Redox regulation of proteins via cysteine residue oxidation is involved in the control of various cellular signal pathways. Pyruvate kinase M2 (PKM2), a rate-limiting enzyme in glycolysis, is critical for the metabolic shift from glycolysis to the pentose phosphate pathway under oxidative stress in cancer cell growth. The PKM2 tetramer is required for optimal pyruvate kinase (PK) activity, whereas the inhibition of inter-subunit interaction of PKM2 induced by Cys358 oxidation has reduced PK activity. In the present study, we identified three oxidation-sensitive cysteine residues (Cys358, Cys423 and Cys424) responsible for four oxidation forms via the thiol oxidant diamide and/or hydrogen peroxide (H 2 O 2 ). Possibly due to obstruction of the dimer-dimer interface, H 2 O 2 -induced sulfenylation (-SOH) and diamide-induced modification at Cys424 inhibited tetramer formation and PK activity. Cys423 is responsible for intermolecular disulfide bonds with heterologous proteins via diamide. Additionally, intramolecular polysulphide linkage (–Sn–, n ≧ 3) between Cys358 and an unidentified PKM2 Cys could be induced by diamide. We observed that cells expressing the oxidation-resistant PKM2 (PKM2 C358,424A ) produced more intracellular reactive oxygen species (ROS) and exhibited greater sensitivity to ROS-generating reagents and ROS-inducible anti-cancer drugs compared with cells expressing wild-type PKM2. These results highlight the possibility that PKM2 inhibition via Cys358 and Cys424 oxidation contributes to eliminating excess ROS and oxidative stress.

The antagonism between thiol oxidation and reduction enables efficient control of protein function and is used as central mechanism in cellular regulation. The best-studied mechanism is the dithiol-disulfide transition in the Calvin Benson Cycle in photosynthesis, including mixed disulfide formation by glutathionylation. The adjustment of the proper thiol redox state is a fundamental property of all cellular compartments. The glutathione redox potential of the cytosol, stroma, matrix and nucleoplasm usually ranges between −300 and −320 mV. Thiol reduction proceeds by short electron transfer cascades consisting of redox input elements and redox transmitters such as thioredoxins. Thiol oxidation ultimately is linked to reactive oxygen species (ROS) and reactive nitrogen species (RNS). Enhanced ROS production under stress shifts the redox network to more positive redox potentials. ROS do not react randomly but primarily with few specific redox sensors in the cell. The most commonly encountered reaction within the redox regulatory network however is the disulfide swapping. The thiol oxidation dynamics also involves transnitrosylation. This review compiles present knowledge on this network and its central role in sensing environmental cues with focus on chloroplast metabolism.

Thiol-based redox regulation is crucial for adjusting chloroplast functions under fluctuating light environments. We recently discovered that the thioredoxin-like2 (TrxL2)/2-Cys peroxiredoxin (2CP) redox cascade supports oxidative thiol modulation by using hydrogen peroxide (H 2 O 2 ) as an oxidizing force. This system plays a key role in switching chloroplast metabolism (e.g. Calvin–Benson cycle) during light to dark transitions; however, information on its function is still limited. In this study, we report a novel protein-activation mechanism based on the TrxL2/2CP redox cascade. Glucose-6-phosphate dehydrogenase (G6PDH) catalyzes the first step of the oxidative pentose phosphate pathway (OPPP). Biochemical studies, including redox state determination and measurement of enzyme activity, suggested that the TrxL2/2CP pathway is involved in the oxidative activation of G6PDH. It is thus likely that the TrxL2/2CP redox cascade shifts chloroplast metabolism to night mode by playing a dual role, namely, down-regulation of the Calvin–Benson cycle and up-regulation of OPPP. G6PDH was also directly oxidized and activated by H 2 O 2 , particularly when H 2 O 2 concentration was elevated. Therefore, G6PDH is thought to be finely tuned by H 2 O 2 levels in both direct and indirect manners.

Glucose 6-phosphate dehydrogenase (G6PDH) catalyzes the first reaction in the oxidative pentose phosphate pathway. In green plant chloroplasts, G6PDH is a unique redox-regulated enzyme, since it is inactivated under the reducing conditions. This regulation is accomplished using a redox-active cysteine pair, which is conserved in plant G6PDH. The inactivation of this enzyme under conditions of light must be beneficial to prevent release of CO 2 from the photosynthetic carbon fixation cycle. In the filamentous, heterocyst-forming, nitrogen-fixing cyanobacterium Anabaena sp. PCC 7120 ( Anabaena 7120), G6PDH plays a pivotal role in providing reducing power for nitrogenase, and its activity is also reported to be suppressed by reduction, though Anabaena G6PDH does not conserve the critical cysteines for regulation. Based on the thorough analyses of the redox regulation mechanisms of G6PDH from Anabaena 7120 and its activator protein OpcA, we found that m -type thioredoxin regulates G6PDH activity by changing the redox states of OpcA. Mass spectrometric analysis and mutagenesis studies indicate that Cys 393 and Cys 399 of OpcA are responsible for the redox regulation property of this protein. Moreover, in vivo analyses of the redox states of OpcA showed that more than half of the OpcA is present as an oxidized form, even under conditions of light, when cells are cultured under the nitrogen-fixing conditions. This redox regulation of OpcA might be necessary to provide reducing power for nitrogenase by G6PDH in heterocysts even during the day.

