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Project Area 2 - Unknown thiol switches

 

 

Becker

 

 

 

Katja Becker, Gießen

 

The glutathione redox couple as a thiol switch operator in the malaria parasite Plasmodium falciparum

As the malaria parasite Plasmodium falciparum has high proliferation rates, digests hemoglobin, and lives in prooxidant environments, antioxidant defense, redox regulation, and redox signaling play a crucial role for parasite host cell homeostasis. Furthermore redox metabolism represents a major target for chemotherapeutic interventions and has been shown to be involved in drug resistance. It has been demonstrated, that Plasmodium possesses a complex redox system comprising glutathione and thioredoxin-based components, specialized selenoproteins and peroxiredoxins, and the thioredoxin-like protein plasmoredoxin.

During the first funding period of the SPP 1710, we studied the glutathione dependent cytosolic and subcellular redox potential as well as hydrogen peroxide signaling in malaria parasites under oxidative and pharmacological stress. We further gained insight into redox-regulatory thiol modifications in P. falciparum and characterized targets of S-glutathionylation and S-nitrosylation in various cellular pathways.

Within the second funding period of our SPP 1710 project, we aim to further elucidate the thiol switch-based redox sensing and interaction of proteins in malaria parasites. Genetically encoded redox probes shall be employed to study mechanisms of drug action and drug resistance. Furthermore, quantitative and qualitative changes in S-glutathionylation and S-sulfenylation under oxidative stress shall be elucidated. To identify the redox interactome of peroxiredoxins, a mixed-disulfide fishing approach will be used. With surface plasmon resonance, the interactions between redox active proteins and their redoxins will be studied.

Project details…

 

 

Berndt Project area 2

Carsten Berndt und Eva- Maria Hanschmann, Düsseldorf

 

Intra- and intercellular functions of redoxins during neuroinflammation

Reversible oxidative thiol modifications are posttranslational modifications that control signal transduction making thiol switches of utmost significance for cellular functions. These switches are tightly regulated by redoxins - oxidoreductases of the thioredoxin family.

Based on our experiences in neurology and inflammation, we will synergistically analyze the functions of redoxins in neuroinflammation combining in vitro (cell cultures), ex vivo (primary cells, organotypic slice cultures), and in vivo models (zebrafish, mice) as well as samples from multiple sclerosis patients (brain slices, serum, cerebrospinal fluid). Within our project we will investigate redoxin-regulated processes and their underlying molecular mechanisms in oligodendrocytes, astrocytes, and microglia during regeneration and immunomodulation.

The regeneration from inflammation-induced brain damage and thereby the protection against neurological deficits depends on the migration of oligodendroglial progenitor cells towards lesions and their remyelinating capacity. We hypothesize that an identified glutaredoxin 2-induced differentiation block enhances regeneration of neurons in different inflammation paradigms (autoimmunity, traumatic injury) and that secreted redoxins have immunomodulatory functions.

In summary, our project provides the first comprehensive investigation of redoxin functions in glial cells. Choosing the pathological condition of neuroinflammation, with emphasis on multiple sclerosis, our proposal will provide both translational results with direct clinical impact and fundamental mechanistic and functional insights into redoxins and redox regulation of intra- and intercellular signaling.

Project details

Thomas Dickmeis, Eppendorf-Leopoldshafen

Redox reactions are fundamental reactions in biology. Reactive oxygen species (ROS) arising from these reactions must be tightly controlled to prevent damage to nucleic acids, proteins and lipids. Excessive ROS production is a feature of a number of diseases, including endocrine and metabolic diseases. However, ROS also serve signaling or regulatory functions in the organism, notably via reversible modifications of thiols in cysteine residues of proteins. It is well documented that such reversible modifications change protein conformation, localization and activity. Nevertheless, regulation of only a few of these “thiol switches” has been well characterized. In addition, knowledge on the spatiotemporal distribution of ROS in cells and tissues of multicellular organisms is limited. This information is needed to map sites and processes where thiol switches normally operate and to allow us a better grasp of causal relationships between shifts in ROS homeostasis and (patho)physiological processes.

