DOCSLIB.ORG

Mechanistic Characterization of Isoform Selective Inhibitors Of

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text

Load more

MECHANISTIC CHARACTERIZATION OF ISOFORM SELECTIVE INHIBITORS OF MAMMALIAN PHOSPHOLIPASE D By PAIGE ELIZABETH SELVY Dissertation Submitted to the Faculty of the Graduate School of Vanderbilt University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY in Pharmacology December, 2011 Nashville, Tennessee Approved by Professor H. Alex Brown Professor Craig W. Lindsley Professor Vsevolod Gurevich Professor Tina M. Iverson Professor Ethan Lee i TO MY DEAR FAMILY FOR THEIR CONTINUED SUPPORT AND LOVE, AND TO MY WONDERFUL HUSBAND WHO IS MY BEST FRIEND AND CONSTANT SOURCE OF ENCOURAGEMENT ii TABLE OF CONTENTS DEDICATION……………………………...………………………………………..…...….ii LIST OF TABLES………………………….…..……………………………………………v LIST OF FIGURES…………………..………..……………………………………....…...vi Chapter I. INTRODUCTION AND OVERVIEW OF PHOSPHOLIPASE D SUPERFAMILY……….…….1 Enzymes with phospholipase D activity……………………………….....…4 Non-HKD enzymes…………………………….……………….……...5 Streptomyces chromofuscus PLD…………………….…..….5 Autotaxin…………………...…………………………….…..…8 HKD enzymes.………………………………………...…...…….……12 Sequence…………………………………………………...…13 Structure…………………………………………………...…..15 Mechanism: hydrolysis versus transphosphatidylation......18 Interfacial kinetics………………………………………….…24 In vitro activity assays……………………………………..…26 Cellular activity assays………………………………….....…30 Prokaryotic PLD………………………………………………………...…….31 Bacterial endonucleases………………………………………........31 Bacterial PLD as virulence factors…………..……………………..34 Streptomyces PLD……………………………………………………35 Plant PLD………………………………………………………………………40 Classes of plant PLD enzymes………………………….…...……..42 Signaling………………………………………………………………47 Fungal PLD……………………………………………………………………48 Budding yeast Spo14………………………………………………..50 Dictyostelium PLD………….…………………………………………56 Zebrafish PLD………………………………………..………………………..59 Mammalian PLD………………………………….……………………….…..61 Isoforms…………………………………..………………….…...…...61 Tissue expression and subcellular localization……….………….. 66 iii Regulation…………………………………………………......……..70 Recombinant protein expression and purification….…………….87 Signaling pathways………………………………...………………..89 Receptor-mediated signaling………………………………...…….90 Vesicular trafficking …………………………………..……………100 PLD inhibitors………………………………………………...……………..101 Indirect inhibitors of PLD activity………………………………….103 Direct inhibitors of PLD activity……………………………...……105 PLD as a potential therapeutic target………………….………..………..108 II. IDENTIFICATION AND DEVELOPMENT OF NOVEL ISOFORM-SELECTIVE PLD INHIBITORS………………………………………..…...110 Halopemide………………………………………………………………….111 Development of novel isoform-selective compounds……..…..……...…112 Structure-Activity Relationship Characterization of Novel PLD Inhibitors………………………………………….…………..…117 Initial Demonstration VU-series Inhibitors Act Directly…………..…..….120 Cellular Ramifications for use of VU-series PLD Inhiibitors…….…..….123 III. MOLECULAR MECHANISM OF ISOFORM-SELECTIVE PHOSPHOLIPASE D INHIBITORS………………………………..……………….....124 Backscattering interferometry measures protein-ligand interactions……………………………………………………...…………….126 Development of novel human PLD1 construct facilitates in vitro studies……………………….…...……………………………….…129 VU-series PLD inhibitors directly interact with enzyme……..….……….131 Small molecule PLD inhibitors block lipid binding……….…..…..…..…..131 VU-series compounds inhibit catalytic activity…..……………..………...136 Protein truncation constructs illuminate the small molecule binding site……………………………………………..……...….138 Confirmation of small molecule binding site with point mutation constructs………………………………………..………………...141 Small molecule PLD inhibitors block cell signal-mediated translocation……………………………………………………………….…145 Model of VU-series mechanism of inhibtion……………………………….146 IV. CONCLUSIONS AND FUTURE DIRECTIONS……………………………….………..155 iv LIST OF TABLES TABLES PAGE 1. Non HKD PLDs………………………………………………… …….…….6 2. Alignment of catalytic motifs for PLD superfamily…….……………….14 3. Bacterial and viral PLDs………………………………………....….…….32 4. Plant PLD enzymes…………………...……………… ……………….…44 5. Mammalian PLD regulatory domains…………...….……...………..…..82 v LIST OF FIGURES FIGURES PAGE 1. Schematic of the various enzyme-catalyzed reactions that form PA……………………………..…………………….…3 2. Mechnism of PLD enzyme activities.…………………..…………………9 3. Comparison of domain alignments for different PLD superfamily enzymes……………………………………..16 4. Crystal structure of PMF PLD………………...………….………………19 5. Proposed PLD superfamily reaction mechanism………….......………21 6. Substrate presentation……………………………………………..……..27 7. Conserved domains of mammalian PLD………………………..………64 8. G-protein coupled receptor activation of PLD…………..…..………….93 9. Activation of PLD through epidermal growth factor receptor……….…97 10. The role of PLD in endocytosis and receptor internalization..…….