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ORM-Like Protein (ORMDL) – Annäherung an Die Funktion Über Die Interaktion

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ORM-Like Protein (ORMDL) – Annäherung an Die Funktion Über Die Interaktion ORM-like protein (ORMDL) - Annäherung an die Funktion über die Interaktion Julian Klingbeil München 2019 Aus der Kinderklinik und Kinderpoliklinik im Dr. von Haunerschen Kinderspital der Ludwig–Maximilians–Universität München Direktor: Prof. Dr. med. Dr. sci. nat. C. Klein ORM-like protein (ORMDL) – Annäherung an die Funktion über die Interaktion Dissertation zum Erwerb des Doktorgrades der Medizin an der Medizinischen Fakultät der Ludwig–Maximilians–Universität zu München vorgelegt von Julian Malte Klingbeil aus Berlin 2019 Mit Genehmigung der Medizinischen Fakultät der Universität München Berichterstatterin: Prof. Dr. Ania Muntau Mitberichterstatter: Prof. Dr. Katja Radon PD Dr. Anne Hilgendorff Prof. Dr. Jürgen Behr Prof. Dr. Ortrud Steinlein Mitbetreuung durch den promovierten Mitarbeiter: Prof. Dr. Søren Gersting Dekan: Prof. Dr. med. dent. Reinhard Hickel Tag der mündlichen Prüfung: 10.10.2019 Inhaltsverzeichnis Zusammenfassung xiii 1 Einleitung 1 1.1 Asthma bronchiale . .1 1.1.1 Epidemiologie, Pathogenese und Klassifikation . .2 1.1.2 Therapie . .4 1.1.3 Gen-Umwelt-Interaktionen und Genomweite Assoziationsstudien5 1.2 Das neue Asthma-Risikogen ORMDL . .8 1.3 Der β2-Adrenorezeptor . 11 1.4 Protein-Protein-Interaktionen . 13 1.4.1 Biolumineszenz-Resonanz-Energie-Transfer . 15 1.5 Zielsetzung der Arbeit . 18 2 Material und Methoden 19 2.1 Material . 19 2.1.1 Laborgeräte . 19 2.1.2 Allgemeine Verbrauchsmaterialien, Chemikalien und Reagenzien . 21 2.1.3 Lösungen, Reagenzien-Kits und Puffer . 26 2.1.4 Medium . 28 2.1.5 Zelllinien und Bakterienstämme . 28 2.1.6 Antikörper . 29 2.1.7 β-Sympathomimetika . 29 2.1.8 Restriktionsenzyme . 30 2.1.9 Vektoren und DNA . 30 2.1.10 Software . 39 2.2 Methoden . 40 2.2.1 Polymerase-Kettenreaktion (PCR) . 40 2.2.2 Site-directed Mutagensis (SDM) . 40 R 2.2.3 Recombinantes Klonieren mittels Gateway Cloning System ..... 41 2.2.4 Hitzeschock-Transformation von Escherichia coli ........... 44 2.2.5 Herstellung von Glycerinkulturen . 45 2.2.6 DNA-Aufreinigung . 45 2.2.7 Restriktionsverdau von Plasmid-DNA und Gelelektrophorese . 46 v vi INHALTSVERZEICHNIS 2.2.8 Zellkultur . 47 2.2.9 Transfektion . 48 2.2.10 Biolumineszenz-Resonanz-Energietransfer (BRET) . 49 2.2.11 Koimmunpräzipitation (Co-IP) . 55 2.2.12 In-Cell/On-Cell ELISA . 56 2.2.13 Statistische Analyse . 57 3 Ergebnisse 58 3.1 Die Erweiterung des ORMDL-Interaktoms . 58 3.1.1 Literaturrecherche zum ORM-Interaktom . 58 3.1.2 Nachweis der Homo- und Heterooligomerisierung von ORMDL mittels Koimmunpräzipitation . 60 3.1.3 Bestätigung der Homo- und Heterooligomerisierung von ORMDL mittels BRET . 60 3.1.4 Übertragung auf humane Homologe der ORM-Interaktionspartner 63 3.1.5 Keine Erweiterung des Interaktoms über Gen-Gen-Interaktionen . 64 3.1.6 Interaktion mit relevanten immunologischen Proteinen . 65 3.1.7 Interaktion mit Membranproteinen . 68 3.1.8 Interaktion mit molekularen Chaperonen . 69 3.1.9 Interaktion mit Proteinen des γ-Sekretase-Komplexes . 73 3.1.10 Bestätigung bekannter Interaktionen mit SPTLC1 und SERCA . 