IAPs Regulate Distinct Innate Immune Pathways to Co-ordinate the Response to Bacterial Peptidoglycans

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Trichodysplasia spinulosa-Associated Polyomavirus Uses a Displaced Binding Site on VP1 to Engage Sialylated Glycolipids

Trichodysplasia spinulosa-associated Polyomavirus (TSPyV) was isolated from a patient suffering from trichodysplasia spinulosa, a skin disease that can appear in severely immunocompromised patients. While TSPyV is one of the five members of the polyomavirus family that are directly linked to a human disease, details about molecular recognition events, the viral entry pathway, and intracellular trafficking events during TSPyV infection remain unknown. Here we have used a structure-function approach to shed light on the first steps of TSPyV infection. We established by cell binding and pseudovirus infection studies that TSPyV interacts with sialic acids during attachment and/or entry. Subsequently, we solved high-resolution X-ray structures of the major capsid protein VP1 of TSPyV in complex with three different glycans, the branched GM1 glycan, and the linear trisaccharides α2,3- and α2,6-sialyllactose. The terminal sialic acid of all three glycans is engaged in a unique binding site on TSPyV VP1, which is positioned about 18 Å from established sialic acid binding sites of other polyomaviruses. Structure-based mutagenesis of sialic acid-binding residues leads to reduction in cell attachment and pseudovirus infection, demonstrating the physiological relevance of the TSPyV VP1-glycan interaction. Furthermore, treatments of cells with inhibitors of N-, O-linked glycosylation, and glycosphingolipid synthesis suggest that glycolipids play an important role during TSPyV infection. Our findings elucidate the first molecular recognition events of cellular infection with TSPyV and demonstrate that receptor recognition by polyomaviruses is highly variable not only in interactions with sialic acid itself, but also in the location of the binding site.     

The Discovery of a Novel Macrophage Binding Site

1. During the course of studies directed to determine the transport of Angiotensin II AT(2) receptors in the rat brain, we found that stab wounds to the brain revealed a binding site recognized by the AT(2) receptor ligand CGP42112 but not by Angiotensin II. 2. We localized this novel site to macrophages/microglia associated with physical or chemical injuries of the brain. 3. The non-Angiotensin II site was also highly localized to inflammatory lesions of peripheral arteries. 4. In rodent tissues, high binding expression was limited to the spleen and to circulating monocytes. A high-affinity binding site was also characterized in human monocytes. 5. Lack of affinity for many ligands binding to known macrophage receptors indicated the possibility that the non-Angiotensin II CGP42112 binding corresponds to a novel site.6. CGP42112 enhanced cell attachment to fibronectin and collagen and metalloproteinase-9 secretion from human monocytes incubated in serum-free medium but did not promote cytokine secretion. 7. When added in the presence of lipopolysaccharide, CGP42112 reduced the lipopolysaccharide-stimulated secretion of the pro-inflammatory cytokines TNF-alpha, IL-1, IL-1 beta, and IL-6, and increased protein kinase A. 8. Molecular modeling revealed that a CGP42112 derivative was selective for the novel macrophage site and did not recognize the Angiotensin II AT(2) receptor. 9. These results demonstrate that CGP42112, previously considered as a selective Angiotensin II AT(2) ligand, recognizes an additional non-Angiotensin II site different from AT(2) receptors. 10. Our observations indicate that CGP42112 or related molecules could be considered of interest as potential anti-inflammatory compounds.     

The First Nucleotide Binding Domain of Cystic Fibrosis Transmembrane Conductance Regulator Is a Site of Stable Nucleotide Interaction, whereas the Second Is a Site of Rapid Turnover

