PhD Scientific Days 2017

Oral Presentations: Basic Sciences II.

Binding capacity of pathogenic and non-pathogenic NPHS2 heterodimers

Előadó neve

Balogh, Eszter

Előadó munkahelye

1st Department of Pediatrics, Semmelweis University, Budapest

Előadó telefonszáma

+36 20 773 86 23

Előadó e-mail címe

baloghesztermail@gmail.com

Az előadás címe

Binding capacity of pathogenic and non-pathogenic NPHS2 heterodimers

Szerző(k) neve és munkahelye

Eszter Balogh, 1st Department of Pediatrics/Semmelweis University, Budapest
Mikó Ágnes, 1st Department of Pediatrics/Semmelweis University, Budapest

Témacsoport

molecular sciences

Szekció

Oral Presentations: Basic Sciences II.

Data of the presenter

Doctoral School of Clinical Medicine
Prevention of Chronic Diseases in Childhood
Kalman Tory, Gusztáv Schay
tory.kalman@med.semmelweis-univ.hu
schay.gusztav@med.semmelweis.univ.hu

Text of the abstract

NPHS2, encoding podocin, is the most frequently mutated gene in steroid-resistant nephrotic syndrome. We recently showed that the pathogenicity of its frequent R229Q variant depends on the trans-associated mutation providing the first example of mutation-dependent recessive inheritance in human genetics. Based on molecular modeling, we predicted that the pathogenic heterodimers have an altered structure.
We aimed to determine the binding capacity of pathogenic and non-pathogenic heterodimers of R229Q and wt podocin. Podocin variants (wt, R229Q, R286Tfs*17, A317Lfs*31, F344* and F344Lfs*4) were transiently expressed in HEK293 cells, extracted by immunoprecipitation, eluted by competitive peptide elution and stained by either Alexa 488 and 555 maleimides. Differently stained podocin variants were mixed two by two and FRET was measured by fluorescence lifetime spectrometer in the Department of Biophysics.
We found the heterodimerization of both R229Q and wt podocin to be highly dependent on the C-terminal truncation of the interacting podocin: while the R286Tfs*17 podocin – lacking all three C-terminal helical regions – had no significant effect on fluorescence lifetime, A317Lfs*31, F344* and F344Lfs*4 all significantly reduced that of both wt and R229Q podocin. The amino acid changes encoded by the frameshift sequence of the F344Lfs*4 podocin exerted a different effect on the non-pathogenic wt-F344Lfs*4 and the pathogenic R229Q-F344Lfs*4 heterodimers by increasing that of the former and decreasing the latter. Nevertheless, we found no clear-cut difference in the binding capacity of the pathogenic and the non-pathogenic dimers.
In conclusion, in contrast to previous reports, the dimerization capacity of podocin is determined exclusively by its C-terminal part. As the R286Tfs*17 podocin is localized to the plasma membrane, the dimerization of podocin is not necessary for the membrane-targeting. The binding capacity on its own does not explain the pathogenicity of some podocin dimers.

Kind

Szabad

Előadás fájl jóváhagyás

nem rendelkezett róla

Előadó

1297

Authors (legacy)

Eszter Balogh, 1st Department of Pediatrics/Semmelweis University, Budapest
Mikó Ágnes, 1st Department of Pediatrics/Semmelweis University, Budapest