Hypochlorous acid reversibly inhibits caspase-3: a potential regulator of apoptosis.

Caspase-3 is involved in apoptosis. Here, we examine whether hypochlorous acid (HOCl), a final product of myeloperoxidase (MPO), is a modulator of caspase-3 at relatively low concentrations and also its application on metaphase II mouse oocytes. We utilised caspase-3 activity assay, TUNEL assay, the CellEvent caspase 3/7 fluorescent assay, and the MPO/hydrogen peroxide (H2O2) system on mouse oocytes with and without cumulus cells to examine whether low concentrations of HOCl mediate apoptosis by inhibition of caspase-3. A UV-visible spectrophotometer was used to study caspase-3 activity. To determine whether HOCl mediates apoptosis in mouse oocytes, two different concentrations (10 and 100 µM) of HOCl generated by the MPO/H2O2 system were used as treatments (10 µM had little effect on oocyte quality, while 100 µM showed significant deterioration). Induction of apoptotic cell death was determined by TUNEL Assay and the CellEvent caspase 3/7. HOCl mediates caspase-3 inactivation in a dose dependent manner. Subsequent addition of dithiothreitol caused recovery of caspase-3 activity indicating involvement of the oxidation of the Cys-thiol group. Accumulation of HOCl generated by MPO in the presence of caspase-3 also inhibits MPO but requires higher HOCl concentrations, indicating specificity of lower HOCl concentrations to inhibition of caspase-3. Exposure of oocytes to lower HOCl concentrations generated by MPO-H2O2 system prevents MPO-mediated apoptosis whereas exposure to higher HOCl (100 µM) showed apoptosis. Similar results were observed by using the CellEvent caspase 3/7 assay. Low concentrations of HOCl inhibit caspase-3 activity, and may play a role in regulating apoptosis, thus affecting oocyte quality.HighlightsCaspase-3 is involved in apoptosis pathway and loss of this regulation is seen in several diseases.These conditions are associated with inflammation and higher myeloperoxidase (MPO) activity.We examined whether hypochlorous acid (HOCl), generated by MPO, is a modulator of caspase-3.Caspase-3 activity showed a dose dependent decrease with HOCl and this reaction was reversible.HOCl modulates caspase-3 activity and may play a physiological role in regulating apoptosis.

Potent antioxidative activity of lycopene: A potential role in scavenging hypochlorous acid.

Lycopene, a carotenoid found in tomatoes, is a proven antioxidant that may lower the risk of certain disorders including heart disease and cancer. Hypochlorous acid (HOCl) is an oxidant linked to tissue oxidation in cardiovascular disease and other inflammatory disorders through its ability to modify proteins, deoxyribonucleic acid, ribonucleic acid, and lipids. Here we show that lycopene can function as a potent scavenger of HOCl at a wide range of concentrations that span various pathophysiological and supplemental ranges. The oxidation of lycopene by HOCl was accompanied by a marked change in color, from red to colorless, of the lycopene solution, suggesting lycopene degradation. HPLC and LC-MS analysis showed that the exposure of lycopene to increasing concentrations of HOCl gave a range of metabolites resulting from oxidative cleavage of one or more C=C. The degree of degradation of lycopene (as assessed by the number and chain lengths of the various oxidative metabolites of lycopene) depends mainly on the ratio of HOCl to lycopene, suggesting that multiple molecules of HOCl are consumed per molecule of lycopene. Collectively, this work demonstrates a direct link between lycopene and HOCl scavenging and may assist in elucidating the mechanism of the protective function exerted by lycopene.

Analysis of the mechanism by which tryptophan analogs inhibit human myeloperoxidase.

Myeloperoxidase (MPO) catalyzes the formation of oxidants that have been implicated in the pathogenesis of various diseases, including cardiovascular and pulmonary diseases and cancer. Inhibition of MPO oxidant-generating activity represents an attractive target for preventing the progression of inflammatory conditions. Peroxidation and chlorination catalytic activity were utilized to screen for the most effective tryptophan analog that inhibits MPO. Rapid kinetic measurements were performed to determine the mechanisms through which these compounds inhibit the catalytic activity of MPO. Substituents on the amino and carboxyl termini of tryptophan enhance its affinity toward MPO compared to a substituent on the indole ring. Hydrogen-bond donor capabilities and a positive charge on the amino group are not required for MPO inhibition. Hydroxyl-containing indoles did not inhibit MPO H(2)O(2)-consumption activity. Elimination of the negative charge from the carboxyl terminus by introducing a hydrophobic character significantly enhanced tryptophan analog affinity for MPO and improved its inhibitory properties. Further mechanistic studies indicated that indole compounds inhibited MPO activity through the accumulation of compound II, an inactive MPO intermediate. Our results show that specific structural features of tryptophan analogs are involved in increasing the affinity for MPO and providing a significantly greater inhibition of MPO's catalytic activities.

Myeloperoxidase interaction with peroxynitrite: chloride deficiency and heme depletion.

