Exploring the binding dynamics of BAR proteins

We used a continuum model based on the Helfrich free energy to investigate the binding dynamics of a lipid bilayer to a BAR domain surface of a crescent-like shape of positive (e.g. I-BAR shape) or negative (e.g. F-BAR shape) intrinsic curvature. According to structural data, it has been suggested that negatively charged membrane lipids are bound to positively charged amino acids at the binding interface of BAR proteins, contributing a negative binding energy to the system free energy. In addition, the cone-like shape of negatively charged lipids on the inner side of a cell membrane might contribute a positive intrinsic curvature, facilitating the initial bending towards the crescent-like shape of the BAR domain. In the present study, we hypothesize that in the limit of a rigid BAR domain shape, the negative binding energy and the coupling between the intrinsic curvature of negatively charged lipids and the membrane curvature drive the bending of the membrane. To estimate the binding energy, the electric potential at the charged surface of a BAR domain was calculated using the Langevin-Bikerman equation. Results of numerical simulations reveal that the binding energy is important for the initial instability (i.e. bending of a membrane), while the coupling between the intrinsic shapes of lipids and membrane curvature could be crucial for the curvature-dependent aggregation of negatively charged lipids near the surface of the BAR domain. In the discussion, we suggest novel experiments using patch clamp techniques to analyze the binding dynamics of BAR proteins, as well as the possible role of BAR proteins in the fusion pore stability of exovesicles.     

The neurotoxic phospholipase A2 associates,through a non-phosphorylated binding motif,with 14-3-3 protein gamma and epsilon isoforms

Two novel acceptors for ammodytoxin C, a presynaptically neurotoxic phospholipase A(2) from snake venom, have been purified from porcine cerebral cortex by a toxin-affinity-based procedure. Using tandem mass spectrometry, the isolated acceptors were identified as 14-3-3 gamma and epsilon isoforms, highly conserved cytoplasmic proteins involved in the regulation of numerous physiological processes. The interaction between ammodytoxin C and 14-3-3 proteins is direct and not mediated by calmodulin, a high-affinity acceptor for both ammodytoxin C and 14-3-3 proteins, as demonstrated in pull-down experiments and by surface plasmon resonance. The latter technique gave an apparent dissociation constant of 1.0+/-0.2 microM for the interaction between chip-immobilized 14-3-3 and ammodytoxin C. 14-3-3 usually interacts with proteins through specific phospho-Ser/Thr motifs. Ammodytoxin C is not a phospho-protein, therefore the interaction must occur through a non-phosphorylated binding site, most probably the KEESEK sequence at its C-terminal end. The interaction we describe suggests an explanation for the pathophysiological effects evoked by some secreted phospholipases A(2), such as the inhibition of protein phosphorylation, of terminal ion currents, and of neurotransmission, as well as the initiation of neuronal cell death, all processes regulated by 14-3-3 proteins.     

Seroprevalence of Toxocara antibodies among patients suspected of ocular toxocariasis in Slovenia

Ocular toxocariasis named also ocular larva migrans is caused by larvae of the roundworm Toxocara spp. The purpose of this study was to find out the seroprevalence of Toxocara antibodies in patients suspected of ocular toxocariasis. Between January 2001 and December 2003, sera from 239 ocular patients, aged 3 to 80 years, were examined by ELISA and confirmed by Western blot test. Out of the 239 patients, 172 (72%) were seronegative and 67 (28%) were Toxocara seropositive; 95% CI (22-34%). The median age of Toxocara seropositive patients was 37.6 years. There was no significant difference in the number of Toxocara positive sera between the younger age group (< or = 14 years) and the older age group (> 14 years), p > 0.05. A high rate of Toxocara seropositivity in ocular patients should alert the ophthalmologists in Slovenia to include toxocariasis in the differential diagnosis of eye diseases more frequently.     

