Abstract: TH-OR57
COP9 Signalosome CSN4 and CSN5 Subunits Protect NKCC2 against Endoplasmic Reticulum Stress and Cellular Hypoxia
Session Information
- Fluid, Electrolyte, and Acid-Base Disorders: Back to the Basics
October 24, 2024 | Location: Room 4, Convention Center
Abstract Time: 04:50 PM - 05:00 PM
Category: Fluid, Electrolytes, and Acid-Base Disorders
- 1101 Fluid, Electrolyte, and Acid-Base Disorders: Basic
Authors
- Laghmani, Kamel, Centre de Recherche des Cordeliers, Paris, France
- Kharma, Stephanie, Centre de Recherche des Cordeliers, Paris, France
- Demaretz, Sylvie, Centre de Recherche des Cordeliers, Paris, France
- Bakhos Al Douaihy, Dalal, Centre de Recherche des Cordeliers, Paris, France
Background
MAGE-D2 mutations cause a severe but transient form of antenatal Bartter syndrome (tBS) associated with impaired expression of the sodium–chloride cotransporters NKCC2 and NCC. Cellular hypoxia is particularly interesting to elucidate the molecular basis of the disease because it is one of the major differences between the antenatal symptomatic period of MAGE-D2-related tBS and the post-natal asymptomatic stage. In support of this, MAGE-D2 was found to be required for appropriate induction of HIF-1α and the protection of NKCC2 against ER stress during hypoxia. The present study aimed to explore further the molecular mechanisms involved in the protection of NKCC2 by MAGE-D2 against ER stress and hypoxia.
Methods
A yeast two-hybrid system (Y2H) was used to identify novel NKCC2 binding partners. Protein-protein interactions were analyzed using co-immunoprecipitation (CO-IP) and co-immunolocalization assays. Cellular hypoxia was induced chemically by incubating HEK cells for 16 hours with cobalt chloride (CoCl2). NKCC2 protein expression was monitored by immunoblotting in HEK cells transfected with the co-transporter. NKCC2 stability was assessed by cycloheximide chase assay.
Results
Y2H identified the COP9 signalosome CSN4 and CSN5 subunits as novel binding partners of NKCC2. This is of particular interest because CSN5 is a MAGE-D2 interactor and also a regulator of HIF-1α stability. Co-IP and co-immunolocalization experiments confirmed CSN4 and CSN5 interactions with NKCC2. Similar to MAGE-D2, CSN4 or CSN5 overexpression increased total and surface NKCC2 abundance. Conversely, CSN4 or CSN5 knockdown enhanced the ER-associated degradation (ERAD) of the cotransporter. Subjecting HEK cells transiently with NKCC2 to cellular hypoxia, significantly decreased NKCC2 stability and maturation. Most importantly, like MAGE-D2, the effect of hypoxia on NKCC2 stability and maturation was more severe following CSN4 or CSN5 knockdown.
Conclusion
Our data reveal that CSN4 and CSN5 are novel key players in the ERAD of NKCC2 under ER stress conditions. Most importantly, our findings strongly suggest that MAGE-D2 cooperates with these COP9 signalosome subunits to protect NKCC2 against ERAD induced by cellular hypoxia, which could contribute to the transient nature of antenatal Bartter’s syndrome in carriers of MAGED2 mutations.
Funding
- Government Support – Non-U.S.