204 research outputs found
Introduction: What we do and do not know regarding redox processes of thiols in signaling pathways
Due to their susceptibility towards redox modification, protein thiols represent primary targets for the modulation of protein activity, conformation and oligomerization. Until fairly recently, such modifications were considered “damage” as a result of oxidative stress, before researchers recognized their physiological importance for biologic signaling. This paradigm shift, and the associated necessity to accurately characterize and quantify the various pathways of thiol redox modifications not only for specific proteins, but also within the cellular environment, has enticed researchers to take a close look at the impact of environment and molecular (protein) structure on these reactions. This Special Issue on Redox Biology of Thiols in Signaling Pathways is the result of a workshop organized at the 2013 Annual Meeting of the Society for Free Radical Biology and Medicine in San Antonio, Texas, summarizing the contributions from many of the presenters. It will provide a stimulating synopsis on what is known, and what is not known, about the reaction mechanisms which underlie the role of thiols and oxidative processes in signaling pathways
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Tuning of Peroxiredoxin Catalysis for Various Physiological Roles
Peroxiredoxins (Prxs) make up an ancient family of enzymes that are the predominant peroxidases for nearly all organisms and play essential roles in reducing hydrogen peroxide, organic hydroperoxides, and peroxynitrite. Even between distantly related organisms, the core protein fold and key catalytic residues related to its cysteine-based catalytic mechanism have been retained. Given that these enzymes appeared early in biology, Prxs have experienced over a billion years of optimization for specific ecological niches. Although their basic enzymatic function remains the same, Prxs have diversified and are involved in roles such as protecting DNA against mutation, defending pathogens against host immune responses, suppressing tumor formation, and—for eukaryotes—helping regulate peroxide signaling via hyperoxidation of their catalytic Cys. Here, we review the current understanding of the physiological roles of Prxs by analyzing knockout and knockdown studies from ca. twenty-five different species. We also review what is known about the structural basis for the sensitivity of some eukaryotic Prxs to inactivation by hyperoxidation. In considering the physiological relevance of hyperoxidation, we explore the distribution across species of sulfiredoxin (Srx), the enzyme responsible for rescuing hyperoxidized Prxs. We unexpectedly find that among eukaryotes appearing to have a “sensitive” Prx isoform, some do not contain Srx. Also, as Prxs are suggested to be promising targets for drug design, we discuss the rationale behind recently proposed strategies for their selective inhibition
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Peroxiredoxins in Parasites
Significance: Parasite survival and virulence relies on effective defenses against reactive oxygen and nitrogen species produced by the host immune system. Peroxiredoxins (Prxs) are ubiquitous enzymes now thought to be central to such defenses and, as such, have potential value as drug targets and vaccine antigens. Recent Advances: Plasmodial and kinetoplastid Prx systems are the most extensively studied, yet remain inadequately understood. For many other parasites our knowledge is even less well developed. Through parasite genome sequencing efforts, however, the key players are being discovered and characterized. Here we describe what is known about the biochemistry, regulation, and cell biology of Prxs in parasitic protozoa, helminths, and fungi. At least one Prx is found in each parasite with a sequenced genome, and a notable theme is the common patterns of expression, localization, and functionality among sequence-similar Prxs in related species. Critical Issues: The nomenclature of Prxs from parasites is in a state of disarray, causing confusion and making comparative inferences difficult. Here we introduce a systematic Prx naming convention that is consistent between organisms and informative about structural and evolutionary relationships. Future Directions: The new nomenclature should stimulate the crossfertilization of ideas among parasitologists and with the broader redox research community. The diverse parasite developmental stages and host environments present complex systems in which to explore the variety of roles played by Prxs, with a view toward parlaying what is learned into novel therapies and vaccines that are urgently needed. Antioxid. Redox Signal. 17, 608-633.Keywords:
Entamoeba histolytica,
Plasmodium falciparum,
Alkyl hydroperoxide reductase,
Falciparum 1 Cys Peroxiredoxin,
Thiol specific antioxidant,
One conserved cysteine,
Protozoan trichomonas vaginalis,
Amebic liver abscess,
Schistosoma mansoni thioredoxin,
Redox sensitive oligomerizatio
Peroxiredoxin Catalysis at Atomic Resolution
