PhD Scientific Days 2024

Poster Session A - Molecular Medicine 1.

Investigation of the interaction between the ubiquitin ligase adapter protein Ndfip1 and the TRESK channel

Előadó neve

Pergel, Enikő, PhD

Neptun code

BJHJLW

Előadó munkahelye

Semmelweis University

Előadó telefonszáma

+36309469473

Előadó e-mail címe

enikopergel@gmail.com

Az előadás címe

Investigation of the interaction between the ubiquitin ligase adapter protein Ndfip1 and the TRESK channel

Szerző(k) neve és munkahelye

Enikő Pergel, PhD¹, Gábor Czirják¹

1: Semmelweis University

Bemutatás módja

Poszter

Szekció

Poster Session A - Molecular Medicine 1.

Language of the presentation

Hungarian

Preferred session

Molecular Medicine

Összefoglaló szövege

Investigation of the interaction between the Ndfip1 protein and the TRESK channel

Enikő Pergel, Gábor Czirják
Semmelweis University, Budapest

Introduction
TRESK belongs to the K2P channel family. Its operation creates a background K+ current, which contributes to the maintenance of the resting membrane potential of sensory neurons. We tested TRESK interaction partners found in a public database, which were predicted by a high-throughput assay, and the Ndfip1 (Nedd4 Family Interacting Protein 1) seemed to have an effect on TRESK.
Aims
I would like to contribute to a detailed understanding of the regulation of the TRESK channel, by mapping possible protein interaction partners, and by examining mechanisms related to ubiquitination.
Method
The coding sequences of Ndfip1 and NEDD4 were amplified from human RNA samples by PCR followed by a reverse transcription reaction, and then inserted into a suitable Xenopus laevis oocyte expression vector by molecular cloning. Plasmids encoding mutant proteins were created using Quikchange PCR-based mutagenesis. Subsequently, cRNA was produced from the constructs by in vitro transcription, with which I injected the oocytes. I measured the current of the expressed channel using the two-electrode voltage clamp method. The effect of Ndfip1 co-expression was also investigated by Western blot.
Results
Co-expression of Ndfip1 with TRESK significantly reduced the K+ current. Co-expression of a mutant Ndfip1 - unable to bind NEDD4 (Neuronal precursor cell-expressed developmentally downregulated 4) - could not exert a reducing effect on TRESK current. I compared the effect of the wild type and the catalytically inactive dominant negative mutant NEDD4 on cells coexpressing both TRESK and Ndfip1. Co-expression of wild-type NEDD4 enhanced the current-reducing effect of Ndfip1, while co-expressing the mutant NEDD4 alongside TRESK and Ndfip1 prevented the effect of Ndfip1. I showed with Western Blot that the amount of TRESK protein decreased in the group that also co-expressed Ndfip1. In addition, co-expression of Ndfip1 significantly increased the ubiquitination of TRESK.
Conclusion
We can conclude that Ndfip1 can interact with the TRESK protein and we can prove with further experiments that as an adaptor, it can contribute to the ubiquitination and subsequent degradation of TRESK by NEDD4.
Funding
ÚNKP-23-4-II
NKFIH K-127988

University

Semmelweis University

Supervisor

Gábor Czirják

Publication of my abstract

I do not give consent to the publication of my abstract on the website of the congress.

Kind

Szabad

Status

elfogadva

Accepted presentation method

poszter

Előadás fájl jóváhagyás

nem rendelkezett róla

Előadó

8066

Start

15:25

End

15:28