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Project Area 3 - Engineered thiol switches

 

 

 

 

Haike Antelmann, Berlin

 

Protein S-bacillithiolations and real-time redox imaging of the bacillithiol redox potential in Staphylococcus Aureus

 

Glutathione serves as the major thiol-redox buffer in the defense against Reactive Oxygen Species (ROS) in eukaryotes. Firmicutes bacteria including Bacillus and Staphylococcus species utilize as thiol redox buffer bacillithiol (Cys-GlcN-Mal, BSH). In eukaryotes, proteins are post-translationally modified to S-glutathionylated proteins in response to oxidative stress. S-glutathionylation has emerged as major redox-regulatory mechanism and protects cysteine residues against overoxidation to sulfonic acids. Using thiol-redox proteomics and mass spectrometry (MS) we have discovered proteome-wide protein S-bacillithiolations as mixed BSH protein disulfides in response to oxidative stress in different industrially related Bacillus species. Protein S-bacillithiolation controls the activity of the redox-sensing OhrR repressor and of the methionine synthase MetE and protects active site cysteine residues of metabolic enzymes, antioxidant function proteins and translation factors. We also found that BSH plays an important role in Staphylococcus aureus in the defense against the host immune system in an phagocytosis assay in S. aureus strain NCTC8325 with complemented bshC gene. However, the role of protein S-bacillithiolation for redox control and the changes of the BSH redox potential under infection-related conditions are unknown in S. aureus. In this project, we propose that protein S-bacillithiolation controls virluence, pathomechanisms and adaptation to the host immune defense in the major human pathogen S. aureus. Major aims are (1) to elucidate the physiological role of BSH for post-translational thiol-modifications of proteins in S. aureus; (2) to monitor in real-time the changes in the BSH redox potential in S. aureus using genetically encoded Brx-roGFP2 biosensors under ROS stress and infection-related conditions. In aim (3) we will functionally characterize novel thiol-based redox switches that are controlled by S-bacillithiolation in S. aureus. We propose that our results will be of major importance to understand the defense mechanisms against the host immune system in S. aureus.

 

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…