The Experts below are selected from a list of 294 Experts worldwide ranked by ideXlab platform
Jasper Rine - One of the best experts on this subject based on the ideXlab platform.
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Ras2 and Ras1 Protein phosphorylation in saccharomyces cerevisiae
Journal of Biological Chemistry, 1997Co-Authors: Jennifer L Whistler, Jasper RineAbstract:Abstract This work describes the phosphorylation ofSaccharomyces cerevisiae Ras Proteins and explores the physiological role of the phosphorylation of Ras2 Protein. Proteins expressed from activated alleles of Ras were less stable and less phosphorylated than Proteins from cells expressing wild-type alleles of Ras. This difference in phosphorylation level did not result from increased signaling through the Ras-cAMP pathway or reflect the primarily GTP-bound nature of activated forms of Ras Protein per se. In addition, phosphorylation of Ras Protein was not dependent on proper localization of the Ras2 Protein to the plasma membrane nor on the interaction of Ras2p with its exchange factor, Cdc25p. The preferred phosphorylation site on Ras2 Protein was identified as serine 214. This site, when mutated to alanine, led to promiscuous phosphorylation of Ras2 Protein on nearby serine residues. A decrease in phosphorylation may lead to a decrease in signaling through the Ras-cAMP pathway.
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Prenylation of mammalian Ras Protein in Xenopus oocytes.
Molecular and Cellular Biology, 1990Co-Authors: Jasper RineAbstract:Abstract Ras Protein requires an intermediate of the cholesterol biosynthetic pathway for posttranslational modification and membrane anchorage. This step is necessary for biological activity. Maturation of Xenopus laevis oocytes induced by an oncogenic human Ras Protein can be inhibited by lovastatin or compactin, inhibitors of the synthesis of mevalonate, an intermediate of cholesterol biosynthesis. This inhibition can be overcome by mevalonic acid or farnesyl diphosphate, a cholesterol biosynthetic intermediate downstream of mevalonate, but not by squalene, an intermediate after farnesyl pyrophosphate in the pathway. This study supports the idea that in Xenopus oocytes, the Ras Protein is modified by a farnesyl moiety or its derivative. Furthermore, an octapeptide with the sequence similar to the C-terminus of the c-H-Ras Protein inhibits the biological activity of Ras Proteins in vivo, suggesting that it competes for the enzyme or enzymes responsible for transferring the isoprenoid moiety (prenylation) in the oocytes. This inhibition of Ras prenylation by the peptide was also observed in vitro, using both Saccharomyces cerevisiae and Xenopus oocyte extracts. These observations show that Xenopus oocytes provide a convenient in vivo system for studies of inhibitors of the posttranslational modification of the Ras Protein, especially for inhibitors such as peptides that do not penetrate cell membranes.
Daniel Huster - One of the best experts on this subject based on the ideXlab platform.
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interaction of the human n Ras Protein with lipid raft model membranes of varying degrees of complexity
Biological Chemistry, 2014Co-Authors: Alexander Vogel, Herbert Waldmann, Daniel Huster, Gemma Triola, Roland Winter, Jorg Nikolaus, Katrin Weise, Andreas HerrmannAbstract:Ternary lipid mixtures composed of cholesterol, saturated (frequently with sphingosine backbone), and unsaturated phospholipids show stable phase separa- tion and are often used as model systems of lipid rafts. Yet, their ability to reproduce raft properties and function is still debated. We investigated the properties and func- tional aspects of three lipid raft model systems of vary- ing degrees of biological relevance - PSM/POPC/Chol, DPPC/POPC/Chol, and DPPC/DOPC/Chol - using 2 H solid- state nuclear magnetic resonance (NMR) spectroscopy, fluorescence microscopy, and atomic force microscopy. While some minor differences were observed, the gen- eral behavior and properties of all three model mixtures were similar to previously investigated influenza envelope lipid membranes, which closely mimic the lipid composi- tion of biological membranes. For the investigation of the functional aspects, we employed the human N-Ras pro- tein, which is posttranslationally modified by two lipid modifications that anchor the Protein to the membrane. It was previously shown that N-Ras preferentially resides in liquid-disordered domains and exhibits a time-depend- ent accumulation in the domain boundaries of influenza envelope lipid membranes. For all three model mixtures, we observed the same membrane partitioning behavior for N-Ras. Therefore, we conclude that even relatively sim- ple models of raft membranes are able to reproduce many of their specific properties and functions.
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Membrane binding of a lipidated N-Ras Protein studied in lipid monolayers.
