We developed a high-throughput mass spectrometry-based method to simultaneously quantify numerous small-molecule thiols and disulfides in blood plasma. Application of this assay to analyze plasma from patients with known oxidative stress (sickle cell disease and sepsis) and from a patient with sickle cell disease treated with the antioxidant N-acetylcysteine suggests that cysteine disulfides, in particular protein-bound cysteine, serve as sensitive plasma biomarkers for the extent of oxidative stress and effectiveness of antioxidant treatment.

Disulfides from Allium stipitatum, commonly known as Persian shallot, were previously reported to possess antibacterial properties. Analogues of these compounds, produced by S-methylthiolation of appropriate thiols using S-methyl methanethiosulfonate, exhibited antimicrobial activity, with one compound inhibiting the growth of Mycobacterium tuberculosis at 17 µM (4 mg L-1) and other compounds inhibiting Escherichia coli and multi-drug-resistant (MDR) Staphylococcus aureus at concentrations ranging between 32-138 µM (8-32 mg L-1). These compounds also displayed moderate inhibitory effects on Klebsiella and Proteus species. Whole-cell phenotypic bioassays such as the spot-culture growth inhibition assay (SPOTi), drug efflux inhibition, biofilm inhibition and cytotoxicity assays were used to evaluate these compounds. Of particular note was their ability to inhibit mycobacterial drug efflux and biofilm formation, while maintaining a high selectivity towards M. tuberculosis H37Rv. These results suggest that methyl disulfides are novel scaffolds which could lead to the development of new drugs against tuberculosis (TB).

Disulfide bonds play a key role in stabilizing protein structures, with disruption strongly associated with loss of protein function and activity. Previous data have suggested that disulfides show only modest reactivity with oxidants. In the current study, we report kinetic data indicating that selected disulfides react extremely rapidly, with a variation of 104 in rate constants. Five-membered ring disulfides are particularly reactive compared with acyclic (linear) disulfides or six-membered rings. Particular disulfides in proteins also show enhanced reactivity. This variation occurs with multiple oxidants and is shown to arise from favorable electrostatic stabilization of the incipient positive charge on the sulfur reaction center by remote groups, or by the neighboring sulfur for conformations in which the orbitals are suitably aligned. Controlling these factors should allow the design of efficient scavengers and high-stability proteins. These data are consistent with selective oxidative damage to particular disulfides, including those in some proteins.

Gaussia luciferase (Gluc)-with its many favorable traits such as small size, bright emission, and exceptional stability-has become a prominent reporter protein for a wide range of bioluminescence-based detection applications. The ten internal cysteine residues crucial to functional structure formation, however, make expression of high quantities of soluble protein in bacterial systems difficult. In addition to this challenge, the current lack of structural data further complicates the use of Gluc for in vitro applications, such as biosensors, or cellular delivery, both of which rely heavily on robust and reproducible bioconjugation techniques. While Gluc is already appreciably small for a luciferase, a reduction in size that still retains significant bioluminescent activity, in conjunction with a more reproducible bioorthogonal method of chemical modification and facile expression in bacteria, would be very beneficial in biosensor design and cellular transport studies. We have developed truncated variants of Gluc, which maintain attractive bioluminescent features, and have characterized their spectral and kinetic properties. These variants were purified in high quantities from a bacterial system. Additionally, a C-terminal linker has been incorporated into these variants that can be used for reliable, specific modification through tyrosine-based bioconjugation techniques, which leave the sensitive network of cysteine residues undisturbed.