Here, we wish to exploit the advantages of zebrafish embryos and larvae for the imaging of in vivo redox events across embryonic development and under pathological conditions mimicking endocrine and metabolic diseases. To this end, we will use zebrafish transgenic lines expressing engineered thiol-switch biosensors for the monitoring of H2O2 dynamics across cytosol and mitochondria of embryonic tissues during development. FACS of cells showing different redox signal levels followed by next generation sequencing analysis of their transcriptomes will give hints as to the regulatory and functional changes linked with different redox states, which will be further studied by manipulating redox levels via chemical treatment or genetic manipulation.

To examine redox changes in an endocrine disease model, we will introduce the sensor lines into rx3 strong mutants, a zebrafish model of adrenal insufficiency in which we have previously described numerous glucocorticoid dependent changes in transcriptional and metabolic dynamics. We will also examine embryos treated with glucocorticoids to create an excess of glucocorticoid signalling, as observed in Cushing’s syndrome. The data will reveal the consequences of both a lack and an excess of glucocorticoids on ROS levels and dynamics in various tissues. This information will be particularly valuable given the scarcity of reports on redox changes in human patients suffering from disorders of the glucocorticoid system. Importantly, the project will provide tools and concepts for the study of redox biology in a model organism highly amenable to in vivo drug screening approaches.

 

Project details

 

 

Dietz Eye catcher

Karl-Josef Dietz, Bielefeld

 

The role of cyclophilin 20-3 in the regulatory feedback loop of plant cell redox homeostasis

Redox homeostasis represents a fundamental characteristic of all living cells and is under control of the redox regulatory network. This network is composed of protein elements that are partly conserved among all organisms. In plants the network has evolved to particularly high complexity as judged from the expansion of involved gene families and targeted processes. Thus redox regulation affects e.g. metabolism, signaling, transcription, and translation and thereby (co-)controls development and acclimation. In this project we want to scrutinize the role of a particular player, the chloroplast cyclophilin Cyp20-3, which appears to hold a central role in regulating cell redox homeostasis and links oxylipin signaling to redox regulation, redox homeostasis and stress acclimation. Recently it could be shown that oxophytodienoic acid (OPDA), a precursor of jasmonic acid, tightly binds to Cyp20-3. OPDA accumulates in leaves upon pathogen attack, wounding and in response to abiotic stresses. The OPDA-liganded Cyp20-3 binds and activates cysteine synthase complex. Increasing cysteine and glutathione levels shift the cell redox potential to more negative values and this allows for activation of defense gene expression. Knock out lines devoid of Cyp20-3 lack this response and are more sensitive to infection. Cyp20-3 contains four Cys residues. Its function as rotamase is redox sensitive, and it interacts with the chloroplast 2-cysteine peroxiredoxin (2-CysPrx) which was suggested to act as thiol redox sensor and adopts different conformations depending on the redox state of two catalytic Cys. Further we could show that Cyp20-3 and 2-CysPrx are early targets of oxidation in vitro. All these data were combined to a novel working hypothesis where the thiol redox state of Cyp20-3 and 2-CysPrx controls the OPDA-dependent activation of Cys synthesis and the transient overreduction of the cell to activate defense gene expression under stress. We want to analyze the various steps of feed forward activation and feedback inhibition. This includes the biochemical analysis of activation and inactivation, e.g. by addressing the role of each Cys in Cyp20-3, the redox control of OPDA-binding, the interaction between Cyp20-3 and 2-CysPrx in vitro, in vivo, or ex vivo and the identification of thiol reductants. We will explore the significance of this thiol switch under different stresses and clarify pathways using mutants. The thiol mechanisms will be dissected by complementing the cyp20-3 knock out line with Cys- or rotamase-free variants. The consequences of these manipulations will be explored on the transcriptome and proteome level. The proposed project should allow us to answer the question whether Cyp20-3 functions as master thiol switch that regulates stress-dependent thiol synthesis in the plastids and photosynthesizing cell. This should clarify a mechanism of in our eyes fundamental significance for plant stress acclimation.

