Cloning and biochemical characterization of the fucanase FcnA: definition of a novel glycoside hydrolase family specific for sulfated fucans

Sulfated fucans are matrix polysaccharides from marine brown algae, consisting of an alpha-L-fucose backbone substituted by sulfate-ester groups, masked with ramifications, and containing other monosaccharide residues. We here report on the characterization of a novel glycoside hydrolase (FcnA) specific for the degradation of sulfated fucans. This glycoside hydrolase was purified to electrophoretic homogeneity from a Flavobacteriaceae referred to as SW5. The gene fcnA was cloned and sequenced (3021 nucleotides), and the protein (1007 amino acids) was produced in Escherichia coli. FcnA exhibited a modular architecture consisting of a 400-residue-long N-terminal domain followed by three repeated domains predicted to adopt an immunoglobulin fold and by an 80-amino acid-long C-terminal domain. A truncated recombinant protein encompassing the N-terminal domain and the immunoglobulin-like repeats was shown to retain the enzyme activity. The N-terminal catalytic domain shared approximately 25% of sequence identity with two patented fucanase genes, and these three fucanases delineate a new family of glycoside hydrolases. As shown by size-exclusion chromatography (SEC) and 1H-NMR analyses, the fucanase FcnA proceeds according to an endolytic mode of action and cleaves the alpha-(1-->4) glycosidic linkages within the blocks of repeating motifs [-->4)-alpha-L-fucopyranosyl-2,3-disulfate-(1-->3)-alpha-L-fucopyranosyl-2-sulfate-(1-->]n.     

FGF21 N- and C-termini play different roles in receptor interaction and activation

Fibroblast growth factor-21 (FGF21) signaling requires the presence of β-Klotho, a co-receptor with a very short cytoplasmic domain. Here we show that FGF21 binds directly to β-Klotho through its C-terminus. Serial C-terminal truncations of FGF21 weakened or even abrogated its interaction with β-Klotho in a Biacore assay, and led to gradual loss of potency in a luciferase reporter assay but with little effect on maximal response. In contrast, serial N-terminal truncations of FGF21 had no impact on β-Klotho binding. Interestingly, several of them exhibited characteristics of partial agonists with minimal effects on potency. These data demonstrate that the C-terminus of FGF21 is critical for binding to β-Klotho and the N-terminus is critical for fibroblast growth factor receptor (FGFR) activation.

Structured summary

MINT-6799939: FGFR1c (uniprotkb:P11362) binds (MI:0407) to β-Klotho (uniprotkb: Q86Z14) by surface plasmon resonance (MI:0107)MINT-6799907, MINT-6799922: FGF21 (uniprotkb: Q9NSA1) binds (MI:0407) to β-Klotho (uniprotkb: Q86Z14) by surface plasmon resonance (MI:0107)     

Effects of high-altitude hypoxia on the hormonal response to hypothalamic factors

Acute and chronic exposure to high altitude induces various physiological changes, including activation or inhibition of various hormonal systems. In response to activation processes, a desensitization of several pathways has been described, especially in the adrenergic system. In the present study, we aimed to assess whether the hypophyseal hormones are also subjected to a hypoxia-induced decrease in their response to hypothalamic factors. Basal levels of hormones and the responses of TSH, thyroid hormones, prolactin, sex hormones, and growth hormone to the injection of TRH, gonadotropin-releasing hormone, and growth hormone-releasing hormone (GHRH) were studied in eight men in normoxia and on prolonged exposure (3-4 days) to an altitude of 4,350 m. Thyroid hormones were elevated at altitude (+16 to +21%), while TSH levels were unchanged, and follicle-stimulating hormone and prolactin decreased, while leutinizing hormone was unchanged. Norepinephrine and cortisol levels were elevated, while no change was observed in levels of epinephrine, dopamine, growth hormone (GH), IGF-1, and IGFBP-3. The mean response to hypothalamic factors was similar in both altitudes for all studied hormones, although total T4 was lower in hypoxia during 45 to 60 min after injection. The effect of hypoxia on the hypophyseal response to hypothalamic factors was similar among subjects, except for the GH response to GHRH administration. We conclude that prolonged exposure to high-altitude hypoxia induces contrasted changes in hormonal levels, but the hypophyseal response to hypothalamic factors does not appear to be blunted.     

