Generation Of Disulfide Bonds Research Articles

Reducing oxidative protein folding alleviates senescence by minimizing ER-to-nucleus H2 O2 release.

Oxidative protein folding occurs in the endoplasmic reticulum (ER) to generate disulfide bonds, and the by-product is hydrogen peroxide (H2 O2 ). However, the relationship between oxidative protein folding and senescence remains uncharacterized. Here, we find that the protein disulfide isomerase (PDI), a key oxidoreductase that catalyzes oxidative protein folding, accumulated in aged human mesenchymal stem cells (hMSCs) and deletion of PDI alleviated hMSCs senescence. Mechanistically, knocking out PDI slows the rate of oxidative protein folding and decreases the leakage of ER-derived H2 O2 into the nucleus, thereby decreasing the expression of SERPINE1, which was identified as a key driver of cell senescence. Furthermore, we show that depletion of PDI alleviated senescence in various cell models of aging. Our findings reveal a previously unrecognized role of oxidative protein folding in promoting cell aging, providing a potential target for aging and aging-related disease intervention.

Impact of salt and homogenization on yield, aggregation, and mechanical characteristics of wheat gluten separated by batter procedure

We investigated the effects of salt addition (0%, 1%, 2%, 3%) and homogenization speed (4000, 6000, 8000, 10,000 rpm) on the yield and aggregation characteristics of gluten fractions separated through a laboratory-scale batter procedure. The gluten yield was linearly correlated with the A-starch yield and increased with salt concentration and the homogenization rate. Adding salt and increasing the homogenization rate reduced the surface hydrophobicity and enhanced the hydration of gluten. The difference in the water-binding capacity of gluten, A- and B-starch granules is the basis for the centrifugal separation. The increase in gluten water-binding capacity promotes the separation of the three phases. Meanwhile, hydration shifted the secondary structure of gluten to β-turn, which enhanced its tensile resistance. In the presence of salt and mechanical forces, free sulfhydryl groups undergo cross-linking reactions to generate disulfide bonds, leading to the polymerization of glutenin macropolymer and the increase of gliadin in SDS-extractable proteins.

Ultrathin organic polymer with p-π conjugated structure for simultaneous photocatalytic disulfide bond generation and CO2 reduction

Combining photocatalytic organic reactions with CO2 reduction is an efficient solar energy utilization mode, but it is still limited by the organic species that can be matched and the low conversion. Herein, ultrathin organic polymer with p-π conjugated structure (TPP) was rationally designed and prepared, and showed a high yield of CO (15.2 mmol g−1) and conversion of SS coupled products (100%), far exceeding the organic polymer with PO structure. The enhanced photoredox activity of TPP is ascribed to the orbital interaction between the p-orbital on phosphorus and the π-orbitals of aromatic, which can accelerate the photoinduced charge carrier separation and improve the CO2 adsorption capacity. TPP can also be used for the dehydrocoupling of various benzyl mercaptans to the corresponding SS bond products. This work provides a new concept for the efficient synthesis of disulfide bonds combined with CO2 reduction in a photoreaction system.

C8J_1298, a bifunctional thiol oxidoreductase of Campylobacter jejuni, affects Dsb (disulfide bond) network functioning.

