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Electrode-confined molecular catalysts are promising systems to enable the efficient conversion of CO 2 to useful products. Here, we describe the development of an original molecular cathode for CO 2 reduction to CO based on the noncovalent integration of a tetraazamacrocyclic Co complex to a carbon nanotube-based matrix. Aqueous electrochemical characterization of the modified electrode allowed for clear observation of a change of redox behavior of the Co center as surface concentration was tuned, highlighting the impact of the catalyst microenvironment on its redox properties. The molecular cathode enabled efficient CO 2 -to-CO conversion in fully aqueous conditions, giving rise to a turnover number (TON CO ) of up to 20 × 10 3 after 2 h of constant electrolysis at a mild overpotential (η = 450 mV) and with a faradaic efficiency for CO of about 95%. Post operando measurements using electrochemical techniques, inductively coupled plasma, X-ray photoelectron spectroscopy and X-ray absorption spectroscopy characterization of the films demonstrated that the catalysis remained of molecular nature, making this Co-based electrode a new promising alternative for molecular electrocatalytic conversion of CO 2 -to-CO in fully aqueous media.

Iron-sulfur (Fe-S) clusters are essential inorganic cofactors dedicated to a wide range of biological functions, including electron transfer and catalysis. Specialized multiprotein machineries present in all types of organisms support their biosynthesis. These machineries encompass a scaffold protein, on which Fe-S clusters are assembled before being transferred to cellular targets. Here, we describe the first characterization of the native Fe-S cluster of the anaerobically purified SufBC 2 D scaffold from Escherichia coli by XAS and Mössbauer, UV-visible absorption, and EPR spectroscopies. Interestingly, we propose that SufBC 2 D harbors two iron-sulfur-containing species, a [2Fe-2S] cluster and an as-yet unidentified species. Mutagenesis and biochemistry were used to propose amino acid ligands for the [2Fe-2S] cluster, supporting the hypothesis that both SufB and SufD are involved in the Fe-S cluster ligation. The [2Fe-2S] cluster can be transferred to ferredoxin in agreement with the SufBC 2 D scaffold function. These results are discussed in the context of Fe-S cluster biogenesis.

