Characterization of an APEX2-p53 Fusion Protein for the Identification of New p53 Interactants

Details

Supervisors

Dumont, Patrick R.

Faculty

Faculté des sciences

Degree label

Master [120] en biochimie et biologie moléculaire et cellulaire, à finalité approfondie

Abstract

The tumor suppressor p53 is nicknamed the guardian of the genome and is involved in many processes that maintain cell integrity. Under stress, p53 is activated to exert its crucial functions. The gene encoding for p53, TP53, is mutated in 50% of human cancers. The protein p53 executes its role of tumor suppressor through the induction of cell cycle arrest or cell death. It does this by firstly acting as a transcription factor, regulating the expression of a variety of genes encoding for proteins which promote cell cycle arrest or apoptosis. A second pro-apoptotic activity of p53 implies its mitochondrial localization in stressed cells. At the mitochondrial outer membrane, it has direct interactions with various members of the BCL-2 protein family, triggering mitochondrial outer membrane permeabilization. This phenomenon is seen as the point of no return in apoptotic cell death, as it provokes the release of mitochondrial apoptosis effectors. Recently, our lab showed that the phosphorylation of serine 392 is important for the mitochondrial localization of p53 in cells exposed to genotoxic stress. The role of S392 phosphorylation is yet unknown, but two hypotheses exist: (1) S392 phosphorylation is induced by stress signals and promotes interaction between p53 and a cytosolic interaction partner that helps in localizing p53 to the mitochondria; (2) In absence of stress, the cytosolic fraction of p53 is sequestered by one or multiple proteins. Upon stress, S392 is phosphorylated and disrupts the inhibitory complex, allowing p53 to reach the mitochondria. The identification of these unknown cytosolic interaction partners of p53 is the ultimate goal of this project. This will allow us to explain the role of S392 phosphorylation in p53 mitochondrial localization and apoptosis induction. For this, a proximity-dependent labeling method is employed. APEX2 (ascorbate peroxidase 2), has been fused to the N-terminus of p53. The construct has been stably transfected in the H1299 (p53-/-) cell line and the expression of APEX2-p53 is inducible by doxycycline via the Tet-On system. Here, we will characterize the fusion protein: the induction of its expression, its subcellular localization, its transcriptional activity, and its ability to trigger apoptotic cell death. While induction of expression and mitochondrial localization of the APEX2-p53 fusion protein are successful, we failed to see any transcriptional activity. Moreover, APEX2-p53 appears unable to trigger cell cycle arrest or apoptosis. Characterization of our cell lines using immunofluorescence microscopy showed that a small proportion of cells correctly express the APEX2-p53 fusion protein. We conclude that currently, the cell lines generated are not suitable for future experiments and we propose different approaches to solve the issues encountered.