Showing papers by "Laboratory of Molecular Biology published in 2000"
TL;DR: Evidence that the appropriate and inappropriate production of oxidants, together with the ability of organisms to respond to oxidative stress, is intricately connected to ageing and life span is reviewed.
Abstract: Living in an oxygenated environment has required the evolution of effective cellular strategies to detect and detoxify metabolites of molecular oxygen known as reactive oxygen species. Here we review evidence that the appropriate and inappropriate production of oxidants, together with the ability of organisms to respond to oxidative stress, is intricately connected to ageing and life span.
8,665 citations
TL;DR: The crystal structure of the 30S subunit from Thermus thermophilus, refined to 3 Å resolution, is reported, which will facilitate the interpretation in molecular terms of lower resolution structural data on several functional states of the ribosome from electron microscopy and crystallography.
Abstract: Genetic information encoded in messenger RNA is translated into protein by the ribosome, which is a large nucleoprotein complex comprising two subunits, denoted 30S and 50S in bacteria. Here we report the crystal structure of the 30S subunit from Thermus thermophilus, refined to 3 A resolution. The final atomic model rationalizes over four decades of biochemical data on the ribosome, and provides a wealth of information about RNA and protein structure, protein–RNA interactions and ribosome assembly. It is also a structural basis for analysis of the functions of the 30S subunit, such as decoding, and for understanding the action of antibiotics. The structure will facilitate the interpretation in molecular terms of lower resolution structural data on several functional states of the ribosome from electron microscopy and crystallography.
2,008 citations
TL;DR: This manuscript presents a meta-analyses of the determinants of infectious disease in eight operation theatres of the immune system and shows clear patterns of disease progression that are consistent with previous studies of immune checkpoint disease.
1,557 citations
TL;DR: The functional implications of the high-resolution 30S crystal structure are described, and details of the interactions between the 30S subunit and its tRNA and mRNA ligands are inferred, which lead to a model for the role of the universally conserved 16S RNA residues A1492 and A1493 in the decoding process.
Abstract: The 30S ribosomal subunit has two primary functions in protein synthesis. It discriminates against aminoacyl transfer RNAs that do not match the codon of messenger RNA, thereby ensuring accuracy in translation of the genetic message in a process called decoding. Also, it works with the 50S subunit to move the tRNAs and associated mRNA by precisely one codon, in a process called translocation. Here we describe the functional implications of the high-resolution 30S crystal structure presented in the accompanying paper, and infer details of the interactions between the 30S subunit and its tRNA and mRNA ligands. We also describe the crystal structure of the 30S subunit complexed with the antibiotics paromomycin, streptomycin and spectinomycin, which interfere with decoding and translocation. This work reveals the structural basis for the action of these antibiotics, and leads to a model for the role of the universally conserved 16S RNA residues A1492 and A1493 in the decoding process.
1,508 citations
TL;DR: A method for obtaining dopaminergic (DA) and serotonergic neurons in high yield from mouse ES cells in vitro is presented and it is demonstrated that the ES cells can be obtained in unlimited numbers and that these neuron types are generated efficiently.
Abstract: Embryonic stem (ES) cells are clonal cell lines derived from the inner cell mass of the developing blastocyst that can proliferate extensively in vitro and are capable of adopting all the cell fates in a developing embryo. Clinical interest in the use of ES cells has been stimulated by studies showing that isolated human cells with ES properties from the inner cell mass or developing germ cells can provide a source of somatic precursors. Previous studies have defined in vitro conditions for promoting the development of specific somatic fates, specifically, hematopoietic, mesodermal, and neurectodermal. In this study, we present a method for obtaining dopaminergic (DA) and serotonergic neurons in high yield from mouse ES cells in vitro. Furthermore, we demonstrate that the ES cells can be obtained in unlimited numbers and that these neuron types are generated efficiently. We generated CNS progenitor populations from ES cells, expanded these cells and promoted their differentiation into dopaminergic and serotonergic neurons in the presence of mitogen and specific signaling molecules. The differentiation and maturation of neuronal cells was completed after mitogen withdrawal from the growth medium. This experimental system provides a powerful tool for analyzing the molecular mechanisms controlling the functions of these neurons in vitro and in vivo, and potentially for understanding and treating neurodegenerative and psychiatric diseases.
