Differential Effects of STCH and Stress-Inducible Hsp70 on the Stability and Maturation of NKCC2

Abstract

Mutations in the Na-K-2Cl co-transporter NKCC2 lead to type I Bartter syndrome, a life-threatening kidney disease. We previously showed that export from the ER constitutes the limiting step in NKCC2 maturation and cell surface expression. Yet, the molecular mechanisms involved in this process remain obscure. Here, we report the identification of chaperone stress 70 protein (STCH) and the stress-inducible heat shock protein 70 (Hsp70), as two novel binding partners of the ER-resident form of NKCC2. STCH knock-down increased total NKCC2 expression whereas Hsp70 knock-down or its inhibition by YM-01 had the opposite effect. Accordingly, overexpressing of STCH and Hsp70 exerted opposite actions on total protein abundance of NKCC2 and its folding mutants. Cycloheximide chase assay showed that in cells over-expressing STCH, NKCC2 stability and maturation are heavily impaired. In contrast to STCH, Hsp70 co-expression increased NKCC2 maturation. Interestingly, treatment by protein degradation inhibitors revealed that in addition to the proteasome, the ER associated degradation (ERAD) of NKCC2 mediated by STCH, involves also the ER-to-lysosome-associated degradation pathway. In summary, our data are consistent with STCH and Hsp70 having differential and antagonistic effects with regard to NKCC2 biogenesis. These findings may have an impact on our understanding and potential treatment of diseases related to aberrant NKCC2 trafficking and expression.

New insights into the role of endoplasmic reticulum‐associated degradation in Bartter Syndrome Type 1

“…The above findings strongly suggest that in cells expressing NKCC2 variants, the defect in the cotransporter maturation arises from ER retention and associated degradation. Given that NKCC2 protein is modified by N ‐linked glycosylation (Benziane et al, 2007) and that several studies have shown that mannose trimming is required for ER‐associated proteasomal degradation of glycoproteins (Bakhos‐Douaihy et al, 2021; Tokunaga et al, 2000; Wang et al, 2011), we compared the effect of MG132 to that of kifunensine on NKCC2 variants. Of note, kifunensine is commonly used to inhibits α‐mannosidases I (including ER mannosidase I and ER Degradation Enhancing Alpha‐Mannosidase Like Protein [EDEM]) that are required for the early steps of ERAD misfolded glycoprotein recognition (Bakhos‐Douaihy et al, 2021; Gong et al, 2005; Groisman et al, 2011; Tokunaga et al, 2000; Wang et al, 2011).…”

“…(a) Effect of MG132 on protein levels of wild‐type and mutants NKCC2 proteins in HEK cells. HEK cells transiently transfected with wild‐type NKCC2 or NKCC2 mutants were treated without (C) or with 2 µM MG132 (M) for 6 for 12 h prior cell lysis, as previously described (Bakhos‐Douaihy et al, 2021). The cell lysates were subjected to SDS‐PAGE and immunoblotted with anti‐Myc antibody.…”

“…In this regard, it is worth noting that export from the ER constitutes the limiting step in the maturation and cell surface expression of the NKCC2 (Zaarour et al, 2012). Despite this, only a few studies performed by our group, have dealt with this phenomenon (Bakhos-Douaihy et al, 2021, #1683Zaarour et al, 2009Zaarour et al, , 2012. In particular, we previously demonstrated that the ER lectin, OS9, interacts with the C-terminal tail of the immature form of NKCC2, an interaction that takes place at the ER (Seaayfan et al, 2016).…”

“…Given the high degree of identity at the protein level between human and mouse NKCC2 (> 90%), with all mutations occurring in residues that are fully conserved among species (Figure 1b), we generated and characterized these independent mutations in a mouse NKCC2 construct, that we previously used in several reports (Bakhos-Douaihy et al, 2021;Laghmani et al, 2016;Seaayfan et al, 2016;Trudu et al, 2013;Zaarour et al, 2009Zaarour et al, , 2011Zaarour et al, , 2012. To characterize the expression and function of WT and mutant NKCC2, we transiently transfected N-terminally tagged WT or mutant proteins into cultured renal cells.…”

Molecular Basis, Diagnostic Challenges and Therapeutic Approaches of Bartter and Gitelman Syndromes: A Primer for Clinicians

“…The second postulates that the mutations in MAGED2 prevent sufficient concentrations of cyclic adenosine monophosphate (cAMP) for the correct function of the antidiuretic hormone (ADH), causing the mislocalization of the channels [ 70 ]. It is essential to bear in mind that ERAD, lysosomal degradation and the specific ubiquitination of unfolded and immature NCC and NKCC2 have also been widely described as key mechanisms of protein expression and localization [ 72 , 73 , 74 , 75 ], for which further study of Bartter’s disease type 5 will contribute to a greater understanding of these fundamental processes. Similarly, the presence of mutations in the MAGED2 gene could explain the cases of idiopathic polyhydramnios and unresolved prenatal tubulopathies [ 76 ].…”