Purine biosynthesis in archaea: variations on a theme

<p>Abstract</p> <p>Background</p> <p>The ability to perform <it>de novo </it>biosynthesis of purines is present in organisms in all three domains of life, reflecting the essentiality of these molecules to life. Although the pathway is quite similar in eukaryotes and bacteria, the archaeal pathway is more variable. A careful manual curation of genes in this pathway demonstrates the value of manual curation in archaea, even in pathways that have been well-studied in other domains.</p> <p>Results</p> <p>We searched the Integrated Microbial Genome system (IMG) for the 17 distinct genes involved in the 11 steps of <it>de novo </it>purine biosynthesis in 65 sequenced archaea, finding 738 predicted proteins with sequence similarity to known purine biosynthesis enzymes. Each sequence was manually inspected for the presence of active site residues and other residues known or suspected to be required for function.</p> <p>Many apparently purine-biosynthesizing archaea lack evidence for a single enzyme, either glycinamide ribonucleotide formyltransferase or inosine monophosphate cyclohydrolase, suggesting that there are at least two more gene variants in the purine biosynthetic pathway to discover. Variations in domain arrangement of formylglycinamidine ribonucleotide synthetase and substantial problems in aminoimidazole carboxamide ribonucleotide formyltransferase and inosine monophosphate cyclohydrolase assignments were also identified.</p> <p>Manual curation revealed some overly specific annotations in the IMG gene product name, with predicted proteins without essential active site residues assigned product names implying enzymatic activity (21 proteins, 2.8% of proteins inspected) or Enzyme Commission (E. C.) numbers (57 proteins, 7.7%). There were also 57 proteins (7.7%) assigned overly generic names and 78 proteins (10.6%) without E.C. numbers as part of the assigned name when a specific enzyme name and E. C. number were well-justified.</p> <p>Conclusions</p> <p>The patchy distribution of purine biosynthetic genes in archaea is consistent with a pathway that has been shaped by horizontal gene transfer, duplication, and gene loss. Our results indicate that manual curation can improve upon automated annotation for a small number of automatically-annotated proteins and can reveal a need to identify further pathway components even in well-studied pathways.</p> <p>Reviewers</p> <p>This article was reviewed by Dr. Céline Brochier-Armanet, Dr Kira S Makarova (nominated by Dr. Eugene Koonin), and Dr. Michael Galperin.</p

Adrenomedullin Function in Vascular Endothelial Cells: Insights from Genetic Mouse Models

Adrenomedullin is a highly conserved peptide implicated in a variety of physiological processes ranging from pregnancy and embryonic development to tumor progression. This review highlights past and present studies that have contributed to our current appreciation of the important roles adrenomedullin plays in both normal and disease conditions. We provide a particular emphasis on the functions of adrenomedullin in vascular endothelial cells and how experimental approaches in genetic mouse models have helped to drive the field forward

Decoy Receptor CXCR7 Modulates Adrenomedullin-Mediated Cardiac and Lymphatic Vascular Development

Atypical 7-transmembrane receptors, often called decoy receptors, act promiscuously as molecular sinks to regulate ligand bioavailability and consequently temper the signaling of canonical G protein-coupled receptor (GPCR) pathways. Loss of mammalian CXCR7, the most recently described decoy receptor, results in postnatal lethality due to aberrant cardiac development and myocyte hyperplasia. Here, we provide the molecular underpinning for this proliferative phenotype by demonstrating that the dosage and signaling of adrenomedullin (Adm = gene, AM = protein)—a mitogenic peptide-hormone required for normal cardiovascular development—is tightly controlled by CXCR7. To this end, Cxcr7−/− mice exhibit gain-of-function cardiac and lymphatic vascular phenotypes which can be reversed upon genetic depletion of adrenomedullin ligand. In addition to identifying a biological ligand accountable for the phenotypes of Cxcr7−/− mice, these results reveal a previously underappreciated role for decoy receptors as molecular rheostats in controlling the timing and extent of GPCR-mediated cardiac and vascular development

Characteristics of Multi-Organ Lymphangiectasia Resulting from Temporal Deletion of <em>Calcitonin Receptor-Like Receptor</em> in Adult Mice