1,346 citations
TL;DR: Interestingly, LY294002 and the lead compound on which it was designed, quercetin, as well as the closely related flavonoid myricetin bind PI3K in remarkably different orientations that are related to each other by 180 degrees rotations.
1,140 citations
1,124 citations
TL;DR: In vitro the mechanism by which the critical region of one prion protein, Sup35, is initially unstructured in solution and then forms self-seeded amyloid fibers is examined, which may be applicable to other protein assembly processes.
Abstract: Prion proteins can serve as genetic elements by adopting distinct physical and functional states that are self-perpetuating and heritable. The critical region of one prion protein, Sup35, is initially unstructured in solution and then forms self-seeded amyloid fibers. We examined in vitro the mechanism by which this state is attained and replicated. Structurally fluid oligomeric complexes appear to be crucial intermediates in de novo amyloid nucleus formation. Rapid assembly ensues when these complexes conformationally convert upon association with nuclei. This model for replicating protein-based genetic information, nucleated conformational conversion, may be applicable to other protein assembly processes.
971 citations
TL;DR: The recently determined atomic structure of the 30S ribosomal subunit is used to determine the structures of its complexes with the antibiotics tetracycline, pactamycin, and hygromycin B to suggest a mechanism for its effects on ribosome function.
848 citations
TL;DR: Circular dichroism spectroscopy indicated that alpha-synuclein undergoes a conformational change from random coil to beta-sheet structure during assembly, and X-ray diffraction and electron diffraction of the alpha- synuclein assemblies showed a cross-beta conformation characteristic of amyloid.
Abstract: Filamentous inclusions made of alpha-synuclein constitute the defining neuropathological characteristic of Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy. Rare familial cases of Parkinson's disease are associated with mutations A53T and A30P in alpha-synuclein. We report here the assembly properties and secondary structure characteristics of recombinant alpha-synuclein. Carboxy-terminally truncated human alpha-synuclein (1-87) and (1-120) showed the fastest rates of assembly, followed by human A53T alpha-synuclein, and rat and zebra finch alpha-synuclein. Wild-type human alpha-synuclein and the A30P mutant showed slower rates of assembly. Upon shaking, filaments formed within 48 h at 37 degrees C. The related proteins beta- and gamma-synuclein only assembled after several weeks of incubation. Synthetic human alpha-synuclein filaments were decorated by an antibody directed against the carboxy-terminal 10 amino acids of alpha-synuclein, as were filaments extracted from dementia with Lewy bodies and multiple system atrophy brains. Circular dichroism spectroscopy indicated that alpha-synuclein undergoes a conformational change from random coil to beta-sheet structure during assembly. X-ray diffraction and electron diffraction of the alpha-synuclein assemblies showed a cross-beta conformation characteristic of amyloid.
745 citations
TL;DR: The results indicate that antibody arrays can be used to identify differentially expressed proteins.
Abstract: We have developed a novel technique for high-throughput screening of recombinant antibodies, based on the creation of antibody arrays. Our method uses robotic picking and high-density gridding of bacteria containing antibody genes followed by filter-based enzyme-linked immunosorbent assay (ELISA) screening to identify clones that express binding antibody fragments. By eliminating the need for liquid handling, we can thereby screen up to 18,342 different antibody clones at a time and, because the clones are arrayed from master stocks, the same antibodies can be double spotted and screened simultaneously against 15 different antigens. We have used our technique in several different applications, including isolating antibodies against impure proteins and complex antigens, where several rounds of phage display often fail. Our results indicate that antibody arrays can be used to identify differentially expressed proteins.
TL;DR: Increased proliferation, reduced cell death, and enhanced dopamine neuron generation in lowered O2 are demonstrated, making this method an important advance in the ex vivogeneration of specific neurons for brain repair.
