C-terminal Oligomerization of Podocin Mediates Interallelic Interactions

Biochim Biophys Acta Mol Basis Dis. 2018 Jul;1864(7):2448-2457. doi: 10.1016/j.bbadis.2018.04.008. Epub 2018 Apr 13.

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 (r2 = 0.68, P = 9.2 × 10-32). 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.

Keywords: Complementation; Dominant negative effect; Endocytosis; Membrane targeting; Nephrotic syndrome; Podocin.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Substitution
  • Cell Line, Transformed
  • Fluorescence Resonance Energy Transfer
  • Humans
  • Intracellular Signaling Peptides and Proteins* / chemistry
  • Intracellular Signaling Peptides and Proteins* / genetics
  • Intracellular Signaling Peptides and Proteins* / metabolism
  • Kidney Diseases* / genetics
  • Kidney Diseases* / metabolism
  • Kidney Diseases* / pathology
  • Membrane Proteins* / chemistry
  • Membrane Proteins* / genetics
  • Membrane Proteins* / metabolism
  • Mutation, Missense*
  • Podocytes / metabolism*
  • Podocytes / pathology
  • Protein Domains
  • Protein Multimerization / genetics*

Substances

  • Intracellular Signaling Peptides and Proteins
  • Membrane Proteins
  • NPHS2 protein