PhD Scientific Days 2025

Molecular Medicine I.

A comprehensive proteomic analysis of the GADD34 (growth arrest and DNA damage [GADD]‐inducible34) protein

Name of the presenter

Kálmán Eszter

Institute/workplace of the presenter

1Department of Molecular Biology at the Institute of Biochemistry and Molecular Biology, Semmelweis University

Authors

Eszter Kálmán¹, Orsolya Kapuy¹, Marianna Holczer¹, Mariam Vakhtangishvili¹, Nikolett Szilárd¹, Krisztina Ella², Krisztina Veszelyi³, Anna Hajdara⁴, Krisztina Futosi², Karolina Pircs³, Balázs Kiss³, Aladár Pettkó-Szandtner⁵, Norbert Gyöngyösi¹

1: Department of Molecular Biology at the Institute of Biochemistry and Molecular Biology, Semmelweis University, Budapest, Hungary
2: Department of Physiology, Semmelweis University, Budapest, Hungary
3: Institute of Translational Medicine, Semmelweis University, Budapest, Hungary
4: VRG Therapeutics Zrt., Budapest, Hungary
5: Laboratory of Proteomics Research, Biological Research Centre, Szeged, Hungary

Text of the abstract

The GADD34 protein, as one of the regulatory subunits of Protein phosphatase 1 (PP1), is involved in several important processes in eukaryotic cells for example in the regulation of translation by dephosphorylating the eukaryotic initiation factor 2α (eIF2α) resulting in translational recovery after stress.
The approved antihypertensive drug, Guanabenz and it’s derivative Sephin 1 can both inhibit GADD34, and have significant therapeutic potential, for example in the treatment of neurodegenerative diseases.

Our aim was to investigate the interactome of GADD34 protein using the proximity-dependent biotin labeling method, Turbo-ID. We have created GADD34-TURBO chimeric proteins, containing a mutant prokaryotic biotin ligase (TURBO) fused to our protein of interest, allowing to label the potential interaction partners in vivo. After this, these proteins can be identified by mass spectrometry (MS). To ensure the homogenic overexpression of our proteins, we have generated stable clones expressing the wild type GADD34-TURBO and a deletion mutant GADD34Δ(578-617)–TURBO chimeric protein, from human embryonic kidney cells. We compared the interactome of both the wild type and mutant GADD34-TURBO under control conditions and after treatments with the SERCA inhibitor thapsigargin and the mTOR inhibitor rapamycin. Additionally, co-immunoprecipitation studies with both chimeras followed by MS were conducted. Interactome data was further investigated by bioinformatics tools, and the role of GADD34 in several cellular pathways were validated by functional tests.

We found numerous novel interaction partners of GADD34 wich can help to unveil new insights into several cellular pathways, and into the development of neurodegenerative disorders. According to our functional assays, inhibition of GADD34 glucose-dependently alters circadian rhythm, and overexpression of GADD34 affects cell cycle and cell fitness in a nutrient-dependent manner. Finally, Guanabenz supresses functionning of human T cells and neutrophils dose-dependently. Our data contributes to a deeper understanding of various cellular processes, including cell cycle regulation, autophagy, apoptosis and functions of immun cells, beside opening new horizons in drug research focusing on GADD34 inhibition.

This project was founded by OTKA NKFI-1 FK 132474.