Browse
Submit Data
Databases
API
Help

Dataset Information

8 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

The function of SARS-CoV-2 spike protein is impaired by disulfide-bond disruption with mutation at cysteine-488 and by thiol-reactive N-acetyl-cysteine and glutathione

ABSTRACT: Viral spike proteins play important roles in the viral entry process, facilitating attachment to cellular receptors and fusion of the viral envelope with the cell membrane. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein binds to the cellular receptor angiotensin converting enzyme-2 (ACE2) via its receptor-binding domain (RBD). The cysteine residue at position 488, consisting of a disulfide bridge with cysteine 480 is located in an important structural loop at ACE2-binding surface of RBD, and is highly conserved among SARS-related coronaviruses. We showed that the substitution of Cys-488 with alanine impaired pseudotyped SARS-CoV-2 infection, syncytium formation, and cell-cell fusion triggered by SARS-CoV-2 spike expression. Consistently, in vitro binding of RBD and ACE2, spike-mediated cell-cell fusion, and pseudotyped viral infection of VeroE6/TMPRSS2 cells were inhibited by the thiol-reactive compounds N-acetylcysteine (NAC) and a reduced form of glutathione (GSH). Furthermore, we demonstrated that the activity of variant spikes from the SARS-CoV-2 alpha and delta strains were also suppressed by NAC and GSH. Taken together, these data indicate that Cys-488 in spike RBD is required for SARS-CoV-2 spike functions and infectivity, and could be a target of anti-SARS-CoV-2 therapeutics.

SUBMITTER: Murae M

PROVIDER: S-EPMC8800159 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

A single disulfide bond disruption in the ?3 integrin subunit promotes thiol/disulfide exchange, a molecular dynamics study.

Project description:The integrins are a family of membrane receptors that attach a cell to its surrounding and play a crucial function in cell signaling. The combination of internal and external stimuli alters a folded non-active state of these proteins to an extended active configuration. The ?3 subunit of the platelet ?IIb?3 integrin is made of well-structured domains rich in disulfide bonds. During the activation process some of the disulfides are re-shuffled by a mechanism requiring partial reduction of some of these bonds; any disruption in this mechanism can lead to inherent blood clotting diseases. In the present study we employed Molecular Dynamics simulations for tracing the sequence of structural fluctuations initiated by a single cysteine mutation in the ?3 subunit of the receptor. These simulations showed that in-silico protein mutants exhibit major conformational deformations leading to possible disulfide exchange reactions. We suggest that any mutation that prevents Cys560 from reacting with one of the Cys(567)-Cys(581) bonded pair, thus disrupting its ability to participate in a disulfide exchange reaction, will damage the activation mechanism of the integrin. This suggestion is in full agreement with previously published experiments. Furthermore, we suggest that rearrangement of disulfide bonds could be a part of a natural cascade of thiol/disulfide exchange reactions in the ?IIb?3 integrin, which are essential for the native activation process.

| S-EPMC3601072 | biostudies-literature

Thiol-based chemical probes exhibit antiviral activity against SARS-CoV-2 via allosteric disulfide disruption in the spike glycoprotein.

Project description:The development of small-molecules targeting different components of SARS-CoV-2 is a key strategy to complement antibody-based treatments and vaccination campaigns in managing the COVID-19 pandemic. Here, we show that two thiol-based chemical probes that act as reducing agents, P2119 and P2165, inhibit infection by human coronaviruses, including SARS-CoV-2, and decrease the binding of spike glycoprotein to its receptor, the angiotensin-converting enzyme 2 (ACE2). Proteomics and reactive cysteine profiling link the antiviral activity to the reduction of key disulfides, specifically by disruption of the Cys379-Cys432 and Cys391-Cys525 pairs distal to the receptor binding motif in the receptor binding domain (RBD) of the spike glycoprotein. Computational analyses provide insight into conformation changes that occur when these disulfides break or form, consistent with an allosteric role, and indicate that P2119/P2165 target a conserved hydrophobic binding pocket in the RBD with the benzyl thiol-reducing moiety pointed directly toward Cys432. These collective findings establish the vulnerability of human coronaviruses to thiol-based chemical probes and lay the groundwork for developing compounds of this class, as a strategy to inhibit the SARS-CoV-2 infection by shifting the spike glycoprotein redox scaffold.

| S-EPMC8833197 | biostudies-literature

SARS-CoV-2 Spike Protein Mutation at Cysteine-488 Impairs Its Golgi Localization and Intracellular S1/S2 Processing.

