Showing papers by "Laboratory of Molecular Biology published in 1999"
TL;DR: In this paper, the authors performed an analysis of the recognition sites seen in 75 protein-protein complexes of known three-dimensional structure: 24 protease-inhibitor, 19 antibody-antigen and 32 other complexes, including nine enzymeinhibitors and 11 that are involved in signal transduction.
1,945 citations
TL;DR: These equations highlight the importance of the contribution of the X-ray background to the standard error and give an estimate of the improvement which can be achieved by profile fitting.
Abstract: The objective of any modern data-processing program is to produce from a set of diffraction images a set of indices (hkls) with their associated intensities (and estimates of their uncertainties), together with an accurate estimate of the crystal unit-cell parameters. This procedure should not only be reliable, but should involve an absolute minimum of user intervention. The process can be conveniently divided into three stages. The first (autoindexing) determines the unit-cell parameters and the orientation of the crystal. The unit-cell parameters may indicate the likely Laue group of the crystal. The second step is to refine the initial estimate of the unit-cell parameters and also the crystal mosaicity using a procedure known as post-refinement. The third step is to integrate the images, which consists of predicting the positions of the Bragg reflections on each image and obtaining an estimate of the intensity of each reflection and its uncertainty. This is carried out while simultaneously refining various detector and crystal parameters. Basic features of the algorithms employed for each of these three separate steps are described, principally with reference to the program MOSFLM.
1,330 citations
TL;DR: These findings represent the first germline loss-of-function mutations in PPARγ and provide compelling genetic evidence that this receptor is important in the control of insulin sensitivity, glucose homeostasis and blood pressure in man.
Abstract: Thiazolidinediones are a new class of antidiabetic agent that improve insulin sensitivity and reduce plasma glucose and blood pressure in subjects with type 2 diabetes. Although these agents can bind and activate an orphan nuclear receptor, peroxisome proliferator-activated receptor gamma (PPARgamma), there is no direct evidence to conclusively implicate this receptor in the regulation of mammalian glucose homeostasis. Here we report two different heterozygous mutations in the ligand-binding domain of PPARgamma in three subjects with severe insulin resistance. In the PPARgamma crystal structure, the mutations destabilize helix 12 which mediates transactivation. Consistent with this, both receptor mutants are markedly transcriptionally impaired and, moreover, are able to inhibit the action of coexpressed wild-type PPARgamma in a dominant negative manner. In addition to insulin resistance, all three subjects developed type 2 diabetes mellitus and hypertension at an unusually early age. Our findings represent the first germline loss-of-function mutations in PPARgamma and provide compelling genetic evidence that this receptor is important in the control of insulin sensitivity, glucose homeostasis and blood pressure in man.
1,320 citations
TL;DR: An electron density map obtained from crystals of a subcomplex of yeast mitochondrial ATP synthase shows a ring of 10 c subunits whose extensive contact between the c ring and the stalk suggests that they may rotate as an ensemble during catalysis.
Abstract: Adenosine triphosphate (ATP) synthase contains a rotary motor involved in biological energy conversion. Its membrane-embedded F 0 sector has a rotation generator fueled by the proton-motive force, which provides the energy required for the synthesis of ATP by the F 1 domain. An electron density map obtained from crystals of a subcomplex of yeast mitochondrial ATP synthase shows a ring of 10 c subunits. Each c subunit forms an α-helical hairpin. The interhelical loops of six to seven of the c subunits are in close contact with the γ and δ subunits of the central stalk. The extensive contact between the c ring and the stalk suggests that they may rotate as an ensemble during catalysis.
1,244 citations
TL;DR: The kinetics of partitioning between chaperones and proteases determines whether a protein will be destroyed before it folds properly, and when both quality control options fail, damaged proteins accumulate as aggregates, a process associated with amyloid diseases.
Abstract: Polypeptides emerging from the ribosome must fold into stable three-dimensional structures and maintain that structure throughout their functional lifetimes. Maintaining quality control over protein structure and function depends on molecular chaperones and proteases, both of which can recognize hydrophobic regions exposed on unfolded polypeptides. Molecular chaperones promote proper protein folding and prevent aggregation, and energy-dependent proteases eliminate irreversibly damaged proteins. The kinetics of partitioning between chaperones and proteases determines whether a protein will be destroyed before it folds properly. When both quality control options fail, damaged proteins accumulate as aggregates, a process associated with amyloid diseases.
