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Characterization of the cold stress-induced cyanobacterial DEAD-box protein CrhC as an RNA helicase

NUCLEIC ACIDS RESEARCH 2000年第20期28卷 3926-3934页

作者： Yu, E Owttrim, GW Univ Alberta Dept Biol Sci Edmonton AB T6G 2E9 Canada

We have shown previously that CrhC is a unique member of the DEAD-box family of RNA helicases whose expression occurs specifically under conditions of cold stress. Here we show that recombinant His-tagged CrhC, purified from Escherichia coli, is an ATP-independent RNA binding protein possessing RNA-dependent ATPase activity which is stimulated most efficiently by rRNA and polysome preparations. RNA strand displacement assays indicate that CrhC possesses RNA unwinding activity that is adenosine nucleotide specific. Unwinding of partially duplexed RNA proceeds in the 5'-->3' but not the 3'-->5' direction using standard assay conditions. Immunoprecipitation and far-western analysis indicate that CrhC is a component of a multisubunit complex, interacting specifically with a 37 kDa polypeptide. We propose that CrhC unwinds cold-stabilized secondary structure in the 5'-UTR of RNA during cold stress.

关键词：腺苷三磷酸酶类/化学腺苷三磷酸酶类/遗传学腺苷三磷酸酶类/分离和提纯腺苷三磷酸酶类/代谢腺苷三磷酸/代谢氨基酸特征结构鱼腥藻属/酶学催化作用阳离子/代谢色谱法亲和辅酶类/代谢冷温度大分子物质分子量肽类/化学肽类/代谢沉淀素试验蛋白质结合 RNA/遗传学 RNA/代谢 RNA解螺旋酶类/化学 RNA解螺旋酶类/遗传学 RNA解螺旋酶类/分离和提纯 RNA解螺旋酶类/代谢 RNA 双链/遗传学 RNA 双链/代谢 RNA结合蛋白质类/化学 RNA结合蛋白质类/遗传学 RNA结合蛋白质类/分离和提纯 RNA结合蛋白质类/代谢重组融合蛋白质类/化学重组融合蛋白质类/遗传学重组融合蛋白质类/分离和提纯重组融合蛋白质类/代谢

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Crystallization and preliminary X-ray analysis of the catalytic domain of the adenylate cyclase GRESAG4.1 from Trypanosoma brucei

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ACTA CRYSTALLOGRAPHICA SECTION D-STRUCTURAL BIOLOGY 2000年第Pt 3期56卷 359-362页

作者： Bieger, B Essen, LO Max Planck Inst Biochem Dept Membrane Biochem D-82152 Martinsried Germany

Adenylate cyclases (ACs) are involved in signal transduction by generating the second messenger, cAMP. In Trypanosoma brucei, 3',5'-cyclic adenosine monophosphate (cAMP) controls the life cycle of this unicellular parasite. cAMP is generated by a class of adenylate cyclases which are either constitutively (GRESAG4.1-4.3) or transiently expressed (ESAG4) during the life cycle. Unlike mammalian ACs, the trypanosomal ACs have a simple topology comprising of a large extracellular region, a transmembrane helix and a cytosolic catalytic region. Two orthorhombic crystal forms of the catalytic AC domain of GRESAG4.1 (residues Ala884-Thr1132) were generated by the hanging-drop vapour-diffusion method. X-ray diffraction data from GRESAG4.1 crystals were collected at 1.9 Angstrom resolution using synchrotron radiation. Furthermore, two heavy-metal derivative data sets were collected from crystal form A;heavy-atom sites were subsequently located in difference Patterson maps.