Posttranslational generation of disulfide bonds catalyzed by bacterial Dsb (disulfide bond) enzymes is essential for the oxidative folding of many proteins. Although we now have a good understanding of the Escherichia coli disulfide bond formation system, there are significant gaps in our knowledge concerning the Dsb systems of other bacteria, including Campylobacter jejuni, a food-borne, zoonotic pathogen. We attempted to gain a more complete understanding of the process by thorough analysis of C8J_1298 functioning in vitro and in vivo. C8J_1298 is a homodimeric thiol-oxidoreductase present in wild type (wt) cells, in both reduced and oxidized forms. The protein was previously described as a homolog of DsbC, and thus potentially should be active in rearrangement of disulfides. Indeed, biochemical studies with purified protein revealed that C8J_1298 shares many properties with EcDsbC. However, its activity in vivo is dependent on the genetic background, namely, the set of other Dsb proteins present in the periplasm that determine the redox conditions. In wt C. jejuni cells, C8J_1298 potentially works as a DsbG involved in the control of the cysteine sulfenylation level and protecting single cysteine residues from oxidation to sulfenic acid. A strain lacking only C8J_1298 is indistinguishable from the wild type strain by several assays recognized as the criteria to determine isomerization or oxidative Dsb pathways. Remarkably, in C. jejuni strain lacking DsbA1, the protein involved in generation of disulfides, C8J_1298 acts as an oxidase, similar to the homodimeric oxidoreductase of Helicobater pylori, HP0231. In E. coli, C8J_1298 acts as a bifunctional protein, also resembling HP0231. These findings are strongly supported by phylogenetic data. We also showed that CjDsbD (C8J_0565) is a C8J_1298 redox partner.

Endoplasmic reticulum–resident protein 57 (ERp57) oxidatively inactivates human transglutaminase 2

Transglutaminase 2 (TG2) is a ubiquitously expressed, intracellular as well as extracellular protein with multiple modes of post-translational regulation, including an allosteric disulfide bond between Cys-370-Cys-371 that renders the enzyme inactive in the extracellular matrix. Although recent studies have established that extracellular TG2 is switched "on" by the redox cofactor protein thioredoxin-1 (TRX), it is unclear how TG2 is switched "off." Here, we demonstrate that TG2 oxidation by small-molecule biological oxidants, including glutathione, cystine, and hydrogen peroxide, is unlikely to be the inactivation mechanism. Instead, endoplasmic reticulum (ER)-resident protein 57 (ERp57), a protein in the ER that promotes folding of nascent proteins and is also present in the extracellular environment, has the cellular and biochemical characteristics for inactivating TG2. We found that ERp57 colocalizes with extracellular TG2 in cultured human umbilical vein endothelial cells (HUVECs). ERp57 oxidized TG2 with a rate constant that was 400-2000-fold higher than those of the aforementioned small molecule oxidants. Moreover, its specificity for TG2 was also markedly higher than those of other secreted redox proteins, including protein disulfide isomerase (PDI), ERp72, TRX, and quiescin sulfhydryl oxidase 1 (QSOX1). Lastly, siRNA-mediated ERp57 knockdown in HUVECs increased TG2-catalyzed transamidation in the extracellular environment. We conclude that, to the best of our knowledge, the disulfide bond switch in human TG2 represents the first example of a post-translational redox regulatory mechanism that is reversibly and allosterically modulated by two distinct proteins (ERp57 and TRX).

Evidence against Stable Protein S-Nitrosylation as a Widespread Mechanism of Post-translational Regulation

SummaryS-nitrosation, commonly referred to as S-nitrosylation, is widely regarded as a ubiquitous, stable post-translational modification that directly regulates many proteins. Such a widespread role would appear to be incompatible with the inherent lability of the S-nitroso bond, especially its propensity to rapidly react with thiols to generate disulfide bonds. As anticipated, we observed robust and widespread protein S-nitrosation after exposing cells to nitrosocysteine or lipopolysaccharide. Proteins detected using the ascorbate-dependent biotin switch method are typically interpreted to be directly regulated by S-nitrosation. However, these S-nitrosated proteins are shown to predominantly comprise transient intermediates leading to disulfide bond formation. These disulfides are likely to be the dominant end effectors resulting from elevations in nitrosating cellular nitric oxide species. We propose that S-nitrosation primarily serves as a transient intermediate leading to disulfide formation. Overall, we conclude that the current widely held perception that stable S-nitrosation directly regulates the function of many proteins is significantly incorrect.

Endoplasmic reticulum stress regulates glutathione metabolism and activities of glutathione related enzymes in Arabidopsis.