Integration of molecular catalysts inside polymeric scaffolds has gained substantial attention over the past decade, as it provides a path towards generating systems with enhanced stability as well as enzyme-like morphologies and properties. In the context of solar fuels research and chemical energy conversion, this approach has been found to improve both rates and energy efficiencies of a range of catalytic reactions. However, system performance still needs to be improved to reach technologically relevant currents and stability, parameters that are heavily influenced by the nature of the incorporated molecular catalyst. Here, we have focused on the integration of a biomimetic {Fe 2 (μ-adt)(CO) 6 } (-CH 2 NHCH 2 S-, azadithiolate or adt 2- ) based active site ("[2Fe2S] adt "), inspired by the catalytic cofactor of [FeFe] hydrogenases, within a synthetic polymeric scaffold using free radical polymerization. The resulting metallopolymers [2Fe2S] adt k [DMAEMA] l [PyBMA] m (DMAEMA = dimethylaminoethyl methacrylate as water soluble monomer; PyBMA = 4-(pyren-1-yl)-butyl methacrylate as hydrophobic anchor for heterogenization) were found to be active for electrochemical H 2 production in neutral aqueous media. The pyrene content was varied to optimize durability and activity. Following immobilization on multiwalled carbon nanotubes (MWNT) the most active metallopolymer, containing ∼2.3 mol% of PyBMA, could reach a turnover number for hydrogen production (TON H 2 ) of ∼0.4 ×10 5 over 20 hours of electrolysis at an overpotential of 0.49 V, two orders of magnitude higher than the isolated catalyst counterpart. The study provides a synthetic methodology for incorporating catalytic units featuring second coordination sphere functional groups, and highlights the benefit of the confinement within the polymer matrix for catalytic performance., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Pigments are among the oldest nanoparticulate products known to mankind, and their use in tattoos is also very old. Nowadays, 25% of American people aged 18 to 50 are tattooed, which poses the question of the delayed effects of tattoos. In this article, we investigated three cobalt [Pigment Violet 14 (purple color)] or cobalt alloy pigments [Pigment Blue 28 (blue color), Pigment Green 14 (green color)], and one zinc pigment [Pigment White 4 (white color)] which constitute a wide range of colors found in tattoos. These pigments contain microparticles and a significant proportion of submicroparticles or nanoparticles (in either aggregate or free form). Because of the key role of macrophages in the scavenging of particulate materials, we tested the effects of cobalt- and zinc-based pigments on the J774A.1 macrophage cell line. In order to detect delayed effects, we compared two exposure schemes: acute exposure for 24 hours and an exposure for 24 hours followed by a 3-day post-exposure recovery period. The conjunction of these two schemes allowed for the investigation of the delayed or sustained effects of pigments. All pigments induced functional effects on macrophages, most of which were pigment-dependent. For example, Pigment Green 19, Pigment Blue 28, and Pigment White 4 showed a delayed alteration of the phagocytic capacity of cells. Moreover, all the pigments tested induced a slight but significant increase in tumor necrosis factor secretion. This effect, however, was transitory. Conversely, only Pigment Blue 28 induced both a short and sustained increase in interleukin 6 secretion. Results showed that in response to bacterial stimuli (LPS), the secretion of tumor necrosis factor and interleukin 6 declined after exposure to pigments followed by a recovery period. For chemoattractant cytokines (MCP-1 or MIP-1α), delayed effects were observed with a secretion decreased in presence of Pigment Blue 28 and Pigment violet 14, both with or without LPS stimuli. The pigments also induced persisting changes in some important macrophage membrane markers such as CD11b, an integrin contributing to cell adhesion and immunological tolerance. In conclusion, the pigments induced functional disorders in macrophages, which, in some cases, persist long after exposure, even at non-toxic doses., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Devcic, Dussol, Collin-Faure, Pérard, Fenel, Schoehn, Carrière, Rabilloud and Dalzon.)

An original 1-acetato-4-(1-pyrenyl)-1,4,7-triazacyclononane (AcPyTACN) was synthesized for the immobilization of a His-tagged recombinant CODH from Rhodospirillum rubrum (RrCODH) on carbon-nanotube electrodes. The strong binding of the enzyme at the Ni-AcPyTACN complex affords a high current density of 4.9 mA cm -2 towards electroenzymatic CO 2 reduction and a high stability of more than 6×10 6  TON when integrated on a gas-diffusion bioelectrode., (© 2022 Wiley-VCH GmbH.)

Sulfs represent a class of unconventional sulfatases which provide an original post-synthetic regulatory mechanism for heparan sulfate polysaccharides and are involved in multiple physiopathological processes, including cancer. However, Sulfs remain poorly characterized enzymes, with major discrepancies regarding their in vivo functions. Here we show that human Sulf-2 (HSulf-2) harbors a chondroitin/dermatan sulfate glycosaminoglycan (GAG) chain, attached to the enzyme substrate-binding domain. We demonstrate that this GAG chain affects enzyme/substrate recognition and tunes HSulf-2 activity in vitro and in vivo. In addition, we show that mammalian hyaluronidase acts as a promoter of HSulf-2 activity by digesting its GAG chain. In conclusion, our results highlight HSulf-2 as a proteoglycan-related enzyme and its GAG chain as a critical non-catalytic modulator of the enzyme activity. These findings contribute to clarifying the conflicting data on the activities of the Sulfs., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Some carboxydotrophs like Rhodospirillum rubrum are able to grow with CO as their sole source of energy using a Carbone monoxide dehydrogenase (CODH) and an Energy conserving hydrogenase (ECH) to perform anaerobically the so called water-gas shift reaction (WGSR) (CO + H 2 O → CO 2  + H 2 ). Several studies have focused at the biochemical and biophysical level on this enzymatic system and a few OMICS studies on CO metabolism. Knowing that CO is toxic in particular due to its binding to heme iron atoms, and is even considered as a potential antibacterial agent, we decided to use a proteomic approach in order to analyze R. rubrum adaptation in term of metabolism and management of the toxic effect. In particular, this study allowed highlighting a set of proteins likely implicated in ECH maturation, and important perturbations in term of cofactor biosynthesis, especially metallic cofactors. This shows that even this CO tolerant microorganism cannot avoid completely CO toxic effects associated with its interaction with metallic ions. SIGNIFICANCE: This proteomic study highlights the fact that even in a microorganism able to handle carbon monoxide and in some way detoxifying it via the intrinsic action of the carbon monoxide dehydrogenase (CODH), CO has important effects on metal homeostasis, metal cofactors and metalloproteins. These effects are direct or indirect via transcription regulation, and amplified by the high interdependency of cofactors biosynthesis., (Copyright © 2021. Published by Elsevier B.V.)