关键词：氨基酸序列催化作用结晶结晶学 X线大肠杆菌质谱分析法模型分子分子序列数据肽碎片/化学肽碎片/分离和提纯蛋白质构象蛋白质折叠蛋白质结构三级原虫蛋白质类/化学原虫蛋白质类/分离和提纯重组融合蛋白质类/化学重组融合蛋白质类/分离和提纯序列比对序列同源性氨基酸种特异性布鲁斯锥虫布鲁斯亚种/酶学动物

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Characterization of Drosophila OVO protein DNA binding specificity using random DNA oligomer selection suggests zinc finger degeneration

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NUCLEIC ACIDS RESEARCH 2000年第3期28卷 826-834页

作者： Lee, S Garfinkel, MD IIT Div Biol Chicago IL 60616 USA

The Drosophila melanogaster ovo locus codes for several tissue- and stage-specific proteins that all possess a common C-terminal array of four C2H2 zinc fingers. Three fingers conform to the motif framework and are evolutionarily conserved;the fourth diverges considerably. The ovo genetic function affects germ cell viability, sex identity and oogenesis, while the overlapping svb function is a key selector for epidermal structures under the control of wnt and EGF receptor signaling. We isolated synthetic DNA oligomers bound by the OVO zinc finger array from a high complexity starting population and derived a statistically significant 9 bp long DNA consensus sequence, which is nearly identical to a consensus derived from several Drosophila genes known or suspected of being regulated by the ovo function in vivo. The DNA consensus recognized by Drosophila OVO protein is atypical for zinc finger proteins in that it does not conform to many of the 'rules' for the interaction of amino acid contact residues and DNA bases. Additionally, our results suggest that only th ree of the OVO zinc fingers contribute to DNA-binding specificity.

关键词：氨基酸序列碱基序列结合部位克隆分子共有序列/遗传学保守序列/遗传学 DNA/化学合成 DNA/化学 DNA/遗传学 DNA/代谢 DNA结合蛋白质类/化学 DNA结合蛋白质类/遗传学 DNA结合蛋白质类/分离和提纯 DNA结合蛋白质类/代谢果蝇蛋白质类黑腹果蝇进化分子基因昆虫/遗传学分子序列数据分子量寡脱氧核糖核苷酸类/化学合成寡脱氧核糖核苷酸类/化学寡脱氧核糖核苷酸类/遗传学寡脱氧核糖核苷酸类/代谢启动区遗传/遗传学蛋白质结合重组融合蛋白质类/化学重组融合蛋白质类/遗传学重组融合蛋白质类/分离和提纯重组融合蛋白质类/代谢序列比对转录因子/化学转录因子/遗传学转录因子/分离和提纯转录因子/代谢锌指/遗传学锌指/生理学动物

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Baculovirus-transfer vector for eukaryotic expression and immuno-affinity purification of Gal4-fusion proteins

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BIOTECHNIQUES 1999年第5期26卷 808-+页

作者： Chen, GQ Courey, AJ Univ Calif Los Angeles Dept Chem & Biochem Los Angeles CA 90095 USA

No abstract available

关键词：氨基酸序列杆状病毒科/遗传学碱基序列碱性螺旋-环-螺旋转录因子类生物技术细胞系克隆分子 DNA引物/遗传学 DNA 重组/遗传学 DNA结合蛋白质类/遗传学真菌蛋白质类/遗传学真菌蛋白质类/分离和提纯基因表达遗传载体免疫印迹法重组融合蛋白质类/遗传学重组融合蛋白质类/分离和提纯阻遏蛋白质类/遗传学限制性内切酶图谱法酿酒酵母蛋白质类斜纹夜蛾属转录因子/遗传学转录因子/分离和提纯动物

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Purification of human interleukin-2 fusion protein produced in insect larvae is facilitated by fusion with green fluorescent protein and metal affinity ligand

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BIOTECHNOLOGY PROGRESS 1999年第2期15卷 283-286页

作者： Cha, HJ Dalal, NG Pham, MQ Bentley, WE Univ Maryland Dept Chem Engn College Pk MD 20742 USA Univ Maryland Inst Biotechnol Agr Biotechnol Ctr College Pk MD 20742 USA

The fusion protein of green fluorescent protein (GFP) and human interleukin-2 (hIL-2) was produced in insect Trichoplusia ni larvae infected with recombinant baculovirus derived from the Autographa californica nuclear polyhedrosis virus (AcNPV). This fusion protein was composed of a metal ion binding site (His)(6) for rapid one-step purification using immobilized metal affinity chromatography (IMAC), UV-optimized GFP (GFPuv), enterokinase cleavage site for recovering hIL-2 from purified fusion protein, and hIL-8 protein. The additional histidine residues on fusion protein enabled the efficient purification of fusion protein based on immobilized metal affinity chromatography. In addition to advantages of GFP as a fusion marker, GFP was able to be used as a selectable purification marker;we easily determined the correct purified fusion protein sample fraction by simply detecting GFP fluorescence.