Stress conditions generate an extra load on protein folding machinery in the endoplasmic reticulum (ER) and if the ER cannot overcome this load, unfolded proteins accumulate in the ER lumen, causing ER stress. ER lumen localised protein disulfide isomerase (PDI) catalyses the generation of disulfide bonds in conjugation with ER oxidoreductase1 (ERO1) during protein folding. Mismatched disulfide bonds are reduced by the conversion of GSH to GSSG. Under prolonged ER stress, GSH pool is oxidised and H2O2 is produced via increased activity of PDI-ERO1. However, it is not known how glutathione metabolism is regulated under ER stress in plants. So, in this study, ER stress was induced with tunicamycin (0.15, 0.3, 0.45μg mL-1 Tm) in Arabidopsis thaliana (L.) Heynh. Glutathione content was increased by ER stress, which was accompanied by induction of glutathione biosynthesis genes (GSH1, GSH2). Also, the apoplastic glutathione degradation pathway (GGT1) was induced. Further, the activities of glutathione reductase (GR), dehydroascorbate reductase (DHAR), glutathione peroxidase (GPX) and glutathione S-transferase (GST) were increased under ER stress. Results also showed that chloroplastic GPX genes were specifically downregulated with ER stress. This is the first report on regulation of glutathione metabolism and glutathione related enzymes in response to ER stress in plants.

86 - Are S-Nitrosothiols Predominantly Stable End-Effectors of Protein Function or Transient Intermediates Leading to Disulfide Formation?

S-nitrosation is considered to be a ubiquitous, stable post-translational modification that directly targets and modulates a vast number of proteins. This widespread role appears incompatible with the inherent lability of the nitroso bond and its propensity to rapidly react with thiols to generate disulfide bonds. We present evidence that S-nitrosothiols primarily exist as transient intermediates leading to disulfides. Exposure of primary rat aortic smooth muscle cells (SMCs) to the nitrosating reagent S-nitrosocysteine (CysNO) generated robust and widespread protein S-nitrosation. However, CysNO also induced disulfides in multiple candidate proteins which preceded a significant increase in widespread protein S-nitrosation. Quantitative analysis of SMCs exposed to CysNO demonstrated that disulfides, not S-nitrosothiols, were the dominant modification that accumulated. S-glutathiolation and S-cysteinylation were significantly increased after exposure of SMCs to biotinylated forms of CysNO or S-nitrosoglutathione respectively. 10 μM CysNO depleted non-protein thiols, predominantly reduced glutathione, 7-fold as measured by the Ellman's assay. Furthermore, this intervention also reduced the activity of NAPDH-dependent enzymes (MTS assay) 2-fold, which together indicates CysNO compromises the cells reducing system (p

N-glycosylation-negative catalase: a useful tool for exploring the role of hydrogen peroxide in the endoplasmic reticulum

Disulfide bond formation during protein folding of nascent proteins is associated with the generation of H2O2 in the endoplasmic reticulum (ER). Approaches to quantifying H2O2 directly within the ER failed because of the oxidative environment in the ER lumen, and ER-specific catalase expression to detoxify high H2O2 concentrations resulted in an inactive protein owing to N-glycosylation. Therefore, the N-glycosylation motifs at asparagine-244 and -439 of the human catalase protein were deleted by site-directed mutagenesis. The ER-targeted expression of these variants revealed that the deletion of the N-glycosylation motif only at asparagine-244 (N244) was associated with the maintenance of full enzymatic activity in the ER. Expression of catalase N244 in the ER (ER-Catalase N244) was ER-specific and protected the cells significantly against exogenously added H2O2. With the expression of ER-Catalase N244, a highly effective H2O2 inactivation within the ER was achieved for the first time. Catalase has a high H2O2-inactivation capacity without the need of reducing cofactors, which might interfere with the ER redox homeostasis, and is not involved in protein folding. With these characteristics ER-Catalase N244 is an ideal tool to explore the impact of ER-generated H2O2 on the generation of disulfide bonds or to study the induction of ER-stress pathways through protein folding overload and accumulation of H2O2.