Reactive sulfur species (RSS) modulate protein functions via S - polysulfidation of reactive Cys residues. Here, we report that Ca 2+ /calmodulin (CaM)-dependent protein kinase IV (CaMKIV) was reversibly inactivated by RSS via polysulfidation of the active-site Cys residue. CaMKIV is phosphorylated at Thr 196 by its upstream CaMK kinase (CaMKK), resulting in the induction of its full activity. In vitro incubation of CaMKIV with the exogenous RSS donors Na 2 S n ( n = 2–4) resulted in dose-dependent inhibition of the CaMKK-induced phospho-Thr 196 and consequent inactivation of the enzyme activity. Conversely, mutated CaMKIV (C198V) was refractory to the Na 2 S n -induced enzyme inhibition. A biotin-polyethylene glycol-conjugated maleimide capture assay revealed that Cys 198 in CaMKIV represents a target for S-polysulfidation. Furthermore, phosho-Thr 196 and CaMKIV activity were inhibited by incubation with cysteine hydropersulfide, a newly identified RSS that is generated from cystine by cystathionine-γ-lyase. In transfected cells expressing CaMKIV, ionomycin-induced CaMKIV phosphorylation at Thr 196 was decreased upon treatment with either Na 2 S 4 or the endoplasmic reticulum (ER) stress inducer thapsigargin, whereas cells expressing mutant CaMKIV (C198V) were resistant to this treatment. In addition, the ionomycin-induced phospho-Thr 196 of endogenous CaMKIV was also inhibited by treatment either with Na 2 S 4 or thapsigargin in Jurkat T lymphocytes. Taken together, these data define a novel signaling function for intracellular RSS in inhibiting CaMKIV activity via S - polysulfidation of its Cys 198 during the response to ER stress.

Kinase-mediated phosphorylation is a pivotal regulatory process in stomatal responses to stresses. Through a redox proteomics study, a sucrose non-fermenting 1-related protein kinase (SnRK2.4) was identified to be redox-regulated in Brassica napus guard cells upon abscisic acid treatment. There are six genes encoding SnRK2.4 paralogs in B. napus . Here, we show that recombinant BnSnRK2.4-1C exhibited autophosphorylation activity and preferentially phosphorylated the N-terminal region of B. napus slow anion channel (BnSLAC1-NT) over generic substrates. The in vitro activity of BnSnRK2.4-1C requires the presence of manganese (Mn 2+ ). Phosphorylation sites of autophosphorylated BnSnRK2.4-1C were mapped, including serine and threonine residues in the activation loop. In vitro BnSnRK2.4-1C autophosphorylation activity was inhibited by oxidants such as H 2 O 2 and recovered by active thioredoxin isoforms, indicating redox regulation of BnSnRK2.4-1C. Thiol-specific isotope tagging followed by mass spectrometry analysis revealed specific cysteine residues responsive to oxidant treatments. The in vivo activity of BnSnRK2.4-1C is inhibited by 15 min of H 2 O 2 treatment. Taken together, these data indicate that BnSnRK2.4-1C, an SnRK preferentially expressed in guard cells, is redox-regulated with potential roles in guard cell signal transduction.

Thiol-based redox regulation is considered to support light-responsive control of various chloroplast functions. The redox cascade via ferredoxin–thioredoxin reductase (FTR)/thioredoxin (Trx) has been recognized as a key to transmitting reducing power; however, Arabidopsis thaliana genome sequencing has revealed that as many as five Trx subtypes encoded by a total of 10 nuclear genes are targeted to chloroplasts. Because each Trx isoform seems to have a distinct target selectivity, the electron distribution from FTR to multiple Trxs is thought to be the critical branch point for determining the consequence of chloroplast redox regulation. In the present study, we aimed to comprehensively characterize the kinetics of electron transfer from FTR to 10 Trx isoforms. We prepared the recombinant FTR protein from Arabidopsis in the heterodimeric form containing the Fe–S cluster. By reconstituting the FTR/Trx system in vitro , we showed that FTR prepared here was enzymatically active and suitable for uncovering biochemical features of chloroplast redox regulation. A series of redox state determinations using the thiol-modifying reagent, 4-acetamido-4′-maleimidylstilbene-2,2′-disulfonate, indicated that all chloroplast Trx isoforms are commonly reduced by FTR; however, significantly different efficiencies were evident. These differences were apparently correlated with the distinct midpoint redox potentials among Trxs. Even when the experiments were performed under conditions of hypothetical in vivo stoichiometry of FTR and Trxs, a similar trend in distinguishable electron transfers was observed. These data highlight an aspect of highly organized circuits in the chloroplast redox regulation network.

Selenophosphate synthetase (SPS) was initially detected in bacteria and was shown to synthesize selenophosphate, the active selenium donor. However, mammals have two SPS paralogues, which are designated SPS1 and SPS2. Although it is known that SPS2 catalyses the synthesis of selenophosphate, the function of SPS1 remains largely unclear. To examine the role of SPS1 in mammals, we generated a Sps1 -knockout mouse and found that systemic SPS1 deficiency led to embryos that were clearly underdeveloped by embryonic day (E)8.5 and virtually resorbed by E14.5. The knockout of Sps1 in the liver preserved viability, but significantly affected the expression of a large number of mRNAs involved in cancer, embryonic development and the glutathione system. Particularly notable was the extreme deficiency of glutaredoxin 1 (GLRX1) and glutathione transferase Omega 1 (GSTO1). To assess these phenotypes at the cellular level, we targeted the removal of SPS1 in F9 cells, a mouse embryonal carcinoma (EC) cell line, which affected the glutathione system proteins and accordingly led to the accumulation of hydrogen peroxide in the cell. Furthermore, we found that several malignant characteristics of SPS1-deficient F9 cells were reversed, suggesting that SPS1 played a role in supporting and/or sustaining cancer. In addition, the overexpression of mouse or human GLRX1 led to a reversal of observed increases in reactive oxygen species (ROS) in the F9 SPS1/GLRX1-deficient cells and resulted in levels that were similar to those in F9 SPS1-sufficient cells. The results suggested that SPS1 is an essential mammalian enzyme with roles in regulating redox homoeostasis and controlling cell growth.

