Calcium-dependent regulation of protein kinase D revealed by a genetically encoded kinase activity reporter

Protein kinase D (PKD) regulates many diverse cellular functions in response to diacylglycerol. To monitor PKD signaling in live cells, we generated a genetically encoded fluorescent reporter for PKD activity, DKAR (D kinase activity reporter). DKAR expressed in mammalian cells undergoes reversible fluorescence resonance energy transfer changes upon activation and inhibition of endogenous PKD. Surprisingly, we find that agonist-evoked activation of PKD is driven not only by diacylglycerol production, but by Ca(2+). Furthermore, elevation of intracellular Ca(2+), in the absence of any other stimulus, is sufficient to activate PKD. Concurrent imaging of Ca(2+), diacylglycerol, and PKD activity reveals that thapsigargin-mediated elevation of intracellular Ca(2+) is closely followed by a robust increase in diacylglycerol production, in turn followed by PKD activation. The Ca(2+)-induced production of diacylglycerol and accompanying PKD activation is dependent on phospholipase C activity. These data reveal that Ca(2+) is a major contributor to the initiation of PKD signaling through positive feedback regulation of diacylglycerol production, unveiling a new mechanism in PKD activation.     

Molecular analysis of the sea anemone toxin Av3 reveals selectivity to insects and demonstrates the heterogeneity of receptor site-3 on voltage-gated Na+ channels

Most known organisms encode proteases that are crucial for constitutive proteolytic events. In the present paper, we describe a method to define these events in proteomes from Escherichia coli to humans. The method takes advantage of specific N-terminal biotinylation of protein samples, followed by affinity enrichment and conventional LC (liquid chromatography)-MS/MS (tandem mass spectrometry) analysis. The method is simple, uses conventional and easily obtainable reagents, and is applicable to most proteomics facilities. As proof of principle, we demonstrate profiles of proteolytic events that reveal exquisite in vivo specificity of methionine aminopeptidase in E. coli and unexpected processing of mitochondrial transit peptides in yeast, mouse and human samples. Taken together, our results demonstrate how to rapidly distinguish real proteolysis that occurs in vivo from the predictions based on in vitro experiments.     

Identification of arylsulfonamides as Aquaporin 4 inhibitors

Carbonic anhydrase inhibitors AZA, EZA, and 4-acetamidobenzsulfonamide were found to inhibit human AQP4-M23 mediated water transport by 80%, 68%, and 23%, respectively, at 20 microM in an in vitro functional assay. AZA was found to have an IC50 against AQP4 of 0.9 microM. Phloretin was inactive under the same conditions.     

Synthesis and characterization of styrylchromone derivatives as beta-amyloid imaging agents

Several promising agents have been synthesized and evaluated for in vivo imaging probes of beta-amyloid plaques in Alzheimer's disease (AD) brain. Recently, we have developed flavone derivatives, which possess the basic structure of the 2-phenylchromone, as useful candidates for amyloid imaging agents. In an attempt to further develop novel tracers, we synthesized and evaluated a series of 2-styrylchromone derivatives, which replace the 2-phenyl substituent of flavone backbone with the 2-styryl. A series of radioiodinated styrylchromone derivatives were designed and synthesized. The binding affinities for amyloid plaques were assessed by in vitro binding assay using pre-formed synthetic Abeta(1-40) aggregates. The new series of styrylchromone derivatives showed high binding affinity to Abeta aggregates at the K(d) values of 32.0, 17.5 and 8.7nM for [(125)I]6, [(125)I]9, and [(125)I]12, respectively. In biodistribution studies using normal mice, [(125)I]6 and [(125)I]9 examined in normal mice displayed high brain uptakes with 4.9 and 2.8%ID/g at 2min post injection. The radioactivity washed out from the brain rapidly (1.6 and 1.0%ID/g at 60min post injection for [(125)I]6 and [(125)I]9, respectively). But [(125)I]12 did not show marked brain uptake, and the washout rate from the brain was relatively slow throughout the time course (1.1 and 1.4%ID/g at 2 and 30min post injection, respectively). Although additional modifications are necessary to improve the brain uptake and rapid clearance of non-specifically bound radiotracer, the styrylchromone backbone may be useful as a backbone structure to develop novel beta-amyloid imaging agents.     

TNF-alpha modulates hepatic Na+-K+ ATPase activity via PGE2 and EP2 receptors

The effect of TNF-alpha on liver Na(+)-K(+) ATPase was studied in Sprague-Dawley rats and in HepG2 cells. TNF-alpha was injected intraperitoneally to rats and 4h later the liver was isolated and the activity and protein expression of hepatic Na(+)-K(+) ATPase studied. The cytokine caused a significant down-regulation of the ATPase and a decrease in its activity. This effect disappeared in presence of indomethacin, an inhibitor of COX enzymes, and PGE2 injected to the animals imitated the effect of TNF-alpha. The observed in vivo effects of TNF and PGE2 on the pump appeared again when HepG2 cells were treated with the cytokine or the prostaglandin. The application of different agonist and antagonist to EP receptors showed that the effect of PGE2 is mediated via EP2 receptors. It was concluded that TNF-alpha induces in hepatocytes, PGE2 production which in turn reduces the activity and protein expression of the Na(+)-K(+) ATPase by activating EP2 receptors.     

