Synthetic amorphous silica is a common food additive and a popular cosmetic ingredient. Mesoporous silica particles are also widely studied for their potential use in drug delivery and imaging applications because of their unique properties, such as tunable pore sizes, large surfaces areas, and assumed biocompatibility. Such a nanomaterial, when consisting of pure silicon dioxide, is generally considered to be chemically inert, but in this study, we showed that oxidation yields for different compounds were facilitated by simply incubating aqueous solutions with pure silica particles. Three thiol-containing molecules, L-cysteine, glutathione, and D-penicillamine, were studied separately, and it was found that more than 95% of oxidation happened after incubating any of these compounds with mesoporous silica particles in the dark for a day at room temperature. Oxidation increased over incubation time, and more oxidation was found for particles having larger surface areas. For nonporous silica particles at submicron ranges, yields of oxidation were different based on the structures of molecules, correlating with steric hindrance while accessing surfaces. We propose that the silyloxy radical (SiO•) on silica surfaces is what facilitates oxidation. Density functional theory calculations were conducted for total energy changes for reactions between different aqueous species and silicon dioxide surfaces. These calculations identified two most plausible pathways of the lowest energy to generate SiO• radicals from water radical cations H2O•+ and hydroxyl radicals •OH, previously known to exist at water interfaces.

New unsymmetrical monoterpenylhetaryl disulfides based on heterocyclic disulfides and monoterpene thiols were synthesized for the first time in 48-88% yields. Hydrolysis of disulfides with fragments of methyl esters of 2-mercaptonicotinic acid was carried out in 73-95% yields. The obtained compounds were evaluated for antioxidant, antibacterial, antifungal activity, cytotoxicity and mutagenicity.

The translation inhibitor and tumor suppressor Pdcd4 was reported to be lost in various tumors and put forward as prognostic marker in tumorigenesis. Decreased Pdcd4 protein stability due to PI3K-mTOR-p70S6K1 dependent phosphorylation of Pdcd4 followed by β-TrCP1-mediated ubiquitination, and proteasomal destruction of the protein was characterized as a major mechanism contributing to the loss of Pdcd4 expression in tumors. In an attempt to identify stabilizers of Pdcd4, we used a luciferase-based high-throughput compatible cellular assay to monitor phosphorylation-dependent proteasomal degradation of Pdcd4 in response to mitogen stimulation. Following a screen of approximately 2000 compounds, we identified 1,2-bis(4-chlorophenyl)disulfide as a novel Pdcd4 stabilizer. To determine an initial structure-activity relationship, we used 3 additional compounds, synthesized according to previous reports, and 2 commercially available compounds for further testing, in which either the linker between the aryls was modified (compounds 2-4) or the chlorine residues were replaced by groups with different electronic properties (compounds 5 and 6). We observed that those compounds with alterations in the sulfide linker completely lost the Pdcd4 stabilizing potential. In contrast, modifications in the chlorine residues showed only minor effects on the Pdcd4 stabilizing activity. A reporter with a mutated phospho-degron verified the specificity of the compounds for stabilizing the Pdcd4 reporter. Interestingly, the active diaryl disulfides inhibited proliferation and viability at concentrations where they stabilized Pdcd4, suggesting that Pdcd4 stabilization might contribute to the anti-proliferative properties. Finally, computational modelling indicated that the flexibility of the disulfide linker might be necessary to exert the biological functions of the compounds, as the inactive compound appeared to be energetically more restricted.

