The cell death protease Kex1p is essential for hypochlorite-induced apoptosis in yeast

Following microbial pathogen invasion, the human immune system of activated phagocytes generates and releases the potent oxidant hypochlorous acid (HOCl), which contributes to the killing of menacing microorganisms. Though tightly controlled, HOCl generation by the myeloperoxidase-hydrogen peroxide-chloride system of neutrophils/monocytes may occur in excess and lead to tissue damage. It is thus of marked importance to delineate the molecular pathways underlying HOCl cytotoxicity in both microbial and human cells. Here, we show that HOCl induces the generation of reactive oxygen species (ROS), apoptotic cell death and the formation of specific HOCl-modified epitopes in the budding yeast Saccharomyces cerevisiae. Interestingly, HOCl cytotoxicity can be prevented by treatment with ROS scavengers, suggesting oxidative stress to mediate the lethal effect. The executing pathway involves the pro-apoptotic protease Kex1p, since its absence diminishes HOCl-induced production of ROS, apoptosis and protein modification. By characterizing HOCl-induced cell death in yeast and identifying a corresponding central executor, these results pave the way for the use of Saccharomyces cerevisiae in HOCl research, not least given that it combines both being a microorganism as well as a model for programmed cell death in higher eukaryotes.     

Influence of HOCl…O3 and HOCl…HOCl interactions on the stability of O3(HOCl)2 complexes: a theoretical study

We have analysed the role of HOCl…O₃ and HOCl…HOCl interactions on the stability of four estimated O₃(HOCl)₂ complexes by means of ab initio molecular orbital calculations. It is predicted that the O₃(HOCl) + HOCl reaction is more energetically favourable than (HOCl)₂ + O₃ one. In all complexes, HOCl…HOCl interaction is stronger than HOCl…O₃ one. The results show that the HOCl…O₃ interaction strengthens the HOCl…HOCl one. On the other hand, O…H interaction in HOCl…O₃ moiety is strengthened when it interacts with HOCl. Quantum theory of atoms in molecules predicts that the weak interactions in O₃(HOCl)₂ complexes have electrostatic characteristic. In all complexes, the charge transfer from O₃ to (HOCl)₂ is expected from natural bond orbital analysis.     

Phospholipid chlorohydrins cause ATP depletion and toxicity in human myeloid cells

Chlorohydrins of stearoyl-oleoyl phosphatidylcholine (SOPC), stearoyl-linoleoyl phosphatidylcholine, and stearoyl-arachidonyl phosphatidylcholine were incubated with cultured myeloid cells (HL60) for 24 h, and the cellular ATP level was measured using a bioluminescent assay. The chlorohydrins caused significant depletion of cellular ATP in the range 10–100 μM. The ATP depletion by the phospholipid chlorohydrins was slightly less than that of 4-hydroxy-2-nonenal, but greater than that of hexanal, trans-2-nonenal, and autoxidised palmitoyl-arachidonoyl phosphatidylcholine. SOPC chlorohydrin was also found to cause loss of viability in U937 cells, and thus phospholipid chlorohydrins could contribute to the formation of a necrotic core in advanced atherosclerotic lesions.     

Studies on the role of goat heart galectin-1 as a tool for detecting post-malignant changes in glycosylation pattern

Galectins are mammalian lectins established to play a crucial role in the progression of various cancer types by the virtue of their differential expression in normal and cancerous cells. In the present study, goat heart galectin-1 (GHG-1) was purified and investigated for its potential role in the detection of post-malignant changes in glycosylation pattern. When exposed to superoxide radicals generated from a pyrogallol auto-oxidation system, GHG-1 treated erythrocyte suspension released higher amount of oxyhemoglobin than the unagglutinated erythrocytes. The extent of erythrocyte hemolysis was found to be directly proportional to concentrations of hypochlorous acid. GHG-1 was used to detect the change in the β-galactoside expression pattern in erythrocyte membrane from human donors suffering from prostate and breast cancer. No significant change was observed in the hemolysis of lectin agglutinated erythrocytes collected from pre-operated breast cancer patients, whereas significant increase was observed in normal healthy control and post-operated samples. Findings of this study proclaim GHG-1 as an important tool for the detection of post-malignant changes in glycosylation pattern.Abbreviations: Gal-1, galectin-1; GHG-1, goat heart galectin-1; HOCl, hypochlorous acid; OxyHb, oxyhemoglobin     

