TXNDC12 knockdown promotes ferroptosis by modulating SLC7A11 expression in glioma.

Ferroptosis is an iron-dependent cell death process mainly triggered by reactive oxygen species (ROS) and lipid peroxidation. Thioredoxin domain protein 12 (TXNDC12) promotes the development of some tumors; however, its function in tumor ferroptosis remains unclear. In this study, we found that knockdown of TXNDC12 promoted erastin-induced increase in ROS, lipid peroxidation, and Fe2+ levels, and decreased glutathione content. TXNDC12 is involved in ferroptosis by regulating SLC7A11. Further studies showed that TXNDC12 knockdown promoted an erastin-induced decrease in glioma cell viability. Overall, TXNDC12 played a significant role in ferroptosis by modulating SLC7A11 expression. Thus, TXNDC12 and ferroptosis may provide new targets for the treatment of gliomas.

The mammalian peroxisomal membrane is permeable to both GSH and GSSG - Implications for intraperoxisomal redox homeostasis.

Despite the large amounts of H2O2 generated in mammalian peroxisomes, cysteine residues of intraperoxisomal proteins are maintained in a reduced state. The biochemistry behind this phenomenon remains unexplored, and simple questions such as "is the peroxisomal membrane permeable to glutathione?" or "is there a thiol-disulfide oxidoreductase in the organelle matrix?" still have no answer. We used a cell-free in vitro system to equip rat liver peroxisomes with a glutathione redox sensor. The organelles were then incubated with glutathione solutions of different redox potentials and the oxidation/reduction kinetics of the redox sensor was monitored. The data suggest that the mammalian peroxisomal membrane is promptly permeable to both reduced and oxidized glutathione. No evidence for the presence of a robust thiol-disulfide oxidoreductase in the peroxisomal matrix could be found. Also, prolonged incubation of organelle suspensions with glutaredoxin 1 did not result in the internalization of the enzyme. To explore a potential role of glutathione in intraperoxisomal redox homeostasis we performed kinetic simulations. The results suggest that even in the absence of a glutaredoxin, glutathione is more important in protecting cysteine residues of matrix proteins from oxidation by H2O2 than peroxisomal catalase itself.

A cryptic oxidoreductase safeguards oxidative protein folding in Corynebacterium diphtheriae.

In many gram-positive Actinobacteria, including Actinomyces oris and Corynebacterium matruchotii, the conserved thiol-disulfide oxidoreductase MdbA that catalyzes oxidative folding of exported proteins is essential for bacterial viability by an unidentified mechanism. Intriguingly, in Corynebacterium diphtheriae, the deletion of mdbA blocks cell growth only at 37 °C but not at 30 °C, suggesting the presence of alternative oxidoreductase enzyme(s). By isolating spontaneous thermotolerant revertants of the mdbA mutant at 37 °C, we obtained genetic suppressors, all mapped to a single T-to-G mutation within the promoter region of tsdA, causing its elevated expression. Strikingly, increased expression of tsdA-via suppressor mutations or a constitutive promoter-rescues the pilus assembly and toxin production defects of this mutant, hence compensating for the loss of mdbA. Structural, genetic, and biochemical analyses demonstrated TsdA is a membrane-tethered thiol-disulfide oxidoreductase with a conserved CxxC motif that can substitute for MdbA in mediating oxidative folding of pilin and toxin substrates. Together with our observation that tsdA expression is upregulated at nonpermissive temperature (40 °C) in wild-type cells, we posit that TsdA has evolved as a compensatory thiol-disulfide oxidoreductase that safeguards oxidative protein folding in C. diphtheriae against thermal stress.

Thiol-disulfide oxidoreductase PDI1;1 regulates actin structures in Oryza sativa root cells.

The polarized and dynamic actin cytoskeleton is essential for root cell growth. Here, we report the key role of thiol-disulfide oxidoreductase PDI1;1 in actin structures. Microscopic analyses revealed that after Oryza sativa roots were exposed to H2 O2 , both actin and PDI1;1 were depolarized and arranged in a meshwork. In H2 O2 -exposed cells, actin formed intermolecularly disulfide-bonded high-molecular-weight structures, which were thiol-trapped by PDI1;1. Recombinant PDI1;1 exhibited the ability to recognize actin in an in vitro binding assay. During recovery from H2 O2 exposure, the amount of disulfide-bonded high-molecular-weight structures of actin decreased over time, but deficiency of PDI1;1 inhibited the decrease. These results suggest a PDI1;1-dependent pathway that reduces disulfide bonds in high-molecular-weight structures of actin, thus promoting their degradation.