The channel-forming component of anthrax toxin, (PA(63))(7), is a heptameric water-soluble protein at neutral pH, but under acidic conditions it spontaneously inserts into lipid bilayers to form a 14-stranded beta-barrel ion-conducting channel. This channel plays a vital role in anthrax pathogenesis because it serves as a conduit for the membrane translocation of the two enzymatic components of anthrax toxin, lethal factor and edema factor. Anthrax channels open and close in response to changes in transmembrane voltage, a property shared by several other pore-forming toxins. We have discovered an unexpected phenomenon in cysteine-substituted channels that provides a window into this gating process: their normal voltage-dependent gating can be abolished by reaction with methanethiosulfonate (MTS) reagents or exposure to oxidizing conditions. Remarkably, this perturbation is seen with cysteines substituted at sites all along the approximately 100 A length of the channel's beta-barrel. In contrast, reaction with N-ethylmaleimide, a thiol-reactive compound that does not form a mixed disulfide, does not affect gating at any of the sites tested. These findings, coupled with our biochemical detection of dimers, have led us to conclude that MTS reagents are catalyzing the formation of intersubunit disulfide bonds that lock channels in a conducting state, and that voltage gating requires a conformational change that involves the entire beta-barrel.

Protein disulfide bonds link pairs of cysteine sulfur atoms and are either structural or functional motifs. The allosteric disulfides control the function of the protein in which they reside when cleaved or formed. Here, we identify potential allosteric disulfides in all Protein Data Bank X-ray structures from bonds that are present in some molecules of a protein crystal but absent in others, or present in some structures of a protein but absent in others. We reasoned that the labile nature of these disulfides signifies a propensity for cleavage and so possible allosteric regulation of the protein in which the bond resides. A total of 511 labile disulfide bonds were identified. The labile disulfides are more stressed than the average bond, being characterized by high average torsional strain and stretching of the sulfur-sulfur bond and neighbouring bond angles. This pre-stress likely underpins their susceptibility to cleavage. The coagulation, complement and oxygen-sensing hypoxia inducible factor-1 pathways, which are known or have been suggested to be regulated by allosteric disulfides, are enriched in proteins containing labile disulfides. The identification of labile disulfide bonds will facilitate the study of this post-translational modification.

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.

Environmental degradation of transition metal disulfides (TMDs) is a key stumbling block in a range of applications. We show that a simple one-pot non-covalent pyrene coating process protects TMDs from both photoinduced oxidation and environmental aging. Pyrene is immobilized non-covalently on the basal plane of exfoliated MoS2 and WS2. The optical properties of TMD/pyrene are assessed via electronic absorption and fluorescence emission spectroscopy. High-resolution scanning transmission electron microscopy coupled with electron energy loss spectroscopy confirms extensive pyrene surface coverage, with density functional theory calculations suggesting a strongly bound stable parallel-stacked pyrene coverage of ~2-3 layers on the TMD surfaces. Raman spectroscopy of exfoliated TMDs while irradiating at 0.9 mW/4 μm2 under ambient conditions shows new and strong Raman bands due to oxidized states of Mo and W. Yet remarkably, under the same exposure conditions TMD/pyrene remain unperturbed. The current findings demonstrate that pyrene physisorbed on MoS2 and WS2 acts as an environmental barrier, preventing oxidative surface reactions in the TMDs catalyzed by moisture, air, and assisted by laser irradiation. Raman spectroscopy confirms that the hybrid materials stored under ambient conditions for two years remained structurally unaltered, corroborating the beneficial role of pyrene for not only hindering oxidation but also inhibiting aging.

The synthesis of precise gene delivery vehicles by solid-supported chemistry is an effective way to establish structure-activity relationships and optimize existing transfection carriers. Sequence-defined cationic oligomers with different topologies were modified with twin disulfide-forming cysteine-arginine-cysteine (CRC) motifs. The influence of this motif versus single disulfide on the biophysical properties and biological performance of polyplexes was investigated, with pDNA and siRNA as nucleic acid cargoes. Clear differences between structures with isolated cysteines and CRC motifs were observed with respect to properties like nucleic acid binding, serum stability, response to reducing agents, and gene transfer/silencing. The main observed effect of the CRC motif was to increase polyplex stability. The consequences for nucleic acid delivery were less predictable and depended on oligomer topology. For some oligomers intrinsically forming stable polyplexes (i.e., already in the absence of CRC motif), this further stabilization resulted in a reduction or even loss in transfection efficiency. For PEGylated and targeted oligomers with intrinsically less stable polyplex structures, this modification led to a significant enhancement in transfection efficiency.

