Peroxiredoxin interaction with the cytoskeletal-regulatory protein CRMP2: Investigation of a putative redox relay

Pace, Paul E.; Peskin, Alexander V.; Königstorfer, Andreas; Jasoni, Christine J.; Winterbourn, Christine C.; Hampton, Mark B.

doi:10.1016/j.freeradbiomed.2018.10.407

Cited by 21 publications

(13 citation statements)

References 42 publications

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“…Initially, we attempted to perform DSP-IP with endogenous Prdx2 using Dynabeads coupled to an anti-Prdx2 antibody, as in Pace et al [ 49 ]. However, this approach proved unsuccessful (data not shown) presumably due to insufficiently high Prdx2 levels in HEK293 cells.…”

Section: Resultsmentioning

confidence: 99%

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

et al. 2021

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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.

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Section: Resultsmentioning

confidence: 99%

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

et al. 2021

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“…The next question we asked is whether Prx2 is guided towards STAT3 by virtue of a direct protein-protein-interaction. We failed to reconstitute a Prx2-STAT3 relay from pure proteins in vitro, and the recently attempted in vitro reconstitution of another redox relay (Prx2-CRMP2) also failed 8 . These negative results can be taken to suggest that one (or more) additional proteins are needed to scaffold the interaction.…”

Section: Discussionmentioning

confidence: 99%

“…The first known example was the Orp1-Yap1 redox relay discovered in yeast 5 . More recently, examples of peroxiredoxin-based redox relays were found in mammalian cells 6 – 8 , and there is emerging evidence for a role of cytosolic peroxiredoxins in transmitting oxidation to a multitude of target proteins 9 .…”

Section: Introductionmentioning

confidence: 99%

A role for annexin A2 in scaffolding the peroxiredoxin 2–STAT3 redox relay complex

2020

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Hydrogen peroxide (H2O2) is recognized to act as a signaling molecule. Peroxiredoxins (Prxs) have the ability to transfer H2O2-derived oxidizing equivalents to redox-regulated target proteins, thus facilitating the transmission of H2O2 signals. It has remained unclear how Prxs and their target proteins are brought together to allow for target-specific protein thiol oxidation. Addressing the specific case of Prx2-dependent STAT3 oxidation, we here show that the association of the two proteins occurs prior to Prx oxidation and depends on a scaffolding protein, the membrane chaperone annexin A2. Deletion or depletion of annexin A2 interrupts the transfer of oxidizing equivalents from Prx2 to STAT3, which is observed to take place on membranes. These findings support the notion that the Prx2-STAT3 redox relay is part of a highly organized membrane signaling domain.

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“…The discovery of AnxA2 as a scaffolding protein for Prdx2:STAT3 [35] was hailed as one that brings us closer to solving the mystery of what dictates the specificity of Prdx redoxrelays. The results of the investigation of the Prdx2:CRMP2 interaction then dampened those hopes, as they suggested that the organization of the same redox-relay was cell typespecific [68]. The findings presented here, showing that scaffolding proteins are not needed for mediating all redox-relays, and that Prdx2 can interact with Prdx1 interactors, further suggest that the molecular details of redox-relays are more complex than we anticipated and that we are still a long way away from understanding how Prdxs and their targets find each other in the "molecularly crowded" environment of the cell.…”

Section: Discussionmentioning

confidence: 99%

Prdx1 Interacts with ASK1 upon Exposure to H2O2 and Independently of a Scaffolding Protein

Pueyo

Wahni

et al. 2021

Antioxidants

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Hydrogen peroxide (H2O2) is a key redox signaling molecule that selectively oxidizes cysteines on proteins. It can accomplish this even in the presence of highly efficient and abundant H2O2 scavengers, peroxiredoxins (Prdxs), as it is the Prdxs themselves that transfer oxidative equivalents to specific protein thiols on target proteins via their redox-relay functionality. The first evidence of a mammalian cytosolic Prdx-mediated redox-relay—Prdx1 with the kinase ASK1—was presented a decade ago based on the outcome of a co-immunoprecipitation experiment. A second such redox-relay—Prdx2:STAT3—soon followed, for which further studies provided insights into its specificity, organization, and mechanism. The Prdx1:ASK1 redox-relay, however, has never undergone such a characterization. Here, we combine cellular and in vitro protein–protein interaction methods to investigate the Prdx1:ASK1 interaction more thoroughly. We show that, contrary to the Prdx2:STAT3 redox-relay, Prdx1 interacts with ASK1 at elevated H2O2 concentrations, and that this interaction can happen independently of a scaffolding protein. We also provide evidence of a Prdx2:ASK1 interaction, and demonstrate that it requires a facilitator that, however, is not annexin A2. Our results reveal that cytosolic Prdx redox-relays can be organized in different ways and yet again highlight the differentiated roles of Prdx1 and Prdx2.

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Peroxiredoxin interaction with the cytoskeletal-regulatory protein CRMP2: Investigation of a putative redox relay

Cited by 21 publications

References 42 publications

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

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

A role for annexin A2 in scaffolding the peroxiredoxin 2–STAT3 redox relay complex

Prdx1 Interacts with ASK1 upon Exposure to H2O2 and Independently of a Scaffolding Protein

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