012 Project funding

Abstract

Excess salt intake is a major risk factors of hypertension, one of the most common diseases in the human population, affecting over 1 billion individuals worldwide. The aldosterone-sensitive distal nephron (ASDN) is able to finely tune the Na(+ ) balance in extracellular fluids (including the blood) by regulating transepithelial Na(+) transport. In this context the epithelial Na(+) channel (ENaC) is highly regulated by a number of hormonal pathways, including aldosterone, vasopressin, and IGF/insulin. We have significantly contributed to the understanding of these regulatory pathways by demonstrating that ubiquitylation plays a central role in ENaC regulation. We showed that the ubiquitin-protein ligase Nedd4-2, interacts with and ubiquitylates ENaC, leading to channel internalization. We then demonstrated that this mechanism is impaired in Liddle’s syndrome, an inherited form of human hypertension, in which mutations in the genes encoding the ENaC subunits inactivate the binding sites for Nedd4-2, and consequently cause accumulation of channels at the cell surface. We have also provided evidence that ENaC ubiquitylation is regulated by several, potentially synergistic pathways: 1) aldosterone-induced Sgk1 kinase phosphorylates Nedd4-2, thereby promoting 14-3-3 binding and interference with Nedd4-2/ENaC binding, 2) dietary Na+ dependant variation of Nedd4-2 expression in the ASDN, and 3) aldosterone-induced expression of Usp2-45, a deubiquitylating enzyme able to deubiquitylate ENaC, and to promote increased cell surface expression as well as proteolytic activation of ENaC. Recently, a Nedd4-2 KO mouse generated by our collaborators proofed the concept that Nedd4-2 plays a role in salt-dependant hypertension. However, other questions remain pertinent such as the in vivo contribution of each Sgk1 and/or Usp2-45 in ENaC regulation, or the involved molecular mechanisms which we just begin to understand. Another, so far neglected mechanism in ENaC regulation involves the assembly, quality control and ER associated degradation (ERAD), which we believe to play another important role to control ENaC targeting to the cell surface. Hypothesis:ENaC is regulated by 2 different mechanisms of ubiquitylation: one involving regulated Nedd4-2 dependant multi-mono-ubiquitylation, and the other one implying ERAD dependant polyubiquitylation, important for channel export to the plasma membrane.Specific aims:1) To establish the molecular mechanism and the role of Usp2-45 in transepithelial Na+ transport both in cells derived from the CCD and in vivo models and to study the relation between Sgk1 and Usp2-45 in Na+ homeostasis in vivo in novel mice models.Methods: Gene expression/suppression in cell lines. Investigation of renal parameters of KO mice2) To characterize the molecular machinery that is involved in ER associated retention and degradation of the epithelial Na+ channel ENaC and its possible role in ENaC regulation.Methods: Identification and characterization of proteins involved in ER associated retention and degradation of ENaC. Use of various cell lines (including renal epithelial cells) for functional and biochemical analysis.Expected value of project:The project addresses fundamental issues with respect to the physiological/pathophysiological regulation of renal Na(+) homeostasis (and its consequences for maintenance of blood pressure), and it treats novel cell biological issues with respect to ubiquitylation/deubiquitylation and ERAD in the control of ion channels.
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