Lactoferrin, a multifunctional iron-binding protein containing 16 disulfides, is actively studied for its antibacterial and anti-carcinogenic properties. However, scarce information is nowadays available about its oxidative folding starting from the reduced and unfolded status. This study discovers unusual properties when this protein is examined in its reduced molten globule-like conformation. Using kinetic, CD and fluorescence analyses together with mass spectrometry, we found that a few cysteines display astonishing hyper-reactivity toward different thiol reagents. In details, four cysteines (i.e. 668, 64, 512 and 424) display thousands of times higher reactivity toward GSSG but normal against other natural disulfides. The formation of these four mixed-disulfides with glutathione probably represents the first step of its folding in vivo. A widespread low pKa decreases the reactivity of other 14 cysteines toward GSSG limiting their involvement in the early phase of the oxidative folding. The origin of this hyper-reactivity was due to transient lactoferrin-GSSG complex, as supported by fluorescence experiments. Lactoferrin represents another disulfide containing protein in addition to albumin, lysozyme, ribonuclease, chymotrypsinogen, and trypsinogen which shows cysteines with an extraordinary and specific hyper-reactivity toward GSSG confirming the discovery of a fascinating new feature of proteins in their nascent phase.

The report presents the first method for simultaneous determination of plasma 2-(3-hydroxy-5-phosphonooxymethyl-2-methyl-4-pyridyl)-1,3-thiazolidine-4-carboxylic acid (HPPTCA), an adduct of cysteine (Cys) and active form of vitamin B6 pyridoxal 5'-phosphate (PLP), as well as total low molecular-weight thiols content, including Cys, homocysteine (Hcy), cysteinyl-glycine (Cys-Gly), and glutathione (GSH). The assay is based on high performance liquid chromatography coupled with ultraviolet detection (HPLC-UV) and involves disulfides reduction with tris(2-carboxyethyl)phosphine (TCEP), derivatization with 2-chloro-1-methylquinolinium tetrafluoroborate (CMQT) followed by sample deproteinization with perchloric acid (PCA). The chromatographic separation of obtained stable UV-absorbing derivatives is achieved on ZORBAX SB-C18 (150 × 4.6 mm, 5.0 µm) column using gradient elution with eluent consisted of 0.1 mol/L trichloroacetic acid (TCA), pH 1.7 and acetonitrile (ACN), delivered at a flow rate 1 mL/min. Under these conditions, the analytes are separated within 14 min at room temperature, and quantified by monitoring at 355 nm. Regarding HPPTCA, the assay linearity was demonstrated within a 1-100 µmol/L in plasma and the lowest concentration on the calibration curve was recognized as the limit of quantification (LOQ). The accuracy ranged from 92.74 to 105.57% and 95.43 to 115.73%, while precision varied from 2.48 to 6.99% and 0.84 to 6.98% for intra- and inter-day measurements, respectively. The utility of the assay was proved by application to plasma samples delivered by apparently healthy donors (n = 18) in which the HPPTCA concentration ranged from 19.2 to 65.6 µmol/L. The HPLC-UV assay provides complementary tool for routine clinical analysis, facilitating further studies on the role of aminothiols and HPPTCA in living systems.

Many proteins provided with disulfide bridges in the native state undergo amorphous irreversible aggregation when these bonds are not formed. Here we show that egg lysozyme displays a clever strategy to prevent this deleterious aggregation during the nascent phase when disulfides are still absent. In fact, when the reduced protein assembles into a molten globule state, its cysteines acquire strong hyper-reactivity towards natural disulfides. The most reactive residue, Cys94, reacts with oxidized glutathione (GSSG) 3000 times faster than an unperturbed protein cysteine. A low pKa of its sulfhydryl group (6.6/7.1) and a productive complex with GSSG (KD = 0.3 mM), causes a fast glutathionylation of this residue (t1/2 = 3 s) and a complete inhibition of the protein aggregation. Other six cysteines display 70 times higher reactivity toward GSSG. The discovery of extreme hyper-reactivity in cysteines only devoted to structural roles opens new research fields for Alzheimer's and Parkinson diseases.