Estrogen receptor beta promotes lung cancer invasion via increasing CXCR4 expression.

Lung cancer is one of the most lethal malignant tumors in the world. The high recurrence and mortality rate make it urgent for scientists and clinicians to find new targets for better treatment of lung cancer. Early studies indicated that estrogen receptor ß (ERß) might impact the progression of non-small-cell lung cancer (NSCLC). However, the detailed mechanisms, especially its linkage to the CXCR4-mediated cell invasion, remain unclear. Here we found that ERß could promote NSCLC cell invasion via increasing the circular RNA (circRNA), circ-TMX4, expression via directly binding to the 5' promoter region of its host gene TMX4. ERß-promoted circ-TMX4 could then sponge and inhibit the micro RNA (miRNA, miR), miR-622, expression, which can then result in increasing the CXCR4 messenger RNA translation via a reduced miRNA binding to its 3' untranslated region (3'UTR). The preclinical study using an in vivo mouse model with orthotopic xenografts of NSCLC cells confirmed the in vitro data, and the human NSCLC database analysis and tissue staining also confirmed the linkage of ERß/miR-622/CXCR4 signaling to the NSCLC progression. Together, our findings suggest that ERß can promote NSCLC cell invasion via altering the ERß/circ-TMX4/miR-622/CXCR4 signaling, and targeting this newly circ-TMX4/miR-622/CXCR4 signaling may help us find new treatment strategies to better suppress NSCLC progression.

Heterologous production of active form of beta-lytic protease by Bacillus subtilis and improvement of staphylolytic activity by protein engineering.

BACKGROUND: Most of the proteases classified into the M23 family in the MEROPS database exhibit staphylolytic activity and have potential as antibacterial agents. The M23 family is further classified into two subfamilies, M23A and M23B. Proteases of the M23A subfamily are thought to lack the capacity for self-maturation by auto-processing of a propeptide, which has been a challenge in heterologous production and application research. In this study, we investigated the heterologous expression, in Bacillus subtilis, of the Lysobacter enzymogenes beta-lytic protease (BLP), a member of the M23A subfamily. RESULTS: We found that B. subtilis can produce BLP in its active form. Two points were shown to be important for the production of BLP in B. subtilis. The first was that the extracellular proteases produced by the B. subtilis host are essential for BLP maturation. When the host strain was deficient in nine extracellular proteases, pro-BLP accumulated in the supernatant. This observation suggested that BLP lacks the capacity for self-maturation and that some protease from B. subtilis contributes to the cleavage of the propeptide of BLP. The second point was that the thiol-disulfide oxidoreductases BdbDC of the B. subtilis host are required for efficient secretory production of BLP. We infer that intramolecular disulfide bonds play an important role in the formation of the correct BLP conformation during secretion. We also achieved efficient protein engineering of BLP by utilizing the secretory expression system in B. subtilis. Saturation mutagenesis of Gln116 resulted in a Q116H mutant with enhanced staphylolytic activity. The minimum bactericidal concentration (MBC) of the wild-type BLP and the Q116H mutant against Staphylococcus aureus NCTC8325 was 0.75 µg/mL and 0.375 µg/mL, respectively, and the MBC against Staphylococcus aureus ATCC43300 was 6 µg/mL and 3 µg/mL, respectively. CONCLUSIONS: In this study, we succeeded in the secretory production of BLP in B. subtilis. To our knowledge, this work is the first report of the successful heterologous production of BLP in its active form, which opens up the possibility of industrial use of BLP. In addition, this study proposes a new strategy of using the extracellular proteases of B. subtilis for the maturation of heterologous proteins.

Clinical Value of TXNDC12 Combined With IDH and 1p19q as Biomarkers for Prognosis of Glioma.

