Members of the diverse bacterial phylum Bacteroidetes have colonized virtually all types of habitats on Earth. They are among the major members of the microbiota of animals, especially in the ...gastrointestinal tract, can act as pathogens and are frequently found in soils, oceans and freshwater. In these contrasting ecological niches, Bacteroidetes are increasingly regarded as specialists for the degradation of high molecular weight organic matter, i.e., proteins and carbohydrates. This review presents the current knowledge on the role and mechanisms of polysaccharide degradation by Bacteroidetes in their respective habitats. The recent sequencing of Bacteroidetes genomes confirms the presence of numerous carbohydrate-active enzymes covering a large spectrum of substrates from plant, algal, and animal origin. Comparative genomics reveal specific Polysaccharide Utilization Loci shared between distantly related members of the phylum, either in environmental or gut-associated species. Moreover, Bacteroidetes genomes appear to be highly plastic and frequently reorganized through genetic rearrangements, gene duplications and lateral gene transfers (LGT), a feature that could have driven their adaptation to distinct ecological niches. Evidence is accumulating that the nature of the diet shapes the composition of the intestinal microbiota. We address the potential links between gut and environmental bacteria through food consumption. LGT can provide gut bacteria with original sets of utensils to degrade otherwise refractory substrates found in the diet. A more complete understanding of the genetic gateways between food-associated environmental species and intestinal microbial communities sheds new light on the origin and evolution of Bacteroidetes as animals' symbionts. It also raises the question as to how the consumption of increasingly hygienic and processed food deprives our microbiota from useful environmental genes and possibly affects our health.
Cancer cells utilize the main de novo pathway and the alternative salvage pathway for deoxyribonucleotide biosynthesis to achieve adequate nucleotide pools. Deoxycytidine kinase is the rate-limiting ...enzyme of the salvage pathway and it has recently emerged as a target for anti-proliferative therapies for cancers where it is essential. Here, we present the development of a potent inhibitor applying an iterative multidisciplinary approach, which relies on computational design coupled with experimental evaluations. This strategy allows an acceleration of the hit-to-lead process by gradually implementing key chemical modifications to increase affinity and activity. Our lead compound, OR0642, is more than 1000 times more potent than its initial parent compound, masitinib, previously identified from a drug repositioning approach. OR0642 in combination with a physiological inhibitor of the de novo pathway doubled the survival rate in a human T-cell acute lymphoblastic leukemia patient-derived xenograft mouse model, demonstrating the proof-of-concept of this drug design strategy.
Summary
The genomic data on heterotrophic marine bacteria suggest the crucial role that microbes play in the global carbon cycle. However, the massive presence of hypothetical proteins hampers our ...understanding of the mechanisms by which this carbon cycle is carried out. Moreover, genomic data from marine microorganisms are essentially annotated in the light of the biochemical knowledge accumulated on bacteria and fungi which decompose terrestrial plants. However marine algal polysaccharides clearly differ from their terrestrial counterparts, and their associated enzymes usually constitute novel protein families. In this study, we have applied a combination of bioinformatics, targeted activity screening and structural biology to characterize a hypothetical protein from the marine bacterium Zobellia galactanivorans, which is distantly related to GH43 family. This protein is in fact a 1,3‐α‐3,6‐anhydro‐l‐galactosidase (AhgA) which catalyses the last step in the degradation pathway of agars, a family of polysaccharides unique to red macroalgae. AhgA adopts a β‐propeller fold and displays a zinc‐dependent catalytic machinery. This enzyme is the first representative of a new family of glycoside hydrolases, especially abundant in coastal waters. Such genes of marine origin have been transferred to symbiotic microbes associated with marine fishes, but also with some specific human populations.
