Identifizierung und Charakterisierung von Brd4 Interaktionspartnern in Krebszellen und DNA Tumorvirus-infizierten Zellen
Virologie
Zusammenfassung der Projektergebnisse
Infectious agents like bacteria, viruses and parasites have been reported to be the underlying cause of approximately 15% of new cancer cases worldwide. Infections with DNA tumorviruses such as Human papillomaviruses (HPV), Epstein-Barr virus (EBV), Kaposi-Sarcoma-Herpesvirus (KSHV) are responsible for over 30% of the cancers caused by infections. The protein Brd4 has been shown to interact with a variety of proteins expressed by DNA-tumorviruses, like the E2 protein of HPV, the EBNA1 protein of EBV, the LANA1 protein of KSHV as well as the large T-antigen of Merkel cell polyomavirus and may contribute to the oncogenic potential of these viruses. In this study I performed a mass-spectrometry based approach to identify changes in the Brd4 interactome in the presence of different viral proteins. The screen revealed several new interaction partners of Brd4 in complex with the tested viral proteins. In the presence of LANA1 Brd4 for example gained the ability to bind to SCAI, a tumorsupressor protein that has been implicated in cancer cell invasion. Follow-up experiments verified the interaction of Brd4 and SCAI in the presence of LANA1. As SCAI has been shown to act as a transcriptional repressor on Serum Responsive Factor mediated transcription while LANA1 has been shown to act as a major activator it seems possible that LANA1 counteracts this repressor function of SCAI during KSHV infections. Experiments to investigate the effect of LANA1 on SCAI function are ongoing. Brd4 is only one example of a host protein whose function is altered by the binding of a viral protein. A second example is the E3 ubiquitin ligase E6AP. E6AP binds to the E6 protein of high risk HPVs and in complex with E6 gains the ability to bind the tumorsupressor p53 resulting in p53 ubiquitination and proteasomal degradation, a step thought to be a major factor in HPV induced cancer development. In collaboration with the Vidal laboratory at the Dana-Farber Cancer Institute I established and performed a yeast-three-hybrid screen that allowed the identification of novel E6AP interaction partners in the absence and presence of HPV16E6. We were able to show that the system is capable to identify proteins interacting with E6AP alone as well as proteins binding to the E6AP/16E6 complex like p53. By comparing the interactome of catalytically active and inactive E6AP variants we were also able to predict which novel E6AP/E6 interactors are potential E6AP substrates. The screen identified three novel potential E6AP/E6 substrates that were identified to interact with E6AP only in the presence of HPV16E6. Experiments to further investigate the biological consequences of these interactions are still ongoing. In summary, the conduction of different protein/protein interaction screens allowed us to identify several new host/virus interactions and a detailed study of those might contribute to our understanding of the molecular mechanisms of infections with these DNA tumorviruses and the subsequent cancer development.
