Genome mining and characterisation of a novel transaminase with remote stereoselectivity.

Microbial enzymes from pristine niches can potentially deliver disruptive opportunities in synthetic routes to Active Pharmaceutical Ingredients and intermediates in the Pharmaceutical Industry. Advances in green chemistry technologies and the importance of stereochemical control, further underscores the application of enzyme-based solutions in chemical synthesis. The rich tapestry of microbial diversity in the oceanic ecosystem encodes a capacity for novel biotransformations arising from the chemical complexity of this largely unexplored bioactive reservoir. Here we report a novel ω-transaminase discovered in a marine sponge Pseudovibrio sp. isolate. Remote stereoselection using a transaminase has been demonstrated for the first time using this novel protein. Application to the resolution of an intermediate in the synthesis of sertraline highlights the synthetic potential of this novel biocatalyst discovered through genomic mining. Integrated chemico-genomics revealed a unique substrate profile, while molecular modelling provided structural insights into this 'first in class' selectivity at a remote chiral centre.

Development and application of spherically curved charge-coupled device imagers.

Operation of a CCD imager on a curved focal surface offers advantages to flat focal planes, especially for lightweight, relatively simple optical systems. The first advantage is that the modulation transfer function can approach diffraction-limited performance for a spherical focal surface employed in large field-of-view or large-format imagers. The second advantage is that a curved focal surface maintains more uniform illumination as a function of radius from the field center. Examples of applications of curved imagers, described here, include a small compact imager and the large curved array used in the Space Surveillance Telescope. The operational characteristics and mechanical limits of an imager deformed to a 15 mm radius are also described.

Aspirated bile: a major host trigger modulating respiratory pathogen colonisation in cystic fibrosis patients.

Chronic respiratory infections are a leading global cause of morbidity and mortality. However, the molecular triggers that cause respiratory pathogens to adopt persistent and often untreatable lifestyles during infection remain largely uncharacterised. Recently, bile aspiration caused by gastro-oesophageal reflux (GOR) has emerged as a significant complication associated with respiratory disease, and cystic fibrosis (CF) in particular. Based on our previous finding that the physiological concentrations of bile influence respiratory pathogens towards a chronic lifestyle in vitro, we investigated the impact of bile aspiration on the lung microbiome of respiratory patients. Sputum samples (n = 25) obtained from a cohort of paediatric CF patients were profiled for the presence of bile acids using high-resolution liquid chromatography-mass spectrometry (LC-MS). Pyrosequencing was performed on a set of ten DNA samples that were isolated from bile aspirating (n = 5) and non-bile aspirating (n = 5) patients. Both denaturing gradient gel electrophoresis (DGGE) and pyrosequencing revealed significantly reduced biodiversity and richness in the sputum samples from bile aspirating patients when compared with non-aspirating patients. Families and genera associated with the pervasive CF microbiome dominated aspirating patients, while bacteria associated with the healthy lung were most abundant in non-aspirating patients. Bile aspiration linked to GOR is emerging as a major host trigger of chronic bacterial infections. The markedly reduced biodiversity and increased colonisation by dominant proteobacterial CF-associated pathogens observed in the sputum of bile aspirating patients suggest that bile may play a major role in disease progression in CF and other respiratory diseases.

Mapping the Anopheles gambiae odorant binding protein 1 (AgamOBP1) using modeling techniques, site directed mutagenesis, circular dichroism and ligand binding assays.

The major malaria vector in Sub-Saharan Africa is the Anopheles gambiae mosquito. This species is a key target of malaria control measures. Mosquitoes find humans primarily through olfaction, yet the molecular mechanisms associated with host-seeking behavior remain largely unknown. To further understand the functionality of A. gambiae odorant binding protein 1 (AgamOBP1), we combined in silico protein structure modeling and site-directed mutagenesis to generate 16 AgamOBP1 protein analogues containing single point mutations of interest. Circular dichroism (CD) and ligand-binding assays provided data necessary to probe the effects of the point mutations on ligand binding and the overall structure of AgamOBP1. Far-UV CD spectra of mutated AgamOBP1 variants displayed both substantial decreases to ordered α-helix structure (up to22%) and increases to disordered α-helix structure(up to 15%) with only minimal changes in random coil (unordered) structure. In mutations Y54A, Y122A and W114Q, aromatic side chain removal from the binding site significantly reduced N-phenyl-1-naphthylamine binding. Several non-aromatic mutations (L15T, L19T, L58T, L58Y, M84Q, M84K, H111A, Y122A and L124T) elicited changes to protein conformation with subsequent effects on ligand binding. This study provides empirical evidence for the in silico predicted functions of specific amino acids in AgamOBP1 folding and ligand binding characteristics.

