PhD Scientific Days 2024

Molecular Medicine II.

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

Text of the abstract

Protein phosphatase 1 (PP1) stands as a prominent serine-threonine phosphatase in eukaryotic cells, owning approximately two hundred regulatory subunits which are responsible for the specificity to its substrates. It regulates cell cycle, glycogen metabolism, and management of endoplasmic reticulum (ER) stress. Among regulatory subunits, GADD34 is very important due to its influence on translation regulation by dephosphorylating the eukaryotic initiation factor 2α, a critical step for translational recovery after stress. Additionally, guanabenz, an approved antihypertensive drug inhibiting GADD34, exhibits therapeutic potential for neurodegenerative disorders alongside its cardiovascular benefits via α-2 adrenergic receptors.

Our study aims to investigate the interactome of GADD34 protein under varied ER stress conditions. Employing the proximity-dependent biotin labeling method with GADD34-TURBO chimera proteins, we used a mutant prokaryotic biotin ligase (TURBO) fused to the protein of interest to label potential interaction partners in vivo, enabling their purification and identification via mass spectrometry (MS). This approach is adept at revealing even transient and weak interactions. Stable clones expressing the chimeric constructs of wild type GADD34-TURBO and a deletion mutant lacking an evolutionarily conserved region were generated from human embryonic kidney cells (adHEK). We compared the interactome of wild type and mutant GADD34-TURBO under control conditions and following ER stress induction via thapsigargin or rapamycin treatement. Additionally, co-immunoprecipitation studies with both chimeras followed by MS were conducted.

Our investigation found numerous novel interaction partners unveiling new insights into several cellular functions pathways, and diseases in which GADD34 might play a role. Identification of new GADD34 interactors involved in neurodegenerative diseases could shed light on guanabenz's mechanism of action in such conditions. Furthermore, our findings suggest potential new indications for guanabenz beyond its current approvals, while also opening avenues for drug research focusing on GADD34 inhibition. Ultimately, our data contributes to a deeper understanding of various cellular processes, including cell cycle regulation, autophagy, apoptosis and functions of immun cells.

This project was founded by OTKA NKFI-1 FK 132474