Myeloperoxidase (MPO) is a hemoprotein involved in the leukocyte-mediated defense mechanism and uses hydrogen peroxide (H2O2) and chloride (Cl(-)) to produce hypochlorous acid. In human saliva and in hypochloremic alkalosis syndrome occurring in breast-fed infants, the MPO-H2O2 system functions in a lower Cl(-) concentration (10-70 mM) compared to plasma levels (100 mM) as part of the antibacterial defense system. The impact of low Cl(-) concentration and exposure to high peroxynitrite (ONOO(-)) synthesized from cigarette smoke or oxidative stress on MPO function is still unexplored. Rapid mixing of ONOO(-) and MPO caused immediate formation of a transient intermediate MPO Compound II, which then decayed to MPO-Fe(III). Double mixing of MPO with ONOO(-) followed by H2O2 caused immediate formation of Compound II, followed by MPO heme depletion, a process that occurred independent of ONOO(-) concentration. Peroxynitrite/H2O2-mediated MPO heme depletion was confirmed by HPLC analysis, and in-gel heme staining showing 60-70% less heme content compared to the control. A nonreducing denaturing SDS-PAGE showed no fragmentation or degradation of protein. Myeloperoxidase heme loss was completely prevented by preincubation of MPO with saturating amounts of Cl(-). Chloride binding to the active site of MPO constrains ONOO(-) binding by filling the space directly above the heme moiety or by causing a protein conformational change that constricts the distal heme pocket, thus preventing ONOO(-) from binding to MPO heme iron. Peroxynitrite interaction with MPO may serve as a novel mechanism for modulating MPO catalytic activity, influencing the regulation of local inflammatory and infectious events in vivo.

Mutants in DsbB that appear to redirect oxidation through the disulfide isomerization pathway.

Disulfide bond formation occurs in secreted proteins in Escherichia coli when the disulfide oxidoreductase DsbA, a soluble periplasmic protein, nonspecifically transfers a disulfide to a substrate protein. The catalytic disulfide of DsbA is regenerated by the inner-membrane protein DsbB. To help identify the specificity determinants in DsbB and to understand the nature of the kinetic barrier preventing direct oxidation of newly secreted proteins by DsbB, we imposed selective pressure to find novel mutations in DsbB that would function to bypass the need for the disulfide carrier DsbA. We found a series of mutations localized to a short horizontal alpha-helix anchored near the outer surface of the inner membrane of DsbB that eliminated the need for DsbA. These mutations changed hydrophobic residues into nonhydrophobic residues. We hypothesize that these mutations may act by decreasing the affinity of this alpha-helix to the membrane. The DsbB mutants were dependent on the disulfide oxidoreductase DsbC, a soluble periplasmic thiol-disulfide isomerase, for complementation. DsbB is not normally able to oxidize DsbC, possibly due to a steric clash that occurs between DsbC and the membrane adjacent to DsbB. DsbC must be in the reduced form to function as an isomerase. In contrast, DsbA must remain oxidized to function as an oxidizing thiol-disulfide oxidoreductase. The lack of interaction that normally exists between DsbB and DsbC appears to provide a means to separate the DsbA-DsbB oxidation pathway and the DsbC-DsbD isomerization pathway. Our mutants in DsbB may act by redirecting oxidant flow to take place through the isomerization pathway.

Identification of redox sensitive thiols of protein disulfide isomerase using isotope coded affinity technology and mass spectrometry.

Regulation of the redox state of protein disulfide isomerase (PDI) is critical for its various catalytic functions. Here we describe a procedure utilizing isotope-coded affinity tag (ICAT) technology and mass spectrometry that quantitates relative changes in the dynamic thiol and disulfide states of human PDI. Human PDI contains six cysteine residues, four present in two active sites within the a and a' domains, and two present in the b' domain. ICAT labeling of human PDI indicates a difference between the redox state of the two active sites. Furthermore, under auto-oxidation conditions an approximately 80% decrease in available thiols within the a domain was detected. Surprisingly, the redox state of one of the two cysteines, Cys-295, within the b' domain was altered between the fully reduced and the auto-oxidized state of PDI while the other b' domain cysteine remained fully reduced. An interesting mono- and dioxidation modification of an invariable tryptophan residue, Trp-35, within the active site was also mapped by tandem mass spectrometry. Our findings indicate that ICAT methodology in conjunction with mass spectrometry represents a powerful tool to monitor changes in the redox state of individual cysteine residues within PDI under various conditions.

Antioxidant and antiplatelet effects of rosuvastatin in a hamster model of prediabetes.

