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The CULT Domain of Cereblon: a Pharmacological Target and Teratogenicity Gateway

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The CULT Domain of Cereblon: a Pharmacological Target and Teratogenicity Gateway The CULT domain of cereblon: a pharmacological target and teratogenicity gateway Dissertation der Mathematisch-Naturwissenschaftlichen Fakultät der Eberhard Karls Universität Tübingen zur Erlangung des Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.) vorgelegt von Iuliia Boichenko aus Schevchenkove, Kiliya Region Odesa Gebiet, Ukraine Tübingen 2016 Tag der mündlichen Qualifikation: 06.06.2016 Dekan: Prof. Dr. Wolfgang Rosenstiel 1. Berichterstatter: Prof. Dr. Andrei Lupas 2. Berichterstatter: Prof. Dr. Alfred Nordheim 2 SUMMARY Half a century ago thalidomide caused one of the biggest pharmaceutical tragedies known by now. It was widely prescribed to pregnant women as a sedative, but displayed teratogenic properties, causing limb malformations and other developmental defects in more than 10,000 babies worldwide. Nevertheless, thalidomide and its derivatives are used nowadays in treatment of leprosy and multiple myeloma. The protein cereblon was identified as a primary target of thalidomide in the cell. As a substrate receptor, cereblon (CRBN) is linked via the damaged DNA binding protein 1 (DDB1) and cullin 4A to the E3 ubiquitin ligase machinery. The drug binds to the C-terminal region of cereblon, also referred to as CULT domain (cereblon domain of unknown activity, binding cellular ligands and thalidomide). This domain represents the most conserved part of cereblon and is also found solely in single-domain proteins in bacteria and, as a secreted form, in eukaryotes. Based on its ligand binding properties, its high degree of conservation and its intracellular as well as extracellular localization, a common interest arose in understanding the functional role of the CULT domain in vivo. The CULT domain carries a number of highly conserved cysteine and tryptophan residues within its amino acid sequence. In the solved crystal structure of the bacterial CULT domain, four conserved cysteines stabilize the protein fold by coordinating a zinc ion. Three invariant tryptophan residues build an aromatic cage, to which the ligand binds. Considering the structural similarity of uridine and thalidomide, we tested uridine binding to the hydrophobic pocket and could show an identical mode of binding. So far, uridine represents the only natural ligand, for which an interaction with the CULT domain has been shown. Further studies demonstrated that parts of the CULT domain fold upon ligand binding, thus stabilizing the protein. The pocket is highly similar to the aromatic cages found in histone readers that recognize methylated lysine or arginine residues in chromatin. This structural similarity suggests analogous ligands for cereblon, which include a distinct type of post-translational modifications. We developed a FRET-based in vitro assay for testing and characterizing ligand binding to cereblon. The determination and comparison of the substrate affinities for three CULT domains, from Homo sapiens, Magnetospirillum gryphiswaldense, and the secreted Caenorhabditis elegans, revealed similar values for the same ligands on a relative scale. This study convincingly confirmed the bacterial protein as a robust model system for both: (i) testing the specificity of the CULT domain to its ligands; and (ii) deciphering the function of cereblon proteins. Using the FRET-assay, we showed that various therapeutically relevant pharmaceuticals display affinities to the CULT domain with binding constants in the micromolar range. These off- 3 target effects were further validated by applying an in vivo assay in zebrafish embryos to test the teratogenic potential of these compounds mediated via their interaction with cereblon. Searching for proteins interacting with cereblon in vivo, we identified transcription termination factor Rho to bind to the bacterial CULT domain. The assumption of a possible role of cereblon in transcriptional regulation is further supported by the fact that hCRBN interacts with LSD1, a demethylase which is essential for transcriptional regulation in multicellular organisms. Taken together, our data imply a potential function of cereblon in transcriptional repression and/or activation, particularly during limb formation. 