Intracellular Part Research Articles

To be or not to be alive: How recent discoveries challenge the traditional definitions of viruses and life

Three major discoveries have recently profoundly modified our perception of the viral world: molecular ecologists have shown that viral particles are more abundant than cells in natural environments; structural biologists have shown that some viruses from the three domains of life, Bacteria, Eukarya and Archaea, are evolutionarily related, and microbiologists have discovered giant viruses that rival with cells in terms of size and gene content. I discuss here the scientific and philosophical impact of these discoveries on the debates over the definition, nature (living or not), and origin of viruses. I suggest that viruses have often been considered non-living, because they are traditionally assimilated to their virions. However, the term virus describes a biological process and should integrate all aspects of the viral reproduction cycle. It is especially important to focus on the intracellular part of this cycle, the virocell, when viral information is actively expressed and reproduced, allowing the emergence of new viral genes. The virocell concept theoretically removes roadblocks that prevent defining viruses as living organisms. However, defining a “living organism” remains challenging, as indicated by the case of organelles that evolved from intracellular bacteria. To bypass this problem, I suggest considering that all biological entities that actively participate in the process of life are living.

Multiple Metal Bridges at the Intracellular Gate of a Voltage Activated Potassium Channel Prevent Closing

Voltage-gated potassium (Kv) channels control K+ permeation in excitable cells. Kv channels are tetramers of α subunit, each composed of six transmembrane segments (S1-S6). The S5, P-loop and the S6 segments form the channel's permeation pathway. At the inner end of the S6, an intracellular gate opens or closes in response to voltage. A valine to cysteine mutation at position 476, near the gate, traps the mutant channel in the open state when Cd2+ is added intracellularly. It has been previously shown that four metal bridges are formed between the cysteine at position 476 of one subunit and a native histidine at position 486 in an adjacent subunit. To understand the contribution of individual bridges, we constructed a concatemer Kv channel with all subunits linked at the DNA level. After introducing the mutation V476C, ionic currents were measured using excised inside-out patches to access the intracellular part of the channel. In the absence of Cd2+, V476C concatemer mutants open and close normally. In the presence of Cd2+, successive addition of V476C mutations to the subunits of the concatemer increasingly slowed down the closing of the channel at −120 mV. With all four V476C mutations, channels are locked open with Cd2+.

Aberrant Activation of the PI3K/mTOR Pathway Promotes Resistance to Sorafenib in AML

