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Thiol-based redox regulation is central to adjusting chloroplast functions under varying light conditions. A redox cascade via the ferredoxin-thioredoxin reductase (FTR)/thioredoxin (Trx) pathway has been well recognized to mediate the light-responsive reductive control of target proteins; however, the molecular basis for reoxidizing its targets in the dark remains unidentified. Here, we report a mechanism of oxidative thiol modulation in chloroplasts. We biochemically characterized a chloroplast stroma-localized atypical Trx from Arabidopsis, designated as Trx-like2 (TrxL2). TrxL2 had redox-active properties with an unusually less negative redox potential. By an affinity chromatography-based method, TrxL2 was shown to interact with a range of chloroplast redox-regulated proteins. The direct discrimination of thiol status indicated that TrxL2 can efficiently oxidize, but not reduce, these proteins. A notable exception was found in 2-Cys peroxiredoxin (2CP); TrxL2 was able to reduce 2CP with high efficiency. We achieved a complete in vitro reconstitution of the TrxL2/2CP redox cascade for oxidizing redox-regulated proteins and draining reducing power to hydrogen peroxide (H₂O₂). We further addressed the physiological relevance of this system by analyzing protein-oxidation dynamics. In Arabidopsis plants, a decreased level of 2CP led to the impairment of the reoxidation of redox-regulated proteins during light–dark transitions. A delayed response of protein reoxidation was concomitant with the prolonged accumulation of reducing power in TrxL2. These results suggest an in vivo function of the TrxL2/2CP redox cascade for driving oxidative thiol modulation in chloroplasts.

Reactive oxygen species exacerbate nonalcoholic steatohepatitis (NASH) by oxidizing macromolecules; yet how they promote NASH remains poorly understood. Here, we show that peroxidase activity of global hepatic peroxiredoxin (PRDX) is significantly decreased in NASH, and palmitic acid (PA) binds to PRDX1 and inhibits its peroxidase activity. Using three genetic models, we demonstrate that hepatic PRDX1 protects against NASH in male mice. Mechanistically, PRDX1 suppresses STAT signaling and protects mitochondrial function by scavenging hydrogen peroxide, and mitigating the oxidation of protein tyrosine phosphatases and lipid peroxidation. We further identify rosmarinic acid (RA) as a potent agonist of PRDX1. As revealed by the complex crystal structure, RA binds to PRDX1 and stabilizes its peroxidatic cysteine. RA alleviates NASH through specifically activating PRDX1’s peroxidase activity. Thus, beyond revealing the molecular mechanism underlying PA promoting oxidative stress and NASH, our study suggests that boosting PRDX1’s peroxidase activity is a promising intervention for treating NASH. Oxidative stress is closely linked with nonalcoholic steatohepatitis (NASH). Here, the authors show that palmitic acid stimulates NASH by inhibiting PRDX1 to increase oxidative stress, while rosmarinic acid improves NASH by activating PRDX1 to reduce oxidative stress.

Brain cells that die after stroke release intracellular proteins into their environment. Akihiko Yoshimura and his colleagues demonstrate that peroxiredoxin proteins released from dying cells induce inflammatory cytokine expression and drive brain damage after stroke. Post-ischemic inflammation is an essential step in the progression of brain ischemia-reperfusion injury. However, the mechanism that activates infiltrating macrophages in the ischemic brain remains to be clarified. Here we demonstrate that peroxiredoxin (Prx) family proteins released extracellularly from necrotic brain cells induce expression of inflammatory cytokines including interleukin-23 in macrophages through activation of Toll-like receptor 2 (TLR2) and TLR4, thereby promoting neural cell death, even though intracellular Prxs have been shown to be neuroprotective. The extracellular release of Prxs in the ischemic core occurred 12 h after stroke onset, and neutralization of extracellular Prxs with antibodies suppressed inflammatory cytokine expression and infarct volume growth. In contrast, high mobility group box 1 (HMGB1), a well-known damage-associated molecular pattern molecule, was released before Prx and had a limited role in post-ischemic macrophage activation. We thus propose that extracellular Prxs are previously unknown danger signals in the ischemic brain and that its blocking agents are potent neuroprotective tools.

The mammalian endoplasmic reticulum (ER) harbors disulfide bond-generating enzymes, including Ero1α and peroxiredoxin 4 (Prx4) and nearly 20 members of the protein disulfide isomerase family (PDIs), which together constitute a suitable environment for oxidative protein folding. Here, we clarified the Prx4 preferential recognition of two PDI family proteins, P5 and ERp46 and the mode of interaction between Prx4 and P5 thioredoxin domain. Detailed analyses of oxidative folding catalyzed by the reconstituted Prx4–PDIs pathways demonstrated that, while P5 and ERp46 are dedicated to rapid, but promiscuous, disulfide introduction, PDI is an efficient proofreader of non-native disulfides. Remarkably, the Prx4-dependent formation of native disulfide bonds was accelerated when PDI was combined with ERp46 or P5, suggesting that PDIs work synergistically to increase the rate and fidelity of oxidative protein folding. Thus, the mammalian ER seems to contain highly systematized oxidative networks for the efficient production of large quantities of secretory proteins.