Aim and Objective: Nowadays, developing effective antibiotics for bacterial control has become difficult due to increased resistance to the available medicines in the market. Essential oils possess interesting biological properties as some of their components have very powerful antiviral and antibacterial properties. Carthamus caeruleus is a plant that has antibacterial and antioxidant activity due to the presence of an acetylenic compound, Carlina oxide. The aim of this work was to provide, for the first time, the chemical modifications to the structure of Carlina oxide and the insilico study of these analogues., Materials and Methods: The essential oil of Carthamus caeruleus was extracted by steam distillation in a Clevenger-type apparatus. Carlina oxide component was separated by column chromatography. Five new analogues were synthetized and identified by spectroscopic analyses (RMN, IR and SM). Molecular docking simulation study was performed using Molecular Operating Environment software (MOE) on three enzymes of bacterial origin (Streptococcus pyogenesis and Enterococcus faecalis)., Results: Five new compounds derived from Carlina oxide were synthesized (IM8-IM12), and their structures were characterized by infrared (IR), 1H and 13C nuclear magnetic resonance (NMR). The new synthesized compounds were evaluated as mSpeB, DHFR from Enterococcus faecalis and DNA gyrase inhibitors by a docking analysis using MOE. These results show interesting ligand interactions with the three enzymes, and the best result was attributed to the complexes formed with IM9, which had the lowest score., Conclusion: In fact, these new compounds could lead to powerful approaches for the research and development of new antibiotics., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Siderophores are iron chelators produced by bacteria to access iron, an essential nutrient. The pathogen Pseudomonas aeruginosa produces two siderophores, pyoverdine and pyochelin, the former with a high affinity for iron and the latter with a lower affinity. Furthermore, the production of both siderophores involves a positive auto-regulatory loop: the presence of the ferri-siderophore complex is essential for their large production. Since pyochelin has a lower affinity for iron it was hard to consider the role of pyochelin in drastic competitive environments where the host or the environmental microbiota produce strong iron chelators and may inhibit iron chelation by pyochelin. We showed here that the pyochelin pathway overcomes this difficulty through a more complex regulating mechanism for pyochelin production than previously described. Indeed, in the absence of pyoverdine, and thus higher difficulty to access iron, the bacteria are able to produce pyochelin independently of the presence of ferri-pyochelin. The regulation of the pyochelin pathway appeared to be more complex than expected with a more intricate tuning between repression and activation. Consequently, when the bacteria cannot produce pyoverdine they are able to produce pyochelin even in the presence of strong iron chelators. Such results support a more complex and varied role for this siderophore than previously described, and complexify the battle for iron during P. aeruginosa infection.