As in other adenine nucleotide binding cassette (ABC) proteins the nucleotide binding domains of the cystic fibrosis transmembrane conductance regulator (CFTR) bind and hydrolyze ATP and in some manner regulate CFTR ion channel gating. Unlike some other ABC proteins, however, there are preliminary indications that the two domains of CFTR are nonequivalent in their nucleotide interactions (Szabo, K., Szakacs, G., Hegeds, T., and Sarkadi, B. (1999) J. Biol. Chem. 274, 12209-12212; Aleksandrov, L., Mengos, A., Chang, X., Aleksandrov, A., and Riordan, J. R. (2001) J. Biol. Chem. 276, 12918-12923). We have now characterized the interactions of the 8-azido-photoactive analogues of ATP, ADP, and 5'-adenyl-beta,gamma-imidodiphosphate (AMP-PNP) with the two domains of functional membrane-bound CFTR. The results show that the two domains appear to act independently in the binding and hydrolysis of 8-azido-ATP. At NBD1 binding does not require a divalent cation. This binding is followed by minimal Mg(2+)-dependent hydrolysis and retention of the hydrolysis product, 8-azido-ADP, but not as a vanadate stabilized post-hydrolysis transition state complex. In contrast, at NBD2, MgN(3)ATP is hydrolyzed as rapidly as it is bound and the nucleoside diphosphate hydrolysis product dissociates immediately. Confirming this characterization of NBD1 as a site of more stable nucleotide interaction and NBD2 as a site of fast turnover, the non-hydrolyzable N(3)AMP-PNP bound preferentially to NBD1. This demonstration of NBD2 as the rapid nucleotide turnover site is consistent with the strong effect on channel gating kinetics of inactivation of this domain by mutagenesis.     

The Response of Three Bacterial Populations to Pollution in a Stream

Abstract Practical methods for biomonitoring of natural systems are still under development. Bacteria are potentially useful indicators of water quality because of their species diversity and ability to rapidly respond to changing environmental conditions. In this study, bacterial populations from unpolluted and polluted stream sites in two watersheds were compared to determine their suitability for use as environmental indicators. Upper Three Runs Creek and Four Mile Creek headwaters have had little anthropogenic disturbance, as opposed to lower Four Mile Creek which received thermal, radioactive, and chemical perturbations. Chemical and physical measurements provided evidence that seepage from holding ponds polluted Four Mile Creek. Polluted sites did not have altered total bacterial numbers but had decreased numbers of colony-forming units. Abundances of three bacterial species, Acinetobacter calcoaceticus, Burkholderia cepacia, and Pseudomonas putida, were determined by colony hybridization with species-specific rDNA probes. Contribution of A. calcoaceticus to the assemblage was higher at polluted sites, which indicated either tolerance of polluted conditions or the ability to utilize compounds existing at these sites to reach larger populations. No differences in B. cepacia populations were detected, and differences in P. putida populations could not be attributed solely to disturbance. The pollution of Four Mile Creek induced differences in bacterial populations that could be monitored using the described approach. Received: 24 September 1996; Accepted: 20 December 1996     

Sulfide Oxidation Coupled to Arsenate Reduction by a Diverse Microbial Community in a Soda Lake

We characterized the arsenate-reducing, sulfide-oxidizing population of Mono Lake, California, by analyzing the distribution and diversity of rrnA, cbbL, and dissimilatory arsenate reductase (arrA) genes in environmental DNA, arsenate-plus sulfide-amended lake water, mixed cultures, and isolates. The arsenate-reducing community was diverse. An organism represented by an rrnA sequence previously retrieved from Mono Lake and affiliated with the Desulfobulbaceae (Deltaproteobacteria) appears to be an important member of the arsenate-reducing, sulfide-oxidizing community. Sulfide oxidation coupled with arsenate reduction appears to proceed via a two-electron transfer, resulting in the production of arsenite and an intermediate S compound that is subsequently disproportionated. A realgar-like As/S mineral was formed in some experiments.     

The Picrotoxinin Binding Site and Its Relationship to the GABA Receptor Complex

The binding characteristics of [3H] alpha-dihydropicrotoxinin to the picrotoxinin binding site were investigated in membrane preparations of adult rat forebrain and living cultures of rat cerebral cortex. The binding of [3H]alpha-dihydropicrotoxinin to rat forebrain was decreased by lysing, treating with Triton X-100, and heating. Coincubation with gamma-aminobutyric acid (GABA), benzodiazepines, or alterations in the Na+ or Cl- composition of the media had no effect on the binding to the rat brain preparation. However, in the living neurons in tissue culture both GABA and diazepam significantly decreased the binding of [3H]alpha-dihydropicrotoxinin. The dose-response relationships for GABA antagonism of [3H]alpha-dihydropicrotoxinin binding and for picrotoxinin antagonism of the GABA enhancement of [3H]flunitrazepam binding in cultured cortical neurons were also investigated. The Hill coefficients for these actions were reciprocal, suggesting that they result from complementary interactions between the binding sites for GABA and picrotoxinin. These data support the association of the picrotoxinin binding site with the postsynaptic GABA receptor complex.     

