Hydrogen peroxide-dependent oxidation of ERK2 within its D-recruitment site alters its substrate selection.

Extracellular signal-regulated kinases 1 and 2 (ERK1/2) are dysregulated in many pervasive diseases. Recently, we discovered that ERK1/2 is oxidized by signal-generated hydrogen peroxide in various cell types. Since the putative sites of oxidation lie within or near ERK1/2's ligand-binding surfaces, we investigated how oxidation of ERK2 regulates interactions with the model substrates Sub-D and Sub-F. These studies revealed that ERK2 undergoes sulfenylation at C159 on its D-recruitment site surface and that this modification modulates ERK2 activity differentially between substrates. Integrated biochemical, computational, and mutational analyses suggest a plausible mechanism for peroxide-dependent changes in ERK2-substrate interactions. Interestingly, oxidation decreased ERK2's affinity for some D-site ligands while increasing its affinity for others. Finally, oxidation by signal-generated peroxide enhanced ERK1/2's ability to phosphorylate ribosomal S6 kinase A1 (RSK1) in HeLa cells. Together, these studies lay the foundation for examining crosstalk between redox- and phosphorylation-dependent signaling at the level of kinase-substrate selection.

Glutathionylation of pyruvate dehydrogenase complex E2 and inflammatory cytokine production during acute inflammation are magnified by mitochondrial oxidative stress.

Lipopolysaccharide (LPS) is a known inducer of inflammatory signaling which triggers generation of reactive oxygen species (ROS) and cell death in responsive cells like THP-1 promonocytes and freshly isolated human monocytes. A key LPS-responsive metabolic pivot point is the 9 MDa mitochondrial pyruvate dehydrogenase complex (PDC), which provides pyruvate dehydrogenase (E1), lipoamide-linked transacetylase (E2) and lipoamide dehydrogenase (E3) activities to produce acetyl-CoA from pyruvate. While phosphorylation-dependent decreases in PDC activity following LPS treatment or sepsis have been deeply investigated, redox-linked processes have received less attention. Data presented here demonstrate that LPS-induced reversible oxidation within PDC occurs in PDCE2 in both THP-1 cells and primary human monocytes. Knockout of PDCE2 by CRISPR and expression of FLAG-tagged PDCE2 in THP-1 cells demonstrated that LPS-induced glutathionylation is associated with wild type PDCE2 but not mutant protein lacking the lipoamide-linking lysine residues. Moreover, the mitochondrially-targeted electrophile MitoCDNB, which impairs both glutathione- and thioredoxin-based reductase systems, elevates ROS similar to LPS but does not cause PDCE2 glutathionylation. However, LPS and MitoCDNB together are highly synergistic for PDCE2 glutathionylation, ROS production, and cell death. Surprisingly, the two treatments together had differential effects on cytokine production; pro-inflammatory IL-1ß production was enhanced by the co-treatment, while IL-10, an important anti-inflammatory cytokine, dropped precipitously compared to LPS treatment alone. This new information may expand opportunities to understand and modulate PDC redox status and activity and improve the outcomes of pathological inflammation.

Glutathionylation of Pyruvate Dehydrogenase Complex E2 and Inflammatory Cytokine Production During Acute Inflammation Are Magnified By Mitochondrial Oxidative Stress.

Lipopolysaccharide (LPS) is a known inducer of inflammatory signaling which triggers generation of reactive oxygen species (ROS) and cell death in responsive cells like THP-1 promonocytes and freshly isolated human monocytes. A key LPS-responsive metabolic pivot point is the 9 megadalton mitochondrial pyruvate dehydrogenase complex (PDC), which provides pyruvate dehydrogenase (E1), lipoamide-linked transacetylase (E2) and lipoamide dehydrogenase (E3) activities to produce acetyl-CoA from pyruvate. While phosphorylation-dependent decreases in PDC activity following LPS treatment or sepsis have been deeply investigated, redox-linked processes have received less attention. Data presented here demonstrate that LPS-induced reversible oxidation within PDC occurs in PDCE2 in both THP-1 cells and primary human monocytes. Knockout of PDCE2 by CRISPR and expression of FLAG-tagged PDCE2 in THP-1 cells demonstrated that LPS-induced glutathionylation is associated with wild type PDCE2 but not mutant protein lacking the lipoamide-linking lysine residues. Moreover, the mitochondrially-targeted electrophile MitoCDNB, which impairs both glutathione- and thioredoxin-based reductase systems, elevates ROS similar to LPS but does not cause PDCE2 glutathionylation. However, LPS and MitoCDNB together are highly synergistic for PDCE2 glutathionylation, ROS production, and cell death. Surprisingly, the two treatments together had differential effects on cytokine production; pro-inflammatory IL-1ß production was enhanced by the co-treatment, while IL-10, an important anti-inflammatory cytokine, dropped precipitously compared to LPS treatment alone. This new information may expand opportunities to understand and modulate PDC redox status and activity and improve the outcomes of pathological inflammation.

