Identification of the prokaryotic ligand-gated ion channels and their implications for the mechanisms and origins of animal Cys-loop ion channels

Background  

Acetylcholine receptor type ligand-gated ion channels (ART-LGIC; also known as Cys-loop receptors) are a superfamily of proteins that include the receptors for major neurotransmitters such as acetylcholine, serotonin, glycine, GABA, glutamate and histamine, and for Zn²⁺ ions. They play a central role in fast synaptic signaling in animal nervous systems and so far have not been found outside of the Metazoa.     

Metallic Zinc Reduction of Disulfide Bonds between Cysteine Residues in Peptides and Proteins

The use of powdered metallic zinc in acidic solution for the reduction of disulfide bonds in peptides and proteins has been investigated. The method has several advantages over the traditional mercapto based reducing methods currently used; the reducing agent is readily available and inexpensive; reduction can be performed in weakly acidic solutions of water and/or acetonitrile; work up simply consists of a centrifugation step followed by pipeting the supernatant from the metal pellet, thereby greatly diminishing the risk of reoxidation as a more elaborate work up procedure could result in. As no mercapto compounds are added, there is no risk that the reducing agent will interfere in subsequent modification of the thiol functionality. Disulfides in a model peptide are reduced within 5 min in any mixture of water/acetonitrile containing 1% TFA, all disulfides in insulin is reduced within 1 h in any mixture of water/acetonitrile containing 5% acetic acid. To stress the convenience of the metallic zinc reduction method, the resulting thiol compound was subjected to two commonly employed reactions in peptide chemistry: Cys(Npys) directed disulfide formation (70% yield) and native chemical ligation between the reduced model peptide and Boc-Ala-p-metylthiobenzyl ester (65% yield of the ligation product plus disulfide formation between Cys and p-thiocresol).     

Expression, purification, and characterization of Bacillus subtilis cytochromes P450 CYP102A2 and CYP102A3: flavocytochrome homologues of P450 BM3 from Bacillus megaterium

The cyp102A2 and cyp102A3 genes encoding the two Bacillus subtilis homologues (CYP102A2 and CYP102A3) of flavocytochrome P450 BM3 (CYP102A1) from Bacillus megaterium have been cloned, expressed in Escherichia coli, purified, and characterized spectroscopically and enzymologically. Both enzymes contain heme, flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) cofactors and bind a variety of fatty acid molecules, as demonstrated by conversion of the low-spin resting form of the heme iron to the high-spin form induced by substrate-binding. CYP102A2 and CYP102A3 catalyze the fatty acid-dependent oxidation of reduced nicotinamide adenine dinucleotide phosphate (NADPH) and reduction of artificial electron acceptors at high rates. Binding of carbon monoxide to the reduced forms of both enzymes results in the shift of the heme Soret band to 450 nm, confirming the P450 nature of the enzymes. Reverse-phase high-performance liquid chromatography (HPLC) of products from the reaction of the enzymes with myristic acid demonstrates that both catalyze the subterminal hydroxylation of this substrate, though with different regioselectivity and catalytic rate. Both P450s 102A2 and 102A3 show kinetic and binding preferences for long-chain unsaturated and branched-chain fatty acids over saturated fatty acids, indicating that the former two molecule types may be the true substrates. P450s 102A2 and 102A3 exhibit differing substrate selectivity profiles from each other and from P450 BM3, indicating that they may fulfill subtly different cellular roles. Titration curves for binding and turnover kinetics of several fatty acid substrates with P450s 102A2 and 102A3 are better described by sigmoidal (rather than hyperbolic) functions, suggesting binding of more than one molecule of substrate to the P450s, or possibly cooperativity in substrate binding. Comparison of the amino acid sequences of the three flavocytochromes shows that several important amino acids in P450 BM3 are not conserved in the B. subtilis homologues, pointing to differences in the binding modes for the substrates that may explain the unusual sigmoidal kinetic and titration properties.     