Metabolomic Investigation of the Bacterial Response to a Metal Challenge

Pseudomonas pseudoalcaligenes KF707 is naturally resistant to the toxic metalloid tellurite, but the mechanisms of resistance are not known. In this study we report the isolation of a KF707 mutant (T5) with hyperresistance to tellurite. In order to characterize the bacterial response and the pathways leading to tolerance, we utilized Phenotype MicroArray technology (Biolog) and a metabolomic technique based on nuclear magnetic resonance spectroscopy. The physiological states of KF707 wild-type and T5 cells exposed to tellurite were also compared in terms of viability and reduced thiol content. Our analyses showed an extensive change in metabolism upon the addition of tellurite to KF707 cultures as well as different responses when the wild-type and T5 strains were compared. Even in the absence of tellurite, T5 cells displayed a “poised” physiological status, primed for tellurite exposure and characterized by altered intracellular levels of glutathione, branched-chain amino acids, and betaine, along with increased resistance to other toxic metals and metabolic inhibitors. We conclude that hyperresistance to tellurite in P. pseudoalcaligenes KF707 is correlated with the induction of the oxidative stress response, resistance to membrane perturbation, and reconfiguration of cellular metabolism.     

The SMN Protein is a Key Regulator of Nuclear Architecture in Differentiating Neuroblastoma Cells

The cell nucleus contains two closely related structures, Cajal bodies (CBs) and gems. CBs are the first site of accumulation of newly assembled splicing snRNPs (small nuclear ribonucleoproteins) following their import into the nucleus, before they form their steady-state localization in nuclear splicing speckles. Gems are the nuclear site of accumulation of survival motor neurons (SMNs), an insufficiency of which leads to the inherited neurodegenerative condition, spinal muscular atrophy (SMA). SMN is required in the cytoplasm for the addition of core, Sm, proteins to new snRNPs and is believed to accompany snRNPs to the CB. In most cell lines, gems are indistinguishable from CBs, although the structures are often separate in vivo . The relationship between CBs and gems is not fully understood, but there is evidence that symmetrical dimethylation of arginine residues in the CB protein coilin brings them together in HeLa cells. During neuronal differentiation of the human neuroblastoma cell line SH-SY5Y, CBs and gems increase their colocalization, mimicking changes seen during foetal development. This does not result from alterations in the methylation of coilin, but from increased levels of SMN. Expression of exogenous SMN results in an increased efficiency of snRNP transport to nuclear speckles. This suggests different mechanisms are present in different cell types and in vivo that may be significant for the tissue-specific pathology of SMA.     

The Marburgvirus-Neutralizing Human Monoclonal Antibody MR191 Targets a Conserved Site to Block Virus Receptor Binding

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Inhibition of HCO(3) Binding to Photosystem II by Atrazine at a Low-Affinity Herbicide Binding Site

In maize chloroplasts, the ratio of HCO₃⁻ (anion) binding sites to high-affinity atrazine binding sites is unity. In the dark, atrazine noncompetitively inhibits the binding of half of the HCO₃⁻ to the photosystem II (PSII) complexes. The inhibition of binding saturates at 5 micromolar atrazine, little inhibition is seen at 0.5 micromolar atrazine, although the high-affinity herbicide binding sites are nearly filled at this concentration. This means that HCO₃⁻ and atrazine interact noncompetitively at a specific low-affinity herbicide binding site that exists on a portion of the PSII complexes. Light abolishes the inhibitory effects of atrazine on HCO₃⁻ binding. Based on the assumption that there is one high-affinity atrazine binding site per PSII complex, we conclude that there is also only one binding site for HCO₃⁻ with a dissociation constant near 80 micromolar. The location of the HCO₃⁻ binding site, and the low-affinity atrazine binding site, is not known.     

