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

Protein phosphatase 1 (PP1) is one of the most important serine-threonine phosphatases in
eukaryotic cells, owning approximately two hundred regulatory subunits which are
responsible for the specificity of its substrates. Among its regulatory subunits, GADD34
plays a well-known substantial role by influencing translation via dephosphorylation of the
eucaryotic initiation factor 2α (eiF2α) which is a key reaction for recovery of protein expression
after stress. Guanabenz, an approved antihypertensive drug acting on α-2 adrenergic
receptors, inhibits GADD34 as well. Guanabnez also exhibits therapeutic potential for
neurodegenerative disorders with repurposing Phase 1 studies ongoing.
We aimed to investigate the interactome of GADD34 protein under different ER stress
conditions. We performed proximity-dependent biotin labelling method using wt and mutant
GADD34-TURBO-2XFLAG chimera proteins in parallel to co-IP experiments. The mutant
fusion protein (GADD34(Δ578-617)-TURBO-2XFLAG) lacked an evolutionary conserved
region, including the known binding site for eiF2α. We generated 3-3 independent stable cell
lines for both types of chimaeras from adherent human embryonic kidney cells (adHEK).
We compared the interactome of wt GADD34-TURBO to that of the deletion mutant both under
control and ER stress condition, i.e after treatment with the SERCA-inhibitor thapsigargin, or
mTOR-inhibitor rapamycin. We labelled potential interaction partners in vivo, followed by
identification via mass spectrometry (MS). In addition, co-immunoprecipitation studies with
both chimaeras followed by MS were conducted.
Our investigation found numerous novel interaction partners providing new insights into
different cellular functions, including e.g. cell cycle, apoptosis, autophagy, cytoskeletal
system/motor proteins and vesicular trafficking. Significant Gene Ontology enrichment
revealed the involvement of the GADD34/PP1 complex in different immune functions and
neurodegenerative diseases as well. Our findings suggest potential new indications for
guanabenz beyond its current approvals, while also opening new perspectives for drug research
focusing on GADD34 inhibition. A new computational model based on our hits fits well with
literature data for the regulation of autophagy via the mTOR-AMPK-GADD34 triangle. We
suggest that the GADD34/PP1 complex is a main switch in the cell to counteract stress-induced
phosphorylation in main cellular functions, promoting cell proliferation, that has been
validated by functional tests as well. Treatment of human T cells and neutrophils by
Guanabenz also validates our proteomic finding. We suggest new mechanisms of action for
antiinflammatory effects and beneficial outcomes in neurodegenerative diseases of
Guanabenz treatment. In summary, our data contributes to a deeper understanding of various
cellular processes, including cell cycle regulation, autophagy, apoptosis and functions of
immune cells besides its pharmacologic relevance regarding GADD34 inhibition.