Rhodanese-Fold Containing Proteins in Humans: Not Just Key Players in Sulfur Trafficking.

The Rhodanese-fold is a ubiquitous structural domain present in various protein subfamilies associated with different physiological functions or pathophysiological conditions in humans. Proteins harboring a Rhodanese domain are diverse in terms of domain architecture, with some representatives exhibiting one or several Rhodanese domains, fused or not to other structural domains. The most famous Rhodanese domains are catalytically active, thanks to an active-site loop containing an essential cysteine residue which allows for catalyzing sulfur transfer reactions involved in sulfur trafficking, hydrogen sulfide metabolism, biosynthesis of molybdenum cofactor, thio-modification of tRNAs or protein urmylation. In addition, they also catalyse phosphatase reactions linked to cell cycle regulation, and recent advances proposed a new role into tRNA hydroxylation, illustrating the catalytic versatility of Rhodanese domain. To date, no exhaustive analysis of Rhodanese containing protein equipment from humans is available. In this review, we focus on structural and biochemical properties of human-active Rhodanese-containing proteins, in order to provide a picture of their established or putative key roles in many essential biological functions.

Metal-chelating activity of soy and pea protein hydrolysates obtained after different enzymatic treatments from protein isolates.

Soy and pea proteins are two rich sources of essential amino acids. The hydrolysis of these proteins reveals functional and bioactive properties of the produced small peptide mixtures. In our study, we employed the hydrolysis of soy and pea protein isolates with the endopeptidases Alcalase® and Protamex®, used alone or followed by the exopeptidase Flavourzyme®. The sequential enzyme treatments were the most efficient regarding the degree of hydrolysis. Then, soy and pea protein hydrolysates (SPHs and PPHs, respectively) were ultrafiltrated in order to select peptides of molecular weight ≤ 1 kDa. Whatever the protein source or the hydrolysis treatment, the hydrolysates showed similar molecular weight distributions and amino acid compositions. In addition, all the ultrafiltrated hydrolysates possess metal-chelating activities, as determined by UV-spectrophotometry and Surface Plasmon Resonance (SPR). However, the SPR data revealed better chelating affinities in SPHs and PPHs when produced by sequential enzymatic treatment.

Lipolysis-Stimulated Lipoprotein Receptor Acts as Sensor to Regulate ApoE Release in Astrocytes.

Astroglia play an important role, providing de novo synthesized cholesterol to neurons in the form of ApoE-lipidated particles; disruption of this process can increase the risk of Alzheimer's disease. We recently reported that glia-specific suppression of the lipolysis-stimulated lipoprotein receptor (LSR) gene leads to Alzheimer's disease-like memory deficits. Since LSR is an Apo-E lipoprotein receptor, our objective of this study was to determine the effect of LSR expression modulation on cholesterol and ApoE output in mouse astrocytes expressing human ApoE3. qPCR analysis showed that siRNA-mediated lsr knockdown significantly increased expression of the genes involved in cholesterol synthesis, secretion, and metabolism. Analysis of media and lipoprotein fractions showed increased cholesterol and lipidated ApoE output in HDL-like particles. Further, lsr expression could be upregulated when astrocytes were incubated 5 days in media containing high levels (two-fold) of lipoprotein, or after 8 h treatment with 1 µM LXR agonist T0901317 in lipoprotein-deficient media. In both conditions of increased lsr expression, the ApoE output was repressed or unchanged despite increased abca1 mRNA levels and cholesterol production. We conclude that LSR acts as a sensor of lipoprotein content in the medium and repressor of ApoE release, while ABCA1 drives cholesterol efflux, thereby potentially affecting cholesterol load, ApoE lipidation, and limiting cholesterol trafficking towards the neuron.

Anti-Inflammatory Activity of Bryophytes Extracts in LPS-Stimulated RAW264.7 Murine Macrophages.

