Showing papers by "Laboratory of Molecular Biology published in 2008"

Structure of a beta1-adrenergic G-protein-coupled receptor.

Abstract: G-protein-coupled receptors have a major role in transmembrane signalling in most eukaryotes and many are important drug targets. Here we report the 2.7 A resolution crystal structure of a beta(1)-adrenergic receptor in complex with the high-affinity antagonist cyanopindolol. The modified turkey (Meleagris gallopavo) receptor was selected to be in its antagonist conformation and its thermostability improved by earlier limited mutagenesis. The ligand-binding pocket comprises 15 side chains from amino acid residues in 4 transmembrane alpha-helices and extracellular loop 2. This loop defines the entrance of the ligand-binding pocket and is stabilized by two disulphide bonds and a sodium ion. Binding of cyanopindolol to the beta(1)-adrenergic receptor and binding of carazolol to the beta(2)-adrenergic receptor involve similar interactions. A short well-defined helix in cytoplasmic loop 2, not observed in either rhodopsin or the beta(2)-adrenergic receptor, directly interacts by means of a tyrosine with the highly conserved DRY motif at the end of helix 3 that is essential for receptor activation.

Mechanisms of membrane fusion: disparate players and common principles

Abstract: Membrane fusion can occur between cells, between different intracellular compartments, between intracellular compartments and the plasma membrane and between lipid-bound structures such as viral particles and cellular membranes In order for membranes to fuse they must first be brought together The more highly curved a membrane is, the more fusogenic it becomes We discuss how proteins, including SNAREs, synaptotagmins and viral fusion proteins, might mediate close membrane apposition and induction of membrane curvature to drive diverse fusion processes We also highlight common principles that can be derived from the analysis of the role of these proteins

Action potential generation requires a high sodium channel density in the axon initial segment.

Abstract: The axon initial segment (AIS) is a specialized region in neurons where action potentials are initiated. It is commonly assumed that this process requires a high density of voltage-gated sodium (Na(+)) channels. Paradoxically, the results of patch-clamp studies suggest that the Na(+) channel density at the AIS is similar to that at the soma and proximal dendrites. Here we provide data obtained by antibody staining, whole-cell voltage-clamp and Na(+) imaging, together with modeling, which indicate that the Na(+) channel density at the AIS of cortical pyramidal neurons is approximately 50 times that in the proximal dendrites. Anchoring of Na(+) channels to the cytoskeleton can explain this discrepancy, as disruption of the actin cytoskeleton increased the Na(+) current measured in patches from the AIS. Computational models required a high Na(+) channel density (approximately 2,500 pS microm(-2)) at the AIS to account for observations on action potential generation and backpropagation. In conclusion, action potential generation requires a high Na(+) channel density at the AIS, which is maintained by tight anchoring to the actin cytoskeleton.

Structural Biology of the Tumor Suppressor p53

Abstract: The tumor suppressor protein p53 induces or represses the expression of a variety of target genes involved in cell cycle control, senescence, and apoptosis in response to oncogenic or other cellular stress signals. It exerts its function as guardian of the genome through an intricate interplay of independently folded and intrinsically disordered functional domains. In this review, we provide insights into the structural complexity of p53, the molecular mechanisms of its inactivation in cancer, and therapeutic strategies for the pharmacological rescue of p53 function in tumors. p53 emerges as a paradigm for a more general understanding of the structural organization of modular proteins and the effects of disease-causing mutations.

Genetically encoding N|[epsi]|-acetyllysine in recombinant proteins

Abstract: N(epsilon)-acetylation of lysine (1) is a reversible post-translational modification with a regulatory role that rivals that of phosphorylation in eukaryotes. No general methods exist to synthesize proteins containing N(epsilon)-acetyllysine (2) at defined sites. Here we demonstrate the site-specific incorporation of N(epsilon)-acetyllysine in recombinant proteins produced in Escherichia coli via the evolution of an orthogonal N(epsilon)-acetyllysyl-tRNA synthetase/tRNA(CUA) pair. This strategy should find wide applications in defining the cellular role of this modification.