Background: Glioma is the primary malignant tumor of the central nervous system and presents high mortality and disability rates under existing treatment measures. Thioredoxin domain-containing 12 (TXNDC12) has been shown to play an important role in various malignant tumors. Therefore, we explored the clinicopathological characteristics of TXNDC12 in glioma to bring to light new ideas in its treatment. Methods: We obtained data packages related to TXNDC12 expression status in gliomas from public databases. We analyzed glioma TXNDC12 expression and patient survival status and validated the above results using glioma specimens from our institution. Next, we analyzed the value of TXNDC12 in combination with 1p19q and isocitrate dehydrogenase (IDH) on the prognosis of glioma by regression model and receiver operating characteristic curve (ROC). Finally, we explored the function of related genes by GO analysis and KEGG analysis. Results: Compared with normal brain tissue, the expression of TXNDC12 in glioma cells, regarding both mRNA and protein levels, was significantly upregulated. The survival time of patients with high-expression of TXNDC12 in glioma cells was shortened. In the World Health Organization pathological classification, IDH status, 1p19q status, and IDH combined with 1p19q subgroups, the expression of TXNDC12 increased with the deterioration of the above indicators. Tumor local immune analysis showed that the immune cell infiltration in TXNDC12 high-expressing glioma tissue increased, the tumor purity was reduced. GO and KEGG analyses indicated that TXNDC12 may be involved in the malignant prognosis of glioma through glycosylation and antigen processing and presentation. Conclusion: We showed that TXNDC12 is significantly highly expressed in gliomas. This high expression predicts the poor prognosis of glioma patients and is related to the gliomas' local immune microenvironment. As a tumor-related gene, TXNDC12 may be used as a new prognostic judgment molecule.

Identification of a Thiol-Disulfide Oxidoreductase (SdbA) Catalyzing Disulfide Bond Formation in the Superantigen SpeA in Streptococcus pyogenes.

Mechanisms of disulfide bond formation in the human pathogen Streptococcus pyogenes are currently unknown. To date, no disulfide bond-forming thiol-disulfide oxidoreductase (TDOR) has been described and at least one disulfide bonded protein is known in S. pyogenes. This protein is the superantigen SpeA, which contains 3 cysteine residues (Cys 87, Cys90, and Cys98) and has a disulfide bond formed between Cys87 and Cys98. In this study, candidate TDORs were identified from the genome sequence of S. pyogenes MGAS8232. Using mutational and biochemical approaches, one of the candidate proteins, SpyM18_2037 (named here SdbA), was shown to be the catalyst that introduces the disulfide bond in SpeA. SpeA in the culture supernatant remained reduced when sdbA was inactivated and restored to the oxidized state when a functional copy of sdbA was returned to the sdbA-knockout mutant. SdbA has a typical C46XXC49 active site motif commonly found in TDORs. Site-directed mutagenesis experiments showed that the cysteines in the CXXC motif were required for the disulfide bond in SpeA to form. Interactions between SdbA and SpeA were examined using cysteine variant proteins. The results showed that SdbAC49A formed a mixed disulfide with SpeAC87A, suggesting that the N-terminal Cys46 of SdbA and the C-terminal Cys98 of SpeA participated in the initial reaction. SpeA oxidized by SdbA displayed biological activities suggesting that SpeA was properly folded following oxidation by SdbA. In conclusion, formation of the disulfide bond in SpeA is catalyzed by SdbA and the findings represent the first report of disulfide bond formation in S. pyogenes. IMPORTANCE Here, we reported the first example of disulfide bond formation in Streptococcus pyogenes. The results showed that a thiol-disulfide oxidoreductase, named SdbA, is responsible for introducing the disulfide bond in the superantigen SpeA. The cysteine residues in the CXXC motif of SdbA are needed for catalyzing the disulfide bond in SpeA. The disulfide bond in SpeA and neighboring amino acids form a disulfide loop that is conserved among many superantigens, including those from Staphylococcus aureus. SpeA and staphylococcal enterotoxins lacking the disulfide bond are biologically inactive. Thus, the discovery of the enzyme that catalyzes the disulfide bond in SpeA is important for understanding the biochemistry of SpeA production and presents a target for mitigating the virulence of S. pyogenes.