Heart failure is the most common cause of morbidity and hospitalization in the western civilization. Protein phosphatases play a key role in the basal cardiac contractility and in the responses to β-adrenergic stimulation with type-1 phosphatase (PP-1) being major contributor. We propose here that formation of transient disulfide bridges in PP-1α might play a leading role in oxidative stress response. First, we established an optimized workflow, the so-called "cross-over-read" search method, for the identification of disulfide-linked species using permutated databases. By applying this method, we demonstrate the formation of unexpected transient disulfides in PP-1α to shelter against over-oxidation. This protection mechanism strongly depends on the fast response in the presence of reduced glutathione. Our work points out that the dimerization of PP-1α involving Cys39 and Cys127 is presumably important for the protection of PP-1α active surface in the absence of a substrate. We finally give insight into the electron transport from the PP-1α catalytic core to the surface. Our data suggest that the formation of transient disulfides might be a general mechanism of proteins to escape from irreversible cysteine oxidation and to prevent their complete inactivation.

Bispecific antibodies (BsAbs), with the ability to recognize two different epitopes simultaneously, offer remarkable advantages in bioassays, cancer therapy, biosensors, and enzyme electrodes. Preparation and purification of BsAbs in adequate quantities remains a major hurdle in their use in various applications. Poor yield is also the principal limitation in the preparation of BsAbs by the redox procedure. IgG with reduced inter-heavy chain disulfides do not dissociate into half molecules at neutral pH. In this study, we report that the dissociation occurs in presence of sodium dodecyl sulphate (SDS) and inclusion of the detergent during the redox procedure results in remarkable increase in the formation of the BsAbs. Exposure of antibodies to 0.1% (w/v) SDS causes only minor loss in secondary/tertiary structure and the ability to bind the antigen. The BsAbs prepared using the modified redox procedure that recognize the antigens HRP and α-LA were prepared and successfully employed for detecting α-LA in milk/dairy products by ELISA and dot blot techniques. BsAbs were also prepared from partially purified immunoglobulin gamma (IgG). This work shows for the first time that SDS, by dissociating IgG with reduced inter-heavy chain disulfides into half molecules, markedly enhances the formation of BsAbs by the redox procedure.

Human immunodeficiency virus (HIV-1) entry is initiated by the binding between the viral envelope glycoprotein gp120 and the host receptor CD4, and followed by reduction of structural disulfides of gp120 and CD4. The host thioredoxin-1 (Trx1) efficiently reduces disulfides of gp120 and CD4 in vitro, and recently CD4-dependent HIV-1 entry was shown to be inhibited by anti-Trx1-antibodies, indicating a central role for Trx1. 1-methylpropyl-2-imidazolyl disulfide (PX-12) is a reversible inhibitor of the Trx1 system that may also cause a slow irreversible thioalkylation of Trx1. It was developed as an antitumor agent, however, the current study aimed to determine if it also has an anti-HIV-1 effect. We show that PX-12 has anti-HIV-1(IIIB) activity in TZM-bl cells, in fact, no virus was detected inside the cells in the presence of 10 µM PX-12. Moreover, PX-12 inhibited the enzymatic activity of Trx1 and the Trx1-dependent disulfide reduction of gp120. Microtubule polymerization and formation of acetylated microtubules were also inhibited, activities shown to be required for HIV-1 life cycle propagation. In conclusion, our data strengthens the notion that the early steps of the HIV-1 life cycle depends on the Trx1 system and indicate that the Trx1 system may be a rational drug target for HIV-1 treatment.

The mammalian endoplasmic reticulum (ER) harbors disulfide bond-generating enzymes, including Ero1α and peroxiredoxin 4 (Prx4), and nearly 20 members of the protein disulfide isomerase family (PDIs), which together constitute a suitable environment for oxidative protein folding. Here, we clarified the Prx4 preferential recognition of two PDI family proteins, P5 and ERp46, and the mode of interaction between Prx4 and P5 thioredoxin domain. Detailed analyses of oxidative folding catalyzed by the reconstituted Prx4-PDIs pathways demonstrated that, while P5 and ERp46 are dedicated to rapid, but promiscuous, disulfide introduction, PDI is an efficient proofreader of non-native disulfides. Remarkably, the Prx4-dependent formation of native disulfide bonds was accelerated when PDI was combined with ERp46 or P5, suggesting that PDIs work synergistically to increase the rate and fidelity of oxidative protein folding. Thus, the mammalian ER seems to contain highly systematized oxidative networks for the efficient production of large quantities of secretory proteins.