Bryophytes produce rare and bioactive compounds with a broad range of therapeutic potential, and many species are reported in ethnomedicinal uses. However, only a few studies have investigated their potential as natural anti-inflammatory drug candidate compounds. The present study investigates the anti-inflammatory effects of thirty-two species of bryophytes, including mosses and liverworts, on Raw 264.7 murine macrophages stimulated with lipopolysaccharide (LPS) or recombinant human peroxiredoxin (hPrx1). The 70% ethanol extracts of bryophytes were screened for their potential to reduce the production of nitric oxide (NO), an important pro-inflammatory mediator. Among the analyzed extracts, two moss species significantly inhibited LPS-induced NO production without cytotoxic effects. The bioactive extracts of Dicranum majus and Thuidium delicatulum inhibited NO production in a concentration-dependent manner with IC50 values of 1.04 and 1.54 µg/mL, respectively. The crude 70% ethanol and ethyl acetate extracts were then partitioned with different solvents in increasing order of polarity (n-hexane, diethyl ether, chloroform, ethyl acetate, and n-butanol). The fractions were screened for their inhibitory effects on NO production stimulated with LPS at 1 ng/mL or 10 ng/mL. The NO production levels were significantly affected by the fractions of decreasing polarity such as n-hexane and diethyl ether ones. Therefore, the potential of these extracts to inhibit the LPS-induced NO pathway suggests their effective properties in attenuating inflammation and could represent a perspective for the development of innovative therapeutic agents.

Electrically Switchable Nanolever Technology for the Screening of Metal-Chelating Peptides in Hydrolysates.

Metal-chelating peptides (MCP) are considered as indirect antioxidants due to their capacity to inhibit radical chain reaction and oxidation. Here, we propose a new proof of concept for the screening of MCPs present in protein hydrolysates for valorizing their antioxidant properties by using the emerging time-resolved molecular dynamics technology, switchSENSE. This method unveils possible interactions between MCPs and immobilized nickel ions using fluorescence and electro-switchable DNA chips. The switchSENSE method was first set up on synthetic peptides known for their metal-chelating properties. Then, it was applied to soy and tilapia viscera protein hydrolysates. Their Cu2+-chelation capacity was, in addition, determined by UV-visible spectrophotometry as a reference method. The switchSENSE method has displayed a high sensitivity to evidence the presence of MCPs in both hydrolysates. Hence, we demonstrate for the first time that this newly introduced technology is a convenient methodology to screen protein hydrolysates in order to determine the presence of MCPs before launching time-consuming separations.

Chromatographic separation simulation of metal-chelating peptides from surface plasmon resonance binding parameters.

Some metal-chelating peptides have antioxidant properties, with potential nutrition, health, and cosmetics applications. This study aimed to simulate their separation on immobilized metal ion affinity chromatography from their affinity constant for immobilized metal ion determined in surface plasmon resonance, both technics are based on peptide-metal ion interactions. In our approach, first, the affinity constant of synthetic peptides was determined by surface plasmon resonance and used as input data to numerically simulate the chromatographic separation with a transport-dispersive model based on Langmuir adsorption isotherm. Then, chromatographic separation was applied on the same peptides to determine their retention time and compare this experimental tR with the simulated tR obtained from simulation from surface plasmon resonance data. For the investigated peptides, the relative values of tR were comparable. Hence, our study demonstrated the pertinence of such numerical simulation correlating immobilized metal ion affinity chromatography and surface plasmon resonance.

CRDSAT Generated by pCARGHO: A New Efficient Lectin-Based Affinity Tag Method for Safe, Simple, and Low-Cost Protein Purification.

Purification of recombinant proteins remains a bottleneck for downstream processing. The authors engineered a new galectin 3 truncated form (CRDSAT ), functionally and structurally characterized, with preserved solubility and lectinic activity. Taking advantage of these properties, the authors designed an expression vector (pCARGHO), suitable for CRDSAT -tagged protein expression in prokaryotes. CRDSAT binds to lactose-Sepharose with a high specificity and facilitates solubilization of fusion proteins. This tag is structurally stable and can be easily removed from fusion proteins using TEV protease. Furthermore, due to their basic isoelectric point (pI), CRDSAT , and TEV are efficiently eliminated using cationic exchange chromatography. When pI of the protein of interest (POI) and CRDSAT are close, other chromatographic methods are successfully tested. Using CRDSAT tag, the authors purified several proteins from prokaryote and eukaryote origin and demonstrated as examples, the preservation of both Escherichia coli Thioredoxin 1 and human CDC25Bcd activities. Overall, yields of proteins obtained after tag removal are about 5-50 mg per litre of bacterial culture. Our purification method displays various advantages described herein that may greatly interest academic laboratories, biotechnology, and pharmaceutical companies.