…102 11. Reported indirect PLD inhibitors………… ………………..………….104 12. Reported direct PLD inhibitors……………………………...…………..107 13. In vitro inhibition of recombinant PLD with select compounds…..…113 14. Focused lead optimization to improve isoform potency and selectivity…………………………………………………...……….115 15. Inhibition of Cell Based Activity with Select Compounds……...…....116 16. Progression from halopemide to PLD1-selective inhibitors…......….119 17. Progression from halopemide to PLD2-selective inhibitors..…...…..119 vi 18. Direct small molecule inhibition of PLD………………...……………..121 19. Backscattering interferometry technique…….……………..………...127 20. Illustration of the domains of the human PLD1 splice variants…..…130 21. PLD1.d311 directly binds VU-series PLD inhibitors….……….….....132 22. VU-Series PLD inhibitors block lipid binding…..…...…………..……135 23. VU-Series compounds inhibit PLD1 regardless of substrate presentation…………………………………...137 24. Truncation constructs suggest VU-series PLD inhibitors directly bind PLD in a loop region……………………………….……...140 25. Point mutations confirm VU-Series PLD inhibitors directly bind PLD in a loop region………….…………………………………….144 26. PLD1 subcellular localization with VU-series inhibitors………….…..147 27. Model of molecular mechanism of action for the VU-series PLD inhibitors…………………………………..……………149 28. Crystal structures demonstrate the mechanism of orthosteric or allosteric Akt ihibition……............................................151 29. Homology model of human PLD1………………..……..……………..158 30. Preliminary mechanistic studies performed with the SERM class of PLD inhibitors…………………...…..…………………160 vii CHAPTER I INTRODUCTION AND OVERVIEW OF PHOSPHOLIPASE D SUPERFAMILY Phosphatidic acid (PA) is a critical phospholipid constituent in eukaryotic cell membranes, that accounts for 1-4 % of the total lipid [1]. This lipophilic glycerophospholipid has a phosphate head group, and as such serves not only a structural capacity in lipid bilayers, but also participates both as an intermediate in lipid metabolism and as a signaling molecule. Because of the small head group, PA facilitates changes in lipid bilayer curvature that are important for membrane fusion events, such as vesicular trafficking and endocytosis [2]. PA is also a precursor to other lipid signaling molecules including diacylglycerol (DAG) and lysophosphatidic acid (LPA). As a lipid second messenger, PA activates signaling proteins and acts as a node within the membrane to which signaling proteins translocate. Several signaling proteins, including Raf-1 [3], [4] and mTOR [5], directly bind PA to mediate translocation or activation, respectively. PA has been implicated in signaling cascades involving cell growth, proliferation, and survival. Aberrant PA signaling has been identified in multiple cancers [6], neurodegeneration [7], and platelet aggregation [8], which makes proteins that mediate cellular levels of PA attractive as potential therapeutic targets. 1 PA can be generated de novo [9], [10], [11] by sequential enzyme- catalyzed acylations of glycerol-3-phosphate, or in response to cell signaling pathways (Figure 1). Every glycerophospholipid generated in eukaryotic membranes transitions through PA, a pathway characterized by Eugene Kennedy and his colleagues more than half a century ago [11], [12]. Signal generated PA is formed by enzymes that modify existing lipids. These enzymes include lysophosphatidic acid acyltransferase (LPAAT) which acylates LPA, DAG kinase which phosphorylates DAG at the sn-3 position, and phospholipase D (PLD) which hydrolyzes the headgroup of a phospholipid, generally phosphatidylcholine (PC), triggering the release of choline. PLD activity, enzyme catalyzed hydrolysis of a phosphodiester bond, was first described in plants [13], [14], [15], [16] and subsequently many enzymes from a range of viral, prokaryotic and eukaryotic organisms have been described as possessing PLD activity. To date, more than 4000 PLD enzymes have been entered in NCBI GenBank. The majority of these enzymes hydrolyze phosphodiester bonds within phospholipids such as PC (classified as EC 3.1.4.4 [17]), but there are other enzymes ascribed to having PLD activity that hydrolyze neutral lipids and even polynucleotide backbone. A large subset of enzymes with PLD activity share a conserved HxKxxxxDx6GSxN motif (HKD motif) [18], or a variation thereof, that is responsible for catalytic activity. These enzymes are members of the PLD superfamily, and are proposed to follow a similar reaction mechanism in which a nucleophilic histidine residue initiates the reaction and generates a covalent intermediate, and a water or short alcohol completes the 2 Figure 1. A schematic of the various enzyme-catalyzed reactions that results in the formation of phosphatidic
Recommended publications
  • Endogenous Reverse Transcriptase and Rnase H-Mediated Antiviral Mechanism in Embryonic Stem Cells

