Helical structure of dermaseptin B2 in a membrane-mimetic environment

Dermaseptins are antimicrobial peptides from frog skin that have high membrane-lytic activity against a broad spectrum of microorganisms. The structure of dermaseptin B2 in aqueous solution, in TFE/water mixtures, and in micellar and nonmicellar SDS was analyzed by CD, FTIR, fluorescence, and NMR spectroscopy combined with molecular dynamics calculations. Dermaseptin B2 is unstructured in water, but helical conformations, mostly in segment 3-18, are stabilized by addition of TFE. SDS titration showed that dermaseptin B2 assumes nonhelical structures at SDS concentrations far below the critical micellar concentration and helical structures at micellar concentrations. Dermaseptin B2 bound to SDS micelles (0.4 mM peptide, 80 mM SDS) adopts a well-defined amphipathic helix between residues 11-31 connected to a more flexible helical segment spanning residues 1-8 by a flexible hinge region around Val9 and Gly10. Experiments using paramagnetic probes showed that dermaseptin B2 lies near the surface of SDS micelles and that residue Trp3 is buried in the SDS micelle, but close to the surface. A slow exchange equilibrium occurs at higher peptide/SDS ratios (2 mM peptide, 80 mM SDS) between forms having distinct sets of resonances in the N-terminal 1-11 segment. This equilibrium could reflect different oligomeric states of dermaseptin B2 interacting with SDS micelles. Structure-activity studies on dermaseptin B2 analogues showed that the N-terminal 1-11 segment is an absolute requirement for antibacterial activity, while the C-terminal 10-33 region is also important for full antibiotic activity.     

Thyrotropin and iodide regulate sulfate concentration in thyroid cells. Relationship to thyroglobulin sulfation

Thyroglobulin (Tg), the thyroid hormone precursor, is sulfated both on tyrosines and on carbohydrates. We showed recently that sulfated tyrosines were involved in thyroid hormone synthesis. Moreover, we also reported that Tg sulfation is downregulated by thyrotropin (TSH), especially on tyrosines. This control may occur at each step in the sulfation process. In this paper, we studied the regulation of the concentration of cytosolic inorganic sulfate, the first substrate, in porcine thyroid cells stimulated by TSH with or without iodide. The amounts of cytosolic sulfate and the cytosolic volumes measured showed that the sulfate concentration depends only on cytosolic volume changes in response to TSH and iodide treatment. After the cells were labelled with [35S]-sulfate, the specific radioactivity (SRA) of cytosolic sulfate was determined. When cells were treated with only TSH, the concentration and SRA of cytosolic sulfate decreased by 30%, and by about 15% when cells were incubated with both TSH and iodide. TSH decreased more conspicuously the rate of [35S]-sulfate incorporation into Tg (by 57% without iodide, by 43% with iodide) than the concentration and SRA of cytosolic sulfate, while iodide altered these parameters to the same extent (15%). These findings suggest that TSH regulates other steps in the sulfation process, such as specific substrate and enzyme levels, while iodide controls mainly the sulfate concentration.     

Discrimination of three mutational events that result in a disruption of the R122 primary autolysis site of the human cationic trypsinogen (PRSS1) by denaturing high performance liquid chromatography

Background  

R122, the primary autolysis site of the human cationic trypsinogen (PRSS1), constitutes an important "self-destruct" or "fail-safe" defensive mechanism against premature trypsin activation within the pancreas. Disruption of this site by a missense mutation, R122H, was found to cause hereditary pancreatitis. In addition to a c.365G>A (CGC>CAC) single nucleotide substitution, a c.365~366GC>AT (CGC>CAT) gene conversion event in exon 3 of PRSS1 was also found to result in a R122H mutation. This imposes a serious concern on the genotyping of pancreatitis by a widely used polymerase chain reaction-restriction fragment length polymorphism assay, which could only detect the commonest c.365G>A variant.     

The serotonin binding site of human and murine 5-HT2B receptors: molecular modeling and site-directed mutagenesis

Bacteriorhodopsin and rhodopsin crystal structures were used as templates to build structural models of the mouse and human serotonin (5-HT)-2B receptors (5-HT(2B)Rs). Serotonin was docked to the receptors, and the amino acids predicted to participate to its binding were subjected to mutagenesis. 5-HT binding affinity and 5-HT-induced inositol triphosphate production were measured in LMTK(-) cells transfected with either wild-type or mutated receptor genes. According to these measurements, the bacteriorhodopsin-based models of the 5-HT(2B)Rs appear more confident than the rhodopsin-based ones. Residues belonging to the transmembrane domains 3 and 6, i.e. Asp(3.32), Ser(3.36), Phe(6.52), and Asn(6.55), make direct contacts with 5-HT. In addition, Trp(3.28), Phe(3.35), Phe(6.52), and Phe(7.38) form an aromatic box surrounding 5-HT. The specificity of human and mouse 5-HT(2B)Rs may be reflected by different rearrangements of the aromatic network upon 5-HT binding. Two amino acids close to Pro(5.50) in the human transmembrane domain 5 sequence were permuted to introduce a "mouse-like" sequence. This change was enough to confer the human 5-HT(2B)R properties similar to those of the mouse. Taken together, the computed models and the site-directed mutagenesis experiments give a structural explanation to (i) the different 5-HT pK(D) values measured with the human and mouse 5-HT(2B)Rs (7.9 and 5.8, respectively) and (ii) the specificity of 5-HT binding to 5-HT(2B)Rs as compared with other serotonergic G-protein coupled receptors.     