Therapy directed against oncogenic FLT3 has been shown to induce response in patients with AML, but these responses are almost always transient. To address the mechanism of FLT3 inhibitor resistance, we generated two resistant MV4-11 and MOLM-13 cell lines by sustained treatment with the FLT3 inhibitor sorafenib. MV4-11 cells express only FLT3-ITD, while MOLM-13 cells express wild-type FLT3 and FLT3-ITD. Both cell lines are dependent on FLT3 activation as sorafenib, PKC-412, and AC220, but not imatinib, dasatinib, nilotinib or bosutinib, inhibit cell survival in both cell lines. After treatment with sorafenib for 90 days, we observed that both cell lines displayed resistance to sorafenib as well as to AC220 suggesting that sustained treatment with an FLT3 inhibitor results in acquired resistance to multiple FLT3 inhibitors.To test whether sorafenib was still effective in FLT3 inhibition, we treated sensitive and resistant cells with sorafenib or DMSO and then stimulated with FLT3 ligand (FL). While DMSO-treated resistant cells responded to FL as expected, sorafenib-treated cells displayed poor FLT3 activation, suggesting that sorafenib was still capable of inhibiting FLT3 activation in the resistant cells. Surprisingly we observed that the resistant cells treated with DMSO had a much more robust response to ligand in terms of FLT3 activation. Similar results were observed with AKT and ERK activation, as sorafenib-treated cells poorly responded to FL-stimulation. Furthermore, resistant cells treated with sorafenib could still form colonies similar to DMSO-treated cells indicating that these cells were no longer dependent on FLT3 activation, although sorafenib could partially block FLT3 activation.To determine whether secondary mutations occurred in FLT3, we sequenced the whole coding region using Sanger sequencing. Except for a mutation in the extracellular domain in all four cell lines no mutations were detected in the inhibitor binding site. Similar to Sanger sequencing, mass spectroscopic analysis of affinity-enriched FLT3 indicated no differences in the intracellular part of FLT3 between sensitive and resistant cells. Because we observed an unexpected activation of FLT3 in resistant cells stimulated with FL, we hypothesized that certain FLT3 residues remain hyper-tyrosine phosphorylated. To test that we used phospho-specific antibodies against known FLT3 tyrosine phosphorylation sites. Although we observed an increase in total FLT3 tyrosine phosphorylation, we were unable to identify a single site that was selectively hyper-phosphorylated. Instead, all sites remained slightly more phosphorylated compared to control cells.Gene expression analysis of mRNA from sensitive and resistant cell lines using ANOVA and significance analysis of microarrays (SAM) suggested an enrichment of the PI3K/mTOR pathway in the resistant phenotype. In addition to pathway enrichment, using a phospho-protein antibody array, we found that phosphorylation of the mTOR substrates S6K and AKT were selectively increased in resistant cells. We also observed an increase in STAT3 phosphorylation. Elevated STAT3 phosphorylation was probably due to the previously described upregulation of JAK3 expression in sorafenib-resistant AML. We also observed enrichment of an mTOR signature in AML blasts from eight patients with sorafenib-resistant AML (GSE35907) and in AML blasts from patients expressing FLT3-ITD compared to those lacking FLT3-ITD (525 samples, GSE14468).Furthermore, a selective PI3K/mTOR inhibitor, gedatolisib, efficiently blocked proliferation, colony and tumor formation, and induced apoptosis in the resistant cell lines. Treatment of cells with a higher concentration of gedatolisib did not alter the phosphorylation of other signaling proteins except for AKT and S6K which was detected by a phospho-specific antibody array. The array data was further verified with western blotting using phospho-specific antibodies against AKT, ERK1/2, p38 and S6K. These results suggest that gedatolisib efficiently blocks the downstream effectors PI3K/mTOR without affecting other signaling pathways.Taken together, our data suggest that aberrant activation of the PI3K/mTOR pathway in FLT3-ITD dependent AML results in resistance to drugs targeting FLT3 and treatment with selective PI3K/mTOR inhibitors are likely to be effective in overcoming acquired resistance in AML patients. DisclosuresStegmaier:Novartis Pharmaceuticals: Consultancy.

Identification and characterization of miRNAs in the ovaries of a highly prolific sheep breed.

Until recently, there have been few studies concerning miRNAs or miRNA-mediated biological processes in sheep (Ovis aries). In the present study, we used a deep-sequencing approach to examine ovarian miRNAs and the mRNA transcriptomes in two ewes of a highly prolific breed, Finnsheep. We identified 113 known sheep miRNAs, 131 miRNAs conserved in other mammals and 60 novel miRNAs, the expression levels of which accounted for 78.22%, 21.73% and 0.05% of the total respectively. Furthermore, the 10 most abundantly expressed miRNAs in the two libraries were characterized in detail, and the putative target genes of these miRNAs were annotated using GO annotation and KEGG pathway enrichment analyses. Among the target genes, intracellular transducers (SMAD1, SMAD4, SMAD5 and SMAD9) and bone morphogenetic protein (BMP) receptors (BMPR1B and BMPR2) were involved in the transforming growth factor β (TGFβ) signaling pathway in the reproductive axis, and the most significant GO terms were intracellular part (GO:0044424), binding (GO:0005488) and biological_process (GO:0008150) for cellular component, molecular function and biological process respectively. Thus, these results expanded the sheep miRNA database and provided additional information on the prolificacy trait regulated through specific miRNAs in sheep and other mammals.