74 3.1.11 Interaktionen mit peroxisomalen Proteinen . 75 3.1.12 Validierung des ORMDL-Interaktoms mittels eines zufälligen Re- ferenzdatensatzes . 77 3.1.13 Zusammenfassung zur Erweiterung des ORMDL-Interaktoms . 79 3.1.14 Ergänzung eines hypothesenfreien Ansatzes mittels Koimmun- präzipitation zur Erweiterung des ORMDL-Interaktoms . 80 3.2 ORMDL und der β2-AR ............................. 81 3.2.1 Charakterisierung der Interaktion von ORMDL mit β2-AR mittels BRET-Sättigungsexperimenten . 82 3.2.2 Bestätigung der Interaktion von ORMDL mit β2-AR mittels Koim- munpräzipitation . 83 3.2.3 Untersuchung der β2-AR Oligomerisierung in Abhängigkeit der häufigsten Polymorphismen mittels BRET . 84 3.2.4 Interaktion zwischen ORMDL3 und den β2-AR-Varianten mit un- terschiedlicher Affinität . 86 3.2.5 Modulation der Interaktionen von ORMDL mit β2-AR durch β- Sympathomimetika in Abhängigkeit vom β2-AR-Allel . 88 3.2.6 Modulation der β2-AR Homooligomerisierung durch β-Sympa- thomimetika in Abhängigkeit vom β2-AR-Allel . 88 3.2.7 β2-AR-Oberflächenexpression unter ORMDL-Einfluss im In-Cell/ On-Cell-ELISA . 91 Inhaltsverzeichnis vii 4 Diskussion 94 4.1 Relevanz der ORMDL Homo- und Heterooligomerisierung . 94 4.2 Das erweiterte ORMDL-Interaktom . 97 4.2.1 Bestätigung einer Verbindung zwischen ORMDL und der Sphin- golipidbiosynthese . 97 4.2.2 Bestätigung einer Verbindung zwischen ORMDL und Chapero- nen, der Unfolded-Protein-Response und dem Calciumstoffwechsel 98 4.2.3 Bestätigung einer Verbindung zwischen ORMDL und dem Im- munsystem . 101 4.2.4 Identifikation von ORMDL als Interaktionspartner anderer Mem- branproteine . 104 4.2.5 Bestätigung einer Verbindung zwischen ORMDL und dem γ- Sekretase-Komplex . 105 4.2.6 Qualitätskontrolle des erweiterten ORMDL-Interaktoms . 106 4.2.7 Methodische Aspekte zum erweiterten ORMDL-Interaktom . 106 4.2.8 Identifikation des hub-Proteins ORMDL . 107 4.3 ORMDL und β2-AR................................ 111 4.3.1 Oligomerisierung von β2-AR und ORMDL . 112 4.3.2 Homooligomerisierung von β2-AR . 113 4.3.3 Oligomerisierung von ORMDL3 mit β2-AR wird durch β2-AR- Allele und β-Sympathomimetika beeinflusst . 118 4.3.4 ORMDL3 als akzessorisches Protein für die Oberflächenexpressi- on von β2-AR ............................... 120 4.4 Ausblick zu ORMDL und Asthma . 123 5 Anhang 125 6 Abkürzungsverzeichnis 127 7 Danksagung 133 8 Eidesstattliche Versicherung 134 Abbildungsverzeichnis 1.1 Prädiktion der Tertiärstruktur von ORMDL3 . .8 1.2 BRET-Funktionsprinzip . 16 2.1 Vektorkarten der BRET-Expressionsvektoren . 32 2.2 BP-Reaktion . 42 2.3 LR-Reaktion . 44 2.4 BRET-Markerkombinationen . 50 2.5 BRET-Spektren: Venus und hRluc . 52 2.6 BRET-Sättigungskurve . 54 3.1 ORMDL Homo-/Heterooligomerisierung (Co-IP) . 61 3.2 ORMDL Homo- und Heterooligomerisierung (BRET) . 62 3.3 ORMDL Interaktom: PPI mit SPTLC1 und ATP2A2 (BRET) . 74 3.4 ORMDL Interaktom: subzelluläre Lokalisation . 79 3.5 ORMDL Interaktom: wahrscheinliche Interaktionspartner (Co-IP mit Tandem- Massenspektrometrie) . 80 3.6 ORMDL Interaktom: mögliche Interaktionspartner (Co-IP mit Tandem- Massenspektrometrie) . 81 3.7 ORMDL und β2-AR: Sättigungskurve (BRET) . 82 3.8 ORMDL und β2-AR: Interaktion (Co-IP) . 83 3.9 β2-AR-Allele: Homo-/Heterooligomerisierung (BRET) . 85 3.10 ORMDL und β2-AR-Allele: Sättigungskurven (BRET) . 