Engrailed homeoprotein secretion is a regulated process

Chicken Engrailed 2 homeoprotein is transported between cells in culture. This intercellular transfer is based on unconventional secretion and internalisation mechanisms: Engrailed 2 has access to vesicles but lacks a signal sequence for secretion and is internalised by a non-endocytic process. We show that phosphorylation of a serine-rich domain within Engrailed 2 by the protein kinase CK2 specifically inhibits Engrailed 2 secretion. The availability of the serine-rich domain to CK2 is highly increased when it is displaced from its normal position to the C terminus of Engrailed 2, leading to a constitutive blockage of Engrailed 2 intercellular transfer. This indicates that intercellular transfer of Engrailed 2 is a highly regulated process.     

Adipose tissue selective insulin receptor knockout protects against obesity and obesity-related glucose intolerance

Insulin signaling in adipose tissue plays an important role in lipid storage and regulation of glucose homeostasis. Using the Cre-loxP system, we created mice with fat-specific disruption of the insulin receptor gene (FIRKO mice). These mice have low fat mass, loss of the normal relationship between plasma leptin and body weight, and are protected against age-related and hypothalamic lesion-induced obesity, and obesity-related glucose intolerance. FIRKO mice also exhibit polarization of adipocytes into populations of large and small cells, which differ in expression of fatty acid synthase, C/EBP alpha, and SREBP-1. Thus, insulin signaling in adipocytes is critical for development of obesity and its associated metabolic abnormalities, and abrogation of insulin signaling in fat unmasks a heterogeneity in adipocyte response in terms of gene expression and triglyceride storage.     

Oocyte Ca2+ spike acquisition during in vitro development of early preantral follicles: influence of age and hormonal supplementation

Calcium signalling is involved in important events in oocytes, such as meiotic competence acquisition. We have previously demonstrated the positive influence of animal age and gonadotropin stimulation in vivo regarding the ability of oocytes recovered from preantral follicles to exhibit calcium spikes. In the present work we determined whether preantral follicle development in vitro also allows oocytes to acquire calcium signalling activity. We also aimed to verify the influence of animal age, FSH + LH and/or insulin on oocyte calcium spike acquisition during preantral follicle culture. Early preantral follicles were isolated from 12-day-old and 1- to 3-month-old F1 hybrid mice and cultured individually for either 2 or 6 days. At the end of the culture period the oocytes were processed for calcium imaging by confocal microscopy. We show that oocytes recovered from cultured preantral follicles exhibit variable calcium spike activity rates, depending on animal age, culture duration and hormonal supplementation. Oocytes recovered from adult animals continue to exhibit calcium spikes, and those recovered from juveniles acquire that activity after culture. Insulin and gonadotropins in combination account for an early and maintained inhibitory effect on calcium signalling acquisition by oocytes. Insulin alone also leads to an early inhibitory effect, which, however, disappears with longer culture periods. Contrary to the complex in vivo situation, the acquisition of calcium signalling by oocytes in a controlled in vitro environment does not seem to be dependent on gonadotropins alone.     

Human CDC25B and CDC25C differ by their ability to restore a functional checkpoint response after gene replacement in fission yeast

In fission yeast, inactivation of the Cdc25 phosphatase by checkpoint kinases participates in the signaling cascade that temporarily stops cell cycle progression after DNA damage. In human, CDC25B and C are also known to be targeted by a similar checkpoint machinery. We have examined by homologous recombination, whether CDC25B and CDC25C were able to substitute for the function of fission yeast Cdc25. We demonstrate that (i) CDC25B and C efficiently replace Cdc25 for vegetative growth, (ii) CDC25C is able to restore a functional checkpoint in response to ionizing radiation in both a Chk1- and Cds1-dependent manner, (iii) CDC25B and C are equally efficient in the response to UV irradiation, CDC25B being only dependent on Chk1, while CDC25C depends on both Chk1 and Cds1, and (iv) CDC25C is able to restore a functional DNA replication checkpoint induced by hydroxyurea in a Cds1-dependent manner. The consequences of these findings on our current view of the checkpoint cascade are discussed.