European biophysics journal : EBJ, 2006Co-Authors: Frank Bringezu, Guido Reuther, Jürgen Kuhlmann, Herbert Waldmann, Monika Majerowicz, Daniel HusterAbstract:The adsorption of doubly lipidated full-length N-Ras Protein on 1,2-dipalmitoyl-sn-phosphatidylcholine (DPPC) monolayers was studied by lateral pressure analysis, grazing incidence X-ray diffraction (GIXD), and specular reflectivity (XR). N-Ras Protein adsorbs to the DPPC monolayer (lateral pressure of 20 mN/m) from the subphase thereby increasing the lateral pressure in the monolayer by 4 mN/m. The Protein insertion does not alter the tilt angle and structure of the lipid molecules at the air/water interface but influences the electron density profile of the monolayer. Further, electron density differences into the subphase were observed. The Fresnel normalized reflectivity could be reconstructed in the analysis using box models yielding electron density profiles of the DPPC monolayer in the absence and in the presence of N-Ras Protein. The electron density profiles of the DPPC monolayer in the presence of Ras showed clear intensity variations in the headgroup/glycerol/upper chain region, the so-called interface region where previous bilayer studies had confirmed Ras binding.
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Structural model of the membrane-bound C terminus of lipid-modified human N-Ras Protein
Angewandte Chemie - International Edition, 2006Co-Authors: Guido Reuther, Kui Thong Tan, Julia Köhler, André Pampel, Jürgen Kuhlmann, Christine Nowak, Herbert Waldmann, Klaus Arnold, Daniel HusterAbstract:(Figure Presented) Solid-state NMR spectroscopy was used to determine a structural model of the backbone of the lipid anchor of membrane-bound N-Ras Protein. The fully functional lipid-modified Protein was obtained by ligating the expressed water-soluble N terminus with a chemically synthesized 13 C-labeled lipidated peptide. After the NMR signals had been assigned by correlation experiments, a structural model was calcul ated from torsion angles derived from 1 H and 13 C chemical-shift data. © 2006 Wiley-VCH Verlag GmbH & Co. KGaA.
Shigeyuki Yokoyama - One of the best experts on this subject based on the ideXlab platform.
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Improving cell-free Protein synthesis for stable-isotope labeling
Journal of Biomolecular NMR, 2007Co-Authors: Takayoshi Matsuda, Seizo Koshiba, Eiko Seki, Noriyuki Iwasaki, Takashi Yabuki, Naoya Tochio, Makoto Inoue, Shigeyuki Yokoyama, Takanori KigawaAbstract:Cell-free Protein synthesis is suitable for stable-isotope labeling of Proteins for NMR analysis. The Escherichia coli cell-free system containing potassium acetate for efficient translation (KOAc system) is usually used for stable-isotope labeling, although it is less productive than other systems. A system containing a high concentration of potassium l-glutamate (l-Glu system), instead of potassium acetate, is highly productive, but cannot be used for stable-isotope labeling of Glu residues. In this study, we have developed a new cell-free system that uses potassium d-glutamate (d-Glu system). The productivity of the d-Glu system is approximately twice that of the KOAc system. The cross peak intensities in the 1H–15N HSQC spectrum of the uniformly stable-isotope labeled Ras Protein, prepared with the d-Glu system, were similar to those obtained with the KOAc system, except that the Asp intensities were much higher for the Protein produced with the d-Glu system. These results indicate that the d-Glu system is a highly productive cell-free system that is especially useful for stable-isotope labeling of Proteins.
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cell free synthesis and amino acid selective stable isotope labeling of Proteins for nmr analysis
Journal of Biomolecular NMR, 1995Co-Authors: Takanori Kigawa, Y. Muto, Shigeyuki YokoyamaAbstract:For the application of multidimensional NMR spectroscopy to larger Proteins, it would be useful to perform selective labeling of one of the 20 amino acids. For some amino acids, however, amino acid metabolism dRastically reduces the efficiency and selectivity of labeling in in vivo expression systems. In the present study, a cell-free Protein synthesis system was optimized, so that highly efficient and selective stable isotope labeling of Proteins can be achieved in the absence of amino acid metabolism. The productivity of the E. coli cell-free coupled transcription-translation system was first improved, by about fivefold, by using the T7 RNA polymeRase for transcription and also by improving the translation conditions. Thus, about 0.1 mg Protein per 1 ml reaction mixture was synthesized. Then, this improved cell-free system was used for Asp- or Ser-selective 15N-labeling of the human c-Ha-Ras Protein. With a 15 ml cell-free reaction, using less than 1 mg of 15N-labeled amino acid, 1 mg of the Ras Protein was obtained. 1H-15N HSQC experiments confirmed that the Ras Protein was efficiently labeled with high selectivity. These results indicate that this cell-free Protein synthesis system is useful for NMR studies.
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A glutamic acid residue at position 31 of Ras Protein is essential to the signal transduction for neurite outgrowth of PC12 cells and the stimulation of GTPase activity by GAPRas.