<div><p>Adrenomedullin (AM) and its receptor complexes, <em>calcitonin receptor-like receptor (Calcrl)</em> and <em>receptor activity modifying protein 2/3</em>, are highly expressed in lymphatic endothelial cells and are required for embryonic lymphatic development. To determine the role of <em>Calcrl</em> in adulthood, we used an inducible Cre-loxP system to temporally and ubiquitously delete <em>Calcrl</em> in adult mice. Following tamoxifen injection, <em>Calcrl^fl/fl/CAGGCre-ER™</em> mice rapidly developed corneal edema and inflammation that was preceded by and persistently associated with dilated corneoscleral lymphatics. Lacteals and submucosal lymphatic capillaries of the intestine were also dilated, while mesenteric collecting lymphatics failed to properly transport chyle after an acute Western Diet, culminating in chronic failure of <em>Calcrl^fl/fl/CAGGCre-ER™</em> mice to gain weight. Dermal lymphatic capillaries were also dilated and chronic edema challenge confirmed significant and prolonged dermal lymphatic insufficiency. <em>In vivo</em> and <em>in vitro</em> imaging of lymphatics with either genetic or pharmacologic inhibition of AM signaling revealed markedly disorganized lymphatic junctional proteins ZO-1 and VE-cadherin. The maintenance of AM signaling during adulthood is required for preserving normal lymphatic permeability and function. Collectively, these studies reveal a spectrum of lymphatic defects in adult <em>Calcrl^fl/fl/CAGGCre-ER™</em> mice that closely recapitulate the clinical symptoms of patients with corneal, intestinal and peripheral lymphangiectasia.</p> </div

Acute-onset eye phenotype, eye inflammation, edema, and enlarged lymphatic vessels in <i>Calcrl^fl/fl/CAGGCre-ER™</i> mice. A,B,

<p>Gross eye images indicating normal appearance of the control <i>Calcrl^fl/fl</i> mice (A) and the distinct color change and disruption of the cornea of <i>Calcrl^fl/fl/CAGGCre-ER™</i> (B), (scale = 2 mm). <b>C,D,</b> Hematoxylin and eosin staining of mouse eyes indicating normal histology in <i>Calcrl^fl/fl</i> (C) and disruption of the cornea in <i>Calcrl^fl/fl/CAGG-CreER™</i> mice (D), (4x objective,scale = 500 µm). <b>E,F,</b> Higher magnification of histological sections of eyes from <i>Calcrl^flf/fl</i> mice (E) as compared to <i>Calcrl^fl/fl/CAGGCre-ER™</i> mice (F) exhibiting corneal edema (arrow) and inflammation (arrowhead) (10x objective, scale = 200 µm). Gross anatomy and histology images are representative from <i>Calcrl^flf/fl</i> mice (n = 8) and <i>Calcrl^fl/fl/CAGGCre-ER™</i> mice (n = 9). <b>G,</b> Eye diagram indicating the location of components of the eye (l = lens, c = cornea, ac = anterior chamber, cb = ciliary body, i = iris). <b>H,</b> Lymphatic markers expressed in the eye shown by podoplanin(red) and Lyve-1(green) staining in a control mouse eye (20x objective, scale = 100 µm). <b>I,J,</b> Visualization of lymphatic vessels at the corneoscleral junction in the <i>Calcrl^flf/fl</i> (I) and <i>Calcrl^fl/fl/CAGGCre-ER™</i> mice (J) indicating enlarged lymphatic vessels in <i>Calcrl^fl/fl/CAGGCre-ER™</i> mice (Lyve-1 = green; DAPI = blue; 20x objective, scale = 100 µm). <b>K,</b> Graph representing increased lymphatic vessel area at the corneoscleral junction in <i>Calcrl^fl/fl/CAGGCre-ER™</i> mice compared to control mice calculated using Image J software(*p<0.015). Mice used were 3–4 months of age.</p

Increased lymphatic vascular permeability without change to blood vascular permeability in <i>Calcrl^fl/fl/CAGGCre-ER™</i> mice. A,B,C,D,

<p><i>In vivo</i> lymphatic permeability assay assessing the leakage of Evan’s blue dye from the dermal lymphatic vessels in the ear. Images represent Evan’s blue dye location directly after injection of the dye and 5 minutes post injection. There is an increase in leakage of the dye from the <i>Calcrl^fl/fl/CAGGCre-ER™</i> mice (B,D) relative to <i>Calcrl^fl/fl</i> mice (A,C). Depicted are representative images from four independent experiments (mice 6–8 months of age). <b>E,</b> Blood vascular permeability assay indicating there is no difference in permeability between genotypes in the various tissues (n = 4 per genotype for each tissue).</p