Vanadium haloperoxidases (VHPO) are key enzymes that oxidize halides and are involved in the biosynthesis of organo-halogens. Until now, only chloroperoxidases (VCPO) and bromoperoxidases (VBPO) have ...been characterized structurally, mainly from eukaryotic species. Three putative VHPO genes were predicted in the genome of the flavobacterium Zobellia galactanivorans, a marine bacterium associated with macroalgae. In a phylogenetic analysis, these putative bacterial VHPO were closely related to other VHPO from diverse bacterial phyla but clustered independently from eukaryotic algal VBPO and fungal VCPO. Two of these bacterial VHPO, heterogeneously produced in Escherichia coli, were found to be strictly specific for iodide oxidation. The crystal structure of one of these vanadium-dependent iodoperoxidases, Zg-VIPO1, was solved by multiwavelength anomalous diffraction at 1.8 Å, revealing a monomeric structure mainly folded into α-helices. This three-dimensional structure is relatively similar to those of VCPO of the fungus Curvularia inaequalis and of Streptomyces sp. and is superimposable onto the dimeric structure of algal VBPO. Surprisingly, the vanadate binding site of Zg-VIPO1 is strictly conserved with the fungal VCPO active site. Using site-directed mutagenesis, we showed that specific amino acids and the associated hydrogen bonding network around the vanadate center are essential for the catalytic properties and also the iodide specificity of Zg-VIPO1. Altogether, phylogeny and structure-function data support the finding that iodoperoxidase activities evolved independently in bacterial and algal lineages, and this sheds light on the evolution of the VHPO enzyme family.
Masitinib, a highly selective protein kinase inhibitor, can sensitise gemcitabine-refractory cancer cell lines when used in combination with gemcitabine. Here we report a reverse proteomic approach ...that identifies the target responsible for this sensitisation: the deoxycytidine kinase (dCK). Masitinib, as well as other protein kinase inhibitors, such as imatinib, interact with dCK and provoke an unforeseen conformational-dependent activation of this nucleoside kinase, modulating phosphorylation of nucleoside analogue drugs. This phenomenon leads to an increase of prodrug phosphorylation of most of the chemotherapeutic drugs activated by this nucleoside kinase. The unforeseen dual activity of protein kinase inhibition/nucleoside kinase activation could be of great therapeutic benefit, through either reducing toxicity of therapeutic agents by maintaining effectiveness at lower doses or by counteracting drug resistance initiated via down modulation of dCK target.
Protein-protein interactions (PPIs) represent an enormous source of opportunity for therapeutic intervention. We and others have recently pinpointed key rules that will help in identifying the next ...generation of innovative drugs to tackle this challenging class of targets within the next decade. We used these rules to design an oriented chemical library corresponding to a set of diverse "PPI-like" modulators with cores identified as privileged structures in therapeutics. In this work, we purchased the resulting 1664 structurally diverse compounds and evaluated them on a series of representative protein-protein interfaces with distinct "druggability" potential using homogeneous time-resolved fluorescence (HTRF) technology. For certain PPI classes, analysis of the hit rates revealed up to 100 enrichment factors compared with nonoriented chemical libraries. This observation correlates with the predicted "druggability" of the targets. A specific focus on selectivity profiles, the three-dimensional (3D) molecular modes of action resolved by X-ray crystallography, and the biological activities of identified hits targeting the well-defined "druggable" bromodomains of the bromo and extraterminal (BET) family are presented as a proof-of-concept. Overall, our present study illustrates the potency of machine learning-based oriented chemical libraries to accelerate the identification of hits targeting PPIs. A generalization of this method to a larger set of compounds will accelerate the discovery of original and potent probes for this challenging class of targets.
