C-terminal oligomerization of podocin mediates interallelic interactions

Abstract

Interallelic interactions of membrane proteins are not taken into account while evaluating the pathogenicity of sequence variants in autosomal recessive disorders. Podocin, a membrane-anchored component of the slit diaphragm, is encoded by NPHS2, the major gene mutated in hereditary podocytopathies. We formerly showed that its R229Q variant is only pathogenic when trans-associated to specific 3' mutations and suggested the causal role of an abnormal C-terminal dimerization. Here we show by FRET analysis and size exclusion chromatography that podocin oligomerization occurs exclusively through the C-terminal tail (residues 283-382): principally through the first C-terminal helical region (H1, 283-313), which forms a coiled coil as shown by circular dichroism spectroscopy, and through the 332-348 region. We show the principal role of the oligomerization sites in mediating interallelic interactions: while the monomer-forming R286Tfs*17 podocin remains membranous irrespective of the coexpressed podocin variant identity, podocin variants with an intact H1 significantly influence each other's localization (r = 0.68, P = 9.2 × 10). The dominant negative effect resulting in intracellular retention of the pathogenic F344Lfs*4-R229Q heterooligomer occurs in parallel with a reduction in the FRET efficiency, suggesting the causal role of a conformational rearrangement. On the other hand, oligomerization can also promote the membrane localization: it can prevent the endocytosis of F344Lfs*4 or F344* podocin mutants induced by C-terminal truncation. In conclusion, C-terminal oligomerization of podocin can mediate both a dominant negative effect and interallelic complementation. Interallelic interactions of NPHS2 are not restricted to the R229Q variant and have to be considered in compound heterozygous individuals.

The mutation-dependent pathogenicity ofNPHS2p.R229Q: A guide for clinical assessment

“…Since the CTT contains a number of hydrophobic amino acids in a distribution reminiscent of the interaction motifs of a coiled‐coil association (a superhelix of helices), we proposed that the dimers are formed by the antiparallel inter‐twine of the H1 helices, reinforced by the wrapping H2 helices (Figure a) (Tory et al., ). In a recent study, we could gain experimental support for this notion, showing that the H1 segment in itself forms a coiled coil, oligomers are assembled from dimeric pairs, and that while dimerization is driven by the H1 and H2 helices, oligomerization is mediated by the short connecting segment between H2 and H3 and H3 itself (Straner et al., ). Using a model based on these findings, we demonstrated that the altered pliability of p.R229Q podocin affects the evolving dimer structures, but this effect is only significant if the associated mutation falls on the winding interaction surface of the coiled‐coiled CTTs (as in positions 284, 288, 295, 297, and 327, but not the outward facing 290) (Figure a,b).…”

“…Analysis of the pathogenic p.R229Q associations allowed us to understand the cellular basis of the pathogenic interactions. In cell culture experiments, we found all the examined dominant negative mutations (p.A284V, p.A288T, p.R291W, p.A297V, p.E310K, p.F344Lfs*4) to mislocalize the encoded podocin and to also retain the coexpressed p.R229Q podocin in intracellular compartments (Straner et al., ; Tory et al., ). Accordingly, the dominant negative mutations are all expected to be pathogenic in the homozygous state, they affect evolutionary conserved amino acids, and with the exception of p.Q328R, all the missense ones are predicted to be pathogenic by Polyphen‐2 (Table ).…”

“…The c.686G>A, p.R229Q variant of the NPHS2 (MIM# 600995) gene, encoding podocin, is the first human variant discovered with a mutation‐dependent pathogenicity; it is only pathogenic when trans‐associated to specific mutations ([p.R229Q];[mut] pat ) (Tory et al., ). These mutations exert a dominant negative effect on p.R229Q podocin by blocking its membrane trafficking through an altered heterooligomerization (Straner et al., ; Tory et al., ). Mutations of NPHS2 cause typically early‐onset steroid‐resistant nephrotic syndrome (SRNS), which leads to end‐stage renal disease before the age of 10 years (Boute et al., ; Hinkes et al., ).…”

“…Given that only a few mutations are pathogenic with p.R229Q, and these are rare among the NPHS2 mutations, a significant enrichment can be easily achieved by a relatively small number of patients with [p.R229Q];[mut] (currently n ≥ 2 out of 106; Table ). This approach allowed us to prove the dominant negative effect of p.A284V, p.A288T, p.R291W, p.A297V, p.E310K, p.E310V, p.L327F, p.Q328R, and p.F344Lfs*4 (Straner et al., ; Tory et al., ).…”

“…According to our structural model, which is primarily based on the crystal structure of the partially homologous stomatin (Tory et al., ), the residues 161–332 of podocin form a globular PHB domain (161–282) and a mainly helical tail region (C‐terminal tail: CTT) comprising two helical segments (H1: 282–313, H2: 317–330). Secondary structure prediction indicates that the final stretch of the C‐terminal contains a further helix (H3: 344–350) and an unstructured loop (351–383) at the terminus (Straner et al., ). The p.R229 is located on the PHB domain, in a position facing the CTT (Figure a).…”