Prdx1 Interacts with ASK1 upon Exposure to H2O2 and Independently of a Scaffolding Protein.

Hydrogen peroxide (H2O2) is a key redox signaling molecule that selectively oxidizes cysteines on proteins. It can accomplish this even in the presence of highly efficient and abundant H2O2 scavengers, peroxiredoxins (Prdxs), as it is the Prdxs themselves that transfer oxidative equivalents to specific protein thiols on target proteins via their redox-relay functionality. The first evidence of a mammalian cytosolic Prdx-mediated redox-relay-Prdx1 with the kinase ASK1-was presented a decade ago based on the outcome of a co-immunoprecipitation experiment. A second such redox-relay-Prdx2:STAT3-soon followed, for which further studies provided insights into its specificity, organization, and mechanism. The Prdx1:ASK1 redox-relay, however, has never undergone such a characterization. Here, we combine cellular and in vitro protein-protein interaction methods to investigate the Prdx1:ASK1 interaction more thoroughly. We show that, contrary to the Prdx2:STAT3 redox-relay, Prdx1 interacts with ASK1 at elevated H2O2 concentrations, and that this interaction can happen independently of a scaffolding protein. We also provide evidence of a Prdx2:ASK1 interaction, and demonstrate that it requires a facilitator that, however, is not annexin A2. Our results reveal that cytosolic Prdx redox-relays can be organized in different ways and yet again highlight the differentiated roles of Prdx1 and Prdx2.

Peroxiredoxins wear many hats: Factors that fashion their peroxide sensing personalities.

Peroxiredoxins (Prdxs) sense and assess peroxide levels, and signal through protein interactions. Understanding the role of the multiple structural and post-translational modification (PTM) layers that tunes the peroxiredoxin specificities is still a challenge. In this review, we give a tabulated overview on what is known about human and bacterial peroxiredoxins with a focus on structure, PTMs, and protein-protein interactions. Armed with numerous cellular and atomic level experimental techniques, we look at the future and ask ourselves what is still needed to give us a clearer view on the cellular operating power of Prdxs in both stress and non-stress conditions.

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).

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.

Reactive oxygen species, aging and articular cartilage homeostasis.

Chondrocytes are responsible for the maintenance of the articular cartilage. A loss of homeostasis in cartilage contributes to the development of osteoarthritis (OA) when the synthetic capacity of chondrocytes is overwhelmed by processes that promote matrix degradation. There is evidence for an age-related imbalance in reactive oxygen species (ROS) production relative to the anti-oxidant capacity of chondrocytes that plays a role in cartilage degradation as well as chondrocyte cell death. The ROS produced by chondrocytes that have received the most attention include superoxide, hydrogen peroxide, the reactive nitrogen species nitric oxide, and the nitric oxide derived product peroxynitrite. Excess levels of these ROS not only cause oxidative-damage but, perhaps more importantly, cause a disruption in cell signaling pathways that are redox-regulated, including Akt and MAP kinase signaling. Age-related mitochondrial dysfunction and reduced activity of the mitochondrial superoxide dismutase (SOD2) are associated with an increase in mitochondrial-derived ROS and are in part responsible for the increase in chondrocyte ROS with age. Peroxiredoxins (Prxs) are a key family of peroxidases responsible for removal of H2O2, as well as for regulating redox-signaling events. Prxs are inactivated by hyperoxidation. An age-related increase in chondrocyte Prx hyperoxidation and an increase in OA cartilage has been noted. The finding in mice that deletion of SOD2 or the anti-oxidant gene transcriptional regulator nuclear factor-erythroid 2- related factor (Nrf2) result in more severe OA, while overexpression or treatment with mitochondrial targeted anti-oxidants reduces OA, further support a role for excessive ROS in the pathogenesis of OA. Therefore, new therapeutic strategies targeting specific anti-oxidant systems including mitochondrial ROS may be of value in reducing the progression of age-related OA.

H2O2 oxidation of cysteine residues in c-Jun N-terminal kinase 2 (JNK2) contributes to redox regulation in human articular chondrocytes.

Reactive oxygen species (ROS), in particular H2O2, regulate intracellular signaling through reversible oxidation of reactive protein thiols present in a number of kinases and phosphatases. H2O2 has been shown to regulate mitogen-activated protein kinase (MAPK) signaling depending on the cellular context. We report here that in human articular chondrocytes, the MAPK family member c-Jun N-terminal kinase 2 (JNK2) is activated by fibronectin fragments and low physiological levels of H2O2 and inhibited by oxidation due to elevated levels of H2O2 The kinase activity of affinity-purified, phosphorylated JNK2 from cultured chondrocytes was reversibly inhibited by 5-20 µm H2O2 Using dimedone-based chemical probes that react specifically with sulfenylated cysteines (RSOH), we identified Cys-222 in JNK2, a residue not conserved in JNK1 or JNK3, as a redox-reactive site. MS analysis of human recombinant JNK2 also detected further oxidation at Cys-222 and other cysteines to sulfinic (RSO2H) or sulfonic (RSO3H) acid. H2O2 treatment of JNK2 resulted in detectable levels of peptides containing intramolecular disulfides between Cys-222 and either Cys-213 or Cys-177, without evidence of dimer formation. Substitution of Cys-222 to alanine rendered JNK2 insensitive to H2O2 inhibition, unlike C177A and C213A variants. Two other JNK2 variants, C116A and C163A, were also resistant to oxidative inhibition. Cumulatively, these findings indicate differential regulation of JNK2 signaling dependent on H2O2 levels and point to key cysteine residues regulating JNK2 activity. As levels of intracellular H2O2 rise, a switch occurs from activation to inhibition of JNK2 activity, linking JNK2 regulation to the redox status of the cell.

Novel hyperoxidation resistance motifs in 2-Cys peroxiredoxins.

2-Cys peroxiredoxins (Prxs) modulate hydrogen peroxide (H2O2)-mediated cell signaling. At high H2O2 levels, eukaryotic Prxs can be inactivated by hyperoxidation and are classified as sensitive Prxs. In contrast, prokaryotic Prxs are categorized as being resistant to hyperoxidation and lack the GGLG and C-terminal YF motifs present in the sensitive Prxs. Additional molecular determinants that account for the subtle differences in the susceptibility to hyperoxidation remain to be identified. A comparison of a new, 2.15-Å-resolution crystal structure of Prx2 in the oxidized, disulfide-bonded state with the hyperoxidized structure of Prx2 and Prx1 in complex with sulfiredoxin revealed three structural regions that rearrange during catalysis. With these regions in hand, focused sequence analyses were performed comparing sensitive and resistant Prx groups. From this combinatorial approach, we discovered two novel hyperoxidation resistance motifs, motifs A and B, which were validated using mutagenesis of sensitive human Prxs and resistant Salmonella enterica serovar Typhimurium AhpC. Introduction and removal of these motifs, respectively, resulted in drastic changes in the sensitivity to hyperoxidation with Prx1 becoming 100-fold more resistant to hyperoxidation and AhpC becoming 800-fold more sensitive to hyperoxidation. The increased sensitivity of the latter AhpC variant was also confirmed in vivo These results support the function of motifs A and B as primary drivers for tuning the sensitivity of Prxs to different levels of H2O2, thus enabling the initiation of variable signaling or antioxidant responses in cells.