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

doi:10.1016/j.freeradbiomed.2018.10.407

Free Radical Biology and Medicine

Volume 129, December 2018, Pages 383-393

https://doi.org/10.1016/j.freeradbiomed.2018.10.407 Get rights and content

Highlights

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Cytosolic peroxiredoxins constitutively interact with CRMP2 in several different cell types.
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Addition of H₂O₂ to Jurkat T lymphoma cells results in CRMP2 oxidation.
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Recombinant CRMP2 has low reactivity with H₂O₂.
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CRMP2 remained sensitive to oxidation in single peroxiredoxin knockout Jurkat cells.
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The data suggest a cell-type specific mechanism to increase the sensitivity of CRMP2 to oxidation.

Abstract

Hydrogen peroxide (H₂O₂) acts as a signaling molecule in cells by oxidising cysteine residues in regulatory proteins such as phosphatases, kinases and transcription factors. It is unclear exactly how many of these proteins are specifically targeted by H₂O₂ because they appear too unreactive to be directly oxidised. One proposal is that peroxiredoxins (Prxs) initially react with H₂O₂ and then oxidise adjacent proteins via a thiol relay mechanism. The aim of this study was to identify constitutive interaction partners of Prx2 in Jurkat T-lymphoma cells, in which thiol protein oxidation occurs at low micromolar concentrations of H₂O₂. Immunoprecipitation and proximity ligation assays identified a physical interaction between collapsin response mediator protein 2 (CRMP2) and cytoplasmic Prx2. CRMP2 regulates microtubule structure during lymphocyte migration and neuronal development. Exposure of Jurkat cells to low micromolar levels of H₂O₂ caused rapid and reversible oxidation of CRMP2, in parallel with Prx2 oxidation, despite purified recombinant CRMP2 protein reacting slowly with H₂O₂ (k~1 M⁻¹s⁻¹). Lowering Prx expression should inhibit oxidation of proteins oxidised by a relay mechanism, however knockout of Prx2 had no effect on CRMP2 oxidation. CRMP2 also interacted with Prx1, suggesting redundancy in single knockout cells. Prx 1 and 2 double knockout Jurkat cells were not viable. An interaction between Prx2 and CRMP2 was also detected in other human and rodent cells, including primary neurons. However, low concentrations of H₂O₂ did not cause CRMP2 oxidation in these cells. This indicates a cell-type specific mechanism for promoting CRMP2 oxidation in Jurkat cells, with insufficient evidence to attribute oxidation to a Prx-dependent redox relay.

Graphical abstract

Introduction

Hydrogen peroxide (H₂O₂) activates or modulates a range of signaling pathways by oxidation of specific cysteine residues in target proteins. While these proteins are susceptible to oxidation, their reactivity to H₂O₂ is far lower than specialist peroxidases such as the peroxiredoxins (Prxs) [1]. It has been proposed that Prxs regulate the oxidation of less reactive target proteins either by controlling local concentrations of H₂O₂, or by a facilitated oxidation reaction in which the Prx sulfenic acid or disulfide reacts with the reduced cysteine of an adjacent target protein [2]. This mechanism, termed a redox relay, provides a layer of selectivity that is dependent on Prx interaction partners. The first reported example of this mechanism was the yeast thiol peroxidase Orp1, which was shown to oxidise its binding partner Yap1, promoting nuclear retention and transcriptional activity of Yap1 [3]. Human Prx1 was shown to interact with and oxidise the protein kinase Ask1, thereby facilitating the phosphorylation of p38 [4], and Prx2 has also been shown to oxidise and inactivate the transcription factor STAT3 [5].

Studies that uncovered putative redox relays did so by capturing transient mixed disulfides between Prxs and target proteins. As an alternative approach we searched for interaction partners of Prx2, reasoning that a physical association favours participation in a redox relay. Human Jurkat T-lymphoma cells were selected as the cell line as we previously characterised the oxidation of several thiol proteins with low micromolar boluses of H₂O₂ [6]. Prx2 was immunoprecipitated from resting cells as previously described [7], but those proteins interacting by disulfides were removed with DTT, enabling focus on those with constitutive non-covalent interactions poised to participate in a redox relay.

We have identified collapsin response mediator protein 2 (CRMP2) as an interaction partner of Prx2. Human CRMP2 is ubiquitously expressed and regulates the assembly and disassembly of microtubules [8], [9]. This function is important for the development and maintenance of neurite extensions [10] and for lymphocyte migration [11]. Cys504 of CRMP2 is susceptible to oxidation, enabling covalent CRMP2 homodimer formation [12], [13]. CRMP2 oxidation, in parallel with phosphorylation of Ser522, is proposed to facilitate further CRMP2 phosphorylation by glycogen synthase kinase 3 β (GSK3β) and loss of binding to tubulin, leading to disassembly of microtubules and neuronal retraction [14]. In lymphocytes, CRMP2 accumulates in the uropod in response to chemokine signaling via phosphorylation of Tyr479, which suppresses GSK3β-dependent CRMP2 phosphorylation but promotes T-cell migration [15]. We show that recombinant CRMP2 is relatively resistant to direct oxidation by H₂O₂. However, Prx2 and CRMP2 are oxidised simultaneously in Jurkat cells treated with H₂O₂, consistent with a relay mechanism. The same physical interaction between Prx2 and CRMP2 was detectable in a range of other cell types, but significant CRMP2 oxidation was only observed in Jurkat cells, indicating cell-type specific regulation.

