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Quantifying changes in the bacterial thiol redox proteome during host-pathogen interaction

ABSTRACT: In this study, we used a quantitative redox proteomic method (OxICAT) to assess the in vivo thiol oxidation status of phagocytized E. coli. The majority (65.5%) of identified proteins harbored thiols that were significantly oxidized (>30%) upon phagocytosis. A substantial number of these proteins are from major metabolic pathways or are involved in cell detoxification and stress response, suggesting a systemic breakdown of the bacterial cysteine proteome in phagocytized bacteria.

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Escherichia Coli

SUBMITTER: Lars Leichert

LAB HEAD: Lars I. Leichert

PROVIDER: PXD011386 | Pride | 2019-01-07

REPOSITORIES: Pride

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			Action	DRS
	Evaluation.xlsx	Xlsx
	OEI20787.raw	Raw
	OEI20787andromeda.zip	Other
	OEI20787txt.zip	Other
	OEI20793.raw	Raw

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Publications

Quantifying changes in the bacterial thiol redox proteome during host-pathogen interaction.

Xie Kaibo K Bunse Christina C Marcus Katrin K Leichert Lars I LI

Redox biology 20181219

Phagocyte-derived production of a complex mixture of different oxidants is a major mechanism of the host defense against microbial intruders. On the protein level, a major target of these oxidants is the thiol group of the amino acid cysteine in proteins. Oxidation of thiol groups is a widespread regulatory post-translational protein modification. It is used by bacteria to respond to and to overcome oxidative stress. Numerous redox proteomic studies have shown that protein thiols in bacteria, su ...[more]

PMID: 30682706

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Analysis of ageing-dependent thiol oxidation reveals early oxidation of proteins involved in core proteostasis functions

Project description:Oxidants have a profound impact on biological systems in physiology and under pathological conditions. Oxidative post-translational modifications of protein thiols are well-recognized as a readily occurring alteration of proteins. Changes in protein thiol redox state can modify the function of proteins and thus can control cellular processes. However, chronic oxidative stress causes oxidative damage to proteins with detrimental consequences for cellular function and organismal health. The development of techniques enabling the site-specific and quantitative assessment of protein thiol oxidation on a proteome-wide scale significantly expanded the number of known oxidation-sensitive protein thiols. However, lacking behind are large-scale data on the redox state of proteins during ageing, a physiological process accompanied by increased levels of endogenous oxidants. Here, we present the landscape of protein thiol oxidation in chronologically aged wild-type Saccharomyces cerevisiae in a time-dependent manner. Our data determine early oxidation targets in key biological processes governing the de novo production of proteins, folding, and protein degradation. Comparison to existing datasets reveals evolutionary conservation of early oxidation targets. To facilitate accessibility and cross-species comparison of the experimental data obtained, we created the OxiAge Database, a free online tool for the research community that integrates current datasets on thiol redoxomes in aged yeast, nematode Caenorhabditis elegans, fruit fly Drosophila melanogaster, and mouse Mus musculus.

2024-02-07 | PXD042047 | Pride

Regulation of titin-based cardiac stiffness by unfolded domain oxidation (UnDOx)

Project description:The relationship between oxidative stress and cardiac stiffness is thought to involve modifications to the giant muscle protein titin, which in turn can determine the progression of heart disease. In vitro studies have shown that S-glutathionylation and disulfide bonding of titin fragments could alter the elastic properties of titin; however, whether and where titin becomes oxidized in vivo is less certain. Here we demonstrate, using multiple models of oxidative stress in conjunction with mechanical loading, that immunoglobulin domains preferentially from the distal titin spring region become oxidized in vivo through the mechanism of unfolded domain oxidation (UnDOx). Via oxidation type-specific modification of titin, UnDOx modulates human cardiomyocyte passive force bidirectionally. UnDOx also enhances titin phosphorylation and, importantly, promotes non-constitutive folding and aggregation of unfolded domains. We propose a mechanism whereby UnDOx enables the controlled homotypic interactions within the distal titin spring to stabilize this segment and regulate myocardial passive stiffness.

