Structural, biochemical and biophysical characterisation of human transcription factor RBP-Jκ

Henning, Karen (2006): Structural, biochemical and biophysical characterisation of human transcription factor RBP-Jκ. Dissertation, LMU München: Fakultät für Biologie

Abstract

RBP-Jκ serves as interaction partner for both cellular and viral proteins. The protein mediates cellular and Epstein-Barr viral signal transduction, which in both cases results in dedifferentiation or immortalization of the cell. The intracellular part of the human Notch1 protein (Notch-IC) and of the Epstein-Barr viral protein EBNA2 binds to RBP-Jκ and expels a corepressor complex to activate transcription. The two proteins have similar functions and their binding regions on RBP-Jκ lie in close vicinity or partially overlap. An important step towards a better understanding of the biology of both signal transduction pathways is to find differences in the properties of Notch and EBNA2, such as binding affinities, stability or binding sites on RBP-Jκ. The aim of the present work was therefore to characterize the interaction of RBP-Jκ with DNA, proteins and relevant peptides using biochemical, biophysical and structural methods. Expression and purification protocols were developed, which enabled us to obtain sufficiently large amounts of each complex partner. A high biological activity of the individual components was obtained by using different expression systems. After the characterization of RBP-Jκ expressed in different systems and of potentially more soluble truncations, which may be easier to crystallize, using EMSA and CD spectroscopy, all subsequent studies were carried out with recombinant RBP-Jκ proteins obtained from insect cells. In comparison with RBP-Jκ proteins expressed in bacteria, these had a higher affinity for DNA as well as for Notch proteins. In contrast, according to EMSA, a high biological activity of Notch and EBNA2 proteins expressed in bacteria was found. There is some controversy in the literature concerning the parts of Notch-IC involved in the binding to RBP-Jκ. The detailed characterization of the interaction of RBP-Jκ with the strongest interacting component, NotchRam, and the naturally occurring fusion of Ram with seven ankyrin repeats, NotchRamANK, using isothermal titration calorimetry (ITC), EMSA and small angle x-ray scattering with binary and ternary complexes allowed us to create models, which unambiguously exclude the participation of the ankyrin repeats in the binding of RBP-Jκ in a system consisting only of the highly purified components of the complex. CD spectroscopy revealed that free Ram is largely unfolded and folds into largely α-helical structures upon binding to RBP-Jκ. Cell biological methods usually provide indirect information about interactions but do not provide quantitative data regarding the strength. The controlled reaction systems developed in the present study enabled us to detect a 20- to 50-fold higher affinity of EBNA291-355 for RBP-Jκ compared to NotchRamANK. Interestingly, first results indicate that the CR6 region of EBNA2, which is described as the most important region interacting with RBP-Jκ cannot account for the higher affinity. A precise description of the binding sites of the interaction partners would require crystals of RBP-Jκ in complex with proteins from Notch-IC and/or DNA. The many attempts at obtaining suitable crystals were hitherto unsuccessful although we were able to narrow down the area of likely crystallization conditions. The results obtained by different methods help to clarify the role of the interaction partners of RBP-Jκ in the context of infections by the Epstein-Barr virus, which may lead to malignant tumours because of both the similarities and functional differences of Notch and EBNA2. Furthermore, with the results of the present study the discussion of whether a therapeutic attack on the level of the RBP-Jκ-EBNA2 interaction is useful has to be resumed.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Keywords: RBP-Jκ, EBNA2, Notch
• Themengebiete: 500 Naturwissenschaften und Mathematik > 570 Biowissenschaften, Biologie ; 500 Naturwissenschaften und Mathematik
• Fakultäten: Fakultät für Biologie
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 19. Oktober 2006
• 1. Bericht­erstatter:in: Kempkes, Bettina