Mark B. Hampton

Thiol chemistry and specificity in redox signaling

Exposure of cells to sublethal oxidative stress results in the modulation of various signaling pathways. Oxidants can activate and inactivate transcription factors, membrane channels, and metabolic enzymes, and regulate calcium-dependent and phosphorylation signaling pathways. Oxidation and reduction of thiol proteins are thought to be the major mechanisms by which reactive oxidants integrate into cellular signal transduction pathways. This review focuses on mechanisms for sensing and transmitting redox signals, from the perspective of their chemical reactivity with specific oxidants. We discuss substrate preferences for different oxidants and how the kinetics of these reactions determines how each oxidant will react in a cell. This kinetic approach helps to identify initial oxidant-sensitive targets and elucidate mechanisms involved in transmission of redox signals. It indicates that only those proteins with very high reactivity, such as peroxiredoxins, are likely to be direct targets for hydrogen peroxide. Other more modestly reactive thiol proteins such as protein tyrosine phosphatases are more likely to become oxidized by an indirect mechanism. The review also examines oxidative changes observed during receptor-mediated signaling, the strengths and limitations of detection methods for reactive oxidant production, and the evidence for hydrogen peroxide acting as the second messenger. We discuss areas where observations in cell systems can be rationalized with the reactivity of specific oxidants and where further work is needed to understand the mechanisms involved.

Dual regulation of caspase activity by hydrogen peroxide: implications for apoptosis

The induction of apoptosis in Jurkat T‐lymphocytes with 50 μM hydrogen peroxide was associated with caspase activation. Caspase activity was first detected 3 h after treatment, and the morphological features of apoptosis were apparent by 6 h. At higher concentrations of hydrogen peroxide there was no detectable caspase activity, and the cells died by necrosis. Cells treated with hydrogen peroxide were impaired in their ability to undergo Fas‐mediated apoptosis. This appeared to be the result of direct inhibition of the cysteine‐dependent caspases. The cells were able to recover and undergo apoptosis at later times. Therefore, hydrogen peroxide has two distinct effects. It initially inhibits the caspases and delays apoptosis. Then, depending on the degree of the initial oxidative stress, the caspases are activated and the cells die by apoptosis, or they remain inactive and necrosis occurs. We discuss the physiological implications of cells having to maintain a reducing environment during apoptosis to allow the caspases to function.

Mitochondrial peroxiredoxin involvement in antioxidant defence and redox signalling.

Prxs (peroxiredoxins) are a family of proteins that are extremely effective at scavenging peroxides. The Prxs exhibit a number of intriguing properties that distinguish them from conventional antioxidants, including a susceptibility to inactivation by hyperoxidation in the presence of excess peroxide and the ability to form complex oligomeric structures. These properties, combined with a high cellular abundance and reactivity with hydrogen peroxide, have led to speculation that the Prxs function as redox sensors that transmit signals as part of the cellular response to oxidative stress. Multicellular organisms express several different Prxs that can be categorized by their subcellular distribution. In mammals, Prx 3 and Prx 5 are targeted to the mitochondrial matrix. Mitochondria are a major source of hydrogen peroxide, and this oxidant is implicated in the damage associated with aging and a number of pathologies. Hydrogen peroxide can also act as a second messenger, and is linked with signalling events in mitochondria, including the induction of apoptosis. A simple kinetic competition analysis estimates that Prx 3 will be the target for up to 90% of hydrogen peroxide generated in the matrix. Therefore, mitochondrial Prxs have the potential to play a major role in mitochondrial redox signalling, but the extent of this role and the mechanisms involved are currently unclear.

The High Reactivity of Peroxiredoxin 2 with H2O2 Is Not Reflected in Its Reaction with Other Oxidants and Thiol Reagents

Peroxiredoxin 2 is a member of the mammalian peroxiredoxin family of thiol proteins that is important in antioxidant defense and redox signaling. We have examined its reactivity with various biological oxidants, in order to assess its ability to act as a direct physiological target for these species. Human erythrocyte peroxiredoxin 2 was oxidized stoichiometrically to its disulfide-bonded homodimer by hydrogen peroxide, as monitored electrophoretically under nonreducing conditions. The protein was highly susceptible to oxidation by adventitious peroxide, which could be prevented by treating buffers with low concentrations of catalase. However, this did not protect peroxiredoxin 2 against oxidation by added H2O2. Experiments measuring inhibition of dimerization indicated that at pH 7.4 catalase and peroxiredoxin 2 react with hydrogen peroxide at comparable rates. A rate constant of 1.3 × 107 m-1 s-1 for the peroxiredoxin reaction was obtained from competition kinetic studies with horseradish peroxidase. This is 100-fold faster than is generally assumed. It is sufficiently high for peroxiredoxin to be a favored cellular target for hydrogen peroxide, even in competition with catalase or glutathione peroxidase. Reactions of t-butyl and cumene hydroperoxides with peroxiredoxin were also fast, but amino acid chloramines reacted much more slowly. This contrasts with other thiol compounds that react many times faster with chloramines than with hydrogen peroxide. The alkylating agent iodoacetamide also reacted extremely slowly with peroxiredoxin 2. These results demonstrate that peroxiredoxin 2 has a tertiary structure that facilitates reaction of the active site thiol with hydrogen peroxide while restricting its reactivity with other thiol reagents.