Nuclear-localized, iron-bound superoxide dismutase-2 antagonizes epithelial lineage programs to promote stemness of breast cancer cells via a histone demethylase activity.

The dichotomous behavior of superoxide dismutase-2 (SOD2) in cancer biology has long been acknowledged and more recently linked to different posttranslational forms of the enzyme. However, a distinctive activity underlying its tumor-promoting function is yet to be described. Here, we report that acetylation, one of such posttranslational modifications (PTMs), increases SOD2 affinity for iron, effectively changing the biochemical function of this enzyme from that of an antioxidant to a demethylase. Acetylated, iron-bound SOD2 localizes to the nucleus, promoting stem cell gene expression via removal of suppressive epigenetic marks such as H3K9me3 and H3K927me3. Particularly, H3K9me3 was specifically removed from regulatory regions upstream of Nanog and Oct-4, two pluripotency factors involved in cancer stem cell reprogramming. Phenotypically, cells expressing nucleus-targeted SOD2 (NLS-SOD2) have increased clonogenicity and metastatic potential. FeSOD2 operating as H3 demethylase requires H2O2 as substrate, which unlike cofactors of canonical demethylases (i.e., oxygen and 2-oxoglutarate), is more abundant in tumor cells than in normal tissue. Therefore, our results indicate that FeSOD2 is a demethylase with unique activities and functions in the promotion of cancer evolution toward metastatic phenotypes.

Bioinformatic Analyses of Peroxiredoxins and RF-Prx: A Random Forest-Based Predictor and Classifier for Prxs.

Peroxiredoxins (Prxs) are a protein superfamily, present in all organisms, that play a critical role in protecting cellular macromolecules from oxidative damage but also regulate intracellular and intercellular signaling processes involving redox-regulated proteins and pathways. Bioinformatic approaches using computational tools that focus on active site-proximal sequence fragments (known as active site signatures) and iterative clustering and searching methods (referred to as TuLIP and MISST) have recently enabled the recognition of over 38,000 peroxiredoxins, as well as their classification into six functionally relevant groups. With these data providing so many examples of Prxs in each class, machine learning approaches offer an opportunity to extract additional information about features characteristic of these protein groups.In this study, we developed a novel computational method named "RF-Prx" based on a random forest (RF) approach integrated with K-space amino acid pairs (KSAAP) to identify peroxiredoxins and classify them into one of six subgroups. Our process performed in a superior manner compared to other machine learning classifiers. Thus the RF approach integrated with K-space amino acid pairs enabled the detection of class-specific conserved sequences outside the known functional centers and with potential importance. For example, drugs designed to target Prx proteins would likely suffer from cross-reactivity among distinct Prxs if targeted to conserved active sites, but this may be avoidable if remote, class-specific regions could be targeted instead.

Oncogenic KRAS G12C: Kinetic and redox characterization of covalent inhibition.