Rumination of different-sized particles in muskoxen (Ovibos moschatus) and moose (Alces alces) on grass and browse diets,and implications for rumination in different ruminant feeding types

The obligatory, periodic regurgitation of forestomach material and its subsequent re-mastication is the hallmark of the most diverse extant large herbivore group, the ruminants. Although the process of rumination is well understood in domestic species, differences between free-ranging wild ruminant species, for example of different body size or different feeding type, remain speculative to date. Here we investigate the proportion of plastic particles of varying size (1, 10 and 20 mm) and density (1.03, 1.20 and 1.44 mg/ml) that are recovered intact or ruminated-upon after insertion into the reticulorumen (RR) of domestic cattle (Bos primigenius f. taurus) on grass silage, and of muskoxen (Ovibos moschatus; n = 4) and moose (Alces alces; n = 2) both fed browse and grass diets. In the three species, the proportion of particles leaving the RR intact depended on particle size, with density showing no effect in this study. The major proportion of 1 mm particles was excreted intact, whereas intact 10–20 mm particles were only excreted sporadically, and not in all animals. Intact particles were mostly found in the initial samples after marker application, and mean retention times of intact particles were always shorter than those of ruminated particles. There were no differences between moose and muskoxen, but diet had a significant effect, with a higher proportion of 1 mm particles ruminated upon on the grass diet in both species, indicating a higher ‘filter-bed effect’ with entrapment of small particles in a fibre mat in the RR on a grass diet. Given that less particles were ruminated on the grass diet, one could either assume that free-ranging browsers ruminate less than grazers on similar food intakes (or that they have higher food intakes at similar levels of rumination). The existing data on time-budgets of free-ranging ruminants, however, does not suffice to test these hypotheses. The fact that indication of a ‘filter-bed effect’ was also detectable in moose raises the question whether adaptations described in ‘cattle-type’ ruminants really serve to re-inforce the processes of RR contents stratification and the ‘filter-bed’, or whether they are not rather directed at other aims, such as maximizing microbial yield from the RR.     

FGF21 Can Be Mimicked In Vitro and In Vivo by a Novel Anti-FGFR1c/β-Klotho Bispecific Protein

The endocrine hormone FGF21 has attracted considerable interest as a potential therapeutic for treating diabetes and obesity. As an alternative to the native cytokine, we generated bispecific Avimer polypeptides that bind with high affinity and specificity to one of the receptor and coreceptor pairs used by FGF21, FGFR1c and β-Klotho. These Avimers exhibit FGF21-like activity in in vitro assays with potency greater than FGF21. In a study conducted in obese male cynomolgus monkeys, animals treated with an FGFR1c/β-Klotho bispecific Avimer showed improved metabolic parameters and reduced body weight comparable to the effects seen with FGF21. These results not only demonstrate the essential roles of FGFR1c and β-Klotho in mediating the metabolic effects of FGF21, they also describe a first bispecific activator of this unique receptor complex and provide validation for a novel therapeutic approach to target this potentially important pathway for treating diabetes and obesity.     

A Comparison of Morphological and Molecular-Based Surveys to Estimate the Species Richness of Chaetoceros and Thalassiosira (Bacillariophyta), in the Bay of Fundy

The goal of this study was to compare the ability of morphology and molecular-based surveys to estimate species richness for two species-rich diatom genera, Chaetoceros Ehrenb. and Thalassiosira Cleve, in the Bay of Fundy. Phytoplankton tows were collected from two sites at intervals over two years and subsampled for morphology-based surveys (2010, 2011), a culture-based DNA reference library (DRL; 2010), and a molecular-based survey (2011). The DRL and molecular-based survey utilized the 3′ end of the RUBISCO large subunit (rbcL-3P) to identify genetic species groups (based on 0.1% divergence in rbcL-3P), which were subsequently identified morphologically to allow comparisons to the morphology-based survey. Comparisons were compiled for the year (2011) by site (n = 2) and by season (n = 3). Of the 34 taxa included in the comparisons, 50% of taxa were common to both methods, 35% were unique to the molecular-based survey, and 12% were unique to the morphology-based survey, while the remaining 3% of taxa were unidentified genetic species groups. The morphology-based survey excelled at identifying rare taxa in individual tow subsamples, which were occasionally missed with the molecular approach used here, while the molecular methods (the DRL and molecular-based survey), uncovered nine cryptic species pairs and four previously overlooked species. The last mentioned were typically difficult to identify and were generically assigned to Thalassiosira spp. during the morphology-based survey. Therefore, for now we suggest a combined approach encompassing routine morphology-based surveys accompanied by periodic molecular-based surveys to monitor for cryptic and difficult to identify taxa. As sequencing technologies improve, molecular-based surveys should become routine, leading to a more accurate representation of species composition and richness in monitoring programs.     

