Lars I. Leichert

Quantifying changes in the thiol redox proteome upon oxidative stress in vivo

Antimicrobial levels of reactive oxygen species (ROS) are produced by the mammalian host defense to kill invading bacteria and limit bacterial colonization. One main in vivo target of ROS is the thiol group of proteins. We have developed a quantitative thiol trapping technique termed OxICAT to identify physiologically important target proteins of hydrogen peroxide (H2O2) and hypochlorite (NaOCl) stress in vivo. OxICAT allows the precise quantification of oxidative thiol modifications in hundreds of different proteins in a single experiment. It also identifies the affected proteins and defines their redox-sensitive cysteine(s). Using this technique, we identified a group of Escherichia coli proteins with significantly (30–90%) oxidatively modified thiol groups, which appear to be specifically sensitive to either H2O2 or NaOCl stress. These results indicate that individual oxidants target distinct proteins in vivo. Conditionally essential E. coli genes encode one-third of redox-sensitive proteins, a finding that might explain the bacteriostatic effect of oxidative stress treatment. We identified a select group of redox-regulated proteins, which protect E. coli against oxidative stress conditions. These experiments illustrate that OxICAT, which can be used in a variety of different cell types and organisms, is a powerful tool to identify, quantify, and monitor oxidative thiol modifications in vivo.

Proteomic Approach to Understanding Antibiotic Action

ABSTRACT We have used proteomic technology to elucidate the complex cellular responses of Bacillus subtilis to antimicrobial compounds belonging to classical and emerging antibiotic classes. We established on two-dimensional gels a comprehensive database of cytoplasmic proteins with pIs covering a range of 4 to 7 that were synthesized during treatment with antibiotics or agents known to cause generalized cell damage. Although each antibiotic showed an individual protein expression profile, overlaps in the expression of marker proteins reflected similarities in molecular drug mechanisms, suggesting that novel compounds with unknown mechanisms of action may be classified. Indeed, one such substance, a structurally novel protein synthesis inhibitor (BAY 50-2369), could be classified as a peptidyltransferase inhibitor. These results suggest that this technique gives new insights into the bacterial response toward classical antibiotics and hints at modes of action of novel compounds. Such a method should prove useful in the process of antibiotic drug discovery.

Protein Thiol Modifications Visualized In Vivo

Thiol-disulfide interconversions play a crucial role in the chemistry of biological systems. They participate in the major systems that control the cellular redox potential and prevent oxidative damage. In addition, thiol-disulfide exchange reactions serve as molecular switches in a growing number of redox-regulated proteins. We developed a differential thiol-trapping technique combined with two-dimensional gel analysis, which in combination with genetic studies, allowed us to obtain a snapshot of the in vivo thiol status of cellular proteins. We determined the redox potential of protein thiols in vivo, identified and dissected the in vivo substrate proteins of the major cellular thiol-disulfide oxidoreductases, and discovered proteins that undergo thiol modifications during oxidative stress. Under normal growth conditions most cytosolic proteins had reduced cysteines, confirming existing dogmas. Among the few partly oxidized cytosolic proteins that we detected were proteins that are known to form disulfide bond intermediates transiently during their catalytic cycle (e.g., dihydrolipoyl transacetylase and lipoamide dehydrogenase). Most proteins with highly oxidized thiols were periplasmic proteins and were found to be in vivo substrates of the disulfide-bond-forming protein DsbA. We discovered a substantial number of redox-sensitive cytoplasmic proteins, whose thiol groups were significantly oxidized in strains lacking thioredoxin A. These included detoxifying enzymes as well as many metabolic enzymes with active-site cysteines that were not known to be substrates for thioredoxin. H2O2-induced oxidative stress resulted in the specific oxidation of thiols of proteins involved in detoxification of H2O2 and of enzymes of cofactor and amino acid biosynthesis pathways such as thiolperoxidase, GTP-cyclohydrolase I, and the cobalamin-independent methionine synthase MetE. Remarkably, a number of these proteins were previously or are now shown to be redox regulated.

Global Characterization of Disulfide Stress in Bacillus subtilis

We used DNA macroarray and proteome analysis to analyze the regulatory networks in Bacillus subtilis that are affected by disulfide stress. To induce disulfide stress, we used the specific thiol oxidant diamide. After addition of 1 mM diamide to an exponentially growing culture, cell growth stopped until the medium was cleared of diamide. Global analysis of the mRNA expression pattern during growth arrest revealed 350 genes that were induced by disulfide stress by greater than threefold. Strongly induced genes included known oxidative stress genes that are under the control of the global repressor PerR and heat shock genes controlled by the global repressor CtsR. Other genes that were strongly induced encode putative regulators of gene expression and proteins protecting against toxic elements and heavy metals. Many genes were substantially repressed by disulfide stress, among them most of the genes belonging to the negative stringent response. Two-dimensional gels of radioactively labeled protein extracts allowed us to visualize and quantitate the massive changes in the protein expression pattern that occurred in response to disulfide stress. The observed dramatic alteration in the protein pattern reflected the changes found in the transcriptome experiments. The response to disulfide stress seems to be a complex combination of different regulatory networks, indicating that redox-sensing cysteines play a key role in different signaling pathways sensing oxidative stress, heat stress, toxic element stress, and growth inhibition.