Common variable immunodeficiency and natural killer cell lymphopenia caused by Ets-binding site mutation in the IL-2 receptor γ (IL2RG) gene promoter

Common variable immunodeficiency and natural killer cell lymphopenia caused by Ets-binding site mutation in the IL-2 receptor γ (IL2RG) gene promoter

NMR Dynamics of Transmembrane and Intracellular Domains of p75NTR in Lipid-Protein Nanodiscs

P75NTR is a type I integral membrane protein that plays a key role in neurotrophin signaling. However, structural data for the receptor in various functional states are sparse and controversial. In this work, we studied the spatial structure and mobility of the transmembrane and intracellular parts of p75NTR, incorporated into lipid-protein nanodiscs of various sizes and compositions, by solution NMR spectroscopy. Our data reveal a high level of flexibility and disorder in the juxtamembrane chopper domain of p75NTR, which results in the motions of the receptor death domain being uncoupled from the motions of the transmembrane helix. Moreover, none of the intracellular domains of p75NTR demonstrated a propensity to interact with the membrane or to self-associate under the experimental conditions. The obtained data are discussed in the context of the receptor activation mechanism.

Identification of six transcripts encoding putative receptor-like protein kinase (RLK) and their expression profiles in Vitis flexuosa infected with pathogens

Grapevines are exposed to various diseases, resulting in serious problems. However, cultivation of grapevines carrying genes related to disease resistance is a potential strategy for management of diseases during grape production. Receptor-like protein kinase (RLK) is an important gene related to disease resistance in plants. We identified six transcripts of receptor-like protein kinase genes showing different expression patterns, VfRLK586 (Vitis flexuosa Receptor-Like Kinase 586), VfRLK2422, VfRLK5099, VfRLK29473, VfRLK29610, and VfRLK55257 from transcriptome analysis based on the Next Generation Sequencing (NGS) of V. flexuosa inoculated with Elsinoe ampelina. The lengths of these genes were 1531, 2800, 2968, 2446, 3548 and 3468bp, and their encoded proteins composed of 401, 625, 918, 642, 1039 and 1012 amino acids, respectively. The predicted amino acid sequences showed typical protein kinase domain in all six transcripts. Among these, VfRLK2422, VfRLK5099, VfRLK29473, VfRLK29610 and VfRLK55257 were typical receptor-like kinase genes containing both intracellular and extracellular parts, while VfRLK586 was receptor-like cytoplasmic kinase (RLCK) gene. Of the six transcripts, only VfRLK2422 showed upregulated expression against all tested pathogens at all time points. VfRLK5099 showed upregulation against Rhizobium vitis and Colletotrichum gloesporioides and downregulated expression against E. ampelina and Botrytis cinerea at later time points. VfRLK29473 showed downregulated expression in later time points against all pathogens. The expression of VfRLK29610 was upregulated against R. vitis and C. gloeosporioides, but downregulated against B. cinerea. All tested genes showed responses with high and differential expression against pathogens, indicating that all RLK genes may be involved in plant resistance responses against different pathogens in grapevines.

Identification of the Mind Bomb1 Interaction Domain in Zebrafish DeltaD.

Ubiquitylation promotes endocytosis of the Notch ligands like Delta and Serrate and is essential for them to effectively activate Notch in a neighboring cell. The RING E3 ligase Mind bomb1 (Mib1) ubiquitylates DeltaD to facilitate Notch signaling in zebrafish. We have identified a domain in the intracellular part of the zebrafish Notch ligand DeltaD that is essential for effective interactions with Mib1. We show that elimination of the Mind bomb1 Interaction Domain (MID) or mutation of specific conserved motifs in this domain prevents effective Mib1-mediated ubiquitylation and internalization of DeltaD. Lateral inhibition mediated by Notch signaling regulates early neurogenesis in zebrafish. In this context, Notch activation suppresses neurogenesis, while loss of Notch-mediated lateral inhibition results in a neurogenic phenotype, where too many cells are allowed to become neurons. While Mib1-mediated endocytosis of DeltaD is essential for effective activation of Notch in a neighboring cell (in trans) it is not required for DeltaD to inhibit function of Notch receptors in the same cell (in cis). As a result, forms of DeltaD that have the MID can activate Notch in trans and suppress early neurogenesis when mRNA encoding it is ectopically expressed in zebrafish embryos. On the other hand, when the MID is eliminated/mutated in DeltaD, its ability to activate Notch in trans fails but ability to inhibit in cis is retained. As a result, ectopic expression of DeltaD lacking an effective MID results in a failure of Notch-mediated lateral inhibition and a neurogenic phenotype.