Project description:The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein binds to the cellular receptor-angiotensin-converting enzyme-2 (ACE2) as the first step in viral cell entry. SARS-CoV-2 spike protein expression in the ACE2-expressing cell surface induces cell-cell membrane fusion, thus forming syncytia. To exert its fusogenic activity, the spike protein is typically processed at a specific site (the S1/S2 site) by cellular proteases such as furin. The C488 residue, located at the spike-ACE2 interacting surface, is critical for the fusogenic and infectious roles of the SARS-CoV-2 spike protein. We have demonstrated that the C488 residue of the spike protein is involved in subcellular targeting and S1/S2 processing. C488 mutant spike localization to the Golgi apparatus and cell surface were impaired. Consequently, the S1/S2 processing of the spike protein, probed by anti-Ser-686-cleaved spike antibody, markedly decreased in C488 mutant spike proteins. Moreover, brefeldin-A-mediated endoplasmic-reticulum-to-Golgi traffic suppression also suppressed spike protein S1/S2 processing. As brefeldin A treatment and C488 mutation inhibited S1/S2 processing and syncytia formation, the C488 residue of spike protein is required for functional spike protein processing.

| S-EPMC9779352 | biostudies-literature

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

Project description: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.

| S-EPMC7089426 | biostudies-literature

2,2'-Dipyridyl diselenide: A chemoselective tool for cysteine deprotection and disulfide bond formation.

Project description:There are many examples of bioactive, disulfide-rich peptides and proteins whose biological activity relies on proper disulfide connectivity. Regioselective disulfide bond formation is a strategy for the synthesis of these bioactive peptides, but many of these methods suffer from a lack of orthogonality between pairs of protected cysteine (Cys) residues, efficiency, and high yields. Here, we show the utilization of 2,2'-dipyridyl diselenide (PySeSePy) as a chemical tool for the removal of Cys-protecting groups and regioselective formation of disulfide bonds in peptides. We found that peptides containing either Cys(Mob) or Cys(Acm) groups treated with PySeSePy in trifluoroacetic acid (TFA) (with or without triisopropylsilane (TIS) were converted to Cys-S-SePy adducts at 37 °C and various incubation times. This novel Cys-S-SePy adduct is able to be chemoselectively reduced by five-fold excess ascorbate at pH 4.5, a condition that should spare already installed peptide disulfide bonds from reduction. This chemoselective reduction by ascorbate will undoubtedly find utility in numerous biotechnological applications. We applied our new chemistry to the iodine-free synthesis of the human intestinal hormone guanylin, which contains two disulfide bonds. While we originally envisioned using ascorbate to chemoselectively reduce one of the formed Cys-S-SePy adducts to catalyze disulfide bond formation, we found that when pairs of Cys(Acm) residues were treated with PySeSePy in TFA, the second disulfide bond formed spontaneously. Spontaneous formation of the second disulfide is most likely driven by the formation of the thermodynamically favored diselenide (PySeSePy) from the two Cys-S-SePy adducts. Thus, we have developed a one-pot method for concomitant deprotection and disulfide bond formation of Cys(Acm) pairs in the presence of an existing disulfide bond.

| S-EPMC7509986 | biostudies-literature

Cysteine [2,4] Disulfide Bond as a New Modifiable Site of α-Conotoxin TxIB.

Project description:α-Conotoxin TxIB, a selective antagonist of α6/α3β2β3 nicotinic acetylcholine receptor, could be a potential therapeutic agent for addiction and Parkinson's disease. As a peptide with a complex pharmacophoric conformation, it is important and difficult to find a modifiable site which can be modified effectively and efficiently without activity loss. In this study, three xylene scaffolds were individually reacted with one pair of the cysteine residues ([1,3] or [2,4]), and iodine oxidation was used to form a disulfide bond between the other pair. Overall, six analogs were synthesized with moderate isolated yields from 55% to 65%, which is four times higher than the traditional two-step oxidation with orthogonal protection on cysteines. The cysteine [2,4] modified analogs, with higher stability in human serum than native TxIB, showed obvious inhibitory effect and selectivity on α6/α3β2β3 nicotinic acetylcholine receptors (nAChRs), which was 100 times more than the cysteine [1,3] modified ones. This result demonstrated that the cysteine [2,4] disulfide bond is a new modifiable site of TxIB, and further modification can be a simple and feasible strategy for the exploitation and utilization of α-Conotoxin TxIB in drug discovery.

| S-EPMC7926623 | biostudies-literature

Oxidation of Human Copper Chaperone Atox1 and Disulfide Bond Cleavage by Cisplatin and Glutathione.