Tight regulation of unstructured proteins: from transcript synthesis to protein degradation.

Abstract: Altered abundance of several intrinsically unstructured proteins (IUPs) has been associated with perturbed cellular signaling that may lead to pathological conditions such as cancer. Therefore, it is important to understand how cells precisely regulate the availability of IUPs. We observed that regulation of transcript clearance, proteolytic degradation, and translational rate contribute to controlling the abundance of IUPs, some of which are present in low amounts and for short periods of time. Abundant phosphorylation and low stochasticity in transcription and translation indicate that the availability of IUPs can be finely tuned. Fidelity in signaling may require that most IUPs be available in appropriate amounts and not present longer than needed.

Conformational thermostabilization of the β1-adrenergic receptor in a detergent-resistant form

Abstract: There are ≈350 non-odorant G protein-coupled receptors (GPCRs) encoded by the human genome, many of which are predicted to be potential therapeutic targets, but there are only two structures available to represent the whole of the family. We hypothesized that improving the detergent stability of these receptors and simultaneously locking them into one preferred conformation will greatly improve the chances of crystallization. We developed a generic strategy for the isolation of detergent-solubilized thermostable mutants of a GPCR, the β1-adrenergic receptor. The most stable mutant receptor, βAR-m23, contained six point mutations that led to an apparent Tm 21°C higher than the native protein, and, in the presence of bound antagonist, βAR-m23 was as stable as bovine rhodopsin. In addition, βAR-m23 was significantly more stable in a wide range of detergents ideal for crystallization and was preferentially in an antagonist conformation in the absence of ligand.

Targeted polypharmacology: discovery of dual inhibitors of tyrosine and phosphoinositide kinases

Abstract: The clinical success of multitargeted kinase inhibitors has stimulated efforts to identify promiscuous drugs with optimal selectivity profiles. It remains unclear to what extent such drugs can be rationally designed, particularly for combinations of targets that are structurally divergent. Here we report the systematic discovery of molecules that potently inhibit both tyrosine kinases and phosphatidylinositol-3-OH kinases, two protein families that are among the most intensely pursued cancer drug targets. Through iterative chemical synthesis, X-ray crystallography and kinome-level biochemical profiling, we identified compounds that inhibit a spectrum of new target combinations in these two families. Crystal structures revealed that the dual selectivity of these molecules is controlled by a hydrophobic pocket conserved in both enzyme classes and accessible through a rotatable bond in the drug skeleton. We show that one compound, PP121, blocks the proliferation of tumor cells by direct inhibition of oncogenic tyrosine kinases and phosphatidylinositol-3-OH kinases. These molecules demonstrate the feasibility of accessing a chemical space that intersects two families of oncogenes.

IL-33 Induces Antigen-Specific IL-5 + T Cells and Promotes Allergic-Induced Airway Inflammation Independent of IL-4

Abstract: Type 2 cytokines (IL-4, IL-5, and IL-13) play a pivotal role in helminthic infection and allergic disorders. CD4+ T cells which produce type 2 cytokines can be generated via IL-4-dependent and -independent pathways. Although the IL-4-dependent pathway is well documented, factors that drive IL-4-independent Th2 cell differentiation remain obscure. We report here that the new cytokine IL-33, in the presence of Ag, polarizes murine and human naive CD4+ T cells into a population of T cells which produce mainly IL-5 but not IL-4. This polarization requires IL-1R-related molecule and MyD88 but not IL-4 or STAT6. The IL-33-induced T cell differentiation is also dependent on the phosphorylation of MAPKs and NF-κB but not the induction of GATA3 or T-bet. In vivo, ST2−/− mice developed attenuated airway inflammation and IL-5 production in a murine model of asthma. Conversely, IL-33 administration induced the IL-5-producing T cells and exacerbated allergen-induced airway inflammation in wild-type as well as IL-4−/− mice. Finally, adoptive transfer of IL-33-polarized IL-5+IL-4−T cells triggered airway inflammation in naive IL-4−/− mice. Thus, we demonstrate here that, in the presence of Ag, IL-33 induces IL-5-producing T cells and promotes airway inflammation independent of IL-4.