Loss-of-Function Variants in a Hungarian Cohort Reveal Structural Insights on TSH Receptor Maturation and Signaling.

Congenital hypothyroidism (CH) is one of the most common inborn endocrine disorders with genetic background. Despite the well-established newborn CH screening program in Hungary, no systematic examination of the underlying genetic alterations has been performed as yet. We aimed to explore TSH receptor (TSHR) mutations in a cohort of Hungarian patients with CH. Eighty-five unrelated patients with permanent primary CH, all diagnosed at newborn screening, were selected. Coding exons of the TSHR gene were sequenced and evaluated together with the thyroid-specific clinical parameters. Functional features of the novel mutations were experimentally examined, and their comparative molecular models were built. In four patients (one heterozygous and three compound heterozygous), seven TSHR mutations were identified. Among these, N432(1.50)D and P449(2.39)L are novel missense alterations. Importantly, the N432(1.50) residue is highly conserved among G protein-coupled receptors, and its function has not been examined yet in human glycoprotein hormone receptors. Our results indicate that the N432(1.50)D mutation disrupts important, architecture-stabilizing intramolecular interactions and ultimately leads to the complete intracellular retention of the receptor. On the other hand, P449(2.39) is located in the intracellular part of the receptor, which is important in G protein coupling. The P449(2.39)L mutation results in signaling impairment, with a more profound effect on the Gq/11 pathway. TSHR mutations are common among Hungarian patients with CH. The novel genetic alterations revealed an important structural role of the N432(1.50) and the P449(2.39) residues in receptor expression and signaling, respectively.

Interaction of the Clostridium difficile Binary Toxin CDT and Its Host Cell Receptor, Lipolysis-stimulated Lipoprotein Receptor (LSR)

CDT (Clostridium difficile transferase) is a binary, actin ADP-ribosylating toxin frequently associated with hypervirulent strains of the human enteric pathogen C. difficile, the most serious cause of antibiotic-associated diarrhea and pseudomembranous colitis. CDT leads to the collapse of the actin cytoskeleton and, eventually, to cell death. Low doses of CDT result in the formation of microtubule-based protrusions on the cell surface that increase the adherence and colonization of C. difficile. The lipolysis-stimulated lipoprotein receptor (LSR) is the host cell receptor for CDT, and our aim was to gain a deeper insight into the interplay between both proteins. We show that CDT interacts with the extracellular, Ig-like domain of LSR with an affinity in the nanomolar range. We identified LSR splice variants in the colon carcinoma cell line HCT116 and disrupted the LSR gene in these cells by applying the CRISPR-Cas9 technology. LSR truncations ectopically expressed in LSR knock-out cells indicated that intracellular parts of LSR are not essential for plasma membrane targeting of the receptor and cellular uptake of CDT. By generating a series of N- and C-terminal truncations of the binding component of CDT (CDTb), we found that amino acids 757-866 of CDTb are sufficient for binding to LSR. With a transposon-based, random mutagenesis approach, we identified potential LSR-interacting epitopes in CDTb. This study increases our understanding about the interaction between CDT and its receptor LSR, which is key to the development of anti-toxin strategies for preventing cell entry of the toxin.

Differentially expressed genes of LPS febrile symptom in rabbits and that treated with Bai-Hu-tang, a classical anti-febrile Chinese herb formula