Project description:Cancer cells cope with high oxidative stress levels, characterized by a shift toward the oxidized form (GSSG) of glutathione (GSH) in the redox couple GSSG/2GSH. Under these conditions, the cytosolic copper chaperone Atox1, which delivers Cu(I) to the secretory pathway, gets oxidized, i.e., a disulfide bond is formed between the cysteine residues of the Cu(I)-binding CxxC motif. Switching to the covalently-linked form, sulfur atoms are not able to bind the Cu(I) ion and Atox1 cannot play an antioxidant role. Atox1 has also been implicated in the resistance to platinum chemotherapy. In the presence of excess GSH, the anticancer drug cisplatin binds to Cu(I)-Atox1 but not to the reduced apoprotein. With the aim to investigate the interaction of cisplatin with the disulfide form of the protein, we performed a structural characterization in solution and in the solid state of oxidized human Atox1 and explored its ability to bind cisplatin under conditions mimicking an oxidizing environment. Cisplatin targets a methionine residue of oxidized Atox1; however, in the presence of GSH as reducing agent, the drug binds irreversibly to the protein with ammine ligands trans to Cys12 and Cys15. The results are discussed with reference to the available literature data and a mechanism is proposed connecting platinum drug processing to redox and copper homeostasis.

| S-EPMC6769983 | biostudies-literature

Missing links in understanding redox signaling via thiol/disulfide modulation: how is glutathione oxidized in plants?

Project description:Glutathione is a small redox-active molecule existing in two main stable forms: the thiol (GSH) and the disulphide (GSSG). In plants growing in optimal conditions, the GSH:GSSG ratio is high in most cell compartments. Challenging environmental conditions are known to alter this ratio, notably by inducing the accumulation of GSSG, an effect that may be influential in the perception or transduction of stress signals. Despite the potential importance of glutathione status in redox signaling, the reactions responsible for the oxidation of GSH to GSSG have not been clearly identified. Most attention has focused on the ascorbate-glutathione pathway, but several other candidate pathways may couple the availability of oxidants such as H2O2 to changes in glutathione and thus impact on signaling pathways through regulation of protein thiol-disulfide status. We provide an overview of the main candidate pathways and discuss the available biochemical, transcriptomic, and genetic evidence relating to each. Our analysis emphasizes how much is still to be elucidated on this question, which is likely important for a full understanding of how stress-related redox regulation might impinge on phytohormone-related and other signaling pathways in plants.

| S-EPMC3838956 | biostudies-literature

Effect of cysteine mutagenesis on the function and disulfide bond formation of human ABCG2.

Project description:ABCG2 is a member of the ATP-binding cassette (ABC) transporter superfamily. Its overexpression causes multidrug resistance in cancer chemotherapy. Based on its apparent half size in sequence when compared with other traditional ABC transporters, ABCG2 has been thought to exist and function as a homodimer linked by intermolecular disulfide bonds. However, recent evidence suggests that ABCG2 may exist as a higher form of oligomers due to noncovalent interactions. In this study, we attempted to create a cysless mutant ABCG2 as a tool for further characterization of this molecule. However, we found that the cysless mutant ABCG2 is well expressed but not functional. Mapping of the cysteine residues showed that three cysteine residues (Cys284, Cys374, and Cys438) are required concurrently for the function of ABCG2 and potentially for intramolecular disulfide bond formation. We also found that the cysteine residues (Cys592, Cys603, and Cys608) in the third extracellular loop are involved in forming intermolecular disulfide bonds and that mutation of these residues does not affect the expression or drug transport activity of human ABCG2. Thus, we conclude that Cys284, Cys374, and Cys438, which may be involved in intramolecular disulfide bond formation, are concurrently required for ABCG2 function, whereas Cys592, Cys603, and Cys608, potentially involved in intermolecular disulfide bond formation, are not required.

| S-EPMC2632310 | biostudies-literature

Cysteine sulfenic acid as an intermediate in disulfide bond formation and nonenzymatic protein folding.

Project description:As a posttranslational protein modification, cysteine sulfenic acid (Cys-SOH) is well established as an oxidative stress-induced mediator of enzyme function and redox signaling. Data presented herein show that protein Cys-SOH forms spontaneously in air-exposed aqueous solutions of unfolded (disulfide-reduced) protein in the absence of added oxidizing reagents, mediating the oxidative disulfide bond formation process key to in vitro, nonenzymatic protein folding. Molecular oxygen (O(2)) and trace metals [e.g., copper(II)] are shown to be important reagents in the oxidative refolding process. Cys-SOH is also shown to play a role in spontaneous disulfide-based dimerization of peptide molecules containing free cysteine residues. In total, the data presented expose a chemically ubiquitous role for Cys-SOH in solutions of free cysteine-containing protein exposed to air.

| S-EPMC2945302 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data