Peroxiredoxins (Prxs) are ubiquitous cysteine-based peroxidases that guard cells against oxidative damage, are virulence factors for pathogens, and are involved in eukaryotic redox regulatory pathways. We have analyzed catalytically active crystals to capture atomic resolution snapshots of a PrxQ-subfamily enzyme (from Xanthomonas campestris) proceeding through thiolate, sulfenate, and sulfinate species. These analyses provide structures of unprecedented accuracy for seeding theoretical studies, and show novel conformational intermediates giving insight into the reaction pathway. Based on a highly non-standard geometry seen for the sulfenate intermediate, we infer that the sulfenate formation itself can strongly promote local unfolding of the active site to enhance productive catalysis. Further, these structures reveal that preventing local unfolding, in this case via crystal contacts, results in facile hyperoxidative inactivation even for Prxs normally resistant to such inactivation. This supports previous proposals that conformation-specific inhibitors may be useful for achieving selective inhibition of Prxs that are drug targets
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Evaluating peroxiredoxin sensitivity towards inactivation by peroxide substrates
Peroxiredoxins (Prxs) are very effective peroxide reducing enzymes, but also are susceptible to being
oxidatively inactivated by their own substrates. The level of sensitivity to such hyperoxidation varies
depending both on the enzyme involved and the type of peroxide substrate. For some Prxs, the
hyperoxidation has physiological relevance, so it is important to define approaches that can be used
to quantify sensitivity. Here we describe three distinct approaches that can be used to obtain
quantitative or semiquantitative estimates of Prx sensitivity and define C[subscript hyp1%] as a simple way of quantifying sensitivity so that values can easily be compared.Keywords: mass spectrometry,
cysteine sulfenic acid,
oxidative inactivation,
peroxidase,
cysteine sulfinic acid,
peroxide signaling,
redox signaling,
hyperoxidatio
Extracellular SOD-Derived H2O2 Promotes VEGF Signaling in Caveolae/Lipid Rafts and Post-Ischemic Angiogenesis in Mice
Reactive oxygen species (ROS), in particular, H2O2, is essential for full activation of VEGF receptor2 (VEGFR2) signaling involved in endothelial cell (EC) proliferation and migration. Extracellular superoxide dismutase (ecSOD) is a major secreted extracellular enzyme that catalyzes the dismutation of superoxide to H2O2, and anchors to EC surface through heparin-binding domain (HBD). Mice lacking ecSOD show impaired postnatal angiogenesis. However, it is unknown whether ecSOD-derived H2O2 regulates VEGF signaling. Here we show that gene transfer of ecSOD, but not ecSOD lacking HBD (ecSOD-ΔHBD), increases H2O2 levels in adductor muscle of mice, and promotes angiogenesis after hindlimb ischemia. Mice lacking ecSOD show reduction of H2O2 in non-ischemic and ischemic limbs. In vitro, overexpression of ecSOD, but not ecSOD-ΔHBD, in cultured medium in ECs enhances VEGF-induced tyrosine phosphorylation of VEGFR2 (VEGFR2-pY), which is prevented by short-term pretreatment with catalase that scavenges extracellular H2O2. Either exogenous H2O2 (<500 µM), which is diffusible, or nitric oxide donor has no effect on VEGF-induced VEGFR2-pY. These suggest that ecSOD binding to ECs via HBD is required for localized generation of extracellular H2O2 to regulate VEGFR2-pY. Mechanistically, VEGF-induced VEGFR2-pY in caveolae/lipid rafts, but non-lipid rafts, is enhanced by ecSOD, which localizes at lipid rafts via HBD. One of the targets of ROS is protein tyrosine phosphatases (PTPs). ecSOD induces oxidation and inactivation of both PTP1B and DEP1, which negatively regulates VEGFR2-pY, in caveolae/lipid rafts, but not non-lipid rafts. Disruption of caveolae/lipid rafts, or PTPs inhibitor orthovanadate, or siRNAs for PTP1B and DEP1 enhances VEGF-induced VEGFR2-pY, which prevents ecSOD-induced effect. Functionally, ecSOD promotes VEGF-stimulated EC migration and proliferation. In summary, extracellular H2O2 generated by ecSOD localized at caveolae/lipid rafts via HBD promotes VEGFR2 signaling via oxidative inactivation of PTPs in these microdomains. Thus, ecSOD is a potential therapeutic target for angiogenesis-dependent cardiovascular diseases
Crystal structure of alkyl hydroperoxidase D like protein PA0269 from Pseudomonas aeruginosa: Homology of the AhpD-like structural family
