Thiol-disulphide independent in-cell trapping for the identification of peroxiredoxin 2 interactors
Ting Luo,
Julia Malo Pueyo,
Khadija Wahni,
Charlotte Yvanoff,
Tamas Lazar,
Sébastien Pyr dit Ruys,
Didier Vertommen,
Daria Ezeriņa,
Joris Messens
Affiliations
Ting Luo
VIB-VUB Center for Structural Biology, Vlaams Instituut voor Biotechnologie, B-1050, Brussels, Belgium; Brussels Center for Redox Biology, Vrije Universiteit Brussel, B-1050, Brussels, Belgium; Structural Biology Brussels, Vrije Universiteit Brussel, B-1050, Brussels, Belgium
Julia Malo Pueyo
VIB-VUB Center for Structural Biology, Vlaams Instituut voor Biotechnologie, B-1050, Brussels, Belgium; Brussels Center for Redox Biology, Vrije Universiteit Brussel, B-1050, Brussels, Belgium; Structural Biology Brussels, Vrije Universiteit Brussel, B-1050, Brussels, Belgium
Khadija Wahni
VIB-VUB Center for Structural Biology, Vlaams Instituut voor Biotechnologie, B-1050, Brussels, Belgium; Brussels Center for Redox Biology, Vrije Universiteit Brussel, B-1050, Brussels, Belgium; Structural Biology Brussels, Vrije Universiteit Brussel, B-1050, Brussels, Belgium
Charlotte Yvanoff
Structural Biology Brussels, Vrije Universiteit Brussel, B-1050, Brussels, Belgium; International Joint Research Group VUB-EPFL NanoBiotechnology & NanoMedicine (NANO), Vrije Universiteit Brussel, Brussels, Belgium and Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland
Tamas Lazar
VIB-VUB Center for Structural Biology, Vlaams Instituut voor Biotechnologie, B-1050, Brussels, Belgium; Structural Biology Brussels, Vrije Universiteit Brussel, B-1050, Brussels, Belgium
Sébastien Pyr dit Ruys
de Duve Institute, Université Catholique de Louvain, 1200, Brussels, Belgium
Didier Vertommen
de Duve Institute, Université Catholique de Louvain, 1200, Brussels, Belgium
Daria Ezeriņa
VIB-VUB Center for Structural Biology, Vlaams Instituut voor Biotechnologie, B-1050, Brussels, Belgium; Brussels Center for Redox Biology, Vrije Universiteit Brussel, B-1050, Brussels, Belgium; Structural Biology Brussels, Vrije Universiteit Brussel, B-1050, Brussels, Belgium; Corresponding author. VIB-VUB Center for Structural Biology, Vlaams Instituut voor Biotechnologie, B-1050, Brussels, Belgium.
Joris Messens
VIB-VUB Center for Structural Biology, Vlaams Instituut voor Biotechnologie, B-1050, Brussels, Belgium; Brussels Center for Redox Biology, Vrije Universiteit Brussel, B-1050, Brussels, Belgium; Structural Biology Brussels, Vrije Universiteit Brussel, B-1050, Brussels, Belgium; Corresponding author. VIB-VUB Center for Structural Biology, Vlaams Instituut voor Biotechnologie, B-1050, Brussels, Belgium.
Hydrogen peroxide (H2O2) acts as a signalling molecule by oxidising cysteine thiols in proteins. Recent evidence has established a role for cytosolic peroxiredoxins in transmitting H2O2-based oxidation to a multitude of target proteins. Moreover, it is becoming clear that peroxiredoxins fulfil their function in organised microdomains, where not all interactors are covalently bound. However, most studies aimed at identifying peroxiredoxin interactors were based on methods that only detect covalently linked partners. Here, we explore the applicability of two thiol-disulphide independent in-cell trapping methodological approaches in combination with mass spectrometry for the identification of interaction partners of peroxiredoxin 2 (Prdx2). The first is biotin-dependent proximity-labelling (BioID) with a biotin ligase A (BirA*)-fused Prdx2, which has never been applied on redox-active proteins. The second is crosslinker co-immunoprecipitation with an N-terminally His-tagged Prdx2. During the initial characterisation of the tagged Prdx2 constructs, we found that the His-tag, but not BirA*, compromises the peroxidase and signalling activities of Prdx2. Further, the Prdx2 interactors identified with each approach showed little overlap. We therefore concluded that BioID is a more reliable method than crosslinker co-immunoprecipitation. After a stringent mass spec data filtering, BioID identified 13 interactors under elevated H2O2 conditions, including subunit five of the COP9 signalosome complex (CSN5). The Prdx2:CSN5 interaction was further confirmed in a proximity ligation assay. Taken together, our results demonstrate that BioID can be used as a method for the identification of interactors of Prdxs, and that caution should be exercised when interpreting protein-protein interaction results using tagged Prdxs.