 

Project details…
Eble Project Bild

Johannes Eble, Münster

 

Integrins are cell adhesion receptors and mediate various functions, such as cell anchorage, force transmission, and migration. They are alpha-beta heterodimers, which bind to extracellular matrix ligands via their head domains and connect them via their extracellular stalks and transmembrane domains to the intracellular cytoskeleton. By undergoing dramatic conformational changes from a bent to an elongated form, integrins are activated and exert their functions. The conformational change is a global movement of the head and stalk domains around a pivot formed by the alpha subunit hinge domain. This hinge domain, and potentially also the calf2-domain within the stalk of the alpha subunit, contains a cysteine-based thiol switch. This explains the enhanced integrin binding activity upon oxidation with hydrogen peroxide at physiological concentrations. We prototypically showed that the thiol switch within the hinge region of alpha7 beta1 integrin, a laminin receptor, is reversibly redox-modified and involved in redox-regulation of integrin-related cell functions, such as migration. Therefore, integrin-mediated cellular functions depend on the redox environment of cells.

In this project, we will examine the role of redox-active cysteines within the calf2-domain of the integrin alpha7 subunit by point mutations. Moreover, in cooperation with SPP-partners, redox enzymes of the thioredoxin family, which redox-regulate the extracellular integrin domains, will be identified to unravel the redox mechanism. We will determine how conformational changes and molecular force transmission of the integrin depend on the thiol switch(es) within the hinge and calf2-domain by using recombinant integrin ectodomains in protein-chemical interaction assays, high resolution electron microscopy and atomic force microscopy. At the cellular level, fibrosarcoma cells and, in a novel approach, melanoma cells will be transfected with different thiol switch-deleted alpha7 beta1 integrin mutants, after the endogenous integrin has been knocked-out by CRISPR/Cas9-technology. Other laminin-binding integrins will be blocked by inhibiting antibodies. Together with SPP1710 partners, the cells will also be characterized for their repertoire of extracellular redox-modifying enzymes. Under different redox conditions and in the presence of redox-modifying enzymes, the transfected cell lines will reveal the biological consequences of the redox-regulated integrin function on cell spreading, adhesion, and migration in impedance-based adhesion/migration assays as well as by video and fluorescence microscopy. Adhesome formation, another consequence of integrin function, will comparatively be studied by 2D-DiGE. Translationally, we will employ melanoma spheroids as in vitro-tumor models to examine by flow cytometry and life fluorescence microscopy whether and how the hypoxia-affected redox milieu within a tumor core influences alpha7 beta1-dependent melanoma migration and dissemination.

Project details

 

 

Hermann

Johannes Herrmann, Kaiserslautern

 

Protein sulfenylation in mitochondria: biochemistry and physiological relevance

Mitochondria contain two aqueous compartments: the matrix and the intermembrane space (IMS). Presumably due to its evolutionary descent from the bacterial periplasm, many of the 50 to 100 proteins of the IMS reach their native structure by an oxidative folding process. The formation of disulfide bonds in IMS proteins is enzymatically catalyzed by the sulfhydryl oxidase Erv1 and the oxidoreductase Mia40. Interestingly, many mitochondrial proteins contain conserved cysteine residues which are not involved in the formation of structural disulfide bonds. Due to oxidative stress generated by the respiratory chain, these residues are prone to overoxidation which can convert thiol groups to sulfenic acid. It was recently shown that peroxide-dependent sulfenylation can serve as a molecular switch to control protein activity in a redox-dependent manner. The degree to which proteins are sulfenylated in vivo is presumably regulated by specific factors. We will use the yeast Saccharomyces cerevisiae as a model system to address the following questions: (1) Which proteins in the IMS and the matrix of mitochondria contain sulfenylated cysteine residues? (2) Under which conditions are proteins sulfenylated? (3) Which mitochondrial redox enzymes control protein sulfenylation? (4) What is the physiological relevance of these sulfenylation reactions? The yeast protein Yap1 is a transcription factor that interacts specifically with sulfenylated proteins via intermolecular disulfide bonds. Reporter proteins using the Yap1 domain were shown to detect sulfenylated proteins both in vivo and in vitro. For this proposed project we want to target a tagged variant of Yap1 to the matrix or the IMS of mitochondria in order to monitor the degree of protein sulfenylation under different conditions and in different yeast mutants. In addition, we will purify sulfenylated proteins that are bound to Yap1 and analyze them by mass spectrometry. Preliminary results show that the fusion proteins can be specifically targeted to the IMS and the matrix. A number of trapped adducts are found on these Yap1 domains, in particular after exiposure of cells to hydrogen peroxide and in cells lacking components of the mitochondrial quality control system. This points to an interesting role of mitochondrial proteases in the removal of sulfenylated proteins. This project promises mechanistic insights into sulfenylation reactions in living cells and the physiological relevance of this type of thiol switches. These studies will strongly benefit from the interactions with other groups in the framework of this DFG priority program.

