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Screen for Kinases Affecting Amyloidogenic Cleavage by BACE1

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Screen for Kinases Affecting Amyloidogenic Cleavage by BACE1 Screen for kinases affecting amyloidogenic cleavage by BACE1 Dissertation zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.) an der Universität Konstanz Mathematisch-Naturwissenschaftliche Sektion Fachbereich Biologie vorgelegt von Stephan Penzkofer Konstanz, Juli 2011 Tag der mündlichen Prüfung: 24.10.2011 1. Referent: Professor Dr. Marcel Leist 2. Referent: Professor Dr. Daniel Dietrich Summary: The Amyloid β peptide (Aβ) is suspected to be a causal agent for Alzheimer’s disease (AD). Therefore a screen for kinases downregulating the initial step of its production, the cleavage of the Amyloid Precursor Protein (APP) by Beta-site of APP Cleaving Enzyme 1 (BACE1), was conducted in this study. Briefly, HEK293 cells were colipofected with one of in total 1357 siRNAs against 60% of the human kinome and either an APP construct with only the β-cleavage site left or normally cleavable APP as control. Remaining β-cleavage was for logistic reasons firstly measured with an activity-test for secreted alkaline phosphatase (SEAP) fused to both types of APP and subjected to Aβ-ELISA when interesting. Before the screen, the APP-constructs were characterized in the cell types HEK293 and CGCs with regards to cleavage, especially by BACE1. The screen resulted in 38 hits of which one, Testis Specific Serine Kinase 3, was confirmed once more. In a second, bioinformatic project, an initially suspected APLP-like pseudogenic-like sequence in C3orf52 was refuted. Further, analysis of C3orf52 gene expression data hints on a role in myeloid leukemia. Lastly, the phylogenetic relationship of the APP family paralogs was examined, also in comparison to neighboring gene families, and found in the topology (APLP1)(APLP2/APP). Zusammenfassung: Das Amyloid β-Peptid (Aβ) steht im Verdacht, ursächlich für die Alzheimer- Demenz (AD) zu sein. Daher wurden in dieser Studie Kinasen gesucht, die den ersten Schritt seiner Entstehung, Schnitt des Amyloid Precursor Proteins durch Beta-site of APP Cleaving Enzyme 1 (BACE1), herabregulieren. Dafür wurden HEK293 colipofiziert mit einer von insgesamt 1357 siRNAs gegen 60% des menschlichen Kinoms und entweder einem ausschliesslich β-spaltbarem APP- Konstrukt oder normal spaltbarem APP als Kontrolle. Aus logistischen Gründen wurde die übriggebliebene β-Spaltung zunächst mit einem Aktivitätstest für sekretierbare alkaline Phosphatase (SEAP), das an beide APPs fusioniert war, gemessen und falls interessant noch mit Aβ-ELISA. Vor dem Screen wurden die APP-Konstrukte in den Zelltypen HEK293 und CGC mit Hinblick auf Spaltung, v. a. durch BACE1, charakterisiert. Der Screen resultierte in 38 Hits, von denen einer, Testis Specific Serine Kinase 3, nochmals bestätigt wurde. In einem zweiten, bioinformatischen Projekt wurde eine zunächst vermeintlich APLP-ähnliche pseudogenartige Sequenz in C3orf52 widerlegt. Weiter deutet die Analyse von C3orf52-Genexpressionsdaten auf eine Rolle in myeloider Leukämie hin. Zuletzt wurden die phylogenetischen Beziehungen der APP-Familie-Paraloge untersucht, auch im Vergleich mit benachbarten Genfamilien, und in der Topologie (APLP1)(APLP2/APP) gefunden. Abbreviations aa amino acids LINE1 Long Interspersed Nuclear Element1 Aβ Amyloid β peptide LOAD Late-Onset AD AβK16V Aβ with K to V exchange, not mbp mega base pairs cleavable by α-secretases MCA Middle Cerebral Artery ACh Acetyl-Choline MCI Mild Cognitive Impairment ACoA Anterior Communicating Artery MRI Magnetic Resonance Imaging AD Alzheimer's Disease my, mya million years, - ago AICD APP Intracellular Domain µm 10-6 m Alu primate specific repeat class NbMc Nucleus basalis of Meynert complex ANNE APLP-Near Notes Extant NCBI National Center for Biotechnology APLP1 APP-Like Protein 1 Information APLP2 APP-Like Protein 2 NCT nicastrin APP Amyloid Precursor Protein nm 10-9 m APPswe APP with Swedish double mutation NSAID Non-Steroidal Anti-Inflammatory Drug AQD Amino-Quinazoline-Derivative O-GlcNAcylation O-linked-β-N-acetyl- ATCC American Tissue type Culture glucosaminylation Collection PBS Phosphate Buffered Saline AU Absorption Units PFK Phosphofructokinase BACE1 Beta-site of APP Cleaving Enzyme 1 pg picogram BBB Blood Brain Barrier pNPP para-Nitro-Phenyl-Phosphate bp base pairs PS1 preseniline 1 C3orf52 chr 3 open reading frame 52 rev reverse CAA Cerebral Amyloid Angiopathy SD Standard Deviation CArG CC-AT-rich(6)-GG box SEAP Secreted Alkaline Phosphatase CGC Cerebellar Granule Cell siRNA small interfering RNA ChAT choline acetyl transferase SNP Single Nucleotide Polymorphism chr chromosome TFA Tierforschungsanlage CSNK1D Casein Kinase 1 isoform D tko triple knockout ct cycle of threshold TMD Transmembrane Domain CTF C-Terminal