Disulfidptosis is a condition where dysregulated NAPDH levels and abnormal accumulation of cystine and other disulfides occur in cells with high SLC7A11 expression under glucose deficiency. This disrupts normal formation of disulfide bonds among cytoskeletal proteins, leading to histone skeleton collapse and triggering cellular apoptosis. However, the correlation between disulfidptosis and immune responses in relation to glioblastoma survival rates and immunotherapy sensitivity remains understudied. Therefore, we utilized The Cancer Genome Atlas and The Chinese Glioma Genome Atlas to identify disulfidptosis-related immune checkpoint genes and established an overall survival (OS) prediction model comprising six genes: CD276, TNFRSF 14, TNFSF14, TNFSF4, CD40, and TNFRSF18, which could also be used for predicting immunotherapy sensitivity. We identified a cohort of glioblastoma patients classified as high-risk, which exhibited an upregulation of angiogenesis, extracellular matrix remodeling, and epithelial-mesenchymal transition as well as an immunosuppressive tumor microenvironment (TME) enriched with tumor associated macrophages, tumor associated neutrophils, CD8 + T-cell exhaustion. Immunohistochemical staining of CD276 in 144 cases further validated its negative correlation with OS in glioma. Disulfidptosis has the potential to induce chronic inflammation and an immunosuppressive TME in glioblastoma.

Beta-2-glycoprotein I (β2GPI) is a blood protein and the major antigen in the autoimmune disorder antiphospholipid syndrome (APS). β2GPI exists mainly in closed or open conformations and comprises of 11 disulfides distributed across five domains. The terminal Cys288/Cys326 disulfide bond at domain V has been associated with different cysteine redox states. The role of this disulfide bond in conformational dynamics of this protein has not been investigated so far. Here, we report on the enzymatic driven reduction by thioredoxin-1 (recycled by Tris(2-carboxyethyl)phosphine; TCEP) of β2GPI. Specific reduction was demonstrated by Western blot and mass spectrometry analyses confirming majority targeting to the fifth domain of β2GPI. Atomic force microscopy images suggested that reduced β2GPI shows a slightly higher proportion of open conformation and is more flexible compared to the untreated protein as confirmed by modelling studies. We have determined a strong increase in the binding of pathogenic APS autoantibodies to reduced β2GPI as demonstrated by ELISA. Our study is relevant for understanding the effect of β2GPI reduction on the protein structure and its implications for antibody binding in APS patients.

Cell surface aminopeptidase N (APN) is a membrane-bound ectoenzyme that hydrolyzes proteins and peptides and regulates numerous cell functions. APN participates in tumor cell expansion and motility, and is a target for cancer therapies. Small drugs that bind to the APN active site inhibit catalysis and suppress tumor growth. APN is also a major cell entry receptor for coronavirus, which binds to a region distant from the active site. Three crystal structures that we determined of human and pig APN ectodomains defined the dynamic conformation of the protein. These structures offered snapshots of closed, intermediate and open APN, which represent distinct functional states. Coronavirus envelope proteins specifically recognized the open APN form, prevented ectodomain progression to the closed form and substrate hydrolysis. In addition, drugs that bind the active site inhibited both coronavirus binding to cell surface APN and infection; the drugs probably hindered APN transition to the virus-specific open form. We conclude that allosteric inhibition of APN functions occurs by ligand suppression of ectodomain motions necessary for catalysis and virus cell entry, as validated by locking APN with disulfides. Blocking APN dynamics can thus be a valuable approach to development of drugs that target this ectoenzyme.

