Cyclic imine toxins survey in coastal european shellfish samples: Bioaccumulation and mode of action of 28-O-palmitoyl ester of pinnatoxin-G. first report of portimine-A bioaccumulation.

Cyclic imine toxins exhibit fast acting neurotoxicity and lethality by respiratory arrest in mice explained by their potent antagonistic activity against muscular nicotinic acetylcholine receptors. We performed a survey of gymnodimine-A, 13-desmethyl spirolide-C, 13,19-didesmethyl spirolide-C, 20-methyl spirolide-G, pinnatoxin-A, pinnatoxin-G, portimine-A and 28-O-palmitoyl ester of pinnatoxin-G in 36 shellfish samples collected in coastal areas of 8 European countries using a microplate receptor binding assay and UPLC-MS/MS for toxin identification and quantification. The major toxins found in these samples were pinnatoxin-G, 20-methyl spirolide-G, 13-desmethyl spirolide-C, gymnodimine-A and portimine-A. Traces of 13,19-didesmethyl spirolide-C, pinnatoxin-A and 28-O-palmitoyl ester of pinnatoxin-G were also detected. The rapid death of mice was correlated with higher pinnatoxin-G concentrations in mussel digestive gland extracts injected intraperitoneally. Our survey included nontoxic control samples that were found to contain moderate to trace amounts of several cyclic imine toxins. Shellfish may bioaccumulate not only cyclic imine toxins but also a large number of acyl derivatives as a product of metabolic transformation of these neurotoxins. This is the first report in which portimine-A and 28-O-palmitoyl ester of pinnatoxin-G were detected in shellfish extracts from digestive glands of mussels collected in Ingril lagoon. The bioaccumulation of portimine-A is particularly of concern because it is cytotoxic and is able to induce apotosis. The mode of action of 28-O-palmitoyl ester of pinnatoxin-G was studied by receptor binding-assay and by two-electrode voltage clamp electrophysiology. The antagonistic behavior of the acylated pinnatoxin-G towards nicotinic acetylcholine receptor of muscle type is shown here for the first time. Since cyclic imine toxins are not regulated further monitoring of these emerging toxins is needed to improve evidence gathering of their occurrence in shellfish commercialized for human consumption in Europe given their potent antagonism against muscle and neuronal nicotinic acetylcholine receptors.

The three-dimensional structure of the toxic peptide Cl13 from the scorpion Centruroides limpidus.

Cl13 is a toxin purified previously from the venom of the Mexican scorpion Centruroides limpidus. This toxin affects the function of voltage gated Na+-channels, human subtypes Nav1.4, Nav1.5 and Nav1.6 in a similar manner as other known ß-toxins from scorpion venoms. Here, we report a correction of the primary structure of Cl13, previously published. The peptide does contain 66 amino acids, but residue 58 is a tryptophan and the last C-terminal amino acid is an amidated lysine, instead of arginine. The main contribution of this communication is the determination of the 3D-structure of Cl13, by solution NMR, showing that Cl13 has the classical cysteine-stabilized α/ß (CSα/ß) folding. It has a triple stranded antiparallel beta sheet commonly present in scorpion sodium channel ß-toxins. In addition, we report and discuss a comparison of Cl13 structure with two other toxins (Cn2 and Css2) from scorpions of the same genus Centruroides, which shows important surface similarities with the structure reported here. Finally, the lack of neutralization of Cl13 toxin by two single-chain antibody fragments (scFvs), named LR and 10FG2, which are capable of neutralizing various toxins from Mexican scorpions, is revised. In particular, 10FG2 is capable of neutralizing toxins Cll1 and Cll2 of the same scorpion C. limpidus. The reasons why LR and 10FG2 are unable of neutralizing Cl13 toxin are discussed.

2nd PSL Chemical Biology Symposium (2019): At the Crossroads of Chemistry and Biology.

Chemical Biology is the science of designing chemical tools to dissect and manipulate biology at different scales. It provides the fertile ground from which to address important problems of our society, such as human health and environment.

The Dual Activity Responsible for the Elongation and Branching of ß-(1,3)-Glucan in the Fungal Cell Wall.