Abstract: Standard cell culture systems impose environmental oxygen (O2) levels of 20%, whereas actual tissue O2 levels in both developing and adult brain are an order of magnitude lower. To address whether proliferation and differentiation of CNS precursors in vitro are influenced by the O2 environment, we analyzed embryonic day 12 rat mesencephalic precursor cells in traditional cultures with 20% O2 and in lowered O2 (3 ± 2%). Proliferation was promoted and apoptosis was reduced when cells were grown in lowered O2, yielding greater numbers of precursors. The differentiation of precursor cells into neurons with specific neurotransmitter phenotypes was also significantly altered. The percentage of neurons of dopaminergic phenotype increased to 56% in lowered O2 compared with 18% in 20% O2. Together, the increases in total cell number and percentage of dopaminergic neurons resulted in a ninefold net increase in dopamine neuron yield. Differential gene expression analysis revealed more abundant messages for FGF8, engrailed-1, and erythropoietin in lowered O2. Erythropoietin supplementation of 20% O2cultures partially mimicked increased dopaminergic differentiation characteristic of CNS precursors cultured in lowered O2. These data demonstrate increased proliferation, reduced cell death, and enhanced dopamine neuron generation in lowered O2, making this method an important advance in the ex vivogeneration of specific neurons for brain repair.
TL;DR: The results support a model in which linker histones bind dynamically to chromatin in a stop-and-go mode, suggesting a higher rate of exchange upon chromatin remodelling.
Abstract: The linker histone H1 is believed to be involved in chromatin organization by stabilizing higher-order chromatin structure. Histone H1 is generally viewed as a repressor of transcription as it prevents the access of transcription factors and chromatin remodelling complexes to DNA. Determining the binding properties of histone H1 to chromatin in vivo is central to understanding how it exerts these functions. We have used photobleaching techniques to measure the dynamic binding of histone H1-GFP to unperturbed chromatin in living cells. Here we show that almost the entire population of H1-GFP is bound to chromatin at any one time; however, H1-GFP is exchanged continuously between chromatin regions. The residence time of H1-GFP on chromatin between exchange events is several minutes in both euchromatin and heterochromatin. In addition to the mobile fraction, we detected a kinetically distinct, less mobile fraction. After hyperacetylation of core histones, the residence time of H1-GFP is reduced, suggesting a higher rate of exchange upon chromatin remodelling. These results support a model in which linker histones bind dynamically to chromatin in a stop-and-go mode.
TL;DR: Ras activation of phosphoinositide 3-kinase (PI3K) is important for survival of transformed cells and it is found that PI3Kgamma is strongly and directly activated by H-Ras G12V in vivo or by GTPgammaS-loaded H- Ras in vitro.
TL;DR: Inaccessibility of biotinylated concanavalin A to the native elementary unit and partial dissociation of the elementary unit after incubation with excess N-glycosidase F or endoglycosidases H suggest that a single molecule of P25 is located internally and plays an important role in maintaining integrity of the complex.
TL;DR: The effects of ROS on signal transduction pathways, the molecules that regulate intracellular ROS production and the potential protein targets of oxidants are focused on.
TL;DR: The findings suggest that the Gcn5p bromodomain may discriminate between different acetylated lysine residues depending on the context in which they are displayed.
Abstract: The bromodomain is an approximately 110 amino acid module found in histone acetyltransferases and the ATPase component of certain nucleosome remodelling complexes. We report the crystal structure at 1.9 A resolution of the Saccharomyces cerevisiae Gcn5p bromodomain complexed with a peptide corresponding to residues 15-29 of histone H4 acetylated at the zeta-N of lysine 16. We show that this bromodomain preferentially binds to peptides containing an N:-acetyl lysine residue. Only residues 16-19 of the acetylated peptide interact with the bromodomain. The primary interaction is the N:-acetyl lysine binding in a cleft with the specificity provided by the interaction of the amide nitrogen of a conserved asparagine with the oxygen of the acetyl carbonyl group. A network of water-mediated H-bonds with protein main chain carbonyl groups at the base of the cleft contributes to the binding. Additional side chain binding occurs on a shallow depression that is hydrophobic at one end and can accommodate charge interactions at the other. These findings suggest that the Gcn5p bromodomain may discriminate between different acetylated lysine residues depending on the context in which they are displayed.