The stinging capsules of cnidarians, nematocysts, function as harpoon-like organelles with unusual biomechanical properties. The nanosecond discharge of the nematocyst requires a dense protein network of the capsule structure withstanding an internal pressure of up to 150 bar. Main components of the capsule are short collagens, so-called minicollagens, that form extended polymers by disulfide reshuffling of their cysteine-rich domains (CRDs). Although CRDs have identical cysteine patterns, they exhibit different structures and disulfide connectivity at minicollagen N and C-termini. We show that the structurally divergent CRDs have different cross-linking potentials in vitro and in vivo. While the C-CRD can participate in several simultaneous intermolecular disulfides and functions as a cystine knot after minicollagen synthesis, the N-CRD is monovalent. Our combined experimental and computational analyses reveal the cysteines in the C-CRD fold to exhibit a higher structural propensity for disulfide bonding and a faster kinetics of polymerization. During nematocyst maturation, the highly reactive C-CRD is instrumental in efficient cross-linking of minicollagens to form pressure resistant capsules. The higher ratio of C-CRD folding types evidenced in the medusozoan lineage might have fostered the evolution of novel, predatory nematocyst types in cnidarians with a free-swimming medusa stage.

Chymotrypsinogen, when reduced and taken to its molten globule-like conformation, displays a single cysteine with an unusual kinetic propensity toward oxidized glutathione (GSSG) and other organic thiol reagents. A single residue, identified by mass spectrometry like Cys1, reacts with GSSG about 1400 times faster than an unperturbed protein cysteine. A reversible protein-GSSG complex and a low pKa (8.1 ± 0.1) make possible such astonishing kinetic property which is absent toward other natural disulfides like cystine, homocystine and cystamine. An evident hyper-reactivity toward 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) and 1-chloro-2,4-dinitrobenzene (CDNB) was also found for this specific residue. The extraordinary reactivity toward GSSG is absent in two proteins of the thermophilic archaeon Sulfolobus solfataricus, an organism lacking glutathione: the Protein Disulphide Oxidoreductase (SsPDO) and the Bacterioferritin Comigratory Protein 1 (Bcp1) that displays Cys residues with an even lower pKa value (7.5 ± 0.1) compared to chymotrypsinogen. This study, which also uses single mutants in Cys residues for Bcp1, proposes that this hyper-reactivity of a single cysteine, similar to that found in serum albumin, lysozyme, ribonuclease, may have relevance to drive the "incipit" of the oxidative folding of proteins from organisms where the glutathione/oxidized glutathione (GSH/GSSG) system is present.

The inherited form of open angle glaucoma arises due to a toxic gain-of-function intracellular misfolding event involving a mutated myocilin olfactomedin domain (OLF). Mutant myocilin is recognized by the endoplasmic reticulum (ER)-resident heat shock protein 90 paralog, glucose regulated protein 94 (Grp94), but their co-aggregation precludes mutant myocilin clearance by ER-associated degradation. When the Grp94-mutant myocilin interaction is abrogated by inhibitors or siRNA, mutant myocilin is efficiently degraded. Here we dissected Grp94 into component domains (N, NM, MC) to better understand the molecular factors governing its interaction with OLF. We show that the Grp94 N-terminal nucleotide-binding N domain is responsible for accelerating OLF aggregation in vitro. Upon inhibiting the isolated N domain pharmacologically or removing the Pre-N terminal 57 residues from full-length Grp94, OLF aggregation rates revert to those seen for OLF alone, but only pharmacological inhibition rescues co-aggregation. The Grp94-OLF interaction is below the detection limit of fluorescence polarization measurements, but chemical crosslinking paired with mass spectrometry analyses traps a reproducible interaction between OLF and the Grp94 N domain, as well as between OLF and the Grp94 M domain. The emerging molecular-level picture of quinary interactions between Grp94 and myocilin points to a role for the far N-terminal sequence of the Grp94 N domain and a cleft in the M domain. Our work further supports drug discovery efforts to inhibit these interactions as a strategy to treat myocilin-associated glaucoma.

