(Re-)Writing the Ubiquitin Code – Manipulating Polyubiquitin Chain Linkage to Investigate Ubiquitin Signalling in Genome Maintenance and Beyond

Introduction

Ubiquitylation is an essential posttranslational modification that governs the activities and interactions of cellular proteins. The structural diversity of polyubiquitin chains, collectively called the ‘ubiquitin code’, is thought to determine the fate of the modified proteins.

Current Limitations

Although many analytical and inhibitory ubiquitin-specific reagents exist, we lack the tools to create polyubiquitin chains of defined linkage on a protein of interest in cells to investigate their signalling functions.

Proposed Methodology

We recently established a method to induce substrate-specific polyubiquitylation via three major linkages:

1. M1
2. K48
3. K63

Based on this technology, we now propose to develop a new generation of research tools for the scientific community. This will include:

• The identification and design of custom enzymes for assembling the rarer, still poorly characterised non-canonical linkages.
• Functionalisation with modules for chain editing, branching, and the identification of effectors.

Application of Tools

In a combination of biochemical, cell biological, and proteomic approaches, we will then apply these tools to major genome maintenance pathways with prominent roles in the defence against disorders such as cancer and premature ageing.

Focus Areas

Specifically, we will analyse:

• The significance of non-canonical polyubiquitylation in the response to DNA double-strand breaks.
• The interplay between degradative and non-degradative ubiquitylation in the regulation of essential DNA replication and repair factors.

Expected Outcomes

We envision that our research will not only provide new insight into ubiquitin signalling in genome maintenance, but the new technology developed in this project will facilitate future investigations of polyubiquitin chains, their readers, and their writers in other signalling pathways.

Conclusion

Situated at the interface between proteostasis and genome maintenance, this project will thus advance our understanding of two essential cellular surveillance systems with critical functions in human health and well-being.