Much data shows that biological metals other than Fe 3+ can interfere with Fe 3+ acquisition by siderophores in bacteria. Siderophores are small Fe 3+ chelators produced by the microorganisms to obtain access to Fe 3+ . Here, we show that Co 2+ is imported into Pseudomonas aeruginosa cells in a complex with the siderophore pyochelin (PCH) by the ferri-PCH outer membrane transporter FptA. Moreover, the presence of Co 2+ in the bacterial environment strongly affects the production of PCH. Proteomic and transcriptomic approaches showed that a decrease of PCH production is associated with repression of the expression of the genes involved in PCH biosynthesis. We used various molecular biology approaches to show that this repression is not Fur-(ferric uptake transcriptional regulator) dependent but due to competition of PCH-Co with PCH-Fe for PchR (transcriptional activator), thus inhibiting the formation of PchR-PCH-Fe and consequently the expression of the PCH genes. We observed a similar mechanism of repression of PCH production, but to a lesser extent, by Ni 2+ , but not for Zn 2+ , Cu 2+ , or Mn 2+ . Here, we show, for the first time at a molecular level, how the presence of a contaminant metal can interfere with Fe 3+ acquisition by the siderophores PCH and PVD.

Activation of nickel enzymes requires specific accessory proteins organized in multiprotein complexes controlling metal transfer to the active site. Histidine-rich clusters are generally present in at least one of the metallochaperones involved in nickel delivery. The maturation of carbon monoxide dehydrogenase in the proteobacterium Rhodospirillum rubrum requires three accessory proteins, CooC, CooT, and CooJ, dedicated to nickel insertion into the active site, a distorted [NiFe 3 S 4 ] cluster coordinated to an iron site. Previously, CooJ from R. rubrum ( Rr CooJ) has been described as a nickel chaperone with 16 histidines and 2 cysteines at its C terminus. Here, the X-ray structure of a truncated version of Rr CooJ, combined with small-angle X-ray scattering data and a modeling study of the full-length protein, revealed a homodimer comprising a coiled coil with two independent and highly flexible His tails. Using isothermal calorimetry, we characterized several metal-binding sites (four per dimer) involving the His-rich motifs and having similar metal affinity ( K D = 1.6 μm). Remarkably, biophysical approaches, site-directed mutagenesis, and X-ray crystallography uncovered an additional nickel-binding site at the dimer interface, which binds Ni(II) with an affinity of 380 nm Although Rr CooJ was initially thought to be a unique protein, a proteome database search identified at least 46 bacterial CooJ homologs. These homologs all possess two spatially separated nickel-binding motifs: a variable C-terminal histidine tail and a strictly conserved H(W/F) X 2 H X 3 H motif, identified in this study, suggesting a dual function for CooJ both as a nickel chaperone and as a nickel storage protein., (© 2019 Alfano et al.)

FUR (Ferric Uptake Regulator) protein is a global transcriptional regulator that senses iron status and controls the expression of genes involved in iron homeostasis, virulence, and oxidative stress. Ubiquitous in Gram-negative bacteria and absent in eukaryotes, FUR is an attractive antivirulence target since the inactivation of the fur gene in various pathogens attenuates their virulence. The characterization of 13-aa-long anti-FUR linear peptides derived from the variable part of the anti-FUR peptide aptamers, that were previously shown to decrease pathogenic E. coli strain virulence in a fly infection model, is described herein. Modeling, docking, and experimental approaches in vitro (activity and interaction assays, mutations) and in cells (yeast two-hybrid assays) were combined to characterize the interactions of the peptides with FUR, and to understand their mechanism of inhibition. As a result, reliable structure models of two peptide-FUR complexes are given. Inhibition sites are mapped in the groove between the two FUR subunits where DNA should also bind. Another peptide behaves differently and interferes with the dimerization itself. These results define these novel small peptide inhibitors as lead compounds for inhibition of the FUR transcription factor.

