The Neurospora crassa blind mutant white collar-1 (wc-1) is pleiotropically defective in all blue light-induced phenomena, establishing a role for the wc-1 gene product in the signal transduction ...pathway. We report the cloning of the wc-1 gene isolated by chromosome walking and mutant complementation. The elucidation of the wc-1 gene product provides a key piece of the blue light signal transduction puzzle. The wc-1 gene encodes a 125 kDa protein whose encoded motifs include a single class four, zinc finger DNA binding domain and a glutamine-rich putative transcription activation domain. We demonstrate that the wc-1 zinc finger domain, expressed in Escherichia coli, is able to bind specifically to the promoter of a blue light-regulated gene of Neurospora using an in vitro gel retardation assay. Furthermore, we show that wc-1 gene expression is autoregulated and is transcriptionally induced by blue light irradiation.
The presence of correctly formed disulfide bonds is crucial to the structure and function of proteins that are synthesized in the endoplasmic reticulum (ER). Disulfide bond formation occurs in the ER ...owing to the presence of several specialized catalysts and a suitable redox potential. Work in yeast has indicated that the ER resident glycoprotein Ero1p provides oxidizing equivalents to newly synthesized proteins via protein disulfide isomerase (PDI). Here we show that Ero1‐Lα, the human homolog of Ero1p, exists as a collection of oxidized and reduced forms and covalently binds PDI. We analyzed Ero1‐Lα cysteine mutants in the presumed active site C391VGCFKC397. Our results demonstrate that this motif is important for protein folding, structural integrity, protein half‐life and the stability of the Ero1‐Lα–PDI complex.
Astrocyte-enriched populations were established from human embryonic brain analyzed for their ability to synthesize cytokines potentially relevant for mechanisms of inflammation and immunity in the ...brain. Unstimulated astrocytes did not secrete significant IL-6, IL-8, macrophage CSF (M-CSF), granulocyte-macrophage CSF (GM-CSF), or granulocyte-CSF (G-CSF), as determined by specific ELISA and/or bioassay. With the exception of M-CSF mRNA, transcripts for the above factors were not detected in unstimulated astrocytes. On exposure of human astrocytes to IL-1 beta, high levels of IL-6, IL-8, M-CSF, G-CSF, and GM-CSF mRNAs were detected; moreover, active secretion of all the above cytokines was demonstrated. TNF-alpha was also able to stimulate IL-6, IL-8, M-CSF, GM-CSF, and G-CSF synthesis and secretion, but was generally less potent than IL-1 beta. No IL-3 mRNA or protein was detected in unstimulated or cytokine-treated astrocytes. IL-1 alpha and IL-1 beta mRNAs and proteins were not detected in unstimulated astrocytes, but were present in very small amounts after stimulation with TNF-alpha/IL-1 beta. No IL-6, M-CSF, GM-CSF, G-CSF, or IL-8 were induced by IL-1 beta or TNF-alpha in early primary cultures, which mainly contain undifferentiated neuronal/glial progenitor cells. These studies demonstrate for the first time the production of multiple cytokines by normal human astrocytes stimulated in culture by IL-1 beta and TNF-alpha. The capacity of human astrocytes to synthesize and release cytokines active on hemolymphopoietic cells supports the concept that these cells play an important role in the regulation of inflammatory and immune responses in a variety of brain pathologies.
Interleukin‐6 (IL‐6) is a differentiation and growth factor for a variety of cell types and its excessive production plays a major role in the pathogenesis of multiple myeloma and post‐menopausal ...osteoporosis. IL‐6, a four‐helix bundle cytokine, is believed to interact sequentially with two transmembrane receptors, the low‐affinity IL‐6 receptor (IL‐6R alpha) and the signal transducer gp130, via distinct binding sites. In this paper we show that combined mutations in the predicted A and C helices, previously suggested to establish contacts with gp130, give rise to variants with no bioactivity but unimpaired binding to IL‐6R alpha. These mutants behave as full and selective IL‐6 receptor antagonists on a variety of human cell lines. Furthermore, a bifacial mutant was generated (called IL‐6 super‐antagonist) in which the antagonist mutations were combined with amino acid substitutions in the predicted D helix that increase binding for IL‐6R alpha. The IL‐6 super‐antagonist has no bioactivity, but improved first receptor occupancy and, therefore, fully inhibits the wild‐type cytokine at low dosage. The demonstration of functionally independent receptor binding sites on IL‐6 suggests that it could be possible to design super‐antagonists of other helical cytokines which drive the assembly of structurally related multisubunit receptor complexes.