    Endogenous Reverse Transcriptase and Rnase H-Mediated Antiviral Mechanism in Embryonic Stem Cells

    www.nature.com/cr www.cell-research.com ARTICLE Endogenous reverse transcriptase and RNase H-mediated antiviral mechanism in embryonic stem cells Junyu Wu1, Chunyan Wu1, Fan Xing1, Liu Cao1, Weijie Zeng1, Liping Guo1, Ping Li1, Yongheng Zhong1, Hualian Jiang1, Manhui Luo1, Guang Shi2, Lang Bu1, Yanxi Ji1, Panpan Hou1, Hong Peng1, Junjiu Huang2, Chunmei Li1 and Deyin Guo 1 Nucleic acid-based systems play important roles in antiviral defense, including CRISPR/Cas that adopts RNA-guided DNA cleavage to prevent DNA phage infection and RNA interference (RNAi) that employs RNA-guided RNA cleavage to defend against RNA virus infection. Here, we report a novel type of nucleic acid-based antiviral system that exists in mouse embryonic stem cells (mESCs), which suppresses RNA virus infection by DNA-mediated RNA cleavage. We found that the viral RNA of encephalomyocarditis virus can be reverse transcribed into complementary DNA (vcDNA) by the reverse transcriptase (RTase) encoded by endogenous retrovirus-like elements in mESCs. The vcDNA is negative-sense single-stranded and forms DNA/RNA hybrid with viral RNA. The viral RNA in the heteroduplex is subsequently destroyed by cellular RNase H1, leading to robust suppression of viral growth. Furthermore, either inhibition of the RTase activity or depletion of endogenous RNase H1 results in the promotion of virus proliferation. Altogether, our results provide intriguing insights into the antiviral mechanism of mESCs and the antiviral function of endogenized retroviruses and cellular RNase H. Such a natural nucleic acid-based antiviral mechanism in mESCs is referred to as ERASE (endogenous RTase/RNase H-mediated antiviral system), which is an addition to the previously known nucleic acid-based antiviral mechanisms including CRISPR/Cas in bacteria and RNAi in plants and invertebrates.
  • Dynamin Functions and Ligands: Classical Mechanisms Behind

