Characterization of a p53 tumor suppressor mutant deficient for mitochondrial localization

Details

Supervisors

Dumont, Patrick R.

Faculty

Faculté des sciences

Degree label

Master [120] en biochimie et biologie moléculaire et cellulaire, à finalité spécialisée: biotechnologie

Abstract

The p53 protein is the guardian angel of our genome. It participates in several processes by maintaining integrity and correctness because of its diverse activities. The deviation from optimum triggers p53 to react and perform its functions. A mutation of TP53, the gene encoding for p53, is present in 50% of human cancers. Indeed, the tumor suppressor p53 is a potent cell growth inhibitor and cell death inducer. First, p53 can act as a transcription factor to regulate the expression of many genes whose products promote cell cycle arrest or apoptosis. Second, p53 has a pro-apoptotic mitochondrial activity. In stressed cells, a fraction of p53 can localize to mitochondria and directly interact with different BCL-2 family members to trigger mitochondrial outer membrane permeabilization and the subsequent release of the mitochondrial effectors of apoptosis. Many works indicate that post-translational modifications regulate the activity of p53. Previous studies by C. Castrogiovanni show that serine 392 phosphorylation is crucial for p53 mitochondrial localization in stressed cells. By using CRISPR-Cas9, S392 was replaced by an alanine (non phosphorylable amino acid) in HCT-116 colon carcinoma cells. This substitution markedly impaired the p53 mitochondrial localization and its ability to trigger apoptosis, underlying the role of S392 phosphorylation. To explain the role of S392 phosphorylation, two hypotheses were proposed: (1) the phosphorylation of S392, induced by a stress, allows p53 to interact with a cytosolic protein that promotes its mitochondrial localization; (2) in unstressed cells, cytosolic p53 could be sequestered by one or several proteins. S392 phosphorylation, after a stress, leads to disruption of the inhibitory complex, thus allowing p53 to reach the mitochondria. The ultimate goal here is thus to determine the cytosolic interactions of wt p53 and S392A, which can be helpful to explain the relation between S392 phosphorylation and p53 mitochondrial localization. A proximity-dependent labeling method using APEX2, a modified ascorbate peroxidase, will be employed. The APEX2-p53 fusion has been performed already. We also stably transfected the construction in the H1299 (p53-/-) cell line. The further step will include the characterization of the activity of the fusion protein (p53 transcriptional activity, subcellular localization, and ability to trigger apoptosis).