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    Abstract: Ion gradients across intracellular membranes contribute to the physicochemical environment inside compartments. CLC anion transport proteins that localise to intracellular organelles are anion-proton exchangers involved in anion sequestration or vesicular acidification. By homology, the only CLC protein of Saccharomyces cerevisiae, Gef1, belongs to this family of intracellular exchangers. Gef1 localises to the late Golgi and prevacuole and is essential in conditions of iron limitation. In the absence of Gef1, a multicopper oxidase involved in iron uptake, Fet3, fails to acquire copper ion cofactors. The precise role of the exchanger in this physiological context is unknown. Here, we show that the Gef1-containing compartment is adjusted to a more alkaline pH under iron limitation. This depends on the antiport function of Gef1, because an uncoupled mutant of Gef1 (E230A) results in the acidification of the lumen and fails to support Fet3 maturation. Furthermore, we found that Gef1 antiport activity correlates with marked effects on cellular glutathione homeostasis, raising the possibility that the effect of Gef1 on Fet3 copper loading is related to the control of compartmental glutathione concentration or redox status. Mutational inactivation of a conserved ATP-binding site in the cytosolic cystathione beta-synthetase domain of Gef1 (D732A) suggests that Gef1 activity is regulated by energy metabolism.
    Type of Publication: Journal article published
    PubMed ID: 20530571
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  • 3
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    Keywords: OXIDATIVE STRESS ; ENDOPLASMIC-RETICULUM ; EPIDERMAL-GROWTH-FACTOR ; DISULFIDE BOND FORMATION ; STRUCTURAL BASIS ; REACTIVE OXYGEN ; GLUTATHIONE BIOSYNTHESIS ; ARTERY SMOOTH-MUSCLE ; INTRACELLULAR HYDROGEN-PEROXIDE ; OXYR TRANSCRIPTION FACTOR
    Abstract: Abstract Redox biochemistry is increasingly recognized as an integral component of cellular signal processing and cell fate decision making. Unfortunately, our capabilities to observe and measure clearly defined redox processes in the natural context of living cells, tissues, or organisms are woefully limited. The most advanced and promising tools for specific, quantitative, dynamic and compartment-specific observations are genetically encoded redox probes derived from green fluorescent protein (GFP). Within only few years from their initial introduction, redox-sensitive yellow FP (rxYFP), redox-sensitive GFPs (roGFPs), and HyPer have generated enormous interest in applying these novel tools to monitor dynamic redox changes in vivo. As genetically encoded probes, these biosensors can be specifically targeted to different subcellular locations. A critical advantage of roGFPs and HyPer is their ratiometric fluorogenic behavior. Moreover, the probe scaffold of redox-sensitive fluorescent proteins (rxYFP and roGFPs) is amenable to molecular engineering, offering fascinating prospects for further developments. In particular, the engineering of redox relays between roGFPs and redox enzymes allows control of probe specificity and enhancement of sensitivity. Genetically encoded redox probes enable the functional analysis of individual proteins in cellular redox homeostasis. In addition, redox biosensor transgenic model organisms offer extended opportunities for dynamic in vivo imaging of redox processes. Antioxid. Redox Signal. 13, 000-000.