Ethnopharmacological relevanceBai-Hu-Tang (BHT) has been traditionally used to clear heat and engender fluids. Aim of the studyTo reveal the alteration of differentially expressed genes (DEGs) between lipopolysaccharide (LPS) febrile syndrome in rabbits and treatment with BHT which is a classical anti-febrile formula in traditional Chinese medicine. Materials and methodsFebrile model was induced by LPS injection (i.v.) in rabbits, and BHT was gavaged to another group of febrile rabbits. After sacrifice of animals, total RNA of liver tissue was isolated, processed, and hybridized to rabbit cDNA microarrays obtained from Agilent Co. The data of DEGs were obtained by lazer scanning and analyzed with Cluster program 3.0. Then bioinformatic analysis of DEGs was conducted through gene ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. In addition, expression levels of four relative genes were detected by quantitative real time ployenzyme chain reaction (qRT-PCR) to validate the accuracy of microarrays. ResultThe results demonstrated that genes expression pattern could be clustered into three groups significantly, and there were 606 up-regulated genes and 859 down-regulated genes in the model group, and 106 up-regulated genes and 429 down-regulated genes in BHT treated group. There were 286 DEGs existed as the common in two experimental groups. Enrichment analysis of GO annotations indicated that DEGs in model and BHT treated animals mainly referred catalytic activity and oxidoreductase activity for metabolic processes located in the membrane system at intracellular part, and binding activities increased significantly in treatment with BHT. Enrichment of KEGG analysis showed that the pathways of phagosome and protein processing in endoplasmic reticulum contained the most altered genes in the LPS group, but the percentage of phagosome pathway almost doubled in BHT group. Most DEGs involved in the LPS signal recognition system was up-regulated in LPS group, but partly decreased in BHT group. RT-PCR results of eight relative genes were consistent with the results of microarrays. ConclusionDEGs of LPS febrile syndrome mainly involved oxidoreductase and catalytic activity of the metabolic processes, and pathways of processing protein for pyrotoxin recognition; BHT mostly regulated the DEGs in the phagosome pathway to clear LPS in the liver, and partly interfered with gene expression in LPS recognition system. The study provided an important pioneering result on gene expression profiling research, and will facilitate the clinical care or further studies of the formula.

Conserved movement of TMS11 between occluded conformations of LacY and XylE of the major facilitator superfamily suggests a similar hinge-like mechanism.

The Δ-distance maps can detect local remodeling that is difficult to accurately determine using superimpositions. Transmembrane segments (TMSs) 11 in both LacY and XylE of the major facilitator superfamily uniquely contribute the greatest amount of mobile surface area in the outward-occluded state and undergo analogous movements. The intracellular part of TMS11 moves away from the C-terminal domain and into the substrate cavity during the conformational change from the outward-occluded to the inward-occluded state. A difference was noted between LacY and XylE when they assumed the inward open state after releasing a substrate to the inside in which TMS11 of LacY moved further into the substrate release space, whereas in XylE, TMS11 slightly retracted into the C-terminal domain. Independent movement of the N-terminal half of TMS11 suggests that it is flexible in the middle. Repeat-swapped homology modeling was used to discover that a loop connecting TMSs 10 and 11 in LacY probably moves during the transition between the unavailable outward-open state and the outward-occluded state. TMSs 11 and the other elements displaying a notable domain-independent movement colocalize with the interdomain linker, suggesting that these elements could drive the alternating access movement between the domain halves. Preliminary evidence indicates that analogous movements occur in other members of the major facilitator superfamily.

On the modularity of the intrinsic flexibility of the µ opioid receptor: a computational study.

The µ opioid receptor (µOR), the principal target to control pain, belongs to the G protein-coupled receptors (GPCRs) family, one of the most highlighted protein families due to their importance as therapeutic targets. The conformational flexibility of GPCRs is one of their essential characteristics as they take part in ligand recognition and subsequent activation or inactivation mechanisms. It is assessed that the intrinsic mechanical properties of the µOR, more specifically its particular flexibility behavior, would facilitate the accomplishment of specific biological functions, at least in their first steps, even in the absence of a ligand or any chemical species usually present in its biological environment. The study of the mechanical properties of the µOR would thus bring some indications regarding the highly efficient ability of the µOR to transduce cellular message. We therefore investigate the intrinsic flexibility of the µOR in its apo-form using all-atom Molecular Dynamics simulations at the sub-microsecond time scale. We particularly consider the µOR embedded in a simplified membrane model without specific ions, particular lipids, such as cholesterol moieties, or any other chemical species that could affect the flexibility of the µOR. Our analyses highlighted an important local effect due to the various bendability of the helices resulting in a diversity of shape and volume sizes adopted by the µOR binding site. Such property explains why the µOR can interact with ligands presenting highly diverse structural geometry. By investigating the topology of the µOR binding site, a conformational global effect is depicted: the correlation between the motional modes of the extra- and intracellular parts of µOR on one hand, along with a clear rigidity of the central µOR domain on the other hand. Our results show how the modularity of the µOR flexibility is related to its pre-ability to activate and to present a basal activity.