Assembly reflects evolution of protein complexes

Abstract: A homomer is formed by self-interacting copies of a protein unit. This is functionally important, as in allostery, and structurally crucial because mis-assembly of homomers is implicated in disease. Homomers are widespread, with 50-70% of proteins with a known quaternary state assembling into such structures. Despite their prevalence, their role in the evolution of cellular machinery and the potential for their use in the design of new molecular machines, little is known about the mechanisms that drive formation of homomers at the level of evolution and assembly in the cell. Here we present an analysis of over 5,000 unique atomic structures and show that the quaternary structure of homomers is conserved in over 70% of protein pairs sharing as little as 30% sequence identity. Where quaternary structure is not conserved among the members of a protein family, a detailed investigation revealed well-defined evolutionary pathways by which proteins transit between different quaternary structure types. Furthermore, we show by perturbing subunit interfaces within complexes and by mass spectrometry analysis, that the (dis)assembly pathway mimics the evolutionary pathway. These data represent a molecular analogy to Haeckel's evolutionary paradigm of embryonic development, where an intermediate in the assembly of a complex represents a form that appeared in its own evolutionary history. Our model of self-assembly allows reliable prediction of evolution and assembly of a complex solely from its crystal structure.

cAMP-dependent signaling as a core component of the mammalian circadian pacemaker.

Abstract: The mammalian circadian clockwork is modeled as transcriptional and posttranslational feedback loops, whereby circadian genes are periodically suppressed by their protein products. We show that adenosine 3',5'-monophosphate (cAMP) signaling constitutes an additional, bona fide component of the oscillatory network. cAMP signaling is rhythmic and sustains the transcriptional loop of the suprachiasmatic nucleus, determining canonical pacemaker properties of amplitude, phase, and period. This role is general and is evident in peripheral mammalian tissues and cell lines, which reveals an unanticipated point of circadian regulation in mammals qualitatively different from the existing transcriptional feedback model. We propose that daily activation of cAMP signaling, driven by the transcriptional oscillator, in turn sustains progression of transcriptional rhythms. In this way, clock output constitutes an input to subsequent cycles.

Direct measurement of somatic voltage clamp errors in central neurons.

Abstract: The somatic voltage clamp technique has revolutionized understanding of synaptic physiology and the excitability of neurons. Although computer simulations have indicated that the somatic voltage clamp poorly controls voltage in the dendritic tree of neurons, where the majority of synaptic contacts are made, there has not been an experimental description of the performance of the somatic voltage clamp. Here, we directly quantify errors in the measurement of dendritic synaptic input by the somatic voltage clamp using simultaneous whole-cell recordings from the soma and apical dendrite of rat neocortical pyramidal neurons. The somatic voltage clamp did not control voltage at sites other than the soma and distorted measurement of the amplitude, kinetics, slope conductance and reversal potential of synaptic inputs in a dendritic distance-dependent manner. These errors question the use of the somatic voltage clamp as a quantitative tool in dendritic neurons.

Mediation, Modulation, and Consequences of Membrane-Cytoskeleton Interactions

Abstract: Elements of the cytoskeleton interact intimately and communicate bidirectionally with cellular membranes. Such interactions are critical for a host of cellular processes. Here we focus on the many types of interactions that exist between the cytoskeleton and the plasma membrane to illustrate why these cellular components can never truly be studied in isolation in vivo. We discuss how membranecytoskeleton interactions are mediated and modulated, and how many proteins involved in these interactions are disrupted in human disease. We then highlight key molecular and physical variables that must be considered in order to mechanistically dissect events associated with changes in plasma membrane morphology. These considerations are integrated into the context of cell migration, filopodia formation, and clathrin-mediated endocytosis to show how a holistic view of the plasma membrane-cytoskeleton interface can allow for the appropriate interpretation of experimental findings and provide novel mechanistic insight into these important cellular events.