The glutathione/cysteine exporter CydDC maintains redox balance in Escherichia coli . A cydD mutant strain was used to probe the influence of CydDC upon reduced thiol export, gene expression, metabolic perturbations, intracellular pH homoeostasis and tolerance to nitric oxide (NO). Loss of CydDC was found to decrease extracytoplasmic thiol levels, whereas overexpression diminished the cytoplasmic thiol content. Transcriptomic analysis revealed a dramatic up-regulation of protein chaperones, protein degradation (via phenylpropionate/phenylacetate catabolism), β-oxidation of fatty acids and genes involved in nitrate/nitrite reduction. 1 H NMR metabolomics revealed elevated methionine and betaine and diminished acetate and NAD + in cydD cells, which was consistent with the transcriptomics-based metabolic model. The growth rate and ΔpH, however, were unaffected, although the cydD strain did exhibit sensitivity to the NO-releasing compound NOC-12. These observations are consistent with the hypothesis that the loss of CydDC-mediated reductant export promotes protein misfolding, adaptations to energy metabolism and sensitivity to NO. The addition of both glutathione and cysteine to the medium was found to complement the loss of bd -type cytochrome synthesis in a cydD strain (a key component of the pleiotropic cydDC phenotype), providing the first direct evidence that CydDC substrates are able to restore the correct assembly of this respiratory oxidase. These data provide an insight into the metabolic flexibility of E. coli , highlight the importance of bacterial redox homoeostasis during nitrosative stress, and report for the first time the ability of periplasmic low molecular weight thiols to restore haem incorporation into a cytochrome complex.

Brassinosteroid insensitive 1 (BR1)-associated receptor-like kinase 1 (BAK1) is spontaneously glutathionylated in vitro by GSSG or GSH plus H 2 O 2 . The glutaredoxin (GRX) AtGRXC2 (GRX C2) interacts with and can catalyse glutathionylation of the BAK1 in vitro . Potential redox control of BAK1 and a novel activity for plant GRXs are discussed.

The cardiac Ca 2+ release channel [ryanodine receptor type 2 (RyR2)] is modulated by thiol reactive agents, but the molecular basis of RyR2 modulation by thiol reagents is poorly understood. Cys 3635 in the skeletal muscle RyR1 is one of the most hyper-reactive thiols and is important for the redox and calmodulin (CaM) regulation of the RyR1 channel. However, little is known about the role of the corresponding cysteine residue in RyR2 (Cys 3602 ) in the function and regulation of the RyR2 channel. In the present study, we assessed the impact of mutating Cys 3602 (C 3602 A) on store overload-induced Ca 2+ release (SOICR) and the regulation of RyR2 by thiol reagents and CaM. We found that the C 3602 A mutation suppressed SOICR by raising the activation threshold and delayed the termination of Ca 2+ release by reducing the termination threshold. As a result, C 3602 A markedly increased the fractional Ca 2+ release. Furthermore, the C 3602 A mutation diminished the inhibitory effect of N-ethylmaleimide on Ca 2+ release, but it had no effect on the stimulatory action of 4,4′-dithiodipyridine (DTDP) on Ca 2+ release. In addition, Cys 3602 mutations (C 3602 A or C 3602 R) did not abolish the effect of CaM on Ca 2+ -release termination. Therefore, RyR2–Cys 3602 is a major site mediating the action of thiol alkylating agent N-ethylmaleimide, but not the action of the oxidant DTDP. Our data also indicate that residue Cys 3602 plays an important role in the activation and termination of Ca 2+ release, but it is not essential for CaM regulation of RyR2.

In chloroplasts, redox regulation of enzyme activities by TRXs (thioredoxins) allows the co-ordination of light/dark metabolisms such as the reductive (so-called Calvin–Benson) pathway and the OPPP (oxidative pentose phosphate pathway). Although the molecular mechanisms underlying the redox regulation of several TRX-regulated enzymes have been investigated in detail, only partial information was available for plastidial G6PDH (glucose-6-phosphate dehydrogenase) catalysing the first and rate-limiting step of the OPPP. In the present study, we investigated changes in catalytic and structural properties undergone by G6PDH1 from Arabidopsis thaliana upon treatment with TRX f1, the most efficient regulator of the enzyme that did not show a stable interaction with its target. We found that the formation of the regulatory disulfide bridge that leads to activation of the enzyme allows better substrate accessibility to the active site and strongly modifies the cofactor-binding properties. Structural modelling and data from biochemical and biophysical studies of site-directed mutant proteins support a mechanism in which the positioning/function of the highly conserved Arg 131 in the cofactor-binding site can be directly influenced by the redox state of the adjacent regulatory disulfide bridge. These findings constitute another example of modifications to catalytic properties of a chloroplastic enzyme upon redox regulation, but by a mechanism unique to G6PDH.