The ferric uptake regulator (Fur) belongs to the family of the DNA-binding metal-responsive transcriptional regulators. Fur is a global regulator found in all proteobacteria. It controls the transcription of a wide variety of genes involved in iron metabolism but also in oxidative stress or virulence factor synthesis. When bound to ferrous iron, Fur can bind to specific DNA sequences, called Fur boxes. This binding triggers the repression or the activation of gene expression, depending on the regulated genes. As a general view, Fur proteins are considered to be dimeric proteins both in solution and when bound to DNA. In this study, we have purified Fur from four pathogenic strains (Pseudomonas aeruginosa, Francisella tularensis, Yersinia pestis, and Legionella pneumophila) and compared them to Fur from Escherichia coli (EcFur), the best characterized of this family. By using a series of "in solution" techniques, including multiangle laser light scattering and small-angle X-ray scattering, as well as cross-linking experiments, we have shown that the Fur proteins can be classified into two groups, according to their quaternary structure. The group of dimers is represented by EcFur and YpFur and the group of very stable tetramers by PaFur, FtFur, and LpFur. Using PaFur as a case study, we also showed that the dissociation of the tetramers into dimers is necessary for binding of Fur to DNA, and that this dissociation requires the combined effect of metal ion binding and DNA proximity.

MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression through RNA interference. Human miRNAs are generated through a series of enzymatic processing steps. The precursor miRNA (pre-miRNA) is recognized and cleaved by a complex containing Dicer and several non-catalytic accessory proteins. HIV TAR element binding protein (TRBP) is a constituent of the Dicer complex, which augments complex stability and potentially functions in substrate recognition and product transfer to the RNA-induced silencing complex. Here we have analysed the interaction between the RNA-binding region of TRBP and an oncogenic human miRNA, miR-155, at different stages in the biogenesis pathway. We show that the region of TRBP that binds immature miRNAs comprises two independent double-stranded RNA-binding domains connected by a 60-residue flexible linker. No evidence of contact between the two double-stranded RNA-binding domains was observed either in the apo- or RNA-bound state. We establish that the RNA-binding region of TRBP interacts with both pre-miR-155 and the miR-155/miR-155* duplex through the same binding surfaces and with similar affinities, and that two protein molecules can simultaneously interact with each immature miRNA. These data suggest that TRBP could play a role before and after processing of pre-miRNAs by Dicer.

The 5'-untranslated region of the hepatitis C virus genome contains an internal ribosome entry site (IRES) that initiates cap-independent translation of the viral RNA. Until now, the structural characterization of the entire (IRES) remained limited to cryo-electron microscopy reconstructions of the (IRES) bound to different cellular partners. Here we report an atomic model of free full-length hepatitis C virus (IRES) refined by selection against small-angle X-ray scattering data that incorporates the known structures of different fragments. We found that an ensemble of conformers reproduces small-angle X-ray scattering data better than a single structure suggesting in combination with molecular dynamics simulations that the hepatitis C virus (IRES) is an articulated molecule made of rigid parts that move relative to each other. Principal component analysis on an ensemble of physically accessible conformers of hepatitis C virus (IRES) revealed dominant collective motions in the molecule, which may underlie the conformational changes occurring in the (IRES) molecule upon formation of the initiation complex.

C1q, a key component of the classical complement pathway, is a major player in the response to microbial infection and has been shown to detect noxious altered-self substances such as apoptotic cells. In this work, using complementary experimental approaches, we identified the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a C1q partner when exposed at the surface of human pathogenic bacteria Streptococcus pneumoniae and human apoptotic cells. The membrane-associated GAPDH on HeLa cells bound the globular regions of C1q as demonstrated by pulldown and cell surface co-localization experiments. Pneumococcal strains deficient in surface-exposed GAPDH harbored a decreased level of C1q recognition when compared with the wild-type strains. Both recombinant human and pneumococcal GAPDHs interacted avidly with C1q as measured by surface plasmon resonance experiments (K(D) = 0.34-2.17 nm). In addition, GAPDH-C1q complexes were observed by transmission electron microscopy after cross-linking. The purified pneumococcal GAPDH protein activated C1 in an in vitro assay unlike the human form. Deposition of C1q, C3b, and C4b from human serum at the surface of pneumococcal cells was dependent on the presence of surface-exposed GAPDH. This ability of C1q to sense both human and bacterial GAPDHs sheds new insights on the role of this important defense collagen molecule in modulating the immune response.