1,099 citations
TL;DR: Transplantation in a rat model of a human myelin disease shows that ES cell-derived precursors interact with host neurons and efficiently myelinate axons in brain and spinal cord.
Abstract: Self-renewing, totipotent embryonic stem (ES) cells may provide a virtually unlimited donor source for transplantation. A protocol that permits the in vitro generation of precursors for oligodendrocytes and astrocytes from ES cells was devised. Transplantation in a rat model of a human myelin disease shows that these ES cell-derived precursors interact with host neurons and efficiently myelinate axons in brain and spinal cord. Thus, ES cells can serve as a valuable source of cell type-specific somatic precursors for neural transplantation.
1,040 citations
TL;DR: Fibrates inhibit the vascular inflammatory response via PPARα by interfering with the NF-κB and AP-1 transactivation capacity involving direct protein-protein interaction with p65 and c-Jun.
1,030 citations
TL;DR: It is shown that reducing corticosteroid levels in aged rats restored the rate of cell proliferation, resulting in increased numbers of new granule neurons, indicating that the neuronal precursor population in the dentate gyrus remains stable into old age, but that neurogenesis is normally slowed by high levels of Corticosteroids.
Abstract: The production of hippocampal granule neurons continues throughout adulthood but dramatically decreases in old age Here we show that reducing corticosteroid levels in aged rats restored the rate of cell proliferation, resulting in increased numbers of new granule neurons This result indicates that the neuronal precursor population in the dentate gyrus remains stable into old age, but that neurogenesis is normally slowed by high levels of corticosteroids The findings further suggest that decreased neurogenesis may contribute to age-related memory deficits associated with high corticosteroids, and that these deficits may be reversible
733 citations
TL;DR: The properly mutated C‐terminus of Bax can target a non‐relevant protein to the mitochondria, showing that specific conformations of this domain alone allow mitochondrial docking.
Abstract: Bax, a pro-apoptotic member of the Bcl-2 family, translocates from the cytosol to the mitochondria during programmed cell death. We report here that both gain-of-function and loss-of-function mutations can be achieved by altering a single amino acid in the Bax hydrophobic C-terminus. The properly mutated C-terminus of Bax can target a non-relevant protein to the mitochondria, showing that specific conformations of this domain alone allow mitochondrial docking. These data along with N-terminus epitope exposure experiments suggest that the C- and the N-termini interact and that upon triggering of apoptosis, Bax changes conformation, exposing these two domains to insert into the mitochondria and regulate the cell death machinery.
728 citations
TL;DR: The crystal structure of the T = 4 capsid has been solved at 3.3 A resolution, revealing a largely helical protein fold that is unusual for icosahedral viruses.
615 citations
TL;DR: Observations suggest that the core of HIV is organized on the principles of a fullerene cone, in analogy to structures recently observed for elemental carbon.
Abstract: The genome of the human immunodeficiency virus (HIV) is packaged within an unusual conical core particle located at the center of the infectious virion. The core is composed of a complex of the NC (nucleocapsid) protein and genomic RNA, surrounded by a shell of the CA (capsid) protein. A method was developed for assembling cones in vitro using pure recombinant HIV-1 CA-NC fusion proteins and RNA templates. These synthetic cores are capped at both ends and appear similar in size and morphology to authentic viral cores. It is proposed that both viral and synthetic cores are organized on conical hexagonal lattices, which by Euler's theorem requires quantization of their cone angles. Electron microscopic analyses revealed that the cone angles of synthetic cores were indeed quantized into the five allowed angles. The viral core and most synthetic cones exhibited cone angles of approximately 19 degrees (the narrowest of the allowed angles). These observations suggest that the core of HIV is organized on the principles of a fullerene cone, in analogy to structures recently observed for elemental carbon.
TL;DR: This analysis shows that, if the surface water molecules are included in the calculations, the overall packing efficiency throughout the protein interior is high and fairly uniform, but if the water structure is removed, the packing efficiency in peripheral regions of theprotein interior is underestimated.
TL;DR: It is shown that mice with targeted deletion of the SAP gene spontaneously develop antinuclear autoimmunity and severe glomerulonephritis, a phenotype resembling human systemic lupus erythematosus, a serious autoimmune disease, and that degradation of long chromatin is retarded in the presence of SAP both in vitro and in vivo.
