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Project Area 3 - Engineered thiol switches

 

 

 

 

Antelmann Projektbild

Haike Antelmann, Berlin

 

Functional charcterization of NaOCl-sensitive thiol-switches and their impact on the bacillithiol redox potential in Staphylococcus aureus

Staphylococcus aureus is a major human pathogen that can cause local skin or soft tissue infections, but also life-threatening diseases. During infections, S. aureus has to cope with reactive oxygen species (ROS) and hypochloric acid (HOCl) that are produced by activated macrophages and neutrophils as major killing mechanism. S. aureus uses the low molecular weight thiol bacillithiol (BSH) as protection mechanism against the host immune defense. BSH contributes to virulence of S. aureus and functions in detoxification of ROS, HOCl, toxins, electrophiles and antibiotics. Under hypochlorite stress, BSH forms mixed disulfides with proteins, termed as S‑bacillithiolations as a widespread thiol-protection and redox-switch mechanism. Using the quantitative thiol-redox proteomics approach OxICAT, we recently identified 58 NaOCl-sensitive proteins in S. aureus that could play protective roles against the host immune defense. Among these are five S-bacillithiolated proteins including the glycolytic Gap as the major target. S-bacillithiolation of Gap functions in thiol-protection against overoxidation to irreversible sulfonic acids and redox-regulation under H2O2 and NaOCl stress. The bacilliredoxins BrxA/B were shown to catalyze the reduction of S-bacillithiolated OhrR, MetE in B. subtilis and Gap in S. aureus in vitro. However, the complete Brx redox pathway is unknown. We have further constructed the first genetically encoded bacilliredoxin-fused redox biosensor (Brx-roGFP2) to monitor dynamic changes in the BSH redox potential in S. aureus.

In this project, we will characterize the functions of the interesting NaOCl-sensitive thiol-switches in the defense against oxidative stress. These thiol-switches include redox regulators, bacilliredoxins and other thiol-disulfide reductases, the nitric oxide synthase, the virulence factor SsaA2 and the metabolic enzymes Gap and AldA. In addition, novel Brx-roGFP2 and Tpx-roGFP2 biosensors will be applied to monitor the changes in the BSH redox potential and intracellular H2O2 generation in the S. aureus thiol-switch mutant backgrounds.

 

Project details…

 

 

Dick

Tobias Dick, Heidelberg


Exploiting peroxidatic thiol switches as ultra-sensitive real-time probes to investigate cellular H2O2 homeostasis

We aim to establish genetically encoded H2O2 probes that are as sensitive as the most sensitive 2-Cys-peroxiredoxins. We want to be able to monitor in real-time the small changes in H2O2 levels that are sensed by those peroxiredoxins exhibiting second order rate constants on the order of ~107 M-1s-1. We estimate that this sensitivity corresponds to the sensing of intracellular H2O2 concentration changes taking place between the picomolar and lower nanomolar range.

 

By characterizing these probes in mechanistic detail we aim to refine our knowledge about structure-function relationships in the Prx family.

We aim to establish how these probes work in mechanistic detail and how they can be further manipulated to change their kinetic properties, e.g. the rate of probe reduction which determines overall H2O2 consumption by the probe. For example, we want to learn if and how subunit cooperativity and the environment of the CP-SOH influence the in vivo real-time dynamics of oxidation and reduction of peroxiredoxins.

 

We aim to understand how H2O2 probes and endogenous H2O2 consumers influence each other in the cellular context.

We want to understand if and how the ectopically expressed H2O2 probes influence other H2O2 consuming systems in the cells and - if necessary - find ways to minimize the impact of the probe on other processes. More generally, we want to know how H2O2 consumers kinetically compete with each other and with the introduced probe, i.e. how the H2O2 flux is distributed in vivo. Another aspect to be addressed is the in vivo occurrence and influence of hyperoxidation on probe responses.

 

We aim to use new probes to obtain insight into metabolic H2O2 homeostasis.

Making use of real-time probes with unprecedented H2O2 sensitivity, we aim to investigate how cytosolic H2O2 levels respond to changes in metabolic parameters including carbon source and O2 levels and to identify the important factors mediating this regulation. Another important aspect will be the study of population diversity with respect to H2O2 levels.

 

 

 

Meyer Teilprojekt

Andreas Meyer , Bonn

 

Regulation of oxidative protein folding by thiol switching in the ER of Arabidopsis thaliana

Secreted proteins are essential for a cell to interact with its environment. The folding of most secretory proteins in the endoplasmic reticulum (ER) strictly requires the catalyzed formation of intramolecular disulfide bonds between cysteine residues. Those disulfides are critical for protein structure and function, which makes their formation indispensable for cell survival. Protein disulfide isomerase (PDI) and ER oxidoreductase (ERO) together make up the oxidative protein folding machinery of the ER. They constitute a disulfide relay system for the transfer of electrons from cysteines of immature protein substrates to molecular oxygen. This machinery needs to be dynamically regulated, as the demand for oxidative protein folding in the ER can vary dramatically, depending on developmental stage and environmental conditions. As sessile organisms plants are frequently exposed to particularly severe environmental changes, which necessitate rapid acclimation responses on cellular level. In yeast and mammals, the activity of the folding machinery is modulated by regulatory thiol switches on the EROs, allowing rapid posttranslational control. While the control of oxidative protein folding is not understood in plants, the ERO isoforms of plants contain several additional cysteines as compared to their yeast and mammalian counterparts. Based on their position in the ERO protein these cysteines are likely to constitute an additional, plant-specific level of ERO redox control by stepwise activation of multiple thiol switches or the formation of alternative disulfides. A combination of knockout and knockdown for the two ERO isoforms in Arabidopsis results in a severe dwarf phenotype indicating that ERO function is indispensable for disulfide formation and cannot be backed up by other systems. The goal of this project is to identify and to characterize regulatory thiol switches in Arabidopsis ERO proteins and their interaction with PDIs as switch operators. Biochemical analysis of ERO regulation will be combined with in vivo studies to understand its physiological significance. Dissection of ERO function in planta builds on the isolation of mutants and transgenic plants expressing constitutively active ERO variants, and analysis of the thiol redox poise in the ER. As dynamic redox measurements in the ER are still limited by a lack of suitable sensors, improved fluorescent protein-based sensor variants will be developed for oxidizing redox environments. Being able to image redox state and H2O2 levels in oxidizing compartments will provide a major step forward for quantitative redox analysis in general. Specifically, it will generate novel depth of insight into the role of ERO and its regulatory thiols in setting ER redox homeostasis.

 

Project details…