Identification of endosomal epidermal growth factor receptor signaling targets by functional organelle proteomics

Epidermal growth factor (EGF) receptor (EGFR) signal transduction is organized by scaffold and adaptor proteins, which have specific subcellular distribution. On a way from the plasma membrane to the lysosome EGFRs are still in their active state and can signal from distinct subcellular locations. To identify organelle-specific targets of EGF receptor signaling on endosomes a combination of subcellular fractionation, two-dimensional DIGE, fluorescence labeling of phosphoproteins, and MALDI-TOF/TOF mass spectrometry was applied. All together 23 EGF-regulated (phospho)proteins were identified as being differentially associated with endosomal fractions by functional organelle proteomics; among them were proteins known to be involved in endosomal trafficking and cytoskeleton rearrangement (Alix, myosin-9, myosin regulatory light chain, Trap1, moesin, cytokeratin 8, septins 2 and 11, and CapZbeta). Interestingly R-Ras, a small GTPase of the Ras family that regulates cell survival and integrin activity, was associated with endosomes in a ligand-dependent manner. EGF-dependent association of R-Ras with late endosomes was confirmed by confocal laser scanning immunofluorescence microscopy and Western blotting of endosomal fractions. EGFR tyrosine kinase inhibitor gefitinib was used to confirm EGF-dependent regulation of all identified proteins. EGF-dependent association of signaling molecules, such as R-Ras, with late endosomes suggests signaling specification through intracellular organelles.     

Characteristics of rat ceruloplasmin from the serum of animals, which received salts of silver with food

Abiogenic Ag(I) ions have electronic structure, similar to Cu(I) ions and can compete with Cu(I) for binding sites of proteins which transport copper from extracellular media to sites of cuproenzyme formation in the cell. Rodents receiving Ag-salts with food develop extracellular deficiency of copper associated with ceruloplasmin (Cp, the major copper-transporting protein in blood serum of vertebrates). The present work focuses on the studies of biochemical and physicochemical properties of Cp, obtained from blood serum of rats, which received AgCl with food for 4 weeks (Ag-rats). Cp-fractions from blood serum of Ag-rats (Ag-Cp) were obtained by ion-exchange chromatography with stepped gradient of NaCl. Each fraction was tested for oxidase and ferroxidase activities by direct measurement of catalytic activity in the gel, and for specific activity in holo-Cp in oxidation of chromogenic substrate. Molecular mass, electrophoretic mobility and ratio of apo- and holo-forms in Ag-Cp fractions were evaluated by immunoblotting. Ag-Cp samples did not contain products of spontaneous partial proteolytic degradation, characteristic of holo-Cp samples. Fractions of Ag-Cp and holo-Cp (from blood serum of control rats) were compared by optical spectra, tertiary structure, susceptibility to thermal denaturation, and by atomic Cu and Ag content. Ag-Cp contained 1-2% Cp, which is similar by spectral and catalytic properties with holo-Cp. [Ag]:[Cu] ration in Ag-Cp samples was about 4:1. As evidenced by circular dichroism and differential scanning calorimetric studies, the major apo-fraction of Ag-Cp lacked tertiary structure of native Cp and was significantly misfolded, which might explain its resistance to spontaneous partial proteolytic degradation.     

The CONJUDOR pipeline for multiplexed knockdown of gene pairs identifies RBBP-5 as a germ cell reprogramming barrier in C. elegans

Multiple gene activities control complex biological processes such as cell fate specification during development and cellular reprogramming. Investigating the manifold gene functions in biological systems requires also simultaneous depletion of two or more gene activities. RNA interference-mediated knockdown (RNAi) is commonly used in Caenorhabditis elegans to assess essential genes, which otherwise lead to lethality or developmental arrest upon full knockout. RNAi application is straightforward by feeding worms with RNAi plasmid-containing bacteria. However, the general approach of mixing bacterial RNAi clones to deplete two genes simultaneously often yields poor results. To address this issue, we developed a bacterial conjugation-mediated double RNAi technique ‘CONJUDOR’. It allows combining RNAi bacteria for robust double RNAi with high-throughput. To demonstrate the power of CONJUDOR for large scale double RNAi screens we conjugated RNAi against the histone chaperone gene lin-53 with more than 700 other chromatin factor genes. Thereby, we identified the Set1/MLL methyltransferase complex member RBBP-5 as a novel germ cell reprogramming barrier. Our findings demonstrate that CONJUDOR increases efficiency and versatility of RNAi screens to examine interconnected biological processes in C. elegans with high-throughput.     

