טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentLivne Adi
SubjectAnalysis of the Mechanism Underlying Induction of Apoptosis
by Adenovirus E4orf4 Protein
DepartmentDepartment of Medicine
Supervisors Professor Adi Salzberg
Professor Tamar Kleinberger


Abstract

 Adenovirus type 5 E4 open-reading-frame 4 (E4orf4) protein is a multifunctional viral regulator shown to down-regulate gene expression of E1A-activated genes, to control alternative splicing of adenovirus mRNAs, and to induce p53-independent apoptosis. Induction of apoptosis by E4orf4 is transformed cell-specific, suggesting that this protein has a terapeutic potential for cancer. When investigating the mechanisms underlying E4orf4 action, we found that the E4orf4 protein binds several cellular proteins, including protein phosphatase 2A (PP2A). we further showed that an interaction of E4orf4 with a subpopulation of PP2A heterotrimers containing the Bα subunit, but not the B’ subunits, is required for induction of apoptosis.

Different stages in the apoptotic pathway occur through the activation and function of caspases, a highly conserved family of cysteine proteases with specificity for aspartic acid residues in their substrates. It was previously reported that E4orf4-induced apoptosis did not involve induction of known caspases in CHO cells. However, recent results from our laboratory show that some apoptotic morphologies induced by E4orf4 are caspase-dependent in H1299 and 293T cells, since their appearance can be inhibited by the general caspase inhibitor zVAD-fmk. In these cells E4orf4 partially activastes the mitochondrial apoptotic pathway, and furthermore, we have shown that E4orf4 operates, in 293T cells, through the death receptor pathway (FAS/TNF), to activate the DISC (death inducing signaling complex).

Reactive Oxygen Species (ROS) have been reported to be important players in apoptosis. We show that E4orf4 induces accumulation of ROS, and that these high levels of ROS are required for E4orf4-induced apoptosis.

E4orf4 Cellular localization may also play a role in E4orf4-induced apoptosis. Observations from our laboratory show that E4orf4, which is present in the nucleus prior to, or at an early stage of the apoptotic process, translocates to the cell periphery later on. We show that E4orf4 goes through unique cellular localization changes which may be required for E4orf4-induced apoptosis.