A Proposed EPR Approach to Evaluating Agonist Binding Site of a Peptide Receptor

Angiotensin II (Ang II) and its transmembrane AT₁ receptor were selected in order to test an innovative strategy that might allow the assessment of the agonist binding site in the receptor molecule. With the use of the 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC) paramagnetic probe, a biologically active agonist (TOAC¹-Ang II), as well as an inactive control (TOAC⁴-Ang II) analogs were mixed in solution with various synthesized AT₁ fragments. Comparative intermolecular interactions, as estimated by analyzing the EPR spectra of solutions, suggested the existence of an agonist binding site containing a sequence composed of portions of the N-terminal (13-17) and the third extracellular loop (266-278) fragments of the AT₁ molecule. Therefore, this combined EPR-TOAC approach shows promise as an alternative for use also in other applications related to specific intermolecular association processes. In memoriam of Professor Paiva.     

The Site of Calcium Binding in Relation to the Activation of Myofibrillar Contraction

Skeletal muscle myofibrils, in the presence of 2 mM MgCl₂ at pH 7.0, were found to have two classes of calcium-binding sites with apparent affinity constants of 2.1 x 10⁶ M ^-1 (class 1) and ∼3 x 10⁴ M ^-1 (class 2), respectively. At free calcium concentrations essential for the activation of myofibrillar contraction (∼10^-6 M) there would be significant calcium binding only to the class 1 sites. These sites could bind about 1.3 µmoles of calcium per g protein. Extraction of myosin from the myofibrils did not alter their calcium-binding parameters. Myosin A, under identical experimental conditions, had little affinity for calcium. The class 1 sites are, therefore, presumed to be located in the I filaments. The class 1 sites could only be detected in F actin and myosin B preparations which were contaminated with the tropomyosin-troponin complex. Tropomyosin bound very little calcium. Troponin, which in conjunction with tropomyosin confers calcium sensitivity on actomyosin systems, could bind 22 µmoles of calcium per g protein with an apparent affinity constant of 2.4 x 10⁶ M ^-1. In view of the identical affinity constants of the myofibrils and troponin and the much greater number of calcium-binding sites on troponin it is suggested that calcium activates myofibrillar contraction by binding to the troponin molecule.     

The Transient Response and Impedance Locus of a Mobile Site Membrane

The kinetic properties of a mobile site membrane are analyzed theoretically and the results are compared with experimental data obtained from a simple model system. In the model system the measured response to a step change in electric current is a single relaxation process, in excellent agreement with the theoretically predicted behavior. To this relaxation process corresponds an anomalous capacitance whose properties are obtained by performing a frequency analysis of the system. By plotting the impedance locus of this mobile site membrane, the locus is shown to exhibit a 45° phase angle at high frequencies, a phenomenon which is generally associated with depletion layers.     

The Architecture of a Prototypical Bacterial Signaling Circuit Enables a Single Point Mutation to Confer Novel Network Properties

Even a single mutation can cause a marked change in a protein''s properties. When the mutant protein functions within a network, complex phenotypes may emerge that are not intrinsic properties of the protein itself. Network architectures that enable such dramatic changes in function from a few mutations remain relatively uncharacterized. We describe a remarkable example of this versatility in the well-studied PhoQ/PhoP bacterial signaling network, which has an architecture found in many two-component systems. We found that a single point mutation that abolishes the phosphatase activity of the sensor kinase PhoQ results in a striking change in phenotype. The mutant responds to stimulus in a bistable manner, as opposed to the wild-type, which has a graded response. Mutant cells in on and off states have different morphologies, and their state is inherited over many generations. Interestingly, external conditions that repress signaling in the wild-type drive the mutant to the on state. Mathematical modeling and experiments suggest that the bistability depends on positive autoregulation of the two key proteins in the circuit, PhoP and PhoQ. The qualitatively different characteristics of the mutant come at a substantial fitness cost. Relative to the off state, the on state has a lower fitness in stationary phase cultures in rich medium (LB). However, due to the high inheritance of the on state, a population of on cells can be epigenetically trapped in a low-fitness state. Our results demonstrate the remarkable versatility of the prototypical two-component signaling architecture and highlight the tradeoffs in the particular case of the PhoQ/PhoP system.