Going through the Barrier: COUPLED DISULFIDE EXCHANGE REACTIONS PROMOTE EFFICIENT CATALYSIS IN QUIESCIN SULFHYDRYL OXIDASE

The quiescin sulfhydryl oxidase (QSOX) family of enzymes generates disulfide bonds in peptides and proteins with the reduction of oxygen to hydrogen peroxide. Determination of the potentials of the redox centers in Trypanosoma brucei QSOX provides a context for understanding catalysis by this facile oxidant of protein thiols. The CXXC motif of the thioredoxin domain is comparatively oxidizing (E'0 of -144 mV), consistent with an ability to transfer disulfide bonds to a broad range of thiol substrates. In contrast, the proximal CXXC disulfide in the ERV (essential for respiration and vegetative growth) domain of TbQSOX is strongly reducing (E'0 of -273 mV), representing a major apparent thermodynamic barrier to overall catalysis. Reduction of the oxidizing FAD cofactor (E'0 of -153 mV) is followed by the strongly favorable reduction of molecular oxygen. The role of a mixed disulfide intermediate between thioredoxin and ERV domains was highlighted by rapid reaction studies in which the wild-type CGAC motif in the thioredoxin domain of TbQSOX was replaced by the more oxidizing CPHC or more reducing CGPC sequence. Mixed disulfide bond formation is accompanied by the generation of a charge transfer complex with the flavin cofactor. This provides thermodynamic coupling among the three redox centers of QSOX and avoids the strongly uphill mismatch between the formal potentials of the thioredoxin and ERV disulfides. This work identifies intriguing mechanistic parallels between the eukaryotic QSOX enzymes and the DsbA/B system catalyzing disulfide bond generation in the bacterial periplasm and suggests that the strategy of linked disulfide exchanges may be exploited in other catalysts of oxidative protein folding.

Disulfide bond generation in mammalian blood serum: detection and purification of quiescin-sulfhydryl oxidase

A sensitive new plate-reader assay has been developed showing that adult mammalian blood serum contains circulating soluble sulfhydryl oxidase activity that can introduce disulfide bonds into reduced proteins with the reduction of oxygen to hydrogen peroxide. The activity was purified 5000-fold to >90% homogeneity from bovine serum and found by mass spectrometry to be consistent with the short isoform of quiescin-sulfhydryl oxidase 1 (QSOX1). This FAD-dependent enzyme is present at comparable activity levels in fetal and adult commercial bovine sera. Thus cell culture media that are routinely supplemented with either fetal or adult bovine sera will contain this facile catalyst of protein thiol oxidation. QSOX1 is present at approximately 25nM in pooled normal adult human serum. Examination of the unusual kinetics of QSOX1 toward cysteine and glutathione at low micromolar concentrations suggests that circulating QSOX1 is unlikely to significantly contribute to the oxidation of these monothiols in plasma. However, the ability of QSOX1 to rapidly oxidize conformationally mobile protein thiols suggests a possible contribution to the redox status of exofacial and soluble proteins in blood plasma. Recent proteomic studies showing that plasma QSOX1 can be utilized in the diagnosis of pancreatic cancer and acute decompensated heart failure, together with the overexpression of this secreted enzyme in a number of solid tumors, suggest that the robust QSOX assay developed here may be useful in the quantitation of enzyme levels in a wide range of biological fluids.

The multiple antibiotic resistance regulator MarR is a copper sensor in Escherichia coli

The widely conserved multiple antibiotic resistance regulator (MarR) family of transcription factors modulates bacterial detoxification in response to diverse antibiotics, toxic chemicals or both. The natural inducer for Escherichia coli MarR, the prototypical transcription repressor within this family, remains unknown. Here we show that copper signaling potentiates MarR derepression in E. coli. Copper(II) oxidizes a cysteine residue (Cys80) on MarR to generate disulfide bonds between two MarR dimers, thereby inducing tetramer formation and the dissociation of MarR from its cognate promoter DNA. We further discovered that salicylate, a putative MarR inducer, and the clinically important bactericidal antibiotics norfloxacin and ampicillin all stimulate intracellular copper elevation, most likely through oxidative impairment of copper-dependent envelope proteins, including NADH dehydrogenase-2. This membrane-associated copper oxidation and liberation process derepresses MarR, causing increased bacterial antibiotic resistance. Our study reveals that this bacterial transcription regulator senses copper(II) as a natural signal to cope with stress caused by antibiotics or the environment.