Redox Regulation of the Caspases during Apoptosisa

ABSTRACT: Apoptosis is now widely recognized as being a distinct process of importance both in normal physiology and pathology. In the current paradigm for apoptotic cell death, the activity of a family of proteases, caspases, related to interleukin‐1β‐converting enzyme (ICE) orchestrates the multiple downstream events, such as cell shrinkage, membrane blebbing, glutathione (GSH) efflux, and chromatin degradation that constitute apoptosis. Recent studies suggest that mitochondria could be the principle sensor and that the release of mitochondrial factors, such as cytochrome c, is the critical event governing the fate of the cell. One of the most reproducible inducers of apoptosis is mild oxidative stress, although it is unclear how an oxidative stimulus can activate the caspase cascade. Oxidative modification of proteins and lipids has also been observed in cells undergoing apoptosis in response to nonoxidative stimuli, suggesting that intracellular oxidation may be a general feature of the effector phase of apoptosis. The caspases themselves are cysteine‐dependent enzymes and, as such, appear to be redox sensitive. Indeed, our recent work on hydrogen peroxide‐mediated apoptosis suggests that prolonged or excessive oxidative stress can actually prevent caspase activation. A physiological example of this is the NADPH oxidase‐derived oxidants generated by stimulated neutrophils that prevent caspase activation in these cells. Pursuant to these findings, stimulated neutrophils appear to use a specialized caspase‐independent pathway to initiate phosphatidylserine (PS) exposure and subsequent phagocytic clearance. The possible implications of these dual roles for reactive oxygen species in apoptosis, that is, induction and inhibition of caspases, are discussed in the present review.

Cytochrome c release and caspase activation in hydrogen peroxide‐ and tributyltin‐induced apoptosis

The ability of H2O2 and tributyltin (TBT) to trigger pro‐caspase activation via export of cytochrome c from mitochondria to the cytoplasm was investigated. Treatment of Jurkat T lymphocytes with H2O2 resulted in the appearance of cytochrome c in the cytosol within 2 h. This was at least 1 h before caspase activation was observed. TBT caused cytochrome c release already after 5 min, followed by caspase activation within 1 h. Measurement of mitochondrial membrane potential (ΔΨm) showed that both H2O2 and TBT dissipated ΔΨm, but with different time courses. TBT caused a concomitant loss of ΔΨm and release of cytochrome c, whereas cytochrome c release and caspase activation preceded any apparent ΔΨm loss in H2O2‐treated cells. Thus, our results suggest that different mechanisms are involved in triggering cytochrome c release with these apoptosis‐inducing agents.

Requirements for NADPH oxidase and myeloperoxidase in neutrophil extracellular trap formation differ depending on the stimulus

Release of NETs by neutrophils is linked with immune protection and host damage. A variety of stimuli promotes NET formation. However, findings from different laboratories often vary, and it is possible that more than one mechanism of NET formation exists. NET formation induced by PMA has been shown to require NADPH oxidase activity, and there is evidence that the granule enzyme MPO is also involved. However, requirements for NADPH oxidase or MPO with other stimuli are less well established. We investigated the role of oxidants in NET formation by human neutrophils induced with PMA, several bacterial genera, and the calcium ionophore ionomycin. With the use of inhibitors of the NADPH oxidase and MPO, oxidant scavengers, and cells from a MPO‐deficient individual, we observed that requirements for oxidant generation depend on the stimulus. NADPH oxidase activity was required with PMA and bacterial stimulation but not with ionomycin. Whereas MPO was required for efficient NET formation with PMA, incubation with bacteria induced NETs independently of MPO activity. Although the specific mechanisms whereby oxidants participate in NET formation remain to be clarified, it is possible that other stimuli that mobilize calcium act like ionomycin via an oxidant‐independent mechanism, and it cannot be inferred from results with PMA that MPO is required with more physiological stimuli.