The recent development of mutant-selective inhibitors for the oncogenic KRASG12C allele has generated considerable excitement. These inhibitors covalently engage the mutant C12 thiol located within the phosphoryl binding loop of RAS, locking the KRASG12C protein in an inactive state. While clinical trials of these inhibitors have been promising, mechanistic questions regarding the reactivity of this thiol remain. Here, we show by NMR and an independent biochemical assay that the pKa of the C12 thiol is depressed (pKa ∼7.6), consistent with susceptibility to chemical ligation. Using a validated fluorescent KRASY137W variant amenable to stopped-flow spectroscopy, we characterized the kinetics of KRASG12C fluorescence changes upon addition of ARS-853 or AMG 510, noting that at low temperatures, ARS-853 addition elicited both a rapid first phase of fluorescence change (attributed to binding, Kd = 36.0 ± 0.7 µM) and a second, slower pH-dependent phase, taken to represent covalent ligation. Consistent with the lower pKa of the C12 thiol, we found that reversible and irreversible oxidation of KRASG12C occurred readily both in vitro and in the cellular environment, preventing the covalent binding of ARS-853. Moreover, we found that oxidation of the KRASG12C Cys12 to a sulfinate altered RAS conformation and dynamics to be more similar to KRASG12D in comparison to the unmodified protein, as assessed by molecular dynamics simulations. Taken together, these findings provide insight for future KRASG12C drug discovery efforts, and identify the occurrence of G12C oxidation with currently unknown biological ramifications.

Mitigation of doxorubicin-induced cardiotoxicity with an H2O2-Activated, H2S-Donating hybrid prodrug.

Doxorubicin (DOX) is one of the most effective anticancer agents in clinical oncology. Its continued use, however, is severely limited by its dose-dependent cardiotoxicity which stems, in part, from its overproduction of reactive oxygen species (ROS) and often manifests itself as full-blown cardiomyopathy in patients, years after the cessation of treatment. Therefore, identifying DOX analogs, or prodrugs, with a diminished cardiotoxic profile is highly desirable. Herein, we describe a novel, H2O2-responsive DOX hybrid codrug (mutual prodrug) that has been rationally designed to concurrently liberate hydrogen sulfide (H2S), a purported cardioprotectant with anticancer activity, in an effort to maintain the antitumor effects of DOX while simultaneously reducing its cardiotoxic side effects. Experiments with cardiomyoblast cells in culture demonstrated a rapid accumulation of prodrug into the cells, but diminished apoptotic effects compared with DOX, dependent upon its release of H2S. Cells treated with the prodrug exhibited significantly higher Nrf2 activation relative to DOX-treated cells. Preliminary indications, using a mouse triple-negative breast cancer cell line sensitive to DOX treatment, are that the prodrug maintains considerable toxicity against the tumor-inducing cell line, suggesting significant promise for this prodrug as a cardioprotective chemotherapeutic to replace DOX.

Cysteine oxidation of copper transporter CTR1 drives VEGFR2 signalling and angiogenesis.

Vascular endothelial growth factor receptor type 2 (VEGFR2, also known as KDR and FLK1) signalling in endothelial cells (ECs) is essential for developmental and reparative angiogenesis. Reactive oxygen species and copper (Cu) are also involved in these processes. However, their inter-relationship is poorly understood. Evidence of the role of the endothelial Cu importer CTR1 (also known as SLC31A1) in VEGFR2 signalling and angiogenesis in vivo is lacking. Here, we show that CTR1 functions as a redox sensor to promote angiogenesis in ECs. CTR1-depleted ECs showed reduced VEGF-induced VEGFR2 signalling and angiogenic responses. Mechanistically, CTR1 was rapidly sulfenylated at Cys189 at its cytosolic C terminus after stimulation with VEGF, which induced CTR1-VEGFR2 disulfide bond formation and their co-internalization to early endosomes, driving sustained VEGFR2 signalling. In vivo, EC-specific Ctr1-deficient mice or CRISPR-Cas9-generated redox-dead Ctr1(C187A)-knockin mutant mice had impaired developmental and reparative angiogenesis. Thus, oxidation of CTR1 at Cys189 promotes VEGFR2 internalization and signalling to enhance angiogenesis. Our study uncovers an important mechanism for sensing reactive oxygen species through CTR1 to drive neovascularization.

Native state fluctuations in a peroxiredoxin active site match motions needed for catalysis.

