Chronic obstructive pulmonary disease (COPD) includes chronic bronchitis and emphysema. Environmental exposure, primarily cigarette smoking, can cause high oxidative stress and is the main factor of COPD development. Cigarette smoke also contributes to the imbalance of oxidant/antioxidant due to exogenous reactive oxygen species (ROS). Moreover, endogenously released ROS during the inflammatory process and mitochondrial dysfunction may contribute to this disease progression. ROS and reactive nitrogen species (RNS) can oxidize different biomolecules such as DNA, proteins, and lipids leading to epithelial cell injury and death. Various detoxifying enzymes and antioxidant defense systems can be involved in ROS removal. In this review, we summarize the main findings regarding the biological role of ROS, which may contribute to COPD development, and cytoprotective mechanisms against this disease progression.

DJ-1 is a multifunctional protein with cytoprotective functions. It is localized in the cytoplasm, nucleus, and mitochondria. The conserved cysteine residue at position 106 (Cys106) within DJ-1 serves as a sensor of redox state and can be oxidized to both the sulfinate (-SO 2 − ) and sulfonate (-SO 3 − ) forms. DJ-1 with Cys106-SO 2 − has cytoprotective activity but high levels of reactive oxygen species can induce its overoxidation to Cys106-SO 3 − . We found increased oxidative stress in alveolar type II (ATII) cells isolated from emphysema patients as determined by 4-HNE expression. DJ-1 with Cys106-SO 3 − was detected in these cells by mass spectrometry analysis. Moreover, ubiquitination of Cys106-SO 3 − DJ-1 was identified, which suggests that this oxidized isoform is targeted for proteasomal destruction. Furthermore, we performed controlled oxidation using H 2 O 2 in A549 cells with DJ-1 knockout generated using CRISPR-Cas9 strategy. Lack of DJ-1 sensitized cells to apoptosis induced by H 2 O 2 as detected using Annexin V and propidium iodide by flow cytometry analysis. This treatment also decreased both mitochondrial DNA amount and mitochondrial ND1 (NADH dehydrogenase 1, subunit 1) gene expression, as well as increased mitochondrial DNA damage. Consistent with the decreased cytoprotective function of overoxidized DJ-1, recombinant Cys106-SO 3 − DJ-1 exhibited a loss of its thermal unfolding transition, mild diminution of secondary structure in CD spectroscopy, and an increase in picosecond–nanosecond timescale dynamics as determined using NMR. Altogether, our data indicate that very high oxidative stress in ATII cells in emphysema patients induces DJ-1 overoxidation to the Cys106-SO 3 − form, leading to increased protein flexibility and loss of its cytoprotective function, which may contribute to this disease pathogenesis.

Peroxiredoxins from the Prx1 subfamily (Prx) are moonlighting peroxidases that operate in peroxide signaling and are regulated by sulfinylation. Prxs offer a major model of protein–thiol oxidative modification. They react with H 2 O 2 to form a sulfenic acid intermediate that either engages into a disulfide bond, committing the enzyme into its peroxidase cycle, or again reacts with peroxide to produce a sulfinic acid that inactivates the enzyme. Sensitivity to sulfinylation depends on the kinetics of these two competing reactions and is critically influenced by a structural transition from a fully folded (FF) to locally unfolded (LU) conformation. Analysis of the reaction of the Tsa1 Saccharomyces cerevisiae Prx with H 2 O 2 by Trp fluorescence-based rapid kinetics revealed a process linked to the FF/LU transition that is kinetically distinct from disulfide formation and suggested that sulfenate formation facilitates local unfolding. Use of mutants of distinctive sensitivities and of different peroxide substrates showed that sulfinylation sensitivity is not coupled to the resolving step kinetics but depends only on the sulfenic acid oxidation and FF-to-LU transition rate constants. In addition, stabilization of the active site FF conformation, the determinant of sulfinylation kinetics, is only moderately influenced by the Prx C-terminal tail dynamics that determine the FF → LU kinetics. From these two parameters, the relative sensitivities of Prxs toward hyperoxidation with different substrates can be predicted, as confirmed by in vitro and in vivo patterns of sulfinylation.

Edited by Barry HalliwellKeywords: Sulfiredoxin Rate-limiting step Peroxiredoxin ATP Sulfinic acid Phosphoryl anhydride intermediate a b s t r a c tThe eukaryotic sulfiredoxin (Srx) catalyzes the reduction of overoxidized typical 2-Cys peroxiredoxins PrxSO 2 via ATP/Mg 2+ -dependent phosphorylation of the sulfinic acid group, followed by formation of a PrxSO-SSrx thiolsulfinate intermediate. Using real-time kinetics of wild-type and C84A Srxs, and pH-rate profiles with ATP/Mg 2+ analogues, we show that the rate-limiting step of the reaction is associated with the chemical process of transfer of the c-phosphate of ATP to the sulfinic acid, in contrast to that described by Jönsson et al. [7]. Two pK apps of 6.2 and 7.5 were extracted from the bell-shaped pH-rate profile, corresponding to the c-phosphate of ATP, and to an acid-base catalyst, respectively.

Aims: Typical 2-Cys peroxiredoxins (2-Cys Prxs) are Cys peroxidases that undergo inactivation by hyperoxidation of the catalytic Cys, a modification reversed by ATP-dependent reduction by sulfiredoxin (Srx). Such an attribute is thought to provide regulation of 2-Cys Prxs functions. The initial steps of the Srx catalytic mechanism lead to a Prx/Srx thiolsulfinate intermediate that must be reduced to regenerate Srx. In Saccharomyces cerevisiae Srx, the thiolsulfinate is resolved by an extra Cys (Cys48) that is absent in mammalian, plant, and cyanobacteria Srxs (1-Cys Srxs). We have addressed the mechanism of reduction of 1-Cys Srxs using S. cerevisiae Srx mutants lacking Cys48 as a model. Results: We have tested the recycling of Srx by glutathione (GSH) by a combination of in vitro steady-state and single-turnover kinetic analyses, using enzymatic coupled assays, Prx fluorescence, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and reverse-phase chromatography coupled to mass spectrometry. We demonstrate that GSH reacts directly with the thiolsulfinate intermediate, by following saturation kinetics with an apparent dissociation constant of 34 lM, while producing S-glutathionylated Srx as a catalytic intermediate which is efficiently reduced by the glutaredoxin/glutathione reductase system. Total cellular depletion of GSH impacted the recycling of Srx, confirming in vivo that GSH is the physiologic reducer of 1-Cys Srx. Innovation: Our study suggests that GSH binds to the thiolsulfinate complex, thus allowing non-rate limiting reduction. Such a structural recognition of GSH enables an efficient catalytic reduction, even at very low GSH cellular levels. Conclusion: This study provides both in vitro and in vivo evidence of the role of GSH as the primary reducer of 1-Cys Srxs. Antioxid. Redox Signal. 22,[731][732][733][734][735][736][737][738][739][740][741][742][743]