The replication machinery of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is closely associated with the endoplasmic reticulum (ER) in host cells. Activation of the unfolded protein response (UPR) is a strategy hijacked by coronavirus to facilitate its replication and suppress host innate immunity. Here, we have found that SARS-CoV-2 ORF8 protein accumulates in the ER and escapes the degradation system by forming mixed disulfide complexes with ER oxidoreductases. ORF8 induces the activation of three UPR pathways through targeting key UPR components, remodels ER morphology and accelerates protein trafficking. Moreover, small molecule reducing agents release ORF8 from the mixed disulfide complexes and facilitate its degradation, therefore mitigate ER stress. Our study reveals a unique mechanism by which SARS-CoV-2 ORF8 escapes degradation by host cells and regulates ER reshaping. Targeting ORF8-involved mixed disulfide complexes could be a new strategy to alleviate SARS-CoV-2 induced ER stress and related diseases.

Polymerizations of phenylamines with a disulfide transfer reagent to yield poly[N,N-(phenylamino) disulfides] (poly-NADs) were investigated due to their unique repeat units that resulted in conjugation along the backbone that was perturbed by the aromatic rings and gave different colors for the polymers. These polymers were synthesized from 10 different anilines and sulfur monochloride in a step-growth polymerization. The polymers were characterized by nuclear magnetic resonance spectroscopy, size exclusion chromatography-multiangle light scattering, and UV-vis spectroscopy. These polymers possessed a polymeric backbone solely consisting of nitrogen and sulfur [-N(R)SS-], which was conjugated and yielded polymers of moderate molecular weight. Most notably, these polymers were an array of colors ranging from pale yellow to a deep purple depending on the substitution of the aromatic ring. The more electron-poor systems produced lighter yellow polymers, while the electron-rich systems gave orange, green, red, and even purple polymers.

APOBEC3G (A3G) is a member of cytosine deaminase family with a variety of innate immune functions. It displays activities against retrovirus and retrotransposon by inhibition of virus infectivity factor (Vif)-deficient HIV-1 replication. The interaction between A3G N-terminal domain and Vif directs the cellular Cullin 5 E3-ubiquitin ligase complex to ubiquitinate A3G, and leads to A3G proteasomal degradation, which is a potential target for anti-HIV drug. Currently, there are very few reports about stable small molecules targeting the interaction between A3G and Vif. In this study, we screened two series of small molecules containing carbamyl sulfamide bond or disulfide bond as bridges of two different aromatic rings. Five asymmetrical disulfides were successfully identified against interaction between A3G and Vif with the IC 50 values close to or smaller than 1 μM, especially, not through covalently binding with A3G or Vif. They restore the A3G expression in the presence of Vif by inhibiting Vif-induced A3G ubiquitination and degradation. This study opens a way to the discovery of new anti-HIV drugs.

Despite their ubiquity, self-assembled monolayers (SAMs) of thiols on coinage metals are difficult to study and are still not completely understood, particularly with respect to the nature of thiol-metal bonding. Recent advances in molecular electronics have highlighted this deficiency due to the sensitivity of tunneling charge-transport to the subtle differences in the overall composition of SAMs and the chemistry of their attachment to surfaces. These advances have also challenged assumptions about the spontaneous formation of covalent thiol-metal bonds. This paper describes a series of experiments that correlate changes in the physical properties of SAMs to photoelectron spectroscopy to unambiguously assign binding energies of noncovalent interactions to physisorbed disulfides. These disulfides can be converted to covalent metal-thiolate bonds by exposure to free thiols, leading to the remarkable observation of the total loss and recovery of length-dependent tunneling charge-transport. The identification and assignment of physisorbed disulfides solve a long-standing mystery and reveal new, dynamic properties in SAMs of thiols.