TL;DR: Concomitant decrease of the tissue thiol content after selenate treatment was consistent with the suggested role of glutathione (GSH) as a repressive effector for the expression of sulphate transporter genes.
Abstract: Summary
To investigate the uptake and long-distance translocation of sulphate in plants, we have characterized three cell-type-specific sulphate transporters, Sultr1;1, Sultr2;1 and Sultr2;2 in Arabidopsis thaliana. Heterologous expression in the yeast sulphate transporter mutant indicated that Sultr1;1 encodes a high-affinity sulphate transporter (Km for sulphate 3.6 ± 0.6 μm), whereas Sultr2;1 and Sultr2;2 encode low-affinity sulphate transporters (Km for sulphate 0.41 ± 0.07 m m and ≥ 1.2 m m, respectively). In Arabidopsis plants expressing the fusion gene construct of the Sultr1;1 promoter and green fluorescent protein (GFP), GFP was localized in the lateral root cap, root hairs, epidermis and cortex of roots. β-glucuronidase (GUS) expressed with the Sultr2;1 promoter was specifically accumulated in the xylem parenchyma cells of roots and leaves, and in the root pericycles and leaf phloem. Expression of the Sultr2;2 promoter–GFP fusion gene showed specific localization of GFP in the root phloem and leaf vascular bundle sheath cells. Plants continuously grown with low sulphate concentrations accumulated high levels of Sultr1;1 and Sultr2;1 mRNA in roots and Sultr2;2 mRNA in leaves. The abundance of Sultr1;1 and Sultr2;1 mRNA was increased remarkably in roots by short-term stress caused by withdrawal of sulphate. Addition of selenate in the sulphate-sufficient medium increased the sulphate uptake capacity, tissue sulphate content and the abundance of Sultr1;1 and Sultr2;1 mRNA in roots. Concomitant decrease of the tissue thiol content after selenate treatment was consistent with the suggested role of glutathione (GSH) as a repressive effector for the expression of sulphate transporter genes.
TL;DR: It is shown that genotoxic stress blocks cell proliferation and induces apoptosis of germ cells in the nematode C. elegans and implicates rad1 homologs in DNA damage-induced apoptosis in animals.
TL;DR: This work has highlighted the importance of positive and negative feedback loops in the dynamic regulation of developmental signalling, which can impart precision, robustness and versatility to intercellular signals.
Abstract: The intercellular communication that regulates cell fate during animal development must be precisely controlled to avoid dangerous errors. How is this achieved? Recent work has highlighted the importance of positive and negative feedback loops in the dynamic regulation of developmental signalling. These feedback interactions can impart precision, robustness and versatility to intercellular signals. Feedback failure can cause disease.
TL;DR: It is clearly demonstrated that T1/ST2 expression plays a role in the development of Th2-like cytokine responses and indicate that effector functions are inhibited in its absence.
Abstract: We have generated mice with a deficiency in T1/ST2 expression to clarify the roles of T1/ST2 in T helper cell type 2 (Th2) responses. Using immunological challenges normally characterized by a Th2-like response, we have compared the responses of T1/ST2-deficient mice with those generated by wild-type mice. Using a primary pulmonary granuloma model, induced with Schistosoma mansoni eggs, we demonstrate that granuloma formation, characterized by eosinophil infiltration, is abrogated in T1/ST2-deficient mice. Furthermore, we clearly demonstrate that in the absence of T1/ST2 expression, the levels of Th2 cytokine production are severely impaired after immunization. Thus, in a secondary pulmonary granuloma model, draining lymph node cells from the T1/ST2-deficient animals produced significantly reduced levels of IL-4 and IL-5, despite developing granulomas of a magnitude similar to those of wild-type mice and comparable antigen-specific immunoglobulin isotype production. These data clearly demonstrate that T1/ST2 expression plays a role in the development of Th2-like cytokine responses and indicate that effector functions are inhibited in its absence.
TL;DR: The reconstitution of microbial rocketing motility in vitro with purified proteins with a dendritic pattern of nucleation controlled by the Arp2/3 complex provides an efficient pushing force for lamellipodial motility.
TL;DR: A molecular model for the HIV-1 capsid is proposed that follows the principles of a fullerene cone, in which the body of the cone is composed of curved hexagonal arrays of CA rings and the ends are closed by inclusion of 12 pentagonal ‘defects’.