Membrane binding and translocation of cell-penetrating peptides

Cell-penetrating peptides (CPPs) have been extensively studied during the past decade, because of their ability to promote the cellular uptake of various cargo molecules, e.g., oligonucleotides and proteins. In a recent study of the uptake of several analogues of penetratin, Tat(48-60) and oligoarginine in live (unfixed) cells [Thorén et al. (2003) Biochem. Biophys. Res. Commun. 307, 100-107], it was found that both endocytotic and nonendocytotic uptake pathways are involved in the internalization of these CPPs. In the present study, the membrane interactions of some of these novel peptides, all containing a tryptophan residue to facilitate spectroscopic studies, are investigated. The peptides exhibit a strong affinity for large unilamellar vesicles (LUVs) containing zwitterionic and anionic lipids, with binding constants decreasing in the order penetratin > R(7)W > TatP59W > TatLysP59W. Quenching studies using the aqueous quencher acrylamide and brominated lipids indicate that the tryptophan residues of the peptides are buried to a similar extent into the membrane, with an average insertion depth of approximately 10-11 A from the bilayer center. The membrane topology of the peptides was investigated using an assay based on resonance energy transfer between tryptophan and a fluorescently labeled lysophospholipid, lysoMC, distributed asymmetrically in the membranes of LUVs. By determination of the energy transfer efficiency when peptide was added to vesicles with lysoMC present exclusively in the inner leaflet, it was shown that none of the peptides investigated is able to translocate across the lipid membranes of LUVs. By contrast, confocal laser scanning microscopy studies on carboxyfluorescein-labeled peptides showed that all of the peptides rapidly traverse the membranes of giant unilamellar vesicles (GUVs). The choice of model system is thus crucial for the conclusions about the ability of CPPs to translocate across lipid membranes. Under the conditions used in the present study, peptide-lipid interactions alone cannot explain the different cellular uptake characteristics exhibited by these peptides.     

Statistically integrated metabonomic-proteomic studies on a human prostate cancer xenograft model in mice

A novel statistically integrated proteometabonomic method has been developed and applied to a human tumor xenograft mouse model of prostate cancer. Parallel 2D-DIGE proteomic and 1H NMR metabolic profile data were collected on blood plasma from mice implanted with a prostate cancer (PC-3) xenograft and from matched control animals. To interpret the xenograft-induced differences in plasma profiles, multivariate statistical algorithms including orthogonal projection to latent structure (OPLS) were applied to generate models characterizing the disease profile. Two approaches to integrating metabonomic data matrices are presented based on OPLS algorithms to provide a framework for generating models relating to the specific and common sources of variation in the metabolite concentrations and protein abundances that can be directly related to the disease model. Multiple correlations between metabolites and proteins were found, including associations between serotransferrin precursor and both tyrosine and 3-D-hydroxybutyrate. Additionally, a correlation between decreased concentration of tyrosine and increased presence of gelsolin was also observed. This approach can provide enhanced recovery of combination candidate biomarkers across multi-omic platforms, thus, enhancing understanding of in vivo model systems studied by multiple omic technologies.     

A theoretical study on nitric oxide reductase activity in a ba(3)-type heme-copper oxidase

The mechanism of nitric oxide reduction in a ba(3)-type heme-copper oxidase has been investigated using density functional theory (B3LYP). Four possible mechanisms have been studied and free energy surfaces for the whole catalytic cycle including proton and electron transfers have been constructed by comparison to experimental data. The first nitric oxide coordinates to heme a(3) and is partly reduced having some nitroxyl anion character ((3)NO(-)), and it is thus activated toward the attack by the second N-O. In this reaction step a cyclic hyponitrous acid anhydride intermediate with the two oxygens coordinating to Cu(B) is formed. The cyclic hyponitrous acid anhydride is quite stable in a local minimum with high barriers for both the backward and forward reactions and should thus be observable experimentally. To break the N-O bond and form nitrous oxide, the hyponitrous acid anhydride must be protonated, the latter appearing to be an endergonic process. The endergonicity of the proton transfer makes the barrier of breaking the N-O bond directly after the protonation too high. It is suggested that an electron should enter the catalytic cycle at this stage in order to break the N-O bond and form N(2)O at a feasible rate. The cleavage of the N-O bond is the rate limiting step in the reaction mechanism and it has a barrier of 17.3 kcal/mol, close to the experimental value of 19.5 kcal/mol. The overall exergonicity is fitted to experimental data and is 45.6 kcal/mol.     

Calling is an honest indicator of paternal genetic quality in poison frogs

Several competing hypotheses have been put forward to explain why females of many species mate preferentially with males possessing the most conspicuous signals (e.g., ornaments, displays, or songs). We performed a laboratory experiment using two species of poison frogs, Dendrobates leucomelas and Epipedobates tricolor, to test the hypothesis that male calling performance is an honest indicator of parental quality. Our analyses are based on data from behavioral observations of mating activities of captive-reared individuals (and their offspring) that were housed in terraria for four consecutive breeding seasons. Male mating success increased with male calling rate and chirp duration in both species, suggesting that females preferred males with more elaborate calls. Because calling performance improved with age in D. leucomelas, female poison frogs that prefer males with more elaborate calls in the wild may end up mating with older males that have already proven their ability to survive. Females that mated with good callers obtained higher quality offspring. Eggs fertilized by males with high calling rates and long chirp durations had higher hatching success and produced tadpoles that were more likely to metamorphose into surviving frogs. As a consequence, females that mated with males with high calling performance obtained more surviving offspring per egg, compared to females that mated with poor callers. Collectively, our findings comply with the notion that female poison frogs prefer to mate with good callers because calling performance is a reliable predictor of offspring quality. The possible influence of maternal allocation and reasons for the strong effect size compared to previous studies are discussed.     