The cyclic five-membered disulfide 1,2-dithiolane has been widely used in chemical biology and in redox probes. Contradictory reports have described it either as nonspecifically reduced in cells, or else as a highly specific substrate for thioredoxin reductase (TrxR). Here we show that 1,2-dithiolane probes, such as "TRFS" probes, are nonspecifically reduced by thiol reductants and redox-active proteins, and their cellular performance is barely affected by TrxR inhibition or knockout. Therefore, results of cellular imaging or inhibitor screening using 1,2-dithiolanes should not be interpreted as reflecting TrxR activity, and previous studies may need re-evaluation. To understand 1,2-dithiolanes' complex behaviour, probe localisation, environment-dependent fluorescence, reduction-independent ring-opening polymerisation, and thiol-dependent cellular uptake must all be considered; particular caution is needed when co-applying thiophilic inhibitors. We present a general approach controlling against assay misinterpretation with reducible probes, to ensure future TrxR-targeted designs are robustly evaluated for selectivity, and to better orient future research.

The intermediate filament protein keratin 14 (K14) provides vital structural support in basal keratinocytes of epidermis. Recent studies evidenced a role for K14-dependent disulfide bonding in the organization and dynamics of keratin IFs in skin keratinocytes. Here we report that knock-in mice harboring a cysteine-to-alanine substitution at Krt14's codon 373 (C373A) exhibit alterations in disulfide-bonded K14 species and a barrier defect secondary to enhanced proliferation, faster transit time and altered differentiation in epidermis. A proteomics screen identified 14-3-3 as K14 interacting proteins. Follow-up studies showed that YAP1, a transcriptional effector of Hippo signaling regulated by 14-3-3sigma in skin keratinocytes, shows aberrant subcellular partitioning and function in differentiating Krt14 C373A keratinocytes. Residue C373 in K14, which is conserved in a subset of keratins, is revealed as a novel regulator of keratin organization and YAP function in early differentiating keratinocytes, with an impact on cell mechanics, homeostasis and barrier function in epidermis.

Congenital FXIII deficiency is a rare bleeding disorder in which mutations are detected in F13A1 and F13B genes that express the two subunits of coagulation FXIII, the catalytic FXIII-A, and protective FXIII-B. Mutations in FXIII-B subunit are considerably rarer compared to FXIII-A. Three mutations in the F13B gene have been reported on its structural disulfide bonds. In the present study, we investigate the structural and functional importance of all 20 structural disulfide bonds in FXIII-B subunit. All disulfide bonds were ablated by individually mutating one of its contributory cysteine's, and these variants were transiently expressed in HEK293t cell lines. The expression products were studied for stability, secretion, the effect on oligomeric state, and on FXIII-A activation. The structural flexibility of these disulfide bonds was studied using classical MD simulation performed on a FXIII-B subunit monomer model. All 20 FXIII-B were found to be important for the secretion and stability of the protein since ablation of any of these led to a secretion deficit. However, the degree of effect that the disruption of disulfide bond had on the protein differed between individual disulfide bonds reflecting a functional hierarchy/diversity within these disulfide bonds.

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).

Chemical vapor deposition (CVD) has been widely used to produce high quality 2D transitional metal dichalcogenides (2D TMDCs). However, violent evaporation and large diffusivity discrepancy of metal and chalcogen precursors at elevated temperatures often result in poor regulation on X:M molar ratio (M = Mo, W etc.; X = S, Se, and Te), and thus it is rather challenging to achieve the desired products of 2D TMDCs. Here, a modified spatially confined strategy (MSCS) is utilized to suppress the rising S vapor concentration between two aspectant substrates, upon which the lateral/vertical growth of 2D WS2 can be selectively regulated via proper S:W zones correspond to greatly broadened time/growth windows. An S:W-time (SW-T) growth diagram was thus proposed as a mapping guide for the general understanding of CVD growth of 2D WS2 and the design of growth routes for the desired 2D WS2 . Consequently, a comprehensive growth management of atomically thin WS2 is achieved, including the versatile controls of domain size, layer number, and lateral/vertical heterostructures (MoS2 -WS2 ). The lateral heterostructures show an enhanced hydrogen evolution reaction performance. This study advances the substantial understanding to the growth kinetics and provides an effective MSCS protocol for growth design and management of 2D TMDCs.

