ヒトCereblonタンパク質とそのキイロショウジョウバエ相同体の機能解析

早大学位記番号:新7514早稲田大

Nuclear cereblon binds to the transcription factor Ikaros and modulates its transcriptional activity

早大学位記番号:新7335早稲田大

The CULT domain of cereblon: a pharmacological target and teratogenicity gateway

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-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

ßaßßß-Modulproteine im Stoffwechsel und in der Resistenz von Pseudomonas aeruginosa

The opportunistic pathogen Pseudomonas aeruginosa is a growing threat to human health care due to the increase in multidrug-resistant strains, which requires the urgent development of new therapeutics. Possible new drug targets could, for example, be so far uncharacterized ßaßßß-module proteins. Despite their conserved fold, these proteins are able to perform a broad range of molecular functions, but the majority of previously characterized representatives are involved in the mediation of resistance mechanisms. P. aeruginosa possesses 22 ßaßßß-module proteins. Based on conserved amino acids in the active centre or in the binding pocket they can be assigned to three classes. Class I contains three conserved metal complexing residues and the majority of the characterized metalloenzymes catalyzes the degradation of toxic compounds. In class II, two aromatic side chains complex toxic aromatics to neutralize them. The remaining proteins have no conserved residues and are assigned to class III. In this study the physiological and molecular functions of the class III proteins PA1358, PA1672, PA2721, PA4183 and PA4518 were investigated using microbiological, biophysical and X-ray crystallographic methods. This revealed that PA1672 has a low affinity to phenothiazines. Ligand structures of PA2721 showed the binding of N-benzyloxycarbonyl-L-proline and that a proposed active site cysteine is covalently modified by the antimicrobial compounds dichlofluanid and captan. P. aeruginosa strains, in which the genes of PA1358, PA4183 and PA4518 were mutated, showed altered sensitivity to a variety of different antimicrobial compounds. Furthermore, high-resolution crystal structures of the class III protein PA4183 and the proteins PA1359, PA2722 and PA2723, which are encoded in the same operon as class III proteins, were determined. Thus, this study elucidated previously undetermined protein structures and provided concrete starting points for the functional characterization of PA2721 and PA1672. In addition, the results of the chemical sensitivity tests seem to support the assumption that at least some of the class III ßaßßß-module proteins are involved in the mediation of resistance mechanisms.Der opportunistische Erreger Pseudomonas aeruginosa stellt, durch die Zunahme von multiresistenten Stämmen, eine wachsende Bedrohung für die menschliche Gesundheitsversorgung dar, wodurch die Entwicklung von neuen Therapeutika dringend benötig wird. Mögliche neue Wirkstoffziele könnten zum Beispiel bisher uncharakterisierte ßaßßß-Modulproteine darstellen. Trotz ihrer konservierten Faltung können diese Proteine vielfältige molekulare Funktionen ausüben, wobei der Großteil der bisher charakterisierten Vertreter an der Vermittlung von Resistenzmechanismen beteiligt ist. P. aeruginosa besitzt 22 ßaßßß-Modulproteine. Diese können aufgrund von konservierten Aminosäuren im aktiven Zentrum oder in der Bindetasche in drei Klassen unterteilt werden. Klasse I enthält drei konservierte Metall-komplexierende Reste, wobei der Großteil der charakterisierten Metalloenzyme den Abbau von toxischen Verbindungen katalysiert. In Klasse II komplexieren zwei aromatische Seitenketten toxische Aromaten, um diese zu neutralisieren. Die übrigen Proteine haben keine konservierten Reste und werden der Klasse III zugeordnet. In dieser Studie wurden die physiologischen und molekularen Funktionen der Klasse III Proteine PA1358, PA1672, PA2721, PA4183 und PA4518 mit mikrobiologischen, biophysikalischen und röntgenkristallographischen Methoden untersucht. Dabei stellte sich heraus, dass PA1672 eine geringe Affinität zu Phenothiazinen aufweist. Ligandenstrukturen von PA2721 zeigten die Bindung von N-Benzyloxycarbonyl-L-Prolin und dass ein Cystein im potentiellen aktiven Zentrum, durch die antimikrobiellen Verbindungen Dichlofluanid und Captan, kovalent modifiziert wird. P. aeruginosa Stämme, in denen die Gene von PA1358, PA4183 und PA4518 mutiert wurden, hatten eine veränderte Empfindlichkeit gegenüber einer Vielzahl verschiedener antimikrobieller Verbindungen. Weiterhin wurden hochauflösende Kristallstrukturen des Klasse III Proteins PA4183, sowie der Proteine PA1359, PA2722 und PA2723, die im gleichen Operon wie Klasse III Proteine kodiert sind, bestimmt. Somit wurden in dieser Studie bisher unbestimmte Proteinstrukturen aufgeklärt und konkrete Ansatzpunkte für die funktionelle Charakterisierung von PA2721 und von PA1672 geschaffen. Außerdem scheinen die Ergebnisse der chemischen Sensitivitätstests die Vermutung zu stützen, dass zumindest einige der Klasse III ßaßßß-Modulproteine an der Vermittlung von Resistenzmechanismen beteiligt sind