NO binds to the receptor sGC (soluble guanylyl cyclase), stimulating cGMP production. The NO–sGC–cGMP pathway is a key component in the cardiovascular system. Discrepancies in sGC activation and deactivation in vitro compared with in vivo have led to a search for endogenous factors that regulate sGC or assist in cellular localization. In our previous work, which identified Hsp (heat-shock protein) 70 as a modulator of sGC, we determined that PDI (protein disulfide-isomerase) bound to an sGC-affinity matrix. In the present study, we establish and characterize this interaction. Incubation of purified PDI with semi-purified sGC, both reduced and oxidized, resulted in different migration patterns on non-reducing Western blots indicating a redox component to the interaction. In sGC-infected COS-7 cells, transfected FLAG-tagged PDI and PDI CXXS (redox active site ‘trap mutant’) pulled down sGC. This PDI–sGC complex was resolved by reductant, confirming a redox interaction. PDI inhibited NO-stimulated sGC activity in COS-7 lysates, however, a PDI redox-inactive mutant PDI SXXS did not. Together, these data unveil a novel mechanism of sGC redox modulation via thiol-disulfide exchange. Finally, in SMCs (smooth muscle cells), endogenous PDI and sGC co-localize by in situ proximity ligation assay, which suggests biological relevance. PDI-dependent redox regulation of sGC NO sensitivity may provide a secondary control over vascular homoeostasis.

IP 3 R (IP 3 [inositol 1,4,5-trisphosphate] receptors) and ryanodine receptors are the most widely expressed intracellular Ca 2+ channels and both are regulated by thiol reagents. In DT40 cells stably expressing single subtypes of mammalian IP 3 R, low concentrations of thimerosal (also known as thiomersal), which oxidizes thiols to form a thiomercurylethyl complex, increased the sensitivity of IP 3 -evoked Ca 2+ release via IP 3 R1 and IP 3 R2, but inhibited IP 3 R3. Activation of IP 3 R is initiated by IP 3 binding to the IBC (IP 3 -binding core; residues 224–604) and proceeds via re-arrangement of an interface between the IBC and SD (suppressor domain; residues 1–223). Thimerosal (100 μM) stimulated IP 3 binding to the isolated NT (N-terminal; residues 1–604) of IP 3 R1 and IP 3 R2, but not to that of IP 3 R3. Binding of a competitive antagonist (heparin) or partial agonist (dimeric-IP 3 ) to NT1 was unaffected by thiomersal, suggesting that the effect of thimerosal is specifically related to IP 3 R activation. IP 3 binding to NT1 in which all cysteine residues were replaced by alanine was insensitive to thimerosal, so too were NT1 in which cysteine residues were replaced in either the SD or IBC. This demonstrates that thimerosal interacts directly with cysteine in both the SD and IBC. Chimaeric proteins in which the SD of the IP 3 R was replaced by the structurally related A domain of a ryanodine receptor were functional, but thimerosal inhibited both IP 3 binding to the chimaeric NT and IP 3 -evoked Ca 2+ release from the chimaeric IP 3 R. This is the first systematic analysis of the effects of a thiol reagent on each IP 3 R subtype. We conclude that thimerosal selectively sensitizes IP 3 R1 and IP 3 R2 to IP 3 by modifying cysteine residues within both the SD and IBC and thereby stabilizing an active conformation of the receptor.

Glutathione is a thiol-containing tripeptide that plays important roles in redox-related processes. The first step in glutathione biosynthesis is catalysed by γ-GCS (γ-glutamylcysteine synthetase). The crystal structure of Escherichia coli γ-GCS has revealed the presence of a disulfide bond. As the disulfide-bonding cysteine residues Cys 372 and Cys 395 are not well conserved among γ-GCS enzymes in this lineage, we have initiated a biochemical genetic strategy to investigate the functional importance of these and other cysteine residues. In a cysteine-free γ-GCS that was non-functional, suppressor analysis yielded combinations of cysteine and aromatic residues at the position of the disulfide bond, and one mutant that lacked any cysteine residues. Kinetic analysis of the wild-type and mutant enzymes revealed that the disulfide bond was not involved in determining the affinity of the enzyme towards its substrate, but had an important role in determining the stability of the protein, and its catalytic efficiency. We show that in vivo the γ-GCS enzyme can also exist in a reduced form and that the mutants lacking the disulfide bond show a decreased half-life. These results demonstrate a novel means of regulation of γ-GCS by the redox environment that works by an alteration in its stability.

ATP-hydrolysis and proton pumping by the V-ATPase (vacuolar proton-translocating ATPase) are subject to redox regulation in mammals, yeast and plants. Oxidative inhibition of the V-ATPase is ascribed to disulfide-bond formation between conserved cysteine residues at the catalytic site of subunit A. Subunits containing amino acid substitutions of one of three conserved cysteine residues of VHA-A were expressed in a vha-A null mutant background in Arabidopsis . In vitro activity measurements revealed a complete absence of oxidative inhibition in the transgenic line expressing VHA-A C256S, confirming that Cys 256 is necessary for redox regulation. In contrast, oxidative inhibition was unaffected in plants expressing VHA-A C279S and VHA-A C535S, indicating that disulfide bridges involving these cysteine residues are not essential for oxidative inhibition. In vivo data suggest that oxidative inhibition might not represent a general regulatory mechanism in plants.

TEM5 (tumour endothelial marker 5; also known as GPR124) is an adhesion G-protein-coupled receptor containing a cryptic RGD motif in its extracellular domain. TEM5 is expressed in endothelial cells and pericytes during angiogenesis. In the present paper, we report that thrombin mediates shedding of an N-terminal TEM5 fragment of 60 kDa (termed N60) containing the RGD motif in an open conformation. Thrombin directly cleaved rsTEM5 (recombinant soluble TEM5) 5 and 34 residues downstream of the RGD motif, resulting in formation of N60 and its C-terminal counterpart (termed C50). Interestingly, N60 derived from thrombin cleavage of rsTEM5 was covalently linked to C50 by disulfide bonds, whereas N60 shed from thrombin-treated cells was not associated with its membrane-bound C-terminal counterpart. Inhibition of the reducing function of cell-surface PDI (protein disulfide-isomerase) abrogated thrombin-induced N60 shedding. Conversely, addition of reduced PDI enhanced N60 shedding. Furthermore, thrombin cleavage of rsTEM5 was increased by reduced PDI and resulted in dissociation of the N60–C50 heterodimer. We conclude that PDI regulates thrombin-induced shedding of N60 and exposure of the TEM5 RGD motif by catalysing the reduction of crucial disulfide bonds of TEM5 on the cell surface. Binding of N60 to RGD-dependent integrins may modulate cellular functions such as adhesion and migration during angiogenesis.