Abstract: The type 1 human immunodeficiency virus (HIV-1) contains a conical capsid comprising approximately 1,500 CA protein subunits, which organizes the viral RNA genome for uncoating and replication in a new host cell. In vitro, CA spontaneously assembles into helical tubes and cones that resemble authentic viral capsids. Here we describe electron cryo-microscopy and image reconstructions of CA tubes from six different helical families. In spite of their polymorphism, all tubes are composed of hexameric rings of CA arranged with approximate local p6 lattice symmetry. Crystal structures of the two CA domains were 'docked' into the reconstructed density, which showed that the amino-terminal domains form the hexameric rings and the carboxy-terminal dimerization domains connect each ring to six neighbours. We propose a molecular model for the HIV-1 capsid that follows the principles of a fullerene cone, in which the body of the cone is composed of curved hexagonal arrays of CA rings and the ends are closed by inclusion of 12 pentagonal 'defects'.
TL;DR: The central stalk in ATP synthase is made of γ, δ and ɛ subunits in the mitochondrial enzyme, and with crystals of F1-ATPase inhibited with dicyclohexylcarbodiimide, the complete structure was revealed.
Abstract: The central stalk in ATP synthase, made of γ, δ and ɛ subunits in the mitochondrial enzyme, is the key rotary element in the enzyme's catalytic mechanism. The γ subunit penetrates the catalytic (αβ)3 domain and protrudes beneath it, interacting with a ring of c subunits in the membrane that drives rotation of the stalk during ATP synthesis. In other crystals of F1-ATPase, the protrusion was disordered, but with crystals of F1-ATPase inhibited with dicyclohexylcarbodiimide, the complete structure was revealed. The δ and ɛ subunits interact with a Rossmann fold in the γ subunit, forming a foot. In ATP synthase, this foot interacts with the c-ring and couples the transmembrane proton motive force to catalysis in the (αβ)3 domain.
07 Jul 2000
TL;DR: The crystal structure of a complex between importin-beta residues 1-442 (Ib442) and five FxFG nucleoporin repeats from Nsp1p is described, providing direct evidence for the functional significance of the import in-beta-FxFG interaction.
Abstract: We describe the crystal structure of a complex between importin-beta residues 1-442 (Ib442) and five FxFG nucleoporin repeats from Nsp1p. Nucleoporin FxFG cores bind on the convex face of Ib442 to a primary site between the A helices of HEAT repeats 5 and 6, and to a secondary site between HEAT repeats 6 and 7. Mutations at importin-beta Ile178 in the primary FxFG binding site reduce both binding and nuclear protein import, providing direct evidence for the functional significance of the importin-beta-FxFG interaction. The FxFG binding sites on importin-beta do not overlap with the RanGTP binding site. Instead, RanGTP may release importin-beta from FxFG nucleoporins by generating a conformational change that alters the structure of the FxFG binding site.
TL;DR: Despite the diversity of substrate specificities, members of the GNAT superfamily demonstrate remarkable similarity in protein topology and mode of acetyl coenzyme A binding, likely reflecting a conserved catalytic mechanism.
Abstract: ▪ Abstract Hundreds of acetyltransferases exist. All use a common acetyl donor—acetyl coenzyme A—and each exhibits remarkable specificity for acetyl acceptors, which include small molecules and proteins. Analysis of the primary sequences of these enzymes indicates that they can be sorted into several superfamilies. This review covers the three-dimensional structures of members of one of these superfamilies, now referred to in the literature as the GCN5-related N-acetyltransferases (GNAT), reflecting the importance of one functional category, the histone acetyltransferases. Despite the diversity of substrate specificities, members of the GNAT superfamily demonstrate remarkable similarity in protein topology and mode of acetyl coenzyme A binding, likely reflecting a conserved catalytic mechanism.
TL;DR: The G x U wobble base pair is a fundamental unit of RNA secondary structure that is present in nearly every class of RNA from organisms of all three phylogenetic domains and has comparable thermodynamic stability to Watson-Crick base pairs and is nearly isomorphic to them.