Transcription factor p53 plays a critical role in the cellular response to stress stimuli. We have seen that p53 dissociates selectively from various promoter sites as a result of oxidation at long-range through DNA-mediated charge transport (CT). Here, we examine this chemical oxidation and determine the residues in p53 that are essential for oxidative dissociation, focusing on the network of cysteine residues adjacent to the DNA-binding site. Of the eight mutants studied, only the C275S mutation shows decreased affinity for the Gadd45 promoter site. However, both mutations C275S and C277S result in substantial attenuation of oxidative dissociation, with C275S causing the most severe attenuation. Differential thiol labeling was used to determine the oxidation states of cysteine residues within p53 after DNA-mediated oxidation. Reduced cysteines were iodoacetamide-labeled, whereas oxidized cysteines participating in disulfide bonds were (13)C2D2-iodoacetamide-labeled. Intensities of respective iodoacetamide-modified peptide fragments were analyzed by mass spectrometry. A distinct shift in peptide labeling toward (13)C2D2-iodoacetamide-labeled cysteines is observed in oxidized samples, confirming that chemical oxidation of p53 occurs at long range. All observable cysteine residues trend toward the heavy label under conditions of DNA CT, indicating the formation of multiple disulfide bonds among the cysteine network. On the basis of these data, it is proposed that disulfide formation involving C275 is critical for inducing oxidative dissociation of p53 from DNA.

ERdj5 is a member of the protein disulfide isomerase family of proteins localized to the endoplasmic reticulum (ER) of mammalian cells. To date, only a limited number of substrates for ERdj5 are known. Here we identify a number of endogenous substrates that form mixed disulfides with ERdj5, greatly expanding its client repertoire. ERdj5 previously had been thought to exclusively reduce disulfides in proteins destined for dislocation to the cytosol for degradation. However, we demonstrate here that for one of the identified substrates, the low-density lipoprotein receptor (LDLR), ERdj5 is required not for degradation, but rather for efficient folding. Our results demonstrate that the crucial role of ERdj5 is to reduce non-native disulfides formed during productive folding and that this requirement is dependent on its interaction with BiP. Hence, ERdj5 acts as the ER reductase, both preparing misfolded proteins for degradation and catalyzing the folding of proteins that form obligatory non-native disulfides.

Reduction of intramolecular disulfides in the HIV-1 envelope protein gp120 occurs after its binding to the CD4 receptor. Protein disulfide isomerase (PDI) catalyzes the disulfide reduction in vitro and inhibition of this enzyme blocks viral entry. PDI belongs to the thioredoxin protein superfamily that also includes human glutaredoxin-1 (Grx1). Grx1 is secreted from cells and the protein has also been found within the HIV-1 virion. We show that Grx1 efficiently catalyzes gp120, and CD4 disulfide reduction in vitro, even at low plasma levels of glutathione. Grx1 catalyzes the reduction of two disulfide bridges in gp120 in a similar manner as PDI. Purified anti-Grx1 antibodies were shown to inhibit the Grx1 activity in vitro and block HIV-1 replication in cultured peripheral blood mononuclear cells. Also, the polyanion PRO2000, that was previously shown to prevent HIV entry, inhibits the Grx1- and PDI-dependent reduction of gp120 disulfides. Our findings suggest that Grx1 activity is important for HIV-1 entry and that Grx1 and the gp120 intramolecular disulfides are novel pharmacological targets for rational drug development.