87 3.11 ORMDL und β2-AR-Allele: BRET50 und BRETmax (BRET) . 87 3.12 ORMDL3 und β2-AR: Sättigungskurven und BRET50 unter Sympathomi- metikaeinfluss (BRET) . 89 3.13 β2-AR Homooligomerisierung unter Sympathomimetikaeinfluss (BRET) . 90 3.14 ORMDL und β2-AR: Oberflächenexpression von β2-AR (ELISA) . 92 3.15 ORMDL und β2-AR-Allele: Oberflächenexpression der β2-AR-Allele (ELI- SA) ......................................... 92 4.1 ORMDL Interaktom . 108 ix Tabellenverzeichnis 2.1 Verwendete Geräte . 19 2.2 Verwendete Chemikalien und Verbrauchsmaterialien . 21 2.3 Lösungen und Pufffer............................... 26 2.4 Zellkulturmedien . 28 2.5 Zelllinien und Bakterienstämme . 28 2.6 Antikörper . 29 2.7 β-Sympathomimetika . 29 2.8 Restriktionsenzyme . 30 2.9 Plasmid-Vektoren . 30 2.10 Venus- und hRluc-Sequenz . 33 2.11 Übersicht aller verwendeten BRET-Vektoren . 34 2.12 Primersequenzen für die Sequenzanalyse von cDNA im pDEST-Vektor . 38 2.13 Primersequenzen für die Sequenzanalyse von cDNA im pENTR-Vektor . 38 2.14 Primersequenzen für die SDM der β2AR . 38 2.15 Verwendete Softwareprogramme und Datenbanken . 39 3.1 ORMDL Interaktom: humane Homologe der ORM-Interaktionspartner . 59 3.2 ORMDL Homo-/Heterodimerisierung: BRET50 (BRET) . 63 3.3 ORMDL-Interaktom: Hefehomologe (BRET) . 64 3.4 ORMDL Interaktom: Gen-Gen-Interaktionen (BRET) . 65 3.5 ORMDL Interaktom: immunologisch relevante Proteine (BRET) . 66 3.6 ORMDL Interaktom: Membranproteine (BRET) . 69 3.7 ORMDL Interaktom: relevante Chaperone (BRET) . 72 3.8 ORMDL Interaktom: γ-Sekretase-Komplex (BRET) . 73 3.9 ORMDL Interaktom: peroxisomale Proteine (BRET) . 76 3.10 ORMDL Interaktom: Screen gegen zufällig ausgewählte Proteine (BRET) . 78 3.11 ORMDL und β2-AR: Übersicht BRET50 (BRET) . 83 3.12 β2-AR-Allele: Übersicht BRET50 (BRET) . 86 5.1 Referenzdatensatz für die Etablierung der Schwellenwerte (BRET) . 125 xi Zusammenfassung Durch genomweite Assoziationsstudien (GWAS) ist die Identifikation von Assoziatio- nen zwischen Krankheiten und Allelen im gesamten Genom möglich. Die Beurteilung der Relevanz identifizierter Einzelnukleotid-Polymorphismen (SNP), das bessere Ver- ständnis der Pathogenese der assoziierten Erkrankung und die Identifikation neuer Therapieansätze erfolgt jedoch über die Kenntnis der Funktion des durch die Genmu- tation in Struktur, Menge oder Funktionalität veränderten Proteins. Ein Beispiel hierfür ist die 2007 erstmals beschriebene Assoziation zwischen kindlichem Asthma bronchiale und dem Genlocus von ORM-like protein 3 (ORMDL3) auf dem Chromosomenabschnitt 17q21, die bei unbekannter ORMDL-Funktion zunächst zu keinem Erkenntnisgewinn hinsichtlich der Pathogenese oder Therapie von Asthma führte. Asthma ist die häu- figste chronische Erkrankung der Atemwege im Kindesalter, doch bisher stehen, auch aufgrund der noch unzureichend verstandenen Pathogenese, nur symptomatische The- rapien zur Verfügung. Ziel dieser Arbeit war es, mittels Biolumineszenz-Resonanzenergietransfer (BRET) Pro- tein-Protein-Interaktionen (PPI) der ORMDL-Proteinfamilie zu identifizieren, um aus dem dadurch entstehenden explorativen Interaktom Rückschlüsse auf die Funktion der ORMDL-Proteine zu ziehen. Im zweiten Teil der Arbeit sollte die Interaktion
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