EFP1 is an ER stress-induced glycoprotein which interacts with the pro-apoptotic protein Par-4

Sarah Appel^1,2,6, Susanne Vetterkind^1,2,6, Ansgar Koplin^1,3, Barbara Maertens^1,4, Meike Boosen^1,5, Ute Preuss¹

¹The Institute of Genetics, University of Bonn, Bonn, Germany; ²Department of Health Sciences, Sargent College of Health and Rehabilitation Sciences, Boston University, Boston, MA, USA; ³Center for Molecular Biology Heidelberg (ZMBH), Heidelberg, Germany; ⁴Institute of Biochemistry II, University of Cologne, Cologne, Germany; ⁵Institute of Pharmacology and Toxicology, University Hospital of Johann Wolfgang Goethe-University Frankfurt am Main, Frankfurt am Main, Germany; 6These authors contributed equally to this work.

Abstract: We have isolated the rat ortholog of EFP1 (EF-hand binding protein 1) as a novel interaction partner of the pro-apoptotic protein Par-4 (prostate apoptosis response-4). Rat EFP1 contains two thioredoxin domains, the COOH-terminal one harboring a CGFC motif, and has a similar protein domain structure as members of the protein disulfide isomerase (PDI) family. In REF52.2 and CHO cells, EFP1 colocalized with the endoplasmic reticulum (ER) marker PDI. Furthermore, EFP1 possesses catalytic activity as demonstrated by an insulin disulfide reduction assay. Western blot analysis revealed two EFP1 protein bands of approximately 136 and 155 kDa, representing different glycosylation states of the protein. Complex formation between EFP1 and Par-4 was confirmed in vitro and in vivo by co-immunoprecipitation, dot blot overlay and pull-down experiments. In CHO cells, coexpression of EFP1 and Par-4 resulted in enhanced Par-4-mediated apoptosis, which required the catalytic activity of EFP1. Interestingly, EFP1 was specifically upregulated in NIH3T3 cells after induction of ER stress by thapsigargin, tunicamycin, and brefeldin A, but not by agents that induce oxidative stress or ER-independent apoptosis. Furthermore, we could show that the induction of apoptosis by Ca²⁺ stress-inducing agents was significantly decreased after siRNA oligonucleotide-mediated knockdown of Par-4. Our data suggest that EFP1 might represent a cell-protective enzyme that could play an important role in the decision between survival and initiation of Par-4-mediated apoptosis.

Keywords: Par-4, EFP1, PDI, thioredoxin, ER stress, apoptosis