Abstract: Serum amyloid P component (SAP), a highly conserved plasma protein named for its universal presence in amyloid deposits1, is the single normal circulating protein that shows specific calcium-dependent binding to DNA and chromatin in physiological conditions2,3. The avid binding of SAP displaces H1-type histones and thereby solubilizes native long chromatin, which is otherwise profoundly insoluble at the physiological ionic strength of extracellular fluids4. Furthermore, SAP binds in vivo both to apoptotic cells5, the surface blebs of which bear chromatin fragments6, and to nuclear debris released by necrosis7. SAP may therefore participate in handling of chromatin exposed by cell death2,3,4,7. Here we show that mice with targeted deletion of the SAP gene8 spontaneously develop antinuclear autoimmunity and severe glomerulonephritis, a phenotype resembling human systemic lupus erythematosus, a serious autoimmune disease. The SAP–/– mice also have enhanced anti-DNA responses to immunization with extrinsic chromatin, and we demonstrate that degradation of long chromatin is retarded in the presence of SAP both in vitro and in vivo. These findings indicate that SAP has an important physiological role, inhibiting the formation of pathogenic autoantibodies against chromatin and DNA, probably by binding to chromatin and regulating its degradation.
TL;DR: It is suggested that the extracellular tunnels are access routes to the binding pockets for ACh, and that the cytoplasmic openings serve as filters to exclude anions and other impermeant species from the vicinity of the pore.
TL;DR: It is concluded that survivin transcript is a defining diagnostic marker for NSCLC that may also yield prognostic information and, as an apoptosis inhibitor, be an important target in cancer therapy.
Abstract: PURPOSE: The survivin gene is a novel apoptosis inhibitor, related to the baculovirus gene, which is believed to play a pivotal role in fetal development and in cancer. We hypothesised that survivin would be expressed in tumors of patients with non–small-cell lung cancer (NSCLC), and we attempted to determine the influence of survivin re-expression on clinical outcome in patients with up to stage IIIA NSCLC who had undergone radical surgery. METHODS: We designed a reverse transcriptase polymerase chain reaction (RT-PCR) assay to study the expression of the survivin gene in 83 NSCLC tumor samples and compared the results with relevant clinical and pathologic data. RESULTS: The RT-PCR identified survivin gene transcript in 71 (85.5%) of the tumor samples and in only 10 (12%) of the paired, histopathologically normal lung samples. There was no relationship between histologic subtype (squamous v nonsquamous) and survivin gene expression. The 12 patients without survivin expression had significantly better ove...
TL;DR: The structure of a complex of the HIV-1 integrase core domain with a novel inhibitor, 5ClTEP, 1-(5-chloroindol-3-yl)-3-hydroxy-3-(2H-tetrazol-5-yl)pro penone, to 2.1-A resolution is determined.
Abstract: HIV integrase, the enzyme that inserts the viral DNA into the host chromosome, has no mammalian counterpart, making it an attractive target for antiviral drug design. As one of the three enzymes produced by HIV, it can be expected that inhibitors of this enzyme will complement the therapeutic use of HIV protease and reverse transcriptase inhibitors. We have determined the structure of a complex of the HIV-1 integrase core domain with a novel inhibitor, 5ClTEP, 1-(5-chloroindol-3-yl)-3-hydroxy-3-(2H-tetrazol-5-yl)-pro penone, to 2.1-A resolution. The inhibitor binds centrally in the active site of the integrase and makes a number of close contacts with the protein. Only minor changes in the protein accompany inhibitor binding. This inhibitor complex will provide a platform for structure-based design of an additional class of inhibitors for antiviral therapy.
TL;DR: It is shown by biochemical analysis that Sprouty is an intracellular protein, associated with the inner surface of the plasma membrane, and is a widespread inhibitor of Ras pathway signal transduction.
TL;DR: A novel Smad-interacting protein, SIP1, which was identified using the yeast two-hybrid system and is likely to be a transcriptional repressor, which may be involved in the regulation of at least one immediate response gene for activin-dependent signal transduction pathways.
TL;DR: The 2.2 Å X-ray crystallographic structure of the catalytic subunit of PI3Kγ, the class I enzyme that is activated by heterotrimeric G-protein βγ subunits and Ras is reported.
