A new oxidative sensing and regulation pathway mediated by the MgrA homologue SarZ in Staphylococcus aureus

Oxidative stress serves as an important host/environmental signal that triggers a wide range of responses from the human pathogen Staphylococcus aureus . Among these, a thiol-based oxidation sensing pathway through a global regulator MgrA controls the virulence and antibiotic resistance of the bacterium. Herein, we report a new thiol-based oxidation sensing and regulation system that is mediated through a parallel global regulator SarZ. SarZ is a functional homologue of MgrA and is shown to affect the expression of ∼87 genes in S. aureus . It uses a key Cys residue, Cys-13, to sense oxidative stress and to co-ordinate the expression of genes involved in metabolic switching, antibiotic resistance, peroxide stress defence, virulence, and cell wall properties. The discovery of this SarZ-mediated regulation, mostly independent from the MgrA-based regulation, fills a missing gap of oxidation sensing and response in S. aureus .     

Cysteine-106 of DJ-1 is the most sensitive cysteine residue to hydrogen peroxide-mediated oxidation in vivo in human umbilical vein endothelial cells

Mutation in DJ-1 gene is the cause of autosomal recessive Parkinson's disease, however, its physiological function remains unclear. The isoelectric point of DJ-1 shows an acidic shift after cells are treated with hydrogen peroxide. This suggests that DJ-1 is modified in response to oxidative stress. Here we report the structural characterization of an acidic isoform of DJ-1 using a proteomic approach with nanospray interface liquid chromatography-electrospray ionization/linear ion trap mass spectrometer. When human umbilical vein endothelial cells were exposed to hydrogen peroxide, all three cysteines in DJ-1 were oxidized to cysteine sulphonic acid. Although a small part of the Cys-46 and Cys-53 were oxidized, Cys-106 was oxidized completely at any hydrogen peroxide concentration used here. These results suggest that Cys-106 is the most sensitive among three cysteine residues to oxidative stress, and that DJ-1 function is regulated, in terms of the intracellular redox state, by oxidation of Cys-106.     

Two vicinal cysteines confer a peculiar redox regulation to low molecular weight protein tyrosine phosphatase in response to platelet-derived growth factor receptor stimulation.

Low molecular weight protein tyrosine phosphatase (LMW-PTP) is an enzyme involved in platelet-derived growth factor (PDGF)-induced mitogenesis and cytoskeleton rearrangement because it is able to bind and dephosphorylate the activated receptor. LMW-PTP presents two cysteines in positions 12 and 17, both belonging to the catalytic pocket; this is a unique feature of LMW-PTP among all protein tyrosine phosphatases. Our previous results demonstrated that in vitro LMW-PTP is oxidized by either H(2)O(2) or nitric oxide with the formation of a disulfide bond between Cys-12 and Cys-17. This oxidation leads to reversible enzyme inactivation because treatment with reductants permits catalytic activity rescue. In the present study we investigated the in vivo inactivation of LMW-PTP by either extracellularly or intracellularly generated H(2)O(2), evaluating its action directly on its natural substrate, PDGF receptor. LMW-PTP is oxidized and inactivated by exogenous oxidative stress and recovers its activity after oxidant removal. LMW-PTP is oxidized also during PDGF signaling, very likely upon PDGF-induced H(2)O(2) production, and recovers its activity within 40 min. Our results strongly suggest that reversibility of in vivo LMW-PTP oxidation is glutathione-dependent. In addition, we propose an intriguing and peculiar role of Cys-17 in the formation of a S-S intramolecular bond, which protects the catalytic Cys-12 from further and irreversible oxidation. On the basis of our results we propose that the presence of an additional cysteine near the catalytic cysteine could confer to LMW-PTP the ability to rapidly recover its activity and finely regulate PDGF receptor activation during both extracellularly and intracellularly generated oxidative stress.     