In eukaryotes, disulfide bonds are formed in the endoplasmic reticulum, facilitated by the Ero1 (endoplasmic reticulum oxidoreductin 1) oxidase/PDI (protein disulfide-isomerase) system. Mammals have two ERO1 genes, encoding Ero1α and Ero1β proteins. Ero1β is constitutively expressed in professional secretory tissues and induced during the unfolded protein response. In the present work, we show that recombinant human Ero1β is twice as active as Ero1α in enzymatic assays. Ero1β oxidizes PDI more efficiently than other PDI family members and drives oxidative protein folding preferentially via the active site in the a ′ domain of PDI. Our results reveal that Ero1β oxidase activity is regulated by long-range disulfide bonds and that Cys 130 plays a critical role in feedback regulation. Compared with Ero1α, however, Ero1β is loosely regulated, consistent with its role as a more active oxidase when massive oxidative power is required.

The redox state of the photosynthetic electron transport chain acts as a critical sensing mechanism by regulating the transcription of key genes involved in the acclimation response to a change in the environment. In the present study we show that the small LuxR-type regulator PedR interacts with Trx (thioredoxin) to achieve photosynthetic electron-transport-dependent transcriptional regulation in the cyanobacterium Synechocystis sp. PCC 6803. TrxM, an isoform of Trx, was isolated as an interacting factor of PedR by pull-down assays. In vitro analysis revealed that the intermolecular disulfide bond formed between Cys 80 residues of the PedR homodimer was reduced by both TrxM and TrxX. It has been shown previously that, although PedR is active under low-light conditions, it becomes transiently inactivated following a shift to high-light conditions, with a concomitant conformational change [Nakamura and Hihara (2006) J. Biol. Chem. 281 , 36758–36766]. In the present study, we found that the conformational change of PedR and the change in the transcript level of its target gene were minimal when mutants of Synechocystis that lack ferredoxin–Trx reductase or NADPH–Trx reductase were exposed to high levels of light. These results indicate that the reduction of PedR by Trx causes transient inactivation of PedR upon the shift of cyanobacterial cells to high-light conditions.

ADAM17 (where ADAM is ‘a disintegrin and metalloproteinase’) can rapidly modulate cell-surface signalling events by the proteolytic release of soluble forms of proligands for cellular receptors. Many regulatory pathways affect the ADAM17 sheddase activity, but the mechanisms for the activation are still not clear. We have utilized a cell-based ADAM17 assay to show that thiol isomerases, specifically PDI (protein disulfide isomerase), could be responsible for maintaining ADAM17 in an inactive form. Down-regulation of thiol isomerases, by changes in the redox environment (for instance as elicited by phorbol ester modulation of mitochondrial reactive oxygen species) markedly enhanced ADAM17 activation. On the basis of ELISA binding studies with novel fragment antibodies against ADAM17 we propose that isomerization of the disulfide bonds in ADAM17, and the subsequent conformational changes, form the basis for the modulation of ADAM17 activity. The shuffling of disulfide bond patterns in ADAMs has been suggested by a number of recent adamalysin crystal structures, with distinct disulfide bond patterns altering the relative orientations of the domains. Such a mechanism is rapid and reversible, and the role of thiol isomerases should be investigated further as a potential factor in the redox regulation of ADAM17.

The ADAM disintegrin metalloproteinases (where ADAM is ‘a disintegrin and metalloproteinase’) are a family of transmembrane cell-surface proteins with essential roles in adhesion and proteolytic processing in all animals. The archetypal family member is ADAM17 {also known as TACE [TNFα (tumour necrosis factor α)-converting enzyme]}, which is involved in processing pro-TNFα and in the activation of ligands for the EGFR [EGF (epidermal growth factor) receptor], as well as cleavage of diverse cell-surface receptors and adhesion molecules. ADAM-mediated shedding is itself influenced via cell signalling pathways. In this issue of the Biochemical Journal , Willems et al. make the observation that phorbol ester activates shedding by ADAM17 by affecting the activity of PDI (protein disulfide isomerase). They propose that PDI maintains ADAM17 in an inactive ‘closed’ state and PMA stimulation generates ROS (reactive oxygen species) and thus an altered redox environment, which in turn inactivates PDI and allows ADAM17 to adopt an ‘open’ active conformation. This activation is accompanied by changes in disulfide bonds in the ADAM17 ectodomain. This is a novel and exciting finding that could help to unlock the actions of ADAM sheddases, as well as a host of other mechanisms that rely upon rapid alterations in protein conformation on the cell surface.

