PhD Scientific Days 2020

Theoretical and Translational III. Posters

Effect of Podocin Variants on the Podocin-Nephrin and Nephrin-Nephrin Interaction

Előadó neve

Dr. Violetta, Antal

Előadó munkahelye

MTA-SE Lendület Nephrogenetic Laboratory; Semmelweis University, Ist Department of Pediatrics

Előadó telefonszáma

+36204638586

Előadó e-mail címe

antal.violetta@med.semmelweis-univ.hu

Az előadás címe

Effect of Podocin Variants on the Podocin-Nephrin and Nephrin-Nephrin Interaction

Szerző(k) neve és munkahelye

Violetta Antal1,4, Máté Kétszeri1,4, Gusztáv Schay2, Anita Ungvári-Veres1, Katalin Csókay2, Miklós Kellermayer2, Dóra Karancsiné Menyhárd3, Kálmán Tory1,4
1 MTA-SE Lendület Nephrogenetic Laboratory, Budapest, Hungary
2 Semmelweis University, Department of Biophysics and Radiation Biology, Budapest, Hungary
3 MTA-ELTE Protein Modeling Research Group and Laboratory of Structural Chemistry and Biology, Eötvös Loránd University, Budapest, Hungary
4 Semmelweis University, Ist Department of Pediatrics, Budapest, Hungary

Szekció

Theoretical and Translational III. Posters

Language of the presentation

Hungarian

Section, first choice

Theoretical and Translational Medicine

Section, second choice

Molecular Sciences

Összefoglaló szövege

Introduction: Podocin is a major component of the glomerular slit diaphragm. It is encoded by NPHS2, the most frequently mutated gene in nephrotic syndrome. We formerly showed that its most frequent non-silent variant, R229Q makes podocin prone to form abnormal oligomers. Podocin binds nephrin, the major, transmembranous component of the slit diaphragm. The shortest dimension of the glomerular pore (3,5-4nm) is determined by the distance between nephrin molecules in cis position. We hypothesized that podocin oligomerization regulates the distance between the nephrin molecules.
Aims: We aimed to explore the effect of podocin variants on the podocin-nephrin and the nephrin-nephrin interaction.
Methods: To assess the effect of podocin variants on the nephrin-nephrin distance, wild type nephrin tagged C-terminally with YPet or with mRuby3 and HA-tagged podocin variants were coexpressed in HEK293 cells. In a second set of experiments the fluorescent tags were inserted in the extracellular, juxtamembranous part of nephrin.
To investigate the effect of R229Q podocin on the nephrin-podocin interaction, C-terminal YPet-tagged nephrin was coexpressed with mRuby3-tagged wild type (wt) podocin and either HA-tagged wt or R229Q podocin in HEK293 cells. The mRuby3-tagged and the HA-tagged podocin were encoded by the same vector (pKK-BI16) allowing the equal expression of the two forms.
Förster resonance energy transfer (FRET) was measured between the fluorescently labelled nephrin or podocin and nephrin molecules in living cells. The fluorescence decay curves measured by time-correlated single photon counting (Chronos BH, ISS Inc.) were decomposed into lifetime components, and the longest lifetime population was used for further calculations.
Results: The FRET efficiency between the nephrin molecules increased when wt podocin was coexpressed (p<0.0005), but remained unaltered in the presence of pathogenic podocin variants (R286Tfs*17, R138Q, A284V, F344Lfs*4). Similar results were obtained with the extracellular nephrin tags.
R229Q podocin reduced the FRET efficiency between nephrin and podocin as compared to the wt podocin (p<0.05).
Conclusion: Podocin regulates the distance between the nephrin molecules: the shortest dimension of the glomerular pore. R229Q podocin induces a change in the conformation or the binding capacity of the nephrin-podocin complex.

Additional Information

Supervisor: Kálmán Tory
E-mail address: tory.kalman@med.semmelweis-univ.hu
Supported by the ÚNKP-19-3-I New National Excellence Program of the Ministry for Innovation and Technology.

Bemutatás módja

Szóbeli

Kind

Szabad

Status

elfogadva

Accepted presentation method

poszter

Előadás fájl jóváhagyás

nem rendelkezett róla

Előadó

4759

Start

11:13

End

11:16

Authors (legacy)

Violetta Antal1,4, Máté Kétszeri1,4, Gusztáv Schay2, Anita Ungvári-Veres1, Katalin Csókay2, Miklós Kellermayer2, Dóra Karancsiné Menyhárd3, Kálmán Tory1,4
1 MTA-SE Lendület Nephrogenetic Laboratory, Budapest, Hungary
2 Semmelweis University, Department of Biophysics and Radiation Biology, Budapest, Hungary
3 MTA-ELTE Protein Modeling Research Group and Laboratory of Structural Chemistry and Biology, Eötvös Loránd University, Budapest, Hungary
4 Semmelweis University, Ist Department of Pediatrics, Budapest, Hungary