Role of GSH and Iron-Sulfur Glutaredoxins in Iron Metabolism—Review

Glutathione (GSH) was initially identified and characterized for its redox properties and later for its contributions to detoxification reactions. Over the past decade, however, the essential contributions of glutathione to cellular iron metabolism have come more and more into focus. GSH is indispensable in mitochondrial iron-sulfur (FeS) cluster biosynthesis, primarily by co-ligating FeS clusters as a cofactor of the CGFS-type (class II) glutaredoxins (Grxs). GSH is required for the export of the yet to be defined FeS precursor from the mitochondria to the cytosol. In the cytosol, it is an essential cofactor, again of the multi-domain CGFS-type Grxs, master players in cellular iron and FeS trafficking. In this review, we summarize the recent advances and progress in this field. The most urgent open questions are discussed, such as the role of GSH in the export of FeS precursors from mitochondria, the physiological roles of the CGFS-type Grx interactions with BolA-like proteins and the cluster transfer between Grxs and recipient proteins

Molecular architecture of Streptococcus pneumoniae surface thioredoxin-fold lipoproteins crucial for extracellular oxidative stress resistance and maintenance of virulence.

19 pags, 8 figs, tabsThe respiratory pathogen Streptococcus pneumoniae has evolved efficient mechanisms to resist oxidative stress conditions and to displace other bacteria in the nasopharynx. Here we character ize at physiological, functional and structural levels two novel surface-exposed thioredoxin-family lipoproteins, Etrx1 and Etrx2. The impact of both Etrx proteins and their r edox partner methionine sulfoxide reductase SpMsrAB2 on pneumococcal pathogenesis was assessed in mouse virulence studies and phagocytosis assays. The results demonstrate that loss of function of either both Etrx proteins or SpMsrAB2 dramatically attenuated pneumococcal virulence in the acute mouse pneumonia model and that Etrx proteins compensate each other. The deficiency of Etrx proteins or SpMsrAB2 further enhanced bacterial uptake by macrophages, and accelerated pneumococcal killing by H2O2 or free methionine sulfoxides (MetSO). Moreover, the absence of both Etrx redox pathways provokes an accumulation of oxidized SpMsrAB2 in vivo. Taken together our results reveal insights into the role of two extracellular electron pathways required for reduction of SpMsrAB2 and surface-exposed MetSO. Identification of this system and its target proteins paves the w ay for the design of novel a ntimicrobialsThe authors thank the PXIII beamline at SLS and the ESRF beamline ID14‐1 for access to synchrotron radiation. We are also grateful to Kristine Sievert‐Giermann, Nadine Gotzmann and Melanie Skibbe (Department of Genetics, University of Greifswald, Germany) for technical assistance. This work was supported by grants from the Deutsche Forschungsgemeinschaft DFG HA3125/4‐2 (to S.H.), DFG AN746/3‐1 (to H.A.), BFU2011‐25326 and S2010/BMD‐2457 (to J.A.H.) and EU FP7 CAREPNEUMO Grant EU‐CP223111 from the European Union (to J.A.H. and S.H.