    Dynamin Functions and Ligands: Classical Mechanisms Behind

    1521-0111/91/2/123–134$25.00 http://dx.doi.org/10.1124/mol.116.105064 MOLECULAR PHARMACOLOGY Mol Pharmacol 91:123–134, February 2017 Copyright ª 2017 by The American Society for Pharmacology and Experimental Therapeutics MINIREVIEW Dynamin Functions and Ligands: Classical Mechanisms Behind Mahaveer Singh, Hemant R. Jadhav, and Tanya Bhatt Department of Pharmacy, Birla Institute of Technology and Sciences Pilani, Pilani Campus, Rajasthan, India Received May 5, 2016; accepted November 17, 2016 Downloaded from ABSTRACT Dynamin is a GTPase that plays a vital role in clathrin-dependent pathophysiology of various disorders, such as Alzheimer’s disease, endocytosis and other vesicular trafficking processes by acting Parkinson’s disease, Huntington’s disease, Charcot-Marie-Tooth as a pair of molecular scissors for newly formed vesicles originating disease, heart failure, schizophrenia, epilepsy, cancer, dominant ’ from the plasma membrane. Dynamins and related proteins are optic atrophy, osteoporosis, and Down s syndrome. This review is molpharm.aspetjournals.org important components for the cleavage of clathrin-coated vesicles, an attempt to illustrate the dynamin-related mechanisms involved phagosomes, and mitochondria. These proteins help in organelle in the above-mentioned disorders and to help medicinal chemists division, viral resistance, and mitochondrial fusion/fission. Dys- to design novel dynamin ligands, which could be useful in the function and mutations in dynamin have been implicated in the treatment of dynamin-related disorders. Introduction GTP hydrolysis–dependent conformational change of GTPase dynamin assists in membrane fission, leading to the generation Dynamins were originally discovered in the brain and identi- of endocytic vesicles (Praefcke and McMahon, 2004; Ferguson at ASPET Journals on September 23, 2021 fied as microtubule binding partners.
  • Solitary and Repetitive Binding Motifs for the Ap2 Complex Α-Appendage in Amphiphysin and Other Accessory Proteins

    Solitary and Repetitive Binding Motifs for the Ap2 Complex Α-Appendage in Amphiphysin and Other Accessory Proteins

    SOLITARY AND REPETITIVE BINDING MOTIFS FOR THE AP2 COMPLEX α-APPENDAGE IN AMPHIPHYSIN AND OTHER ACCESSORY PROTEINS Lene E. Olesen*, Eva M. Schmid*, Marijn G. J. Ford#*, Yvonne Vallis, M. Madan Babu, Peter Li, Ian G. Mills∑, Harvey T. McMahon§ and Gerrit J.K. Praefcke♣§ Laboratory of Molecular Biology, Medical Research Council, Neurobiology Division, Hills Road, Cambridge CB2 2QH, UK. ♣ Center for Molecular Medicine Cologne (CMMC), Institute for Genetics, Zülpicher Straße 47, 50674 Köln, Germany. Running Title: Classification of AP2 α-appendage-binding FxDxF and DxF/W motifs § Address correspondence to: Harvey T. McMahon, Email: [email protected]; Tel.: +44(0)1223-402311; Fax: +44(0)1223-402310 or Gerrit J.K. Praefcke, Email [email protected]; Tel.: +49(0)221-470-1561; Fax: +49(0)221-470-6749 Adaptor protein (AP) complexes bind to coated pits, for the platform subdomain of the transmembrane proteins destined for α-appendage. The motif domain of internalisation and to membrane lipids, so amphiphysin1 contains one copy of each of a linking cargo to the accessory internalisation DxF/W and FxDxF motif. We find that the machinery. This machinery interacts with the FxDxF motif is the main determinant for the appendage domains of APs, which have high affinity interaction with the α-adaptin platform and β-sandwich subdomains, appendage. We describe the optimal sequence forming the binding surfaces for interacting of the FxDxF motif using thermodynamic and proteins. Proteins which interact with the structural data and show how sequence subdomains do so via short motifs, usually variation controls the affinities of these motifs found in regions of low structural complexity for the α-appendage.
  • ATP-Induced Focal Adhesion Kinase Activity Is Negatively Modulated by Phospholipase D2 in PC12 Cells

    ATP-Induced Focal Adhesion Kinase Activity Is Negatively Modulated by Phospholipase D2 in PC12 Cells