Identification of the Primary Factors Determining theSpecificity of Human VKORC1 Recognition by Thioredoxin-Fold Proteins.

Redox (reduction-oxidation) reactions control many important biological processes in all organisms, both prokaryotes and eukaryotes. This reaction is usually accomplished by canonical disulphide-based pathways involving a donor enzyme that reduces the oxidised cysteine residues of a target protein, resulting in the cleavage of its disulphide bonds. Focusing on human vitamin K epoxide reductase (hVKORC1) as a target and on four redoxins (protein disulphide isomerase (PDI), endoplasmic reticulum oxidoreductase (ERp18), thioredoxin-related transmembrane protein 1 (Tmx1) and thioredoxin-related transmembrane protein 4 (Tmx4)) as the most probable reducers of VKORC1, a comparative in-silico analysis that concentrates on the similarity and divergence of redoxins in their sequence, secondary and tertiary structure, dynamics, intraprotein interactions and composition of the surface exposed to the target is provided. Similarly, hVKORC1 is analysed in its native state, where two pairs of cysteine residues are covalently linked, forming two disulphide bridges, as a target for Trx-fold proteins. Such analysis is used to derive the putative recognition/binding sites on each isolated protein, and PDI is suggested as the most probable hVKORC1 partner. By probing the alternative orientation of PDI with respect to hVKORC1, the functionally related noncovalent complex formed by hVKORC1 and PDI was found, which is proposed to be a first precursor to probe thiol-disulphide exchange reactions between PDI and hVKORC1.

Emerging Moonlighting Functions of the Branched-Chain Aminotransferase Proteins.

Significance: Unique to the branched-chain aminotransferase (BCAT) proteins is their redox-active CXXC motif. Subjected to post-translational modification by reactive oxygen species and reactive nitrogen species, these proteins have the potential to adopt numerous cellular roles, which may be fundamental to their role in oncogenesis and neurodegenerative diseases. An understanding of the interplay of the redox regulation of BCAT with important cell signaling mechanisms will identify new targets for future therapeutics. Recent Advances: The BCAT proteins have been assigned novel thiol oxidoreductase activity that can accelerate the refolding of proteins, in particular when S-glutathionylated, supporting a chaperone role for BCAT in protein folding. Other metabolic proteins were also shown to have peroxide-mediated redox associations with BCAT, indicating that the cellular function of BCAT is more diverse. Critical Issues: While the role of branched-chain amino acid metabolism and its metabolites has dominated aspects of cancer research, less is known about the role of BCAT. The importance of the CXXC motif in regulating the BCAT activity under hypoxic conditions, a characteristic of tumors, has not been addressed. Understanding how these proteins operate under various cellular redox conditions will become important, in particular with respect to their moonlighting roles. Future Directions: Advances in the quantification of thiols, their measurement, and the manipulation of metabolons that rely on redox-based interactions should accelerate the investigation of the cellular role of moonlighting proteins such as BCAT. Given the importance of cross talk between signaling pathways, research should focus more on these "housekeeping" proteins paying attention to their wider application. Antioxid. Redox Signal. 34, 1048-1067.

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.

TXNDC12 promotes EMT and metastasis of hepatocellular carcinoma cells via activation of ß-catenin.

Metastasis is one of the main contributors to the poor prognosis of hepatocellular carcinoma (HCC). However, the underlying mechanism of HCC metastasis remains largely unknown. Here, we showed that TXNDC12, a thioredoxin-like protein, was upregulated in highly metastatic HCC cell lines as well as in portal vein tumor thrombus and lung metastasis tissues of HCC patients. We found that the enforced expression of TXNDC12 promoted metastasis both in vitro and in vivo. Subsequent mechanistic investigations revealed that TXNDC12 promoted metastasis through upregulation of the ZEB1-mediated epithelial-mesenchymal transition (EMT) process. We subsequently showed that TXNDC12 overexpression stimulated the nuclear translocation and activation of ß-catenin, a positive transcriptional regulator of ZEB1. Accordingly, we found that TXNDC12 interacted with ß-catenin and that the thioredoxin-like domain of TXNDC12 was essential for the interaction between TXNDC12 and ß-catenin as well as for TXNDC12-mediated ß-catenin activation. Moreover, high levels of TXNDC12 in clinical HCC tissues correlated with elevated nuclear ß-catenin levels and predicted worse overall and disease-free survival. In summary, our study demonstrated that TXNDC12 could activate ß-catenin via protein-protein interaction and promote ZEB1-mediated EMT and HCC metastasis.