Solid-State NMR Study of a 41 kDa Membrane Protein Complex DsbA/DsbB

The disulfide bond generation system in E. coli is led by a periplasmic protein, DsbA, and an integral membrane protein, DsbB. Here we present a solid-state NMR (SSNMR) study of a 41 kDa membrane protein complex DsbA/DsbB precipitated in the presence of native lipids to investigate conformational changes and dynamics that occur upon transient complex formation within the electron transfer pathway. Chemical shift changes in the periplasmic enzyme DsbA in three states (wild type, C33S mutant, and in complex with DsbB) reveal structural and/or dynamic information. We report a 4.9 ppm (15)N chemical shift change observed for Pro31 in the active site between the wild type and C33S mutant of DsbA. Additionally, the Pro31 residue remains elusive in the DsbA/DsbB complex, indicating that the dynamics change drastically in the active site between the three states of DsbA. Using three-dimensional SSNMR spectra, partial (13)C and (15)N de novo chemical shift assignments throughout DsbA in the DsbA/DsbB complex were compared with the shifts from DsbA alone to map site-specific chemical shift perturbations. These results demonstrate that there are further structural and dynamic changes of DsbA in the native membrane observed by SSNMR, beyond the differences between the crystal structures of DsbA and the DsbA/DsbB complex.

Diversification of Quiescin sulfhydryl oxidase in a preserved framework for redox relay

BackgroundThe enzyme family Quiescin Sulfhydryl Oxidase (QSOX) is defined by the presence of an amino-terminal thioredoxin-fold (Trx) domain and a carboxy-terminal Erv family sulfhydryl oxidase domain. QSOX enzymes, which generate disulfide bonds and transfer them to substrate proteins, are present in a wide variety of eukaryotic species including metazoans and plants, but are absent from fungi. Plant and animal QSOXs differ in their active-site amino acid sequences and content of non-catalytic domains. The question arises, therefore, whether the Trx-Erv fusion has the same mechanistic significance in all QSOX enzymes, and whether shared features distinguish the functional domains of QSOX from other instances in which these domains occur independently. Through a study of QSOX phylogeny and an analysis of QSOX sequence diversity in light of recently determined three-dimensional structures, we sought insight into the origin and evolution of this multi-domain redox alliance.ResultsAn updated collection of QSOX enzymes was used to confirm and refine the differences in domain composition and active-site sequence motif patterns of QSOXs belonging to various eukaryotic phyla. Beyond the expected phylogenetic distinction of animal and plant QSOX enzymes, trees based on individual redox-active QSOX domains show a particular distinction of the Trx domain early in plant evolution. A comparison of QSOX domains with Trx and Erv domains from outside the QSOX family revealed several sequence and structural features that clearly differentiate QSOXs from other enzymes containing either of these domains. Notably, these features, present in QSOXs of various phyla, localize to the interface between the Trx and Erv domains observed in structures of QSOX that model interdomain redox communication.ConclusionsThe infrastructure for interdomain electron relay, previously identified for animal and parasite QSOXs, is found broadly across the QSOX family, including the plant enzymes. We conclude that the conserved three-dimensional framework of the QSOX catalytic domains accommodates lineage-specific differences and paralog diversification in the amino acid residues surrounding the redox-active cysteines. Our findings indicate that QSOX enzymes are characterized not just by the presence of the two defining domain folds but also by features that promote coordinated activity.