In the present study we report on the identification and characterization of three novel chloroplast-targeted DnaJ-like proteins CDJ3–5, which in addition to their J-domains contain bacterial-type ferredoxin domains. In sequence databases we could identify homologues of CDJ3–5 in green algae, moss and higher plants, but not in cyanobacteria. Phylogenetic analyses allowed us to distinguish two clades containing CDJ3/4 and CDJ5 that must have diverged early in the ancestor of the ‘green lineage’ and have further diversified later on. Molecular and biochemical analysis of CDJ3 and CDJ4 from Chlamydomonas reinhardtii revealed that both proteins are weakly expressed and appear to be localized to the stroma and to thylakoid membranes respectively. The low transcript levels of the CDJ3 and CDJ4 genes declined even further in the initial phase of heat shock, but CDJ3 transcript levels strongly increased after a dark-to-light shift. Accordingly, the Arabidopsis orthologue of CDJ5 was also found to be light-inducible and to be under strong circadian control. CDJ3 and CDJ4 proteins could both be expressed in Escherichia coli and had redox-active Fe–S clusters. In vitro cross-linking studies demonstrated that CDJ3 and CDJ4 interact with chloroplast ATP-bound HSP70B (heat-shock protein 70B), presumably as dimers, and immunoprecipitation studies showed that CDJ3/4 were also in a complex with HSP70B in Chlamydomonas cell extracts. Finally, CDJ3 was found in complexes with apparent molecular masses of approx. 550–2800 kDa, which appeared to contain RNA. We speculate that the CDJ3–5 proteins might represent redox switches that act by recruiting HSP70B for the reorganization of regulatory protein complexes.

In the present paper we show that distinct human RyR2 (ryanodine receptor type 2) inherited mutations expressed in mammalian cells exhibit either unaltered or increased FKBP12.6 (12.6 kDa FK506-binding protein) binding compared with the wild-type. Oxidizing conditions result in decreased FKBP12.6 binding, but to the same extent as for the wild-type. Our findings suggest that FKBP12.6 regulation of RyR2 is unlikely to be the primary defect in inherited arrhythmogenic cardiac disease.

The mechanisms of NO inhibition of CaMK [Ca 2+ /CaM (calmodulin)-dependent protein kinase] II activity were studied. In rat pituitary tumour GH3 cells, TRH [thyrotrophin (TSH)-releasing hormone]-stimulated phosphorylation of nNOS [neuronal NOS (NO synthase)] at Ser 847 was sensitive to an inhibitor of CaMKs, KN-93, and was enhanced by inhibition of nNOS with 7NI (7-nitroindazole). Enzyme activity of CaMKII following in situ treatment with 7NI was also increased. The in vitro activity of CaMKII was inhibited by co-incubation either with nNOS and L -arginine or with NO donors SNAP ( S -nitroso- N -acetyl- DL -penicillamine) and DEA-NONOate [diethylamine-NONOate (diazeniumdiolate)]. Once inhibited by these treatments, CaMKII was observed to undergo full reactivation on the addition of a reducing reagent, DTT (dithiothreitol). In transfected cells expressing CaMKII and nNOS, treatment with the calcium ionophore A23187 further revealed nNOS phosphorylation at Ser 847 , which was enhanced by 7NI and CaMKII S -nitrosylation. Mutated CaMKII (C6A), in which Cys 6 was substituted with an alanine residue, was refractory to 7NI-induced enhancement of nNOS phosphorylation or to CaMKII S -nitrosylation. Furthermore, we could identify Cys 6 as a direct target for S -nitrosylation of CaMKII using MS. In addition, treatment with glutamate caused an increase in CaMKII S -nitrosylation in rat hippocampal slices. This glutamate-induced S -nitrosylation was blocked by 7NI. These results suggest that inactivation of CaMKII mediated by S -nitrosylation at Cys 6 may contribute to NO-induced neurotoxicity in the brain.

The Saccharomyces cerevisiae genome encodes three proteins that display similarities with human GSTOs (Omega class glutathione S-transferases) hGSTO1-1 and hGSTO2-2. The three yeast proteins have been named Gto1, Gto2 and Gto3, and their purified recombinant forms are active as thiol transferases (glutaredoxins) against HED (β-hydroxyethyl disulphide), as dehydroascorbate reductases and as dimethylarsinic acid reductases, while they are not active against the standard GST substrate CDNB (1-chloro-2,4-dinitrobenzene). Their glutaredoxin activity is also detectable in yeast cell extracts. The enzyme activity characteristics of the Gto proteins contrast with those of another yeast GST, Gtt1. The latter is active against CDNB and also displays glutathione peroxidase activity against organic hydroperoxides such as cumene hydroperoxide, but is not active as a thiol transferase. Analysis of point mutants derived from wild-type Gto2 indicates that, among the three cysteine residues of the molecule, only the residue at position 46 is required for the glutaredoxin activity. This indicates that the thiol transferase acts through a monothiol mechanism. Replacing the active site of the yeast monothiol glutaredoxin Grx5 with the proposed Gto2 active site containing Cys 46 allows Grx5 to retain some activity against HED. Therefore the residues adjacent to the respective active cysteine residues in Gto2 and Grx5 are important determinants for the thiol transferase activity against small disulphide-containing molecules.

Tre6P (trehalose 6-phosphate) is implicated in sugar-signalling pathways in plants, but its exact functions in vivo are uncertain. One of the main obstacles to discovering these functions is the difficulty of measuring the amount of Tre6P in plant tissues. We have developed a highly specific assay, using liquid chromatography coupled to MS-Q3 (triple quadrupole MS), to measure Tre6P in the femto-picomole range. The Tre6P content of sucrose-starved Arabidopsis thaliana seedlings in axenic culture increased from 18 to 482 pmol·g −1 FW (fresh weight) after adding sucrose. Leaves from soil-grown plants contained 67 pmol·g −1 FW at the end of the night, which rose to 108 pmol·g −1 FW after 4 h of illumination. Even greater changes in Tre6P content were seen after a 6 h extension of the dark period, and in the starchless mutant, pgm . The intracellular concentration of Tre6P in wild-type leaves was estimated to range from 1 to 15 μM. It has recently been reported that the addition of Tre6P to isolated chloroplasts leads to redox activation of AGPase (ADPglucose pyrophosphorylase) [Kolbe, Tiessen, Schluepmann, Paul, Ulrich and Geigenberger (2005) Proc. Natl. Acad. Sci. U.S.A. 102 , 11118–11123]. Using the new assay for Tre6P, we found that rising sugar levels in plants are accompanied by increases in the level of Tre6P, redox activation of AGPase and the stimulation of starch synthesis in vivo . These results indicate that Tre6P acts as a signalling metabolite of sugar status in plants, and support the proposal that Tre6P mediates sucrose-induced changes in the rate of starch synthesis.