Molecular Mechanisms of the Methionine Sulfoxide Reductase System from Neisseria meningitidis.

Neisseria meningitidis, an obligate pathogenic bacterium in humans, has acquired different defense mechanisms to detect and fight the oxidative stress generated by the host's defense during infection. A notable example of such a mechanism is the PilB reducing system, which repairs oxidatively-damaged methionine residues. This review will focus on the catalytic mechanism of the two methionine sulfoxide reductase (MSR) domains of PilB, which represent model enzymes for catalysis of the reduction of a sulfoxide function by thiols through sulfenic acid chemistry. The mechanism of recycling of these MSR domains by various "Trx-like" disulfide oxidoreductases will also be discussed.

SPR screening of metal chelating peptides in a hydrolysate for their antioxidant properties.

There is a growing need in the industrial sector (health, nutrition and cosmetic) to discover new biomolecules with various physico-chemical and bioactive properties. Various beneficial effects of peptides - notably those produced from protein hydrolysis - are reported in the literature. The antioxidant activity involves various mechanisms, among them metal chelation, studied by UV-visible spectrophotometry. In this paper, we set up an original method of screening metal chelating peptides in a hydrolysate using Surface Plasmon Resonance (SPR) for their antioxidant properties. To date, the empirical approach used several cycles of hydrolysate fractionation and bioactivity evaluation until the isolation of the pure bioactive molecule and its identification. Besides, the detection of metal-chelating peptide is not sensitive enough by spectrophotometry. For the first time, metal chelating peptides were screened in hydrolysates using SPR and a correlation was established between affinity constant determined in SPR and metal chelation capacity determined from UV-visible spectrophotometry.

Biochemical and functional characterization of a periplasmic disulfide oxidoreductase from Neisseria meningitidis essential for meningococcal viability.

TlpAs (thioredoxin-like proteins) are bacterial thioredoxin-like periplasmic disulfide oxidoreductases generally involved in cytochrome c maturation (Ccm) process. They contain a characteristic CXXC active site motif involved in disulfide exchange reaction. In the human pathogenic Neisseria meningitidis species, no TlpA has been characterized so far. In the present study, using an in silico analysis, we identified a putative periplasmic TlpA, called TlpA2. Biochemical and kinetic characterizations of the soluble form of TlpA2, tTlpA2 (truncated TlpA2), were performed. A reduction potential of -0.230 V at pH 7 was calculated, suggesting that TlpA2 acts as a reductant in the oxidative environment of the periplasm. Using a second-order reactive probe, high pKapp (apparent pKa) values were determined for the two cysteines of the SCXXC motif. The tTlpA2 was shown to be efficiently reduced by the N-terminal domain of the DsbD, whereas tTlpA2 reduced a mimetic peptide of cytochrome c' with a catalytic efficiency similar to that observed with other disulfide oxidoreductase like ResA. Moreover, the corresponding gene tlpA2 was shown to be essential for the pathogen viability and able to partially complement a Bordetella pertussis CcsX mutant. Together, these data support an essential role of TlpA2 in the Ccm process in N. meningitidis.

Methionine sulfoxide reductase: chemistry, substrate binding, recycling process and oxidase activity.

Three classes of methionine sulfoxide reductases are known: MsrA and MsrB which are implicated stereo-selectively in the repair of protein oxidized on their methionine residues; and fRMsr, discovered more recently, which binds and reduces selectively free L-Met-R-O. It is now well established that the chemical mechanism of the reductase step passes through formation of a sulfenic acid intermediate. The oxidized catalytic cysteine can then be recycled by either Trx when a recycling cysteine is operative or a reductant like glutathione in the absence of recycling cysteine which is the case for 30% of the MsrBs. Recently, it was shown that a subclass of MsrAs with two recycling cysteines displays an oxidase activity. This reverse activity needs the accumulation of the sulfenic acid intermediate. The present review focuses on recent insights into the catalytic mechanism of action of the Msrs based on kinetic studies, theoretical chemistry investigations and new structural data. Major attention is placed on how the sulfenic acid intermediate can be formed and the oxidized catalytic cysteine returns back to its reduced form.

Kinetic evidence that methionine sulfoxide reductase A can reveal its oxidase activity in the presence of thioredoxin.