 

Project details…

 

 

Krauth-Siegel

Luise Krauth-Siegel, Heidelberg

Genetically encoded biosensors for monitoring redox changes in the trypanothione-based thiol metabolism of trypanosomes

Trypanosomes and Leishmania lack glutathione reductases and thioredoxin reductase. The thiol redox homeostasis of the parasitic protozoa is based on trypanothione, a conjugate composed of the polyamine spermidine and two molecules of glutathione. All enzymes involved in the biosynthesis and reduction of the dithiol are located in the cytosol of the cell. Here, by thiol/disulfide exchange, trypanothione reduces glutathione disulfide as well as redox proteins such tryparedoxin, thioredoxin and glutaredoxins.

Measurements of total glutathione, glutathionylspermidine and trypanothione revealed that the cellular thiol pool undergoes highly distinct changes when the bloodstream form of Trypanosoma brucei , the causative agent of African sleeping sickness and Nagana cattle disease, is exposed to exogenous and endogenous oxidative stresses. This type of analysis, however, cannot provide any information about the specific situation in the different cell compartments.

Aim of this project is to generate cell lines that express various redox active green fluorescent proteins (roGFPs) in the cytosol as well as intermembrane space and matrix of the single mitochondrion of T. brucei and thus allow a compartment-specific analysis of the thiol metabolism under steady state and thiol switch conditions in the intact parasites.

The main questions that will be addressed are: Does the whole cell thiol status reflect the conditions in the cytosol? Is there a cross-talk between the thiol pool of the cytosol and the mitochondrial compartments? How does the thiol redox status in the fully elaborated mitochondrion of the insect stage compare with that in the rudimentary organelle of the infectious bloodstream parasite? How does down-regulation of trypanothione biosynthesis or reduction affect the cytosolic and mitochondrial thiol redox status? Do the two glutaredoxins, localized in the cytosol and mitochondrial intermembrane space, respectively, act as thiol redox switches? The work should give a profound insight into the cytosolic and mitochondrial thiol metabolism of African trypanosomes. In addition, the stable cell lines generated are expected to be attractive tools for future studies of putatively redox regulated processes in the parasites such as differentiation.

 

Project details…

 

 

Leichert

Lars Leichert, Bochum

 

Global changes in the thiol redox state of Escherichia coli in a host pathogen setting