The eukaryotic Engulfment and CellMotility (ELMO) proteins form an evolutionary conserved family of key regulators which play a central role in Rho-dependent biological processes such as engulfment and cell motility/migration. ELMO proteins interact with a subset of Downstream of Crk (DOCK) family members, a new type of guanine exchange factors (GEF) for Rac and cdc42 GTPases. The physiological function of DOCK is to facilitate actin remodeling, a process which occurs only in presence of ELMO. Several studies have determined that the last 200 C-terminal residues of ELMO1 and the first 180 N-terminal residues of DOCK180 are responsible for the ELMO-DOCK interaction. However, the precise role of the different domains and motifs identified in these regions has remained elusive. Divergent functional, biochemical and structural data have been reported regarding the contribution of the C-terminal end of ELMO, comprising its polyproline motif, and of the DOCK SH3 domain. In the present study, we have investigated the contribution of the C-terminal end of ELMO1 to the interaction between ELMO1 and the SH3 domain of DOCK180 using nuclear magnetic resonance spectroscopy and surface plasmon resonance. Our data presented here demonstrate the ability of the SH3 domain of DOCK180 to interact with ELMO1, regardless of the presence of the polyproline-containing C-terminal end. However, the presence of the polyproline region leads to a significant increase in the half-life of the ELMO1-DOCK180 complex, along with a moderate increase on the affinity., (Copyright © 2011 Elsevier Masson SAS. All rights reserved.)

Ficolins and pentraxins are soluble oligomeric pattern-recognition molecules that sense danger signals from pathogens and altered self-cells and might act synergistically in innate immune defense and maintenance of immune tolerance. The interaction of M-ficolin with the long pentraxin pentraxin 3 (PTX3) has been characterized using surface plasmon resonance spectroscopy and electron microscopy. M-ficolin was shown to bind PTX3 with high affinity in the presence of calcium ions. The interaction was abolished in the presence of EDTA and inhibited by N-acetyl-D-glucosamine, indicating involvement of the fibrinogen-like domain of M-ficolin. Removal of sialic acid from the single N-linked carbohydrate of the C-terminal domain of PTX3 abolished the interaction. Likewise, an M-ficolin mutant with impaired sialic acid-binding ability did not interact with PTX3. Interaction was also impaired when using the isolated recognition domain of M-ficolin or the monomeric C-terminal domain of PTX3, indicating requirement for oligomerization of both proteins. Electron microscopy analysis of the M-ficolin-PTX3 complexes revealed that the M-ficolin tetramer bound up to four PTX3 molecules. From a functional point of view, immobilized PTX3 was able to trigger M-ficolin-dependent activation of the lectin complement pathway. These data indicate that interaction of M-ficolin with PTX3 arises from its ability to bind sialylated ligands and thus differs from the binding to the short pentraxin C-reactive protein and from the binding of L-ficolin to PTX3. The M-ficolin-PTX3 interaction described in this study represents a novel case of cross-talk between soluble pattern-recognition molecules, lending further credit to the integrated view of humoral innate immunity that emerged recently.

Many viral mRNAs contain a 5'-UTR RNA element called internal ribosome-entry site (IRES), which bypasses the requirement of some canonical initiation factors allowing cap-independent translation. The IRES of hepatitis-C virus drives translation by directly recruiting 40S ribosomal subunits and binds to eIF3 which plays a critical role in both cap-dependent and cap-independent translation. However, the molecular basis for eIF3 activity in either case remains enigmatic. Here we report that subunit b of the eIF3 complex directly binds to HCV IRES domain III via its N-terminal-RRM. Because eIF3b was previously shown to be involved in eIF3j binding, biological implications are discussed.