The impact of ozone fumigation and fertilization on chlorophyll fluorescence of birch leaves (Betula pendula)

 The impact of ozone fumigation on chlorophyll a fluorescence parameters and chlorophyll content of birch trees grown at high and low fertilization were studied for 6-, 8-, and 12-week old leaves. Fluorescence parameters were measured with a portable fluorometer with its fibre optics tightly inserted in a gas exchange cuvette at light intensities from 0 to 220 μmol photons m⁻² s⁻¹. Ozone caused significant changes of primary photosynthetic reactions: a decrease of the quantum yield of photosystem II and an increase of non-photochemical quenching. In all leaves a biphasic light response of non-photochemical quenching was observed. Ozone fumigation shifted the onset of the second phase from a PFD of about 60 μmol m⁻² s⁻¹ to about 30 μmol m⁻² s⁻¹. While the fertilizer concentration had no influence on this character, high fertilization supply of plants partially reduced O₃-induced damage. The light responses of Ft, Fm′ and NPQ observed in birch leaves grown in O₃-free air indicate the existence of at least two different processes governing energy conversion of the photosynthetic apparatus at PS II in the range of PFD 0–200 μmol photons m⁻² s⁻¹. The first phase was attributed to a rather slowly relaxing type of non-photochemical quenching, which, at least at low PFD, is thought to be related to a state 1–2 transition. The further changes of the fluorescence parameters studied at higher PFD might be explained by an increase of energy-dependent quenching, connected with the energization of the thylakoid membrane and zeaxanthin synthesis. A major effect of ozone treatment was a lowering of PS II quantum yield. This reflects a reduction of PS II electron transport and corresponds to the reduction of CO₂-fixation observed in ozonated leaves. Received: 24 September 1996 / Accepted: 27 January 1999     

Virioimmunoassay for studying the interaction of diphtheria toxin with cell membranes]

A model for studying the interaction of diphtheria toxin with cell membranes includes immobilization of purified cell membranes on Sephadex G-25, adsorbtion of toxin on the membranes in the presence of protective colloid, and subsequent detection of adsorbed toxin by means of virioimmunoassay. Diphtheria toxin adsorbed rapidly on membranes of both sensitive (HeLa, macrophages) and resistent to tis action cells, but not on stromaof human erythrocytes. The rate of interaction depends on the concentration of toxin and the temperature of incubation. Adsorbed toxin may be eluted by acidic buffer, 8 M area and 4 M guanidin. HCl, but not by triton X-100, tween 20 and 80, sodium dodecylaulfate and basic buffer.     

The sensitivity of G protein-activated K+ channels toward halothane is essentially determined by the C terminus

G protein-activated K(+) channels (GIRKs or Kir3.x) are targets for the volatile anesthetic, halothane. When coexpressed with the m(2) acetylcholine (ACh) receptor in Xenopus oocytes, agonist-activated GIRK1(F137S)- and GIRK2-mediated currents are inhibited by halothane, whereas in the absence of ACh, high concentrations of halothane induce GIRK1(F137S)-mediated currents. To elucidate the molecular mechanism of halothane action on GIRK currents of different subunit compositions, we constructed deletion mutants of GIRK1(F137S) (GIRK1(Delta363*)) and GIRK2 (GIRK2(Delta356)) lacking the C-terminal ends, as well as chimeric GIRK channels. Mutated GIRK channels showed normal currents when activated by ACh but exhibited different pharmacological properties toward halothane. GIRK2(Delta356) showed no sensitivity against the inhibitory action of halothane but was activated by halothane in the absence of an agonist. GIRK1(Delta363*) was activated by halothane more efficiently. Currents mediated by chimeric channels were inhibited by anesthetic concentrations that were at least 30-fold lower than those necessary to decrease GIRK2 wild type currents. Glutathione S-transferase pulldown experiments did not show displacement of bound Gbetagamma by halothane, indicating that halothane does not interfere with Gbetagamma binding. Single channel experiments revealed an influence of halothane on the gating of the channels: The agonist-induced currents of GIRK1 and GIRK2, carried mainly by brief openings, were inhibited, whereas higher concentrations of the anesthetic promoted long openings of GIRK1 channels. Because the C terminus is crucial for these effects, an interaction of halothane with the channel seems to be involved in the mechanism of current modulation.