Interleukin‐6 (IL‐6) triggers the formation of a high affinity receptor complex with the ligand binding subunit IL‐6Ralpha and the signal transducing chain gp130. Since the intracytoplasmic region of ...the IL‐6Ralpha does not contribute to signaling, soluble forms of the extracytoplasmic domain (sIL‐6Ralpha), potentiate IL‐6 bioactivity and induce a cytokine‐responsive status in cells expressing gp130 only. This observation, together with the detection of high levels of circulating soluble human IL‐6Ralpha (shIL‐6Ralpha) in sera, suggests that the hIL‐6‐shIL‐6Ralpha complex is an alternative form of the cytokine. Here we describe the generation of human IL‐6 (hIL‐6) variants with strongly enhanced shIL‐6Ralpha binding activity and bioactivity. Homology modeling and site‐directed mutagenesis of hIL‐6 suggested that the binding interface for hIL‐6Ralpha is constituted by the C‐terminal portion of the D‐helix and residues contained in the AB loop. Four libraries of hIL‐6 mutants were generated by each time fully randomizing four different amino acids in the predicted AB loop. These libraries were displayed monovalently on filamentous phage surface and sorted separately for binding to immobilized shIL‐6Ralpha. Mutants were selected which, when expressed as soluble proteins, showed a 10‐ to 40‐fold improvement in shIL‐6Ralpha binding; a further increase (up to 70‐fold) was achieved by combining variants isolated from different libraries. Interestingly, high affinity hIL‐6 variants show strongly enhanced bioactivity on cells expressing gp13O in the presence of shIL‐6Ralpha at concentrations similar to those normally found in human sera.
Oxidative conditions must be generated in the endoplasmic reticulum (ER) to allow disulfide bond formation in secretory proteins. A family of conserved genes, termed ERO for ER oxidoreductins, plays ...a key role in this process. We have previously described the human gene ERO1-L, which complements several phenotypic traits of the yeast thermo- sensitive mutant ero1-1 (Cabibbo, A., Pagani, M., Fabbri, M., Rocchi, M., Farmery, M. R., Bulleid, N. J., and Sitia, R. (2000) J. Biol. Chem. 275, 4827-4833). Here, we report the cloning and characterization of a novel human member of this family, ERO1-L beta . Immunofluorescence, endoglycosidase sensitivity, and in vitro translation/translocation assays reveal that the products of the ERO1-L beta gene are primarily localized in the ER of mammalian cells. The ability to allow growth at 37 degree C and to alleviate the "unfolded protein response" when expressed in ero1-1 cells indicates that ERO1-L beta is involved also in generating oxidative conditions in the ER. ERO1-L and ERO1-L beta display different tissue distributions. Furthermore, only ERO1-L beta transcripts are induced in the course of the unfolded protein response. Our results suggest a complex regulation of ER redox homeostasis in mammalian cells.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Interleukin 6 is a 184-aa polypeptide postulated to belong to the class of helical cytokines. We built a three-dimensional model of human interleukin 6 based on the similarity of its hydrophobicity ...pattern with that of other cytokines and on the x-ray structure of growth hormone, interleukin 2, interleukin 4, interferon β, and granulocyte-macrophage colony-stimulating factor. The resulting model is a bundle of four α-helices and suggests possible alternative conformations for the 9 C-terminal amino acids; in this region, the importance of Arg-182 and Met-184 for biological activity has been demonstrated Lutticken, C., Kruttgen, A., Moller, C., Heinrich, P. C. \& Rose-John, S. (1991) FEBS Lett. 282, 265-267. Therefore, we generated a large collection of single-amino acid variants in residues 175-181. Analysis of their biological activity in two systems and the receptor binding properties of a subset of the mutants indicates that the entire region is involved in forming the receptor binding surface and supports the hypothesis that this region does not assume an α-helical conformation. Remarkably, we also found a mutant with receptor affinity and biological activity much higher than wild type; the potential therapeutical value of this finding is discussed.
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Microstructural evolution due to equal-channel angular-pressing (ECAP) with increasingly severe deformation was investigated in a commercially pure 1200 aluminum alloy. A true strain of eight ...produced sub-micrometer scale grains and very fine subgrains in the grain interior. The deformation process was documented and described using field-emission (FEG) gun scanning and transmission electron microscopy techniques. After eight ECAP passes, the high-angle grain boundaries accounted for ∼70% of all boundaries. The fine spacing resolution of FEG scanning electron microscopy allowed detailed grain and subgrain statistical evaluation in the deformed microstructure; transmission electron microscopic inspection afforded appreciation of the role of very low-angle misorientation boundaries in the microstructure-refining process. ECAP results were compared with those produced by cold rolling. The material's texture evolved in a decreasing trend of Cube {001}〈100〉 intensities in favor of Cube rotated toward the normal-to-pressing direction {001}〈120〉, while Goss {110}〈001〉 and {111}〈110〉, {111}〈112〉 directions slightly increased with strain.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
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