Fragment TTMP TPA-induced Transmembrane Protein dko double knockout us upstream DMSO dimethylsulphoxide WHO World Health Organization DS Down's Syndrome 3'UTR 3' Untranslated Region ds downstream Embry.s.c. Embryonic stem cells ENSEMBL joint project between EMBL - EBI Bestiarium and Wellcome Trust Sanger Institute EOAD Early-Onset AD Ac Anolis carolinensis green anole ER Endoplasmic Reticulum Bt Bos taurus cattle e-value expected value Ce Caenorhabditis elegans worm fwd forward Cf Canis familiaris dog gbp giga base pairs Cm Callorhinchus milii elephant shark GFP Green Fluorescent Protein Dm Drosophila melanogaster fruitfly HBP Hexosamine Biosynthesis Pathway Dr Danio rerio zebrafish HEK293 Human Embryonic Kidney293 cell line Ec Equus caballus horse HIV Human Immunodeficiency Virus Ga Gasterosteus aculeatus stickleback ICD-10 International Classification of Diseases Gg Gallus gallus chicken 10th revision Hs Homo sapiens human inh inhibitor Mm Mus musculus mouse IPAD Iso-Phthal-Amide-Derivative Oa Ornithorhynchus anatinus platypus IQ Intelligence Quotient Oc Oryctolagus cuniculus rabbit kbp kilo base pairs Ol Oryzias latipes medaka kd knockdown Pt Pan troglodytes chimpanzee ko knockout St Silurana tropicalis clawed frog Tn Tetraodon nigroviridis Tr Takifugu rubripes pufferfish Some parts of this thesis have already been published: Poster: Identification of BACE1- and APP-regulating kinases Stephan Penzkofer*, Christiane Volbracht&, Karina Fog&, Kenneth Vielsted& and Marcel Leist*! *:Doerenkamp-Zbinden Chair for alternative in vitro methods, University of Konstanz, Konstanz, Germany; !:corresponding author &:H. Lundbeck A/S, Valby, Denmark Presented at PENS Summer School: “Novel molecular strategies to treat neurodegenerative diseases”, Ofir, Portugal, 7. July 2007 Christiane Volbracht, Stephan Penzkofer, David Mansson, Kenneth Vielsted Christensen, Karina Fog, Stefan Schildknecht, Marcel Leist, Jacob Nielsen, Measurement of cellular β-site of APP cleaving enzyme 1 activity and its modulation in neuronal assay systems, Analytical Biochemistry 387 (2009) 208-220. Curriculum vitae of Stephan Penzkofer 06.06.1978 born in Munich, Germany 09/1984 – 06/1998 attendance of schools in Munich and Erding 07/1998 – 04/1999 compulsory military service 05/1999 – 09/1999 newspaper catering and car rental at the Munich Airport 10/1999 – 09/2000 studies in civil engineering at the Technical University of Munich 10/2000 – 09/2002 basic study period in biology at the University of Regensburg, Germany, with intermediate examinations in physics (oral), chemistry (oral) and biology (written; best result of the semester) 10/2002 – 03/2004 laboratory research courses in genetics, organic chemistry, biophysics and biochemistry followed by diploma examinations in organic chemistry, biochemistry and biophysics 04/2004 – 04/2005 diploma thesis with the Institute of Biophysics and physical Biochemistry at the University of Regensburg: “Characterisation of the PDZ2-PIP-Interaction with NMR-titration studies” 10/2005 – 05/2006 research attachment in cell culture, proteomics with mass spectrometry, capillary electrophoresis and flow cytometry with the Institute of Bioengineering and Nanotechnology, Biopolis, Singapore 09/2006 – 09/2011 doctoral thesis with the Doerenkamp-Zbinden-Chair of alternative in vitro methods at the University of Konstanz, Germany: “Screen for kinases affecting amyloidogenic cleavage by BACE1” eidesstattliche Erklärung: "Ich erkläre hiermit, dass ich die vorliegende Arbeit ohne unzulässige Hilfe Dritter und ohne Benutzung anderer als der angegebenen Hilfsmittel angefertigt habe. Die aus anderen Quellen direkt oder indirekt übernommenen Daten und Konzepte sind unter Angabe der Quelle gekennzeichnet." Table of contents 1 Alzheimer’s disease (AD) and screening for BACE1-affecting kinases 1 1.1 History of AD research and AD in Down’s syndrome 1 I 1.2 Plaques and Aβ 4 N 1.3 APP and the discovery of its cleavage by proteases in HEK293 6 T 1.4 The families of APP and BACE1 11 R 1.5 Diabetes type 2 and the families of APP and BACE1 12 O 1.6 Atrophy in AD and the cholinergic hypothesis 13 D 1.7 Tau modifications and BACE1 transcriptional regulation 17 U 1.8 Influence of kinases on Aβ-generation 23 C 1.9 Difficulties with direct inhibition of BACE1 30 T 1.10 Aims of the study 33 I 2 Evolution of the APP family and characteristics of processed pseudogenes 34 O 2.1 Evolution of the APP family 34 N 2.2 Characteristics of processed pseudogenes 37 3 Screen preparation and execution 42 M 3.1 Cells 42 A 3.2 Molecular material 45 T 3.2.1 APP-constructs 45 E 3.2.2 Inhibitors and other compounds 48 R 3.2.3 Antibodies 49 I 3.2.4 Primers 50 A 3.2.5 small interfering RNA (siRNA) 50 L 3.3 Experimental setup 69 S 3.4 Data collection and analysis 71 3.4.1 SEAP activity measurement 72 & 3.4.2 A40 quantification 72
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