    Type of Publication: Journal article published
    PubMed ID: 20088706
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  • 4
    Keywords: brain ; ANGIOGENESIS ; ACID ; inactivation ; TUMOR-SUPPRESSOR PTEN ; HYDROGEN-SULFIDE ; SULFANE SULFUR ; 3-MERCAPTOPYRUVATE SULFURTRANSFERASE ; S-SULFHYDRATION ; MERCAPTOPYRUVATE
    Abstract: Aims: Hydrogen sulfide (H2S) is suggested to act as a gaseous signaling molecule in a variety of physiological processes. Its molecular mechanism of action was proposed to involve protein S-sulfhydration, that is, conversion of cysteinyl thiolates (Cys-S-) to persulfides (Cys-S-S-). A central and unresolved question is how H2S-that is, a molecule with sulfur in its lowest possible oxidation state (-2)-can lead to oxidative thiol modifications. Results: Using the lipid phosphatase PTEN as a model protein, we find that the "H2S donor" sodium hydrosulfide (NaHS) leads to very rapid reversible oxidation of the enzyme in vitro. We identify polysulfides formed in NaHS solutions as the oxidizing species, and present evidence that sulfane sulfur is added to the active site cysteine. Polysulfide-mediated oxidation of PTEN was induced by all "H2S donors" tested, including sodium sulfide (Na2S), gaseous H2S, and morpholin-4-ium 4-methoxyphenyl(morpholino) phosphinodithioate (GYY4137). Moreover, we show that polysulfides formed in H2S solutions readily modify PTEN inside intact cells. Innovation: Our results shed light on the previously unresolved question of how H2S leads to protein thiol oxidation, and suggest that polysulfides formed in solutions of H2S mediate this process. Conclusion: This study suggests that the effects that have been attributed to H2S in previous reports may in fact have been mediated by polysulfides. It also supports the notion that sulfane sulfur rather than sulfide is the actual in vivo agent of H2S signaling.
    Type of Publication: Journal article published
    PubMed ID: 23646934
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    Keywords: PEPTIDE ; Germany ; SYSTEM ; NEW-YORK ; MOLECULES ; COMPLEX ; COMPLEXES ; QUALITY ; REDUCTION ; ANTIGEN ; BINDING ; SUSCEPTIBILITY ; MOLECULE ; MHC ; QUALITY-CONTROL ; antigen presentation ; CLASS-I ; HLA CLASS-I ; MHC class I ; MHC CLASS-I ; LOADING COMPLEX ; ENDOPLASMIC-RETICULUM ; MAJOR HISTOCOMPATIBILITY COMPLEX ; beta(2)-microglobulin ; DISULFIDE BOND FORMATION ; OXIDOREDUCTASE ERP57 ; PEPTIDE-LOADING COMPLEX ; RE ; regulation ; IMMUNE-SYSTEM ; USA ; function ; immunology ; ERp57
    Abstract: The function of the oxidoreductase ERp57 in the major histocompatibility complex (MHC) class I peptide-loading complex has remained elusive. Here we show that in the absence of tapasin, the alpha(2) disulfide bond in the MHC class I peptide-binding groove was rapidly reduced. Covalent sequestration of ERp57 by tapasin was needed to protect the alpha(2) disulfide bond against reduction and thus to maintain the binding groove in a peptide-receptive state. Allelic variations in MHC class I tapasin dependency reflected their susceptibility to reduction of the alpha(2) disulfide bond. In the absence of sequestration, ERp57 acted directly on the alpha(2) disulfide bond. Our work provides insight into how the immune system customizes 'quality control' in the endoplasmic reticulum to fit the needs of antigen presentation
    Type of Publication: Journal article published
    PubMed ID: 17603488
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  • 7
    Keywords: RECEPTOR ; proliferation ; tumor ; CELL ; FACTOR RECEPTOR ; Germany ; INHIBITION ; PATHWAY ; PATHWAYS ; NEW-YORK ; SITE ; PROTEIN ; PROTEINS ; TUMOR-NECROSIS-FACTOR ; LIGAND ; RESPONSES ; MECHANISM ; BIOLOGY ; MEMBER ; MOLECULAR-BIOLOGY ; SIGNAL ; TARGET ; GLUTATHIONE ; LYMPHOCYTES ; SURFACE ; EXCHANGE ; SUPERFAMILY ; leukocyte ; INVOLVEMENT ; INTERACTS ; EFFECTOR ; signaling ; SINGLE ; CYTOKINE ; molecular biology ; molecular ; RE ; regulation ; REDOX STATE ; interaction ; REDOX REGULATION ; technique ; TUMOR NECROSIS FACTOR ; USA ; function ; SIGNALS ; NECROSIS ; mechanism of action ; CD30 ; DISULFIDE EXCHANGE ; GLUTAREDOXIN ; LEUKEMIA-DERIVED FACTOR ; NEOPLASTIC-CELLS ; thiol-disulfide exchange ; thioredoxin ; TNF receptor superfamily ; TNFR FAMILY