DREF [DRE (DNA replication-related element) binding factor] is an 80 kDa polypeptide homodimer which plays an important role in regulating cell proliferation-related genes. Both DNA binding and dimer formation activities are associated with residues 16–115 of the N-terminal region. However, the mechanisms by which DREF dimerization and DNA binding are regulated remain unknown. Here, we report that the DNA binding activity of DREF is regulated by a redox mechanism, and that the cysteine residues are involved in this regulation. Electrophoretic mobility shift analysis using Drosophila Kc cell extracts or recombinant DREF proteins indicated that the DNA binding domain is sufficient for redox regulation. Site-directed mutagenesis and transient transfection assays showed that Cys 59 and/or Cys 62 are critical both for DNA binding and for redox regulation, whereas Cys 91 is dispensable. In addition, experiments using Kc cells indicated that the DNA binding activity and function of DREF are affected by the intracellular redox state. These findings give insight into the exact nature of DREF function in the regulation of target genes by the intracellular redox state.

Peroxisomes are essential and ubiquitous cell organelles having a key role in mammalian lipid and oxygen metabolism. The presence of flavine oxidases makes them an important intracellular source of H 2 O 2 : an obligate product of peroxisomal redox reactions and a key reactive oxygen species. Peroxisomes proliferate in response to external signals triggered by peroxisome-proliferator-activated receptor signalling pathways. Peroxisome-derived oxidative stress as a consequence of this proliferation is increasingly recognized to participate in pathologies ranging from carcinogenesis in rodents to alcoholic and non-alcoholic steatosis hepatitis in humans. To date, no sensitive approach exists to record H 2 O 2 turnover of peroxisomes in real time. Here, we introduce a sensitive chemiluminescence method that allows the monitoring of H 2 O 2 generation and degradation in real time in suspensions of intact peroxisomes. Importantly, removal, as well as release of, H 2 O 2 can be assessed at nanomolar, non-toxic concentrations in the same sample. Owing to the kinetic properties of catalase and oxidases, H 2 O 2 forms fast steady-state concentrations in the presence of various peroxisomal substrates. Substrate screening suggests that urate, glycolate and activated fatty acids are the most important sources for H 2 O 2 in rodents. Kinetic studies imply further that peroxisomes contribute significantly to the β-oxidation of medium-chain fatty acids, in addition to their essential role in the breakdown of long and very long ones. These observations establish a direct quantitative release of H 2 O 2 from intact peroxisomes. The experimental approach offers new possibilities for functionally studying H 2 O 2 metabolism, substrate transport and turnover in peroxisomes of eukaryotic cells.

Here we report the localization of protein disulphide isomerase (PDI) in the mitochondrial compartments, comparing it with that of thioredoxin reductase. The latter enzyme is present mostly in the matrix, whereas PDI is located at the level of the outer membrane. We characterize the different submitochondrial fractions with specific marker enzymes. PDI, whether isolated from whole mitochondria or from purified outer membranes, exhibits the same electrophoretic mobility, indicating identical molecular masses. Moreover, immunoblot analysis with monoclonal anti-PDI antibody shows immunoreactivity only with the microsomal PDI, indicating the specificity of the mitochondrial isoform. The significance of these findings is discussed with reference to the potential role of PDI and thioredoxin reductase in regulating the mitochondrial functions dependent on the thiol–disulphide transition.

Redox modulation participates in the regulation of intracellular free calcium concentration ([Ca 2+ ] i ) in several cell types. In thyroid cells, including FRTL-5 cells, changes in [Ca 2+ ] i regulate several important functions, including the production of H 2 O 2 (hydrogen peroxide). As H 2 O 2 is of crucial importance for the production of thyroid hormones, we investigated the effects of H 2 O 2 on [Ca 2+ ] i in thyroid FRTL-5 cells. H 2 O 2 itself did not modulate basal [Ca 2+ ] i . However, H 2 O 2 attenuated store-operated calcium entry evoked by thapsigargin, both in a sodium-containing buffer and in a sodium-free buffer. The effect of H 2 O 2 was abrogated by the reducing agent β-mercaptoethanol. H 2 O 2 also attenuated the thapsigargin-evoked entry of barium and manganese. The effect of H 2 O 2 was, at least in part, mediated by activation of protein kinase C (PKC), as H 2 O 2 enhanced the binding of [ 3 H]phorbol 12,13-dibutyrate. H 2 O 2 also stimulated the translocation of the isoenzyme PKCε from the cytosolic fraction to the particulate fraction. Furthermore, H 2 O 2 did not attenuate store-operated calcium entry in cells treated with staurosporine or calphostin C, or in cells with down-regulated PKC. H 2 O 2 depolarized the membrane potential in bisoxonol-loaded cells and when patch-clamp in the whole-cell mode was used. The depolarization was attenuated in cells with down-regulated PKC. This depolarization, at least in part, explained the H 2 O 2 -evoked inhibition of calcium entry. In addition, H 2 O 2 enhanced the extrusion of calcium from cells stimulated with thapsigargin and this effect was abolished in cells with down-regulated PKC and after treatment of the cells with the reducing agent β-mercaptoethanol. In conclusion H 2 O 2 attenuates an increase in [Ca 2+ ] i . As H 2 O 2 is produced in thyroid cells in a calcium-dependent manner, our results suggest that H 2 O 2 may participate in the regulation of [Ca 2+ ] i in these cells via a negative-feedback mechanism involving activation of PKC.