关键词：生物学标记/分析肠肽酶/代谢基因表达绿色荧光蛋白质类指示剂和试剂/代谢昆虫/化学昆虫/生长和发育白细胞介素2/遗传学白细胞介素2/分离和提纯白细胞介素2/代谢幼虫发光蛋白质类/遗传学发光蛋白质类/分离和提纯发光蛋白质类/代谢金属/代谢重组融合蛋白质类/遗传学重组融合蛋白质类/分离和提纯重组融合蛋白质类/代谢动物人类

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Immunopurified mammalian target of rapamycin phosphorylates and activates p70 S6 kinase α in vitro

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JOURNAL OF BIOLOGICAL CHEMISTRY 1999年第48期274卷 34493-34498页

作者： Isotani, S Hara, K Tokunaga, C Inoue, H Avruch, J Yonezawa, K Kobe Univ Biosignal Res Ctr Nada Ku Kobe Hyogo 6518501 Japan Massachusetts Gen Hosp Diabet Unit Boston MA 02129 USA Massachusetts Gen Hosp Med Serv Boston MA 02129 USA Harvard Univ Sch Med Boston MA 02129 USA

p70 S6 kinase alpha (p70 alpha) is activated in vivo through a multisite phosphorylation in response to mitogens if a sufficient supply of amino acids is available or to high concentrations of amino acids per se. The immunosuppressant drug rapamycin inhibits p70 alpha activation in a manner that can be overcome by coexpression of p70 alpha with a rapamycin-resistant mutant of the mammalian target of rapamycin (mTOR) but only if the mTOR kinase domain is intact. We report here that a mammalian recombinant p70 alpha polypeptide, extracted in an inactive form from rapamycin-treated cells, can be directly phosphorylated by the mTOR kinase in vitro predominantly at the rapamycin-sensitive site Thr-412. mTOR-catalyzed p70 alpha phosphorylation in vitro is accompanied by a substantial restoration in p70 alpha kinase activity toward its physiologic substrate, the 40 S ribosomal protein S6. Moreover, sequential phosphorylation of p70 alpha by mTOR and 3-phosphoinositide-dependent protein kinase 1 in vitro resulted in a synergistic stimulation of p70 alpha activity to levels similar to that attained by serum stimulation in vivo. These results indicate that mTOR is likely to function as a direct activator of p70 in vivo, although the relative contribution of mTOR-catalyzed p70 phosphorylation in each of the many circumstances that engender p70 activation remains to be defined.

关键词：细胞系培养基无血清/药理学酶激活/药物作用免疫印迹法有丝分裂原/药理学突变磷酰化/药物作用磷酸转移酶类(醇族体)/遗传学磷酸转移酶类(醇族体)/分离和提纯磷酸转移酶类(醇族体)/代谢沉淀素试验蛋白激酶类蛋白质丝氨酸苏氨酸激酶/遗传学蛋白质丝氨酸苏氨酸激酶/代谢重组融合蛋白质类/遗传学重组融合蛋白质类/分离和提纯重组融合蛋白质类/代谢核糖体蛋白质S6激酶类/遗传学核糖体蛋白质S6激酶类/代谢 TOR丝氨酸-苏氨酸激酶人类

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A highly active, soluble mutant of the membrane-associated (S)-mandelate dehydrogenase from Pseudomonas putida

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BIOCHEMISTRY 1999年第38期38卷 12367-12376页

作者： Xu, Y Mitra, B Wayne State Univ Sch Med Dept Biochem & Mol Biol Detroit MI 48201 USA