Using Quantitative Redox Proteomics to Dissect the Yeast Redoxome

Background: Proteins with redox-sensitive thiols confer rapid response to changes in redox conditions and/or oxidant levels. Results: Quantitative redox proteomics unveiled steady-state thiol oxidation states of ∼5% of yeast proteins and revealed physiologically relevant redox- and peroxide-sensitive proteins in yeast. Conclusion: Redox-sensitive thiols appear structurally distinct from peroxide-sensitive thiols. Significance: Pathways are fine-tuned by protein oxidation under both non-stress and oxidative stress conditions. To understand and eventually predict the effects of changing redox conditions and oxidant levels on the physiology of an organism, it is essential to gain knowledge about its redoxome: the proteins whose activities are controlled by the oxidation status of their cysteine thiols. Here, we applied the quantitative redox proteomic method OxICAT to Saccharomyces cerevisiae and determined the in vivo thiol oxidation status of almost 300 different yeast proteins distributed among various cellular compartments. We found that a substantial number of cytosolic and mitochondrial proteins are partially oxidized during exponential growth. Our results suggest that prevailing redox conditions constantly control central cellular pathways by fine-tuning oxidation status and hence activity of these proteins. Treatment with sublethal H2O2 concentrations caused a subset of 41 proteins to undergo substantial thiol modifications, thereby affecting a variety of different cellular pathways, many of which are directly or indirectly involved in increasing oxidative stress resistance. Classification of the identified protein thiols according to their steady-state oxidation levels and sensitivity to peroxide treatment revealed that redox sensitivity of protein thiols does not predict peroxide sensitivity. Our studies provide experimental evidence that the ability of protein thiols to react to changing peroxide levels is likely governed by both thermodynamic and kinetic parameters, making predicting thiol modifications challenging and de novo identification of peroxide sensitive protein thiols indispensable.

Small RNA-mediated control of the Agrobacterium tumefaciens GABA binding protein

Wounded plants activate a complex defence programme in response to Agrobacterium tumefaciens. They synthesize the non‐proteinogenic amino acid γ‐aminobutyric acid (GABA), which stimulates degradation of the quorum sensing signal N‐(3‐oxo‐octanoyl) homoserine lactone. GABA is transported into A. tumefaciens via an ABC transporter dependent on the periplasmic binding protein Atu2422. We demonstrate that expression of atu2422 and two other ABC transporter genes is downregulated by the conserved small RNA (sRNA) AbcR1 (for ABC regulator). AbcR1 is encoded in tandem with another sRNA, which is similar in sequence and structure. Both sRNAs accumulate during stationary phase but only the absence of AbcR1 resulted in significant accumulation of Atu2422 and increased GABA import. AbcR1 inhibits initiation of atu2422 translation by masking its Shine–Dalgarno sequence and thereby reduces stability of the atu2422 transcript. It is the first described bacterial sRNA that controls uptake of a plant‐generated signalling molecule. Given that similar sRNAs and ABC transporter genes are present in various Rhizobiaceae and in Brucella, it is likely that such sRNA‐mediated control impacts a number of host–microbe interactions.

Heme regulatory motifs in heme oxygenase-2 form a thiol/disulfide redox switch that responds to the cellular redox state

Heme oxygenase (HO) catalyzes the rate-limiting step in heme catabolism to generate CO, biliverdin, and free iron. Two isoforms of HO have been identified in mammals: inducible HO-1 and constitutively expressed HO-2. HO-1 and HO-2 share similar physical and kinetic properties but have different physiological roles and tissue distributions. Unlike HO-1, which lacks cysteine residues, HO-2 contains three Cys-Pro signatures, known as heme regulatory motifs (HRMs), which are known to control processes related to iron and oxidative metabolism in organisms from bacteria to humans. In HO-2, the C-terminal HRMs constitute a thiol/disulfide redox switch that regulates affinity of the enzyme for heme (Yi, L., and Ragsdale, S. W. (2007) J. Biol. Chem. 282, 20156–21067). Here, we demonstrate that the thiol/disulfide switch in human HO-2 is physiologically relevant. Its redox potential was measured to be −200 mV, which is near the ambient intracellular redox potential. We expressed HO-2 in bacterial and human cells and measured the redox state of the C-terminal HRMs in growing cells by thiol-trapping experiments using the isotope-coded affinity tag technique. Under normal growth conditions, the HRMs are 60–70% reduced, whereas oxidative stress conditions convert most (86–89%) of the HRMs to the disulfide state. Treatment with reductants converts the HRMs largely (81–87%) to the reduced dithiol state. Thus, the thiol/disulfide switch in HO-2 responds to cellular oxidative stress and reductive conditions, representing a paradigm for how HRMs can integrate heme homeostasis with CO signaling and redox regulation of cellular metabolism.