Nitric oxide (NO) is a free radical product of cell metabolism that plays diverse and important roles in the regulation of cellular function. S-Nitrosylation is emerging as a specific and fundamental posttranslational protein modification for the transduction of NO bioactivity, but very little is known about its physiological functions in plants. We investigated the molecular mechanism for S-nitrosylation of peroxiredoxin II E (PrxII E) from Arabidopsis thaliana and found that this posttranslational modification inhibits the hydroperoxide-reducing peroxidase activity of PrxII E, thus revealing a novel regulatory mechanism for peroxiredoxins. Furthermore, we obtained biochemical and genetic evidence that PrxII E functions in detoxifying peroxynitrite (ONOO⁻), a potent oxidizing and nitrating species formed in a diffusion-limited reaction between NO and$\\text{O}_{2}{}^{-}$that can interfere with Tyr kinase signaling through the nitration of Tyr residues. S-Nitrosylation also inhibits the ONOO⁻ detoxification activity of PrxII E, causing a dramatic increase of ONOO⁻-dependent nitrotyrosine residue formation. The same increase was observed in a prxII E mutant line after exposure to ONOO⁻, indicating that the PrxII E modulation of ONOO⁻ bioactivity is biologically relevant. We conclude that NO regulates the effects of its own radicals through the S-nitrosylation of crucial components of the antioxidant defense system that function as common triggers for reactive oxygen species- and NO-mediated signaling events.

Nonclassical protein secretion is of major importance as a number of cytokines and inflammatory mediators are secreted via this route. Current evidence indicates that there are several mechanistically distinct methods of nonclassical secretion. We have shown recently that peroxiredoxin (Prdx) 1 and Prdx2 are released by various cells upon exposure to inflammatory stimuli such as lipopolysaccharide (LPS) or tumor necrosis factor alpha (TNF-α). The released Prdx then acts to induce production of inflammatory cytokines. However, Prdx1 and 2 do not have signal peptides and therefore must be secreted by alternative mechanisms, as has been postulated for the inflammatory mediators interleukin-1β (IL-1β) and high mobility group box-1 (HMGB1). We show here that circulating Prdx1 and 2 are present exclusively as disulfide-linked homodimers. Inflammatory stimuli also induce in vitro release of Prdx1 and 2 as disulfide-linked homodimers. Mutation of cysteines Cys51 or Cys172 (but not Cys70) in Prdx2, and Cys52 or Cys173 (but not Cys71 or Cys83) in Prdx1 prevented dimer formation and this was associated with inhibition of their TNF-α-induced release. Thus, the presence and oxidation of key cysteine residues in these proteins are a prerequisite for their secretion in response to TNF-α, and this release can be induced with an oxidant. By contrast, the secretion of the nuclear-associated danger signal HMGB1 is independent of cysteine oxidation, as shown by experiments with a cysteine-free HMGB1 mutant. Release of Prdx1 and 2 is not prevented by inhibitors of the classical secretory pathway, instead, both Prdx1 and 2 are released in exosomes from both human embryonic kidney (HEK) cells and monocytic cells. Serum Prdx1 and 2 also are associated with the exosomes. These results describe a novel pathway of protein secretion mediated by cysteine oxidation that underlines the importance of redox-dependent signaling mechanisms in inflammation.

Cyanobacteria are model organisms for photosynthesis and are attractive for biotechnology applications. To aid investigation of genotype-phenotype relationships in cyanobacteria, we develop an inducible CRISPRi gene repression library in Synechocystis sp. PCC 6803, where we aim to target all genes for repression. We track the growth of all library members in multiple conditions and estimate gene fitness. The library reveals several clones with increased growth rates, and these have a common upregulation of genes related to cyclic electron flow. We challenge the library with 0.1 M L-lactate and find that repression of peroxiredoxin bcp2 increases growth rate by 49%. Transforming the library into an L-lactate-secreting Synechocystis strain and sorting top lactate producers enriches clones with sgRNAs targeting nutrient assimilation, central carbon metabolism, and cyclic electron flow. In many examples, productivity can be enhanced by repression of essential genes, which are difficult to access by transposon insertion. Developing cyanobacteria as CO2-neutral cell factories relies on the knowledge of the regulation mechanisms for growth and metabolism. Here, the authors develop an inducible CRISPRi gene repression library in Synechocystis sp. PCC 6803 and screens genes potentially affecting growth and L-lactate tolerance and production.