ubiI,a New Gene in Escherichia coli Coenzyme Q Biosynthesis,Is Involved in Aerobic C5-hydroxylation

Coenzyme Q (ubiquinone or Q) is a redox-active lipid found in organisms ranging from bacteria to mammals in which it plays a crucial role in energy-generating processes. Q biosynthesis is a complex pathway that involves multiple proteins. In this work, we show that the uncharacterized conserved visC gene is involved in Q biosynthesis in Escherichia coli, and we have renamed it ubiI. Based on genetic and biochemical experiments, we establish that the UbiI protein functions in the C5-hydroxylation reaction. A strain deficient in ubiI has a low level of Q and accumulates a compound derived from the Q biosynthetic pathway, which we purified and characterized. We also demonstrate that UbiI is only implicated in aerobic Q biosynthesis and that an alternative enzyme catalyzes the C5-hydroxylation reaction in the absence of oxygen. We have solved the crystal structure of a truncated form of UbiI. This structure shares many features with the canonical FAD-dependent para-hydroxybenzoate hydroxylase and represents the first structural characterization of a monooxygenase involved in Q biosynthesis. Site-directed mutagenesis confirms that residues of the flavin binding pocket of UbiI are important for activity. With our identification of UbiI, the three monooxygenases necessary for aerobic Q biosynthesis in E. coli are known.     

A new cytochrome P450 belonging to the 107L subfamily is responsible for the efficient hydroxylation of the drug terfenadine by Streptomyces platensis

Fexofenadine, an antihistamine drug used in allergic rhinitis treatment, can be produced by oxidative biotransformation of terfenadine by Streptomyces platensis, which involves three consecutive oxidation reactions. We report here the purification and identification of the enzyme responsible for the first step, a cytochrome P450 (P450)-dependent monooxygenase. The corresponding P450, designated P450_terf, was found to catalyze the hydroxylation of the t-butyl group of terfenadine and exhibited UV–Vis characteristics of a P450. Its interaction with terfenadine led to a shift of its Soret peak from 418 to 390 nm, as expected for the formation of a P450–substrate complex. In combination with spinach ferredoxin:NADP(+) oxidoreductase and ferredoxin, and in the presence of NADPH, it catalyzed the hydroxylation of terfenadine and some of its analogues, such as terfenadone and ebastine, with k_m values at the μM level, and k_cat values around 30 min⁻¹. Sequencing of the p450_terf gene led to a 1206 bp sequence, encoding for a 402 aminoacid polypeptide exhibiting 56–65% identity with the P450s from the 107L family. These results confirmed that P450s from Streptomyces species are interesting tools for the biotechnological production of secondary metabolites, such as antibiotics or antitumor compounds, and in the oxidative biotransformation of xenobiotics, such as drugs.     

The translational repressor 4E-BP called to order by eIF4E: new structural insights by SAXS

eIF4E binding protein (4E-BP) inhibits translation of capped mRNA by binding to the initiation factor eIF4E and is known to be mostly or completely unstructured in both free and bound states. Using small angle X-ray scattering (SAXS), we report here the analysis of 4E-BP structure in solution, which reveals that while 4E-BP is intrinsically disordered in the free state, it undergoes a dramatic compaction in the bound state. Our results demonstrate that 4E-BP and eIF4E form a 'fuzzy complex', challenging current visions of eIF4E/4E-BP complex regulation.     

Thrombopoietin-Increased DNA-PK-Dependent DNA Repair Limits Hematopoietic Stem and Progenitor Cell Mutagenesis in Response to DNA Damage

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Highlights? Mpl loss increases γ-irradiation-induced genomic instability in HSPCs ? TPO promotes DNA repair in vitro and in vivo in HSPCs ? TPO increases DNA-PK activity and NHEJ-mediated repair efficiency in HSPCs ? A single TPO injection before mouse TBI limits long-term HSC injury and mutagenesis