A thiol-disulphide switch in the regulation of cytoskeletal dynamics

Numerous signalling pathways orchestrate the development, the functions, and the survival of cells, mostly in response to external stimuli. An overwhelming amount of data supports the concept of specific, spatio-temporal redox signalling pathways that affect the redox state of protein cysteinyl side chains and thus the biological function of these proteins. Glutaredoxins (Grxs) and thioredoxins (Trxs) catalyse reversible thiol-disulphide exchange reactions. The cytosolic Grx2 isoform Grx2c is essential for brain development and axonal outgrowth. A reversible dithiol-disulphide switch of CRMP2 has been identified as one of the major targets regulated by Grx2c. This CRMP2 redox switch is toggled in neuronal differentiation. Reduction of CRMP2 thiols induces profound conformational changes, modifying interactions and downstream elements of this redox switch. In [article I] and [manuscript V], we identified the Cys504 of CRMP2 to be the redox regulated residue. We used various in vitro assays with recombinant protein and molecular dynamics simulations to characterise the conformational change. The changes involve the solvent accessible surface area of at least one known phosphorylation site at the C-terminus of the protein. In [article III], we analysed the function of Grx2 and Trx1 in a model for perinatal asphyxia. Trx family proteins exhibit a very complex, cell-type and tissue specific expression pattern following hypoxia/ischemia and reoxygenation, especially Trx1 and Grx2. The results imply the clinical relevance for both proteins in perinatal asphyxia as well as many other neurological disorders. In agreement with the results presented in [articleI], Grx2 may be required for the re-establishment of neuronal integrity and connectivity. Cell shape, all forms of intracellular transport, and cell movement depend on the cytoskeleton, particularly on the fine tuned complex regulation of the dynamic re-arrangement of actin filaments and microtubules. In [article IV], we discuss the redox regulation of this dynamic cytoskeletal remodelling. Taking recent discoveries into account, we focus on redox signalling mechanisms, e.g. reversible thiol and methionyl switches. These switches are specifically controlled by enzymes such as Trx1 and Grx2c, for instance, and not the result of random modification by unspecific oxidants. Methionyl sulphoxidation of actin can be reversed by methionyl sulphoxide reductase (MsrA), promoting actin polymerisation. Human cells express two different Msr enzymes (MsrA and MsrB), that can reduce S- and R-methionyl sulphoxide, respectively. In the gram-positive Streptococcus pneumoniae, on the other hand, both Msr genes and thus enzymes were fused during evolution. In [article II], we characterised the surface-exposed thioredoxin family lipoproteins Etrx1 and 2 and regulators of this Msr (SpMsrAB). A loss of function of both Etrx proteins or SpMsrAB dramatically reduced pneumococcal virulence, enhanced the bacterial uptake by macrophages, and accelerated pneumococcal killing by H2O2 or free methionine sulphoxide. Identification and characterisation of components of this redox regulated system may contribute to the design of new antimicrobials. In [manuscript VI], we investigated the effects of Grx2c expression on cell morphology, migration, and invasion behaviour of cancer cells. Grx2c expressing cancer cells developed dramatic changes in phenotype, including alterations in cytoskeletal dynamics and significantly increased motility and invasiveness. We used quantitative proteomics and phopshoproteomic approaches to characterise the underlying mechanisms. Proteins and pathways regulating cytoskeletal dynamics, cell adhesion, and receptor-mediated signal transduction were detected to be specifically altered. We started a clinical pilot study with patients suffering from clear cell renal cell carcinoma (ccRCC). Grx2c was expressed with significantly higher frequency in ccRCC compared to healthy kidney tissue, associated with a strong trend for locally more advanced tumour stages and a clear tendency for a decreased cancer-specific survival, compared to patients without detectable Grx2c. These results were supported by data from "The Cancer Genome Atlas". In synopsis, the results presented and discussed in these articles and manuscripts, support the concept of specific redox signalling in different models and model organisms. They also demonstrate the importance of the specific redox control of signalling pathways that, in the case of errors or misinterpretations, contribute to pathophysiological alterations. The regulation of the CRMP2 redox switch by Grx2c, for instance, is physiologically essential for brain development, but might lead to cancer progression, if "switched on" in adult tissue. Identification of further interaction partners as well as the development of compounds modulating this redox switch and CRMP2s conformations, will be