Abstract: The G·U wobble base pair is a fundamental unit of RNA secondary structure that is present in nearly every class of RNA from organisms of all three phylogenetic domains. It has comparable thermodynamic stability to Watson–Crick base pairs and is nearly isomorphic to them. Therefore, it often substitutes for G·C or A·U base pairs. The G·U wobble base pair also has unique chemical, structural, dynamic and ligand-binding properties, which can only be partially mimicked by Watson–Crick base pairs or other mispairs. These features mark sites containing G·U pairs for recognition by proteins and other RNAs and allow the wobble pair to play essential functional roles in a remarkably wide range of biological processes.
TL;DR: An atomic model for structural changes involved in the vectorial, light-driven transport of protons by bacteriorhodopsin is presented, which provides an ‘opening’ of the protein to protons on the cytoplasmic side of the membrane.
Abstract: Bacteriorhodopsin, a membrane protein with a relative molecular mass of 27,000, is a light driven pump which transports protons across the cell membrane of the halophilic organism Halobacterium salinarum. The chromophore retinal is covalently attached to the protein via a protonated Schiff base. Upon illumination, retinal is isomerized. The Schiff base then releases a proton to the extracellular medium, and is subsequently reprotonated from the cytoplasm. An atomic model for bacteriorhodopsin was first determined by Henderson et al1, and has been confirmed and extended by work in a number of laboratories in the last few years2. Here we present an atomic model for structural changes involved in the vectorial, light-driven transport of protons by bacteriorhodopsin. A ‘switch’ mechanism ensures the vectorial nature of pumping. First, retinal unbends, triggered by loss of the Schiff base proton, and second, a protein conformational change occurs. This conformational change, which we have determined by electron crystallography at atomic (3.2 A in-plane and 3.6 A vertical) resolution, is largely localized to helices F and G, and provides an ‘opening’ of the protein to protons on the cytoplasmic side of the membrane.
TL;DR: It is demonstrated that during a dynamic type 2 cytokine disease process IL-13 is detrimental to survival following infection, whereas IL-4 is beneficial.
Abstract: Experimental Schistosoma mansoni infections of mice lead to a dynamic type 2 cytokine-mediated pathological process. We have used IL-4-deficient, IL-13-deficient, and IL-4/13-deficient mice to dissect the role of these cytokines in the development of immune response and pathology following S. mansoni infection. We demonstrate that while both of these cytokines are necessary to develop a robust Th2 cell-driven, eosinophil-rich granuloma response, they also perform disparate functions that identify novel sites for therapeutic intervention. IL-13-deficient mice demonstrated significantly enhanced survival following infection, which correlated with reduced hepatic fibrosis. In contrast, increased mortality was manifest in IL-4-deficient and IL-4/13-deficient mice, and this correlated with hepatocyte damage and intestinal pathology. Therefore, we demonstrate that during a dynamic type 2 cytokine disease process IL-13 is detrimental to survival following infection, whereas IL-4 is beneficial.
TL;DR: A physiological and heretofore unrecognized role for mitochondrial oxidant release is described and it is suggested that although chronic oxidant production may have deleterious effects, mitochondrial oxidants can also function acutely as signaling molecules to provide communication between the mitochondria and the cytosol.
Abstract: Leakage of mitochondrial oxidants contributes to a variety of harmful conditions ranging from neurodegenerative diseases to cellular senescence. We describe here, however, a physiological and heretofore unrecognized role for mitochondrial oxidant release. Mitochondrial metabolism of pyruvate is demonstrated to activate the c-Jun N-terminal kinase (JNK). This metabolite-induced rise in cytosolic JNK1 activity is shown to be triggered by increased release of mitochondrial H2O2. We further demonstrate that in turn, the redox-dependent activation of JNK1 feeds back and inhibits the activity of the metabolic enzymes glycogen synthase kinase 3β and glycogen synthase. As such, these results demonstrate a novel metabolic regulatory pathway activated by mitochondrial oxidants. In addition, they suggest that although chronic oxidant production may have deleterious effects, mitochondrial oxidants can also function acutely as signaling molecules to provide communication between the mitochondria and the cytosol.