While cellular metabolism impacts the DNA damage response, a systematic understanding of the metabolic requirements that are crucial for DNA damage repair has yet to be achieved. Here, we investigate the metabolic enzymes and processes that are essential for the resolution of DNA damage. By integrating functional genomics with chromatin proteomics and metabolomics, we provide a detailed description of the interplay between cellular metabolism and the DNA damage response. Further analysis identified that Peroxiredoxin 1, PRDX1, contributes to the DNA damage repair. During the DNA damage response, PRDX1 translocates to the nucleus where it reduces DNA damage‐induced nuclear reactive oxygen species. Moreover, PRDX1 loss lowers aspartate availability, which is required for the DNA damage‐induced upregulation of de novo nucleotide synthesis. In the absence of PRDX1, cells accumulate replication stress and DNA damage, leading to proliferation defects that are exacerbated in the presence of etoposide, thus revealing a role for PRDX1 as a DNA damage surveillance factor. Synopsis Genetic screens, proteomics, and metabolomics are performed to investigate the crosstalk between metabolism and the DNA damage response. Integrative analyses identify Peroxiredoxin‐1 (PRDX1) as a DNA damage surveillance factor. Systematic approaches following DNA damage induction by etoposide reveal the aspects of metabolism that are crucial for maintaining genome integrity. Loss of electron transport chain enzymes is synthetically viable with etoposide, and some of these enzymes are partially located on chromatin 24 h after etoposide release. The metabolic enzyme PRDX1 contributes to DNA repair and translocates to the nucleus where it reduces DNA damage‐induced nuclear ROS. Loss of PRDX1 lowers aspartate availability and perturbs de novo nucleotide synthesis, which induces replication stress and limits the DNA repair capacities of the cells. Graphical Abstract Genetic screens, proteomics, and metabolomics are performed to investigate the crosstalk between metabolism and the DNA damage response. Integrative analyses identify Peroxiredoxin‐1 (PRDX1) as a DNA damage surveillance factor.

Ferroptosis is a non-apoptotic, iron-dependent oxidative form of cell death that is specifically induced by erastin in RAS mutant cancer cells. Ferroptotic cell death is the result of membrane lipid peroxide damage caused by the accumulation of hydroxyl radicals derived from H 2 O 2 by the Fenton reaction. Peroxidases are key cellular antioxidant enzymes that block such damaging processes. Few studies have examined the roles of long non-coding RNAs (lncRNAs) in the regulation of cellular oxidative stress, especially in ferroptosis. Here, we demonstrated that erastin upregulated the lncRNA GABPB1-AS1, which downregulated GABPB1 protein levels by blocking GABPB1 translation, leading to the downregulation of the gene encoding Peroxiredoxin-5 (PRDX5) peroxidase and the eventual suppression of the cellular antioxidant capacity. Such effects critically inhibited the cellular antioxidant capacity and cell viability. Additionally, high expression levels of GABPB1 were correlated with poor prognosis of hepatocellular carcinoma (HCC) Patients, while high GABPB1-AS1 levels in HCC patients correlated with improved overall survival. Collectively, these data demonstrate a mechanistic link between GABPB1 and its antisense lncRNA GABPB1-AS1 in erastin-induced ferroptosis and establish GABPB1 and GABPB1-AS1 as attractive therapeutic targets for HCC.

Mitochondria are multifunctional cellular organelles that are major producers of reactive oxygen species (ROS) in eukaryotes; to maintain the redox balance, they are supplemented with different ROS scavengers, including mitochondrial peroxiredoxins (Prdxs). Mitochondrial Prdxs have physiological and pathological significance and are associated with the initiation and progression of various cancer types. In this review, we have focused on signaling involving ROS and mitochondrial Prdxs that is associated with cancer development and progression. An upregulated expression of Prdx3 and Prdx5 has been reported in different cancer types, such as breast, ovarian, endometrial, and lung cancers, as well as in Hodgkin’s lymphoma and hepatocellular carcinoma. The expression of Prdx3 and Prdx5 in different types of malignancies involves their association with different factors, such as transcription factors, micro RNAs, tumor suppressors, response elements, and oncogenic genes. The microenvironment of mitochondrial Prdxs plays an important role in cancer development, as cancerous cells are equipped with a high level of antioxidants to overcome excessive ROS production. However, an increased production of Prdx3 and Prdx5 is associated with the development of chemoresistance in certain types of cancers and it leads to further complications in cancer treatment. Understanding the interplay between mitochondrial Prdxs and ROS in carcinogenesis can be useful in the development of anticancer drugs with better proficiency and decreased resistance. However, more targeted studies are required for exploring the tumor microenvironment in association with mitochondrial Prdxs to improve the existing cancer therapies and drug development.