Abstract: TH-PO016
C-Terminal Oligomerization of Podocin Can Mediate Interallelic Complementation
Session Information
- Complement Your Knowledge of Kidney Disease
November 02, 2017 | Location: Hall H, Morial Convention Center
Abstract Time: 10:00 AM - 10:00 AM
Category: Genetic Diseases of the Kidney
- 802 Non-Cystic Mendelian Diseases
Authors
- Balogh, Eszter, Hungarian Academy of Sciences, Budapest, Hungary
- Straner, Pal, Eötvös Loránd University, Budapest, Hungary
- Schay, Gusztáv, Semmelweis University, Budapest, Hungary
- Arrondel, Christelle, INSERM, Paris, France
- Mikó, Ágnes, Hungarian Academy of Sciences, Budapest, Hungary
- L'aune, Gerda, Hungarian Academy of Sciences, Budapest, Hungary
- Benmerah, Alexandre, INSERM U1163, Imagine Institute, Paris, France
- Perczel, Andras, Eötvös Loránd University, Budapest, Hungary
- Menyhard, Dora K., Eötvös Loránd University, Budapest, Hungary
- Antignac, Corinne, Imagine Institute, Paris, France
- Mollet, Geraldine, Inserm U1163, Paris, France
- Tory, Kalman, Hungarian Academy of Sciences, Budapest, Hungary
Group or Team Name
- MTA-SE Lendulet Nephrogenetic Laboratory
Background
Podocin, a membrane-anchored component of the slit diaphragm, is encoded by NPHS2, the major gene of steroid-resistant nephrotic syndrome. We formerly showed that its most frequent non-silent variant, R229Q, is pathogenic only when trans-associated to specific 3’ missense mutations and suggested that an altered dimerization mediates their dominant negative effect. Here we aimed to determine the membrane targeting and the oligomerization capacity of podocin in function of its C-terminal integrity.
Methods
Podocin localization was studied in cultured podocytes co-transfected with GFP- and HA-tagged podocin variants. The role of endocytosis was studied by the coexpression of a dominant negative dynamin. Binding capacity of the dimers was measured as the FRET efficiency between podocin variants expressed either in HEK293 cells or in E.coli, extracted and stained with fluorescent maleimides. Oligomerization was studied by size exclusion chromatography.
Results
We found neither colocalization, nor FRET between the membranous R286Tfs*17 podocin lacking the C-terminal tail (CTT) and other podocin variants indicating that oligomerization occurs exclusively through the CTT. Podocin variants containing the first helix (H1) of the CTT (273-313) dimerized, and those also containing the 332-348 region, oligomerized. Truncated podocin variants with an intact H1 were significantly influenced in their localization by the coexpressed missense podocin variants. We found the F344Lfs*4 and F344* podocin mutants to be endocytosed. Interestingly, it was inhibited by the coexpression of some membranous podocin variants with an intact CTT. We found the F344Lfs*4 podocin, the first frameshift mutation that is pathogenic with R229Q to similarly retain R229Q podocin as other pathogenic associations. Its dominant negative effect was exerted through the FDL344_346LTY amino acid changes encoded by the frameshift sequence.
Conclusion
Oligomerization of podocin is mediated exclusively by the CTT. Though oligomerization is not prerequisite for membrane-targeting, it may mediate not only a dominant negative effect between podocin variants, but also normalization of the localization, i.e. interallelic complementation. Such a complementation can also modify the pathogenicity of NPHS2 alleles.
Funding
- Other NIH Support