A dynamic view of molecular switch behavior at serotonin receptors: implications for functional selectivity.

Functional selectivity is a property of G protein-coupled receptors that allows them to preferentially couple to particular signaling partners upon binding of biased agonists. Publication of the X-ray crystal structure of serotonergic 5-HT1B and 5-HT2B receptors in complex with ergotamine, a drug capable of activating G protein coupling and β-arrestin signaling at the 5-HT1B receptor but clearly favoring β-arrestin over G protein coupling at the 5-HT2B subtype, has recently provided structural insight into this phenomenon. In particular, these structures highlight the importance of specific residues, also called micro-switches, for differential receptor activation. In our work, we apply classical molecular dynamics simulations and enhanced sampling approaches to analyze the behavior of these micro-switches and their impact on the stabilization of particular receptor conformational states. Our analysis shows that differences in the conformational freedom of helix 6 between both receptors could explain their different G protein-coupling capacity. In particular, as compared to the 5-HT1B receptor, helix 6 movement in the 5-HT2B receptor can be constrained by two different mechanisms. On the one hand, an anchoring effect of ergotamine, which shows an increased capacity to interact with the extracellular part of helices 5 and 6 and stabilize them, hinders activation of a hydrophobic connector region at the center of the receptor. On the other hand, this connector region in an inactive conformation is further stabilized by unconserved contacts extending to the intracellular part of the 5-HT2B receptor, which hamper opening of the G protein binding site. This work highlights the importance of considering receptor capacity to adopt different conformational states from a dynamic perspective in order to underpin the structural basis of functional selectivity.

Non-targeted metabolomic reveals the effect of salt stress on global metabolite of halotolerant yeast Candida versatilis and principal component analysis.

As one of the major microbes in the soy sauce fermentation, Candida versatilis enriches the flavor and improves the quality of soy sauce. In this study, a combination of five different GC-MS and LC-MS-based metabolome analytical approaches was used to analyze the intracellular, extracellular and whole metabolites of C. versatilis. Our results found out that a total of 132, 244 and 267 different metabolites were detectable from the intracellular, extracellular and whole part, respectively. When exposed to 0. 9 and 18% salt, respectively, 114, 123 and 129 different intracellular metabolites, 184, 200 and 178 extracellular metabolites and 177, 188 and 186 whole metabolites were detected, respectively. Our data showed that salt enhances the metabolic capacity of C. versatilis, especially its amino acid and enhances the synthesis and secretion of some metabolites of C. versatilis, especially the aldehydes and phenols, such as vanillin, guaiacol and 5-hydroxymethylfurfural. Our data also showed that special attention has to be paid to the generation of biogenic amines when C. versatilis was treated with salt.

Targeted disruption of the intracellular domain of receptor FgfrL1 in mice.

FgfrL1 is the fifth member of the fibroblast growth factor receptor (Fgfr) family. Studies with FgfrL1 deficient mice have demonstrated that the gene plays an important role during embryonic development. FgfrL1 knock-out mice die at birth as they have a malformed diaphragm and lack metanephric kidneys. Similar to the classical Fgfrs, the FgfrL1 protein contains an extracellular part composed of three Ig-like domains that interact with Fgf ligands and heparin. However, the intracellular part of FgfrL1 is not related to the classical receptors and does not possess any tyrosine kinase activity. Curiously enough, the amino acid sequence of this domain is barely conserved among different species, with the exception of three motifs, namely a dileucine peptide, a tandem tyrosine-based motif YXXΦ and a histidine-rich sequence. To investigate the function of the intracellular domain of FgfrL1, we have prepared genetically modified mice that lack the three conserved sequence motifs, but instead contain a GFP cassette (FgfrL1ΔC-GFP). To our surprise, homozygous FgfrL1ΔC-GFP knock-in mice are viable, fertile and phenotypically normal. They do not exhibit any alterations in the diaphragm or the kidney, except for a slight reduction in the number of glomeruli that does not appear to affect life expectancy. In addition, the pancreas of both FgfrL1ΔC-GFP knock-in and FgfrL1 knock-out mice do not show any disturbances in the production of insulin, in contrast to what has been suggested by recent studies. Thus, the conserved motifs of the intracellular FgfrL1 domain are dispensable for organogenesis and normal life. We conclude that the extracellular domain of the protein must conduct the vital functions of FgfrL1.