Peroxiredoxins are ubiquitous enzymes that detoxify peroxides and regulate redox signaling. During catalysis, a "peroxidatic" cysteine (CP) in the conserved active site reduces peroxide while being oxidized to a CP-sulfenate, prompting a local unfolding event that enables formation of a disulfide with a second, "resolving" cysteine. Here, we use nuclear magnetic resonance spectroscopy to probe the dynamics of the CP-thiolate and disulfide forms of Xanthomonas campestris peroxiredoxin Q. Chemical exchange saturation transfer behavior of the resting enzyme reveals 26 residues in and around the active site exchanging at a rate of 72 s-1 with a locally unfolded, high-energy (2.5% of the population) state. This unequivocally establishes that a catalytically relevant local unfolding equilibrium exists in the enzyme's CP-thiolate form. Also, faster motions imply an active site instability that could promote local unfolding and, based on other work, be exacerbated by CP-sulfenate formation so as to direct the enzyme along a functional catalytic trajectory.

[18F]Fluoro-DCP, a first generation PET radiotracer for monitoring protein sulfenylation in vivo.

Redox metabolism plays essential functions in the pathology of cancer and many other diseases. While several radiotracers for imaging redox metabolism have been developed, there are no reports of radiotracers for in vivo imaging of protein oxidation. Here we take the first step towards this goal and describe the synthesis and kinetic properties of a new positron emission tomography (PET) [18F]Fluoro-DCP radiotracer for in vivo imaging of protein sulfenylation. Time course biodistribution and PET/CT studies using xenograft animal models of Head and Neck Squamous Cell Cancer (HNSCC) demonstrate its capability to distinguish between tumors with radiation sensitive and resistant phenotypes consistent with previous reports of decreased protein sulfenylation in clinical specimens of radiation resistant HNSCC. We envision further development of this technology to aid research efforts towards improving diagnosis of patients with radiation resistant tumors.

Sirtuin 6 (SIRT6) regulates redox homeostasis and signaling events in human articular chondrocytes.

The nuclear localized protein deacetylase, SIRT6, has been identified as a crucial regulator of biological processes that drive aging. Among these processes, SIRT6 can promote resistance to oxidative stress conditions, but the precise mechanisms remain unclear. The objectives of this study were to examine the regulation of SIRT6 activity by age and oxidative stress and define the role of SIRT6 in maintaining redox homeostasis in articular chondrocytes. Although SIRT6 levels did not change with age, SIRT6 activity was significantly reduced in chondrocytes isolated from older adults. Using dimedone-based chemical probes that detect oxidized cysteines, we identified that SIRT6 is oxidized in response to oxidative stress conditions, an effect that was associated with reduced SIRT6 activity. Enhancement of SIRT6 activity through adenoviral SIRT6 overexpression specifically increased the basal levels of two antioxidant proteins, peroxiredoxin 1 (Prx1) and sulfiredoxin (Srx) and decreased the levels of an inhibitor of antioxidant activity, thioredoxin interacting protein (TXNIP). Conversely, in chondrocytes derived from mice with cartilage specific Sirt6 knockout, Sirt6 loss decreased Prx1 levels and increased TXNIP levels. SIRT6 overexpression decreased nuclear-generated H2O2 levels and oxidative stress-induced accumulation of nuclear phosphorylated p65. Our data demonstrate that SIRT6 activity is altered with age and oxidative stress conditions associated with aging. SIRT6 contributes to chondrocyte redox homeostasis by regulating specific members of the Prx catalytic cycle. Targeted therapies aimed at preventing the age-related decline in SIRT6 activity may represent a novel strategy to maintain redox balance in joint tissues and decrease catabolic signaling events implicated in osteoarthritis (OA).

Identification of sulfenylation patterns in trophozoite stage Plasmodium falciparum using a non-dimedone based probe.