The mouse methionine sulfoxide reductase A (MsrA) belongs to the subclass of MsrAs with one catalytic and two recycling Cys corresponding to Cys51, Cys198 and Cys206 in Escherichia coli MsrA, respectively. It was previously shown that in the absence of thioredoxin, the mouse and the E. coli MsrAs, which reduce two mol of methionine-O substrate per mol of enzyme, displays an in vitro S-stereospecific methionine oxidase activity. In the present study carried out with E. coli MsrA, kinetic evidence are presented which show that formation of the second mol of Ac-L-Met-NHMe is rate-limiting in the absence of thioredoxin. In the presence of thioredoxin, the overall rate-limiting step is associated with the thioredoxin-recycling process. Kinetic arguments are presented which support the accumulation of the E. coli MsrA under Cys51 sulfenic acid state in the presence of Trx. Thus, the methionine oxidase activity could be operative in vivo without the action of a regulatory protein in order to block the action of Trx as previously proposed.

Thioredoxin 2 from Escherichia coli is not involved in vivo in the recycling process of methionine sulfoxide reductase activities.

Thioredoxins (Trx) 1 and 2, and three methionine sulfoxide reductases (Msr) whose activities are Trx-dependent, are expressed in Escherichia coli. A metB(1)trxA mutant was shown to be unable to grow on methionine sulfoxide (Met-O) suggesting that Trx2 is not essential in the Msr-recycling process. In the present study, we have determined the kinetic parameters of the recycling process of the three Msrs by Trx2 and the in vivo expression of Trx2 in a metB(1)trxA mutant. The data demonstrate that the lack of growth of the metB(1)trxA mutant on Met-O is due to low in vivo expression of Trx2 and not to the lower catalytic efficiency of Msrs for Trx2.

Structural and biochemical characterization of free methionine-R-sulfoxide reductase from Neisseria meningitidis.

A new family of methionine-sulfoxide reductase (Msr) was recently described. The enzyme, named fRMsr, selectively reduces the R isomer at the sulfoxide function of free methionine sulfoxide (Met-R-O). The fRMsrs belong to the GAF fold family. They represent the first GAF domain to show enzymatic activity. Two other Msr families, MsrA and MsrB, were already known. MsrA and MsrB reduce free Met-S-O and Met-R-O, respectively, but exhibit higher catalytic efficiency toward Met-O within a peptide or a protein context. The fold of the three families differs. In the present work, the crystal structure of the fRMsr from Neisseria meningitidis has been determined in complex with S-Met-R-O. Based on biochemical and kinetic data as well as genomic analyses, Cys(118) is demonstrated to be the catalytic Cys on which a sulfenic acid is formed. All of the structural factors involved in the stereoselectivity of the l-Met-R-O binding were identified and account for why Met-S-O, DMSO, and a Met-O within a peptide are not substrates. Taking into account the structural, enzymatic, and biochemical information, a scenario of the catalysis for the reductase step is proposed. Based on the thiol content before and after Met-O reduction and the stoichiometry of Met formed per subunit of wild type and Cys-to-Ala mutants, a scenario of the recycling process of the N. meningitidis fRMsr is proposed. All of the biochemical, enzymatic, and structural properties of the N. meningitidis fRMsr are compared with those of MsrA and MsrB and are discussed in terms of the evolution of function of the GAF domain.

Methionine sulfoxide reductase B displays a high level of flexibility.

Methionine sulfoxide reductases (Msrs) are enzymes that catalyze the reduction of methionine sulfoxide back to methionine. In vivo, Msrs are essential in the protection of cells against oxidative damage to proteins and in the virulence of some bacteria. Two structurally unrelated classes of Msrs, named MsrA and MsrB, exist. MsrB are stereospecific to R epimer on the sulfur of sulfoxide. All MsrB share a common reductase step with the formation of a sulfenic acid intermediate. For the subclass of MsrB whose recycling process passes through the formation of an intradisulfide bond, the recycling reducer is thioredoxin. In the present study, X-ray structures of Neisseria meningitidis MsrB have been determined. The structures have a fold based on two beta-sheets, similar to the fold already described for other MsrB, with the recycling Cys63 located in a position favorable for disulfide bond formation with the catalytic Cys117. X-ray structures of Xanthomonas campestris MsrB have also been determined. In the C117S MsrB structure with a bound substrate, the recycling Cys31 is far from Ser117, with Trp65 being essential in the reductase step located in between. This positioning prevents the formation of the Cys31-Cys117 disulfide bond. In the oxidized structure, a drastic conformational reorganization of the two beta-sheets due to withdrawal of the Trp65 region from the active site, which remains compatible with an efficient thioredoxin-recycling process, is observed. The results highlight the remarkable structural malleability of the MsrB fold.