ß-(1,3)-Glucan, the major fungal cell wall component, ramifies through ß-(1,6)-glycosidic linkages, which facilitates its binding with other cell wall components contributing to proper cell wall assembly. Using Saccharomyces cerevisiae as a model, we developed a protocol to quantify ß-(1,6)-branching on ß-(1,3)-glucan. Permeabilized S. cerevisiae and radiolabeled substrate UDP-(14C)glucose allowed us to determine branching kinetics. A screening aimed at identifying deletion mutants with reduced branching among them revealed only two, the bgl2Δ and gas1Δ mutants, showing 15% and 70% reductions in the branching, respectively, compared to the wild-type strain. Interestingly, a recombinant Gas1p introduced ß-(1,6)-branching on the ß-(1,3)-oligomers following its ß-(1,3)-elongase activity. Sequential elongation and branching activity of Gas1p occurred on linear ß-(1,3)-oligomers as well as Bgl2p-catalyzed products [short ß-(1,3)-oligomers linked by a linear ß-(1,6)-linkage]. The double S. cerevisiae gas1Δ bgl2Δ mutant showed a drastically sick phenotype. An ScGas1p ortholog, Gel4p from Aspergillus fumigatus, also showed dual ß-(1,3)-glucan elongating and branching activity. Both ScGas1p and A. fumigatus Gel4p sequences are endowed with a carbohydrate binding module (CBM), CBM43, which was required for the dual ß-(1,3)-glucan elongating and branching activity. Our report unravels the ß-(1,3)-glucan branching mechanism, a phenomenon occurring during construction of the cell wall which is essential for fungal life.IMPORTANCE The fungal cell wall is essential for growth, morphogenesis, protection, and survival. In spite of being essential, cell wall biogenesis, especially the core ß-(1,3)-glucan ramification, is poorly understood; the ramified ß-(1,3)-glucan interconnects other cell wall components. Once linear ß-(1,3)-glucan is synthesized by plasma membrane-bound glucan synthase, the subsequent event is its branching event in the cell wall space. Using Saccharomyces cerevisiae as a model, we identified GH72 and GH17 family glycosyltransferases, Gas1p and Bgl2p, respectively, involved in the ß-(1,3)-glucan branching. The sick phenotype of the double Scgas1Δ bgl2Δ mutant suggested that ß-(1,3)-glucan branching is essential. In addition to ScGas1p, GH72 family ScGas2p and Aspergillus fumigatus Gel4p, having CBM43 in their sequences, showed dual ß-(1,3)-glucan elongating and branching activity. Our report identifies the fungal cell wall ß-(1,3)-glucan branching mechanism. The essentiality of ß-(1,3)-glucan branching suggests that enzymes involved in the glucan branching could be exploited as antifungal targets.

Large-scale synthesis and structural analysis of a synthetic glycopeptide dendrimer as an anti-cancer vaccine candidate.

Herein, we report a new process that enables the gram-scale production of a fully synthetic anti-cancer vaccine for human use. This therapeutic vaccine candidate, named MAG-Tn3, is a high-molecular-weight tetrameric glycopeptide encompassing carbohydrate tumor-associated Tn antigen clusters and peptidic CD4+ T-cell epitopes. The synthetic process involves (i) the stepwise solid-phase assembly of protected amino acids, including the high value-added Tn building blocks with only 1.5 equivalents, (ii) a single isolated intermediate, and (iii) the simultaneous deprotection of 36 hindered protective groups. The resulting MAG-Tn3 was unambiguously characterized using a combination of techniques, including a structural analysis by nuclear magnetic resonance spectroscopy. The four peptidic chains are flexible in solution, with a more constrained but extended conformation at the Tn3 antigen motif. Finally, we demonstrate that, when injected into HLA-DR1-expressing transgenic mice, this vaccine induces Tn-specific antibodies that mediate the killing of human Tn-positive tumor cells. These studies led to a clinical batch of the MAG-Tn3, currently investigated in breast cancer patients (phase I clinical trial). The current study demonstrates the feasibility of the multigram-scale synthesis of a highly pure complex glycopeptide, and it opens new avenues for the use of synthetic glycopeptides as drugs in humans.

