Podcasts about rbp j

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.

EBNA-2 is a multifunctional viral oncogene involved in the immortalisation of B-cells by EBV. EBNA-2 regulates transcription of viral and cellular genes in the proliferative phase of the viral life cycle, which in vitro results in the outgrowth of EBV positive B-cells into lymphoblastoid cell lines (LCLs). EBNA-2 transcriptional signalling is mediated by cellular DNA-binding proteins, such as RBP-J and PU.1, since EBNA-2 does not contain its own DNA-binding domain. In order to better characterise EBNA-2 signalling we conducted a mutational analysis of the viral LMP-1 promoter that is strongly induced by EBNA-2 in the EBV-immortalised B-cells. Our mutational analysis of the LMP-1 promoter confirmed that the PU.1 binding site is important for transactivation of the LMP-1 promoter by EBNA-2, whereas RBP-J binding to the LMP-1 promoter leads to repression and EBNA-2 binding to RBP-J is not required for transactivation. These results imply that EBNA-2 transactivates the LMP-1 promoter preferentially by an RBP-J independent mechanism. We further characterised EBNA-2 signalling by dissection of promoter targeting domains in the EBNA-2 protein. Two EBNA-2 mutants, the CR4del and WW mutant, preferentially activated RBP-J dependent and independent signalling indicating that EBNA-2 uses at least two separate signalling pathways. We introduced the characterised EBNA-2 mutants into the EBV genome and produced recombinant viruses carrying specific mutations in the EBNA-2 genes. Primary B-cells were infected with increasing titres of recombinant EBVs lacking the EBNA-2 ORF or carrying the WW or CR4del mutant. Viruses lacking the EBNA-2 ORF or carrying the WW mutant were not able to immortalise primary B-cells even at high viral titres. The CR4 region of EBNA-2 strongly influenced B-cell immortalisation efficiency and growth rate of the immortalised B-cells. These results indicate that EBNA-2 and the RBP-J signalling of EBNA-2 are absolutely essential for B-cell immortalisation by EBV. In contrast, the CR4 EBNA-2 region mediating RBP-J independent signalling is critical, but not absolutely essential for the process of EBV immortalisation.

EBV is a γ-herpes virus which is able to infect human resting B-cells and to transform them into permanently growing lymphoblastoid cell lines (LCLs). EBNA2 (Epstein-Barr virus nuclear antigen 2) is one of the first viral proteins expressed after in vitro infection and interacts with different cellular proteins like RBP-Jκ and PU.1. The EBNA2 protein acts as a transcriptional activator of the viral Latent Membrane Proteins 1 and 2 (LMP1 and LMP2) and the viral nuclear genes EBNA1, EBNA3A, -3B, -3C, EBNA-LP. Additionally EBNA2 is also able to transactivate cellular genes like CD21, CD23 or c-myc. To study the different EBNA2 target genes and the function of EBNA2 a LCL was established (ER/EB2-5 cells, Kempkes et al., 1995) harboring an estrogen-inducible EBNA2. In the presence of estrogen the ER/EBNA2 fusion protein (estrogen receptor binding domain) is located in the nucleus were EBNA2 can transactivate its target genes, whereas in the absence of estrogen the ER/EBNA2 fusion protein is kept in the cytoplasm and therefore inactive. The cells proliferate in the presence of estrogen and they arrest in the absence resulting in a phenotype similar to resting B-lymphocytes. By using the ER/EB2-5 cell line I could clearly show that the cell surface molecule CD83, belonging to the immunoglobuline superfamily (Zhou et al., 1992), is upregulated after the activation of EBNA2. By using a derivative ER/EB2-5 cell line that constitutively expressed LMP1 I could show that CD83 is still expressed even in the absence of functional EBNA2 suggesting that LMP1, the viral target gene of EBNA2, is responsible for the induction of CD83. Therefore I analysed the activation of the CD83 promoter by LMP1. LMP1 is a transmembrane protein with a short intracellular N-terminus, 6 hydrophobic transmembrane domains and a long intracellular C-terminus, containing C-terminal activator regions CTAR1, 2 and 3. The different CTAR regions are responsible for activating genes via NF-κB, ATF, AP1 and STAT signaling pathways. For the activation of its target genes LMP1 uses the same signaling molecules (TRAF, TRADD) as family members of the TNF-R family (CD40, TNF-R1, TNF-R2). The CD83 promoter was activated by LMP1 as shown by promoter luciferase reporter assays in 293-T cells. The induction was not observed in the absence of a NF-κB binding site in a CD83 promoter mutant. Furthermore LMP1 mutants which are mutated in the binding regions for TRAF2 (CTAR1) or TRADD (CTAR2) are not able to transactivate the CD83 promoter. By co-transfection of LMP1 and dominant/negative IκB the CD83 promoter could not be activated because of inactivation of NF-κB. These experiments clearly demonstrate that the CD83 promoter is transactivated by LMP1 via NF-κB. Additionally to the regulation of CD83 I was also interested in the functional role of CD83. Until now only little is known about the function of CD83. CD83 seems to have a specific role in the decision to single positive CD4+ T-cells in the thymus (Fujimoto et al., 2002). I have tested a possible co-stimulatory function of CD83 to CD4+ T-cells by retroviral expression of CD83 in non-professional antigen presenting cells (RCC). Indeed CD83 expression increased the CD4+ response in comparison to CD80 or GFP retroviral infected RCC cells. In mixed lymphocyte reactions this co-stimulatory effect could not be clearly demonstrated although a soluble CD83-Ig showed a small inhibitory influence. The identification of a CD83 ligand molecule could give new insights into the function of CD83. Therefore a CD83-Ig fusion protein as well as a CD83-tetramer construct were generated and used to screen for a potential ligand of CD83. First results showed that the CD83-Ig fusion protein and the CD83-tetramer construct bound to CD4+ and to CD8+ T-cells of isolated PBMCs as well as to activated T-cells in a culture of mixed T-cell populations.