2020-09-15 | PXD018174 | Pride

Effects of the antibacterial compund Allicin from garlic on Escherichia coli

Project description:Allicin (diallyl thiosulfinate) from garlic is a highly potent natural antimicrobial substance. It inhibits growth of a variety of microorganisms, among them antibiotic resistant strains. Here, we show that upon exposure to allicin, growth of Escherichia coli is strongly inhibited. Growth inhibition coincides with a significant drop of total sulfhydryl concentrations and induction of the heat shock and oxidative stress response. In contrast to diamide, a potent inducer of disulfide stress, allicin does not induce cystine bond formation in proteins such as cytoplasmically expressed alkaline phosphatase PhoA. We identified and quantified the allicin-induced modification S-allylmercapto-cysteine for a set of cytoplasmic proteins by using a combination of label-free mass spectrometry and differential OxICAT labeling. Activity of isocitrate lyase AceA, an S-allylmercapto-modified candidate protein, is largely inhibited by allicin treatment in vivo. Our results indicate that the mode of action of allicin is a combination of disturbance of the redox balance and inactivation of crucial metabolic enzymes.

2016-05-02 | PXD003063 | Pride

Monitoring of protein thiol-oxidation in S.aureus USA300 under hypochlorite stress using OxICAT approach.

Project description:During infections, S. aureus has to cope with the oxidative burst of activated macrophages and neutrophils, including reactive oxygen and nitrogen species (RNS, ROS) and the strong oxidant hypochloric acid. We aimed to understand the global thiol-redox state in the major pathogen S. aureus and discover new NaOCl-sensitive proteins driven by thiol-switches. Thus, we performed a quantitative redox proteomics approach based on OxICAT and analyzed the percentages of thiol-oxidation levels in S.aureus before and after sub-lethal doses of 150 µM NaOCl stress. In parallel, we searched for protein S-bacillithiolation.

2016-12-22 | PXD004918 | Pride

Lens Disulfide crosslink - Evidence of highly conserved β-crystallin disulfidome that can be mimicked by in vitro oxidation in age-related human cataract and glutathione depleted LEGSKO mouse lens

Project description:Low glutathione levels are associated with crystallin oxidation in age-related nuclear cataract (ARNC). To understand the role of cysteine residue oxidation, we used the novel approach of comparing human cataracts with glutathione-depleted LEGSKO mouse lenses for intra- vs. intermolecular disulfide crosslinks using 2D-PAGE and proteomics, and then systematically identified in vivo and in vitro all disulfide forming sites using ICAT labeling method coupled with proteomics. Crystallins rich in intramolecular disulfides were abundant at young age in human and WT mouse lens but shifted to multimeric intermolecular disulfides at older age. The shift was ~4x accelerated in LEGSKO lens. Most cysteine disulfides in β-crystallins (except βA4 in human) were highly conserved in mouse and human and could be generated by oxidation with H2O2, while γ-crystallin oxidation selectively affected γC23/42/79/80/154, γD42/33 and γS83/115/130 in human cataracts, and γB79/80/110, γD19/109, γF19/79, γE19, γS83/130 and γN26/128 in mouse. Analysis based on available crystal structure suggests that conformational changes are needed to expose C42, C79/80, C154 in γC; C42, C33 in γD, and C83, C115 and C130 in γS. In conclusion, while the β-crystallin disulfidome is highly conserved in ARNC and LEGSKO mouse, and reproducible by in vitro oxidation, some of the disulfide formation sites in γ-crystallins necessitate prior conformational changes. Overall, the LEGSKO mouse model is closely reminiscent of ARNC.

2015-10-16 | PXD002658 | Pride

Monitoring global protein thiol-oxidation and protein S-mycothiolation in Mycobacterium smegmatis under hypochlorite stress

Project description:Mycothiol (AcCys-GlcN-Ins, MSH) is the major thiol-redox buffer in Actinomycetes, including Mycobacterium and Corynebacterium species. ). Protein S-mycothiolation controls the activities of several redox enzymes that function in detoxification of ROS and methionine sulfoxides, including the thiol peroxidase Tpx, the mycothiol peroxidase Mpx and the methionine sulfoxide reductase MsrA. Here we investigated the level of protein S-mycothiolation in Mycobacterium smegmatis under oxidative stress as well as its NaOCl stress response.