(S)-Mandelate dehydrogenase (MDH) from Pseudomonas putida, a member of the flavin mononucleotide-dependent alpha-hydroxy acid oxidase/dehydrogenase family, is a membrane-associated protein, in contrast to the more well-characterized members of this protein family including glycolate oxidase (GOX) from spinach. In a previous study [Mitra, B., et al. (1993) Biochemistry 32, 12959-12967], the membrane association of MDH was correlated to a 53 amino acid segment in the interior of the primary sequence by construction of a chimeric enzyme, MDH-GOX1, in which the membrane-binding segment in MDH was deleted and replaced with the corresponding 34 amino acid segment from the soluble GOX. Though MDH-GOX1 was soluble, it was an inefficient, nonspecific enzyme that involved a different transition state for the catalyzed reaction from that of the wild-type MDH. In the present study, it is shown that the membrane-binding segment in MDH is somewhat shorter, similar to 39 residues long. Partial or total deletion of this segment disrupts membrane localization of MDH. This segment is not important for substrate oxidation activity. A new chimera, MDH-GOX2, was created by replacing this shorter membrane-binding segment from MDH with the corresponding 20 amino acid segment from GOX. The soluble MDH-GOX2 is very similar to the wild-type membrane-bound enzyme in its spectroscopic properties, substrate specificity, catalytic activity, kinetic mechanism, and lack of reactivity toward oxygen. Therefore, it should prove to be a highly useful model for structural studies of MDH.

关键词：醇氧化还原酶类/拮抗剂和抑制剂醇氧化还原酶类/遗传学醇氧化还原酶类/分离和提纯醇氧化还原酶类/代谢氨基酸序列细胞膜/酶学酶激活/药物作用酶激活/遗传学大肠杆菌/酶学大肠杆菌/遗传学黄素单核苷酸/化学黄素单核苷酸/代谢羟丁酸盐类/化学动力学膜蛋白质类/遗传学膜蛋白质类/分离和提纯膜蛋白质类/代谢分子序列数据肽碎片/遗传学肽碎片/分离和提纯肽碎片/代谢蛋白质结合/遗传学假单胞菌恶臭/酶学假单胞菌恶臭/遗传学重组融合蛋白质类/化学合成重组融合蛋白质类/遗传学重组融合蛋白质类/分离和提纯溶解度立体异构现象

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Analysis of nuclear localization signals using a green fluorescent protein-fusion protein library

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EXPERIMENTAL CELL RESEARCH 1999年第2期251卷 299-306页

作者： Fujii, G Tsuchiya, R Ezoe, E Hirohashi, S Natl Canc Ctr Res Inst Div Pathol Chuo Ku Tokyo 1040045 Japan JST ERATO Hirohashi Cell Configurat Project Ibaraki Osaka 3002635 Japan

We describe here an efficient method for identifying intracellular localization signals in proteins with stereospecific intracellular localizations in culture cells. The method involves rapid fluorescence screening of cells transfected with a cDNA library in which cDNAs are fused to the gene encoding the Aequorea victoria green fluorescent protein (GFP). We analyzed nuclear localization and nuclear localization signals (NLSs) in a model application of this method. As a result, we identified classical MLSs in 75% of nuclear localized proteins. We identified some novel NLS candidates among the classical NLS-negative sequences whose nuclear localization was also identified in another cell line and with other molecular tag sequences. This method will be useful for identifying intracellular localization signals and for more detailed analysis of intracellular architecture. (C) 1999 Academic Press.

关键词：氨基酸序列细胞系克隆分子/方法基因文库遗传载体绿色荧光蛋白质类肝/细胞学发光蛋白质类/遗传学分子序列数据核定位信号核蛋白质类/遗传学核蛋白质类/分离和提纯肽类重组融合蛋白质类/分离和提纯选择(遗传学) β半乳糖苷酶类/遗传学人类

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The Rift Valley fever virus nonstructural protein NSs is phosphorylated at serine residues located in casein kinase II consensus motifs in the carboxy-terminus

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VIROLOGY 1999年第2期263卷 517-525页

作者： Kohl, A di Bartolo, V Bouloy, M Inst Pasteur Unite Arbovirus & Virus Fievres Hemorrag Grp Bunyavirides F-75724 Paris France Inst Pasteur Unite Immunol Mol F-75724 Paris France