Oxidative Protein-Folding Systems in Plant Cells

Plants are unique among eukaryotes in having evolved organelles: the protein storage vacuole, protein body, and chloroplast. Disulfide transfer pathways that function in the endoplasmic reticulum (ER) and chloroplasts of plants play critical roles in the development of protein storage organelles and the biogenesis of chloroplasts, respectively. Disulfide bond formation requires the cooperative function of disulfide-generating enzymes (e.g., ER oxidoreductase 1), which generate disulfide bonds de novo, and disulfide carrier proteins (e.g., protein disulfide isomerase), which transfer disulfides to substrates by means of thiol-disulfide exchange reactions. Selective molecular communication between disulfide-generating enzymes and disulfide carrier proteins, which reflects the molecular and structural diversity of disulfide carrier proteins, is key to the efficient transfer of disulfides to specific sets of substrates. This review focuses on recent advances in our understanding of the mechanisms and functions of the various disulfide transfer pathways involved in oxidative protein folding in the ER, chloroplasts, and mitochondria of plants.

Protein Substrate Discrimination in the Quiescin Sulfhydryl Oxidase (QSOX) Family

This work explores the substrate specificity of the quiescin sulfhydryl oxidase (QSOX) family of disulfide-generating flavoenzymes to provide enzymological context for investigation of the physiological roles of these facile catalysts of oxidative protein folding. QSOX enzymes are generally unable to form disulfide bonds within well-structured proteins. Use of a temperature-sensitive mutant of ubiquitin-conjugating enzyme 4 (Ubc4') as a model substrate shows that QSOX activity correlates with the unfolding of Ubc4' monitored by circular dichroism. Fusion of Ubc4' with the more stable glutathione-S-transferase domain demonstrates that QSOX can selectively introduce disulfides into the less stable domain of the fusion protein. In terms of intermolecular disulfide bond generation, QSOX is unable to cross-link well-folded globular proteins via their surface thiols. However, the construction of a septuple mutant of RNase A, retaining a single cysteine residue, demonstrates that flexible protein monomers can be directly coupled by the oxidase. Steady- and pre-steady-state kinetic experiments, combined with static fluorescence approaches, indicate that while QSOX is an efficient catalyst for disulfide bond formation between mobile elements of structure, it does not appear to have a significant binding site for unfolded proteins. These aspects of protein substrate discrimination by QSOX family members are rationalized in terms of the stringent steric requirements for disulfide exchange reactions.

Balanced Ero1 activation and inactivation establishes ER redox homeostasis

The endoplasmic reticulum (ER) provides an environment optimized for oxidative protein folding through the action of Ero1p, which generates disulfide bonds, and Pdi1p, which receives disulfide bonds from Ero1p and transfers them to substrate proteins. Feedback regulation of Ero1p through reduction and oxidation of regulatory bonds within Ero1p is essential for maintaining the proper redox balance in the ER. In this paper, we show that Pdi1p is the key regulator of Ero1p activity. Reduced Pdi1p resulted in the activation of Ero1p by direct reduction of Ero1p regulatory bonds. Conversely, upon depletion of thiol substrates and accumulation of oxidized Pdi1p, Ero1p was inactivated by both autonomous oxidation and Pdi1p-mediated oxidation of Ero1p regulatory bonds. Pdi1p responded to the availability of free thiols and the relative levels of reduced and oxidized glutathione in the ER to control Ero1p activity and ensure that cells generate the minimum number of disulfide bonds needed for efficient oxidative protein folding.

The Physiological Functions of Mammalian Endoplasmic Oxidoreductin 1: On Disulfides and More

The oxidative process of disulfide-bond formation is essential for the folding of most secretory and membrane proteins in the endoplasmic reticulum (ER). It is driven by electron relay pathways that transfer two electrons derived from the fusion of two adjacent cysteinyl side chains onto various types of chemical oxidants. The conserved, ER-resident endoplasmic oxidoreductin 1 (Ero1) sulfhydryl oxidases that reduce molecular oxygen to generate an active-site disulfide represent one of these pathways. In mammals, two family members exist, Ero1α and Ero1β. The two mammalian Ero1 enzymes differ in transcriptional and post-translational regulation, tissue distribution, and catalytic turnover. A specific protein-protein interaction between either isoform and protein disulfide isomerase (PDI) facilitates the propagation of disulfides from Ero1 via PDI to nascent polypeptides, and inbuilt oxidative shutdown mechanisms in Ero1α and Ero1β prevent excessive oxidation of PDI. Besides disulfide-bond generation, Ero1α also regulates calcium release from the ER and the secretion of disulfide-linked oligomers through its reversible association with the chaperone ERp44. This review explores the functional repertoire and possible redundancy of mammalian Ero1 enzymes. Systematic analyses of different knockout mouse models will be the most promising strategy to shed new light on unique and tissue-specific roles of Ero1α and Ero1β. Moreover, in-depth characterization of the known physical interactions of Ero1 with peroxidases and PDI family members will help broaden our functional and mechanistic understanding of Ero1 enzymes.