Abstract: Phosphoinositide 3-kinases (PI3Ks) are ubiquitous lipid kinases that function both as signal transducers downstream of cell-surface receptors and in constitutive intracellular membrane and protein trafficking pathways. All PI3Ks are dual-specificity enzymes with a lipid kinase activity which phosphorylates phosphoinositides at the 3-hydroxyl, and a protein kinase activity. The products of PI3K-catalysed reactions, phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3), PtdIns(3,4)P2 and PtdIns(3)P, are second messengers in a variety of signal transduction pathways, including those essential to cell proliferation, adhesion, survival, cytoskeletal rearrangement and vesicle trafficking. Here we report the 2.2 A X-ray crystallographic structure of the catalytic subunit of PI3Kgamma, the class I enzyme that is activated by heterotrimeric G-protein betagamma subunits and Ras. PI3Kgamma has a modular organization centred around a helical-domain spine, with C2 and catalytic domains positioned to interact with phospholipid membranes, and a Ras-binding domain placed against the catalytic domain where it could drive allosteric activation of the enzyme.
TL;DR: A short account is given of the physical and chemical studies that have led to an understanding of the structure of the tobacco mosaic virus particle and how it is assembled from its constituent coat protein and RNA.
Abstract: A short account is given of the physical and chemical studies that have led to an understanding of the structure of the tobacco mosaic virus particle and how it is assembled from its constituent coat protein and RNA. The assembly is a much more complex process than might have been expected from the simplicity of the helical design of the particle. The protein forms an obligatory intermediate (a cylindrical disk composed of two layers of protein units), which recognizes a specific RNA hairpin sequence. This extraordinary mechanism simultaneously fulfils the physical requirement for nucleating the growth of the helical particle and the biological requirement for specific recognition of the viral DNA.
TL;DR: Crystal structures suggest that the seven Sm proteins could form a closed ring and the snRNAs may be bound in the positively charged central hole.
TL;DR: The temperature‐dependent dissociation of the large storage form of Hsp26 into a smaller, active species and the subsequent re‐association to a defined large chaperone–substrate complex represents a novel mechanism for the functional activation of a molecular chaperones.
Abstract: Small heat shock proteins (sHsps) are a conserved protein family, with members found in all organisms analysed so far. Several sHsps have been shown to exhibit chaperone activity and protect proteins from irreversible aggregation in vitro. Here we show that Hsp26, an sHsp from Saccharomyces cerevisiae, is a temperature-regulated molecular chaperone. Like other sHsps, Hsp26 forms large oligomeric complexes. At heat shock temperatures, however, the 24mer chaperone complex dissociates. Interestingly, chaperone assays performed at different temperatures show that the dissociation of the Hsp26 complex at heat shock temperatures is a prerequisite for efficient chaperone activity. Binding of non-native proteins to dissociated Hsp26 produces large globular assemblies with a structure that appears to be completely reorganized relative to the original Hsp26 oligomers. In this complex one monomer of substrate is bound per Hsp26 dimer. The temperature-dependent dissociation of the large storage form of Hsp26 into a smaller, active species and the subsequent re-association to a defined large chaperone-substrate complex represents a novel mechanism for the functional activation of a molecular chaperone.
TL;DR: The frequency of tau mutations in a large population-based study of FTD carried out in the Netherlands from January 1994 to June 1998 is reported, finding an intronic mutation at position +33 after exon 9, which is likely to affect the alternative splicing of t Tau.
Abstract: Summary Mutations in microtubule-associated protein tau recently have been identified in familial cases of frontotemporal dementia (FTD). We report the frequency of tau mutations in a large population-based study of FTD carried out in the Netherlands from January 1994 to June 1998. Thirty-seven patients had ≥1 first-degree relative with dementia. A mutation in the tau gene was found in 17.8% of the group of patients with FTD and in 43% of patients with FTD who also had a positive family history of FTD. Three distinct missense mutations (G272V, P301L, R406W) accounted for 15.6% of the mutations. These three missense mutations, and a single amino acid deletion (ΔK280) that was detected in one patient, strongly reduce the ability of tau to promote microtubule assembly. We also found an intronic mutation at position +33 after exon 9, which is likely to affect the alternative splicing of tau. Tau mutations are responsible for a large proportion of familial FTD cases; however, there are also families with FTD in which no mutations in tau have been found, which indicates locus and/or allelic heterogeneity. The different tau mutations may result in disturbances in the interactions of the protein tau with microtubules, resulting in hyperphosphorylation of tau protein, assembly into filaments, and subsequent cell death.