Dilated dermal lymphatic capillaries with exacerbated and prolonged edema. A,B,

<p>Images of dermal lymphatic capillaries in the tail of <i>Calcrl^fl/fl</i>(A) and <i>Calcrl^fl/fl/CAGGCre-ER™</i> (B) mice indicating increased diameter of these lymphatic vessels in <i>Calcrl^fl/fl/CAGGCre-ER™</i> mice (scale = 0.5 mm). <b>C,</b> Graphic representation of the increase in vessel diameter in the <i>Calcrl^fl/fl/CAGGCre-ER™</i> mice with respect to <i>Calcrl^fl/fl</i> mice (*p≤0.05). <b>D,</b> Edema formation assay using hindpaw injections of CFA (4 µg/µl on Day 0). Assessment of paw thickness over 3 weeks (n = 5 for <i>Calcrl^fl/fl</i> and n = 4 for <i>Calcrl^fl/fl/CAGGCre-ER™</i> mice) indicated enhanced and prolonged edema in <i>Calcrl^fl/fl/CAGGCre-ER™</i> mice relative to control mice (***p<0.05, **p<0.01, *p<0.001). Representative images of CFA-injected hindpaws at Day 11 for <i>Calcrl^fl/fl</i> and <i>Calcrl^fl/fl/CAGGCre-ER™</i> mice (scale = 3 mm). Mice used were 6–8 months of age.</p

Inhibition of AM signaling disrupts lymphatic endothelial cell-cell junctions. A,B,C,D,

<p>Confocal images of VE-Cadherin (red) and Lyve-1(green) expression in mesenteric lymphatic vessels of <i>Calcrl^fl/fl</i> (A,C) and <i>Calcrl^fl/fl/CAGGCre-ER™</i> mice (B,D) (scale = 10 µm). Boxed region depicted in C and D. Junctional protein, VE-cadherin, is disorganized in <i>Calcrl^fl/fl/CAGGCre-ER™</i> mice relative to <i>Calcrl^fl/fl</i> mice (representative images from n = 4 per genotype, age 6–8 months). <b>E,F,G,H,</b> Lymphatic endothelial cells stained with VE-Cadherin (green), ZO-1 (red) and DAPI (blue) after various treatments including a no treatment control (A), 10 nm AM (B), 1 µm AM22-52 (C), AM+AM22-52 (D) (arrow refers to inset region). Disorganization of cell-cell junctions occurs with inhibitor treatment (AM22-52) as compared to AM treatment. (VE-cadherin = red, Lyve-1 = green, DAPI = blue, 40x objective, scale = 100 µm; representative images from 3 independent experiments).</p

Dilated lacteals and submucosal lymphatics in <i>Calcrl^fl/fl/CAGGCre-ER™</i> mice and chyle-filled lymphatics after short-term Western diet. A,B,

<p>Hematoxylin and eosin staining of mouse intestine showing normal histology in both <i>Calcrl^fl/fl</i>(A) and <i>Calcrl^fl/fl/CAGGCre-ER™</i> mice(B) (6.3x objective, scale = 500 µm). <b>C,D,E,</b> Lymphatic marker expression in the lacteals and submucosal lymphatic vessels in wildtype mouse. Image was obtained from the jejunum of the intestine. Lyve-1 (C,green) and podoplanin(D,red) colocalize in the lymphatic vessels as seen in the merged image(E) (20x objective; scale = 100 µm). <b>F,G</b> Lyve-1(green) and DAPI(blue) staining in <i>Calcrl^fl/fl</i>(F) and <i>Calcrl^fl/fl/CAGGCre-ER™</i> (G) mice indicating dilated lacteals and submucosal lymphatic vessels with temporal deletion of <i>Calcrl</i> in the jejunum of the intestine (4x objective, scale = 500 µm). Histology and immunofluorescent images are representative from <i>Calcrl^flf/fl</i> mice (n = 7) and <i>Calcrl^fl/fl/CAGGCre-ER™</i> mice (n = 6). <b>H,I,</b> Chyle-filled mesenteric collecting lymphatic vessels in <i>Calcrl^fl/fl/CAGGCre-ER™</i> mice (I) relative to non-chyle filled vessels in control animals (H). Valves are distinctly visible in <i>Calcrl^fl/fl/CAGGCre-ER™</i> mice (arrows; inset refers to enlarged image of valve; scale = 3 mm; n = 4 per genotype). Mice used were 6–8 months of age.</p