Molecular Basis of the Interaction of the Human Protein Tyrosine Phosphatase Non-receptor Type 4 (PTPN4) with the Mitogen-activated Protein Kinase p38γ.

The human protein tyrosine phosphatase non-receptor type 4 (PTPN4) prevents cell death induction in neuroblastoma and glioblastoma cell lines in a PDZ·PDZ binding motifs-dependent manner, but the cellular partners of PTPN4 involved in cell protection are unknown. Here, we described the mitogen-activated protein kinase p38γ as a cellular partner of PTPN4. The main contribution to the p38γ·PTPN4 complex formation is the tight interaction between the C terminus of p38γ and the PDZ domain of PTPN4. We solved the crystal structure of the PDZ domain of PTPN4 bound to the p38γ C terminus. We identified the molecular basis of recognition of the C-terminal sequence of p38γ that displays the highest affinity among all endogenous partners of PTPN4. We showed that the p38γ C terminus is also an efficient inducer of cell death after its intracellular delivery. In addition to recruiting the kinase, the binding of the C-terminal sequence of p38γ to PTPN4 abolishes the catalytic autoinhibition of PTPN4 and thus activates the phosphatase, which can efficiently dephosphorylate the activation loop of p38γ. We presume that the p38γ·PTPN4 interaction promotes cellular signaling, preventing cell death induction.

Structural basis of the signalling through a bacterial membrane receptor HasR deciphered by an integrative approach.

Bacteria use diverse signalling pathways to adapt gene expression to external stimuli. In Gram-negative bacteria, the binding of scarce nutrients to membrane transporters triggers a signalling process that up-regulates the expression of genes of various functions, from uptake of nutrient to production of virulence factors. Although proteins involved in this process have been identified, signal transduction through this family of transporters is not well understood. In the present study, using an integrative approach (EM, SAXS, X-ray crystallography and NMR), we have studied the structure of the haem transporter HasR captured in two stages of the signalling process, i.e. before and after the arrival of signalling activators (haem and its carrier protein). We show for the first time that the HasR domain responsible for signal transfer: (i) is highly flexible in two stages of signalling; (ii) extends into the periplasm at approximately 70-90 Å (1 Å=0.1 nm) from the HasR ß-barrel; and (iii) exhibits local conformational changes in response to the arrival of signalling activators. These features would favour the signal transfer from HasR to its cytoplasmic membrane partners.

Deciphering the unconventional peptide binding to the PDZ domain of MAST2.

Phosphatase and tensin homologue (PTEN) and microtubule-associated serine threonine kinase 2 (MAST2) are key negative regulators of survival pathways in neuronal cells. The two proteins interact via the PDZ (PSD-95, Dlg1, Zo-1) domain of MAST2 (MAST2-PDZ). During infection by rabies virus, the viral glycoprotein competes with PTEN for interaction with MAST2-PDZ and promotes neuronal survival. The C-terminal PDZ-binding motifs (PBMs) of the two proteins bind similarly to MAST2-PDZ through an unconventional network of connectivity involving two anchor points. Combining stopped-flow fluorescence, analytical ultracentrifugation (AUC), microcalorimetry and NMR, we document the kinetics of interaction between endogenous and viral ligands to MAST2-PDZ as well as the dynamic and structural effects of these interactions. Viral and PTEN peptide interactions to MAST2-PDZ occur via a unique kinetic step which involves both canonical C-terminal PBM binding and N-terminal anchoring. Indirect effects induced by the PBM binding include modifications to the structure and dynamics of the PDZ dimerization surface which prevent MAST2-PDZ auto-association. Such an energetic communication between binding sites and distal surfaces in PDZ domains provides interesting clues for protein regulation overall.

(1)H, (13)C and (15)N resonance assignments of the RodA hydrophobin from the opportunistic pathogen Aspergillus fumigatus.

Hydrophobins are fungal proteins characterised by their amphipathic properties and an idiosyncratic pattern of eight cysteine residues involved in four disulphide bridges. The soluble form of these proteins spontaneously self-assembles at hydrophobic/hydrophilic interfaces to form an amphipathic monolayer. The RodA hydrophobin of the opportunistic pathogen Aspergillus fumigatus forms an amyloid layer with a rodlet morphology that covers the surface of fungal spores. This rodlet layer bestows hydrophobicity to the spores facilitating their dispersal in the air and rendering the conidia inert relative to the human immune system. As a first step in the analysis of the solution structure and self-association of RodA, we report the (1)H, (13)C and (15)N resonance assignments of the soluble monomeric form of RodA.