<p>Abstract</p> <p>Background</p> <p>Alkyl hydroperoxidase activity provides an important antioxidant defense for bacterial cells. The catalytic mechanism requires two peroxidases, AhpC and AhpD, where AhpD plays the role of an essential adaptor protein.</p> <p>Results</p> <p>The crystal structure of a putative AhpD from <it>Pseudomonas aeruginosa </it>has been determined at 1.9 Å. The protein has an all-helical fold with a chain topology similar to a known AhpD from <it>Mycobacterium tuberculosis </it>despite a low overall sequence identity of 9%. A conserved two α-helical motif responsible for function is present in both. However, in the <it>P. aeruginosa </it>protein, helices H3, H4 of this motif are located at the N-terminal part of the chain, while in <it>M. tuberculosis </it>AhpD, the corresponding helices H8, H9 are situated at the C-terminus. Residues 24-62 of the putative catalytic region of <it>P. aeruginosa </it>have a higher sequence identity of 33% where the functional activity is supplied by a proton relay system of five residues, Glu36, Cys48, Tyr50, Cys51, and His55, and one structural water molecule. A comparison of five other related hypothetical proteins from various species, assigned to the alkyl hydroperoxidase D-like protein family, shows they contain the same conserved structural motif and catalytic sequence Cys-X-X-Cys. We have shown that AhpD from <it>P. aeruginosa </it>exhibits a weak ability to reduce H<sub>2</sub>O<sub>2 </sub>as tested using a ferrous oxidation-xylenol orange (FOX) assay, and this activity is blocked by thiol alkylating reagents.</p> <p>Conclusion</p> <p>Thus, this hypothetical protein was assigned to the AhpD-like protein family with peroxidase-related activity. The functional relationship of specific oligomeric structures of AhpD-like structural family is discussed.</p
Oxidation state governs structural transitions in peroxiredoxin II that correlate with cell cycle arrest and recovery
Inactivation of eukaryotic 2-Cys peroxiredoxins (Prxs) by hyperoxidation has been proposed to promote accumulation of hydrogen peroxide (H2O2) for redox-dependent signaling events. We examined the oxidation and oligomeric states of PrxI and -II in epithelial cells during mitogenic signaling and in response to fluxes of H2O2. During normal mitogenic signaling, hyperoxidation of PrxI and -II was not detected. In contrast, H2O2-dependent cell cycle arrest was correlated with hyperoxidation of PrxII, which resulted in quantitative recruitment of ∼66- and ∼140-kD PrxII complexes into large filamentous oligomers. Expression of cyclin D1 and cell proliferation did not resume until PrxII-SO2H was reduced and native PrxII complexes were regenerated. Ectopic expression of PrxI or -II increased Prx-SO2H levels in response to oxidant exposure and failed to protect cells from arrest. We propose a model in which Prxs function as peroxide dosimeters in subcellular processes that involve redox cycling, with hyperoxidation controlling structural transitions that alert cells of perturbations in peroxide homeostasis
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Peroxiredoxins: Guardians Against Oxidative Stress and Modulators of Peroxide Signaling
Peroxiredoxins (Prxs) are a ubiquitous family of cysteine-dependent peroxidase enzymes that play dominant roles in regulating peroxide levels within cells. These enzymes, often present at high levels and capable of rapidly clearing peroxides, display a remarkable array of variations in their oligomeric states and susceptibility to regulation by hyperoxidative inactivation and other post-translational modifications. Key conserved residues within the active site promote catalysis by stabilizing the transition state required for transferring the terminal oxygen of hydroperoxides to the active site (peroxidatic) cysteine residue. Extensive investigations continue to expand our understanding of the scope of their importance as well as the structures and forces at play within these critical defense and regulatory enzymes.Keywords: redox signaling, peroxidase, antioxidant enzyme, antioxidant defens
Oxidative Stress Promotes Peroxiredoxin Hyperoxidation and Attenuates Pro-survival Signaling in Aging Chondrocytes
Oxidative stress-mediated post-translational modifications of redox-sensitive proteins are postulated as a key mechanism underlying age-related cellular dysfunction and disease progression. Peroxiredoxins (PRX) are critical intracellular antioxidants that also regulate redox signaling events. Age-related osteoarthritis is a common form of arthritis that has been associated with mitochondrial dysfunction and oxidative stress. The objective of this study was to determine the effect of aging and oxidative stress on chondrocyte intracellular signaling, with a specific focus on oxidation of cytosolic PRX2 and mitochondrial PRX3. Menadione was used as a model to induce cellular oxidative stress. Compared with chondrocytes isolated from young adult humans, chondrocytes from older adults exhibited higher levels of PRX1–3 hyperoxidation basally and under conditions of oxidative stress. Peroxiredoxin hyperoxidation was associated with inhibition of pro-survival Akt signaling and stimulation of pro-death p38 signaling. These changes were prevented in cultured human chondrocytes by adenoviral expression of catalase targeted to the mitochondria (MCAT) and in cartilage explants from MCAT transgenic mice. Peroxiredoxin hyperoxidation was observed in situ in human cartilage sections from older adults and in osteoarthritic cartilage. MCAT transgenic mice exhibited less age-related osteoarthritis. These findings demonstrate that age-related oxidative stress can disrupt normal physiological signaling and contribute to osteoarthritis and suggest peroxiredoxin hyperoxidation as a potential mechanism
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