Specific interactions among odorant-binding proteins of the African malaria vector Anopheles gambiae.

In this report we present results from a comprehensive study undertaken toward the identification of proteins interacting with odourant-binding proteins (OBPs) of the African malaria vector Anopheles gambiae with a focus on the interactions among different OBPs. From an initial screen for proteins that interact with a member of the Plus-C group of OBPs, OBP48, which is primarily expressed in female antennae and downregulated after a blood meal, a number of interacting proteins were identified, which included five classic OBPs and OBP48 itself. The interacting OBPs as well as a number of other classic and Plus-C group OBPs that were not identified in the initial screen, were expressed in lepidopteran cells and subsequently examined for in vitro interactions in the absence of exogenously added ligands. Co-immunoprecipitation and chemical cross-linking studies suggest that OBP48 is capable of homodimerizing, heterodimerizing and forming higher order complexes with those examined examples of classical OBPs identified in the initial screen but not with other classical or Plus-C group OBPs that failed to appear in the screen. The latter OBPs are, however, also capable of forming homodimers in vitro and, at least in the case of two examined classic OBPs, heterodimers as well. These results suggest a previously unsuspected potential of nonrandom combinatorial complexity that may be crucial for odour discrimination by the mosquito.

Sexual dimorphic expression of putative antennal carrier protein genes in the malaria vector Anopheles gambiae.

To obtain a better understanding of the olfactory processes that allow mosquitoes to identify human hosts, a molecular study has been performed to identify and characterize molecules in the olfactory signalling pathway of the African malaria vector Anopheles gambiae. Using cDNA libraries from antennae of females and males, a collection of cDNAs encoding odorant binding proteins and other novel antennal proteins were isolated and characterized, which represent various families of putative carrier proteins with homologues in other insects. Using filter array hybridizations and quantitative RT PCR, regulation and gender specificity of expression of these genes was investigated. Significant differences in steady-state levels of some of these putative carrier protein genes were detected between the sexes and after blood feeding in females.

The Partner of Inscuteable/Discs-large complex is required to establish planar polarity during asymmetric cell division in Drosophila.

Frizzled (Fz) signaling regulates cell polarity in both vertebrates and invertebrates. In Drosophila, Fz orients the asymmetric division of the sensory organ precursor cell (pI) along the antero-posterior axis of the notum. Planar polarization involves a remodeling of the apical-basal polarity of the pI cell. The Discs-large (Dlg) and Partner of Inscuteable (Pins) proteins accumulate at the anterior cortex, while Bazooka (Baz) relocalizes to the posterior cortex. Dlg interacts directly with Pins and regulates the localization of Pins and Baz. Pins acts with Fz to localize Baz posteriorly, but Baz is not required to localize Pins anteriorly. Finally, Baz and the Dlg/Pins complex are required for the asymmetric localization of Numb. Thus, the Dlg/Pins complex responds to Fz signaling to establish planar asymmetry in the pI cell.

Signaling pathways are focused at specialized regions of the plasma membrane by scaffolding proteins of the MAGUK family.

The MAGUKs (membrane-associated guanylate kinase homologs) are a family of proteins that act as molecular scaffolds for signaling pathway components at the plasma membrane of animal cells. They are localized in and required for the formation of several types of cell junctions, including epithelial tight and septate junctions as well as synaptic and neuromuscular junctions. They are also localized at the plasma membrane of other cell types, including erythrocytes, where they contribute to cell shape maintenance. MAGUKs function mainly by binding directly to the cytoplasmic termini of transmembrane proteins as well as to other signal transduction proteins. They appear to hold together elements of individual signaling pathways, thereby contributing to the efficiency and specificity of signaling interactions while simultaneously maintaining the structural specializations of the plasma membrane. BioEssays 1999;21:912-921.

Human CASK/LIN-2 binds syndecan-2 and protein 4.1 and localizes to the basolateral membrane of epithelial cells.