part of our future research.Zahllose Signalwege bestimmen die Entwicklung, die Funktion und das Überleben von Zellen. Eine überwältigende Menge an Daten stützt das Konzept spezifischer Redox-Signalwege, die den Redoxstatus von cysteinyl Seitenketten von Proteinen beeinflussen und somit deren biologische Funktion ändern. Glutaredoxine (Grxe) und Thioredoxine (Trxe) katalysieren reversible Thiol-Disulfid-Austauschreaktionen. Die zytosolische Grx2 Isoform Grx2c ist essentiell für die Gehirnentwicklung und das Auswachsen von Axonen. Ein reversibler Thiol-Disulfid-Schalter in CRMP2, welcher in der neuronalen Differenzierung reguliert wird, konnte als wichtiges Ziel von Grx2c identifiziert werden. Die Reduktion der Thiole in CRMP2 führt zu einer umfassenden Konformationsänderung, welche die Interaktionen und nachgeschalteten Elemente des Redox-Schalters modifiziert. In [Artikel I] und [Manuskript V] haben wir CRMP2‘s Cys504 als redoxregulierten Rest identifiziert. Wir nutzten verschiedene in vitro Analysen mit rekombinantem Protein und Molekulardynamische Simulationen um die Konformationsänderung zu charakterisieren. Die Veränderungen betreffen auch die Lösemittel-zugängliche Oberfläche von mindestens einer bekannten Phosphorylierungsstelle am C-Terminus des Proteins. In [Artikel III] analysierten wir die Funktion von Grx2 und Trx1 in einem Modell für perinatale Asphyxie. Proteine der Trx-Familie zeigen ein sehr komplexes, Zelltyp und Gewebe spezifisches Expressionsmuster nach Hypoxie/Ischämie und Reoxygenierung, vor allem Grx2 und Trx1. Die Ergebnisse lassen eine klinische Relevanz beider Proteine auch in vielen anderen neurologischen Erkrankungen vermuten. Grx2 ist vermutlich notwendig für die Wiederherstellung der neuronalen Integrität und Konnektivität, übereinstimmend mit den in [Artikel I] präsentierten Ergebnissen. Zellform, intrazellulärer Transport und Zellbewegung hängen vom Zytoskelett ab, vor allem von der genau abgestimmten und komplex regulierten Reorganisation von Aktinfilamenten und Mikrotubuli. Wir diskutieren die Redoxregulation der dynamischen Zytoskelett Umformung in [Artikel IV]. Der Fokus liegt auf Redox-Signalmechanismen, wie z.B. reversible Thiol und Methionin Schalter, welche durch Enzyme, z.B. Trx1 und Grx2, spezifisch reguliert werden und keine zufälligen Modifikationen durch unspezifische Oxidationsmittel sind. Methionylsulfoxide in Aktin können durch die Methionylsulfoxid Reduktase (Msr) reduziert werden, was die Aktinpolymerisation fördert. Humane Zellen exprimieren zwei verschieden Msr Enzyme (MsrA/MsrB) während bei Streptococcus pneumoniae beide Msr Gene und ebenso die Enzyme während der Evolution verschmolzen sind. In [Artikel II] charakterisieren wir die Oberflächen-exponierten Lipoproteine Etrx1 und 2 als Regulatoren dieser Msr (SpMsrAB). Ein Funktionsverlust beider Etrx Proteine oder von SpMsrAB reduzierte die Virulenz der Pneumokokken drastisch, erhöhte die Aufnahme durch Makrophagen und beschleunigte das Abtöten durch H2O2 oder freies Methioninsulfoxid. Die Identifikation und Charakterisierung von Komponenten dieses redoxregulierten Systems können zur Entwicklung neuer antimikrobieller Stoffe beitragen. Im [Manuskript VI] untersuchten wir die Effekte der Grx2c Expression in Krebszellen. Grx2c exprimierende Zellen entwickelten drastische Veränderungen des Phänotyps, inklusive Veränderungen der Zytoskelettdynamik und signifikant erhöhter Beweglichkeit und Invasivität. Mit Hilfe quantitativer Proteomics und Phosphoproteomics charakterisierten wir die zugrunde liegenden Mechanismen. Vor allem Proteine und Signalwege, welche die Zytoskelettdynamik, Zelladhäsion und Rezeptor-vermittelte Signaltransduktion regulieren, waren verändert. In einer klinischen Pilotstudie mit Patienten, welche an klarzelligem Nierenzellkarzinom (ccRCC) erkrankt sind, fanden wir Grx2c Expression häufiger in ccRCC verglichen mit gesundem Nierengewebe. Es zeigte sich zudem ein Trend zu fortgeschrittenen Tumorstadien und eine klare Tendenz zu einer geringeren Krebs-spezifischen Überlebensrate in Patienten mit detektierbarem Grx2c verglichen zu denen ohne. Diese Ergebnisse werden von Daten aus „The Cancer Genome Atlas“ gestützt. Zusammenfassend unterstützen die Ergebnisse, welche in den Artikeln und Manuskripten dieser Arbeit präsentiert und diskutiert wurden, das Konzept der spezifischen Redox-Signalwege in verschiedenen Modellen und Modellorganismen. Sie demonstrieren wie wichtig die spezifische Redox-Kontrolle von Signalwegen ist, welche im Fall von Fehlern oder Missinterpretationen, zu pathologischen Veränderungen beitragen. Die Regulation des CRMP2 Redox-Schalters durch Grx2c ist essenziell für die Gehirnentwicklung, trägt aber möglicherweise zu Krebsentwicklung bei, wenn er im Gewebe eines Erwachsenen „eingeschaltet“ wird. Die Identifizierung weiterer Interaktionspartner sowie die Entwicklung von Stoffen die diesen Redox-Schalter und damit die Konformation von CRMP2 modulieren sind Bestandteil unserer zukünftigen Forschung