Regulation of the catalytic activity of the human phosphatase PTPN4 by its PDZ domain.

The human protein tyrosine phosphatase non-receptor type 4 (PTPN4) prevents cells death. Targeting its PDZ domain abrogates this protection and triggers apoptosis. We demonstrate here that the PDZ domain inhibits the phosphatase activity of PTPN4. The mere binding of a PDZ ligand is sufficient to release the catalytic inhibition. We combined analytical ultracentrifugation, small angle X-ray scattering and NMR to understand how the PDZ domain controls PTPN4 activity. We show that the physiologically active PTPN4 two-domain, encompassing the PDZ and the phosphatase domains, adopts a predominant compact conformation in solution. The PDZ ligand binding restores the catalytic competence of PTPN4 disrupting the transient interdomain communication. This study strengthens the emerging notion that PDZ domains can act as regulators of enzyme activity and therefore are active players in the dynamic regulation of signaling pathways.

Non-stoichiometric O-acetylation of Shigella flexneri 2a O-specific polysaccharide: synthesis and antigenicity.

Synthetic functional mimics of the O-antigen from Shigella flexneri 2a are seen as promising vaccine components against endemic shigellosis. Herein, the influence of the polysaccharide non-stoichiometric di-O-acetylation on antigenicity is addressed for the first time. Three decasaccharides, representing relevant internal mono- and di-O-acetylation profiles of the O-antigen, were synthesized from a pivotal protected decasaccharide designed to tailor late stage site-selective O-acetylation. The latter was obtained via a convergent route involving the imidate glycosylation chemistry. Binding studies to five protective mIgGs showed that none of the acetates adds significantly to broad antibody recognition. Yet, one of the five antibodies had a unique pattern of binding. With IC50 in the micromolar to submicromolar range mIgG F22-4 exemplifies a remarkable tight binding antibody against diversely O-acetylated and non-O-acetylated fragments of a neutral polysaccharide of medical importance.

Interaction of a partially disordered antisigma factor with its partner, the signaling domain of the TonB-dependent transporter HasR.

Bacteria use diverse signaling pathways to control gene expression in response to external stimuli. In Gram-negative bacteria, the binding of a nutrient is sensed by an outer membrane transporter. This signal is then transmitted to an antisigma factor and subsequently to the cytoplasm where an ECF sigma factor induces expression of genes related to the acquisition of this nutrient. The molecular interactions involved in this transmembrane signaling are poorly understood and structural data on this family of antisigma factor are rare. Here, we present the first structural study of the periplasmic domain of an antisigma factor and its interaction with the transporter. The study concerns the signaling in the heme acquisition system (Has) of Serratia marcescens. Our data support unprecedented partially disordered periplasmic domain of an anti-sigma factor HasS in contact with a membrane-mimicking environment. We solved the 3D structure of the signaling domain of HasR transporter and identified the residues at the HasS-HasR interface. Their conservation in several bacteria suggests wider significance of the proposed model for the understanding of bacterial transmembrane signaling.

Chemical organization of the cell wall polysaccharide core of Malassezia restricta.

Malassezia species are ubiquitous residents of human skin and are associated with several diseases such as seborrheic dermatitis, tinea versicolor, folliculitis, atopic dermatitis, and scalp conditions such as dandruff. Host-Malassezia interactions and mechanisms to evade local immune responses remain largely unknown. Malassezia restricta is one of the most predominant yeasts of the healthy human skin, its cell wall has been investigated in this paper. Polysaccharides in the M. restricta cell wall are almost exclusively alkali-insoluble, showing that they play an essential role in the organization and rigidity of the M. restricta cell wall. Fractionation of cell wall polymers and carbohydrate analyses showed that the polysaccharide core of the cell wall of M. restricta contained an average of 5% chitin, 20% chitosan, 5% ß-(1,3)-glucan, and 70% ß-(1,6)-glucan. In contrast to other yeasts, chitin and chitosan are relatively abundant, and ß-(1,3)-glucans constitute a minor cell wall component. The most abundant polymer is ß-(1,6)-glucans, which are large molecules composed of a linear ß-(1,6)-glucan chains with ß-(1,3)-glucosyl side chain with an average of 1 branch point every 3.8 glucose unit. Both ß-glucans are cross-linked, forming a huge alkali-insoluble complex with chitin and chitosan polymers. Data presented here show that M. restricta has a polysaccharide organization very different of all fungal species analyzed to date.