Functional and structural characterization of the kinase insert and the carboxy terminal domain in VEGF receptor 2 activation.

Vascular endothelial growth factors (VEGFs) regulate blood and lymphatic vessel development and homeostasis. VEGF receptor 2 (VEGFR-2) is the major receptor involved in vasculogenesis and angiogenesis and regulates endothelial cell survival, migration, and mitogenesis. Ligand-mediated receptor dimerization instigates transmembrane signaling, thereby promoting activation of the intracellular kinase domain. The intracellular part of the receptor comprises the juxtamembrane domain, the catalytic kinase domain, the kinase insert domain (KID), and the carboxy terminal domain (CD). Here we show that the CD inhibits VEGFR-2 activity in the absence of ligand, whereas the KID, particularly a tyrosine residue in this domain (Y951), is indispensable for downstream signaling by the activated kinase. Because of the lack of crystallographic data for the complete kinase domain, we applied size-exclusion chromatography, multiangle laser scattering, analytical ultracentrifugation, and small-angle X-ray scattering to build and functionally validate structural models. Our data show substantial conformational changes of the kinase when it is switched from the inactive, unphosphorylated state to the active, phosphorylated state. Finally, we structurally characterized recombinantly produced protein complexes between VEGFR-2 and T cell-specific adapter protein, a molecule involved in downstream signaling by VEGFR-2.

Protein profiling in the gut of Penaeus monodon gavaged with oral WSSV-vaccines and live white spot syndrome virus.

White spot syndrome virus (WSSV) is a pathogen that causes considerable mortality of the farmed shrimp, Penaeus monodon. Candidate 'vaccines', WSSV envelope protein VP28 and formalin-inactivated WSSV, can provide short-lived protection against the virus. In this study, P. monodon was orally intubated with the aforementioned vaccine candidates, and protein expression in the gut of immunised shrimps was profiled. The alterations in protein profiles in shrimps infected orally with live-WSSV were also examined. Seventeen of the identified proteins in the vaccine and WSSV-intubated shrimps varied significantly compared to those in the control shrimps. These proteins, classified under exoskeletal, cytoskeletal, immune-related, intracellular organelle part, intracellular calcium-binding or energy metabolism, are thought to directly or indirectly affect shrimp's immunity. The changes in the expression levels of crustacyanin, serine proteases, myosin light chain, and ER protein 57 observed in orally vaccinated shrimp may probably be linked to immunoprotective responses. On the other hand, altered expression of proteins linked to exoskeleton, calcium regulation and energy metabolism in WSSV-intubated shrimps is likely to symbolise disturbances in calcium homeostasis and energy metabolism.

Is "capacitive coupling" purely excitatory in the cardiac tissue?