Nitroxyl (HNO) acutely activates the glucose uptake activity of GLUT1

Nitroxyl (HNO) is a molecule of significant interest due to its unique pharmacological properties, particularly within the cardiovascular system. A large portion of HNO biological effects can be attributed to its reactivity with protein thiols, where it can generate disulfide bonds. Evidence from studies in erythrocytes suggests that the activity of GLUT1 is enhanced by the formation of an internal disulfide bond. However, there are no reports that document the effects of HNO on glucose uptake. Therefore, we examined the acute effects of Angeli’s salt (AS), a HNO donor, on glucose uptake activity of GLUT1 in L929 fibroblast cells. We report that AS stimulates glucose uptake with a maximum effective concentration of 5.0 mM. An initial 7.2-fold increase occurs within 2 min, which decreases and plateaus to a 4.0-fold activation after 10 min. About 60% of the 4.0-fold activation recovers within 10 min, and 40% remains after an hour. The activation is blocked by the pretreatment of cells with thiol-reactive compounds, iodoacetamide (0.75 mM), cinnamaldehyde (2.0 mM), and phenylarsine oxide (10 μM). The effects of AS are not additive to the stimulatory effects of other acute activators of glucose uptake in L929 cells, such as azide (5 mM), berberine (50 μM), or glucose deprivation. These data suggest that GLUT1 is acutely activated in L929 cells by the formation of a disulfide bond, likely within GLUT1 itself.

Solid-State NMR Study of the Charge-Transfer Complex between Ubiquinone-8 and Disulfide Bond Generating Membrane Protein DsbB

Ubiquinone (Coenzyme Q) plays an important role in the mitochondrial respiratory chain and also acts as an antioxidant in its reduced form, protecting cellular membranes from peroxidation. De novo disulfide bond generation in the E. coli periplasm involves a transient complex consisting of DsbA, DsbB, and ubiquinone (UQ). It is hypothesized that a charge-transfer complex intermediate is formed between the UQ ring and the DsbB-C44 thiolate during the reoxidation of DsbA, which gives a distinctive ~500 nm absorbance band. No enzymological precedent exists for an UQ-protein thiolate charge-transfer complex, and definitive evidence of this unique reaction pathway for DsbB has not been fully demonstrated. In order to study the UQ-8-DsbB complex in the presence of native lipids, we have prepared isotopically labeled samples of precipitated DsbB (WT and C41S) with endogenous UQ-8 and lipids, and we have applied advanced multidimensional solid-state NMR methods. Double-quantum filter and dipolar dephasing experiments facilitated assignments of UQ isoprenoid chain resonances not previously observed and headgroup sites important for the characterization of the UQ redox states: methyls (~20 ppm), methoxys (~60 ppm), olefin carbons (120-140 ppm), and carbonyls (150-160 ppm). Upon increasing the DsbB(C41S) pH from 5.5 to 8.0, we observed a 10.8 ppm upfield shift for the UQ C1 and C4 carbonyls indicating an increase of electron density on the carbonyls. This observation is consistent with the deprotonation of the DsbB-C44 thiolate at pH 8.0 and provides direct evidence of the charge-transfer complex formation. A similar trend was noted for the UQ chemical shifts of the DsbA(C33S)-DsbB(WT) heterodimer, confirming that the charge-transfer complex is unperturbed by the DsbB(C41S) mutant used to mimic the intermediate state of the disulfide bond generating reaction pathway.