Nucleosome repeat length and linker histone stoichiometry determine chromatin fiber structure

Abstract: To understand how nuclear processes involving DNA are regulated, knowledge of the determinants of chromatin condensation is required. From recent structural studies it has been concluded that the formation of the 30-nm chromatin fiber does not require the linker histone. Here, by comparing the linker histone-dependent compaction of long, reconstituted nucleosome arrays with different nucleosome repeat lengths (NRLs), 167 and 197 bp, we establish that the compaction behavior is both NRL- and linker histone-dependent. Only the 197-bp NRL array can form 30-nm higher-order chromatin structure. Importantly for understanding the regulation of compaction, this array shows a cooperative linker histone-dependent compaction. The 167-bp NRL array displays a limited linker histone-dependent compaction, resulting in a thinner and topologically different fiber. These observations provide an explanation for the distribution of NRLs found in nature.

A Role for the ESCRT System in Cell Division in Archaea

Abstract: Archaea are prokaryotic organisms that lack endomembrane structures. However, a number of hyperthermophilic members of the Kingdom Crenarchaea, including members of the Sulfolobus genus, encode homologs of the eukaryotic endosomal sorting system components Vps4 and ESCRT-III (endosomal sorting complex required for transport–III). We found that Sulfolobus ESCRT-III and Vps4 homologs underwent regulation of their expression during the cell cycle. The proteins interacted and we established the structural basis of this interaction. Furthermore, these proteins specifically localized to the mid-cell during cell division. Overexpression of a catalytically inactive mutant Vps4 in Sulfolobus resulted in the accumulation of enlarged cells, indicative of failed cell division. Thus, the archaeal ESCRT system plays a key role in cell division.

DBD--taxonomically broad transcription factor predictions: new content and functionality.

Abstract: DNA-binding domain (DBD) is a database of predicted sequence-specific DNA-binding transcription factors (TFs) for all publicly available proteomes. The proteomes have increased from 150 in the initial version of DBD to over 700 in the current version. All predicted TFs must contain a significant match to a hidden Markov model representing a sequence-specific DNA-binding domain family. Access to TF predictions is provided through http://transcriptionfactor.org, where new search options are now provided such as searching by gene names in model organisms, searching for all proteins in a particular DBD family and specific organism. We illustrate the application of this type of search facility by contrasting trends of DBD family occurrence throughout the tree of life, highlighting the clear partition between eukaryotic and prokaryotic DBD expansions. The website content has been expanded to include dedicated pages for each TF containing domain assignment details, gene names, links to external databases and links to TFs with similar domain arrangements. We compare the increase in number of predicted TFs with proteome size in eukaryotes and prokaryotes. Eukaryotes follow a slower rate of increase in TFs than prokaryotes, which could be due to the presence of splice variants or an increase in combinatorial control.

A structural explanation for the binding of endocytic dileucine motifs by the AP2 complex

