Unraveling the Mystery of Peroxiredoxin IV Secretion Reveals a Redox‐independent Unconventional Protein Secretory Pathway

Abstract

The peroxiredoxin (Prx) family of proteins functions as leading cellular antioxidants that scavenge hydrogen peroxide as well as contributes to oxidative signaling under both physiological and pathological conditions. As the only member containing a putative signal peptide, Prx4 has been long suspected to be secreted extracellularly to fulfill the role of maintaining local and systemic redox homeostasis. However, the absence of experimental evidence on Prx4 secretion and the lack of mechanistic input compromise the understanding of its role and significance in the pathogenesis of human diseases. The purpose of this study is to unravel the mystery of Prx4 secretion and to determine its secretory mechanism in human normal cells under physiological conditions. Methodologically, immunoblotting was used to examine endogenous and ectopically expressed Prx4 in mouse plasma, tissues, HEK293T cell lysates, extracellular vehicles (EVs) and supernatants free of EVs in the presence or absence of peptidase inhibitors or drugs blocking ER-Golgi transport including brefeldin A and monensin. Subcellular fractionation and live-cell imaging were used to evaluate the localization and release of Prx4/Prx4-EGFP with or without signal peptide, or mutated cleavage site in the presence of ER/Golgi indicators. Calcium ion chelators and ionophores such as ionomycin were used to evaluate the effect of intracellular Ca2+ levels on Prx4 secretion. We demonstrated that Prx4 is only present and secreted as a mature form, of which signal peptide was cleaved during protein synthesis. The secreted Prx4 is not presented in EVs but enriched as a monomer in culture supernatant free of EVs. Mutation of the cleavage site leads to the presence of a full-length, pro-Prx4 with higher molecular weight that can still be secreted efficiently. Mature Prx4 is primarily localized in the ER and lack of distribution in Golgi as shown by imaging, and its secretion is insensitive to the inhibition by blockers of ER-Golgi transport. However, removal of the signal peptide leads to its localization redistributed to cytosol and cannot be secreted. Moreover, presence of oxidative stress, or mutation of catalytic/resolving cysteines, also does not inhibit the secretion of Prx4. Increase of intracellular calcium ionsby ionophoresstimulates the secretion of Prx4, which is completely blocked in the presence of Ca2+ chelators. In conclusion, our data indicate that Prx4 is translocated to ER through the co-translation translocation pathway with the immediate cleavage of the signal peptide upon synthesis. Mature Prx4 is secreted through an unconventional protein secretory route that bypasses Golgi apparatus, and is released in a redox-independent, non-vesicular manner. The unconventional secretory pathway of Prx4 secretion is further stimulated by the influx of intracellular calcium ions, which is presumably mediated through the activation of other intermediators such as protein kinase C (PKC). A model of our current understanding of Prx4 secretion in normal cells is shown in the attached image.