PhD Scientific Days 2023

Budapest, 22-23 June 2023

Molecular Sciences - Posters K

Investigation of the interactions of the GADD34 (growth arrest and DNA damage [GADD]‐inducible34) protein

1 Eszter Éva Kálmán, Department of Molecular Biology at the Institute of Biochemistry and Molecular Biology, Semmelweis University, Budapest, Hungary
2 Orsolya Kapuy, Department of Molecular Biology at the Institute of Biochemistry and Molecular Biology, Semmelweis University, Budapest, Hungary
3 Aladár Pettkó-Szandtner, Laboratory of Proteomics Research, Biological Research Centre, Szeged, Hungary
4 Norbert Gyöngyösi, Department of Molecular Biology at the Institute of Biochemistry and Molecular Biology, Semmelweis University, Budapest, Hungary

Text of the abstract

Eukaryotic protein synthesis is a well-regulated process controlled by reversible phosphorylation of numerous translation factors. Dephosphorylation of the eIF2α-P factor by the holoenzyme GADD34: PP1 (protein phosphatase 1) is a key reaction for translation recovery in cells. PP1 is an important phosphatase in eukaryotic cells and plays a role in the control of circadian rhythm, glycogen metabolism, and endoplasmic reticulum stress (ER), among others. It is also known that GADD34 expression is induced under ER stress conditions.

In our experiments, we aimed to investigate the molecular interactions of the Gadd34 protein under different ER stress conditions. To investigate these interactions, we used the so-called proximity-dependent biotin labeling method using Turbo ID chimeras. This method is based on a mutant prokaryotic biotin ligase fused to the protein of interest that can label potential interaction partners in close proximity in vivo. Biotinylation allows us to purify and identify the potential interactome by mass spectrometry.

We used classical molecular biology techniques such as SDS-PAGE and Western blot. We made a chimeric construct containing the GADD34 protein sequence and a deletion mutant of GADD34 designated GADD34 (578-617) and fused to the coding sequence of an engineered biotin ligase (Turbo ID). We generated stable clones from adherent human embryonic kidney cells (adHEK) to study the interactome under ER stress conditions induced by Thapsigargin.

The GO (Gene Ontology) analysis of our experiments showed a significant enrichment of potential interaction partners in known functions of the GADD34 protein. Thapsigargin treatment showed a significant effect on the GADD34 interactome, but we found no effect on the ATF4 (Activating transcription factor 4) or CHOP (DNA damage-inducible transcript 3, also known as C/EBP homologous protein) proteins, which play a role in the ER stress response.
In summary, our data can serve as a useful list of new putative partners of GADD34. In the following, we plan to investigate the differences between the interactomes of wild-type and mutant versions of GADD34.

This project was founded by OTKA NKFI-1 FK 132474 and GRANT FK 134267 (OK), Grant FK 132474 (NG), EU's Horizon 2020 research and innovation program under grant agreement No. 739593 (AP-S) and GINOP‐2.3.2‐15‐2016‐00032 (AP‐S).