Cardiac muscle is a syncytial tissue of electrically coupled cells in which excitation of one cardiomyocyte spreads to all cardiomyocytes. Mechanisms of cell coupling in myocardial tissue can be categorized in two main groups: mechanisms that connect the intracellular parts of myocytes together (i.e., gap junctions), and mechanisms that act through changes in the small extracellular space of intercalated disks (ICD). The latter mechanisms are electric field coupling, ephaptic coupling, K accumulation, and capacitive coupling (Sperelakis and McConnell, 2002). ICD cleft has a limited space and therefore a small ionic current can change both its electric and diffusion potentials via the change in the ionic concentration. The potassium accumulation, for instance, can change the reversal potential of potassium channels toward more positive values, and as a results of which the potassium current can depolarize the cells. When ionic currents change the electric potential of cleft we have ephaptic or electrical coupling, and when the capacitive current of membrane changes the electric potential we have capacitive coupling. In electrical coupling through gap junctions, both experimental and modeling studies show that the reduction of propagation velocity is proportional to the reduction of gap junction density or conductance. In the case of coupling through ICD field potential, since the experimental data are based on the effect of these mechanisms on conduction velocity (not a direct measurement of the field potential), there are significant discrepancies between different experiments, and between experiments and simulation studies (Rhett et al., 2013). One explanation of these differences is to consider a mixture of excitatory and inhibitory effects for each of these mechanisms and suppose that each experiment grasp one feature of these mechanisms based on the experimental condition. Such a mixed behavior was proposed for the ephaptic coupling in a previous study, where the voltage gated sodium channels of the ICD negatively impacted the conduction velocity unless more than 10% of the gap junctions remained active. Further reduction of gap junction density was reversed their impact (Kucera et al., 2002). In the case of capacitive coupling, also, we suggest that a mixture of excitatory and inhibitory effects take place; so that depending on the compartment on which the membrane potential is measured, different effects may be observed. In capacitive coupling, we have two capacitors that share a voltage node, but each end of these capacitors are connected to different intracellular voltage nodes that provide the main polarity of the capacitors. Since these capacitors have a different polarity (Figure ​(Figure1A),1A), discharge (depolarization) of one of them by action potential generation would charge (hyperpolarize) the other one. Still, these capacitors in series can pass the current, which would increase the potential in the next intracellular voltage node. The hyperpolarization of ICD membrane was reported in a previous modeling study, but due to the complex nature of their model it was impossible to dissociate the electrical field coupling from the capacitive coupling (Ge and Sperelakis, 1992). Figure 1 Capacitive coupling simulation. (A) The equivalent circuit of intercalated disk. (B) Cell arrangement in the final model. Colors show the membrane potential in all cells. (C) Membrane potential and extracellular voltage in different locations of the intercalated ... We have simulated ICD in the NEURON simulation environment (Hines and Carnevale, 1997). In our model, an equivalent circuit of the ICD connects 10 myocytes together in the ICD (Figure ​(Figure1B).1B). Our model could potentially simulate ion channels in the cleft and gap junction. Capacitive coupling is the property of membranes lacking ion channels. Therefore, in the Figure ​Figure1C,1C, we show results of this model where there is no gap junction and ion channels in the ICD. The results of this model clearly show that the capacitive coupling hyperpolarizes the neighboring membrane in the ICD and depolarizes the adjacent myocyte membrane to some extent. Overall, based on the abovementioned simulation we hypothesize that the capacitive coupling has mixed excitatory and inhibitory effects. Therefore, we suggest that action potential propagation models should be revised based on this finding.

Human β-Cell Proliferation and Intracellular Signaling Part 2: Still Driving in the Dark Without a Road Map

Enhancing β-cell proliferation is a major goal for type 1 and type 2 diabetes research. Unraveling the network of β-cell intracellular signaling pathways that promote β-cell replication can provide the tools to address this important task. In a previous Perspectives in Diabetes article, we discussed what was known regarding several important intracellular signaling pathways in rodent β-cells, including the insulin receptor substrate/phosphatidylinositol-3 kinase/Akt (IRS-PI3K-Akt) pathways, glycogen synthase kinase-3 (GSK3) and mammalian target of rapamycin (mTOR) S6 kinase pathways, protein kinase Cζ (PKCζ) pathways, and their downstream cell-cycle molecular targets, and contrasted that ample knowledge to the small amount of complementary data on human β-cell intracellular signaling pathways. In this Perspectives, we summarize additional important information on signaling pathways activated by nutrients, such as glucose; growth factors, such as epidermal growth factor, platelet-derived growth factor, and Wnt; and hormones, such as leptin, estrogen, and progesterone, that are linked to rodent and human β-cell proliferation. With these two Perspectives, we attempt to construct a brief summary of knowledge for β-cell researchers on mitogenic signaling pathways and to emphasize how little is known regarding intracellular events linked to human β-cell replication. This is a critical aspect in the long-term goal of expanding human β-cells for the prevention and/or cure of type 1 and type 2 diabetes.