Original scientific paper
https://doi.org/10.18054/pb.v127i3-4.36109
Investigating KEAP1-DPP3 interaction in live cells: Challenges and considerations in BiFC assay design
Ana Tomašić Paić
orcid.org/0000-0003-0622-7663
; Laboratory for Protein Biochemistry and Molecular Modelling, Division of Organic Chemistry and Biochemistry, Ruđer Bošković Institute, Zagreb, Croatia
Lucija Horvat
; Laboratory of Cell Biophysics, Division of Molecular Biology, Ruđer Bošković Institute, Zagreb, Croatia
Katja Ester
orcid.org/0000-0002-4044-8331
; Laboratory of Experimental Therapy, Division of Molecular Medicine, Ruđer Bošković Institute, Zagreb, Croatia
Mihaela Matovina
orcid.org/0000-0001-7647-3339
; Laboratory for Protein Biochemistry and Molecular Modelling, Division of Organic Chemistry and Biochemistry, Ruđer Bošković Institute, Zagreb, Croatia
*
* Corresponding author.
Abstract
Background and purpose: Kelch-like ECH-associated protein 1 (KEAP1) is a repressor of the transcription factor Nuclear factor erythroid 2-related factor 2 (NRF2), a key regulator of the oxidative stress response. Under basal conditions, the NRF2 monomer is bound by a KEAP1 dimer in a complex with Cullin-3 (CUL3) and E3 ubiquitin-protein ligase RBX1 (RBX1), leading to its ubiquitination and subsequent degradation by the proteasome. One mechanism of NRF2 activation involves competitive protein interactors binding to KEAP1, preventing NRF2 ubiquitination. One such interactor is dipeptidyl peptidase 3 (DPP3), whose binding to KEAP1 reduces NRF2 degradation and promotes its transcriptional activity. We developed bimolecular fluorescence complementation (BiFC) assay for livecell analysis of DPP3-KEAP1 interaction in order to investigate its subcellular localization and determine if BiFC is an appropriate method for the investigation of DPP3’s protein interactions.
Materials and methods: We constructed BiFC vectors containing Nand C-terminal fragments of the Venus fluorescent protein and cloned WT DPP3, KEAP1 and DPP3-ΔETGE in all possible topologies.
Results: All eight WT DPP3 and KEAP1 vector combinations produced positive BiFC signals and the localization of the signal was in the cytosol; however, three out of eight negative control vector combinations (DPP3-ΔETGE-VenusfN/KEAP1-VenusfC, VenusfC-DPP3-ΔETGE/VenusfNKEAP1 and VenusfN- DPP3-ΔETGE/VenusfC-KEAP1) also yielded false positive signals.
Conclusion: BiFC is a useful method for studying protein-protein interactions and their subcellular localization, but careful selection of vector combinations is crucial to avoid false-positive signals.
Keywords
dipeptidyl peptidase 3; DPP3; Kelch-like ECH-associated protein 1; KEAP1; protein-protein interaction; PPI; bimolecular fluorescence complementation; BiFC
Hrčak ID:
347133
URI
Publication date:
13.5.2026.
Visits: 215 *