Metabolites containing amino groups cover multiple pathways and play important roles in redox homeostasis and biosyntheses of proteins, nucleotides and neurotransmitters. Here, we report a new method for simultaneous quantification of 124 such metabolites. This is achieved by derivatization-assisted sensitivity enhancement with 5-aminoisoquinolyl-N-hydroxysuccinimidyl carbamate (5-AIQC) followed with comprehensive analysis using ultra-high performance liquid chromatography and electrospray ionization tandem mass spectrometry (UHPLC-MS/MS). In an one-pot manner, this quantification method enables simultaneous coverage of 20 important metabolic pathways including protein biosynthesis/degradation, biosyntheses of catecholamines, arginine and glutathione, metabolisms of homocysteine, taurine-hypotaurine etc. Compared with the reported ones, this method is capable of simultaneously quantifying thiols, disulfides and other oxidation-prone analytes in a single run and suitable for quantifying aromatic amino metabolites. This method is also much more sensitive for all tested metabolites with LODs well below 50 fmol (at sub-fmol for most tested analytes) and shows good precision for retention time and quantitation with inter-day and intra-day relative standard deviations (RSDs) below 15% and good recovery from renal cancer tissue, rat urine and plasma. The method was further applied to quantify the amino metabolites in silkworm hemolymph from multiple developmental stages showing its applicability in metabolomics and perhaps some clinical chemistry studies.

Despite the mechanisms for endogenous nitroxyl (HNO) production and action being incompletely understood, pharmacological donors show broad therapeutic promise and are in clinical trials. Mass spectrometry and site-directed mutagenesis showed that chemically distinct HNO donors 1-nitrosocyclohexyl acetate or Angeli's salt induced disulfides within cGMP-dependent protein kinase I-alpha (PKGIα), an interdisulfide between Cys42 of the two identical subunits of the kinase and a previously unobserved intradisulfide between Cys117 and Cys195 in the high affinity cGMP-binding site. Kinase activity was monitored in cells transfected with wildtype (WT), Cys42Ser or Cys117/195Ser PKGIα that cannot form the inter- or intradisulfide, respectively. HNO enhanced WT kinase activity, an effect significantly attenuated in inter- or intradisulfide-deficient PKGIα. To investigate whether the intradisulfide modulates cGMP binding, real-time imaging was performed in vascular smooth muscle cells expressing a FRET-biosensor comprising the cGMP-binding sites of PKGIα. HNO induced FRET changes similar to those elicited by an increase of cGMP, suggesting that intradisulfide formation is associated with activation of PKGIα. Intradisulfide formation in PKGIα correlated with enhanced HNO-mediated vasorelaxation in mesenteric arteries in vitro and arteriolar dilation in vivo in mice. HNO induces intradisulfide formation in PKGIα, inducing the same effect as cGMP binding, namely kinase activation and thus vasorelaxation.

Heteropoda venatoria in the family Sparassidae is highly valued in pantropical countries because the species feed on domestic insect pests. Unlike most other species of Araneomorphae, H. venatoria uses the great speed and strong chelicerae (mouthparts) with toxin glands to capture the insects instead of its web. Therefore, H. venatoria provides unique opportunities for venom evolution research. The venom of H. venatoria was explored by matrix-assisted laser desorption/ionization tandem time-of-flight and analyzing expressed sequence tags. The 154 sequences coding cysteine-rich peptides (CRPs) revealed 24 families based on the phylogenetic analyses of precursors and cysteine frameworks in the putative mature regions. Intriguingly, four kinds of motifs are first described in spider venom. Furthermore, combining the diverse CRPs of H. venatoria with previous spider venom peptidomics data, the structures of precursors and the patterns of cysteine frameworks were analyzed. This work revealed the dynamic evolutionary trends of venom CRPs in H. venatoria: the precursor has evolved an extended mature peptide with more cysteines, and a diminished or even vanished propeptides between the signal and mature peptides; and the CRPs evolved by multiple duplications of an ancestral ICK gene as well as recruitments of non-toxin genes.