Aerial exposure to the bacterial volatile compound trimethylamine modifies antibiotic resistance of physically separated bacteria by raising culture medium pH.

UNLABELLED: Bacteria release a wide diversity of small bioactive molecules that often correspond to secondary metabolites. Among them, volatile molecules produced under various growth conditions were shown to mediate cross-kingdom interactions with plants, nematodes, and fungi. Although the role of volatile compounds in bacterial biology is not well understood, recent reports indicated that they could play a role in airborne interactions between bacteria and influence antibiotic resistance, biofilm formation, and virulence. In this study, we investigated long-distance effects of 14 previously described Escherichia coli volatile compounds upon the bacteria E. coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Bacillus subtilis. We show that several of these molecules constitute chemical cues influencing growth, adhesion, and motility in exposed bacteria. Moreover, we show that aerial exposure to trimethylamine (TMA), a volatile compound produced in animal intestines and tissues upon biogenic reduction of trimethylamine oxide (TMAO), modifies the antibiotic resistance profiles of all tested Gram-positive and Gram-negative bacteria. We demonstrate that the TMA mode of action is distinct from that previously described for ammonia and results from nonspecific transient alteration of antibiotic uptake due to pH increase in the environment of bacteria aerially exposed to TMA. Our study therefore presents a new way by which volatile compounds can affect community behavior and structure in physically separated bacteria. It further demonstrates that bacterial gases and volatile compounds mediate chemical interactions, triggering functional responses that play an important role in the development of bacterial communities. IMPORTANCE: Bacteria release many different volatile compounds during food transformation and fermentation. Here we sought to investigate the role of several bacterial volatile molecules released by Escherichia coli during long-distance airborne interactions with other bacteria. While several tested volatiles affect bacterial motility and surface adhesion, we show that aerial exposure to trimethylamine, a molecule produced by E. coli and many other Gram-negative bacteria in animal intestines and infected tissues, also modulates antibiotic resistance in all tested bacteria. We demonstrate that exposure to trimethylamine increases the pH of the growth medium of exposed bacteria, resulting in modifications in antibiotic uptake and transient alteration of antibiotic resistance. Our study therefore presents a new mechanism by which volatile compounds can affect community behavior and structure in physically separated bacteria, and it illustrates how airborne chemical interactions between bacteria contribute to the development of bacterial communities.

¹H, ¹5N and ¹³C resonance assignments of PpdD, a type IV pilin from enterohemorrhagic Escherichia coli.

Bacterial type 4 pili (T4P) are long flexible fibers involved in adhesion, DNA uptake, phage transduction, aggregation and a flagella-independent movement called "twitching motility". T4P comprise thousands of copies of the major pilin subunit, which is initially inserted in the plasma membrane, processed and assembled into dynamic helical filaments. T4P are crucial for host colonization and virulence of many Gram-negative bacteria. In enterohemorrhagic Escherichia coli the T4P, called hemorrhagic coli pili (HCP) promote cell adhesion, motility, biofilm formation and signaling. To understand the mechanism of HCP assembly and function, we analyzed the structure of the major subunit prepilin peptidase-dependent protein D (PpdD) (also called HcpA), a 15 kDa pilin with two potential disulfide bonds. Here we present the (1)H, (15)N and (13)C backbone and side chain resonance assignments of the C-terminal globular domain of PpdD as a first step to its structural determination.

The structure of HasB reveals a new class of TonB protein fold.