Section snippets

Prx2 immunoprecipitates with CRMP2 from Jurkat cells

Anti-Prx2 antibodies irreversibly crosslinked to protein A-coated Dynabeads were incubated with Jurkat cell lysates. Following reduction with DTT to remove covalent interactors, the remaining bound proteins were eluted with pH 3 glycine. SDS-PAGE followed by silver staining consistently revealed a small number of bands either not present or at lower amounts compared with IgG control immunoprecipitations (Fig. 1a). Aside from Prx1 and Prx2, two major bands were identified by mass spectrometry as

Discussion

In this study immunoprecipitation and proximity ligation assays were used to reveal the cytoskeletal regulatory protein CRMP2 as a constitutive interaction partner of cytoplasmic peroxiredoxins in a wide range of cell types. The function of CRMP2 has been predominantly studied in neurons, where axonal guidance molecules such as semaphorins trigger CRMP2 phosphorylation, loss of tubulin binding and microtubule collapse. Oxidation of Cys504 has been previously shown to result in CRMP2 homodimer

Cell culture, treatment and lysis

Jurkat T lymphoma cells (TIB-152 clone E6-1 from ATCC) were maintained in RPMI 1640 medium (RPMI) supplemented with 10% [v/v] FBS (HyClone, GE Healthcare), 100 U/mL penicillin, 100 µg/mL streptomycin (P/S, Invitrogen) (complete RPMI medium) at 0.2–1.0 × 10⁶ cells/mL. HEK293FT (Invitrogen), and MEF cells (kind gifts from the Walter & Eliza Hall Institute, Melbourne, Australia) were maintained in DMEM supplemented with 10% [v/v] FBS + P/S and cultured according to the supplier's instructions.

PC12

Acknowledgements

This work was supported by the Marsden Fund from Government funding (17-UOO-086), administered by Royal Society Te Apārangi, New Zealand, as well as the Canterbury Medical Research Foundation (14/01), New Zealand, and the Neurological Foundation of New Zealand (1622-PG). We thank Dr Abel Ang for his assistance with CRMP2 cloning, Dr Louise Paton and Dr Nina Dickerhof for help with mass spectrometry analyses, and Lorryn Fisher for extracting, culturing and preparing the DRG neurons for analyses.

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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.
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Several mechanisms have been suggested to explain the adverse effects of air pollutants on airway cells. One such explanation is the presence of high concentrations of oxidants and pro-oxidants in environmental pollutants. All animal and plant cells have developed several mechanisms to prevent damage by oxidative molecules. Among these, the peroxiredoxins (PRDXs) are of interest due to a high reactivity with reactive oxygen species (ROS) through the functioning of the thioredoxin/thioredoxin reductase system. This study aimed to verify the gene expression patterns of the PRDX family in bronchial epithelial airway cells (BEAS-2B) cells exposed to diesel exhaust particles (DEPs) at a concentration of 15 μg/mL for 1 or 2 h because this it is a major component of particulate matter in the atmosphere. There was a significant decrease in mRNA fold changes of PRDX2 (0.43 ± 0.34; *p = 0.0220), PRDX5 (0.43 ± 0.34; *p = 0.0220), and PRDX6 (0.33 ± 0.25; *p = 0.0069) after 1 h of exposure to DEPs. The reduction in mRNA levels may consequently lead to a decrease in the levels of PRDX proteins, increasing oxidative stress in bronchial epithelial cells BEAS-2B and thus, negatively affecting cellular functions.
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Original articlePeroxiredoxin interaction with the cytoskeletal-regulatory protein CRMP2: Investigation of a putative redox relay

Highlights

Abstract

Graphical abstract

Introduction

Section snippets

Prx2 immunoprecipitates with CRMP2 from Jurkat cells

Discussion

Cell culture, treatment and lysis

Acknowledgements

Free Radic. Biol. Med.

Cell

Free Radic. Biol. Med.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Blood

Free Radic. Biol. Med.

Biochem. Pharmacol.

Free Radic. Biol. Med.

FEBS Lett.

Free Radic. Biol. Med.

J. Biol. Chem.

Redox Biol.

Free Radic. Biol. Med.

Neuron

J. Biol. Chem.

The conundrum of hydrogen peroxide signaling and the emerging role of peroxiredoxins as redox relay hubs

Antioxid. Redox Signal.

Peroxiredoxin-2 and STAT3 form a redox relay for H2O2 signaling

Nat. Chem. Biol.

Proteomic detection of hydrogen peroxide-sensitive thiol proteins in Jurkat cells

Biochem. J.

Hyperoxidized peroxiredoxin 2 interacts with the protein disulfide- isomerase ERp46

Biochem. J.

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