The yeast tumor suppressor homologue Sro7p is required for targeting of the sodium pumping ATPase to the cell surface

The SRO7/SOP1 encoded tumor suppressor homologue of Saccharomyces cerevisiae is required for maintenance of ion homeostasis in cells exposed to NaCl stress. Here we show that the NaCl sensitivity of the sro7Delta mutant is due to defective sorting of Ena1p, the main sodium pump in yeast. On exposure of sro7Delta mutants to NaCl stress, Ena1p fails to be targeted to the cell surface, but is instead routed to the vacuole for degradation via the multivesicular endosome pathway. SRO7-deficient mutants accumulate post-Golgi vesicles at high salinity, in agreement with a previously described role for Sro7p in late exocytosis. However, Ena1p is not sorted into these post-Golgi vesicles, in contrast to what is observed for the vesicles that accumulate when exocytosis is blocked in sec6-4 mutants at high salinity. These observations imply that Sro7p has a previously unrecognized role for sorting of specific proteins into the exocytic pathway. Screening for multicopy suppressors identified RSN1, encoding a transmembrane protein of unknown function. Overexpression of RSN1 restores NaCl tolerance of sro7Delta mutants by retargeting Ena1p to the plasma membrane. We propose a model in which blocked exocytic sorting in sro7Delta mutants, gives rise to quality control-mediated routing of Ena1p to the vacuole.     

Induction of dendritic cell migration upon Toxoplasma gondii infection potentiates parasite dissemination

The processes leading to systemic dissemination of the obligate intracellular parasite Toxoplasma gondii remain unelucidated. In vitro studies on human and murine dendritic cells (DC) revealed that active invasion of DC by Toxoplasma induces a state of hypermotility in DC, enabling transmigration of infected DC across endothelial cell monolayers in the absence of chemotactic stimuli. Infected DC exhibited upregulation of maturation markers and co-stimulatory molecules. While modulation of cell adhesion molecules CD11/CD18 was similar for Toxoplasma-infected DC and lipopolysaccharide (LPS)-matured DC, Toxoplasma-infected DC did not exhibit upregulation of CD54/ICAM-1. Induction of host cell migration in vitro required live intracellular parasite(s) and was inhibited by uncoupling the Gi-protein signalling pathway with pertussis toxin, but did not depend on CCR5, CCR7 or Toll/interleukin-1 receptor signalling. When migration of Toxoplasma-infected DC was compared with migration of LPS-stimulated DC in vivo, similar or higher numbers of Toxoplasma-infected DC reached the mesenteric lymph nodes and spleen respectively. Adoptive transfer of Toxoplasma-infected DC resulted in more rapid dissemination of parasites to distant organs and in exacerbation of infection compared with inoculation with free parasites. Altogether, these findings show that Toxoplasma is able to subvert the regulation of host cell motility and likely exploits the host's natural pathways of cellular migration for parasite dissemination.     

Resequencing data provide no evidence for a human bottleneck in Africa during the penultimate glacial period

Based on the accumulation of genetic, climatic, and fossil evidence, a central theory in paleoanthropology stipulates that a demographic bottleneck coincided with the origin of our species Homo Sapiens. This theory proposes that anatomically modern humans--which were only present in Africa at the time--experienced a drastic bottleneck during the penultimate glacial age (130-190 kya) when a cold and dry climate prevailed. Two scenarios have been proposed to describe the bottleneck, which involve either a fragmentation of the range occupied by humans or the survival of one small group of humans. Here, we analyze DNA sequence data from 61 nuclear loci sequenced in three African populations using Approximate Bayesian Computation and numerical simulations. In contrast to the bottleneck theory, we show that a simple model without any bottleneck during the penultimate ice age has the greatest statistical support compared with bottleneck models. Although the proposed bottleneck is ancient, occurring at least 130 kya, we can discard the possibility that it did not leave detectable footprints in the DNA sequence data except if the bottleneck involves a less than a 3-fold reduction in population size. Finally, we confirm that a simple model without a bottleneck is able to reproduce the main features of the observed patterns of genetic variation. We conclude that models of Pleistocene refugium for modern human origins now require substantial revision.