4 ZUSAMMENFASSUNG Der Contergan-Skandal in der Mitte des letzten Jahrhunderts war eine der größten Tragödien in der Geschichte der Arzneimittelentwicklung und -zulassung. Die Behandlung von Schwangerschaftsübelkeit mit dem Beruhigungsmittel Contergan basierend auf dem Wirkstoff Thalidomid, führte zu fatalen Fehlbildungen bei über 10000 Neugeborenen weltweit. Aufgrund ihrer sowohl entzündungshemmenden als auch immunsuppressiven Eigenschaften, finden Thalidomid und seine Derivate trotz ihrer teratogenen Eigenschaften auch heute noch eine breite Anwendung in der Behandlung von Lepra und Multiplem Myelom. Fast 60 Jahre dauerte es, bis das Protein Cereblon, als ein zelluläres Target von Thalidomid identifiziert wurde. Als Substratrezeptor ist Cereblon durch Wechselwirkung mit DDB1 (damaged DNA binding protein 1) mit dem Cul4A E3 Ubiquitin-Ligase-Komplex verbunden. Durch die Bindung von Thalidomid an die C-terminale Domäne von Cereblon, auch als CULT-Domäne (cereblon domain of unknown activity, binding cellular ligands and thalidomide) bezeichnet, wird die Substraterkennung von Cereblon moduliert. Die CULT- Domäne ist hochkonserviert. Man findet ausschließlich aus dieser Domäne bestehende Homologe sowohl in Bakterien als auch in einer Gruppe von eukaryotischen sekretierten Proteinen. Aufgrund ihrer Eigenschaft Thalidomid zu binden, ihres hohen Konservierungsgrades und ihres sowohl extrazellulären als auch intrazellulären Vorkommens, besteht großes Interesse, die funktionellen Aspekte der CULT-Domäne in vivo zu verstehen. Mit Lösung der Kristallstruktur einer bakteriellen CULT-Domäne konnten wir zeigen, dass streng konservierte Cysteinreste durch Bindung eines Zinkions zur Stabilisierung des Proteins beitragen. Ebenfalls hoch konservierte Tryptophanreste bilden eine Substratbindetasche, in der Thalidomid gebunden wird. Weitere Untersuchungen ergaben, dass Uridin, welches in seiner Struktur Thalidomid ähnelt, auf die gleiche Art und Weise gebunden wird. Es repräsentiert damit den bisher einzigen bekannten Cereblonliganden natürlicher Herkunft. Strukturelle Studien zeigten, dass Ligandenbindung das Protein stabilisiert, da einige Teile des Proteins erst dadurch falten. Die Bindetasche der CULT- Domäne zeigt eine starke Ähnlichkeit zu ebenfalls aromatischen, käfigartigen Bindestellen in anderen Proteinen mit verschiedenen Arten von Liganden. Dazu gehören z.B. die sogenannten Histone reader, welche methylierte Lysin- bzw. Argininreste binden. 5 Wir haben einen fluoreszenzbasierten in vitro Assay (FRET-Assay) entwickelt, welcher die Identifizierung und nachfolgende Charakterisierung von Cereblonliganden ermöglicht. Mit Hilfe dieses Assays zeigten wir in einer vergleichenden Analyse der CULT-Domänen aus Magnetospirillum gryphiswaldense, Caenorhabditis elegans und dem Menschen ähnliche Ligandenspezifitäten als auch -affinitäten. Dies ermöglicht die Anwendung des, im Vergleich zu seinen Homologen, sehr robusten bakteriellen Proteins als: (i) Modellsystem zur Identifizierung potentieller, pharmakologisch interessanter Cereblonliganden und (ii) zur Entschlüsselung der zellulären Funktion der CULT-Domäne. Unter Nutzung des bakteriellen Modellsystems konnten wir mit Hilfe des FRET-Assays die Bindung von therapeutisch relevanten Pharmaka an die CULT-Domäne mit Affinitäten im mikromolaren Bereich zeigen. Mögliche teratogene Auswirkungen dieser unerwünschten Wechselwirkung zwischen Cereblon und Arzneimittelwirkstoff als auch zwischen Cereblon und weiteren identifizierten Verbindungen wurden anschließend in einem In vivo-Assay in Zebrafischembryonen analysiert. In Bakterien durchgeführte Experimente zur Identifizierung zellulärer Bindeproteine der CULT-Domäne zeigten eine Interaktion mit dem Transcription termination factor Rho. Eine mögliche regulatorische Funktion von Cereblon in der Kontrolle der Transkription wird durch den Befund unterstützt, dass für das humane Homologe eine Interaktion mit LSD1, einer für multizelluläre Organismen essentiellen Demethylase, gezeigt werden konnte. Zusammengefasst lassen alle vorliegenden Ergebnisse und Beobachtungen eine potentielle Beteiligung von Cereblon an Vorgängen der transkriptionellen Regulation, insbesondere während der Embryonalentwicklung, vermuten. 6 CONTENTS SUMMARY............................................................................................................. 3 CONTENTS............................................................................................................ 7 ABREVIATIONS................................................................................................... 10 1 INTRODUCTION................................................................................................. 13 1.1 The teratogenic phenotype induced by thalidomide....................................... 13 1.2 Chemistry and pharmacokinetics of thalidomide........................................... 14 1.3 Development of thalidomide derivatives....................................................... 15 1.4 Hypotheses of thalidomide teratogenicity...................................................... 16 1.5 Species- and tissue-specificity of thalidomide action.................................... 19 1.6 Cereblon is a primary target in myeloma treatment....................................... 21 1.7 Cereblon is a protein with various functions.................................................
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