Abstract: The movement of most transmembrane proteins between the various membranes of the cell is directed by short linear amino acid sequences in their cytoplasmic portions. These sequence motifs are bound by protein components of transport vesicle coats thereby incorporating the transmembrane proteins as vesicle cargo. In the case of clathrin-coated vesicles (CCVs) the motifs are recognised by the AP complexes or GGA clathrin adaptors. The clathrin adaptor AP2 recognizes two major classes of endocytic motifs including an acidic dileucine motif. In this study, Kelly et al. present the crystal structure of AP2 in a complex with the dileucine motif of a cargo protein thereby revealing the mechanism of cargo-adaptor recognition. During clathrin-mediated endocytosis, cargo proteins are recognized by clathrin adaptors. The clathrin adaptor AP2 recognizes two major classes of endocytic motifs, including an acidic dileucine motif. This study presents the crystal structure of AP2 in complex with the diceucine motif of a cargo protein, thereby revealing the mechanism of cargo–adaptor recognition. Most transmembrane proteins are selected as transport-vesicle cargo through the recognition of short, linear amino-acid motifs in their cytoplasmic portions by vesicle coat proteins. For clathrin-coated vesicles, the motifs are recognized by clathrin adaptors. The AP2 adaptor complex (subunits α, β2, μ2 and σ2) recognizes both major endocytic motifs: YxxΦ motifs1 (where Φ can be F, I, L, M or V) and [ED]xxxL[LI] acidic dileucine motifs. Here we describe the binding of AP2 to the endocytic dileucine motif from CD4 (ref. 2). The major recognition events are the two leucine residues binding in hydrophobic pockets on σ2. The hydrophilic residue four residues upstream from the first leucine sits on a positively charged patch made from residues on the σ2 and α subunits. Mutations in key residues inhibit the binding of AP2 to ‘acidic dileucine’ motifs displayed in liposomes containing phosphatidylinositol-4,5-bisphosphate, but do not affect binding to YxxΦ motifs through μ2. In the ‘inactive’ AP2 core structure3 both motif-binding sites are blocked by different parts of the β2 subunit. To allow a dileucine motif to bind, the β2 amino terminus is displaced and becomes disordered; however, in this structure the YxxΦ-binding site on μ2 remains blocked.

miR-122 targeting with LNA/2′-O-methyl oligonucleotide mixmers, peptide nucleic acids (PNA), and PNA–peptide conjugates

Abstract: MicroRNAs are small noncoding RNAs that regulate many cellular processes in a post-transcriptional mode. MicroRNA knockdown by antisense oligonucleotides is a useful strategy to explore microRNA functionality and as potential therapeutics. MicroRNA-122 (miR-122) is a liver-specific microRNA, the main function of which has been linked with lipid metabolism and liver homeostasis. Here, we show that lipofection of an antisense oligonucleotide based on a Locked Nucleic Acids (LNA)/2′-O-methyl mixmer or electroporation of a Peptide Nucleic Acid (PNA) oligomer is effective at blocking miR-122 activity in human and rat liver cells. These oligonucleotide analogs, evaluated for the first time in microRNA inhibition, are more effective than standard 2′-O-methyl oligonucleotides in binding and inhibiting microRNA action. We also show that microRNA inhibition can be achieved without the need for transfection or electroporation of the human or rat cell lines, by conjugation of an antisense PNA to the cell-penetrating peptide R6-Penetratin, or merely by linkage to just four Lys residues, highlighting the potential of PNA for future therapeutic applications as well as for studying microRNA function.

Structural Basis of CXCR4 Sulfotyrosine Recognition by the Chemokine SDF-1/CXCL12

Abstract: Stem cell homing and breast cancer metastasis are orchestrated by the chemokine stromal cell-derived factor 1 (SDF-1) and its receptor CXCR4. Here, we report the nuclear magnetic resonance structure of a constitutively dimeric SDF-1 in complex with a CXCR4 fragment that contains three sulfotyrosine residues important for a high-affinity ligand-receptor interaction. CXCR4 bridged the SDF-1 dimer interface so that sulfotyrosines sTyr7 and sTyr12 of CXCR4 occupied positively charged clefts on opposing chemokine subunits. Dimeric SDF-1 induced intracellular Ca2+ mobilization but had no chemotactic activity; instead, it prevented native SDF-1-induced chemotaxis, suggesting that it acted as a potent partial agonist. Our work elucidates the structural basis for sulfotyrosine recognition in the chemokine-receptor interaction and suggests a strategy for CXCR4-targeted drug development.