The S segment of Rift Valley fever Virus (Bunyaviridae, Phlebovirus) codes for two proteins, the nucleoprotein N and the nonstructural protein NSs. The NSs protein is a phosphoprotein of unknown function that is localized in the cytoplasm and the nuclei of infected cells where it forms filamentous structures. To characterize further the protein expressed in VC10 cells infected with the MP12 strain, we analyzed its phosphorylation states and showed that phosphorylated forms were found in both compartments. Cytoplasmic and nuclear NSs were phosphorylated only at serine residues. Phosphopeptide mapping and molecular analysis of mutants obtained by site-directed mutagenesis allowed us to map the major phosphorylation sites of nuclear and cytoplasmic forms of NSs to serine residues 252 and 256, located at the carboxy-terminus in consensus sequences for casein kinase II. A similar map was obtained when the protein was purified from mosquito cells infected with MP12. In addition, we showed that the purified unphosphorylated NSs protein expressed from pET-NSs plasmid in a coupled transcription-translation reaction containing Escherichia coli S30 extracts did not possess autophosphorylation activity but was phosphorylated in vitro after incubation with recombinant casein kinase II. (C) 1999 Academic Press.

关键词：氨基酸特征结构氨基酸序列酪蛋白激酶Ⅱ 细胞系细胞核/代谢共有序列/遗传学蚊科/细胞学蚊科/病毒学溴化氰/代谢细胞质/代谢分子序列数据诱变定点突变肽谱磷酰化磷酸丝氨酸/分析磷酸丝氨酸/代谢蛋白质丝氨酸苏氨酸激酶/分离和提纯蛋白质丝氨酸苏氨酸激酶/代谢重组融合蛋白质类/化学重组融合蛋白质类/分离和提纯重组融合蛋白质类/代谢立夫特山谷热病毒/遗传学立夫特山谷热病毒/代谢丝氨酸/遗传学丝氨酸/代谢病毒非结构蛋白质类/化学病毒非结构蛋白质类/遗传学动物

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Crystal structure of human T cell leukemia virus type 1 gp21 ectodomain crystallized as a maltose-binding protein chimera reveals structural evolution of retroviral transmembrane proteins

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PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 1999年第8期96卷 4319-4324页

作者： Kobe, B Center, RJ Kemp, BE Poumbourios, P St Vincents Inst Med Res Fitzroy Vic 3065 Australia

Retroviral entry into cells depends on envelope glycoproteins, whereby receptor binding to the surface-exposed subunit triggers membrane fusion by the transmembrane protein (TM) subunit. We determined the crystal structure at 2.5-Angstrom resolution of the ectodomain of gp21, the TM from human T cell leukemia virus type 1. The gp21 fragment was crystallized as a maltose-binding protein chimera, and the maltose-binding protein domain was used to solve the initial phases by the method of molecular replacement. The structure of gp21 comprises an N-terminal trimeric coiled coil, an adjacent disulfide-bonded loop that stabilizes a chain reversal, and a C-terminal sequence structurally distinct from HIV type 1/simian immunodeficiency virus gp41 that packs against the coil in an extended antiparallel fashion. Comparison of the gp21 structure with the structures of other retroviral TMs contrasts the conserved nature of the coiled coil-forming region and adjacent disulfide-bonded loop with the variable nature of the C-terminal ectodomain segment. The structure points to these features having evolved to enable the dual roles of retroviral TMs: conserved fusion function and an ability to anchor diverse surface-exposed subunit structures to the virion envelope and infected cell surface. The structure of gp21 implies that the N-terminal fusion peptide is in close proximity to the C-terminal transmembrane domain and likely represents a postfusion conformation.

关键词：氨基酸序列载体蛋白质类/化学载体蛋白质类/分离和提纯计算机制图结晶结晶学 X线/方法进化分子基因产物 env/化学基因产物 env/遗传学基因产物 env/分离和提纯人类嗜T淋巴细胞病毒1型/化学人类嗜T淋巴细胞病毒1型/遗传学大分子物质麦芽糖结合蛋白质类模型分子分子序列数据蛋白质结构二级重组融合蛋白质类/化学重组融合蛋白质类/分离和提纯逆转录病毒科蛋白质类致癌/化学逆转录病毒科蛋白质类致癌/遗传学逆转录病毒科蛋白质类致癌/分离和提纯序列比对序列同源性氨基酸 env基因产物人免疫缺陷病毒人类

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