Pseudomonas aeruginosa OspR is an oxidative stress sensing regulator that affects pigment production, antibiotic resistance and dissemination during infection

Oxidative stress is one of the main challenges bacteria must cope with during infection. Here, we identify a new oxidative stress sensing and response ospR ( o xidative s tress response and p igment production R egulator) gene in Pseudomonas aeruginosa . Deletion of ospR leads to a significant induction in H₂O₂ resistance. This effect is mediated by de-repression of PA2826 , which lies immediately upstream of ospR and encodes a glutathione peroxidase. Constitutive expression of ospR alters pigment production and β-lactam resistance in P. aeruginosa via a PA2826 -independent manner. We further discovered that OspR regulates additional genes involved in quorum sensing and tyrosine metabolism. These regulatory effects are redox-mediated as addition of H₂O₂ or cumene hydroperoxide leads to the dissociation of OspR from promoter DNA. A conserved Cys residue, Cys-24, plays the major role of oxidative stress sensing in OspR. The serine substitution mutant of Cys-24 is less susceptible to oxidation in vitro and exhibits altered pigmentation and β-lactam resistance . Lastly, we show that an ospR null mutant strain displays a greater capacity for dissemination than wild-type MPAO1 strain in a murine model of acute pneumonia. Thus, OspR is a global regulator that senses oxidative stress and regulates multiple pathways to enhance the survival of P. aeruginosa inside host.     

Oxidative stress induced S-glutathionylation and proteolytic degradation of mitochondrial thymidine kinase 2

Protein glutathionylation in response to oxidative stress can affect both the stability and activity of target proteins. Mitochondrial thymidine kinase 2 (TK2) is a key enzyme in mitochondrial DNA precursor synthesis. Using an antibody specific for glutathione (GSH), S-glutathionylated TK2 was detected after the addition of glutathione disulfide (GSSG) but not GSH. This was reversed by the addition of dithiothreitol, suggesting that S-glutathionylation of TK2 is reversible. Site-directed mutagenesis of the cysteine residues and subsequent analysis of mutant enzymes demonstrated that Cys-189 and Cys-264 were specifically glutathionylated by GSSG. These cysteine residues do not appear to be part of the active site, as demonstrated by kinetic studies of the mutant enzymes. Treatment of isolated rat mitochondria with hydrogen peroxide resulted in S-glutathionylation of added recombinant TK2. Treatment of intact cells with hydrogen peroxide led to reduction of mitochondrial TK2 activity and protein levels, as well as S-glutathionylation of TK2. Furthermore, the addition of S-glutathionylated recombinant TK2 to mitochondria isolated from hydrogen peroxide-treated cells led to degradation of the S-glutathionylated TK2, which was not observed with unmodified TK2. S-Glutathionylation on Cys-189 was responsible for the observed selective degradation of TK2 in mitochondria. These results strongly suggest that oxidative damage-induced S-glutathionylation and degradation of TK2 have significant impact on mitochondrial DNA precursor synthesis.     

Role of a cysteine synthase in Staphylococcus aureus

The gram-positive human pathogen Staphylococcus aureus is often isolated with media containing potassium tellurite, to which it has a higher level of resistance than Escherichia coli. The S. aureus cysM gene was isolated in a screen for genes that would increase the level of tellurite resistance of E. coli DH5alpha. The protein encoded by S. aureus cysM is sequentially and functionally homologous to the O-acetylserine (thiol)-lyase B family of cysteine synthase proteins. An S. aureus cysM knockout mutant grows poorly in cysteine-limiting conditions, and analysis of the thiol content in cell extracts showed that the cysM mutant produced significantly less cysteine than wild-type S. aureus SH1000. S. aureus SH1000 cannot use sulfate, sulfite, or sulfonates as the source of sulfur in cysteine biosynthesis, which is explained by the absence of genes required for the uptake and reduction of these compounds in the S. aureus genome. S. aureus SH1000, however, can utilize thiosulfate, sulfide, or glutathione as the sole source of sulfur. Mutation of cysM caused increased sensitivity of S. aureus to tellurite, hydrogen peroxide, acid, and diamide and also significantly reduced the ability of S. aureus to recover from starvation in amino acid- or phosphate-limiting conditions, indicating a role for cysteine in the S. aureus stress response and survival mechanisms.     