Oxidation of Bovine β-Casein by Hypochlorite

We recently observed two 2,4-dinitrophenylhydrazine (DNPH)-reactive proteins of 40 and 120 kDa in the bronchoalveolar lavage fluids of rats exposed to >95% O₂ for 48 h. The N-terminal sequences of these proteins were both identical over 16 amino acids with rat β-casein, which, in addition to its more common association with milk, is produced by cytotoxic T-lymphocytes, and has been found to have proinflammatory properties. Because of the inflammatory response that accompanies hyperoxic lung injury, we investigated the oxidation of bovine β-casein by HOCl. Following exposure to HOCl at 4°C for 15 min, derivatization with DNPH, washing, and digestion with trypsin, the resultant peptides were separated by reverse-phase HPLC. One peptide isolated from a peak absorbing at 365 nm was identified as AVP(Y*)PQR, corresponding to amino acids 177–183 of bovine β-casein. Analysis of the peptide by both electrospray and matrix assisted laser desorption ionization (MALDI) mass spectrometry identified a molecular ion MH⁺ of 1008.5 Da, which represents an increase of 178 Da from the calculated monoisotopic MH⁺ of the unmodified peptide of 830.45 Da. Daughter ion spectra of the doubly charged parent ion of the peptide further support the oxidation of the tyrosine to the quinone methide, with subsequent conversion to the corresponding hydrazone with DNPH. A second pair of products were identified as arising from oxidation of Y¹⁹³ within the tryptic peptide constituted by amino acids 184–202, and the corresponding chymotryptic cleavage side product, 191–202. Exposure of β-casein to increasing amounts of HOCl revealed that M and Y residues were the most susceptible, although bovine β-casein contains no C, and a single W, which would not be detected by our methods. The approach described in the present report can be used to evaluate the contributions of distinct mechanisms of oxidation in other experimental or pathological models. © 1997 Elsevier Science Inc.     

Hydogen peroxide-dependent photocytotoxicity by phloxine B,a xanthene-type food colorant

Background

Phloxine B (PhB; 2′,4′,5′,7′-tetrabromo-4,5,6,7-tetrachloro-fluorescein), an artificial xanthene colorant, has been used as a red coloring agent in drugs and cosmetics as well as foods in some countries. However, little effort has been devoted to the study of this colorant as a potentially useful medicinal agent.

Methods

We investigated the daily light-induced photocytotoxicity of PhB in two human leukemia cells, HL-60 and Jurkat, and its underlying mechanisms by in vitro experiments using antioxidants.

Reuslts and conclusions

PhB inhibited cell proliferation more preferentially to HL-60 cells than to Jurkat cells. Co-treatment of catalase completely blocked the photocytotoxicity by PhB in HL-60 cells, whereas the effect of histidine was only partial, suggesting that hydrogen peroxide (H₂O₂), rather than singlet oxygen, might be a prerequisite for the PhB-induced HL-60 cell death. Actually, PhB produced a significant amount of H₂O₂ in the media as well as in the cells in concentration- and light-dependent manners. Furthermore, methionine, a hypochlorous acid (HOCl) scavenger, also significantly attenuated the cytotoxicity in HL-60 cells, but not in Jurkat cells, indicating the involvement of myeloperoxidase (MPO)-dependent hypohalous acid formation during the photocytotoxicity. In vitro experiments revealed that halogenated tyrosine was generated from the reaction of bovine serum albumin with PhB and HL-60 cell lysate. The present findings suggested that PhB induced a differential photodynamic action in the MPO-containing leukemia cells through an H₂O₂-dependent mechanism.

General significance

Our findings provide new insights into the molecular mechanisms underlying the PhB-induced apoptosis and also evaluated PhB as a promising PDT agent.     

The physiological role of reversible methionine oxidation

Sulfur-containing amino acids such as cysteine and methionine are particularly vulnerable to oxidation. Oxidation of cysteine and methionine in their free amino acid form renders them unavailable for metabolic processes while their oxidation in the protein-bound state is a common post-translational modification in all organisms and usually alters the function of the protein. In the majority of cases, oxidation causes inactivation of proteins. Yet, an increasing number of examples have been described where reversible cysteine oxidation is part of a sophisticated mechanism to control protein function based on the redox state of the protein. While for methionine the dogma is still that its oxidation inhibits protein function, reversible methionine oxidation is now being recognized as a powerful means of triggering protein activity. This mode of regulation involves oxidation of methionine to methionine sulfoxide leading to activated protein function, and inactivation is accomplished by reduction of methionine sulfoxide back to methionine catalyzed by methionine sulfoxide reductases. Given the similarity to thiol-based redox-regulation of protein function, methionine oxidation is now established as a novel mode of redox-regulation of protein function. This article is part of a Special Issue entitled: Thiol-Based Redox Processes.     

Nitrite reduces the effects of HOCl on unsaturated phosphatidylcholines--a MALDI-TOF MS study

The concentration of nitrite (NO₂⁻) increases under inflammatory conditions. However, the physiological role of nitrite is so far controversial discussed: it was reported that effects of HOCl (an important inflammation mediator) on phospholipids (PL) may be enhanced but also reduced in the presence of nitrite.

In this paper a simple model system was used: unsaturated phosphatidylcholine (PC) vesicles were treated with HOCl in the presence of varying NaNO₂ concentrations and the yield of reaction products was determined by MALDI-TOF MS: the extent of chlorohydrin generation was significantly reduced in the presence of NaNO₂ because HOCl is consumed by the oxidation of NO₂⁻ to NO₃⁻.

Similar results were obtained when HOCl was generated by the myeloperoxidase (MPO)/H₂O₂/Cl⁻ system or the experiments were carried out in the presence of a simple peptide. It is concluded that the transient products of the reaction between HOCl and NO₂⁻ do not have a sufficient reactivity to modify PL.