Oncogene, 1992Co-Authors: Mikako Shirouzu, Junko Fujita-yoshigaki, Hiroshi Koide, Susumu Nishimura, Shigeyuki YokoyamaAbstract:: The roles of residues at positions 23-31 adjacent to the 'effector region' and residues at positions 61-65 in a phosphoryl binding loop of the human c-Ha-Ras Protein were studied by changing each residue of the normal (Gly-12 type) and oncogenic (Val-12 type) Ras Proteins to the corresponding residue of the K-rev-1 Protein. Firstly, the signal-transducing activities of the mutant Ras Proteins of Val-12 type were examined by analysis of their ability to induce neurite outgrowth of phaeochromocytoma (PC12) cells upon expression of the mutant Ras gene. Thus, replacement of Glu-31 by Lys was found to impair the signal-transducing activity of the oncogenic Ras Protein. Furthermore, it was shown that expression of the Gly-12----Val/Glu-31----Lys mutant Ras Protein in PC12 cells suppresses neurite outgrowth induced either by microinjection of the oncogenic Ras Protein or by addition of nerve growth factor to the medium. As for the Glu-31----Lys mutant Ras Protein (Gly-12 type), the GTPase activity in the presence of GTPase-activating Protein for Ras (GAPRas) is much lower than that of the normal Ras Protein, whereas the intrinsic GTPase activity is nearly the same as that of the normal Ras Protein. Therefore, Gly-31 is one of the determinants for the signal transduction and the correct interaction with GAPRas. On the other hand, the GTPase activity of the Gln-61----Thr mutant Ras Protein (Gly-12 type) is negligibly low both in the absence and in the presence of GAPRas.
Alfonso Valencia - One of the best experts on this subject based on the ideXlab platform.
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the Ras Protein superfamily evolutionary tree and role of conserved amino acids
Journal of Cell Biology, 2012Co-Authors: Ana M Rojas, Gloria Fuentes, Antonio Rausell, Alfonso ValenciaAbstract:The Ras superfamily is a fascinating example of functional diversification in the context of a preserved structural framework and a prototypic GTP binding site. Thanks to the availability of complete genome sequences of species representing important evolutionary branch points, we have analyzed the composition and organization of this superfamily at a greater level than was previously possible. Phylogenetic analysis of gene families at the organism and sequence level revealed complex relationships between the evolution of this Protein superfamily sequence and the acquisition of distinct cellular functions. Together with advances in computational methods and structural studies, the sequence information has helped to identify features important for the recognition of molecular partners and the functional specialization of different members of the Ras superfamily.
Takanori Kigawa - One of the best experts on this subject based on the ideXlab platform.
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Improving cell-free Protein synthesis for stable-isotope labeling
Journal of Biomolecular NMR, 2007Co-Authors: Takayoshi Matsuda, Seizo Koshiba, Eiko Seki, Noriyuki Iwasaki, Takashi Yabuki, Naoya Tochio, Makoto Inoue, Shigeyuki Yokoyama, Takanori KigawaAbstract:Cell-free Protein synthesis is suitable for stable-isotope labeling of Proteins for NMR analysis. The Escherichia coli cell-free system containing potassium acetate for efficient translation (KOAc system) is usually used for stable-isotope labeling, although it is less productive than other systems. A system containing a high concentration of potassium l-glutamate (l-Glu system), instead of potassium acetate, is highly productive, but cannot be used for stable-isotope labeling of Glu residues. In this study, we have developed a new cell-free system that uses potassium d-glutamate (d-Glu system). The productivity of the d-Glu system is approximately twice that of the KOAc system. The cross peak intensities in the 1H–15N HSQC spectrum of the uniformly stable-isotope labeled Ras Protein, prepared with the d-Glu system, were similar to those obtained with the KOAc system, except that the Asp intensities were much higher for the Protein produced with the d-Glu system. These results indicate that the d-Glu system is a highly productive cell-free system that is especially useful for stable-isotope labeling of Proteins.
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cell free synthesis and amino acid selective stable isotope labeling of Proteins for nmr analysis
Journal of Biomolecular NMR, 1995Co-Authors: Takanori Kigawa, Y. Muto, Shigeyuki YokoyamaAbstract:For the application of multidimensional NMR spectroscopy to larger Proteins, it would be useful to perform selective labeling of one of the 20 amino acids. For some amino acids, however, amino acid metabolism dRastically reduces the efficiency and selectivity of labeling in in vivo expression systems. In the present study, a cell-free Protein synthesis system was optimized, so that highly efficient and selective stable isotope labeling of Proteins can be achieved in the absence of amino acid metabolism. The productivity of the E. coli cell-free coupled transcription-translation system was first improved, by about fivefold, by using the T7 RNA polymeRase for transcription and also by improving the translation conditions. Thus, about 0.1 mg Protein per 1 ml reaction mixture was synthesized. Then, this improved cell-free system was used for Asp- or Ser-selective 15N-labeling of the human c-Ha-Ras Protein. With a 15 ml cell-free reaction, using less than 1 mg of 15N-labeled amino acid, 1 mg of the Ras Protein was obtained. 1H-15N HSQC experiments confirmed that the Ras Protein was efficiently labeled with high selectivity. These results indicate that this cell-free Protein synthesis system is useful for NMR studies.