Ammodytoxin, a neurotoxic secreted phospholipase A(2), can act in the cytosol of the nerve cell

Recent identification of intracellular proteins that bind ammodytoxin (calmodulin, 14-3-3 proteins, and R25) suggests that this snake venom presynaptically active phospholipase A(2) acts intracellularly. As these ammodytoxin acceptors are cytosolic and mitochondrial proteins, the toxin should be able to enter the cytosol of a target cell and remain stable there to interact with them. Using laser scanning confocal microscopy we show here that Alexa-labelled ammodytoxin entered the cytoplasm of the rat hippocampal neuron and subsequently also its nucleus. The transport of proteins into the nucleus proceeds via the cytosol of a cell, therefore, ammodytoxin passed the cytosol of the neuron on its way to the nucleus. Although it is not yet clear how ammodytoxin is translocated into the cytosol of the neuron, our results demonstrate that its stability in the cytosol is not in question, providing the evidence that the toxin can act in this cellular compartment.     

Protein disulphide isomerase binds ammodytoxin strongly: possible implications for toxin trafficking

Ammodytoxin, a group IIA secreted phospholipase A(2) from the venom of the long-nosed viper (Vipera ammodytes ammodytes), is a potent presynaptically acting neurotoxin. It blocks the secretion of neurotransmitter from the nerve cell, thus hindering the communication with the neighbouring neuron or muscle cell. To express the neurotoxicity, ammodytoxin should interact with specific receptors in the axon terminal and express phospholipase activity. Our previous results indicate that, following the association with a receptor on the external side of the presynaptic membrane, the toxin penetrates into the cytosol of the nerve cell. Here, we show that the toxin associates specifically with protein disulphide isomerase, a protein in the lumen of endoplasmic reticulum, which may be crucial for the retention and concentration of the toxin in this cellular compartment and for its subsequent transport across the membrane of endoplasmic reticulum into the cytosol of the nerve cell.     

Common molecular pathways mediate long-term potentiation of synaptic excitation and slow synaptic inhibition

Synaptic plasticity, the cellular correlate for learning and memory, involves signaling cascades in the dendritic spine. Extensive studies have shown that long-term potentiation (LTP) of the excitatory postsynaptic current (EPSC) through glutamate receptors is induced by activation of N-methyl-D-asparate receptor (NMDA-R)--the coincidence detector--and Ca(2+)/calmodulin-dependent protein kinase II (CaMKII). Here we report that the same signaling pathway in the postsynaptic CA1 pyramidal neuron also causes LTP of the slow inhibitory postsynaptic current (sIPSC) mediated by metabotropic GABA(B) receptors (GABA(B)-Rs) and G protein-activated inwardly rectifying K(+) (GIRK) channels, both residing in dendritic spines as well as shafts. Indicative of intriguing differences in the regulatory mechanisms for excitatory and inhibitory synaptic plasticity, LTP of sIPSC but not EPSC was abolished in mice lacking Nova-2, a neuronal-specific RNA binding protein that is an autoimmune target in paraneoplastic opsoclonus myoclonus ataxia (POMA) patients with latent cancer, reduced inhibitory control of movements, and dementia.     

Complete mitochondrial genome of the Verticillium-wilt causing plant pathogen Verticillium nonalfalfae

Verticillium nonalfalfae is a fungal plant pathogen that causes wilt disease by colonizing the vascular tissues of host plants. The disease induced by hop isolates of V. nonalfalfae manifests in two different forms, ranging from mild symptoms to complete plant dieback, caused by mild and lethal pathotypes, respectively. Pathogenicity variations between the causal strains have been attributed to differences in genomic sequences and perhaps also to differences in their mitochondrial genomes. We used data from our recent Illumina NGS-based project of genome sequencing V. nonalfalfae to study the mitochondrial genomes of its different strains. The aim of the research was to prepare a V. nonalfalfae reference mitochondrial genome and to determine its phylogenetic placement in the fungal kingdom. The resulting 26,139 bp circular DNA molecule contains a full complement of the 14 "standard" fungal mitochondrial protein-coding genes of the electron transport chain and ATP synthase subunits, together with a small rRNA subunit, a large rRNA subunit, which contains ribosomal protein S3 encoded within a type IA-intron and 26 tRNAs. Phylogenetic analysis of this mitochondrial genome placed it in the Verticillium spp. lineage in the Glomerellales group, which is also supported by previous phylogenetic studies based on nuclear markers. The clustering with the closely related Verticillium dahliae mitochondrial genome showed a very conserved synteny and a high sequence similarity. Two distinguishing mitochondrial genome features were also found—a potential long non-coding RNA (orf414) contained only in the Verticillium spp. of the fungal kingdom, and a specific fragment length polymorphism observed only in V. dahliae and V. nubilum of all the Verticillium spp., thus showing potential as a species specific biomarker.     