TonB is a key protein in active transport of essential nutrients like vitamin B12 and metal sources through the outer membrane transporters of Gram-negative bacteria. This inner membrane protein spans the periplasm, contacts the outer membrane receptor by its periplasmic domain and transduces energy from the cytoplasmic membrane pmf to the receptor allowing nutrient internalization. Whereas generally a single TonB protein allows the acquisition of several nutrients through their cognate receptor, in some species one particular TonB is dedicated to a specific system. Despite a considerable amount of data available, the molecular mechanism of TonB-dependent active transport is still poorly understood. In this work, we present a structural study of a TonB-like protein, HasB dedicated to the HasR receptor. HasR acquires heme either free or via an extracellular heme transporter, the hemophore HasA. Heme is used as an iron source by bacteria. We have solved the structure of the HasB periplasmic domain of Serratia marcescens and describe its interaction with a critical region of HasR. Some important differences are observed between HasB and TonB structures. The HasB fold reveals a new structural class of TonB-like proteins. Furthermore, we have identified the structural features that explain the functional specificity of HasB. These results give a new insight into the molecular mechanism of nutrient active transport through the bacterial outer membrane and present the first detailed structural study of a specific TonB-like protein and its interaction with the receptor.

Solution structure of an archaeal DNA binding protein with an eukaryotic zinc finger fold.

While the basal transcription machinery in archaea is eukaryal-like, transcription factors in archaea and their viruses are usually related to bacterial transcription factors. Nevertheless, some of these organisms show predicted classical zinc fingers motifs of the C2H2 type, which are almost exclusively found in proteins of eukaryotes and most often associated with transcription regulators. In this work, we focused on the protein AFV1p06 from the hyperthermophilic archaeal virus AFV1. The sequence of the protein consists of the classical eukaryotic C2H2 motif with the fourth histidine coordinating zinc missing, as well as of N- and C-terminal extensions. We showed that the protein AFV1p06 binds zinc and solved its solution structure by NMR. AFV1p06 displays a zinc finger fold with a novel structure extension and disordered N- and C-termini. Structure calculations show that a glutamic acid residue that coordinates zinc replaces the fourth histidine of the C2H2 motif. Electromobility gel shift assays indicate that the protein binds to DNA with different affinities depending on the DNA sequence. AFV1p06 is the first experimentally characterised archaeal zinc finger protein with a DNA binding activity. The AFV1p06 protein family has homologues in diverse viruses of hyperthermophilic archaea. A phylogenetic analysis points out a common origin of archaeal and eukaryotic C2H2 zinc fingers.

(1)H, (13)C and (15)N resonance assignments of the periplasmic signalling domain of HasR, a TonB-dependent outer membrane heme transporter.

TonB-dependent transporters (TBDTs) are bacterial outer membrane proteins that internalize nutrients such as vitamin B12, metal complexes, heme, some carbohydrates, etc. In addition to their transport activity, several TBDTs are also involved in a signalling cascade from the cell surface into the cytoplasm, via their periplasmic signalling domain. Here we report the backbone and side chain resonance assignments of the signalling domain of HasR, a TonB-dependent outer membrane heme transporter from Serratia marcescens as a first step towards its structural study.

Ordered phosphorylation events in two independent cascades of the PTEN C-tail revealed by NMR.

PTEN phosphatase is a tumor suppressor controlling notably cell growth, proliferation and survival. The multisite phosphorylation of the PTEN C-terminal tail regulates PTEN activity and intracellular trafficking. The dynamical nature of such regulatory events represents a crucial dimension for timing cellular decisions. Here we show that NMR spectroscopy allows reporting on the order and kinetics of clustered multisite phosphorylation events. We first unambiguously identify in vitro seven bona fide sites modified by CK2 and GSK3ß kinases and two new sites on the PTEN C-terminal tail. Then, monitoring the formation of transient intermediate phosphorylated states, we determine the sequence of these reactions and calculate their apparent rate constants. Finally, we assess the dynamic formation of these phosphorylation events induced by endogenous kinases directly in extracts of human neuroblastoma cells. Taken together, our data indicate that two cascades of events controlled by CK2 and GSK3ß occur independently on two clusters of sites (S380-S385 and S361-S370) and that in each cluster the reactions follow an ordered model with a distributive kinetic mechanism. Besides emphasizing the ability of NMR to quantitatively and dynamically follow post-translational modifications, these results bring a temporal dimension on the establishment of PTEN phosphorylation cascades.