In Caenorhabditis elegans, mutations in the lin-2 gene inactivate the LET-23 receptor tyrosine kinase/Ras/MAP kinase pathway required for vulval cell differentiation. One function of LIN-2 is to localize LET-23 to the basal membrane domain of vulval precursor cells. LIN-2 belongs to the membrane-associated guanylate kinase family of proteins. We have cloned and characterized the human homolog of LIN-2, termed hCASK, and Northern and Western blot analyses reveal that it is ubiquitously expressed. Indirect immunofluorescence localizes CASK to distinct lateral and/or basal plasma membrane domains in different epithelial cell types. We detect in a yeast two-hybrid screen that the PDZ domain of hCASK binds to the heparan sulfate proteoglycan syndecan-2. This interaction is confirmed using in vitro binding assays and immunofluorescent colocalization. Furthermore, we demonstrate that hCASK binds the actin-binding protein 4.1. Syndecans are known to bind extracellular matrix, and to form coreceptor complexes with receptor tyrosine kinases. We speculate that CASK mediates a link between the extracellular matrix and the actin cytoskeleton via its interaction with syndecan and with protein 4.1. Like other membrane-associated guanylate kinases, its multidomain structure enables it to act as a scaffold at the membrane, potentially recruiting multiple proteins and coordinating signal transduction.

Camguk, Lin-2, and CASK: novel membrane-associated guanylate kinase homologs that also contain CaM kinase domains.

MAGUKs (membrane-associated guanylate kinase homologs) are proteins involved in cell junction organization, tumor suppression, and signalling. Their structure includes one or three copies of a DHR or PDZ domain (discs-large homologous region or PSD-95/SAP90, discs-large ZO-1 homologous domain), an SH3 domain, and a guanylate kinase domain. MAGUKs were classified into two subfamilies: Dlg-like with three DHR/PDZ domains and p55-like with a single DHR/PDZ domain. There is now a new subfamily whose members have a novel domain structure: a calcium/calmodulin-dependent protein kinase domain in the N-terminus as well as the DHR/PDZ, SH3 and GUK domains in the C-terminus. These new MAGUKs may regulate transmembrane molecules that bind calcium, calmodulin, or nucleotides, camguk (cmg) is a Drosophila member of this novel MAGUK subfamily; we report its sequence and domain structure.

Localization of proteins to the apico-lateral junctions of Drosophila epithelia.

We have examined the distribution of proteins in the apico-lateral cell junctions in Drosophila imaginal discs. The subcellular distribution of these proteins in normal and mutant proliferating cells was analyzed with marker antibodies and confocal microscopy. Antibodies to phosphotyrosine (PY), Armadillo (Arm) and Drosophila E-cadherin (DE-cad) as well as FITC phalloidin marking filamentous actin, labeled the site of the adherens junction, whereas antibodies to Discs large (DIg), Fasciclin III (FasIII) and Coracle (Cor) labeled the more basal septate junction. The junctional proteins labeled by these antibodies underwent specific changes in distribution during the cell cycle. We have previously shown that a loss-of-function dlg mutation, which causes neoplastic imaginal disc overgrowth, leads to loss of the septate junctions and the formation of what appear to be ectopic adherens junctions [Woods et al., 1996]. We therefore extended this study to examine the effects of mutations in other genes that also cause imaginal disc overgrowth. Based on staining with PY and DIg antibodies, the apico-lateral junctional complexes appeared normal in tissue from the hyperplastic overgrowth mutants fat, dco, gd and wts. However, imaginal disc tissue from the neoplastic overgrowth mutants dlg and lgl showed abnormal distribution of the junctional markers including a complete loss of apico-basal polarity in loss-of-function dlg mutations. These results support the idea that some of the proteins of apico-lateral junctions are required both for apico-basal cell polarity and for the signalling mechanisms controlling cell proliferation, whereas others are required more specifically in cell-cell signalling.

Organizing a functional junctional complex requires specific domains of the Drosophila MAGUK Discs large.