    EXPERIMENTAL and MOLECULAR MEDICINE, Vol. 33, No. 3, 150-155, September 2001 ATP-induced focal adhesion kinase activity is negatively modulated by phospholipase D2 in PC12 cells Yoe-Sik Bae1 and Sung Ho Ryu1,2 Introduction 1 Division of Molecular and Life Sciences, Pohang University of Purinergic receptors have been reported to play impor- Science and Technology, Pohang 790-784, Korea tant roles on the regulation of neuronal cell functions 2 Corresponding author: Tel, +82-54-279-2292; (Communi et al., 2000; Di Iorio et al., 1998). ATP, a Fax, +82-54-279-2199; E-mail, [email protected] ligand for the receptors modulate various cellular re- sponses such as mitogenic and morphogenic activity in Accepted 18 September 2001 PC12 rat pheochromocytoma cells (Neary et al., 1996; Soltoff et al., 1998; Schindelholz et al., 2000). Stimu- Abbreviations: Fak, focal adhesion kinase; PLD, phospholipase D; lation of cells with ATP induces tyrosine phosphorylation PA, phosphatidic acid; PC, phosphatidylcholine; DAG, diacylglyc- of several cytoskeletal proteins and focal adhesion erol; PBt, phosphatidylbutanol; PKC, protein kinase C; PAP, phos- molecules such as focal adhesion kinase (Fak), proline- phatidic acid phosphohydrolase rich tyrosine kinase (Pyk2), and paxillin (Soltoff et al., 1998; Schindelholz et al., 2000). Since these cytosk- eleton-associated proteins have been regarded as important factors for the regulation of neuronal cell Abstract functions, the study on the regulatory mechanism for the proteins remains an important issue. Extracellular ATP has been known to modulate vari- Phospholipase D (PLD) catalyzes the hydrolysis of ous cellular responses including mitogenesis, secre- phosphatidylcholine (PC) into phosphatidic acid (PA) tion and morphogenic activity in neuronal cells.
  • BIN1/M-Amphiphysin2 Induces Clustering of Phosphoinositides to Recruit Its Downstream Partner Dynamin

    BIN1/M-Amphiphysin2 Induces Clustering of Phosphoinositides to Recruit Its Downstream Partner Dynamin

    ARTICLE Received 19 May 2014 | Accepted 22 Oct 2014 | Published 9 Dec 2014 DOI: 10.1038/ncomms6647 BIN1/M-Amphiphysin2 induces clustering of phosphoinositides to recruit its downstream partner dynamin Laura Picas1, Julien Viaud2, Kristine Schauer1, Stefano Vanni3, Karim Hnia4, Vincent Fraisier5, Aure´lien Roux6, Patricia Bassereau7,Fre´de´rique Gaits-Iacovoni2, Bernard Payrastre2, Jocelyn Laporte4, Jean-Baptiste Manneville1 & Bruno Goud1 Phosphoinositides play a central role in many physiological processes by assisting the recruitment of proteins to membranes through specific phosphoinositide-binding motifs. How this recruitment is coordinated in space and time is not well understood. Here we show that BIN1/M-Amphiphysin2, a protein involved in T-tubule biogenesis in muscle cells and fre- quently mutated in centronuclear myopathies, clusters PtdIns(4,5)P2 to recruit its down- stream partner dynamin. By using several mutants associated with centronuclear myopathies, we find that the N-BAR and the SH3 domains of BIN1 control the kinetics and the accumu- lation of dynamin on membranes, respectively. We show that phosphoinositide clustering is a mechanism shared by other proteins that interact with PtdIns(4,5)P2, but do not contain a BAR domain. Our numerical simulations point out that clustering is a diffusion-driven process in which phosphoinositide molecules are not sequestered. We propose that this mechanism plays a key role in the recruitment of downstream phosphoinositide-binding proteins. 1 Institut Curie and CNRS UMR 144, 26 rue d’Ulm, 75005 Paris, France. 2 INSERM, UMR1048, Universite´ Toulouse III, Institut des Maladies Me´taboliques et Cardiovasculaires, 1 avenue Jean Poulhe`s, 31432 Toulouse, France.
  • Revised Glossary for AQA GCSE Biology Student Book

    Revised Glossary for AQA GCSE Biology Student Book

    Biology Glossary amino acids small molecules from which proteins are A built abiotic factor physical or non-living conditions amylase a digestive enzyme (carbohydrase) that that affect the distribution of a population in an breaks down starch ecosystem, such as light, temperature, soil pH anaerobic respiration respiration without using absorption the process by which soluble products oxygen of digestion move into the blood from the small intestine antibacterial chemicals chemicals produced by plants as a defence mechanism; the amount abstinence method of contraception whereby the produced will increase if the plant is under attack couple refrains from intercourse, particularly when an egg might be in the oviduct antibiotic e.g. penicillin; medicines that work inside the body to kill bacterial pathogens accommodation ability of the eyes to change focus antibody protein normally present in the body acid rain rain water which is made more acidic by or produced in response to an antigen, which it pollutant gases neutralises, thus producing an immune response active site the place on an enzyme where the antimicrobial resistance (AMR) an increasing substrate molecule binds problem in the twenty-first century whereby active transport in active transport, cells use energy bacteria have evolved to develop resistance against to transport substances through cell membranes antibiotics due to their overuse against a concentration gradient antiretroviral drugs drugs used to treat HIV adaptation features that organisms have to help infections; they
  • Supplemental Figure 1. Vimentin