    Abstract: The thiol-disulfide oxidoreductase thioredoxin-1 (Trx1) is known to be secreted by leukocytes and to exhibit cytokine-like properties. Extracellular effects of Trx1 require a functional active site, suggesting a redox-based mechanism of action. However, specific cell surface proteins and pathways coupling extracellular Trx1 redox activity to cellular responses have not been identified so far. Using a mechanism-based kinetic trapping technique to identify disulfide exchange interactions on the intact surface of living lymphocytes, we found that Trx1 catalytically interacts with a single principal target protein. This target protein was identified as the tumor necrosis factor receptor superfamily member 8 (TNFRSF8/CD30). We demonstrate that the redox interaction is highly specific for both Trx1 and CD30 and that the redox state of CD30 determines its ability to engage the cognate ligand and transduce signals. Furthermore, we confirm that Trx1 affects CD30-dependent changes in lymphocyte effector function. Thus, we conclude that receptor-ligand signaling interactions can be selectively regulated by an extracellular redox catalyst
    Type of Publication: Journal article published
    PubMed ID: 17557078
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  • 8
    Keywords: RECEPTOR ; CANCER ; CELLS ; GROWTH ; proliferation ; tumor ; TUMOR-CELLS ; CELL ; FACTOR RECEPTOR ; Germany ; INHIBITION ; PATHWAY ; PATHWAYS ; NEW-YORK ; SITE ; ENZYMES ; PROTEIN ; PROTEINS ; TUMOR-NECROSIS-FACTOR ; ACTIVATION ; LIGAND ; RESPONSES ; MECHANISM ; BIOLOGY ; MEMBER ; MEMBERS ; MOLECULAR-BIOLOGY ; SIGNAL ; FORM ; PARTICLES ; TARGET ; IDENTIFICATION ; GLUTATHIONE ; MEMBRANE ; NUMBER ; FUSION ; LYMPHOCYTES ; SURFACE ; EXCHANGE ; SUPERFAMILY ; INTEGRIN ; leukocyte ; INVOLVEMENT ; INTERACTS ; TUMOR CELLS ; microenvironment ; PROTEOMICS ; EFFECTOR ; MEMBRANE-PROTEIN ; signaling ; SINGLE ; CYTOKINE ; molecular biology ; molecular ; RE ; MOTIF ; INCREASE ; regulation ; REDOX STATE ; interaction ; REDOX REGULATION ; ENZYME ; TUMOR-CELL ; technique ; TUMOR NECROSIS FACTOR ; USA ; function ; cancer research ; SIGNALS ; NECROSIS ; host ; e-mail ; viral ; mechanism of action ; German ; evidence ; VIRAL PARTICLES ; CD30 ; DISULFIDE EXCHANGE ; GLUTAREDOXIN ; LEUKEMIA-DERIVED FACTOR ; NEOPLASTIC-CELLS ; thiol-disulfide exchange ; thioredoxin ; TNF receptor superfamily ; TNFR FAMILY
    Abstract: Abstract: A number of thiol-dependent oxidoreductases are released from cells and act on the cell surface. Correspondingly, several cell-surface processes appear to depend on catalyzed thiol-disulfide exchange, including integrin activation and the fusion of viral particles with the host membrane. Tumor cells frequently increase the abundance of secreted and cell-surface forms of particular oxidoreductases, and evidence suggests that oxidoreductases released from tumor cells promote growth and contribute to the remodeling of the cellular microenvironment. Few cell-surface or membrane proteins that are targeted by extracellular redox enzymes have been identified. One major reason for this slow progress is the highly transient nature of thiol-disulfide exchange, making its detection by conventional techniques difficult or impossible. Here we describe the application of an activity-based proteomics approach, also known as "mechanism-based kinetic trapping," to identify individual cell-surface target proteins that engage in disulfide exchange with thiol-dependent oxidoreductases. Although we have applied this approach to thioredoxin-1, it should also be applicable to other members of the thioredoxin superfamily whose activity is based on the CXXC active-site motif.