Nitrosative stress treatment of E. coli targets distinct set of thiol‐containing proteins

Reactive nitrogen species (RNS) function as powerful antimicrobials in host defence, but so far little is known about their bacterial targets. In this study, we set out to identify Escherichia coli proteins with RNS‐sensitive cysteines. We found that only a very select set of proteins contain cysteines that undergo reversible thiol modifications upon nitric oxide (NO) treatment in vivo. Of the 10 proteins that we identified, six (AtpA, AceF, FabB, GapA, IlvC, TufA) have been shown to harbour functionally important thiol groups and are encoded by genes that are considered essential under our growth conditions. Media supplementation studies suggested that inactivation of AceF and IlvC is, in part, responsible for the observed NO‐induced growth inhibition, indicating that RNS‐mediated modifications play important physiological roles. Interestingly, the majority of RNS‐sensitive E. coli proteins differ from E. coli proteins that harbour H2O2‐sensitive thiol groups, implying that reactive oxygen and nitrogen species affect distinct physiological processes in bacteria. We confirmed this specificity by analysing the activity of one of our target proteins, the small subunit of glutamate synthase. In vivo and in vitro activity studies confirmed that glutamate synthase rapidly inactivates upon NO treatment but is resistant towards other oxidative stressors.

The sulfur carrier protein TusA has a pleiotropic role in Escherichia coli that also affects molybdenum cofactor biosynthesis.

Background: The sulfur carrier protein TusA is involved in sulfur transfer to different biomolecules in the cell. Results: TusA is involved in sulfur transfer for molybdopterin. Conclusion: Direction of sulfur transfer to biomolecules is mediated by the interaction partners of IscS. Significance: This study furthers the understanding of how sulfur transfer is regulated in bacteria. The Escherichia coli l-cysteine desulfurase IscS mobilizes sulfur from l-cysteine for the synthesis of several biomolecules such as iron-sulfur (FeS) clusters, molybdopterin, thiamin, lipoic acid, biotin, and the thiolation of tRNAs. The sulfur transfer from IscS to various biomolecules is mediated by different interaction partners (e.g. TusA for thiomodification of tRNAs, IscU for FeS cluster biogenesis, and ThiI for thiamine biosynthesis/tRNA thiolation), which bind at different sites of IscS. Transcriptomic and proteomic studies of a ΔtusA strain showed that the expression of genes of the moaABCDE operon coding for proteins involved in molybdenum cofactor biosynthesis is increased under aerobic and anaerobic conditions. Additionally, under anaerobic conditions the expression of genes encoding hydrogenase 3 and several molybdoenzymes such as nitrate reductase were also increased. On the contrary, the activity of all molydoenzymes analyzed was significantly reduced in the ΔtusA mutant. Characterization of the ΔtusA strain under aerobic conditions showed an overall low molybdopterin content and an accumulation of cyclic pyranopterin monophosphate. Under anaerobic conditions the activity of nitrate reductase was reduced by only 50%, showing that TusA is not essential for molybdenum cofactor biosynthesis. We present a model in which we propose that the direction of sulfur transfer for each sulfur-containing biomolecule is regulated by the availability of the interaction partner of IscS. We propose that in the absence of TusA, more IscS is available for FeS cluster biosynthesis and that the overproduction of FeS clusters leads to a modified expression of several genes.

Quantitative redox proteomics: the NOxICAT method.

Because of its versatile chemical properties, the amino acid cysteine plays a variety of vital roles in proteins. It can form structure-stabilizing elements (e.g., disulfide bonds), coordinate metal cofactors and is part of the catalytic center of many enzymes. Recently, a new role has been discovered for cysteine: so-called redox-sensitive proteins use the thiol group of cysteine as a specific sensor for Reactive Oxygen Species (ROS) and Reactive Nitrogen Species (RNS). The oxidation of such a redox-active cysteine, e.g., under conditions of elevated cellular ROS or RNS levels (oxidative or nitrosative stress), often results in a reversible thiol modification. This, in turn, might lead to structural changes and altered protein activity. When the oxidative stress subsides, cellular antioxidant systems, including thioredoxin and glutathione can reduce the redox-active cysteine and restore the original structure and activity of the redox-sensitive protein. This makes oxidative thiol modifications an attractive mechanism for cellular redox sensing and signaling.To study the target cysteines of oxidative and nitrosative stress and to quantify the extent of the thiol modifications generated under these conditions, we have recently developed a thiol trapping technique using isotope coded affinity tag (ICAT) chemistry (1). With this method, reduced cysteines are selectively labeled with the isotopically light form of ICAT and oxidized cysteines with the isotopically heavy form of ICAT. Thus we could globally quantify the ratio of reduced and oxidized cysteines in cellular proteins based on the modified peptide masses. Here, we present an expansion of this method, which we term NOxICAT, because it uses ICAT chemistry to detect changes in thiol modifications of proteins upon Nitrosative and Oxidative stress. The NOxICAT-method is a highly specific and quantitative method to study the global changes in the thiol redox state of cellular proteins under a variety of physiological and pathological stress conditions.