COA6 Is Structurally Tuned to Function as a Thiol-Disulfide Oxidoreductase in Copper Delivery to Mitochondrial Cytochrome c Oxidase.

In eukaryotes, cellular respiration is driven by mitochondrial cytochrome c oxidase (CcO), an enzyme complex that requires copper cofactors for its catalytic activity. Insertion of copper into its catalytically active subunits, including COX2, is a complex process that requires metallochaperones and redox proteins including SCO1, SCO2, and COA6, a recently discovered protein whose molecular function is unknown. To uncover the molecular mechanism by which COA6 and SCO proteins mediate copper delivery to COX2, we have solved the solution structure of COA6, which reveals a coiled-coil-helix-coiled-coil-helix domain typical of redox-active proteins found in the mitochondrial inter-membrane space. Accordingly, we demonstrate that COA6 can reduce the copper-coordinating disulfides of its client proteins, SCO1 and COX2, allowing for copper binding. Finally, our determination of the interaction surfaces and reduction potentials of COA6 and its client proteins provides a mechanism of how metallochaperone and disulfide reductase activities are coordinated to deliver copper to CcO.

ERp18 regulates activation of ATF6α during unfolded protein response.

Activation of the ATF6α signaling pathway is initiated by trafficking of ATF6α from the ER to the Golgi apparatus. Its subsequent proteolysis releases a transcription factor that translocates to the nucleus causing downstream gene activation. How ER retention, Golgi trafficking, and proteolysis of ATF6α are regulated and whether additional protein partners are required for its localization and processing remain unresolved. Here, we show that ER-resident oxidoreductase ERp18 associates with ATF6α following ER stress and plays a key role in both trafficking and activation of ATF6α. We find that ERp18 depletion attenuates the ATF6α stress response. Paradoxically, ER stress accelerates trafficking of ATF6α to the Golgi in ERp18-depleted cells. However, the translocated ATF6α becomes aberrantly processed preventing release of the soluble transcription factor. Hence, we demonstrate that ERp18 monitors ATF6α ER quality control to ensure optimal processing following trafficking to the Golgi.

A new oxidative stress indicator: Effect of 5-hydroxytryptophan on thiol-disulfide homeostasis in exercise.

OBJECTIVES: The aim of the present study was to evaluate the relationship between exercise and both 5-hydroxytryptophan and oxidative stress using thiol-disulfide homeostasis via what is likely a novel biomarker. METHODS: Male albino Wistar rats (n = 32) were randomly divided into four groups as follows: control, exercise group, 5-hydroxytryptophan group (5H), and 5-HTP + exercise group (5Hex). Exercise and 5-HTP administration (25mg/kg per d) were performed 5d/wk for 10 wk. After completion of the experimental protocol, to determine oxidative stress parameters, serum total thiol and native thiol concentrations were measured. Dynamic disulfide status, reduced thiol, oxidized thiol (OT), and thiol oxidation reduction percentage ratios were compared between the groups. The methods used in the present study to measure dynamic thiol-disulfide homeostasis as calorimetric and duplex quantities were developed in 2014. These new methods are simple, reliable, and sensitive, with both high linearity and repeatability. RESULTS: Compared with the control group, serum dynamic disulfide levels were significantly lower in the 5H group and highest in the control group. The lowest OT and the highest reduced thiol rates were determined to be in the 5H group. The highest OT value was found in the 5Hex group. Thiol oxidation reduction values were found to be highest in the 5H group and lowest in the 5Hex group. CONCLUSIONS: Both 5-HTP and moderate exercise seem to be significantly effective in inhibiting oxidative damage. In addition, the new oxidative stress measurement method used in this study is a promising practical and useful method to evaluate and improve the performance of athletes.

