Probing life-and-death switches using "designer" p53

Proteins are molecular machines that control most biological processes. Among them, a protein called p53 acts as a safeguard against cancer in that it instructs cancers cells to self-destruct before they can multiply. Accordingly, malfunction of p53 is a prerequisite for most human cancers. It is therefore important to understand the molecular mechanism by which p53 operates, alone and in conjunction with other protein partners. At the molecular level, p53 receives a multitude of stress signals from cells that are deposited onto p53 in the form of covalent biochemical signals, so-called post-translational modifications. These signals form a type of code which p53 then integrates into a defined cellular response ranging from "business as usual" and targeted repair processes all the way to the initiation of cell death protocols. However, how exactly p53 and its protein partners decode post-translational modifications remains enigmatic.

This proposal aims to unravel how p53 makes such life-and-death decisions by determining how specific post-translational modifications change the functional interactions between p53 and its protein partners. We are able to achieve this goal because we have developed chemical tools that allows us to synthesise "designer p53" carrying defined modifications. Upon synthesising new versions of "designer p53", we aim to reconstitute biochemical signalling processes in the test tube, allowing us to measure the effect of post-translational modifications under controlled circumstances. Specifically, we aim to address how p53 modifications act as molecular docking platforms to connect with known and new protein partners and convert these interactions into further biochemical signals. In this way, we will illuminate how p53 acts as a molecular signal integration device which sets the foundation for new quantitative insights into its action as a tumour suppressor.

The tumour suppressor p53 orchestrates a cellular response to various stress signals. Its activity is tightly regulated by a plethora of post-translational modifications (PTMs) which are deposited onto p53 by stress-activated enzyme "writers". These biochemical switches are thought to form a code which is interpreted by p53-interacting proteins ("readers"). However, despite decades of intense research, the molecular details of how p53 PTMs modulate protein-protein interactions remains enigmatic. This dearth in knowledge is in part due to the difficulty in obtaining p53 in defined PTM states (inputs) and the complexity of the multivalent interactions (outputs). To address this limitation, we have developed a strategy to access site-specifically phosphorylated p53 via protein semi-synthesis. In this grant, we propose to take advantage of this technology to generate a small library of biologically relevant phospho-p53 variants, enabling us to measure the consequences of p53 PTMs in defined biochemical assays. Specifically, we aim to carry out a suite of mechanistic biochemistry experiments to elucidate the complex crosstalk between p53 phosphorylation and acetylation, mediated by the acetyltransferase p300 (a "writer"). Semi-synthetic p53 will further enable us to discover new p53 PTM "readers" as well as cascades based on proteins that feature both "reader" and "writer" domains. Collectively, these experiments will establish a biochemical basis for how p53 PTMs are interpreted and lays the foundation for multi-pronged approaches to decode how p53 and its interaction partners make cellular life-and-death decisions.