Genome-Wide Screens in Saccharomyces cerevisiae Highlight a Role for Cardiolipin in Biogenesis of Mitochondrial Outer Membrane Multispan Proteins

A special group of mitochondrial outer membrane (MOM) proteins spans the membrane several times via multiple helical segments. Such multispan proteins are synthesized on cytosolic ribosomes before their targeting to mitochondria and insertion into the MOM. Previous work recognized the import receptor Tom70 and the mitochondrial import (MIM) complex, both residents of the MOM, as required for optimal biogenesis of these proteins. However, their involvement is not sufficient to explain either the entire import pathway or its regulation. To identify additional factors that are involved in the biogenesis of MOM multispan proteins, we performed complementary high-throughput visual and growth screens in Saccharomyces cerevisiae. Cardiolipin (CL) synthase (Crd1) appeared as a candidate in both screens. Our results indeed demonstrate lower steady-state levels of the multispan proteins Ugo1, Scm4, and Om14 in mitochondria from crd1Δ cells. Importantly, MOM single-span proteins were not affected by this mutation. Furthermore, organelles lacking Crd1 had a lower in vitro capacity to import newly synthesized Ugo1 and Scm4 molecules. Crd1, which is located in the mitochondrial inner membrane, condenses phosphatidylglycerol together with CDP-diacylglycerol to obtain de novo synthesized CL molecules. Hence, our findings suggest that CL is an important component in the biogenesis of MOM multispan proteins.     

Probing a label-free local bend in DNA by single molecule tethered particle motion

Being capable of characterizing DNA local bending is essential to understand thoroughly many biological processes because they involve a local bending of the double helix axis, either intrinsic to the sequence or induced by the binding of proteins. Developing a method to measure DNA bend angles that does not perturb the conformation of the DNA itself or the DNA-protein complex is a challenging task. Here, we propose a joint theory-experiment high-throughput approach to rigorously measure such bend angles using the Tethered Particle Motion (TPM) technique. By carefully modeling the TPM geometry, we propose a simple formula based on a kinked Worm-Like Chain model to extract the bend angle from TPM measurements. Using constructs made of 575 base-pair DNAs with in-phase assemblies of one to seven 6A-tracts, we find that the sequence CA₆CGG induces a bend angle of 19° ± 4°. Our method is successfully compared to more theoretically complex or experimentally invasive ones such as cyclization, NMR, FRET or AFM. We further apply our procedure to TPM measurements from the literature and demonstrate that the angles of bends induced by proteins, such as Integration Host Factor (IHF) can be reliably evaluated as well.     

DAP5 associates with eIF2β and eIF4AI to promote Internal Ribosome Entry Site driven translation

Initiation is a highly regulated rate-limiting step of mRNA translation. During cap-dependent translation, the cap-binding protein eIF4E recruits the mRNA to the ribosome. Specific elements in the 5′UTR of some mRNAs referred to as Internal Ribosome Entry Sites (IRESes) allow direct association of the mRNA with the ribosome without the requirement for eIF4E. Cap-independent initiation permits translation of a subset of cellular and viral mRNAs under conditions wherein cap-dependent translation is inhibited, such as stress, mitosis and viral infection. DAP5 is an eIF4G homolog that has been proposed to regulate both cap-dependent and cap-independent translation. Herein, we demonstrate that DAP5 associates with eIF2β and eIF4AI to stimulate IRES-dependent translation of cellular mRNAs. In contrast, DAP5 is dispensable for cap-dependent translation. These findings provide the first mechanistic insights into the function of DAP5 as a selective regulator of cap-independent translation.     

Cellular defibrillation: interaction of micro-scale electric fields with voltage-gated ion channels

We study the effect of micro-scale electric fields on voltage-gated ion channels in mammalian cell membranes. Such micro- and nano-scale electric fields mimic the effects of multiferroic nanoparticles that were recently proposed [1] as a novel way of controlling the function of voltage-sensing biomolecules such as ion channels. This article describes experimental procedures and initial results that reveal the effect of the electric field, in close proximity of cells, on the ion transport through voltage-gated ion channels. We present two configurations of the whole-cell patch-clamping apparatus that were used to detect the effect of external stimulation on ionic currents and discuss preliminary results that indicate modulation of the ionic currents consistent with the applied stimulus.     

CLAUSA restricts tomato leaf morphogenesis and GOBLET expression

Leaf morphogenesis and differentiation are highly flexible processes. The development of compound leaves is characterized by an extended morphogenesis stage compared with that of simple leaves. The tomato mutant clausa (clau) possesses extremely elaborate compound leaves. Here we show that this elaboration is generated by further extension of the morphogenetic window, partly via the activity of ectopic meristems present on clau leaves. Further, we propose that CLAU might negatively affect expression of the NAM/CUC gene GOBLET (GOB), an important modulator of compound‐leaf development, as GOB expression is elevated in clau mutants and reducing GOB expression suppresses the clau phenotype. Expression of GOB is also elevated in the compound leaf mutant lyrate (lyr), and the remarkable enhancement of the clau phenotype by lyr suggests that clau and lyr affect leaf development and GOB in different pathways.