The uncoupling proteins (UCPs) leak protons across the inner mitochondrial membrane, thus uncoupling the proton gradient from ATP synthesis. The main known physiological role for this is heat ...generation by UCP1 in brown adipose tissue. However, UCPs are also believed to be important for protection against reactive oxygen species, fine-tuning of metabolism and have been suggested to be involved in disease states such as obesity, diabetes and cancer. Structural studies of UCPs have long been hampered by difficulties in sample preparation with neither expression in yeast nor refolding from inclusion bodies in
yielding sufficient amounts of pure and stable protein. In this study, we have developed a protocol for cell-free expression of human UCP1, 2 and 3, resulting in 1 mg pure protein per 20 mL of expression media. Lauric acid, a natural UCP ligand, significantly improved protein thermal stability and was therefore added during purification. Secondary structure characterisation using circular dichroism spectroscopy revealed the proteins to consist of mostly α-helices, as expected. All three UCPs were able to bind GDP, a well-known physiological inhibitor, as shown by the Fluorescence Resonance Energy Transfer (FRET) technique, suggesting that the proteins are in a natively folded state.
The production of stable and soluble proteins is one of the most important steps prior to structural and functional studies of biological importance. We investigated the parallel production in a ...medium throughput strategy of genes coding for proteins from various marine organisms, using protocols that involved recombinatorial cloning, protein expression screening and batch purification. This strategy was applied in order to respond to the need for post-genomic validation of the recent success of a large number of marine genomic projects. Indeed, the upcoming challenge is to go beyond the bioinformatic data, since the bias introduced through the genomes of the so called model organisms leads to numerous proteins of unknown function in the still unexplored world of the oceanic organisms.
We present here the results of expression tests for 192 targets using a 96-well plate format. Genes were PCR amplified and cloned in parallel into expression vectors pFO4 and pGEX-4T-1, in order to express proteins N-terminally fused to a six-histidine-tag and to a GST-tag, respectively. Small-scale expression and purification permitted isolation of 84 soluble proteins and 34 insoluble proteins, which could also be used in refolding assays. Selected examples of proteins expressed and purified to a larger scale are presented.
The objective of this program was to get around the bottlenecks of soluble, active protein expression and crystallization for post-genomic validation of a number of proteins that come from various marine organisms. Multiplying the constructions, vectors and targets treated in parallel is important for the success of a medium throughput strategy and considerably increases the chances to get rapid access to pure and soluble protein samples, needed for the subsequent biochemical characterizations. Our set up of a medium throughput strategy applied to genes from marine organisms had a mean success rate of 44% soluble protein expression from marine bacteria, archaea as well as eukaryotic organisms. This success rate compares favorably with other protein screening projects, particularly for eukaryotic proteins. Several purified targets have already formed the base for experiments aimed at post-genomic validation.
The purple pigmented bacterium Chromobacterium violaceum is a dominant component of tropical soil microbiota that can cause rare but fatal septicaemia in humans. Its sequenced genome provides insight ...into the abundant potential of this organism for biotechnological and pharmaceutical applications and allowed an ORF encoding a protein that is 60% identical to the fucose binding lectin (PA-IIL) from Pseudomonas aeruginosa and the mannose binding lectin (RS-IIL) from Ralstonia solanacearum to be identified. The lectin, CV-IIL, has recently been purified from C. violaceum Zinger-Yosovich, K., Sudakevitz, D., Imberty, A., Garber, N. C., and Gilboa-Garber, N. (2006) Microbiology 152, 457-463 and has been confirmed to be a tetramer with subunit size of 11.86 kDa and a binding preference for fucose. We describe here the cloning of CV-IIL and its expression as a recombinant protein. A complete structure-function characterization has been made in an effort to analyze the specificity and affinity of CV-IIL for fucose and mannose. Crystal structures of CV-IIL complexes with monosaccharides have yielded the molecular basis of the specificity. Each monomer contains two close calcium cations that mediate the binding of the monosaccharides, which occurs in different orientations for fucose and mannose. The thermodynamics of binding has been analyzed by titration microcalorimetry, giving dissociation constants of 1.7 and 19 microM for alpha-methyl fucoside and alpha-methyl mannoside, respectively. Further analysis demonstrated a strongly favorable entropy term that is unusual in carbohydrate binding. A comparison with both PA-IIL and RS-IIL, which have binding preferences for fucose and mannose, respectively, yielded insights into the monosaccharide specificity of this important class of soluble bacterial lectins.
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