The objectives of this study were to determine the relationships among Type II diabetes (T2DM)-dependent elevations in platelet-derived reactive oxygen species (ROS), platelet-surface protein disulfide isomerase (psPDI) NO-releasing activity, and platelet aggregation and to evaluate the efficacy of rosuvastatin in normalizing these parameters in primary cells derived from a hamster model of prediabetic insulin resistance induced by fructose feeding. Platelets from rosuvastatin-treated non-fructose-fed (NFF) and fructose-fed (FF) hamsters were analyzed for aggregability and psPDI-denitrosation activity. Platelets from NFF animals treated with xanthine/xanthine oxidase (X/XO) were assessed for the same parameters and primary aortic endothelial cells (AEC) cultivated with a range of [rosuvastatin] +/- mevalonate were analyzed for ROS production. Platelets from FF hamsters displayed statistically significant enhanced ROS production, diminished psPDI-mediated NO-releasing activity, and hyperaggregability. Suggestively, platelets from NFF animals treated with X/XO displayed characteristics similar to platelets from FF animals. Rosuvastatin elicited a normalizing effect on all parameters measured in platelets from FF animals. Further, ROS production in primary AEC from FF animals could be blunted to that of NFF animals by concentrations of rosuvastatin in the range of those achieved in the bloodstream. Diminished psPDI-dependent NO-releasing activity and increased initial aggregation rates of FF platelets may result from elevated vascular ROS production under conditions of insulin resistance. Normalization of ROS production and platelet aggregation by rosuvastatin indicates its potential use as a vasculoprotective agent.

Reversible inhibition of caspase-3 activity by iron(III): potential role in physiological control of apoptosis.

Desferoxamine is known to induce apoptosis in cancer cells, but the mechanisms are still not fully understood. We have shown that iron(III) is a potent caspase-3 inhibitor, and the inhibition is reversible by the iron chelating agent desferoxamine. Also, protein disulfide isomerase (PDI) is capable of activating caspase-3 inhibited by iron(III), likely by formation of iron-sulfur complex through its active site thiols. Data presented here suggests that iron(III) could be a potential inhibitor of apoptosis in vivo, by caspase-3-dependent inhibition with a possibility of recovery through PDI overexpression.

Characterization of the S-denitrosation activity of protein disulfide isomerase.

S-nitrosoglutathione (GSNO) denitrosation activity of recombinant human protein disulfide isomerase (PDI) has been kinetically characterized by monitoring the loss of the S-NO absorbance, using a NO electrode, and with the aid of the fluorogenic NOx probe 2,3-diaminonaphthalene. The initial rates of denitrosation as a function of [GSNO] displayed hyperbolic behavior irrespective of the method used to monitor denitrosation. The Km values estimated for GSNO were 65 +/- 5 microm and 40 +/- 10 microm for the loss in the S-NO bond and NO production (NO electrode or 2,3-diaminonaphthalene), respectively. Hemoglobin assay provided additional evidence that the final product of PDI-dependent GSNO denitrosation was NO*. A catalytic mechanism, involving a nitroxyl disulfide intermediate stabilized by imidazole (His160 a-domain or His589 a'-domain), which after undergoing a one-electron oxidation decomposes to yield NO plus dithiyl radical, has been proposed. Evidence for the formation of thiyl/dithiyl radicals during PDI-catalyzed denitrosation was obtained with 4-((9-acridinecarbonyl)-amino)-2,2,6,6-tetramethylpiperidine-1-oxyl. Evidence has also been obtained showing that in a NO- and O2-rich environment, PDI can form N2O3 in its hydrophobic domains. This "NO-charged PDI" can perform intra- and intermolecular S-nitrosation reactions similar to that proposed for serum albumin. Interestingly, reduced PDI was able to denitrosate S-nitrosated PDI (PDI-SNO) resulting in the release of NO. PDI-SNO, once formed, is stable at room temperature in the absence of reducing agent over the period of 2 h. It has been established that PDI is continuously secreted from cells that are net producers of NO-like endothelial cells. The present demonstration that PDI can be S-nitrosated and that PDI-SNO can be denitrosated by PDI suggests that this enzyme could be intimately involved in the transport of intracellular NO equivalents to the cell surface as well as the previous demonstration of PDI in the transfer of S-nitrosothiol-bound NO to the cytosol.

Platelet cell-surface protein disulphide-isomerase mediated S-nitrosoglutathione consumption.

S-nitrosothiols (RSNOs) regulate several aspects of platelet physiology including inhibition of activation, adhesion and aggregation. PDI (protein disulphide-isomerase) has recently been found to be localized to the cell surface, where it exhibits both disulphide-exchange and denitrosation activities. The disulphide-exchange activity of PDI has been linked to aspects of platelet aggregation. The present study suggests that the metabolism of RSNOs by platelets is a function of PDI denitrosation activity. Exposure of washed human platelets to increasing concentrations of GSNO (S-nitrosoglutathione) resulted in saturable denitrosation kinetics. The presence of known PDI inhibitors phenylarsine oxide and anti-PDI antibodies prevented GSNO denitrosation. The fact that, in the presence of GSNO plus the cell-permeable guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxaline-1-one, the initial rates of ADP-induced platelet aggregation and the maximum DeltaOD were diminished by approximately 40% shows that RSNOs have dual inhibitory effects on platelets, which are mediated through PDI. First, PDI denitrosates RSNOs, releasing NO that, via the guanylate cyclase/G-kinase route, attenuates platelet activation. Secondly, RSNOs are denitrosated at the same PDI-active site that catalyses the disulphide bond formation between integrins and their ligands, thereby attenuating irreversible aggregation.