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 "steady state" 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 almost 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 steady state 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 ΔdsbA mutant. Taken together this suggests the presence of an auxiliary, glutathione-dependent thiol-oxidation system in the bacterial periplasm.

An increase in surface hydrophobicity mediates chaperone activity in N-chlorinated RidA.

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), it turns into a potent chaperone-like holdase that can effectively protect E. coli's proteome during oxidative stress. However, it is unknown, which residues 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 RidA's chaperone-like function. Through LC-MS/MS of digested RidAHOCl, we obtained direct evidence of the chlorination of one arginine residue. To overcome the instability of the N-chloramine modification, 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/or R128 led to a substantial reduction of RidAHOCl's 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.

Hypochlorous acid-modified human serum albumin suppresses MHC class II - dependent antigen presentation in pro-inflammatory macrophages.

Macrophages are innate immune cells that internalize and present exogenous antigens to T cells via MHC class II proteins. They operate at sites of infection in a highly inflammatory environment, generated in part by reactive oxygen species, in particular the strong oxidant hypochlorous acid (HOCl) produced in the neutrophil respiratory burst. HOCl effectively kills a broad range of pathogens but can also contribute to host tissue damage at sites of inflammation. To prevent tissue injury, HOCl is scavenged by human serum albumin (HSA) and other plasma proteins in interstitial fluids, leading to the formation of variously modified advanced oxidation products (AOPPs) with pro-inflammatory properties. Previously, we showed that HOCl-mediated N-chlorination converts HSA and other plasma proteins into efficient activators of the phagocyte respiratory burst, but the role of these AOPPs in antigen presentation by macrophages remained unclear. Here, we show that physiologically relevant amounts of N-chlorinated HSA can strongly impair the capacity of THP-1-derived macrophages to present antigens to antigen-specific T cells via MHC class II proteins at multiple stages. Initially, N-chlorinated HSA inhibits antigen internalization by converting antigens into scavenger receptor (SR) ligands and competing with the modified antigens for binding to SR CD36. Later steps of antigen presentation, such as intracellular antigen processing and MHC class II expression are negatively affected, as well. We propose that impaired processing of pathogens or exogenous antigens by immune cells at an initial stage of infection prevents antigen presentation in an environment potentially hostile to cells of the adaptive immune response, possibly shifting it towards locations removed from the actual insult, like the lymph nodes. On the flip side, excessive retardation or complete inhibition of antigen presentation by N-chlorinated plasma proteins could contribute to chronic infection and inflammation.

Functional metagenomics of the thioredoxin superfamily.

Environmental sequence data of microbial communities now makes up the majority of public genomic information. The assignment of a function to sequences from these metagenomic sources is challenging because organisms associated with the data are often uncharacterized and not cultivable. To overcome these challenges, we created a rationally designed expression library of metagenomic proteins covering the sequence space of the thioredoxin superfamily. This library of 100 individual proteins represents more than 22,000 thioredoxins found in the Global Ocean Sampling data set. We screened this library for the functional rescue of Escherichia coli mutants lacking the thioredoxin-type reductase (ΔtrxA), isomerase (ΔdsbC), or oxidase (ΔdsbA). We were able to assign functions to more than a quarter of our representative proteins. The in vivo function of a given representative could not be predicted by phylogenetic relation but did correlate with the predicted isoelectric surface potential of the protein. Selected proteins were then purified, and we determined their activity using a standard insulin reduction assay and measured their redox potential. An unexpected gel shift of protein E5 during the redox potential determination revealed a redox cycle distinct from that of typical thioredoxin-superfamily oxidoreductases. Instead of the intramolecular disulfide bond formation typical for thioredoxins, this protein forms an intermolecular disulfide between the attacking cysteines of two separate subunits during its catalytic cycle. Our functional metagenomic approach proved not only useful to assign in vivo functions to representatives of thousands of proteins but also uncovered a novel reaction mechanism in a seemingly well-known protein superfamily.

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.

Corrigendum: Characterization of ML-005, a Novel Metaproteomics-Derived Esterase.

[This corrects the article DOI: 10.3389/fmicb.2018.01925.].

Loss of a conserved salt bridge in bacterial glycosyl hydrolase BgIM-G1 improves substrate binding in temperate environments.

Salt bridges are the strongest electrostatic interactions in proteins. They substantially contribute to a protein's structural stability. Thus, mutations of salt bridges are typically selected against. Here, we report on the evolutionary loss of a highly conserved salt bridge in the GH1 family glycosyl hydrolase BglM-G1. BglM-G1's gene was found in the bacterial metagenome of a temperate, seasonally cold marine habitat. In BglM-G1, arginine 75 is replaced by a histidine. While fully retaining ß-glucosidase activity, BglM-G1 is less heat stable than an H75R variant, in which the salt bridge was artificially re-introduced. However, the K m toward its substrates was lower in wild type, leading to an overall higher catalytic efficiency. Our results indicate that this loss of the salt bridge leads to higher flexibility in BglM-G1's active site, trading structural stability at high temperatures, a trait not needed in a temperate, seasonally cold habitat, for a more effective catalytic activity.

Characterization of ML-005, a Novel Metaproteomics-Derived Esterase.

A novel gene encoding for a lipolytic enzyme, designated ML-005, was recently identified using a functional metaproteomics approach. We heterologously expressed this protein in Escherichia coli and biochemically characterized it. ML-005 exhibited lipolytic activity toward short-chained substrates with the preferred substrate being p-nitrophenyl-butyrate, suggesting that ML-005 is an esterase. According to homology analysis and site-directed mutagenesis, the catalytic triad of the enzyme was identified as Ser-99, Asp-164, and His-191. Its optimal pH was determined to be at pH 8. Optimal activity was observed at 45°C. It also exhibited temperature, pH and salt tolerance. Residual relative activity after incubating at 50-60°C for 360 min was above 80% of its initial activity. It showed tolerance over a broad range of pH (5-12) and retained most of its initial activity. Furthermore, incubating ML-005 in 1 - 5M NaCl solution had negligible effect on its activity. DTT, EDTA, and ß-mercaptoethanol had no significant effect on ML-005's activity. However, addition of PMSF led to almost complete inactivation consistent with ML-005 being a serine hydrolase. ML-005 remains stable in the presence of a range of metal ions, but addition of Cu2+ significantly reduces its relative activity. Organic solvents have an inhibitory effect on ML-005, but it retained 21% of activity in 10% methanol. SDS had the most pronounced inhibitory effect on ML-005 among all detergents tested and completely inactivated it. Furthermore, the Vmax of ML-005 was determined to be 59.8 µM/min along with a Km of 137.9 µM. The kcat of ML-005 is 26 s-1 and kcat/Km is 1.88 × 105 M-1 s-1.