Crucial role of neutrophils in the development of mechanical inflammatory hypernociception

Abstract: Neutrophil migration is responsible for tissue damage observed in inflammatory diseases. Neutrophils are also implicated in inflammatory nociception, but mechanisms of their participation have not been elucidated. In the present study, we addressed these mechanisms in the carrageenan-induced mechanical hypernociception, which was determined using a modification of the Randall-Sellito test in rats. Neutrophil accumulation into the plantar tissue was determined by the contents of myeloperoxidase activity, whereas cytokines and PGE(2) levels were measured by ELISA and radioimmunoassay, respectively. The pretreatment of rats with fucoidin (a leukocyte adhesion inhibitor) inhibited carrageenan-induced hypernociception in a dose- and time-dependent manner. Inhibition of hypernociception by fucoidin was associated with prevention of neutrophil recruitment, as it did not inhibit the hypernociception induced by the direct-acting hypernociceptive mediators, PGE(2) and dopamine, which cause hypernociception, independent of neutrophils. Fucoidin had no effect on carrageenan-induced TNF-alpha, IL-1beta, and cytokine-induced neutrophil chemoattractant 1 (CINC-1)/CXCL1 production, suggesting that neutrophils were not the source of hypernociceptive cytokines. Conversely, hypernociception and neutrophil migration induced by TNF-alpha, IL-1beta, and CINC-1/CXCL1 was inhibited by fucoidin, suggesting that neutrophils are involved in the production of direct-acting hypernociceptive mediators. Indeed, neutrophils stimulated in vitro with IL-1beta produced PGE(2), and IL-1beta-induced PGE(2) production in the rat paw was inhibited by the pretreatment with fucoidin. In conclusion, during the inflammatory process, the migrating neutrophils participate in the cascade of events leading to mechanical hypernociception, at least by mediating the release of direct-acting hypernociceptive mediators, such as PGE(2). Therefore, the blockade of neutrophil migration could be a target to development of new analgesic drugs.

IL-9– and mast cell–mediated intestinal permeability predisposes to oral antigen hypersensitivity

Abstract: Previous mouse and clinical studies demonstrate a link between Th2 intestinal inflammation and induction of the effector phase of food allergy. However, the mechanism by which sensitization and mast cell responses occurs is largely unknown. We demonstrate that interleukin (IL)-9 has an important role in this process. IL-9–deficient mice fail to develop experimental oral antigen–induced intestinal anaphylaxis, and intestinal IL-9 overexpression induces an intestinal anaphylaxis phenotype (intestinal mastocytosis, intestinal permeability, and intravascular leakage). In addition, intestinal IL-9 overexpression predisposes to oral antigen sensitization, which requires mast cells and increased intestinal permeability. These observations demonstrate a central role for IL-9 and mast cells in experimental intestinal permeability in oral antigen sensitization and suggest that IL-9–mediated mast cell responses have an important role in food allergy.

Mitochondrial genomes reveal an explosive radiation of extinct and extant bears near the Miocene-Pliocene boundary

Abstract: Despite being one of the most studied families within the Carnivora, the phylogenetic relationships among the members of the bear family (Ursidae) have long remained unclear. Widely divergent topologies have been suggested based on various data sets and methods. We present a fully resolved phylogeny for ursids based on ten complete mitochondrial genome sequences from all eight living and two recently extinct bear species, the European cave bear (Ursus spelaeus) and the American giant short-faced bear (Arctodus simus). The mitogenomic data yield a well-resolved topology for ursids, with the sloth bear at the basal position within the genus Ursus. The sun bear is the sister taxon to both the American and Asian black bears, and this clade is the sister clade of cave bear, brown bear and polar bear confirming a recent study on bear mitochondrial genomes. Sequences from extinct bears represent the third and fourth Pleistocene species for which complete mitochondrial genomes have been sequenced. Moreover, the cave bear specimen demonstrates that mitogenomic studies can be applied to Pleistocene fossils that have not been preserved in permafrost, and therefore have a broad application within ancient DNA research. Molecular dating of the mtDNA divergence times suggests a rapid radiation of bears in both the Old and New Worlds around 5 million years ago, at the Miocene-Pliocene boundary. This coincides with major global changes, such as the Messinian crisis and the first opening of the Bering Strait, and suggests a global influence of such events on species radiations.