Department of Microbiology, Tumor and Cell Biology (MTC)

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Department of Microbiology, Tumor and Cell Biology (MTC)
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Georg Klein Group

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Georg Klein Group

Elena Kashuba Project

EBNA-binding cellular proteins and their role in cell transformation

The discovery of molecular mechanisms of cell transformation is one of the most important and intricate questions of modern cell biology. We are using the Epstein-Barr virus (EBV)-induced B-cell immortalization as a model system. It is clear now that EBV exploits the normal signaling pathways of the B lymphocyte and the six growth transformation associated EBNAs promote cell proliferation and protect from apoptosis. Our project is aimed on elucidation of the role of cellular proteins that bind to EBNAs in cell transformation.

Over the last few years, we have discovered 17 previously unknown interactions between three transforming proteins - EBNA-3, EBNA-5 and EBNA-6 and cellular proteins.

Recently we have shown that trimolecular protein complexes of EBNA-5, MDM2, and p53 were formed, where MDM2 served as a bridge. EBNA-5 inhibited MDM2-dependent polyubiquitination (but not monoubiquitination) and degradation of p53 in vitro. These findings indicate that the high p53 levels in LCL are due to EBNA-5 dependent inhibition of p53 degradation. Tri-molecular complex formation prevents binding of p53 to DNA, and thereby inactivates the p53 dependent apoptotic pathway. We have also found that EBNA-3 protects LCL from VDR-induced apoptosis due to the binding to VDR that represses the transcription of VDR target genes.

We have discovered that the HIF1A protein is stabilized in EBV transformed B-cells, in contrast to mitogen activated B-cells. Lymphoblastoid cells use both the aerobic, oxidative, and non aerobic, glycolytic pathways for energy production, even in the presence of abundant oxygen, thus resemble most solid tumors with regard to the 'Warburg effect'.

Studying the influence of EBV on the RB E2F1 pathway, we have found that EBNA-6 interacts with RB-associated protein, mitochondrial ribosomal protein MRPS18-2 (S18-2), leading to the liberation of E2F1. This stimulates the entry of the cell into the S-phase.

Unexpectedly, overexpression of S18-2 protein in the primary rat embryonic fibroblasts (REFs) led to REF immortalization. A derived cell line (18IM) expressed the embryonic stem cell markers SSEA-1 and Sox2. Pathways involved in oxidative phosphorylation, ubiquinone (Coenzyme Q 10) biosynthesis, fatty acid elongation in mitochondria, PI3K/AKT signaling, a characteristic of rapidly proliferating cells, were upregulated in 18IM. Genes involved in the transcription/translation machinery and redox reactions were upregulated as well. 18IM cells produced more pyruvate, indicating enhanced ATP synthesis.

We are currently investigating the role of S18-2 protein in cell immortalization and embryogenesis. We would like to characterize novel pathways of cell fate regulation and to identify the new putative diagnostic and/or prognostic markers of cancerogenesis.