Breakdown of Arabidopsis thaliana thioredoxins and glutaredoxins based on electrostatic similarity-Leads to common and unique interaction partners and functions.

The reversible reduction and oxidation of protein thiols was first described as mechanism to control light/dark-dependent metabolic regulation in photosynthetic organisms. Today, it is recognized as an essential mechanism of regulation and signal transduction in all kingdoms of life. Proteins of the thioredoxin (Trx) family, Trxs and glutaredoxins (Grxs) in particular, catalyze thiol-disulfide exchange reactions and are vital players in the operation of thiol switches. Various Trx and Grx isoforms are present in all compartments of the cell. These proteins have a rather broad but at the same time distinct substrate specificity. Understanding the molecular basis of their target specificity is central to the understanding of physiological and pathological redox signaling. Electrostatic complementarity of the redoxins with their target proteins has been proposed as a major reason. Here, we analyzed the electrostatic similarity of all Arabidopsis thaliana Trxs, Grxs, and proteins containing such domains. Clustering of the redoxins based on this comparison suggests overlapping and also distant target specificities and thus functions of the different sub-classes including all Trx isoforms as well as the three classes of Grxs, i.e. CxxC-, CGFS-, and CC-type Grxs. Our analysis also provides a rationale for the tuned substrate specificities of both the ferredoxin- and NADPH-dependent Trx reductases

Substrate specificity of thioredoxins and glutaredoxins–towards afunctional classification

The spatio-temporal reduction and oxidation of protein thiols is an essential mechanism in signal transduction inall kingdoms of life. Thioredoxin (Trx) family proteins efficiently catalyze thiol-disulfide exchange reactions andthe proteins are widely recognized for their importance in the operation of thiol switches. Trx family proteinshave a broad and at the same time very distinct substrate specificity–a prerequisite for redox switching. Despiteof multiple efforts, the true nature for this specificity is still under debate. Here, we comprehensively compare theclassification/clustering of various redoxins from all domains of life based on their similarity in amino acidsequence, tertiary structure, and their electrostatic properties. We correlate these similarities to the existence ofcommon interaction partners, identified in various previous studies and suggested by proteomic screenings. Theseanalyses confirm that primary and tertiary structure similarity, and thereby all common classification systems, donot correlate to the target specificity of the proteins as thiol-disulfide oxidoreductases. Instead, a number ofexamples clearly demonstrate the importance of electrostatic similarity for their target specificity, independent oftheir belonging to the Trx or glutaredoxin subfamilie

Molecular Basis for the Interactions of Human Thioredoxins with Their Respective Reductases

The mammalian cytosolic thioredoxin (Trx) system consists of Trx1 and its reductase, the NADPH-dependent seleno-enzyme TrxR1. These proteins function as electron donor for metabolic enzymes, for instance in DNA synthesis, and the redox regulation of numerous processes. In this work, we analysed the interactions between these two proteins. We proposed electrostatic complementarity as major force controlling the formation of encounter complexes between the proteins and thus the efficiency of the subsequent electron transfer reaction. If our hypothesis is valid, formation of the encounter complex should be independent of the redox reaction. In fact, we were able to confirm that also a redox inactive mutant of Trx1 lacking both active site cysteinyl residues (C32,35S) binds to TrxR1 in a similar manner and with similar kinetics as the wild-type protein. We have generated a number of mutants with alterations in electrostatic properties and characterised their interaction with TrxR1 in kinetic assays. For human Trx1 and TrxR1, complementary electrostatic surfaces within the area covered in the encounter complex appear to control the affinity of the reductase for its substrate Trx. Electrostatic compatibility was even observed in areas that do not form direct molecular interactions in the encounter complex, and our results suggest that the electrostatic complementarity in these areas influences the catalytic efficiency of the reduction. The human genome encodes ten cytosolic Trx-like or Trx domain-containing proteins. In agreement with our hypothesis, the proteins that have been characterised as TrxR1 substrates also show the highest similarity in their electrostatic properties

Nucleoredoxin Plays a Key Role in the Maintenance of Retinal Pigmented Epithelium Differentiation

Nucleoredoxin (Nrx) belongs to the Thioredoxin protein family and functions in redox-mediated signal transduction. It contains the dithiol active site motif Cys-Pro-Pro-Cys and interacts and regulates different proteins in distinct cellular pathways. Nrx was shown to be catalytically active in the insulin assay and recent findings indicate that Nrx functions, in fact, as oxidase. Here, we have analyzed Nrx in the mammalian retina exposed to (perinatal) hypoxia-ischemia/reoxygenation, combining ex vivo and in vitro models. Our data show that Nrx regulates cell differentiation, which is important to (i) increase the number of glial cells and (ii) replenish neurons that are lost following the hypoxic insult. Nrx is essential to maintain cell morphology. These regulatory changes are related to VEGF but do not seem to be linked to the Wnt/β-catenin pathway, which is not affected by Nrx knock-down. In conclusion, our results strongly suggest that hypoxia-ischemia could lead to alterations in the organization of the retina, related to changes in RPE cell differentiation. Nrx may play an essential role in the maintenance of the RPE cell differentiation state via the regulation of VEGF release