2017-06-30 | PXD003303 | Pride

Monitoring global protein thiol-oxidation and protein S-mycothiolation in Corynebacterium diphtheriae under hypochlorite stress

Project description:Mycothiol (AcCys-GlcN-Ins, MSH) is the major thiol-redox buffer in Actinomycetes, including Mycobacterium and Corynebacterium species. Protein S-mycothiolation controls the activities of several redox enzymes that function in detoxification of ROS and methionine sulfoxides, including the thiol peroxidase Tpx, the mycothiol peroxidase Mpx and the methionine sulfoxide reductase MsrA. Here we investigated the level of protein S-mycothiolation in Corynebacterium diphtheriae DSM43989 under oxidative stress as well as its NaOCl stress response.

2017-07-17 | PXD003321 | Pride

The Prion Protein (PrP) is involved in translation via RNA granule-like mRNPs, a connection which is impaired during neurodegenerative diseases

Project description:Cytoplasmic RNA granules have emerged as important elements of posttranscriptional and translational regulation. Stress, germinal and neuronal granules contain RNA-binding proteins capable of self-assembly due to prion-like domains. Hyperassembly mediated by these prion-like domains causes several neurodegenerative diseases. Here, we report a subset of the mammalian prion protein (PrP), also prone to self-assembly, propagation and to cause devastating diseases, is a component of naturally occurring messenger ribonucleoproteins (mRNPs) in adult mouse brains. Biomolecules co-purified with PrP revealed a multitude of diverse RNA granule associated proteins and mRNAs encoding members of the translation machinery, indicating a role in a specialized translation process. Importantly, PrP mutations linked to Creutzfeldt-Jakob disease (CJD) or fatal familial insomnia (FFI) severely impaired recovery of mRNPs from preclinical mice, possibly representing a very early pathological process. Thus, mutant PrP may cause dysfunction in RNA regulation, thereby joining the constantly expanding ranks of disease associated RNP granule proteins. The file Enrichment_analysis.xlsx contains mRNAs (FDR < 0.01) co-purified with PrP in both WT sample pools as identified by DESeq2 and the respective gene counts and log2 fold changes for CJD and FFI PrP:IP sample pools. RIP-Seq analysis of mRNAs co-purified with PrP from murine brain cytoplasmic fractions in wild-type (WT), CJD and FFI mice. Each RIP-Seq and control (input) library represents a pool of 12 to 16 co-immunoprecipitation samples out of 3 to 4 mice. To control for post-homogenization artifacts, we conducted an experiment in which we prepared homogenates from WT and PrP-KO (Prnp-/-) mice of different genetic backgrounds (C57BL/6 and 129S4) and identified SNPs in RIP-Seq and control libraries to finally identify specifically co-purified mRNAs.

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Project description:Reactive oxygen species (ROS) can modulate protein function through cysteine oxidation. Identifying targets of ROS can provide insight into uncharacterized ROS-regulated pathways. Several redox-proteomic workflows, such as OxICAT, exist to identify sites of cysteine oxidation. However, determining ROS targets localized within subcellular compartments and ROS hotspots remains challenging with existing workflows. Here, we present a chemoproteomic platform, PL-OxICAT, which combines proximity labeling (PL) with OxICAT to monitor localized cysteine oxidation events. We show that TurboID-based PL-OxICAT can monitor cysteine oxidation events within subcellular compartments such as the mitochondrial matrix and intermembrane space. Furthermore, we use APEX-based PL-OxICAT to monitor oxidation events within ROS hotspots by using endogenous ROS as the source of peroxide for APEX activation. Together, these platforms further hone our ability to monitor cysteine oxidation events within specific subcellular locations and ROS hotspots and provide a deeper understanding of the protein targets of endogenous and exogenous ROS.

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