Discs large (Dlg) was the first identified member of an increasingly important class of proteins called membrane-associated guanylate kinase homologs (MAGUKs), which are often concentrated at cell junctions and contain distinct peptide domains named PDZ1-3, SH3, HOOK, and GUK. Dlg is localized at and required for the formation of both septate junctions in epithelial cells and synaptic junctions in neurons. In the absence of Dlg, epithelia lose their organization and overgrow. We tested the functions of each domain of Dlg in vivo by constructing transgenic flies expressing altered forms of the protein. In the first set of experiments each domain was examined for its ability to correctly target an epitope-tagged Dlg to pre-existing septate junctions. Based on these results the Hook domain is necessary for localization of the protein to the cell membrane and the PDZ2 is required for restricting the protein to the septate junction. In the second set of experiments each domain was tested for its role in growth regulation and organization of epithelial structure. These results show that PDZ1 and GUK are apparently dispensable for function, PDZ2 and PDZ3 are required for growth regulation but not for epithelial structure, and SH3 and HOOK are essential for both aspects of function. The results demonstrate the functional modularity of Dlg and clarify the functions of individual MAGUK domains in regulating the structure and growth of epithelial tissue.

Dlg protein is required for junction structure, cell polarity, and proliferation control in Drosophila epithelia.

The Discs large (Dlg) protein of Drosophila is the prototypic member of a growing family of proteins termed membrane-associated guanylate kinase homologs (MAGUKs). The MAGUKs are composed of a series of peptide domains that include one or three DHR/PDZs, an SH3, and a region homologous to guanylate kinase (GUK). We have previously shown that the product of this gene, the Dlg protein, is localized at the septate junctions between epithelial cells, and that mutations in the gene cause neoplastic overgrowth of the imaginal discs. The dlg locus is therefore defined as a tumor suppressor gene. In this paper, we show that the Dlg protein is localized on the cytoplasmic face of the septate junction and is required for the maintenance of this structure. It is also required for proper organization of the cytoskeleton, for the differential localization of membrane proteins, and for apicobasal polarity of epithelial cells. However, these other functions can be uncoupled from Dlg's role as a tumor suppressor since mutations in two domains of the protein, the SH3 and GUK, cause loss of normal cell proliferation control without affecting the other functions of the protein. These results suggest that, besides regulating cellular proliferation, the Dlg protein is a critical component of the septate junctions and is required for maintaining apicobasal polarity in Drosophila epithelium.

The Drosophila tumor suppressor gene warts encodes a homolog of human myotonic dystrophy kinase and is required for the control of cell shape and proliferation.

Homozygous loss of the warts (wts) gene of Drosophila, caused by mitotic recombination in somatic cells, leads to the formation of cell clones that are fragmented, rounded, and greatly overgrown compared with normal controls. Therefore, the gene is required for the control of the amount and direction of cell proliferation as well as for normal morphogenesis. The absence of wts function also results in apical hypertrophy of imaginal disc epithelial cells. Secretion of cuticle over and between the domed apical surfaces of these cells leads to a honeycomb-like structure and gives the superficial wart-like phenotype of mitotic clones on the adult. One wts allele allows survival of homozygotes to the late larval stage, and these larvae show extensive imaginal disc overgrowth. Because of the excess growth and abnormalities of differentiation that follow homozygous loss, we consider wts to be a tumor suppressor gene. The wts gene is defined by the breakpoints of overlapping deficiencies in the right telomeric region of chromosome 3, region 100A, and by lethal P-element insertions and excisions. It encodes a protein kinase that is most similar to human myotonic dystrophy kinase, the Neurospora cot-1 protein kinase, two cell-cycle regulated kinases of yeast, and several putative kinases from plants. These proteins define a new subfamily of protein kinases that are closely related to but distinct from the cyclic AMP-dependent kinases. Although myotonic dystrophy is defined by a neuromuscular disorder, it is sometimes associated with multiple pilomatrixomas, which are otherwise rare epithelial tumors, and with other tumors including neurofibromas and parathyroid adenomas. Our results raise the possibility that homozygous loss of the myotonic dystrophy kinase may contribute to the development of these tumors.

Tumor suppressor genes and signal transduction in Drosophila.