How fast monoamine oxidases decompose adrenaline? Kinetics of isoenzymes A and B evaluated by empirical valence bond simulation

This work scrutinizes kinetics of decomposition of adrenaline catalyzed by monoamine oxidase (MAO) A and B enzymes, a process controlling the levels of adrenaline in the central nervous system and other tissues. Experimental kinetic data for MAO A and B catalyzed decomposition of adrenaline are reported only in the form of the maximum reaction rate. Therefore, we estimated the experimental free energy barriers form the kinetic data of closely related systems using regression method, as was done in our previous study. By using multiscale simulation on the Empirical Valence Bond (EVB) level, we studied the chemical reactivity of the MAO A catalyzed decomposition of adrenaline and we obtained a value of activation free energy of 17.3 ± 0.4 kcal/mol. The corresponding value for MAO B is 15.7 ± 0.7 kcal/mol. Both values are in good agreement with the estimated experimental barriers of 16.6 and 16.0 kcal/mol for MAO A and MAO B, respectively. The fact that we reproduced the kinetic data and preferential catalytic effect of MAO B over MAO A gives additional support to the validity of the proposed hydride transfer mechanism. Furthermore, we demonstrate that adrenaline is preferably involved in the reaction in a neutral rather than in a protonated form due to considerably higher barriers computed for the protonated adrenaline substrate. The results are discussed in the context of chemical mechanism of MAO enzymes and possible applications of multiscale simulation to rationalize the effects of MAO activity on adrenaline level.     

Broad taxonomic characterization of Verticillium wilt resistance genes reveals an ancient origin of the tomato Ve1 immune receptor

Plant‐pathogenic microbes secrete effector molecules to establish themselves on their hosts, whereas plants use immune receptors to try and intercept such effectors in order to prevent pathogen colonization. The tomato cell surface‐localized receptor Ve1 confers race‐specific resistance against race 1 strains of the soil‐borne vascular wilt fungus Verticillium dahliae which secrete the Ave1 effector. Here, we describe the cloning and characterization of Ve1 homologues from tobacco (Nicotiana glutinosa), potato (Solanum tuberosum), wild eggplant (Solanum torvum) and hop (Humulus lupulus), and demonstrate that particular Ve1 homologues govern resistance against V. dahliae race 1 strains through the recognition of the Ave1 effector. Phylogenetic analysis shows that Ve1 homologues are widely distributed in land plants. Thus, our study suggests an ancient origin of the Ve1 immune receptor in the plant kingdom.     

Impaired αGDI Function in the X-Linked Intellectual Disability: The Impact on Astroglia Vesicle Dynamics

X-linked non-syndromic intellectual disability (XLID) is a common mental disorder recognized by cognitive and behavioral deficits. Mutations in the brain-specific αGDI, shown to alter a subset of RAB GTPases redistribution in cells, are linked to XLID, likely via changes in vesicle traffic in neurons. Here, we show directly that isolated XLID mice astrocytes, devoid of pathologic tissue environment, exhibit vesicle mobility deficits. Contrary to previous studies, we show that astrocytes express two GDI proteins. The siRNA-mediated suppression of expression of αGDI especially affected vesicle dynamics. A similar defect was recorded in astrocytes from the Gdi1 ^-/Y mouse model of XLID and in astrocytes with recombinant mutated human XLID αGDI. Endolysosomal vesicles studied here are involved in the release of gliosignaling molecules as well as in regulating membrane receptor density; thus, the observed changes in astrocytic vesicle mobility may, over the long time-course, profoundly affect signaling capacity of these cells, which optimize neural activity.