Plasmodium falciparum causes the deadliest form of malaria. Adequate redox control is crucial for this protozoan parasite to overcome oxidative and nitrosative challenges, thus enabling its survival. Sulfenylation is an oxidative post-translational modification, which acts as a molecular on/off switch, regulating protein activity. To obtain a better understanding of which proteins are redox regulated in malaria parasites, we established an optimized affinity capture protocol coupled with mass spectrometry analysis for identification of in vivo sulfenylated proteins. The non-dimedone based probe BCN-Bio1 shows reaction rates over 100-times that of commonly used dimedone-based probes, allowing for a rapid trapping of sulfenylated proteins. Mass spectrometry analysis of BCN-Bio1 labeled proteins revealed the first insight into the Plasmodium falciparum trophozoite sulfenylome, identifying 102 proteins containing 152 sulfenylation sites. Comparison with Plasmodium proteins modified by S-glutathionylation and S-nitrosation showed a high overlap, suggesting a common core of proteins undergoing redox regulation by multiple mechanisms. Furthermore, parasite proteins which were identified as targets for sulfenylation were also identified as being sulfenylated in other organisms, especially proteins of the glycolytic cycle. This study suggests that a number of Plasmodium proteins are subject to redox regulation and it provides a basis for further investigations into the exact structural and biochemical basis of regulation, and a deeper understanding of cross-talk between post-translational modifications.

Discovery of Spilanthol Endoperoxide as a Redox Natural Compound Active against Mammalian Prx3 and Chlamydia trachomatis Infection.

Chlamydia trachomatis (Ct) is a bacterial intracellular pathogen responsible for a plethora of diseases ranging from blindness to pelvic inflammatory diseases and cervical cancer. Although this disease is effectively treated with antibiotics, concerns for development of resistance prompt the need for new low-cost treatments. Here we report the activity of spilanthol (SPL), a natural compound with demonstrated anti-inflammatory properties, against Ct infections. Using chemical probes selective for imaging mitochondrial protein sulfenylation and complementary assays, we identify an increase in mitochondrial oxidative state by SPL as the underlying mechanism leading to disruption of host cell F-actin cytoskeletal organization and inhibition of chlamydial infection. The peroxidation product of SPL (SPL endoperoxide, SPLE), envisioned to be the active compound in the cellular milieu, was chemically synthesized and showed more potent anti-chlamydial activity. Comparison of SPL and SPLE reactivity with mammalian peroxiredoxins, demonstrated preferred reactivity of SPLE with Prx3, and virtual lack of SPL reaction with any of the reduced Prx isoforms investigated. Cumulatively, these findings support the function of SPL as a pro-drug, which is converted to SPLE in the cellular milieu leading to inhibition of Prx3, increased mitochondrial oxidation and disruption of F-actin network, and inhibition of Ct infection.

Acyl-lipid desaturases and Vipp1 cooperate in cyanobacteria to produce novel omega-3 PUFA-containing glycolipids.

BACKGROUND: Dietary omega-3 (n-3), long chain (LC-, ≥ 20 carbons), polyunsaturated fatty acids (PUFAs) derived largely from marine animal sources protect against inflammatory processes and enhance brain development and function. With the depletion of natural stocks of marine animal sources and an increasing demand for n-3 LC-PUFAs, alternative, sustainable supplies are urgently needed. As a result, n-3 18-carbon and LC-PUFAs are being generated from plant or algal sources, either by engineering new biosynthetic pathways or by augmenting existing systems. RESULTS: We utilized an engineered plasmid encoding two cyanobacterial acyl-lipid desaturases (DesB and DesD, encoding Δ15 and Δ6 desaturases, respectively) and "vesicle-inducing protein in plastids" (Vipp1) to induce production of stearidonic acid (SDA, 18:4 n-3) at high levels in three strains of cyanobacteria (10, 17 and 27% of total lipids in Anabaena sp. PCC7120, Synechococcus sp. PCC7002, and Leptolyngbya sp. strain BL0902, respectively). Lipidomic analysis revealed that in addition to SDA, the rare anti-inflammatory n-3 LC-PUFA eicosatetraenoic acid (ETA, 20:4 n-3) was synthesized in these engineered strains, and ~ 99% of SDA and ETA was complexed to bioavailable monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) species. Importantly, novel molecular species containing alpha-linolenic acid (ALA), SDA and/or ETA in both acyl positions of MGDG and DGDG were observed in the engineered Leptolyngbya and Synechococcus strains, suggesting that these could provide a rich source of anti-inflammatory molecules. CONCLUSIONS: Overall, this technology utilizes solar energy, consumes carbon dioxide, and produces large amounts of nutritionally important n-3 PUFAs and LC-PUFAs. Importantly, it can generate previously undescribed, highly bioavailable, anti-inflammatory galactosyl lipids. This technology could therefore be transformative in protecting ocean fisheries and augmenting the nutritional quality of human and animal food products.