Since the evolution of photosynthesis, biological systems had to find ways to adapt to the toxicity of oxygen. Bacteria can encounter oxidative stress in numerous ways, be it through generation of reactive oxygen species during respiration or the exposure to environmental toxins. Of particular physiological and pathological importance, however, is the oxidative insult inflicted by the immunological host defense. A highly sensitive target of oxidation in proteins is the thiol group of cysteines. While non-native oxidative thiol modifications most often lead to protein damage, in so-called redox-regulated proteins these modifications can quickly change the protein's structure and activity. Because thiol modifications in low oxidation states are reversible in vivo, they can be either directly reduced by thiol disulfide oxidoreductase systems or transferred to other cysteine containing proteins. This reversibility combined with the high specificity of oxidative thiol modifications to distinct reactive oxygen or nitrogen species enables thiol groups to act as molecular nano-switches that change the activity of proteins upon oxidation. Thus thiol-based redox reactions are the molecular foundation of redox regulation. In this present proposal, we plan to to study the role of thiol-based redox signaling in host pathogen interactions. We propose to focus particularly on the global quantitation of several different thiol modifications in parallel. Specifically, we want to develop a method that enables us to distinguish between nitrosative and oxidative modifications of thiol residues in E. coli proteins. Based on recently published observations that a) E. coli can, as a facultative anaerobe, use nitrate generated in inflammatory processes in the gut as terminal electron acceptor to his advantage and b) that endogenous S-Nitrosylation occurs as a signaling component specifically during these nitrate respiration conditions we hypothesize that redox signaling and S-nitrosylation in particular plays an important role in host pathogen interactions. To test our hypothesis, we plan to: 1. Use a new redox proteomics workflow, based on novel iodo-TMT probes to distinguish and quantify oxidative and nitrosative thiol modifications in the E. coli proteome under low oxygen/high nitrosating conditions, as they occur in the host in the inflamed gut. 2. Establish roGFP2-based probes in our lab, to real-time monitor redox changes in E. coli to guide our redox proteomics experiments in a host pathogen interaction setting. 3. Set up a co-cultivation assay of E. coli with a neutrophil-like cell line to study redox signaling in inflammation and phagocytosis.

 

Project details…

 

 

Riemer

Jan Riemer, Köln

 

Thiol-dependent redox regulation in the mitochondrial intermembrane space

Mitochondria are composed of four subcompartments, the outer and inner membranes, the matrix and the mitochondrial intermembrane space (IMS). IMS proteins fulfill a plethora of functions in protein import, folding and degradation, in apoptosis, the degradation of reactive oxygen species (ROS), the exchange of metabolites and the signaling from mitochondria to the remainder of the cell. In addition the IMS is a key hotspot for redox pathways. It harbors a highly reducing glutathione pool that is in contact with the cytosol, and it contains a number of redox-active enzymes most of them likely at very low amounts. Among them are glutaredoxins 1 and 2 (Grx1/2), thioredoxin 1 (Trx1), glutathione peroxidase 3 (Gpx3), superoxide dismutase 1 (Sod1) and the components of the mitochondrial disulfide relay, Mia40 and Erv1 that oxidatively fold many IMS proteins. Additionally, the IMS is exposed to ROS generated by the respiratory chain or redox cofactor-containing enzymes. The interplay of oxidizing and reducing factors has to be carefully controlled and balanced because deviations might influence or impair IMS-localized processes or redox signaling to the remainder of the cell. We recently identified one process that highlights the importance of IMS redox homeostasis: The disulfide relay machinery is controlled by carefully balanced amounts of IMS-localized Grx1/2. Both deletion and overexpression of Grx1/2 disturb the redox state of Mia40 and delay oxidative proteins folding. In the proposed project we aim to understand the IMS-specific functions of Grx1/2, Trx1 and Gpx3 and to elucidate which IMS proteins are regulated by changes in their thiol redox state. We will therefore first identify targets of redox regulation in the IMS of yeast and mammalian tissue culture cells. To this end, we will solve the interactome of IMS-localized Grx1/2, Trx1 and Gpx3, and additionally we will determine which IMS proteins change their redox state upon mitochondria-specific oxidative stress. Subsequently, we will characterize how the targets of redox regulation are controlled. In this context, we will not only focus on the target proteins but also on the small molecule milieu in the IMS (glutathione, H2O2, pH) and on the mechanisms of distribution of the dually localized Grx1/2, Trx1 and Gpx3 between cytosol and IMS. Additionally, we will in cooperation begin to establish conditional knockout models (fibroblasts and mice) to explore the physiological impact of changes in redox activity in the mitochondrial IMS. This project combines the identification of novel thiol switches and their mechanistic characterization with the application of genetically encoded small molecule sensors and in the future aims at unraveling the physiological significance of the identified thiol switches. We would thus not only greatly benefit from the DFG priority program SPP1710 but also expect to provide valuable contribution to other projects in the consortium.

 

Project details