Formation of the complex between DsbD and PilB N-terminal domains from Neisseria meningitidis necessitates an adaptability of nDsbD.

DsbD transmembrane protein dispatches electrons to periplasmic Trx/DsbE-like partners via specific interactions with its N-terminal domain, nDsbD. In the present study, PilB N-terminal domain (NterPilB) is shown to efficiently accept electrons coming from nDsbD from Neisseria meningitidis. Using an NMR-driven docking approach, we have modeled the structure of a mixed disulfide complex between NterPilB and nDsbD. We show the needed opening of nDsbD cap-loop whereas NterPilB FLHE loop does not seem essential in the formation and stabilization of the complex. Relaxation analysis performed on backbone amide groups highlights a kind of dynamics transfer from nDsbD cap-loop on NterPilB alpha1 helix, suggesting that a mobility contribution is required not only for the formation of the mixed disulfide complex, but also for its disruption. Taking into account previous X-ray data on covalent complexes involving nDsbD, a cartoon of interactions between Trx-like partners and nDsbD is proposed that illustrates the adaptability of nDsbD.

Solution structure and backbone dynamics of the cysteine 103 to serine mutant of the N-terminal domain of DsbD from Neisseria meningitidis.

The DsbD protein is essential for electron transfer from the cytoplasm to the periplasm of Gram-negative bacteria. Its N-terminal domain dispatches electrons coming from cytoplasmic thioredoxin (Trx), via its central transmembrane and C-terminal domains, to its periplasmic partners: DsbC, DsbE/CcmG, and DsbG. Previous structural studies described the latter proteins as Trx-like folds possessing a characteristic C-X-X-C motif able to generate a disulfide bond upon oxidation. The Escherichia coli nDsbD displays an immunoglobulin-like fold in which two cysteine residues (Cys103 and Cys109) allow a disulfide bond exchange with its biological partners.We have determined the structure in solution and the backbone dynamics of the C103S mutant of the N-terminal domain of DsbD from Neisseria meningitidis. Our results highlight significant structural changes concerning the beta-sheets and the local topology of the active site compared with the oxidized form of the E. coli nDsbD. The structure reveals a "cap loop" covering the active site, similar to the oxidized E. coli nDsbD X-ray structure. However, regions featuring enhanced mobility were observed both near to and distant from the active site, revealing a capacity of structural adjustments in the active site and in putative interaction areas with nDsbD biological partners. Results are discussed in terms of functional consequences.

Solution structure and dynamics of the reduced and oxidized forms of the N-terminal domain of PilB from Neisseria meningitidis.

The secreted form of the PilB protein was proposed to be involved in pathogen survival fighting against the defensive host's oxidative burst. PilB protein is composed of three domains. The central and the C-terminal domains display methionine sulfoxide reductase A and B activities, respectively. The N-terminal domain, which possesses a CXXC motif, was recently shown to regenerate in vitro the reduced forms of the methionine sulfoxide reductase domains of PilB from their oxidized forms, as does the thioredoxin 1 from E. coli, via a disulfide bond exchange. The thioredoxin-like N-terminal domain belongs to the cytochrome maturation protein structural family, but it possesses a unique additional segment (99)FLHE (102) localized in a loop. This segment covers one edge of the active site in the crystal structure of the reduced form of the N-terminal domain of PilB. We have determined the solution structure and the dynamics of the N-terminal domain from Neisseria meningitidis, in its reduced and oxidized forms. The FLHE loop adopts, in both redox states, a well-defined conformation. Subtle conformational and dynamic changes upon oxidation are highlighted around the active site, as well as in the FLHE loop. The functional consequences of the cytochrome maturation protein topology and those of the presence of FLHE loop are discussed in relation to the enzymatic properties of the N-terminal domain.