Human Ran cysteine 112 oxidation by pervanadate regulates its binding to keratins

We used a proteomic approach to identify proteins that associate with keratins 8 or 18 (K8/K18) in a pervanadate-dependent manner. Pervanadate triggers Ran-K8/K18 binding and a gel-migration-shift of Ran from 25 to 27 kDa, which does not occur upon exposure to H2O2 or vanadate or if pervanadate is excluded during cell solubilization. Generation of 27-kDa Ran is not related to hyperphosphorylation, is heat-insensitive, but occurs upon conversion of Ran cysteines to cysteic acid. The pervanadate-mediated Ran cysteine --> cysteic acid oxidation and its related gel migration shift affects other proteins including actin. Mutation of the three Ran cysteines (Cys-85, -112, and -120) showed that Ran Cys-112 oxidation generates 27-kDa Ran and accounts for its keratin binding. Proteasome inhibition accentuates Ran-keratin binding after cell exposure to pervanadate. Therefore, cell-free exposure to pervanadate causes cysteine to cysteic acid oxidation of Ran and several other proteins and Ran-K8/K18 association. In cells, stabilization of oxidized Ran by proteasome inhibition promotes Ran-keratin interaction. Keratin sequestration of oxidized Ran may provide a back-up protective mechanism in some cases of oxidative injury.     

Formation of albumin dimers induced by exposure to peroxides in human plasma: a possible biomarker for oxidative stress

Human serum albumin (HSA) has one free thiol residue at Cys-34 that is likely oxidized by various reactive oxygen species (ROS). We attempted to identify the oxidation product of Cys-34 of HSA following exposure of plasma to ROS. Oxidation induced by tert-butyl hydroperoxide (t-BuOOH) of this free cysteine residue in HSA was observed in detail. Analysis of oxidized albumin in a partially purified fraction obtained by affinity column chromatography clearly revealed the formation of albumin disulfide dimers following t-BuOOH exposure. Albumin disulfide dimer formation was observed in normal plasma following treatment with various peroxides, as well as in untreated plasma from patients on hemodialysis using SDS-PAGE and Western blot analysis. The present results indicate that albumin dimers are oxidative products derived from peroxides, and that their presence in plasma might be a marker of oxidative stress as secondary metabolites of peroxidation.     

ATPase activity of p97/valosin-containing protein is regulated by oxidative modification of the evolutionally conserved cysteine 522 residue in Walker A motif

Valosin-containing protein (p97/VCP) has been proposed as playing crucial roles in a variety of physiological and pathological processes such as cancer and neurodegeneration. We previously showed that VCP(K524A), an ATPase activity-negative VCP mutant, induced vacuolization, accumulation of ubiquitinated proteins, and cell death, phenotypes commonly observed in neurodegenerative disorders. However, any regulatory mechanism of its ATPase activity has not yet been clarified. Here, we show that oxidative stress readily inactivates VCP ATPase activity. With liquid chromatography/tandem mass spectrometry, we found that at least three cysteine residues were modified by oxidative stress. Of them, the 522nd cysteine (Cys-522) was identified as the site responsible for the oxidative inactivation of VCP. VCP(C522T), a single-amino acid substitution mutant from cysteine to threonine, conferred almost complete resistance to the oxidative inactivation. In response to oxidative stress, VCP strengthened the interaction with Npl4 and Ufd1, both of which are essential in endoplasmic reticulum-associated protein degradation. Cys-522 is located in the second ATP binding motif and is highly conserved in multicellular but not unicellular organisms. Cdc48p (yeast VCP) has threonine in the corresponding amino acid, and it showed resistance to the oxidative inactivation in vitro. Furthermore, a yeast mutant (delta cdc48 + cdc48[T532C]) was shown to be susceptible to oxidants-induced growth inhibition and cell death. These results clearly demonstrate that VCP ATPase activity is regulated by the oxidative modification of the Cys-522 residue. This regulatory mechanism may play a key role in the conversion of oxidative stress to endoplasmic reticulum stress response in multicellular organisms and also in the pathological process of various neurodegenerative disorders.     