TL;DR: Eight severe inherited neurodegenerative diseases are caused by expansion of glutamine repeats in the affected proteins, whereas proteins with repeats of more than 40 glutamine residues precipitate as insoluble fibres, apparently because of a structural transition associated with the increased length.
TL;DR: Data indicate that a novel, lengthwise (‘spring-like’) conformational change in a dynamin helix may participate in vesicle fission during GTP hydrolysis by dynamin.
Abstract: Nucleotide-dependent conformational changes in dynamin: evidence for
a mechanochemical molecular spring
TL;DR: The isolation of the gene for an enzyme that can hydrolyze the bond between this protein and DNA is described and this gene is conserved in eukaryotes and identifies a family of enzymes that has not been previously recognized.
Abstract: Covalent intermediates between topoisomerase I and DNA can become dead-end complexes that lead to cell death. Here, the isolation of the gene for an enzyme that can hydrolyze the bond between this protein and DNA is described. Enzyme-defective mutants of yeast are hypersensitive to treatments that increase the amount of covalent complexes, indicative of enzyme involvement in repair. The gene is conserved in eukaryotes and identifies a family of enzymes that has not been previously recognized. The presence of this gene in humans may have implications for the effectiveness of topoisomerase I poisons, such as the camptothecins, in chemotherapy.
TL;DR: It is proposed that α-synuclein modulates the phosphorylation of soluble axonal tau and thereby indirectly affects the stability of axonal microtubules.
TL;DR: A crystallographic analysis of the structure of the 30S ribosomal subunit from the bacterium Thermus thermophilus shows double-helical regions of RNA to be identified throughout the subunit, and all seven of the small-subunit proteins of known crystal structure to be positioned in the electron density map.
Abstract: The 30S ribosomal subunit binds messenger RNA and the anticodon stem-loop of transfer RNA during protein synthesis. A crystallographic analysis of the structure of the subunit from the bacterium Thermus thermophilus is presented. At a resolution of 5.5 A, the phosphate backbone of the ribosomal RNA is visible, as are the α-helices of the ribosomal proteins, enabling double-helical regions of RNA to be identified throughout the subunit, all seven of the small-subunit proteins of known crystal structure to be positioned in the electron density map, and the fold of the entire central domain of the small-subunit ribosomal RNA to be determined.
TL;DR: It is concluded that IL-4 and IL-13 cooperate to initiate rapid Th2 cell–driven responses, and that although their functions overlap, they perform additive roles.
Abstract: Using a single vector targeting strategy, we have generated mice with a combined deficiency of interleukin (IL)-4 and IL-13 to clarify their roles in T helper type 2 (Th2) cell responses. Using immunological challenges normally characterized by a Th2-like response, we have compared the responses of the double-deficient mice with those generated by wild-type, IL-4-deficient, and IL-13-deficient mice. Using a pulmonary granuloma model, induced with Schistosoma mansoni eggs, we demonstrate that although eosinophil infiltration, immunoglobulin E, and IL-5 production are reduced in the IL-4-deficient mice and IL-13-deficient mice, they are abolished only in the combined absence of both cytokines. Furthermore, IL-4/13-deficient animals are severely impaired in their ability to expel the gastrointestinal nematode Nippostrongylus brasiliensis. Unexpectedly, N. brasiliensis-infected IL-4/13-deficient mice developed elevated IL-5 and eosinophilia, indicating that compensatory mechanisms exist for the expression of IL-5, although serum IgE remained undetectable. IL-4/13-deficient mice default to a Th1-like phenotype characterized by the expression of interferon gamma and the production of IgG2a and IgG2b. We conclude that IL-4 and IL-13 cooperate to initiate rapid Th2 cell-driven responses, and that although their functions overlap, they perform additive roles.
TL;DR: A comparison of the structural and kinetic results reveals that these enzymes frequently exhibit allosteric interactions that synchronize the reactions to prevent the build-up of excess intermediate (12–16).