Biology subjects

Lupilov, N.

Publications and source records attributed to Lupilov, N..

7 recordsLinked to original sources

bioRxiv2024

Comprehensive elucidation of glutathione import in Escherichia coli

Glutathione is the major thiol-based antioxidant in a wide variety of biological systems, ranging from bacteria to eukaryotes. As a redox couple, consisting of reduced glutathione (GSH) and oxidized glutathione disulfide (GSSG), it is crucial for the maintenance of the cellular redox balance. Glutathione transport out of and into cellular compartments and the extracellular space is a determinant of the thiol-disulfide redox state of the organelles and bodily fluids in question, but is currently not well understood. Here we use the genetically-encoded, glutathione-measuring redox probe Grx1-roGFP2 to comprehensively elucidate the import of extracellular glutathione into the cytoplasm of the model organism Escherichia coli. The elimination of only two ATP-Binding Cassette (ABC) transporter systems, Gsi and Opp, completely abrogates glutathione import into E. colis cytoplasm, both in its reduced and oxidized form. The lack of only one of them, Gsi, completely prevents import of oxidized glutathione (GSSG), while the lack of the other, Opp, substantially retards the uptake of reduced glutathione (GSH).

biochemistry↗

bioRxiv2024

HOCl Forms Lipid N-Chloramines in Cell Membranes of Bacteria and Immune Cells

Neutrophils orchestrate a coordinated attack on bacteria, combining phagocytosis with a potent cocktail of oxidants, including the highly toxic hypochlorous acid (HOCl), renowned for its deleterious effects on proteins. Here, we examined the occurrence of lipid N-chloramines in vivo, their biological activity and neutralization. Using a chemical probe for N-chloramines, we demonstrate their formation in the membranes of bacteria and monocytic cells exposed to physiologically relevant concentrations of HOCl. N-chlorinated model membranes composed of phosphatidylethanolamine, the major membrane lipid in Escherichia coli and an important component of eukaryotic membranes, exhibited oxidative activity towards the redox-sensitive protein roGFP2, suggesting a role for lipid N-chloramines in protein oxidation. Conversely, the cellular antioxidant glutathione neutralized lipid N-chloramines by removing the chlorine moiety. We propose that lipid N-chloramines, like protein N-chloramines, are involved in inflammation and accelerate the host immune response.

microbiology↗

bioRxiv2024

Auranofin induces disulfide bond-mimicking S-Au-S bonds in protein thiol pairs

Auranofin is an inhibitor of human thioredoxin reductase, clinically used in the treatment of rheumatoid arthritis. More recently, it has been shown to possess strong antibacterial activity. Despite the structural dissimilarity and the independent evolutionary origins of human thioredoxin reductase and its bacterial counterpart (TrxB), inhibition of bacterial thioredoxin reductase is often suggested to be a major factor in auranofins antibacterial mode of action. To test this hypothesis, we attempted to determine the mechanism of inhibition of auranofin for bacterial TrxB in the presence of thioredoxin, TrxBs natural substrate. However, the data obtained in these experiments was not consistent with a specific and exclusive interaction between TrxB and auranofin. Instead, it suggested that auranofin directly interacts with the cysteine thiols in thioredoxin, TrxBs substrate. Using the fluorescent redox protein roGFP2, we showed that auranofin does indeed directly interact with cysteine pairs in proteins, forming a thiol modification that is similar to, but clearly distinct from a disulfide bond. The Au:S stoichiometries of auranofin-treated roGFP2 and thioredoxin strongly suggest the presence of an S-Au-S bridge between two cysteines in those proteins. These S-Au-S bonds form independent of thioredoxin reductase at a rate that indicates their pertinence in auranofins antibacterial mode of action.

biochemistry↗

bioRxiv2023

Comparison of the mechanism of antimicrobial action of the gold(I) compound auranofin in Gram-positive and Gram-negative bacteria

The antirheumatic gold(I) compound auranofin has been suggested to exhibit bactericidal activity by disrupting the thiol homeostasis through direct inhibition of bacterial thioredoxin reductase (TrxB). While highly effective at killing Gram-positive bacteria, it lacks significant activity against Gram-negative species for reasons that largely remain unclear. Here, we aimed to elucidate the molecular mechanisms underlying the low susceptibility of the Gram-negative model organism Escherichia coli to auranofin when compared to the Gram-positive model organism Bacillus subtilis. A change in the proteome of E. coli exposed to auranofin suggested that the effect of this gold compound is a combination of inactivation of thiol-containing enzymes, upregulation of proteins involved in basal metabolism, and the consequent induction of systemic oxidative stress. Susceptibility tests in E. coli mutants lacking the proteins upregulated upon auranofin treatment suggested that none of them are directly involved in E. colis high tolerance to auranofin. To elucidate factors that could make Gram-negative bacteria less susceptible to auranofin, we tested E. coli cells lacking the efflux pump component TolC. These cells were more sensitive to auranofin treatment than the wild type, but not to an extent that would fully explain the observed difference in susceptibility of Gram-positive and Gram-negative organisms. We thus tested if E. colis thioredoxin reductase (TrxB) is inherently less sensitive to auranofin than TrxB from B. subtilis, which was not the case. E. coli cells lacking components of the thioredoxin-system were also only marginally more susceptible to auranofin. However, E. coli strains lacking the low molecular weight thiol glutathione, but not glutathione reductase, showed a high susceptibility to auranofin. Bacterial cells expressing the genetically encoded redox probe roGFP2 allowed us to observe the oxidation of cellular protein thiols in situ. In line with their susceptibility, the kinetics of probe oxidation and the degree of oxidation promoted by auranofin is significantly higher in Gram-positive bacteria when compared to Gram-negative bacteria. Based on our findings, we hypothesize that auranofin leads to a global disturbance in the cellular thiol redox homeostasis in bacteria, but Gram-negative bacteria are inherently more resistant due to the presence of drug export systems and high cellular concentrations of glutathione.