    Supplemental Figure 1. Vimentin

    Double mutant specific genes Transcript gene_assignment Gene Symbol RefSeq FDR Fold- FDR Fold- FDR Fold- ID (single vs. Change (double Change (double Change wt) (single vs. wt) (double vs. single) (double vs. wt) vs. wt) vs. single) 10485013 BC085239 // 1110051M20Rik // RIKEN cDNA 1110051M20 gene // 2 E1 // 228356 /// NM 1110051M20Ri BC085239 0.164013 -1.38517 0.0345128 -2.24228 0.154535 -1.61877 k 10358717 NM_197990 // 1700025G04Rik // RIKEN cDNA 1700025G04 gene // 1 G2 // 69399 /// BC 1700025G04Rik NM_197990 0.142593 -1.37878 0.0212926 -3.13385 0.093068 -2.27291 10358713 NM_197990 // 1700025G04Rik // RIKEN cDNA 1700025G04 gene // 1 G2 // 69399 1700025G04Rik NM_197990 0.0655213 -1.71563 0.0222468 -2.32498 0.166843 -1.35517 10481312 NM_027283 // 1700026L06Rik // RIKEN cDNA 1700026L06 gene // 2 A3 // 69987 /// EN 1700026L06Rik NM_027283 0.0503754 -1.46385 0.0140999 -2.19537 0.0825609 -1.49972 10351465 BC150846 // 1700084C01Rik // RIKEN cDNA 1700084C01 gene // 1 H3 // 78465 /// NM_ 1700084C01Rik BC150846 0.107391 -1.5916 0.0385418 -2.05801 0.295457 -1.29305 10569654 AK007416 // 1810010D01Rik // RIKEN cDNA 1810010D01 gene // 7 F5 // 381935 /// XR 1810010D01Rik AK007416 0.145576 1.69432 0.0476957 2.51662 0.288571 1.48533 10508883 NM_001083916 // 1810019J16Rik // RIKEN cDNA 1810019J16 gene // 4 D2.3 // 69073 / 1810019J16Rik NM_001083916 0.0533206 1.57139 0.0145433 2.56417 0.0836674 1.63179 10585282 ENSMUST00000050829 // 2010007H06Rik // RIKEN cDNA 2010007H06 gene // --- // 6984 2010007H06Rik ENSMUST00000050829 0.129914 -1.71998 0.0434862 -2.51672
  • Increased O-Glcnacylation Rapidly Decreases GABAAR Currents in Hippocampus but Depresses Neuronal Output L

    Increased O-Glcnacylation Rapidly Decreases GABAAR Currents in Hippocampus but Depresses Neuronal Output L

    www.nature.com/scientificreports OPEN Increased O-GlcNAcylation rapidly decreases GABAAR currents in hippocampus but depresses neuronal output L. T. Stewart1,3, K. Abiraman1,3, J. C. Chatham2 & L. L. McMahon1 ✉ O-GlcNAcylation, a post-translational modifcation involving O-linkage of β-N-acetylglucosamine to Ser/Thr residues on target proteins, is increasingly recognized as a critical regulator of synaptic function. Enzymes that catalyze O-GlcNAcylation are found at both presynaptic and postsynaptic sites, and O-GlcNAcylated proteins localize to synaptosomes. An acute increase in O-GlcNAcylation can afect neuronal communication by inducing long-term depression (LTD) of excitatory transmission at hippocampal CA3-CA1 synapses, as well as suppressing hyperexcitable circuits in vitro and in vivo. Despite these fndings, to date, no studies have directly examined how O-GlcNAcylation modulates the efcacy of inhibitory neurotransmission. Here we show an acute increase in O-GlcNAc dampens GABAergic currents onto principal cells in rodent hippocampus likely through a postsynaptic mechanism, and has a variable efect on the excitation/inhibition balance. The overall efect of increased O-GlcNAc is reduced synaptically-driven spike probability via synaptic depression and decreased intrinsic excitability. Our results position O-GlcNAcylation as a novel regulator of the overall excitation/inhibition balance and neuronal output. Synaptic integration and spike initiation in neurons is controlled by synaptic inhibition, which strongly infu- ences neuronal output and information processing1. Importantly, the balance of excitation to inhibition (E/I) is crucial to the proper functioning of circuits, and E/I imbalances have been implicated in a number of neurode- velopmental disorders and neurodegenerative diseases including schizophrenia, autism spectrum disorders, and Alzheimer’s disease2–5.
  • Activation and Phosphorylation of the 'Dense-Vesicle' High-Affinity Cyclic AMP Phosphodiesterase by Cyclic AMP-Dependent Protein Kinase