Chlorination of bacterial and neutrophil proteins during phagocytosis and killing of Staphylococcus aureus.

Myeloperoxidase is proposed to play a central role in bacterial killing by generating hypochlorous acid within neutrophil phagosomes. However, it has yet to be demonstrated that these inflammatory cells target hypochlorous acid against bacteria inside phagosomes. In this investigation, we treatedStaphylococcus aureus with varying concentrations of reagent hypochlorous acid and found that even at sublethal doses, it converted some tyrosine residues in their proteins to 3-chlorotyrosine and 3,5-dichlorotyrosine. To determine whether or not ingested bacteria were exposed to hypochlorous acid in neutrophil phagosomes, we labeled their proteins with [13C6]tyrosine and used gas chromatography with mass spectrometry to identify the corresponding chlorinated isotopes after the bacteria had been phagocytosed. Chlorinated tyrosines were detected in bacterial proteins 5 min after phagocytosis and reached levels of approximately 2.5/1000 mol of tyrosine at 60 min. Inhibitor studies revealed that chlorination was dependent on myeloperoxidase. Chlorinated neutrophil proteins were also detected and accounted for 94% of total chlorinated tyrosine residues formed during phagocytosis. We conclude that hypochlorous acid is a major intracellular product of the respiratory burst. Although some reacts with the bacteria, most reacts with neutrophil components.

Proteomic detection of hydrogen peroxide-sensitive thiol proteins in Jurkat cells

Thiol proteins are important in cellular antioxidant defenses and redox signalling. It is postulated that reactive oxidants cause selective thiol oxidation, but relative sensitivities of different cell proteins and critical targets are not well characterized. We exposed Jurkat cells to H2O2 for 10 min and measured changes in reversibly oxidized proteins by labelling with iodoacetamidofluorescein and two-dimensional electrophoresis. At 200 microM H2O2, which caused activation of the MAP (mitogen-activated protein) kinase ERK (extracellular-signal-regulated kinase), growth arrest and apoptosis, relatively few changes were seen. A total of 28 spots were reversibly oxidized (increased labelling intensity) and 24 decreased. The latter included isoforms of peroxiredoxins 1 and 2, which were irreversibly oxidized. Oxidation of GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was striking, and other affected proteins included glutathione S-transferase P1-1, enolase, a regulatory subunit of protein kinase A, annexin VI, the mitotic checkpoint serine/threonine-protein kinase BUB1beta, HSP90beta (heat-shock protein 90beta) and proteosome components. At 20 microM H2O2, changes were fewer, but GAPDH and peroxiredoxin 2 were still modified. Dinitrochlorobenzene treatment, which inhibited cellular thioredoxin reductase and partially depleted GSH, caused reversible oxidation of several proteins, including thioredoxin 1 and peroxiredoxins 1 and 2. Most changes were distinct from those with H2O2, and changes with H2O2 were scarcely enhanced by dinitrochlorobenzene. Relatively few proteins, including deoxycytidine kinase, nucleoside diphosphate kinase and a proteosome activator subunit, responded only to the combined treatment. Thus most of the effects of H2O2 were not linked to thioredoxin oxidation. Our study has identified peroxiredoxin 2 and GAPDH as two of the most oxidant-sensitive cell proteins and has highlighted how readily peroxiredoxins undergo irreversible oxidation.

Detection of oxidant sensitive thiol proteins by fluorescence labeling and two‐dimensional electrophoresis

Oxidants can activate signaling pathways and modulate a variety of cellular activities. Their action at a molecular level involves the post‐translational modification of protein thiols. We have developed a proteomic method to monitor the reduction and oxidation of protein thiols, and identify those thiol proteins most sensitive to oxidation. Cells were disrupted in the presence of N‐ethylmaleimide to block the reduced thiol proteins and dithiothreitol was added to reduce the oxidized thiol proteins before labeling with 5‐iodoacetamidofluorescein. Two‐dimensional (2‐D) electrophoresis was used to resolve the labeled samples. We applied the method to Jurkat T lymphocytes and examined the effect of diamide on the oxidized and reduced thiol protein profiles. A small percentage of protein thiols were already oxidized in untreated cells. Exposure of cells to 2 mM diamide for ten minutes led to a dramatic increase in thiol protein oxidation as seen in the oxidized thiol protein map. However, it was difficult to detect any change in the pattern of reduced thiol proteins. Separation of proteins by 2‐D electrophoresis revealed approximately 200 thiol proteins that were oxidized by diamide treatment. This method will be valuable in elucidating redox signaling pathways.