Several studies in Drosophila have revealed the existence of loss-of-function mutations that lead to overproliferation of epithelial tissues. The molecular analysis of these tumor suppressor genes has provided important clues in the search for signalling mechanisms controlling growth. We discuss here two genes that act to control epithelial cell proliferation. Mutations in the fat locus cause hyperplastic imaginal disc overgrowth, in which the imaginal disc cells retain their epithelial structure, junctional complexes, and ability to differentiate. However, mutations in fat also cause defective cell-cell adhesion, as indicated by the separation of epithelial vesicles from the epithelial sheet in the mutant tissue. The fat gene encodes a giant relative of cadherins, providing the first evidence that this class of junction-associated molecules can act to regulate growth. Loss of the discs large (dlg) gene product results in neoplastic overgrowth of imaginal discs, causes the epithelial cells to lose apical/basal polarity, and prevents them from differentiating. The gene encodes a protein made up of a series of peptide motifs, including an SH3 and a potential enzymatic domain, a region homologous to guanylate kinases. The presence of these domains suggests a role for Dlg in guanine nucleotide-mediated signal transduction. The Dlg protein is restricted to a specific apical junctional complex in Drosophila epithelial cells, the septate junction. It is the first identified member of a new family of junction-associated proteins that includes ZO-1 and ZO-2, major components of vertebrate tight junctions. These results suggest that junctional contacts could play previously unrecognized roles in regulating the growth of epithelial tissues and that ZO-1 and/or ZO-2 may act as tumor suppressor proteins in humans.

Apical junctions and cell signalling in epithelia.

Genetic analysis in Drosophila has led to the identification of several proteins that mediate cell-cell interactions controlling the fate and proliferation of epithelial cells. These proteins are localized or enriched in the adherens and septate junctions at the apical end of the lateral membranes between cells. The proteins localized or enriched at adherens junctions include Notch, which is important for the cell interactions controlling neuroblast and bristle patterning; Boss and sevenless, which are required for the cell interaction that establishes the R7 photoreceptor cell; and Armadillo, required for the wingless-dependent cell interactions that control segment polarity and imaginal disc patterning. Proteins localized at septate junctions include the product of the tumor suppressor gene dlg, which is required for septate junction formation, apical basal cell polarity, and the cell interactions that control proliferation. The results suggest that the cell signalling events important for cell fate determination and for cell proliferation control in epithelia occur at the apical junctions. The migration of the nucleus to the apical surface of the epithelium for mitosis may enable it to interact directly with the junction-associated signalling mechanisms.

Tumor suppressor genes encoding proteins required for cell interactions and signal transduction in Drosophila.

Tumor suppressor genes, whose products are required for the control of cell proliferation, have been identified by their mutant phenotype of tissue overgrowth. Here we describe recent work on the molecular identification of tumor suppressor genes that function in two different cell types of the Drosophila larva: the blood cells, and the undifferentiated epithelial cells of developing imaginal discs. Mutations in the aberrant immune response8 (air8) gene lead to overproduction and precocious differentiation of blood cells. This gene encodes the Drosophila homolog of human ribosomal protein S6. The mutant phenotype is consistent with a role for S6 in the control of cell proliferation, and is compatible with findings from mammalian cells where alterations in S6 expression and phosphorylation are associated with changes in cell proliferation. Mutations in the discs large (dlg) gene cause neoplastic overgrowth of imaginal discs in the larva. The mutant discs show loss of septate junctions and of apical-basal cell polarity, and they also lose the ability to differentiate cuticular structures. The dlg protein product (DlgA) is localized at septate junctions between epithelial cells, and cDNA sequencing indicates that the gene product includes a domain with homology to guanylate kinase (GUK). Two mammalian homologs of this gene have been identified, and one of them (PSD-95/SAP90) encodes a component of synaptic densities in the brain; this protein therefore resembles the DlgA protein in being located in a specialized cell junction that functions in information transfer between cells. Mutations in the fat gene cause hyperplastic imaginal disc overgrowth, in which the overgrowing disc tissue retains its epithelial structure and its ability to differentiate. Some of the excess disc tissue is shed as vesicles suggesting a loss of cell adhesion. In support of this hypothesis, the predicted gene product shows homology to cadherins in its extracellular domain. However, the fat protein is much larger than known cadherins. As in human cancer, somatic loss of the normal alleles of tumor suppressor genes can lead to tumor formation in Drosophila; an example of this is provided by the warts (wts) locus. The wts gene was identified by the dramatic overgrowth of mitotic recombination clones that are homozygous for a wts deletion. In these clones the cuticle intrudes between epithelial cells, suggesting an alteration in cell adhesion. The study of these and other tumor suppressor genes in Drosophila is providing new evidence supporting the critical role of cell interactions and specialized apical junctions in controlling epithelial cell proliferation.