Thioredoxin 1 and glutaredoxin 2 contribute to maintain the phenotype and integrity of neurons following perinatal asphyxia

Background Thioredoxin (Trx) family proteins are crucial mediators of cell functions via regulation of the thiol redox state of various key proteins and the levels of the intracellular second messenger hydrogen peroxide. Their expression, localization and functions are altered in various pathologies. Here, we have analyzed the impact of Trx family proteins in neuronal development and recovery, following hypoxia/ischemia and reperfusion. Methods We have analyzed the regulation and potential functions of Trx family proteins during hypoxia/ischemia and reoxygenation of the developing brain in both an animal and a cellular model of perinatal asphyxia. We have analyzed the distribution of 14 Trx family and related proteins in the cerebellum, striatum, and hippocampus, three areas of the rat brain that are especially susceptible to hypoxia. Using SH-SY5Y cells subjected to hypoxia and reoxygenation, we have analyzed the functions of some redoxins suggested by the animal experiment. Results and conclusions We have described/discovered a complex, cell-type and tissue-specific expression pattern following the hypoxia/ischemia and reoxygenation. Particularly, Grx2 and Trx1 showed distinct changes during tissue recovery following hypoxia/ischemia and reoxygenation. Silencing of these proteins in SH-SY5Y cells subjected to hypoxia-reoxygenation confirmed that these proteins are required to maintain the normal neuronal phenotype. General significance These findings demonstrate the significance of redox signaling in cellular pathways. Grx2 and Trx1 contribute significantly to neuronal integrity and could be clinically relevant in neuronal damage following perinatal asphyxia and other neuronal disorders.Fil: Romero, Juan Ignacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Cardiológicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Cardiológicas; ArgentinaFil: Hanschmann, Eva Maria. Universitätsmedizin Greifswald; AlemaniaFil: Gellert, Manuela. Universitätsmedizin Greifswald; AlemaniaFil: Eitner, Susanne. Universitätsmedizin Greifswald; AlemaniaFil: Holubiec, Mariana Ines. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Cardiológicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Cardiológicas; ArgentinaFil: Blanco Calvo, Eduardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Cardiológicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Cardiológicas; Argentina. Universidad de Lleida; EspañaFil: Lillig, Christopher Horst. Universitätsmedizin Greifswald; AlemaniaFil: Capani, Francisco. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Cardiológicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Cardiológicas; Argentina. Universidad Argentina "John F. Kennedy"; Argentin

Thioredoxin 1 and glutaredoxin 2 contribute to maintain the phenotype and integrity of neurons following perinatal asphyxia

BACKGROUND: Thioredoxin (Trx) family proteins are crucial mediators of cell functions via regulation of the thiol redox state of various key proteins and the levels of the intracellular second messenger hydrogen peroxide. Their expression, localization and functions are altered in various pathologies. Here, we have analyzed the impact of Trx family proteins in neuronal development and recovery, following hypoxia/ischemia and reperfusion. METHODS: We have analyzed the regulation and potential functions of Trx family proteins during hypoxia/ischemia and reoxygenation of the developing brain in both an animal and a cellular model of perinatal asphyxia. We have analyzed the distribution of 14 Trx family and related proteins in the cerebellum, striatum, and hippocampus, three areas of the rat brain that are especially susceptible to hypoxia. Using SH-SY5Y cells subjected to hypoxia and reoxygenation, we have analyzed the functions of some redoxins suggested by the animal experiment. RESULTS AND CONCLUSIONS: We have described/discovered a complex, cell-type and tissue-specific expression pattern following the hypoxia/ischemia and reoxygenation. Particularly, Grx2 and Trx1 showed distinct changes during tissue recovery following hypoxia/ischemia and reoxygenation. Silencing of these proteins in SH-SY5Y cells subjected to hypoxia-reoxygenation confirmed that these proteins are required to maintain the normal neuronal phenotype. GENERAL SIGNIFICANCE: These findings demonstrate the significance of redox signaling in cellular pathways. Grx2 and Trx1 contribute significantly to neuronal integrity and could be clinically relevant in neuronal damage following perinatal asphyxia and other neuronal disorder