    Activation and Phosphorylation of the 'Dense-Vesicle' High-Affinity Cyclic AMP Phosphodiesterase by Cyclic AMP-Dependent Protein Kinase

    Biochem. J. (1989) 260, 27-36 (Printed in Great Britain) 27 Activation and phosphorylation of the 'dense-vesicle' high-affinity cyclic AMP phosphodiesterase by cyclic AMP-dependent protein kinase Elaine KILGOUR, Neil G. ANDERSON and Miles D. HOUSLAY Molecular Pharmacology Group, Department of Biochemistry, University of Glasgow, Glasgow G12 8QQ, Scotland, U.K. Incubation of a hepatocyte particulate fraction with ATP and the isolated catalytic unit of cyclic AMP- dependent protein kinase (A-kinase) selectively activated the high-affinity 'dense-vesicle' cyclic AMP phosphodiesterase. Such activation only occurred if the membranes had been pre-treated with Mg2". Mg2" pre-treatment appeared to function by stimulating endogenous phosphatases and did not affect phosphodiesterase activity. Using the antiserum DV4, which specifically immunoprecipitated the 51 and 57 kDa components of the 'dense-vesicle' phosphodiesterase from a detergent-solubilized membrane extract, we isolated a 32P-labelled phosphoprotein from 32P-labelled hepatocytes. MgCl2 treatment of such labelled membranes removed 32P from the immunoprecipitated protein. Incubation of the Mg2+-pre-treated membranes with [32P]ATP and A-kinase led to the time-dependent incorporation of label into the 'dense- vesicle' phosphodiesterase, as detected by specific immunoprecipitation with the antiserum DV4. The time- dependences of phosphodiesterase activation and incorporation of label were similar. It is suggested (i) that phosphorylation of the 'dense-vesicle' phosphodiesterase by A-kinase leads to its activation, and that such a process accounts for the ability of glucagon and other hormones, which increase intracellular cyclic AMP concentrations, to activate this enzyme, and (ii) that an as yet unidentified kinase can phosphorylate this enzyme without causing any significant change in enzyme activity but which prevents activation and phosphorylation of the phosphodiesterase by A-kinase.
  • Crystal Structure of the Targeting Endonuclease of the Human LINE-1 Retrotransposon

    Crystal Structure of the Targeting Endonuclease of the Human LINE-1 Retrotransposon

    View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Structure, Vol. 12, 975–986, June, 2004, 2004 Elsevier Ltd. All rights reserved. DOI 10.1016/j.str.2004.04.011 Crystal Structure of the Targeting Endonuclease of the Human LINE-1 Retrotransposon Oliver Weichenrieder,1,* Kostas Repanas,1 transcriptase. Depending on the DNA integration mech- and Anastassis Perrakis* anism, two classes of retrotransposons are distin- The Netherlands Cancer Institute guished. The first class contains long terminal repeat Department of Molecular Carcinogenesis-H2 (LTR) retrotransposons and retroviruses. These retroele- Plesmanlaan 121 ments use an integrase that recognizes the LTRs of the 1066 CX Amsterdam double-stranded DNA copy. The second, much larger, The Netherlands and more ancient class includes all non-LTR retro- transposons. Those are thought to integrate via target- primed reverse transcription (TPRT), a process in which Summary reverse transcription and integration are coupled (Eick- bush and Malik, 2002; Kazazian, 2004). An endonuclease The human L1 endonuclease (L1-EN) is encoded by that is part of the same polypeptide chain as the reverse the non-LTR retrotransposon LINE-1 (L1). L1 is re- transcriptase nicks the genomic DNA and hands over sponsible for more than 1.5 million retrotransposition the resulting ribose 3Ј-hydroxyl end as a primer for re- events in the history of the human genome, contribut- verse transcription of associated template RNA (Cost ing more than a quarter to human genomic DNA (L1 et al., 2002; Luan et al., 1993). and Alu elements). L1-EN is related to the well-under- Most non-LTR retrotransposons encode an endonu- stood human DNA repair endonuclease APE1, and its clease located N-terminally of the reverse transcriptase.
  • NADPH-Dependent A-Oxidation of Unsaturated Fatty Acids With