Introduction to approaches and tools for the evaluation of protein cysteine oxidation.

Oxidative modifications of cysteine thiols in cellular proteins are pivotal to the way signal-stimulated reactive oxygen species are sensed and elicit appropriate or sometimes pathological responses, but the dynamic and often transitory nature of these modifications offer a challenge to the investigator trying to identify such sites and the responses they elicit. A number of reagents and workflows have been developed to identify proteins undergoing oxidation and to query the timing, extent and location of such modifications, as described in this minireview. While no approach is perfect to capture all the redox information in a functioning cell, best practices described herein can enable considerable insights into the "redox world" of cells and organisms.

Unraveling the effects of peroxiredoxin 2 nitration; role of C-terminal tyrosine 193.

Peroxiredoxins (Prx) are enzymes that efficiently reduce hydroperoxides through active participation of cysteine residues (CP, CR). The first step in catalysis, the reduction of peroxide substrate, is fast, 107 - 108 M-1s-1 for human Prx2. In addition, the high intracellular concentration of Prx positions them not only as good antioxidants but also as central players in redox signaling pathways. These biological functions can be affected by post-translational modifications that could alter the peroxidase activity and/or interaction with other proteins. In particular, inactivation by hyperoxidation of CP, which occurs when a second molecule of peroxide reacts with the CP in the sulfenic acid form, modulates their participation in redox signaling pathways. The higher sensitivity to hyperoxidation of some Prx has been related to the presence of structural motifs that disfavor disulfide formation at the active site, making the CP sulfenic acid more available for hyperoxidation or interaction with a redox protein target. We previously reported that treatment of human Prx2 with peroxynitrite results in tyrosine nitration, a post-translational modification on non-catalytic residues, yielding a more active peroxidase with higher resistance to hyperoxidation. In this work, studies on various mutants of hPrx2 confirm that the presence of the tyrosyl side-chain of Y193, belonging to the C-terminal YF motif of eukaryotic Prx, is necessary to observe the increase in Prx2 resistance to hyperoxidation. Moreover, our results underline the critical role of this structural motif on the rate of disulfide formation that determines the differential participation of Prx in redox signaling pathways.

Triphenylphosphonium-Derived Protein Sulfenic Acid Trapping Agents: Synthesis, Reactivity, and Effect on Mitochondrial Function.

Redox-mediated protein modifications control numerous processes in both normal and disease metabolism. Protein sulfenic acids, formed from the oxidation of protein cysteine residues, play a critical role in thiol-based redox signaling. The reactivity of protein sulfenic acids requires their identification through chemical trapping, and this paper describes the use of the triphenylphosphonium (TPP) ion to direct known sulfenic acid traps to the mitochondria, a verified source of cellular reactive oxygen species. Coupling of the TPP group with the 2,4-(dioxocyclohexyl)propoxy (DCP) unit and the bicyclo[6.1.0]nonyne (BCN) group produces two new probes, DCP-TPP and BCN-TPP. DCP-TPP and BCN-TPP react with C165A AhpC-SOH, a model protein sulfenic acid, to form the expected adducts with second-order rate constants of k = 1.1 M-1 s-1 and k = 5.99 M-1 s-1, respectively, as determined by electrospray ionization time-of-flight mass spectrometry. The TPP group does not alter the rate of DCP-TPP reaction with protein sulfenic acid compared to dimedone but slows the rate of BCN-TPP reaction compared to a non-TPP-containing BCN-OH control by 4.6-fold. The hydrophobic TPP group may interact with the protein, preventing an optimal reaction orientation for BCN-TPP. Unlike BCN-OH, BCN-TPP does not react with the protein persulfide, C165A AhpC-SSH. Extracellular flux measurements using A549 cells show that DCP-TPP and BCN-TPP influence mitochondrial energetics, with BCN-TPP producing a drastic decrease in basal respiration, perhaps due to its faster reaction kinetics with sulfenylated proteins. Further control experiments with BCN-OH, TPP-COOH, and dimedone provide strong evidence for mitochondrial localization and accumulation of DCP-TPP and BCN-TPP. These results reveal the compatibility of the TPP group with reactive sulfenic acid probes as a mitochondrial director and support the use of the TPP group in the design of sulfenic acid traps.