The activity of the nuclear factor I/CCAAT transcription factor (NFI/CTF) is negatively regulated by oxidative stress. The addition of relatively high (millimolar) H 2 O 2 concentrations inactivates cellular NFI DNA-binding activity whereas lower concentrations can repress NFI/CTF transactivating function. We have investigated the mechanism of this regulation using Gal4 fusion proteins and transfection assays. We show that micromolar H 2 O 2 concentrations repress the transactivating domain of NFI/CTF in a dose-dependent manner and are less or not active on other transcription factors' transactivating domains. Studies using deletions and point mutations pointed to the critical role of Cys-427. Indeed, when this cysteine is mutated into a serine, the repression by H 2 O 2 is totally blunted. Mutation of other cysteine, serine and tyrosine residues within the transactivating domain had no clear effect on the repression by H 2 O 2 . Finally, treatment of cells with the thiol-alkylating reagent N -ethylmaleimide leads to a decrease in the transactivating function, which is dependent on Cys-427. This study shows that transactivating domains of transcription factors can constitute very sensitive targets of oxidative stress and highlights the critical role of these domains.

Human immunodeficiency viruses encode a homodimeric protease that is essential for the production of infectious virus. Previous studies have shown that HIV-1 protease is susceptible to oxidative inactivation at the dimer interface at Cys-95, a process that can be reversed both chemically and enzymically. Here we demonstrate a related yet distinct mechanism of reversible inactivation of the HIV-2 protease. Exposure of the HIV-2 protease to H 2 O 2 resulted in conversion of the two methionine residues (Met-76 and Met-95) to methionine sulphoxide as determined by amino acid analysis and mass spectrometry. This oxidation completely inactivated protease activity. However, the activity could be restored (up to 40%) after exposure of the oxidized protease to methionine sulphoxide reductase. This treatment resulted in the reduction of methionine sulphoxide 95 but not methionine sulphoxide 76 to methionine, as determined by peptide mapping/mass spectrometry. We also found that exposure of immature HIV-2 particles to H 2 O 2 led to the inhibition of polyprotein processing in maturing virus particles comparable to that demonstrated for HIV-1 particles. Thus oxidative inactivation of the HIV protease in vitro and in maturing viral particles is not restricted to the type 1 proteases. These studies indicate that two distinct retroviral proteases are susceptible to inactivation after a very minor modification at residue 95 of the dimer interface and suggest that the dimer interface might be a viable target for the development of novel protease inhibitors.

Mammalian thioredoxin reductase (TR) is a flavoprotein catalysing reduction of oxidized thioredoxin in an NADPH-dependent manner, and contains a selenocysteine (Sec) residue near the C-terminus. We observed that TR activity was decreased in A549 cells by the lowering of the fetal bovine serum content in the culture medium and was recovered by the addition of selenium. To study the role of Sec in TR activity, we have isolated a full-length clone of the rat TR cDNA (3.3 kb) and have expressed it in COS-1 cells in a transient-expression system. TR activities in COS-1 cells expressing rat TR were increased in accordance with supplemented sodium selenite concentrations, whereas levels of TR protein, examined by Western blotting, were not affected by sodium selenite concentrations. We introduced various deletions into the 3ʹ-untranslated region of the TR cDNA to localize and examine the role of a Sec insertion-sequence (SECIS) element in the functional expression of TR. TR activities were observed only in COS-1 cells transfected with the TR cDNAs containing the putative SECIS element located between 1856 and 1915 bp in the correct orientation. We also carried out radiolabelling of proteins by incubation of the cDNA-transfected cells with sodium [ 75 Se]selenite. 75 Se was incorporated into the expressed TR protein of the cells transfected with the SECIS element-containing cDNAs, but not into those without the SECIS element or with an inverted SECIS element. These data clearly showed a requirement of selenium for the formation of functional TR protein.

2-Carboxy- d -arabinitol 1-phosphate (CA1P) phosphatase de- grades CA1P, an inhibitor associated with the regulation of ribulose bisphosphate carboxylase/oxygenase in numerous plant species. CA1P phosphatase purified from Phaseolus vulgaris was partially inactivated by oxidizing conditions during dialysis in air-equilibrated buffer. Phosphatase activity could then be stimulated 1.3-fold by dithiothreitol and also by addition of reduced thioredoxin from Escherichia coli . These effects were enhanced synergistically by the positive effector, fructose 1,6-bisphosphate (FBP). Most notably, CA1P phosphatase activity was stimulated up to 35-fold by glutathione, and was sensitive to the ratio of reduced (GSH) to oxidized (GSSG) forms. At concentrations of glutathione approximating measured levels in chloroplasts of P. vulgaris (5 mM total S), CA1P phosphatase exhibited > 20-fold stimulation by a change in the redox status of glutathione from 60 to 100% GSH. This stimulation was augmented further by reduced E. coli thioredoxin. In contrast, FBP, which activates CA1P phosphatase under reducing conditions, was strongly inhibitory in the presence of GSSG. We propose that glutathione may have an appreciable role in the light/dark regulation of CA1P phosphatase in vivo . A model for the reversible activation of CA1P phosphatase by GSH was derived based upon the various responses of the enzyme's activity to a range of thiol reagents including N -ethylmaleimide, 5,5´-dithiobis-(2-nitrobenzoic acid) and arsenite. These data indicate that the bean enzyme contains two physically distinct sets of thiol groups that are critical to its redox regulation.