    NADPH-Dependent A-Oxidation of Unsaturated Fatty Acids With

    Proc. Natl. Acad. Sci. USA Vol. 89, pp. 6673-6677, August 1992 Biochemistry NADPH-dependent a-oxidation of unsaturated fatty acids with double bonds extending from odd-numbered carbon atoms (5-enoyl-CoA/A3,A2-enoyl-CoA isomerase/A3',52'4-dienoyl-CoA isomerase/2,4-dienoyl-CoA reductase) TOR E. SMELAND*, MOHAMED NADA*, DEAN CUEBASt, AND HORST SCHULZ* *Department of Chemistry, City College of the City University of New York, New York, NY 10031; and tJoined Departments of Chemistry, Manhattan College/College of Mount Saint Vincent, Riverdale, NY 10471 Communicated by Salih J. Wakil, April 13, 1992 ABSTRACT The mitochondrial metabolism of 5-enoyl- a recent observation of Tserng and Jin (5) who reported that CoAs, which are formed during the (3-oxidation of unsaturated the mitochondrial -oxidation of 5-enoyl-CoAs is dependent fatty acids with double bonds extending from odd-numbered on NADPH. Their analysis of metabolites by gas chroma- carbon atoms, was studied with mitochondrial extracts and tography/mass spectrometry led them to propose that the purified enzymes of (3-oxidation. Metabolites were identified double bond of 5-enoyl-CoAs is reduced by NADPH to yield spectrophotometrically and by high performance liquid chro- the corresponding saturated fatty acyl-CoAs, which are then matography. 5-cis-Octenoyl-CoA, a putative metabolite of further degraded by P-oxidation. linolenic acid, was efficiently dehydrogenated by medium- This report addresses the question of how 5-enoyl-CoAs chain acyl-CoA dehydrogenase (EC 1.3.99.3) to 2-trans-5-cis- are chain-shortened by P-oxidation. We demonstrate that octadienoyl-CoA, which was isomerized to 3,5-octadienoyl- 5-enoyl-CoAs, after dehydrogenation to 2,5-dienoyl-CoAs, CoA either by mitochondrial A3,A2-enoyl-CoA isomerase (EC can be isomerized to 2,4-dienoyl-CoAs, which are reduced by 5.3.3.8) or by peroxisomal trifunctional enzyme.
  • PLCG1) Mutations in Sézary Syndrome

    PLCG1) Mutations in Sézary Syndrome

    This electronic thesis or dissertation has been downloaded from the King’s Research Portal at https://kclpure.kcl.ac.uk/portal/ Functional interrogations of Phospholipase C Gamma 1 (PLCG1) mutations in Sézary Syndrome Patel, Varsha Maheshkumar Awarding institution: King's College London The copyright of this thesis rests with the author and no quotation from it or information derived from it may be published without proper acknowledgement. END USER LICENCE AGREEMENT Unless another licence is stated on the immediately following page this work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International licence. https://creativecommons.org/licenses/by-nc-nd/4.0/ You are free to copy, distribute and transmit the work Under the following conditions: Attribution: You must attribute the work in the manner specified by the author (but not in any way that suggests that they endorse you or your use of the work). Non Commercial: You may not use this work for commercial purposes. No Derivative Works - You may not alter, transform, or build upon this work. Any of these conditions can be waived if you receive permission from the author. Your fair dealings and other rights are in no way affected by the above. Take down policy If you believe that this document breaches copyright please contact [email protected] providing details, and we will remove access to the work immediately and investigate your claim. Download date: 11. Oct. 2021 Functional interrogations of Phospholipase C Gamma 1 (PLCG1) mutations in Sézary Syndrome Varsha Maheshkumar Patel Skin Tumour Unit, St John’s Institute of Dermatology, School of Basic and Medical Biosciences, King’s College London.