Chaperone activation and client binding of a 2-cysteine peroxiredoxin.

Many 2-Cys-peroxiredoxins (2-Cys-Prxs) are dual-function proteins, either acting as peroxidases under non-stress conditions or as chaperones during stress. The mechanism by which 2-Cys-Prxs switch functions remains to be defined. Our work focuses on Leishmania infantum mitochondrial 2-Cys-Prx, whose reduced, decameric subpopulation adopts chaperone function during heat shock, an activity that facilitates the transition from insects to warm-blooded host environments. Here, we have solved the cryo-EM structure of mTXNPx in complex with a thermally unfolded client protein, and revealed that the flexible N-termini of mTXNPx form a well-resolved central belt that contacts and encapsulates the unstructured client protein in the center of the decamer ring. In vivo and in vitro cross-linking studies provide further support for these interactions, and demonstrate that mTXNPx decamers undergo temperature-dependent structural rearrangements specifically at the dimer-dimer interfaces. These structural changes appear crucial for exposing chaperone-client binding sites that are buried in the peroxidase-active protein.

Peroxiredoxins in Cancer and Response to Radiation Therapies.

Peroxiredoxins have a long-established cellular function as regulators of redox metabolism by catalyzing the reduction of peroxides (e.g., H2O2, lipid peroxides) with high catalytic efficiency. This activity is also critical to the initiation and relay of both phosphorylation and redox signaling in a broad range of pathophysiological contexts. Under normal physiological conditions, peroxiredoxins protect normal cells from oxidative damage that could promote oncogenesis (e.g., environmental stressors). In cancer, higher expression level of peroxiredoxins has been associated with both tumor growth and resistance to radiation therapies. However, this relationship between the expression of peroxiredoxins and the response to radiation is not evident from an analysis of data in The Cancer Genome Atlas (TCGA) or NCI60 panel of cancer cell lines. The focus of this review is to summarize the current experimental knowledge implicating this class of proteins in cancer, and to provide a perspective on the value of targeting peroxiredoxins in the management of cancer. Potential biases in the analysis of the TCGA data with respect to radiation resistance are also highlighted.

Differential peroxiredoxin hyperoxidation regulates MAP kinase signaling in human articular chondrocytes.

The peroxiredoxin (Prx) family of Cys-dependent peroxidases control intracellular levels of H2O2 and can regulate signal transduction. Inhibition of the Prxs, through hyperoxidation amongst other mechanisms, leads to oxidative stress conditions that can alter homeostatic signaling. To determine the effects oxidation of Prx1-Prx3 has on MAP kinase and IGF-1 signaling events in human chondrocytes, this study used 2-methyl-1,4-naphthoquinone (menadione) and 2,3-dimethyl-1,4-naphthoquinone (DMNQ) as H2O2-generating tools due to their differential mechanisms of action. Menadione and DMNQ generated similar levels of intracellular H2O2 as determined using the biosensor Orp1-roGFP and by measuring Prx redox status. However, menadione generated higher levels of mitochondrial H2O2 associated with Prx3 hyperoxidation and phosphorylation of Prx1 while DMNQ treatment was associated with hyperoxidation of cytosolic Prx1 and Prx2 but not mitochondrial Prx3. Both menadione and DMNQ induced sustained phosphorylation of p38 but only DMNQ activated JNK. Menadione but not DMNQ inhibited IGF-1-induced Akt phosphorylation. Chondrocytes transduced with an adenoviral vector to overexpress Prx3 displayed decreased PrxSO2/3 formation in response to menadione which was associated with restoration of IGF-1-mediated Akt signaling and inhibition of p38 phosphorylation. Prx1 and Prx2 overexpression had no effects on Prx redox status but Prx1 overexpression enhanced basal Akt phosphorylation. These results suggest that hyperoxidation of specific Prx isoforms is associated with distinct cell signaling events and identify Prx3 redox status as an important regulator of anabolic and catabolic signal transduction. Targeted strategies to prevent mitochondrial Prx3 hyperoxidation could be useful in maintaining cellular redox balance and homeostatic signaling.