    Type of Publication: Journal article published
    PubMed ID: 18089859
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  • 9
    Keywords: APOPTOSIS ; CELLS ; PROTEIN ; STRESS ; DAMAGE ; MITOCHONDRIA ; GEL-ELECTROPHORESIS ; free radicals ; DEHYDROGENASE ; REDOX REGULATION ; COMPLEX-I ; THIOREDOXIN SYSTEM ; BOVINE HEART-MITOCHONDRIA ; DT40 CELLS ; Electron transport chain ; Hydrogen peroxide ; Methionyl-tRNA synthetase ; Mitochondrial protein biosynthesis ; Thioredoxin 2
    Abstract: Reactive oxygen species (ROS) are released at the mitochondrial inner membrane by the electron transport chain (ETC). Increasing evidence suggests that mitochondrial H2O2 acts as a signaling molecule and participates in the (feedback) regulation of mitochondrial activity and turnover. It seems likely that key mitochondrial components contain redox-sensitive thiols that help to adapt protein function to changes in electron flow. However, the identity of most redox-regulated mitochondrial proteins remains to be defined. Thioredoxin 2 (Trx2) is the major protein-thiol-reducing oxidoreductase in the mitochondrial matrix. We used in situ mechanism-based kinetic trapping to identify disulfide-exchange interactions of Trx2 within functional mitochondria of intact cells. Mass spectrometry successfully identified known and suspected Trx2 target proteins and, in addition, revealed a set of new candidate target proteins. Our results suggest that the mitochondrial protein biosynthesis machinery is a major target of ETC-derived ROS. In particular, we identified mitochondrial methionyl-tRNA synthetase (mtMetRS) as one of the most prominent Trx2 target proteins. We show that an increase in ETC-derived oxidants leads to an increase in mtMetRS oxidation in intact cells. In conclusion, we find that in situ kinetic trapping provides starting points for future functional studies of intramitochondrial redox regulation.
    Type of Publication: Journal article published
    PubMed ID: 21295137
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  • 10
    Keywords: SACCHAROMYCES-CEREVISIAE ; DROSOPHILA-MELANOGASTER ; resistance ; OXIDATIVE STRESS ; REDUCTASE ; FLUORESCENT PROTEIN INDICATORS ; THIOREDOXIN SYSTEM ; OXIDIZED GLUTATHIONE ; S-GLUTATHIONYLATION ; EUKARYOTIC CYTOSOL
    Abstract: Glutathione is central to cellular redox chemistry. The majority of glutathione redox research has been based on the chemical analysis of whole-cell extracts, which unavoidably destroy subcellular compartment-specific information. Compartment-specific real-time measurements based on genetically encoded fluorescent probes now suggest that the cytosolic glutathione redox potential is about 100 mV more reducing than previously thought. Using these probes in yeast, we show that even during severe oxidative stress, the cytosolic glutathione disulfide (GSSG) concentration is much more tightly regulated than expected and provide a mechanistic explanation for the discrepancy with conventional measurements. GSSG that is not immediately reduced in the cytosol is rapidly transported into the vacuole by the ABC-C transporter Ycf1. The amount of whole-cell GSSG is entirely dependent on Ycf1 and uninformative about the cytosolic glutathione pool. Applying these insights, we identify Trx2 and Grx2 as efficient backup systems to glutathione reductase for cytosolic GSSG reduction.
    Type of Publication: Journal article published
    PubMed ID: 23242256
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