Abstract
Human peroxiredoxin-5 (PRDX5) is a unique redox-sensitive protein that plays a dual role in brain ischemia-reperfusion injury. While intracellular PRDX5 has been reported to act as a neuroprotective antioxidative enzyme by scavenging peroxides, once released extracellularly from necrotic brain cells, the protein aggravates neural cell death by inducing expression of proinflammatory cytokines in macrophages through activation of Toll-like receptor (TLR) 2 (TLR2) and 4 (TLR4). Although recent evidence showed that PRDX5 was able to interact directly with TLR4, little is known regarding the role of the cysteine redox state of PRDX5 on its DAMP function. To gain insights into the role of PRDX5 redox-active cysteine residues in the TLR4-dependent proinflammatory activity of the protein, we used a recombinant human PRDX5 in the disulfide (oxidized) form and a mutant version lacking the peroxidatic cysteine, as well as chemically reduced and hyperoxidized PRDX5 proteins. We first analyzed the oxidation state and oligomerization profile by Western blot, mass spectrometry, and SEC-MALS. Using ELISA, we demonstrate that the disulfide bridge between the enzymatic cysteines is required to allow improved TLR4-dependent IL-8 secretion. Moreover, single-molecule force spectroscopy experiments revealed that TLR4 alone is not sufficient to discriminate the different PRDX5 redox forms. Finally, flow cytometry binding assays show that disulfide PRDX5 has a higher propensity to bind to the surface of living TLR4-expressing cells than the mutant protein. Taken together, these results demonstrate the importance of the redox state of PRDX5 cysteine residues on TLR4-induced inflammation.
Highlights
Peroxiredoxins (PRDXs) constitute a large superfamily of highly diverse and multifunctional peroxidases, expressed as six isoforms in mammals (PRDX1–6)
Our results indicate that all redox forms bind TLR4 with a similar kinetic dissociation rate (k o f f ), with the exception of hyperoxidized PRDX5 that is not able to establish a specific interaction with the receptor
These results provide the basis for better understanding how the redox state of catalytic redox-sensitive cysteine residues (Cys) allows PRDXs to promote inflammation in inflammatory-related diseases, which is of crucial importance for the development of new therapeutic strategies related to ischemia-reperfusion injury
Summary
Peroxiredoxins (PRDXs) constitute a large superfamily of highly diverse and multifunctional peroxidases, expressed as six isoforms in mammals (PRDX1–6). Their intracellular functions are related to the redox state of their catalytic cysteine residues (Cys), of which there are one in PRDX6 and two in PRDX1–5. PRDXs act as cytoprotective peroxidases against oxidative stress by reducing peroxides (hydrogen peroxide (H2 O2 ), alkyl hydroperoxides, and peroxynitrite), as modulators of redox signaling, and as chaperones [1,2,3]. Unlike prosthetic group-dependent enzymes, the PRDX catalytic cycle relies on a conserved Cys, so-called peroxydatic Cys, located in the N-terminal part of the protein. Depending on the PRDX subfamily, the Antioxidants 2021, 10, 1902.
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