Role of Cell Surface Lipids and Thiol-Disulphide Exchange Pathways in Regulating the Encryption and Decryption of Tissue Factor.

Tissue factor (TF), a transmembrane glycoprotein, is the cellular receptor of the coagulation factors VII (FVII) and VIIa (FVIIa). The formation of TF-FVIIa complex triggers the initiation of the blood coagulation pathway. TF plays an essential role in haemostasis, but an aberrant expression of TF activity contributes to thrombotic disorders. In health, TF pro-coagulant activity on cells is controlled tightly to allow sufficient coagulant activity to achieve haemostasis but not to cause thrombosis. It is achieved largely by selective localization of TF in the body and encryption of TF at the cell surface. A vast majority of TF on resting cells exists in an encrypted state with minimal pro-coagulant activity but becomes pro-thrombotic following cell injury or activation. At present, the mechanisms that are responsible for TF encryption and activation (decryption) are not entirely clear, but recent studies provide important mechanistic insights into these processes. To date, externalization of phosphatidylserine to the outer leaflet and thiol-disulphide exchange pathways that either turn on and off the allosteric disulphide bond in TF are shown to play a major role in regulating TF pro-coagulant activity on cell surfaces. Recent studies showed that sphingomyelin, a major phospholipid in the outer leaflet of plasma membrane, plays a critical role in the encryption of TF in resting cells. The present review provides an overview of recent literature on the above-described mechanisms of TF encryption and decryption with a particular emphasis on our recent findings.

Identification of Redox Partners of the Thiol-Disulfide Oxidoreductase SdbA in Streptococcus gordonii.

We previously identified a novel thiol-disulfide oxidoreductase, SdbA, in Streptococcus gordonii that formed disulfide bonds in substrate proteins and played a role in multiple phenotypes. In this study, we used mutational, phenotypic, and biochemical approaches to identify and characterize the redox partners of SdbA. Unexpectedly, the results showed that SdbA has multiple redox partners, forming a complex oxidative protein-folding pathway. The primary redox partners of SdbA that maintain its active site in an oxidized state are a surface-exposed thioredoxin family lipoprotein called SdbB (Sgo_1171) and an integral membrane protein annotated as CcdA2. Inactivation of sdbB and ccdA2 simultaneously, but not individually, recapitulated the sdbA mutant phenotype. The sdbB-ccdA2 mutant had defects in a range of cellular processes, including autolysis, bacteriocin production, genetic competence, and extracellular DNA (eDNA) release. AtlS, the natural substrate of SdbA produced by the sdbB-ccdA2 mutant lacked activity and an intramolecular disulfide bond. The redox state of SdbA in the sdbB-ccdA2 mutant was found to be in a reduced form and was restored when sdbB and ccdA2 were knocked back into the mutant. In addition, we showed that SdbB formed a disulfide-linked complex with SdbA in the cell. Recombinant SdbB and CcdA2 exhibited oxidase activity and reoxidized reduced SdbA in vitro Collectively, our results demonstrate that S. gordonii uses multiple redox partners for oxidative protein folding.IMPORTANCEStreptococcus gordonii is a commensal bacterium of the human dental plaque. Previously, we identified an enzyme, SdbA, that forms disulfide bonds in substrate proteins and plays a role in a number of cellular processes in S. gordonii Here, we identified the redox partners of SdbA. We showed that SdbA has multiple redox partners, SdbB and CcdA2, forming a complex oxidative protein-folding pathway. This pathway is essential for autolysis, bacteriocin production, genetic competence, and extracellular DNA (eDNA) release in S. gordonii These cellular processes are considered to be important for the success of S. gordonii as a dental plaque organism. This is the first example of an oxidative protein-folding pathway in Gram-positive bacteria that consists of an enzyme that uses multiple redox partners to function.

Redox-Driven Signaling: 2-Oxo Acid Dehydrogenase Complexes as Sensors and Transmitters of Metabolic Imbalance.