biochemistry↗

bioRxiv2023

The role of glutathione in periplasmic redox homeostasis and oxidative protein folding in Escherichia coli

The thiol redox balance in the periplasm of E. coli depends on the DsbA/B pair for oxidative power and the DsbC/D system as its complement for isomerization of non-native disulfides. While the standard redox potentials of those systems are known, the in vivo redox potential imposed onto protein thiol disulfide pairs in the periplasm remains unknown. Here, we used genetically encoded redox probes (roGFP2 and roGFP-iL), targeted to the periplasm, to directly probe the thiol redox homeostasis in this compartment. These probes contain two cysteine residues, that are virtually completely reduced in the cytoplasm, but once exported into the periplasm, can form a disulfide bond, a process that can be monitored by fluorescence spectroscopy. Even in the absence of DsbA, roGFP2, exported to the periplasm, was fully oxidized, suggesting the presence of an alternative system for the introduction of disulfide bonds into exported proteins. However, the absence of DsbA shifted the periplasmic thiol-redox potential from -228 mV to a more reducing -243 mV and the capacity to re-oxidize periplasmic roGFP2 after a reductive pulse was significantly decreased. Re-oxidation in a DsbA strain could be fully restored by exogenous oxidized glutathione (GSSG), while reduced GSH accelerated re-oxidation of roGFP2 in the WT. In line, a strain devoid of endogenous glutathione showed a more reducing periplasm, and was significantly worse in oxidatively folding PhoA, a native periplasmic protein and substrate of the oxidative folding machinery. PhoA oxidative folding could be enhanced by the addition of exogenous GSSG in the WT and fully restored in a {Delta}dsbA mutant. Taken together this suggests the presence of an auxiliary, glutathione-dependent thiol-oxidation system in the bacterial periplasm.

biochemistry↗

bioRxiv2021

An increase in surface hydrophobicity mediates chaperone activity in N-chlorinated proteins

Under physiological conditions, Escherichia coli RidA is an enamine/imine deaminase, which promotes the release of ammonia from reactive enamine/imine intermediates. However, when modified by hypochlorous acid (HOCl), as produced by the host defense, RidAHOCl turns into a potent chaperone-like holdase that can effectively protect the proteome of E. coli during oxidative stress. We previously reported that the activation of RidAs chaperone-like function coincides with the addition of at least seven and up to ten chlorine atoms. These atoms are reversibly added to basic amino acids in RidAHOCl and removal by reducing agents leads to inactivation. Nevertheless, it remains unclear, which residues in particular need to be chlorinated for activation. Here, we employ a combination of LC-MS/MS analysis, a chemo-proteomic approach, and a mutagenesis study to identify residues responsible for RidAs chaperone-like function. Through LC-MS/MS of digested RidAHOCl, we obtained direct evidence of the chlorination of one arginine residue (and, coincidentally, two tyrosine residues), while other N- chlorinated residues could not be detected, presumably due to the instability of the modification and its potential interference with a proteolytic digest. Therefore, we established a chemoproteomic approach using 5-(dimethylamino) naphthalene-1-sulfinic acid (DANSO2H) as a probe to label N-chlorinated lysines. Using this probe, we were able to detect the N-chlorination of six additional lysine residues. Moreover, using a mutagenesis study to genetically probe the role of single arginine and lysine residues, we found that the removal of arginines R105 and R128 leads to a substantial reduction of RidAHOCls chaperone activity. These results, together with structural analysis, confirm that the chaperone activity of RidA is concomitant with the loss of positive charges on the protein surface, leading to an increased overall protein hydrophobicity. Molecular modelling of RidAHOCl and the rational design of a RidA variant that shows chaperone activity even in the absence of HOCl further supports our hypothesis. Our data provide a molecular mechanism for HOCl-mediated chaperone activity found in RidA and a growing number of other HOCl-activated chaperones.

biochemistry↗

bioRxiv2019

N-chlorination mediates protective and immunomodulatory effects of oxidized human plasma proteins

Hypochlorous acid (HOCl), a powerful antimicrobial oxidant, is produced by neutrophils to fight infections. Here we show that N-chlorination, induced by HOCl concentrations encountered at sites of inflammation, converts blood plasma proteins into chaperone-like holdases that protect other proteins from aggregation. This chaperone-like conversion was reversible by antioxidants and was abrogated by prior methylation of basic amino acids. Furthermore, reversible N-chlorination of basic amino acid side chains is the major factor that converts plasma proteins into efficient activators of immune cells. Finally, HOCl-modified serum albumin was found to act as a pro-survival molecule that protects neutrophils from cell death induced by highly immunogenic foreign antigens. We propose that activation and enhanced persistence of neutrophils mediated by HOCl-modified plasma proteins, resulting in the increased and prolonged generation of ROS, including HOCl, constitutes a potentially detrimental positive feedback loop that can only be attenuated through the reversible nature of the modification involved.

biochemistry↗