טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentHorowitz Ben
SubjectStudy of the Interactions between the Adenovirus E4ORF4
Protein and its Partners ACP and PP2A
DepartmentDepartment of Medicine
Supervisor Professor Tamar Kleinberger
Full Thesis textFull thesis text - English Version


Abstract

The 14kDa adenovirus E4orf4 protein induces apoptosis in transformed cells upon its individual expression. PP2A is a major serine/threonine phosphatase which is an important E4orf4 partner. Acf1 (Human ATP-utilizing Chromatin assembly and remodeling Factor), a regulatory factor which participates in an ATP-dependent chromatin remodeling complex containing the Snf2h ATPase, is another protein found to be in a physical and functional interaction with E4orf4. PP2A was found to be recruited by E4orf4 to Acf1 and to chromatin. In this work we set out to characterize E4orf4 binding to its known associates Acf1 and PP2A. First, we used GST pull-down assays to map the E4orf4 interaction site within Acf1 to a previously uncharacterized region. However, an Acf1 mutant that we constructed which lacks this site was found to interact with E4orf4 as efficiently as did wild type Acf1, while it did not bind Snf2h. We concluded that E4orf4 may bind Acf1 at more than one site. In the second part we set out to reveal the PP2A-B55α regulatory subunit residues that interact with E4orf4. This was performed by structural and computational methods using the solved B55α structure and a model of E4orf4 structure. Amino acids predicted to participate in the E4orf4-binding site on B55α were mutated and immunoprecipitation experiments revealed that the interaction site included two residues that are part of two α-helices located outside the suggested substrate-binding groove of B55α. This finding might improve our knowledge of the nature of the interaction between the two proteins and its meaning in the context of E4orf4-induced apoptosis. In the third part of the work we performed a wide proteomic search for additional proteins interacting with E4orf4 and for proteins whose interaction with Acf1/Snf2h is altered as a result of E4orf4 expression and tried to characterize the changes in the nuclear phospho-proteome induced by E4orf4 expression. This part was carried out using the SILAC labeling method. The experiments revealed 11 proteins that precipitated with the specific anti-E4orf4 antibody.. The immunoprecipitaion experiments in which anti-Acf1 and anti-Snf2h antibodies were used, identified 68 proteins that precipitated with the anti-Acf1 antibody and 122 proteins that precipitated with the anti-Snf2h antibody. The phospho-peptide analysis of control and E4orf4 expressing cells identified 314 phosphorylated peptides. Out of these peptides, 44 demonstrated an H/L ratio which was significantly different than the population distribution, indicating that these peptides experienced a significant shift in their phosphorylation status upon E4orf4 expression.