SIGNIFICANCE: This article develops a holistic view on production of reactive oxygen species (ROS) by 2-oxo acid dehydrogenase complexes. Recent Advances: Catalytic and structural properties of the complexes and their components evolved to minimize damaging effects of side reactions, including ROS generation, simultaneously exploiting the reactions for homeostatic signaling. CRITICAL ISSUES: Side reactions of the complexes, characterized in vitro, are analyzed in view of protein interactions and conditions in vivo. Quantitative data support prevalence of the forward 2-oxo acid oxidation over the backward NADH oxidation in feeding physiologically significant ROS production by the complexes. Special focus on interactions between the active sites within 2-oxo acid dehydrogenase complexes highlights the central relevance of the complex-bound thiyl radicals in regulation of and signaling by complex-generated ROS. The thiyl radicals arise when dihydrolipoyl residues of the complexes regenerate FADH2 from the flavin semiquinone coproduced with superoxide anion radical in 1e- oxidation of FADH2 by molecular oxygen. FUTURE DIRECTIONS: Interaction of 2-oxo acid dehydrogenase complexes with thioredoxins (TRXs), peroxiredoxins, and glutaredoxins mediates scavenging of the thiyl radicals and ROS generated by the complexes, underlying signaling of disproportional availability of 2-oxo acids, CoA, and NAD+ in key metabolic branch points through thiol/disulfide exchange and medically important hypoxia-inducible factor, mammalian target of rapamycin (mTOR), poly (ADP-ribose) polymerase, and sirtuins. High reactivity of the coproduced ROS and thiyl radicals to iron/sulfur clusters and nitric oxide, peroxynitrite reductase activity of peroxiredoxins and transnitrosylating function of thioredoxin, implicate the side reactions of 2-oxo acid dehydrogenase complexes in nitric oxide-dependent signaling and damage.

Methods to identify the substrates of thiol-disulfide oxidoreductases.

The formation of a disulfide bond is a critical step in the folding of numerous secretory and membrane proteins and catalyzed in vivo. A variety of mechanisms and protein structures have evolved to catalyze oxidative protein folding. Those enzymes that directly interact with a folding protein to accelerate its oxidative folding are mostly thiol-disulfide oxidoreductases that belong to the thioredoxin superfamily. The enzymes of this class often use a CXXC active-site motif embedded in their thioredoxin-like fold to promote formation, isomerization, and reduction of a disulfide bond in their target proteins. Over the past decade or so, an increasing number of substrates of the thiol-disulfide oxidoreductases that are present in the ER of mammalian cells have been discovered, revealing that the enzymes play unexpectedly diverse physiological functions. However, functions of some of these enzymes still remain unclear due to the lack of information on their substrates. Here, we review the methods used by researchers to identify the substrates of these enzymes and provide data that show the importance of using trichloroacetic acid in sample preparation for the substrate identification, hoping to aid future studies. We particularly focus on successful studies that have uncovered physiological substrates and functions of the enzymes in the periplasm of Gram-negative bacteria and the endoplasmic reticulum of mammalian cells. Similar approaches should be applicable to enzymes in other cellular compartments or in other organisms.

Reduction of leucocyte cell surface disulfide bonds during immune activation is dynamic as revealed by a quantitative proteomics workflow (SH-IQ).

Communication through cell surface receptors is crucial for maintaining immune homeostasis, coordinating the immune response and pathogen clearance. This is dependent on the interaction of cell surface receptors with their ligands and requires functionally active conformational states. Thus, immune cell function can be controlled by modulating the structure of either the receptor or the ligand. Reductive cleavage of labile disulfide bonds can mediate such an allosteric change, resulting in modulation of the immune system by a hitherto little studied mechanism. Identifying proteins with labile disulfide bonds and determining the extent of reduction is crucial in elucidating the functional result of reduction. We describe a mass spectrometry-based method-thiol identification and quantitation (SH-IQ)-to identify, quantify and monitor such reduction of labile disulfide bonds in primary cells during immune activation. These results provide the first insight into the extent and dynamics of labile disulfide bond reduction in leucocyte cell surface proteins upon immune activation. We show that this process is thiol oxidoreductase-dependent and mainly affects activatory (e.g. CD132, SLAMF1) and adhesion (CD44, ICAM1) molecules, suggesting a mechanism to prevent over-activation of the immune system and excessive accumulation of leucocytes at sites of inflammation.