Thiol-disulphide independent in-cell trapping for the identification of peroxiredoxin 2 interactors

Abstract

Hydrogen peroxide (H 2 O 2 ) acts as a signalling molecule by oxidising cysteine thiols in proteins. Recent evidence has established a role for cytosolic peroxiredoxins in transmitting H 2 O 2 -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 H 2 O 2 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.

Experimental Approaches for Investigating Disulfide-Based Redox Relays in Cells

“…From a biochemical standpoint, affinity purification procedures, sometimes coupled with amine-reactive cross-linking procedures or, more recently, proximity-mediated biotinylation, have been used to isolate and identify interaction partners of thiol-dependent oxidoreductases. For instance, protein disulfide isomerase interaction partners have been identified using both standard copurification techniques as well as with amine-reactive cross-linkers (which capture weak binding interactions), , whereas the interaction partners of human peroxiredoxin 2 have recently been studied using proximity-mediated biotinylation . As with the genetic approaches described above, interaction partners identified using such procedures are not necessarily engaged in direct redox partnerships.…”

“…For instance, protein disulfide isomerase interaction partners have been identified using both standard copurification techniques as well as with amine-reactive cross-linkers (which capture weak binding interactions), 142,143 whereas the interaction partners of human peroxiredoxin 2 have recently been studied using proximity-mediated biotinylation. 144 As with the genetic approaches described above, interaction partners identified using such procedures are not necessarily engaged in direct redox partnerships. Thus, more direct methods to detect direct disulfide formation between proteins are often needed to confirm such interactions.…”