Yeast thioredoxin peroxidase expression enhances the resistance of Escherichia coli to oxidative stress induced by singlet oxygen

Singlet oxygen ((1)O(2)) is a highly reactive form of molecular oxygen that may harm living systems by oxidizing critical cellular macromolecules. A soluble protein from Saccharomyces cerevisiae specifically provides protection against a thiol-containing metal-catalyzed oxidation system (thiol/Fe(3+)/O(2)) but not against an oxidation system without thiol. This 25 kDa protein acts as a peroxidase but requires the NADPH-dependent thioredoxin system or a thiol-containing intermediate, and was named thioredoxin peroxidase (TPx). The role of TPx in the cellular defense against oxidative stress induced by singlet oxygen was investigated in Escherichia coli containing an expression vector with a yeast genomic DNA fragment that encodes TPx and mutant in which the catalytically essential amino acid cysteine (Cys-47) has been replaced with alanine by a site-directed mutagenesis. Upon exposure to methylene blue and visible light, which generates singlet oxygen, there was a distinct difference between the two strains in regard to growth kinetics, viability, the accumulation of oxidized proteins and lipids, and modulation of activities of superoxide dismutase and catalase. The results suggest that TPx may play an important protective role in a singlet oxygen-mediated cellular damage.     

Yeast thioredoxin peroxidase expression enhances the resistance of Escherichia coli to oxidative stress induced by singlet oxygen

Abstract

Singlet oxygen (¹O₂) is a highly reactive form of molecular oxygen that may harm living systems by oxidizing critical cellular macromolecules. A soluble protein from Saccharomyces cerevisiae specifically provides protection against a thiol-containing metal-catalyzed oxidation system (thiol/Fe³⁺/O₂) but not against an oxidation system without thiol. This 25 kDa protein acts as a peroxidase but requires the NADPH-dependent thioredoxin system or a thiol-containing intermediate, and was named thioredoxin peroxidase (TPx). The role of TPx in the cellular defense against oxidative stress induced by singlet oxygen was investigated in Escherichia coli containing an expression vector with a yeast genomic DNA fragment that encodes TPx and mutant in which the catalytically essential amino acid cysteine (Cys-47) has been replaced with alanine by a site-directed mutagenesis. Upon exposure to methylene blue and visible light, which generates singlet oxygen, there was a distinct difference between the two strains in regard to growth kinetics, viability, the accumulation of oxidized proteins and lipids, and modulation of activities of superoxide dismutase and catalase. The results suggest that TPx may play an important protective role in a singlet oxygen-mediated cellular damage.     

Mitochondrial translocation of oxidized cofilin induces caspase-independent necrotic-like programmed cell death of T cells

Oxidative stress leads to T-cell hyporesponsiveness or death. The actin-binding protein cofilin is oxidized during oxidative stress, which provokes a stiff actin cytoskeleton and T-cell hyporesponsiveness. Here, we show that long-term oxidative stress leads to translocation of cofilin into the mitochondria and necrotic-like programmed cell death (PCD) in human T cells. Notably, cofilin mutants that functionally mimic oxidation by a single mutation at oxidation-sensitive cysteins (Cys-39 or Cys-80) predominately localize within the mitochondria. The expression of these mutants alone ultimately leads to necrotic-like PCD in T cells. Accordingly, cofilin knockdown partially protects T cells from the fatal effects of long-term oxidative stress. Thus, we introduce the oxidation and mitochondrial localization of cofilin as the checkpoint for necrotic-like PCD upon oxidative stress as it occurs, for example, in tumor environments.