Oxidation of peroxiredoxin-4 induces oligomerization and promotes interaction with proteins governing protein folding and endoplasmic reticulum stress

Ester Zito,Evan A Elko,Vikas Anathy,Allison M Manuel,Sheryl White,Yvonne M.W Janssen-Heininger,Albert Van Der Vliet

doi:10.1016/j.jbc.2021.100665

Abstract

Peroxiredoxins (PRDXs) catalyze the reduction of hydrogen peroxide (H2O2). PRDX4 is the only peroxiredoxin located within the endoplasmic reticulum (ER) and is the most highly expressed H2O2 scavenger in the ER. PRDX4 has emerged as an important player in numerous diseases, such as fibrosis and metabolic syndromes, and its overoxidation is a potential indicator of ER redox stress. It is unclear how overoxidation of PRDX4 governs its oligomerization state and interacting partners. Herein, we addressed these questions via nonreducing Western blots, mass spectrometry, and site-directed mutagenesis. We report that the oxidation of PRDX4 in lung epithelial cells treated with tertbutyl hydroperoxide caused a shift of PRDX4 from monomer/dimer to high molecular weight (HMW) species, which contain PRDX4 modified with sulfonic acid residues (PRDX4-SO3), as well as of a complement of ER-associated proteins, including protein disulfide isomerases important in protein folding, thioredoxin domain–containing protein 5, and heat shock protein A5, a key regulator of the ER stress response. Mutation of any of the four cysteines in PRDX4 altered the HMW species in response to tertbutyl hydroperoxide as well as the secretion of PRDX4. We also demonstrate that the expression of ER oxidoreductase 1 alpha, which generates H2O2 in the ER, increased PRDX4 HMW formation and secretion. These results suggest a link between SO3 modification in the formation of HMW PRDX4 complexes in cells, whereas the association of key regulators of ER homeostasis with HMW oxidized PRDX4 point to a putative role of PRDX4 in regulating ER stress responses.

Highlights

Peroxiredoxins (PRDXs) are a family of highly expressed proteins that scavenge cellular hydrogen peroxide (H2O2)
Our findings indicate that PRDX4 high molecular weight (HMW) species interact with endoplasmic reticulum (ER)-associated proteins (thioredoxin domain– containing protein 5 [thioredoxin domain–containing protein 5 (TXNDC5)], endoplasmic reticulum resident protein 44 (ERP44), protein disulfide isomerase A6 [PDIA6], protein disulfide isomerase, and ER chaperone binding immunoglobulin protein as well as the cytosolic localized PRDX1)
These results demonstrate that the HMW species of PRDX4 found in oxidizing conditions contain primarily an overoxidized peroxidatic cysteine and that the catalytic cysteine in the monomeric form of PRDX4 is only found in the reduced state

Summary

Results

H2O2 and tertbutyl hydroperoxide (TBuOOH) have been shown to be substrates for mammalian PRDXs and are capable of inducing inactivation via overoxidation [18]. The peptide containing Cys127 in the HMW PRDX4 species from cells exposed to TBuOOH showed oxidation to a sulfonic acid (−SO3) at the Cys127 residue (Fig. 1, D and G). These results demonstrate that the HMW species of PRDX4 found in oxidizing conditions contain primarily an overoxidized peroxidatic cysteine and that the catalytic cysteine in the monomeric form of PRDX4 is only found in the reduced state. Expression of PRDX4-SBP in C10 cells allowed for the detection of PRDX4-binding partners in monomer, dimer, and HMW forms via MS/MS. Oxidation of PRDX4 with TBuOOH showed an increase in proteins associating with the HMW species compared with dimer or monomer (Fig. 2C). In the region of 40 to 75 kDa, in control cells, PRDX1 and eukaryotic elongation factor 2 were found to interact with PRDX4, whereas in this molecular weight (MW) range, in TBuOOH-treated cells, eukaryotic elongation factor 2 was no longer detected while in addition to PRDX1, TXNDC5, ERP44, and ALDOA were pulled down with

56 Prdx4 - Peroxiredoxin-4

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Discussion

Experimental procedures